The SARS epidemic started in the weeks that the ‘allied forces’ were waging war on Iraq to hunt down Saddam Hussein and his still elusive ‘weapons of mass destruction’.
SARS – Severe Acute Respiratory Syndrome – is a completely new infectious disease spread by human contact. By 20 June 2003, World Health Organisation figures registered 8461 cases in 31 countries worldwide, and 804 deaths. The overall death rate is nearly 10% and could be 20% or higher.
Although there are signs that the disease is under control, there are also fears that it may return.
Mystery of the SARS virus
The World Health Organisation, which played the key role in coordinating the research of a dozen laboratories, formally announced on 16 April that a new pathogen, a member of the coronavirus family never before seen in humans, is the cause of SARS, though lingering doubt has remained. The virus cannot be identified all patients diagnosed with SARS, and it can only be isolated from cultured green monkey kidney cells.
Known coronaviruses are placed in three groups based on similarities in their genomes. Group 1 contains the porcine epidemic diarrhoea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), canine coronavirus (CCV), feline infectious peritonitis virus (FIPV) and human coronovirus 229E (HuCV229E); Group 2 contains the avian infectious bronchitis virus (AIBV) and turkey coronavirus; while Group 3 contains the murine hepatitis virus (MHV) bovine coronavirus (BCV), human coronavirus (HuOC43) and others.
The molecular phylogenies published 10 April in the New England Journal of Medicine, based on small fragments of the polymerase gene, have placed the SARS virus in a separate group somewhere between groups 2 and 3.
More detailed analysis, subsequently published in the New England Journal of Medicine, Science and the Lancet indicate that the new virus is not closely related to any known virus at all, human, mouse, bovine, cat, pig, bird, notwithstanding. It is neither a mutant that switched host, nor a recombinant from existing coronaviruses. It is more complicated than that.
SARS virus - a product of genetic engineering?
Two scientists who have genetic engineered coronaviruses in their laboratories, Holmes and Enjuanes, suggested in a commentary in the journal Science that the SARS virus probably "evolved separately from an ancestor of the known coronavirus, and infected an unidentified animal, bird, or reptile host for a very long time before infecting humans and starting the SARS epidemic." (p.1377)
Following soon afterwards, there was a claim that the SARS virus came from the masked civet cat in south China. But that claim could not be substantiated. An alternative hypothesis entertained in the mainstream journals was that the virus came from outerspace.
There are very unusual features to the SARS virus. Its sequence most closely matches that of mouse hepatitis virus (MHV) and Bovine corona virus (BCV), both in group 3. The match is quite good in the middle third of the genome that’s nearly 30 000nt long, and not good at all for the first third or last third of the sequence.
But, antibodies to the SARS virus cross react with FIPV, HuCV229E and TGEV, all in Group 1. And the SARS virus can grow in Vero green monkey kidney cells, which no other coronavirus can, with the exception of PEDV, another virus in Group 1.
Could the SARS virus have come from genetic engineering? This is a question that Ho and Cummins have put to the scientific community. So far, we have not had a proper reply.
Holmes and Enjuanes stated in their commentary, "SARS-CoV is also unlikely to have been created from known coronaviruses by genetic engineering, because at present it would be impossible to modify 50% of a coronavirus genome without abrogating viral infectivity."
This is a quite a feeble response. The whole point to genetic engineering is that it greatly increases the scope of recombination, and provides selective tools to find the most unlikely recombinants that are still infectious.
Coronaviruses have been subjected to increasing genetic manipulation since the latter half of the 1990s, when P.S. Masters in Wadworth Center, New York State Department of Health and New York State University at Albany, used RNA recombination to introduce extensive changes into the genome of mouse hepatitis virus (MHV). In a review published in 1999, he wrote, "targeted recombination could be used to create extensive substitutions to the cornavirus genome, generating recombinants that could not be made otherwise between two viruses separated by a species barrier." (p.254)
‘Defective interfering RNAs’ – sequences of the viral genome with large deletions as well as mutations and substitutions or insertions - were used as donor sequences to introduce major substitutions and point mutations into the genome of the viruses by RNA recombination.
In the course of such work, researchers have even isolated a recombinant of cororanvirus with the green fluorescent protein (GFP) gene, presumably from cells in which coronaviruses have been cultured, which has become inserted into the spike protein gene. The GFP gene, originally from a jelly-fish, is extensively used in genetic engineering as a marker gene because it makes the cells that have taken up the foreign genes give off a green glow under uv light. The GFP-coronavirus recombinant could only have come about as an unintended by-product of genetic engineering.
In the same review, P.S. Masters showed that both point mutations and large substitutions can readily be transferred to the last third of the genome of MHV and other coronaviruses. He further indicated that similar strategies could be used to mutate and substitute the first third of the genome, though not for the middle third. "A comprehensive genetic study of the highly complex gene for the RNA polymerase and all of its associated activities [encoded by the middle third of the genome] will likely await either the construction of an infectious full-length clone or the development of an innovative scheme for mutant selection." (p.259)
Is that why the middle third of SARS virus genome has retained good homology to MHV and BCV, which were the first coronaviruses to be engineered in this manner, while the other parts are much more different?
Another feature of the SARS virus is that the spike protein, which determines host range, is unlike the spike protein of any known coronavirus. Instead, it appears to have homologies to segments of the human chromosome 7, according to sequence analysis performed by Howard Urnovitz.
Urnovitz believes that the spike protein of the SARS virus is the result of genetic rearrangements provoked by environmental genotoxic agents, much like those he and his colleagues have detected in Gulf War I veterans suffering from Gulf War Syndrome.
But how did the virus get to south China? A possible answer was provided by Urnovitz: Migratory birds that frequent gene-swapping hot spots like southeast China could have carried the SARS virus there.
Urnovitz himself doesn’t think the SARS virus is the real cause of SARS. Instead, it is the piece of reshuffled human chromosome 7 that others are referring to as the spike protein gene of the SARS virus. That alone is sufficient to trigger serious autoimmune responses in people.
Hence, to create vaccines against that ‘spike’ protein is also tantamount to vaccinating people against their own genes (see "Dynamic genomics")
Friday, October 23, 2009
Adult Bone Marrow Cells Mend Heart without Transplant
Sudden blockages of a major artery to the heart cuts off blood supply and lead to rapid death of the muscle cells and blood vessels in the heart. This condition, myocardial infarction, is a common form of heart disease. Despite the demonstration that some of the heart muscle cells can multiply and new vessels formed, regeneration is restricted to the living part of the heart wall. The ‘infarcted’ or dead area is irreversible, and in time, scar tissue is formed. Attempts to replace the dead tissue by transplanting heart muscle cells or skeletal muscle cells have failed to mend the damaged part properly.
In previous experiments on mice, researchers in New York Medical College and the National Institute of Health injected bone marrow cells along the border of the damaged area of the heart, and found that the cells did differentiate into muscle and blood vessels. But this surgical intervention killed a high number of the mice and the grafting success was only 40%. This prompted them to consider a ‘non-invasive’ method, which involved stimulating the mice to overproduce bone marrow cells before and after myocardial infarction was induced .
For the purpose, the mice were given daily injections of two cytokines (small molecules that influence the activities of cells), stem cell factor (SCF) and granulocyte-colony-stimulating factor (G-CSF), which increased the number of circulating stem cells two to three hundred fold.
Mice given cytokines had a survival rate of 73% after the operation, compared with 20% in controls not given cytokines. There were clear signs of repair in the damaged area of the heart in the cytokine-injected group, both new heart muscle and blood vessels were formed, whereas only scar-tissue was found in controls. The hearts of the cytokine-injected group also performed significantly better than the controls.
The experimental results looked impressive enough even though the protocol of inducing myocardial infarction in such large numbers of animals is debatable. In addition, there was an unaccountably small number of experimental animals, only 15 compared to 52 in the group of controls. This may be because the researchers excluded mice that died within 48h of the operation, "to minimize the influence of the surgical trauma". But could it be that the mice died from stress of overproduction of bone marrow cells caused by the cytokines injected? There are certainly more ways to be invasive; and much more effort should be devoted to reducing unnecessary and stressful interventions, both physical and chemical.
In previous experiments on mice, researchers in New York Medical College and the National Institute of Health injected bone marrow cells along the border of the damaged area of the heart, and found that the cells did differentiate into muscle and blood vessels. But this surgical intervention killed a high number of the mice and the grafting success was only 40%. This prompted them to consider a ‘non-invasive’ method, which involved stimulating the mice to overproduce bone marrow cells before and after myocardial infarction was induced .
For the purpose, the mice were given daily injections of two cytokines (small molecules that influence the activities of cells), stem cell factor (SCF) and granulocyte-colony-stimulating factor (G-CSF), which increased the number of circulating stem cells two to three hundred fold.
Mice given cytokines had a survival rate of 73% after the operation, compared with 20% in controls not given cytokines. There were clear signs of repair in the damaged area of the heart in the cytokine-injected group, both new heart muscle and blood vessels were formed, whereas only scar-tissue was found in controls. The hearts of the cytokine-injected group also performed significantly better than the controls.
The experimental results looked impressive enough even though the protocol of inducing myocardial infarction in such large numbers of animals is debatable. In addition, there was an unaccountably small number of experimental animals, only 15 compared to 52 in the group of controls. This may be because the researchers excluded mice that died within 48h of the operation, "to minimize the influence of the surgical trauma". But could it be that the mice died from stress of overproduction of bone marrow cells caused by the cytokines injected? There are certainly more ways to be invasive; and much more effort should be devoted to reducing unnecessary and stressful interventions, both physical and chemical.
Thursday, October 22, 2009
Scrambled Genomes in Human Gene Therapy and Transgenic Plants
Human gene therapy is usually considered separate and distinct from genetic modification (GM) of crops, but this is misleading.
Adeno-associated virus vectors (AAV) are most commonly used in clinical gene-therapy trials. The wild-type adenovirus has terminal repeats that enable it to integrate into human chromosome 19 at a specific site. The AAV vector, however, has no specific integration site. It often integrates into chromosome 19, though not at the integration site of the wild-type virus, and it may also integrate into any of the other human chromosomes. AAV vector is preferred for most gene therapy experiments because the chromosome insertion is more stable and the AAV vector transforms both dividing and non-dividing cells.
Crop GM is achieved using Agrobacterium transformation or direct plasmid transfer using biolistic transformation (gene gun) methods. The Agrobacterium T –DNA vector is flanked by 25 base-pair direct repeats that facilitate integration of plasmid sequences into the plant chromosome.
The common features of gene therapy in human cells and crop GM is the presence of integrating vector sequences flanking transgene(s) each equipped with a promoter to drive expression.
While many studies have been carried out on transgenic DNA in plants, there have been relatively few that analyze host genome at the site of insertion. In a recent issue of Nature Genetics, researchers in the Department of Medicine, University of Washington Seattle, report that integrated AAV are associated with chromosomal deletions and other rearrangements and are frequently located on chromosome 19 (although not at the wildtype AAV integration site).
The researchers analysed the chromosomal DNA flanking the site of vector insertion. By searching the human genome sequence databases, the junctions were located to 6 different chromosomes. Four integrated into genes. Four of nine inserts went into a relatively large, 22-Mb (millions of base pairs) region of chromosome 10, but not the wild type site, which only spans 1kb.
Chromosomal deletions and additions were found, as well as translocations of parts of one chromosome to another. There were also unexpected vector sequences at integration sites.
The study is reminiscent of the recent finding of unexpected sequences and genome scrambling associated with transgenes in GM crops such as soybean (see "Scrambled genome of Roundup Ready Soya", ISIS News 9/10 www.i-sis.org.uk). Further analysis is likely to show that scrambled and unexpected sequences are commonplace in GM crops. We have questioned the legality and safety of approving crops that contain unknown, uncharacterised DNA sequences. If scrambled and unexpected sequences are found even in the most widely distributed and established commercial GM crop, the problems are likely to be worse with newer transgenic crops of corn, cotton or canola, which have yet to be analysed. Certainly, government regulators and their academic satellites seem passive and submissive in dealing with important findings that question the safety of GM crops.
The observation that gene scrambling occurs in both human gene therapy and in GM crops suggest that there is a fundamental flaw in both genetic engineering technology and in the auditing of molecular properties of the modified humans or crops.
The corporate audit of molecular characteristics in gene therapy vectors and those of GM crops may be analogous to the Enron audits of the Arthur Anderson Accounting Firm. Those outside of direct involvement in gene therapy and crop genetic modification should be well enough informed to require full and truthful molecular audits of gene therapy vectors and GM crops.
Adeno-associated virus vectors (AAV) are most commonly used in clinical gene-therapy trials. The wild-type adenovirus has terminal repeats that enable it to integrate into human chromosome 19 at a specific site. The AAV vector, however, has no specific integration site. It often integrates into chromosome 19, though not at the integration site of the wild-type virus, and it may also integrate into any of the other human chromosomes. AAV vector is preferred for most gene therapy experiments because the chromosome insertion is more stable and the AAV vector transforms both dividing and non-dividing cells.
Crop GM is achieved using Agrobacterium transformation or direct plasmid transfer using biolistic transformation (gene gun) methods. The Agrobacterium T –DNA vector is flanked by 25 base-pair direct repeats that facilitate integration of plasmid sequences into the plant chromosome.
The common features of gene therapy in human cells and crop GM is the presence of integrating vector sequences flanking transgene(s) each equipped with a promoter to drive expression.
While many studies have been carried out on transgenic DNA in plants, there have been relatively few that analyze host genome at the site of insertion. In a recent issue of Nature Genetics, researchers in the Department of Medicine, University of Washington Seattle, report that integrated AAV are associated with chromosomal deletions and other rearrangements and are frequently located on chromosome 19 (although not at the wildtype AAV integration site).
The researchers analysed the chromosomal DNA flanking the site of vector insertion. By searching the human genome sequence databases, the junctions were located to 6 different chromosomes. Four integrated into genes. Four of nine inserts went into a relatively large, 22-Mb (millions of base pairs) region of chromosome 10, but not the wild type site, which only spans 1kb.
Chromosomal deletions and additions were found, as well as translocations of parts of one chromosome to another. There were also unexpected vector sequences at integration sites.
The study is reminiscent of the recent finding of unexpected sequences and genome scrambling associated with transgenes in GM crops such as soybean (see "Scrambled genome of Roundup Ready Soya", ISIS News 9/10 www.i-sis.org.uk). Further analysis is likely to show that scrambled and unexpected sequences are commonplace in GM crops. We have questioned the legality and safety of approving crops that contain unknown, uncharacterised DNA sequences. If scrambled and unexpected sequences are found even in the most widely distributed and established commercial GM crop, the problems are likely to be worse with newer transgenic crops of corn, cotton or canola, which have yet to be analysed. Certainly, government regulators and their academic satellites seem passive and submissive in dealing with important findings that question the safety of GM crops.
The observation that gene scrambling occurs in both human gene therapy and in GM crops suggest that there is a fundamental flaw in both genetic engineering technology and in the auditing of molecular properties of the modified humans or crops.
The corporate audit of molecular characteristics in gene therapy vectors and those of GM crops may be analogous to the Enron audits of the Arthur Anderson Accounting Firm. Those outside of direct involvement in gene therapy and crop genetic modification should be well enough informed to require full and truthful molecular audits of gene therapy vectors and GM crops.
Saturday, October 3, 2009
Stem cell
Stem cells are cells found in most, if not all, multi-cellular organisms. They are characterized by the ability to renew themselves through mitotic cell division and differentiating into a diverse range of specialized cell types. Research in the stem cell field grew out of findings by Canadian scientists Ernest A. McCulloch and James E. Till in the 1960s. The two broad types of mammalian stem cells are: embryonic stem cells that are isolated from the inner cell mass of blastocysts, and adult stem cells that are found in adult tissues. In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing specialized cells, but also maintain the normal turnover of regenerative organs, such as blood, skin or intestinal tissues.
Stem cells can now be grown and transformed into specialized cells with characteristics consistent with cells of various tissues such as muscles or nerves through cell culture. Highly plastic adult stem cells from a variety of sources, including umbilical cord blood and bone marrow, are routinely used in medical therapies. Embryonic cell lines and autologous embryonic stem cells generated through therapeutic cloning have also been proposed as promising candidates for future therapies.
Properties
The classical definition of a stem cell requires that it possess two properties:
* Self-renewal - the ability to go through numerous cycles of cell division while maintaining the undifferentiated state.
* Potency - the capacity to differentiate into specialized cell types. In the strictest sense, this requires stem cells to be either totipotent or pluripotent - to be able to give rise to any mature cell type, although multipotent or unipotent progenitor cells are sometimes referred to as stem cells.
Potency definitions
Pluripotent, embryonic stem cells originate as inner mass cells within a blastocyst. The stem cells can become any tissue in the body, excluding a placenta. Only the morula's cells are totipotent, able to become all tissues and a placenta.
Human embryonic stem cells
A: Cell colonies that are not yet differentiated.
B: Nerve cell
Potency specifies the differentiation potential (the potential to differentiate into different cell types) of the stem cell.
* Totipotent (a.k.a omnipotent) stem cells can differentiate into embryonic and extraembryonic cell types. Such cells can construct a complete, viable, organism. These cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent.
* Pluripotent stem cells are the descendants of totipotent cells and can differentiate into nearly all cells, i.e. cells derived from any of the three germ layers.
* Multipotent stem cells can differentiate into a number of cells, but only those of a closely related family of cells.
* Oligopotent stem cells can differentiate into only a few cells, such as lymphoid or myeloid stem cells.
* Unipotent cells can produce only one cell type, their own, but have the property of self-renewal which distinguishes them from non-stem cells (e.g. muscle stem cells).
Identification
The practical definition of a stem cell is the functional definition - a cell that has the potential to regenerate tissue over a lifetime. For example, the gold standard test for a bone marrow or hematopoietic stem cell (HSC) is the ability to transplant one cell and save an individual without HSCs. In this case, a stem cell must be able to produce new blood cells and immune cells over a long term, demonstrating potency. It should also be possible to isolate stem cells from the transplanted individual, which can themselves be transplanted into another individual without HSCs, demonstrating that the stem cell was able to self-renew.
Properties of stem cells can be illustrated in vitro, using methods such as clonogenic assays, where single cells are characterized by their ability to differentiate and self-renew. As well, stem cells can be isolated based on a distinctive set of cell surface markers. However, in vitro culture conditions can alter the behavior of cells, making it unclear whether the cells will behave in a similar manner in vivo. Considerable debate exists whether some proposed adult cell populations are truly stem cells.
Embryonic
Embryonic stem cell lines (ES cell lines) are cultures of cells derived from the epiblast tissue of the inner cell mass (ICM) of a blastocyst or earlier morula stage embryos. A blastocyst is an early stage embryo—approximately four to five days old in humans and consisting of 50–150 cells. ES cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type. They do not contribute to the extra-embryonic membranes or the placenta.
Nearly all research to date has taken place using mouse embryonic stem cells (mES) or human embryonic stem cells (hES). Both have the essential stem cell characteristics, yet they require very different environments in order to maintain an undifferentiated state. Mouse ES cells are grown on a layer of gelatin and require the presence of Leukemia Inhibitory Factor (LIF). Human ES cells are grown on a feeder layer of mouse embryonic fibroblasts (MEFs) and require the presence of basic Fibroblast Growth Factor (bFGF or FGF-2). Without optimal culture conditions or genetic manipulation, embryonic stem cells will rapidly differentiate.
A human embryonic stem cell is also defined by the presence of several transcription factors and cell surface proteins. The transcription factors Oct-4, Nanog, and Sox2 form the core regulatory network that ensures the suppression of genes that lead to differentiation and the maintenance of pluripotency. The cell surface antigens most commonly used to identify hES cells are the glycolipids SSEA3 and SSEA4 and the keratan sulfate antigens Tra-1-60 and Tra-1-81. The molecular definition of a stem cell includes many more proteins and continues to be a topic of research.
After nearly ten years of research, there are no approved treatments using embryonic stem cells. The first human trial was approved by the US Food & Drug Administration in January 2009. ES cells, being pluripotent cells, require specific signals for correct differentiation - if injected directly into another body, ES cells will differentiate into many different types of cells, causing a teratoma. Differentiating ES cells into usable cells while avoiding transplant rejection are just a few of the hurdles that embryonic stem cell researchers still face. Many nations currently have moratoria on either ES cell research or the production of new ES cell lines. Because of their combined abilities of unlimited expansion and pluripotency, embryonic stem cells remain a theoretically potential source for regenerative medicine and tissue replacement after injury or disease.
Stem cells can now be grown and transformed into specialized cells with characteristics consistent with cells of various tissues such as muscles or nerves through cell culture. Highly plastic adult stem cells from a variety of sources, including umbilical cord blood and bone marrow, are routinely used in medical therapies. Embryonic cell lines and autologous embryonic stem cells generated through therapeutic cloning have also been proposed as promising candidates for future therapies.
Properties
The classical definition of a stem cell requires that it possess two properties:
* Self-renewal - the ability to go through numerous cycles of cell division while maintaining the undifferentiated state.
* Potency - the capacity to differentiate into specialized cell types. In the strictest sense, this requires stem cells to be either totipotent or pluripotent - to be able to give rise to any mature cell type, although multipotent or unipotent progenitor cells are sometimes referred to as stem cells.
Potency definitions
Pluripotent, embryonic stem cells originate as inner mass cells within a blastocyst. The stem cells can become any tissue in the body, excluding a placenta. Only the morula's cells are totipotent, able to become all tissues and a placenta.
Human embryonic stem cells
A: Cell colonies that are not yet differentiated.
B: Nerve cell
Potency specifies the differentiation potential (the potential to differentiate into different cell types) of the stem cell.
* Totipotent (a.k.a omnipotent) stem cells can differentiate into embryonic and extraembryonic cell types. Such cells can construct a complete, viable, organism. These cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent.
* Pluripotent stem cells are the descendants of totipotent cells and can differentiate into nearly all cells, i.e. cells derived from any of the three germ layers.
* Multipotent stem cells can differentiate into a number of cells, but only those of a closely related family of cells.
* Oligopotent stem cells can differentiate into only a few cells, such as lymphoid or myeloid stem cells.
* Unipotent cells can produce only one cell type, their own, but have the property of self-renewal which distinguishes them from non-stem cells (e.g. muscle stem cells).
Identification
The practical definition of a stem cell is the functional definition - a cell that has the potential to regenerate tissue over a lifetime. For example, the gold standard test for a bone marrow or hematopoietic stem cell (HSC) is the ability to transplant one cell and save an individual without HSCs. In this case, a stem cell must be able to produce new blood cells and immune cells over a long term, demonstrating potency. It should also be possible to isolate stem cells from the transplanted individual, which can themselves be transplanted into another individual without HSCs, demonstrating that the stem cell was able to self-renew.
Properties of stem cells can be illustrated in vitro, using methods such as clonogenic assays, where single cells are characterized by their ability to differentiate and self-renew. As well, stem cells can be isolated based on a distinctive set of cell surface markers. However, in vitro culture conditions can alter the behavior of cells, making it unclear whether the cells will behave in a similar manner in vivo. Considerable debate exists whether some proposed adult cell populations are truly stem cells.
Embryonic
Embryonic stem cell lines (ES cell lines) are cultures of cells derived from the epiblast tissue of the inner cell mass (ICM) of a blastocyst or earlier morula stage embryos. A blastocyst is an early stage embryo—approximately four to five days old in humans and consisting of 50–150 cells. ES cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type. They do not contribute to the extra-embryonic membranes or the placenta.
Nearly all research to date has taken place using mouse embryonic stem cells (mES) or human embryonic stem cells (hES). Both have the essential stem cell characteristics, yet they require very different environments in order to maintain an undifferentiated state. Mouse ES cells are grown on a layer of gelatin and require the presence of Leukemia Inhibitory Factor (LIF). Human ES cells are grown on a feeder layer of mouse embryonic fibroblasts (MEFs) and require the presence of basic Fibroblast Growth Factor (bFGF or FGF-2). Without optimal culture conditions or genetic manipulation, embryonic stem cells will rapidly differentiate.
A human embryonic stem cell is also defined by the presence of several transcription factors and cell surface proteins. The transcription factors Oct-4, Nanog, and Sox2 form the core regulatory network that ensures the suppression of genes that lead to differentiation and the maintenance of pluripotency. The cell surface antigens most commonly used to identify hES cells are the glycolipids SSEA3 and SSEA4 and the keratan sulfate antigens Tra-1-60 and Tra-1-81. The molecular definition of a stem cell includes many more proteins and continues to be a topic of research.
After nearly ten years of research, there are no approved treatments using embryonic stem cells. The first human trial was approved by the US Food & Drug Administration in January 2009. ES cells, being pluripotent cells, require specific signals for correct differentiation - if injected directly into another body, ES cells will differentiate into many different types of cells, causing a teratoma. Differentiating ES cells into usable cells while avoiding transplant rejection are just a few of the hurdles that embryonic stem cell researchers still face. Many nations currently have moratoria on either ES cell research or the production of new ES cell lines. Because of their combined abilities of unlimited expansion and pluripotency, embryonic stem cells remain a theoretically potential source for regenerative medicine and tissue replacement after injury or disease.
Friday, October 2, 2009
Molecular Genetic Engineers in Junk DNA
Perhaps only 1% of the human genome codes for genes, and that’s what the human genome map contains. The rest is mainly repetitive DNA, commonly known as ‘junk DNA’.
However, evidence has been emerging that lurking within junk DNA are armies of transposons (mobile genetic elements) that play an indispensable role in ‘natural genetic engineering’ the genome. They make up nearly half of the human genome, and serve as ‘recombination hotspots’ for cutting and splicing, and hence reshuffling the genome. They are also a source of ready to use motifs for gene expression, as well as new protein-coding sequences.
These important transposons are scattered throughout the genome. There are two main categories: Long Interspersed Elements (LINEs) about 6.7 kilobasepairs in length and Short Interspersed Elements (SINEs) of several hundred basepairs.
The most abundant SINEs are Alu elements, of which 1.4 million copies exist, comprising 10% of the human genome, and are apparently only found in primates.
Most LINEs are dormant, except for LINE1. But both LINE 1 and other LINEs are abundant in areas of the genome where the bases A and T predominate, which do not have many genes. Alu elements, however, are more common in ‘GC-rich’regions that are also gene-rich. This is quite baffling as Alu itself cannot move, but depends on enzymes encoded by LINE1 in order to insert itself.
Alu elements, like LINE1 are retrotransposons that move and multiply by being transcribed into RNA, then reverse transcribed into DNA copies that jump into new sites.
New research is suggesting that Alu elements may help create new proteins from existing ones. The reasons the human genome contains so few genes – the latest count is just under 25 000, is that more than half the genes are interrupted and subject to alternative splicing.
In other words, the coding sequence of the gene is broken up into segments (exons) interrupted by non-coding segments (introns) that are spliced out from the RNA transcript before it is translated into protein. But there are alternative splicing pathways that join different exons together, with the result that many different proteins can be made from a single gene.
It appears that about 5% of alternatively spliced internal exons in the human genome originate in an Alu sequence. It suggests that Alu elements can actually jump into genes and, instead of destroying that gene, actually contributes a new coding sequence to it.
There are two ways in which this could be done, either by jumping into an existing gene so that the gene gains a new exon and increases its repertoire of possible proteins by alternative splicing, or else, the gene can become duplicated first, with one copy remaining intact, while the other is crafted by the Alu element. Currently, it is estimated that 238 000 Alu elements are located within introns of protein-coding genes and each one can actually or potentially become an exon.
Gil Ast, head of a group in Tel Aviv University, Israel, which has made some of the most significant discoveries about Alu, is understandably pleased. "We believe that Alus allowed the shuffling of genetic information that may have led to the evolution of primates," said Ast. "They may contribute to a lot of disorders we don’t even know about yet. But they have also created genetic diversity."
Alternative splicing is quite precisely regulated, and all other things being equal, depends on the spacing of potential splice sites consisting of the dinucleotide, AG, as well as neighbouring nucleotides. A single base change can destroy alternative splicing, and this can cause a disease, as in the case of Alport syndrome.
Also, gaining an exon is not always a good thing. There are numerous ‘insertion mutations’ associated with the inappropriate insertion of transposons into genes. And even Alu sequences that are already in introns can cause problems. More than a decade ago, it was discovered that a point mutation in an Alu element residing in the third intron of the ornithine aminotransferase gene activated a cryptic splice site, and led to part of the Alu element becoming incorporated into the coding sequence. Unfortunately, it carried a stop codon, which cut the protein short, leading to ornithine aminotransferase deficiency.
There is increasing evidence that physical and chemical stresses to the cell, such as heat shock, chemical poisons and viral infections, tend to activate Alu elements. The resultant gene reshuffling may be responsible for a variety of chronic diseases.
source:- i-sis.org.uk
However, evidence has been emerging that lurking within junk DNA are armies of transposons (mobile genetic elements) that play an indispensable role in ‘natural genetic engineering’ the genome. They make up nearly half of the human genome, and serve as ‘recombination hotspots’ for cutting and splicing, and hence reshuffling the genome. They are also a source of ready to use motifs for gene expression, as well as new protein-coding sequences.
These important transposons are scattered throughout the genome. There are two main categories: Long Interspersed Elements (LINEs) about 6.7 kilobasepairs in length and Short Interspersed Elements (SINEs) of several hundred basepairs.
The most abundant SINEs are Alu elements, of which 1.4 million copies exist, comprising 10% of the human genome, and are apparently only found in primates.
Most LINEs are dormant, except for LINE1. But both LINE 1 and other LINEs are abundant in areas of the genome where the bases A and T predominate, which do not have many genes. Alu elements, however, are more common in ‘GC-rich’regions that are also gene-rich. This is quite baffling as Alu itself cannot move, but depends on enzymes encoded by LINE1 in order to insert itself.
Alu elements, like LINE1 are retrotransposons that move and multiply by being transcribed into RNA, then reverse transcribed into DNA copies that jump into new sites.
New research is suggesting that Alu elements may help create new proteins from existing ones. The reasons the human genome contains so few genes – the latest count is just under 25 000, is that more than half the genes are interrupted and subject to alternative splicing.
In other words, the coding sequence of the gene is broken up into segments (exons) interrupted by non-coding segments (introns) that are spliced out from the RNA transcript before it is translated into protein. But there are alternative splicing pathways that join different exons together, with the result that many different proteins can be made from a single gene.
It appears that about 5% of alternatively spliced internal exons in the human genome originate in an Alu sequence. It suggests that Alu elements can actually jump into genes and, instead of destroying that gene, actually contributes a new coding sequence to it.
There are two ways in which this could be done, either by jumping into an existing gene so that the gene gains a new exon and increases its repertoire of possible proteins by alternative splicing, or else, the gene can become duplicated first, with one copy remaining intact, while the other is crafted by the Alu element. Currently, it is estimated that 238 000 Alu elements are located within introns of protein-coding genes and each one can actually or potentially become an exon.
Gil Ast, head of a group in Tel Aviv University, Israel, which has made some of the most significant discoveries about Alu, is understandably pleased. "We believe that Alus allowed the shuffling of genetic information that may have led to the evolution of primates," said Ast. "They may contribute to a lot of disorders we don’t even know about yet. But they have also created genetic diversity."
Alternative splicing is quite precisely regulated, and all other things being equal, depends on the spacing of potential splice sites consisting of the dinucleotide, AG, as well as neighbouring nucleotides. A single base change can destroy alternative splicing, and this can cause a disease, as in the case of Alport syndrome.
Also, gaining an exon is not always a good thing. There are numerous ‘insertion mutations’ associated with the inappropriate insertion of transposons into genes. And even Alu sequences that are already in introns can cause problems. More than a decade ago, it was discovered that a point mutation in an Alu element residing in the third intron of the ornithine aminotransferase gene activated a cryptic splice site, and led to part of the Alu element becoming incorporated into the coding sequence. Unfortunately, it carried a stop codon, which cut the protein short, leading to ornithine aminotransferase deficiency.
There is increasing evidence that physical and chemical stresses to the cell, such as heat shock, chemical poisons and viral infections, tend to activate Alu elements. The resultant gene reshuffling may be responsible for a variety of chronic diseases.
source:- i-sis.org.uk
Endogenous Viruses and Chronic Disease
Retroviruses are RNA viruses found in all vertebrates. The retrovirus reproduces by reverse transcribing (copying) its RNA into DNA that is then integrated into the host-cell genome to be transcribed into viruses. Many retroviral sequences have become permanently integrated into the human genome as human endogenous retroviruses, or HERVs. The human genome (indeed all genomes) also contains retrovirus-like retrotransposons, mobile elements that multiply by making RNA copies that are reverse transcribed into DNA and integrated into new sites in the genome. The main difference between a retrovirus and a retrotransposon is that the latter lacks an envelope.
Most HERVs and retrotransposons are defective, having lost one or more gene functions; but can nevertheless multiply and move with the help of other elements or infecting viruses.
Which came first, retrovirus or retrotransposon? As viruses depend on cells to reproduce, it seems reasonable to suppose that retroviruses are simply retrotransposons that have gained an envelop, which helps in entering other cells. That idea was first put forward by Howard Temin, who discovered reverse transcriptase, the enzyme that reverse transcribes, encoded by the retrovirus and retrotransposon.
Retrotransposons play an important role in turning genes on or off, and in rearranging and amplifying genes during development and cell differentiation. And evidence has been accumulating that they are the natural molecular genetic engineers of the fluid genome, which are necessary for the survival of the organism .
HERVs are flanked by ‘long terminal repeats’ that contain strong promoters for gene expression. Promoters are stretches of DNA with binding sites for transcription factors of the host cell that boosts transcription, effectively saying to the cell, "make many copies of the message following". HERVs and retrotransposons are regulated by the cell, and ultimately, by the organism as a whole, which stops most of them from being expressed; though they can be activated under a variety of conditions (see below).
Many endogenous viruses show xenotropism, i.e., they are not active in the host cell but can become infectious to cells of non-host species. Xenotropism is one of the major health hazards of xenotransplantation, the transplant of organs and tissues between species, as from pig to humans.
Defective or dormant HERVs, like defective retrotransposons, can become expressed when missing gene-functions are provided by a ‘helper’ virus that happens to infect the cell, and that includes ‘attenuated’ viruses in vaccines. Like retrotransposons, HERVs can also be induced: by X-rays or various chemical agents and drugs, such as inhibitors of protein synthesis, organophosphates and other pesticides, inflammatory cytokines (hormone-like factors that influence cells of the immune system) or steroid hormones, and retinoic acid.
In a comprehensive review published in 1996, virologists Howard Urnovitz and William Murphy raised the possibility that many chronic debilitating diseases may be linked to HERVs. These include leukaemia and other cancers, B-cell immunoglobulin diseases, inflammatory diseases of the nervous system, autoimmune rheumatic and connective tissue disease and chronic fatigue syndrome.
There are several mechanisms linking HERVs with chronic diseases, though it is not at all clear which mechanism comes into effect under different circumstances.
One way in which endogenous viruses can cause disease is for them to move and insert itself next to certain genes, that, when over-expressed, results in uncontrolled cell division, or cancer. This mechanism may be involved in mouse and human leukaemia, breast cancer and teratocarcinoma. This is also the mechanism that causes cancer in gene therapy, when viral vectors integrate next to these same genes.
Another possibility is for an HERV to recombine with an infecting virus to generate new viruses. One theory for the origin of the AIDS virus it that it may have come from early preparations of polioviruses used for vaccines that were propagated in rhesus and African green monkey kidney cells. At least 26 monkey viruses, including adenoviruses, cosackievirus, herpesvirus, echovirus, and possibly other groups of viruses were found as contaminants in such preparations. Current vaccines are presumably free of such contamination, though that does not necessarily make them safe (see below).
Urnovitz and Murphy suggested that human immunodeficiency virus type 1 (HIV-1) may be a chimera between one of the simian (monkey) viruses (simian immunodeficiency virus) and HERV sequences. Simian immunodeficiency virus capable of causing simian AIDS, appears to occur exclusively in African monkeys, particularly in the African green monkey. And DNA sequences related to the highly conserved domain of the HIV reverse transcriptase and glycoprotein gp41 (part of the gp160 polyprotein of the HIV that’s cleaved into gp120 and gp41) have been found in the human genome.
Another way in which disease may arise is when HERV encoded proteins are expressed. This provokes antibodies against the body’s own cells, giving rise to autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, Sjögren’s syndrome, mixed connective tissue diseases and inflammatory neurologic disease. The inflammatory response could be the most important trigger for the development of autoimmune disease, as infecting viruses can strongly activate HERVs to express, resulting in production of HERV protein antigens.
In this context, vaccines came in for special criticism in Unovitz’ testimony to the United States House of Representatives in 1999.
In his view, "there appears to be a limit on how much foreign material to which the human body can be exposed before some level of genetic damage occurs and a chronic disease initiates". Gulf War I veterans (GWIVs) were given large numbers of vaccines (see "Gulf War Syndrome and vaccinations", this series), and vaccine overload is a significant factor in Gulf War Syndrome.
Urnovitz described a case of a woman who died from a mysterious case of AIDS. Over several years, laboratory tests failed to reveal HIV-1 antibodies in her blood. However, she was subsequently tested HIV-1 positive in her urine. The virus was eventually isolated and sequenced; and came to be known as HIV-1 Group O. Analyses of the viral genetic material suggest that the virus originated, in part, from genetic reshuffling of human chromosomal material.
Vaccination against HIV-1 is particularly dangerous, if as Urnovitz and Murphy have suggested, HIV arose from recombination between the simian aids virus and HERV sequences (see also "AIDS vaccines worse than useless?" this series).
"To put it simply, are we embarking on a course that will vaccinate people against their own genes?" Dr. Urnovitz asked.
A second example is the intensive effort directed to creating a vaccine for the hepatitis C virus. Unfortunately, there is no hard evidence in the scientific literature that hepatitis C virus exists. Urnovitz continued, "As a scientist I am compelled to ask, how can we vaccinate people against a disease-causing agent that has not been fully characterised?"
Finally, he drew attention to the contaminated polio vaccines that is now being increasingly implicated in cancer,
"Had my mother and father known that the poliovirus vaccines of the 1950s were heavily contaminated with more than 26 monkey viruses, including the cancer virus SV40, I can say with certainty that they would not have allowed their children and themselves to take those vaccines. Both of my parents might not have developed cancers suspected of being vaccine-related, and might even be alive today."
But even uncontaminated Polio vaccines are of doubtful efficacy in protecting against viral infections and chronic disease. Enteroviruses have been shown to be a major factor in Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome by John Richardson who studied more than 4000 patients over a 50 year period. Vaccination against polio provides protection against only 3 strains of polio leaving no protection against the other 70 or so enteroviruses- coxsackie, echo and others. These viruses have a range of pathological effects on the central nervous system, the cardiovascular system, and endocrine and exocrine glands.
source:- i-sis.org.uk
Most HERVs and retrotransposons are defective, having lost one or more gene functions; but can nevertheless multiply and move with the help of other elements or infecting viruses.
Which came first, retrovirus or retrotransposon? As viruses depend on cells to reproduce, it seems reasonable to suppose that retroviruses are simply retrotransposons that have gained an envelop, which helps in entering other cells. That idea was first put forward by Howard Temin, who discovered reverse transcriptase, the enzyme that reverse transcribes, encoded by the retrovirus and retrotransposon.
Retrotransposons play an important role in turning genes on or off, and in rearranging and amplifying genes during development and cell differentiation. And evidence has been accumulating that they are the natural molecular genetic engineers of the fluid genome, which are necessary for the survival of the organism .
HERVs are flanked by ‘long terminal repeats’ that contain strong promoters for gene expression. Promoters are stretches of DNA with binding sites for transcription factors of the host cell that boosts transcription, effectively saying to the cell, "make many copies of the message following". HERVs and retrotransposons are regulated by the cell, and ultimately, by the organism as a whole, which stops most of them from being expressed; though they can be activated under a variety of conditions (see below).
Many endogenous viruses show xenotropism, i.e., they are not active in the host cell but can become infectious to cells of non-host species. Xenotropism is one of the major health hazards of xenotransplantation, the transplant of organs and tissues between species, as from pig to humans.
Defective or dormant HERVs, like defective retrotransposons, can become expressed when missing gene-functions are provided by a ‘helper’ virus that happens to infect the cell, and that includes ‘attenuated’ viruses in vaccines. Like retrotransposons, HERVs can also be induced: by X-rays or various chemical agents and drugs, such as inhibitors of protein synthesis, organophosphates and other pesticides, inflammatory cytokines (hormone-like factors that influence cells of the immune system) or steroid hormones, and retinoic acid.
In a comprehensive review published in 1996, virologists Howard Urnovitz and William Murphy raised the possibility that many chronic debilitating diseases may be linked to HERVs. These include leukaemia and other cancers, B-cell immunoglobulin diseases, inflammatory diseases of the nervous system, autoimmune rheumatic and connective tissue disease and chronic fatigue syndrome.
There are several mechanisms linking HERVs with chronic diseases, though it is not at all clear which mechanism comes into effect under different circumstances.
One way in which endogenous viruses can cause disease is for them to move and insert itself next to certain genes, that, when over-expressed, results in uncontrolled cell division, or cancer. This mechanism may be involved in mouse and human leukaemia, breast cancer and teratocarcinoma. This is also the mechanism that causes cancer in gene therapy, when viral vectors integrate next to these same genes.
Another possibility is for an HERV to recombine with an infecting virus to generate new viruses. One theory for the origin of the AIDS virus it that it may have come from early preparations of polioviruses used for vaccines that were propagated in rhesus and African green monkey kidney cells. At least 26 monkey viruses, including adenoviruses, cosackievirus, herpesvirus, echovirus, and possibly other groups of viruses were found as contaminants in such preparations. Current vaccines are presumably free of such contamination, though that does not necessarily make them safe (see below).
Urnovitz and Murphy suggested that human immunodeficiency virus type 1 (HIV-1) may be a chimera between one of the simian (monkey) viruses (simian immunodeficiency virus) and HERV sequences. Simian immunodeficiency virus capable of causing simian AIDS, appears to occur exclusively in African monkeys, particularly in the African green monkey. And DNA sequences related to the highly conserved domain of the HIV reverse transcriptase and glycoprotein gp41 (part of the gp160 polyprotein of the HIV that’s cleaved into gp120 and gp41) have been found in the human genome.
Another way in which disease may arise is when HERV encoded proteins are expressed. This provokes antibodies against the body’s own cells, giving rise to autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, Sjögren’s syndrome, mixed connective tissue diseases and inflammatory neurologic disease. The inflammatory response could be the most important trigger for the development of autoimmune disease, as infecting viruses can strongly activate HERVs to express, resulting in production of HERV protein antigens.
In this context, vaccines came in for special criticism in Unovitz’ testimony to the United States House of Representatives in 1999.
In his view, "there appears to be a limit on how much foreign material to which the human body can be exposed before some level of genetic damage occurs and a chronic disease initiates". Gulf War I veterans (GWIVs) were given large numbers of vaccines (see "Gulf War Syndrome and vaccinations", this series), and vaccine overload is a significant factor in Gulf War Syndrome.
Urnovitz described a case of a woman who died from a mysterious case of AIDS. Over several years, laboratory tests failed to reveal HIV-1 antibodies in her blood. However, she was subsequently tested HIV-1 positive in her urine. The virus was eventually isolated and sequenced; and came to be known as HIV-1 Group O. Analyses of the viral genetic material suggest that the virus originated, in part, from genetic reshuffling of human chromosomal material.
Vaccination against HIV-1 is particularly dangerous, if as Urnovitz and Murphy have suggested, HIV arose from recombination between the simian aids virus and HERV sequences (see also "AIDS vaccines worse than useless?" this series).
"To put it simply, are we embarking on a course that will vaccinate people against their own genes?" Dr. Urnovitz asked.
A second example is the intensive effort directed to creating a vaccine for the hepatitis C virus. Unfortunately, there is no hard evidence in the scientific literature that hepatitis C virus exists. Urnovitz continued, "As a scientist I am compelled to ask, how can we vaccinate people against a disease-causing agent that has not been fully characterised?"
Finally, he drew attention to the contaminated polio vaccines that is now being increasingly implicated in cancer,
"Had my mother and father known that the poliovirus vaccines of the 1950s were heavily contaminated with more than 26 monkey viruses, including the cancer virus SV40, I can say with certainty that they would not have allowed their children and themselves to take those vaccines. Both of my parents might not have developed cancers suspected of being vaccine-related, and might even be alive today."
But even uncontaminated Polio vaccines are of doubtful efficacy in protecting against viral infections and chronic disease. Enteroviruses have been shown to be a major factor in Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome by John Richardson who studied more than 4000 patients over a 50 year period. Vaccination against polio provides protection against only 3 strains of polio leaving no protection against the other 70 or so enteroviruses- coxsackie, echo and others. These viruses have a range of pathological effects on the central nervous system, the cardiovascular system, and endocrine and exocrine glands.
source:- i-sis.org.uk
Dynamic Genomics
During the Persian Gulf War, some 700 000 individuals were exposed to a whole range of environmental hazards, including low-level chemical warfare agents, investigational drugs (inclucing pyridostigmine bromide, used as a prophylactic against nerve agents), organophosphate, carbamate, and other pesticides and insect repellents, low levels of nuclear and electromagnetic radiation, toxic combustion products from oilwell fires, diesel exhaust products and airborne particles, all collectively known to be genotoxic, or capable of causing harm through effects on the genetic material. The veterans were also exposed to multiple vaccinations, also of questionable safety. A significant proportion of the veterans developed a pattern of symptoms that have been referred to as Persian Gulf War-Related Illnesses, or Gulf War Syndrome (GWS): rash, fatigue, muscle and joint pain, headache, irritability, depression, unrefreshing sleep, gastrointestinal and respiratory disorders and cognitive defects. These were eventually defined as a clinical entity in 1998.
Most Gulf War I veterans (GWIVs) received oral poliovirus vaccine before deployment. Persistent enterovirus infection has been implicated in the chronic fatigue syndrome, one of the major disorders of GWIVs. There has already been a report that enterovirus-specific RNA was found in the sera of patients with chronic fatigue syndrome. For these reasons, Howard Urnovitz, Scientific Director of the Chronic Illness Research Foundation, and his colleagues decided to search for virus-specific nucleic acids in the sera of the GWIVs by using virus-specific primers to amplify RNA sequence. The sera from 24 GWIV with GWS deployed approximately 5 years previously were compared with serum samples from 50 controls, for the most part matched by age, sex and race.
When the amplified RNAs were separated according to size by running the mixtures through an agarose gel in an electric field, a striking difference between the GWIVs and controls was seen. Controls typically gave no more than three faint RNA bands, all less than 350 nucleotides (nt) in length. The sera from GWIVs, in contrast, contained numerous bright bands of very large RNAs, most of them longer than 750nt and especially longer than 2 000 nt. Most of the bands, moreover, did not belong to either the poliovirus or enterovirus. Both viral RNAs tended to be found more frequently in the sera of GWIVs, but the differences from controls were not significant.
The team sequenced two of the many bands that were found only in GWIVs, one 414nt and the other, 759nt, from three different samples. They were 99% identical between samples, but unrelated to each other, and were not homologous (similar) to any sequence found the public DNA database GenBank. However, short stretches of 14 to 15nt were homologous to segments in a region on the short arm of chromosome 22, 22q11.2. It is as though something had chopped up that region into pieces, shuffled them, and joined them up together again.
Thus, 3 sequences of 15nt and 8 of 14nt in the 759nt RNA had 100% homology to short segments of chromosome 22q11.2. Five of these segments occur only on chromosome 22q11.2. For the 414nt RNA, there were 2 sequences of 15nt and 4 of 14nt with 100% homology to the 22q11.2 region, but these segments also occur on other chromosomes, so it cannot be excluded that other chromosome regions were also involved in this gene shuffling exercise. Another important feature is that 6 of the segments in the 759nt RNA and 2 of those in the 414nt RNA occur near, between, or in Alu elements ("Molecular genetic engineers in junk DNA?", this series) that are capable of multiplying and jumping around the genome, and are hence thought to be involved in genetic recombination or gene shuffling.
This is a surprising finding. After all, GWS is generally considered to be a ‘multifactorial’ disease, ie, a disease due to multiple causes, possibly one for each of the symptoms. And yet, for the first time, Urnovitz and his colleagues have demonstrated that there could be a common molecular marker for the disease.
Not only that, using the same techniques, Urnovitz and colleagues were able to identify another unique RNA molecular marker in patients with multiple myeloma (malignant transformation of blood plasma precursor cells) and related disorders. They analysed 65 patients with multiple myeoloma (MM) 3 with Waldenstrom’s macroglobulinemia (WM), 2 with monoclonal gammopathy of undetermined significance (MGUS), and 50 healthy controls.
A 713nt plasma RNA occurred in 16/18 of MM patients in relapse, 5/8 MM patients who were untreated, 2/3 WM patients and ½ MGUS patients. None of the MM patients in remission, nor the 50 healthy controls was positive. The homology of the 713nt RNA between four samples was > 99.7% and matched (99.6%) a 704ng sequence of the flanking region of the peroxisome proliferator activator receptor gene, located in the same genome region, chromosome 22q11.2. A 255nt sequence within the 713nt RNA had a 90.2% homology with an Alu consensus sequence.
There is reasonable evidence that multiple myeloma is associated with exposure to industrial chemicals, pesticides or other environmental insults, as in the case of GWS.
This raises key questions: what is the origin of these RNAs? What is the possible role of these RNAs and of chromosome 22q11.2 in these diseases? Have environmental genotoxins played a role in causing disease? And finally, could the RNA molecular markers offer diagnostic tools for the diseases?
Chromosome 22q11.2 has been identified as a region full of hotspots for genetic deletions and translocations correlated with multiple myelomas and related disorders, as well as with rearrangements of the immunoglobulin lambda light chains in the normal immune response. Chromosome 22 appears to be involved in the so-called Goldenhar complex, a birth defect possibly associated with GWS.
That region is full of Alu sequences, previously thought to be nothing but junk DNA. But it is becoming increasingl clear that they have important regulatory functions. Alu expression is induced when cells are stressed by heat shock, or genotoxic agents, and may be part of the detoxification response. Alu sequences are known to be involved in genetic recombination or gene shuffling. Alu-Alu rcombinants are generated by both extrachromosomal and chromosomal genetic mechanisms.
Thus, it seems reasonable to conclude that exposure to toxic substances had activated retrotransposable Alu elements, possibly in specific parts of the genome, which results in gene shuffling to produce the unique sequences of RNAs circulating in the serum.
These circulating RNAs appear to be derived from white blood cells that have died, and are enclosed in proteolipid vesicles that protect them from being broken down. There is evidence that such plasma RNAs account for at least some of the illnesses. They are capable of transforming the blood cells of healthy animals in a mouse model, and are associated with immune suppression, making them more susceptible to infections.
At a conference celebrating the Centennial of the University of Michigan Department of Microbiology and Immunology in May 2003, Urnovitz, presented the new concept of "the dynamic genome", the idea that the genome contains "an operating system that instructs the organism how to both use and adapt genomic elements to the constant challenges of a dynamic environment."
This concept led to a practical breakthough, surrogate marker blood tests for yet another condition, mad cow disease, which can be performed on live animals. And, he also mentioned potential public health application for understanding the role of the genome in epidemics ranging from influenza-like pandemics (SARS) to "Gulf War syndrome, chronic fatigue syndrome, and AIDS".
What led him to the idea of the dynamic genome is the discovery that blood borne particles, or "microvesicles" contain "non-blueprint" RNA. In the past, they were assumed to be foreign, and hence mistaken as viruses.
He rejects the theory that a coronavirus is the cause of SARS. The virus was isolated from lab cultures that showed sick and dying cells. "Transmissible factors don’t have to kill a cell to be part of the disease," Urnovitz says, "they could just dysregulate cell function without killing the host cell."
He has carried out his own analysis on the so-called SARS-related coronavirus gene sequence. "Frankly, I do not see a virus. I see a unique and complete rearrangement of genomic elements. For example, when I look at what is believed to be the gene sequence coding for the spike protein of this coronavirus, I see a complicated gene rearrangement of a region of human chromosome 7." As with the Gulf War Syndrome, gene rearrangements like this immediately says to him, "search for an associated catastrophic environmental event that could have caused such genomic rearrangement."
He sees a correlation between nuclear and chemical weapons deployment over the last 100 years and the associated occurrence of flu-like pandemics. He postulates that when animals are exposed to nuclear or chemical weapons, entirely new regulatory gene set are expressed and packaged into non-viral RNA regulatory microvesicles. The risk of turning an epidemic into a pandemic is increased when the exposed animals are migratory birds that frequent gene-swapping hot spots like southeast China. He says, "The recent sightings in eastern China and Hong Kong of rare migratory birds – white cranes, grey cranes, and swans – that spend significant time feeding in the radioactive-contaminated regions of Siberia suggest that international efforts should be focussed on not only hunting for weapons of mass destruction but also on cleaning up the ones that have already been released into the environment."
He rejects the common belief that vaccines are the key to stopping epidemics: "While the current dogma states that vaccines stop viral epidemics, the historical data do not support that claim. From smallpox to polio to HIV, all vaccine attempts have been ineffective or hazardous to the vaccinee."
His company, Chronix Biomedical, develop screening and diagnostic tests based on the detection of non-viral RNA regulatory microvesicles for both veterinary and human diseases. Is it making a profit? "Not yet," he answered.
The blood test for mad cow disease, or bovine spongiform encephalitis (BSE) —the first that can be performed on live animals—is under development in the laboratory of Professor Bertram Brenig, Director of the Institute of Veterinary Medicine, Georg-August University, Göttingen, Germany. Urnovitz’s collaboration with Brenig’s laboratory has resulted in the detection of a specific RNA unique to cows at risk for developing, or that have confirmed cases of BSE.
Urnovitz claims that the BSE blood test is 100% sensitive on all 6 BSE cows confirmed with a licensed prion test, and 100% specific on all 46 animals from known healthy herds. They found that 3.5% of cohort animals (two animals out of 57) showed a positive response in the surrogate blood marker for BSE. Cohorts are animals born and/or raised in the same herd as a confirmed BSE case within approximately 12 months before and after the date of birth of the BSE case. Positive cohort cases may represent animals at risk for developing BSE.
If Urnovitz is right, we have to seriously rethink environmental health.
source:- i-sis.org.uk
Most Gulf War I veterans (GWIVs) received oral poliovirus vaccine before deployment. Persistent enterovirus infection has been implicated in the chronic fatigue syndrome, one of the major disorders of GWIVs. There has already been a report that enterovirus-specific RNA was found in the sera of patients with chronic fatigue syndrome. For these reasons, Howard Urnovitz, Scientific Director of the Chronic Illness Research Foundation, and his colleagues decided to search for virus-specific nucleic acids in the sera of the GWIVs by using virus-specific primers to amplify RNA sequence. The sera from 24 GWIV with GWS deployed approximately 5 years previously were compared with serum samples from 50 controls, for the most part matched by age, sex and race.
When the amplified RNAs were separated according to size by running the mixtures through an agarose gel in an electric field, a striking difference between the GWIVs and controls was seen. Controls typically gave no more than three faint RNA bands, all less than 350 nucleotides (nt) in length. The sera from GWIVs, in contrast, contained numerous bright bands of very large RNAs, most of them longer than 750nt and especially longer than 2 000 nt. Most of the bands, moreover, did not belong to either the poliovirus or enterovirus. Both viral RNAs tended to be found more frequently in the sera of GWIVs, but the differences from controls were not significant.
The team sequenced two of the many bands that were found only in GWIVs, one 414nt and the other, 759nt, from three different samples. They were 99% identical between samples, but unrelated to each other, and were not homologous (similar) to any sequence found the public DNA database GenBank. However, short stretches of 14 to 15nt were homologous to segments in a region on the short arm of chromosome 22, 22q11.2. It is as though something had chopped up that region into pieces, shuffled them, and joined them up together again.
Thus, 3 sequences of 15nt and 8 of 14nt in the 759nt RNA had 100% homology to short segments of chromosome 22q11.2. Five of these segments occur only on chromosome 22q11.2. For the 414nt RNA, there were 2 sequences of 15nt and 4 of 14nt with 100% homology to the 22q11.2 region, but these segments also occur on other chromosomes, so it cannot be excluded that other chromosome regions were also involved in this gene shuffling exercise. Another important feature is that 6 of the segments in the 759nt RNA and 2 of those in the 414nt RNA occur near, between, or in Alu elements ("Molecular genetic engineers in junk DNA?", this series) that are capable of multiplying and jumping around the genome, and are hence thought to be involved in genetic recombination or gene shuffling.
This is a surprising finding. After all, GWS is generally considered to be a ‘multifactorial’ disease, ie, a disease due to multiple causes, possibly one for each of the symptoms. And yet, for the first time, Urnovitz and his colleagues have demonstrated that there could be a common molecular marker for the disease.
Not only that, using the same techniques, Urnovitz and colleagues were able to identify another unique RNA molecular marker in patients with multiple myeloma (malignant transformation of blood plasma precursor cells) and related disorders. They analysed 65 patients with multiple myeoloma (MM) 3 with Waldenstrom’s macroglobulinemia (WM), 2 with monoclonal gammopathy of undetermined significance (MGUS), and 50 healthy controls.
A 713nt plasma RNA occurred in 16/18 of MM patients in relapse, 5/8 MM patients who were untreated, 2/3 WM patients and ½ MGUS patients. None of the MM patients in remission, nor the 50 healthy controls was positive. The homology of the 713nt RNA between four samples was > 99.7% and matched (99.6%) a 704ng sequence of the flanking region of the peroxisome proliferator activator receptor gene, located in the same genome region, chromosome 22q11.2. A 255nt sequence within the 713nt RNA had a 90.2% homology with an Alu consensus sequence.
There is reasonable evidence that multiple myeloma is associated with exposure to industrial chemicals, pesticides or other environmental insults, as in the case of GWS.
This raises key questions: what is the origin of these RNAs? What is the possible role of these RNAs and of chromosome 22q11.2 in these diseases? Have environmental genotoxins played a role in causing disease? And finally, could the RNA molecular markers offer diagnostic tools for the diseases?
Chromosome 22q11.2 has been identified as a region full of hotspots for genetic deletions and translocations correlated with multiple myelomas and related disorders, as well as with rearrangements of the immunoglobulin lambda light chains in the normal immune response. Chromosome 22 appears to be involved in the so-called Goldenhar complex, a birth defect possibly associated with GWS.
That region is full of Alu sequences, previously thought to be nothing but junk DNA. But it is becoming increasingl clear that they have important regulatory functions. Alu expression is induced when cells are stressed by heat shock, or genotoxic agents, and may be part of the detoxification response. Alu sequences are known to be involved in genetic recombination or gene shuffling. Alu-Alu rcombinants are generated by both extrachromosomal and chromosomal genetic mechanisms.
Thus, it seems reasonable to conclude that exposure to toxic substances had activated retrotransposable Alu elements, possibly in specific parts of the genome, which results in gene shuffling to produce the unique sequences of RNAs circulating in the serum.
These circulating RNAs appear to be derived from white blood cells that have died, and are enclosed in proteolipid vesicles that protect them from being broken down. There is evidence that such plasma RNAs account for at least some of the illnesses. They are capable of transforming the blood cells of healthy animals in a mouse model, and are associated with immune suppression, making them more susceptible to infections.
At a conference celebrating the Centennial of the University of Michigan Department of Microbiology and Immunology in May 2003, Urnovitz, presented the new concept of "the dynamic genome", the idea that the genome contains "an operating system that instructs the organism how to both use and adapt genomic elements to the constant challenges of a dynamic environment."
This concept led to a practical breakthough, surrogate marker blood tests for yet another condition, mad cow disease, which can be performed on live animals. And, he also mentioned potential public health application for understanding the role of the genome in epidemics ranging from influenza-like pandemics (SARS) to "Gulf War syndrome, chronic fatigue syndrome, and AIDS".
What led him to the idea of the dynamic genome is the discovery that blood borne particles, or "microvesicles" contain "non-blueprint" RNA. In the past, they were assumed to be foreign, and hence mistaken as viruses.
He rejects the theory that a coronavirus is the cause of SARS. The virus was isolated from lab cultures that showed sick and dying cells. "Transmissible factors don’t have to kill a cell to be part of the disease," Urnovitz says, "they could just dysregulate cell function without killing the host cell."
He has carried out his own analysis on the so-called SARS-related coronavirus gene sequence. "Frankly, I do not see a virus. I see a unique and complete rearrangement of genomic elements. For example, when I look at what is believed to be the gene sequence coding for the spike protein of this coronavirus, I see a complicated gene rearrangement of a region of human chromosome 7." As with the Gulf War Syndrome, gene rearrangements like this immediately says to him, "search for an associated catastrophic environmental event that could have caused such genomic rearrangement."
He sees a correlation between nuclear and chemical weapons deployment over the last 100 years and the associated occurrence of flu-like pandemics. He postulates that when animals are exposed to nuclear or chemical weapons, entirely new regulatory gene set are expressed and packaged into non-viral RNA regulatory microvesicles. The risk of turning an epidemic into a pandemic is increased when the exposed animals are migratory birds that frequent gene-swapping hot spots like southeast China. He says, "The recent sightings in eastern China and Hong Kong of rare migratory birds – white cranes, grey cranes, and swans – that spend significant time feeding in the radioactive-contaminated regions of Siberia suggest that international efforts should be focussed on not only hunting for weapons of mass destruction but also on cleaning up the ones that have already been released into the environment."
He rejects the common belief that vaccines are the key to stopping epidemics: "While the current dogma states that vaccines stop viral epidemics, the historical data do not support that claim. From smallpox to polio to HIV, all vaccine attempts have been ineffective or hazardous to the vaccinee."
His company, Chronix Biomedical, develop screening and diagnostic tests based on the detection of non-viral RNA regulatory microvesicles for both veterinary and human diseases. Is it making a profit? "Not yet," he answered.
The blood test for mad cow disease, or bovine spongiform encephalitis (BSE) —the first that can be performed on live animals—is under development in the laboratory of Professor Bertram Brenig, Director of the Institute of Veterinary Medicine, Georg-August University, Göttingen, Germany. Urnovitz’s collaboration with Brenig’s laboratory has resulted in the detection of a specific RNA unique to cows at risk for developing, or that have confirmed cases of BSE.
Urnovitz claims that the BSE blood test is 100% sensitive on all 6 BSE cows confirmed with a licensed prion test, and 100% specific on all 46 animals from known healthy herds. They found that 3.5% of cohort animals (two animals out of 57) showed a positive response in the surrogate blood marker for BSE. Cohorts are animals born and/or raised in the same herd as a confirmed BSE case within approximately 12 months before and after the date of birth of the BSE case. Positive cohort cases may represent animals at risk for developing BSE.
If Urnovitz is right, we have to seriously rethink environmental health.
source:- i-sis.org.uk
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