Adrienne Dellwo, Fibromyalgia & CFS Blog
ABSTRACT
NEWSBRIEF: A virus suspected of triggering some cases of chronic fatigue syndrome (CFS or ME/CFS) can be passed genetically from parent to child, according to an article published in the journal Pediatrics.
Experts used to believe that mothers passed human herpesvirus 6 (HHV-6) to their babies through blood exchanged during childbirth. HHV-6 causes roseola in infected children. New evidence shows the virus can come from either parent in the genetic material.
Nearly everyone is infected with HHV-6 at some point in their lives, but researchers are now investigating what it means to have a virus integrated into your genetic matter -- whether it can activate and cause problems, or cause an immune response just by being there.
Guide note: Could this be causing a genetic predisposition to chronic fatigue syndrome or autoimmune diseases? If so, genetic testing may someday be able to diagnose the condition and reveal who is at risk for it.
Nov 28, 2008
HHV-6 is Passed to Children Through DNA
Brief to Genetic Mapping
What is genetic mapping?
Developing new and better tools to make gene hunts faster, cheaper and practical for any scientist was a primary goal of the Human Genome Project (HGP).
One of these tools is genetic mapping, the first step in isolating a gene. Genetic mapping - also called linkage mapping - can offer firm evidence that a disease transmitted from parent to child is linked to one or more genes. It also provides clues about which chromosome contains the gene and precisely where it lies on that chromosome.
Genetic maps have been used successfully to find the single gene responsible for relatively rare inherited disorders, like cystic fibrosis and muscular dystrophy. Maps have also become useful in guiding scientists to the many genes that are believed to interact to bring about more common disorders, such as asthma, heart disease, diabetes, cancer and psychiatric conditions.
How do researchers create a genetic map?
To produce a genetic map, researchers collect blood or tissue samples from family members where a certain disease or trait is prevalent. Using various laboratory techniques, the scientists isolate DNA from these samples and examine it for the unique patterns of bases seen only in family members who have the disease or trait. These characteristic molecular patterns are referred to as polymorphisms, or markers.
Before researchers identify the gene responsible for the disease or trait, DNA markers can tell them roughly where the gene is on the chromosome. This is possible because of a genetic process known as recombination. As eggs or sperm develop within a person's body, the 23 pairs of chromosomes within those cells exchange - or recombine - genetic material. If a particular gene is close to a DNA marker, the gene and marker will likely stay together during the recombination process, and be passed on together from parent to child. So, if each family member with a particular disease or trait also inherits a particular DNA marker, chances are high that the gene responsible for the disease lies near that marker.
The more DNA markers there are on a genetic map, the more likely it is that one will be closely linked to a disease gene - and the easier it will be for researchers to zero-in on that gene. One of the first major achievements of the HGP was to develop dense maps of markers spaced evenly across the entire collection of human DNA.
Markers themselves usually consist of DNA that does not contain a gene, however they can tell a researcher the identity of the person a DNA sample came from. This makes markers extremely valuable for tracking inheritance of traits through generations of a family, and markers have also proven useful in criminal investigations and other forensic applications.
Although there are several different types of genetic markers, the type most used on genetic maps today is known as a microsatellite map. However, maps of even higher resolution are being constructed using single-nucleotide polymorphisms, or SNPs (pronounced "snips"). Both types of markers are easy to use with automated laboratory equipment, so researchers can rapidly map a disease or trait in a large number of family members.
The development of high-resolution, easy-to-use genetic maps, coupled with the HGP's successful sequencing and physical mapping of the entire human genome, has revolutionized genetics research. The improved quality of genetic data has reduced the time required to identify a gene from a period of years to, in many cases, a matter of months or even weeks. Genetic mapping data generated by the HGP's laboratories is freely accessible to scientists through databases maintained by the National Institutes of Health and the National Library of Medicine's National Center for Biotechnology Information.
Nov 23, 2008
DNA Microchip Technology
What is a DNA microchip?
Scientists know that a mutation - or alteration - in a particular gene's DNA often results in a certain disease. However, it can be very difficult to develop a test to detect these mutations, because most large genes have many regions where mutations can occur. For example, researchers believe that mutations in the genes BRCA1 and BRCA2 cause as many as 60 percent of all cases of hereditary breast and ovarian cancers. But there is not one specific mutation responsible for all of these cases. Researchers have already discovered over 800 different mutations in BRCA1 alone.
The DNA microchip is a mew tool used to identify mutations in genes like BRCA1 and BRCA2. The chip, which consists of a small glass plate encased in plastic, is manufactured somewhat like a computer microchip. On the surface, each chip contains thousands of short, synthetic, single-stranded DNA sequences, which together add up to the normal gene in question.
What is a DNA microchip used for?
Because chip technology is still relatively new, it is currently only a research tool. Scientists use it to conduct large-scale population studies - for example, to determine how often individuals with a particular mutation actually develop breast cancer.
As we gain more insight into the mutations that underlie various diseases, researchers will likely produce new chips to help assess individual risks for developing different cancers as well as heart disease, diabetes and other diseases.
How does a DNA microchip work?
To determine whether an individual possesses a mutation for BRCA1 or BRCA2, a scientist first obtains a sample of DNA from the patient's blood as well as a control sample - one that does not contain a mutation in either gene.
The researcher then denatures the DNA in the samples - a process that separates the two complementary strands of DNA into single-stranded molecules. The next step is to cut the long strands of DNA into smaller, more manageable fragments and then to label each fragment by attaching a fluorescent dye. The individual's DNA is labeled with green dye and the control - or normal - DNA is labeled with red dye. Both sets of labeled DNA are then inserted into the chip and allowed to hybridize - or bind - to the synthetic BRCA1 or BRCA2 DNA on the chip. If the individual does not have a mutation for the gene, both the red and green samples will bind to the sequences on the chip.
If the individual does possess a mutation, the individual's DNA will not bind properly in the region where the mutation is located. The scientist can then examine this area more closely to confirm that a mutation is present.
Last Reviewed: November 3, 2008 （Source：National Human Genomic Program）
DNA Microchips
Perhaps the best-known type of surface attachment involves DNA oligonucleotide microarrays (so called gene chips). Their importance to the study of genetics was illustrated by the appearance in (1999) of an entire supplemental issue of Nature Genetics dedicated to the subject. These chips have been used to enable rapid sequencing of DNA, for the detection of RFLPs (Hacia & Collins, 1999) , for monitoring the presence of microorganisms in the food industry (Kuipers, 1999; Kuipers et al., 1999) , or the environment (Guschin et al., 1997; Oh & Liao, 2000) and to assay gene expression in a wide range of organisms (Harrington et al., 2000) . More recently, chips have been manufactured with a three-dimensional array based on the use of a polyacrylamide matrix (Guschin et al., 1997) . These are likely to lead to faster screening techniques and the use of larger “databases” of genes on the chips for expression studies. Microchip development has also spawned a vast collection of associated industries producing clone-sets, arrayers, scanners and software for data analysis (Epstein & Butlow, 2000) . It seems likely that single chips will get smaller, moving toward the nanoscale and the detection/use of single DNA molecules.
Currently, there are two major variants of DNA chip technology based on the type of immobilised DNA. In the first format, the immobilised probe consists of cDNA (500-5000bp DNA fragments produced by copying expressed genes) spotted onto a surface using robotics. The second format, consists of 20-25-mer oligonucleotides (or peptide nucleic acid - PNA) synthesised in situ (such as the gene chips produced by Affymetrix), or by conventional synthesis and then immobilised. The oligonucleotides are of known DNA sequence allowing detection of complementary sequences. Therefore, the DNA on the chip acts as a probe in a hybridisation reaction (Souteyrand et al., 2000; Southern et al., 1999) . The arrays come in various sizes from macroarrays to microarrays and beyond. Macroarrays are generally produced by hand, or using simple robots to pipette DNA solutions onto prepared surfaces and contains “tens to hundreds” of spots over an area of 500 mm or more. Microarrays contain many thousands of spots over an area of less than 500 mm.
Gene chips for cancer detection
DNA microchips have been used extensively for research purposes, but they also have a powerful use as a biosensor. For example, the onset of prostate cancer involves the differential expression of approximately 500 genes. A prototype chip for prostate cancer detection is already under development by the biotechnology firm Darwin Molecular (Bothell, Washington USA). They predict that within a year they will have a system that can distinguish slow-growing cancers that do not require immediate treatment and those aggressive tumours that require surgery (Zajtchuk, 1999).
Detection of hybridisation usually involves the use of PCR amplified, fluorescently-tagged DNA. Generally, for gene expression studies, this involves amplification of a pair of mRNA samples (i.e. a control and an experimental sample) using different fluorophores for each sample. Identical amounts of the amplified cDNA are hybridised to the same DNA chip and fluorescent intensities compared (Winzeler et al., 1999) . The use of two fluorophores overcomes problems associated with different amounts of probe DNA deposited at the specific spots on the chip.
The first consideration in the design of a DNA chip is the support material and the production of simple arrays in a research environment was discussed by Cheung et al. (1999) . Most usually, this involves the use of glass. However, recently silicon has also been employed as a support allowing the use of its semiconducting properties (Souteyrand et al., 2000) . Electrochemical deposition of oligonucleotides, via pyrrole groups is used to attach the DNA to the surface of electrodes (Livache et al., 1998a) . These electrodes can interact with the silicon allowing for the development of new techniques for detecting hybridisation based on the semiconductor properties of the silicon. A typical 128-sample grid is shown in Figure 17. The chip uses polarisation of the electrodes (Souteyrand et al., 2000) to prevent diffusion, allowing the samples to concentrate ensuring hybridisation occurs in a few seconds. After hybridisation, a short burst of the opposite polarisation will release weakly bound molecules improving signal-noise ratios. Finally, the charge on the DNA strands allows detection of hybridisation without using fluorophores, through the field-effects on the electrodes.
Perhaps the company most associated with DNA microchips, Affymetrix, believes that the capacity of DNA microchips is following Moore’s Law, which predicts a two-fold increase in computer chip memory every 18 months. Affymetrix’s prototype chips held 20,000 oligonucleotide DNA probes. By 1997, this number had increased to 65,000 probes and it has recently introduced a chip with 400,000 probes. Ten such chips could scan the entire human genome for ‘malfunctioning’ genes.
（Source：Keith Firman, Nanonet)
Deoxyribonucleic Acid (DNA)
What is DNA?
We all know that elephants only give birth to little elephants, giraffes to giraffes, dogs to dogs and so on for every type of living creature. But why is this so?
The answer lies in a molecule called deoxyribonucleic acid (DNA), which contains the biological instructions that make each species unique. DNA, along with the instructions it contains, is passed from adult organisms to their offspring during reproduction.
DNA is found inside a special area of the cell called the nucleus. Because the cell is very small, and because organisms have many DNA molecules per cell, each DNA molecule must be tightly packaged. This packaged form of the DNA is called a chromosome.
DNA spends a lot of time in its chromosome form. But during cell division, DNA unwinds so it can be copied and the copies transferred to new cells. DNA also unwinds so that its instructions can be used to make proteins and for other biological processes.
Researchers refer to DNA found in the cell's nucleus as nuclear DNA. An organism's complete set of nuclear DNA is called its genome.
Besides the DNA located in the nucleus, humans and other complex organisms also have a small amount of DNA in cell structures known as mitochondria. Mitochondria generate the energy the cell needs to function properly.
In sexual reproduction, organisms inherit half of their nuclear DNA from the male parent and half from the female parent. However, organisms inherit all of their mitochondrial DNA from the female parent. This occurs because only egg cells, and not sperm cells, keep their mitochondria during fertilization.
DNA is made of chemical building blocks called nucleotides. These building blocks are made of three parts: a phosphate group, a sugar group and one of four types of nitrogen bases. To form a strand of DNA, nucleotides are linked into chains, with the phosphate and sugar groups alternating.
The four types of nitrogen bases found in nucleotides are: adenine (A), , thymine (T), guanine (G) and cytosine (C). The order, or sequence, of these bases determines what biological instructions are contained in a strand of DNA. For example, the sequence ATCGTT might instruct for blue eyes, while ATCGCT might instruct for brown.
Each DNA sequence that contains instructions to make a protein is known as a gene. The size of a gene may vary greatly, ranging from about 1,000 bases to 1 million bases in humans.
The complete DNA instruction book, or genome, for a human contains about 3 billion bases and about 20,000 genes on 23 pairs of chromosomes.
DNA contains the instructions needed for an organism to develop, survive and reproduce. To carry out these functions, DNA sequences must be converted into messages that can be used to produce proteins, which are the complex molecules that do most of the work in our bodies.
How are DNA sequences used to make proteins?
DNA's instructions are used to make proteins in a two-step process. First, enzymes read the information in a DNA molecule and transcribe it into an intermediary molecule called messenger ribonucleic acid, or mRNA.
Next, the information contained in the mRNA molecule is translated into the "language" of amino acids, which are the building blocks of proteins. This language tells the cell's protein-making machinery the precise order in which to link the amino acids to produce a specific protein. This is a major task because there are 20 types of amino acids, which can be placed in many different orders to form a wide variety of proteins.
The German biochemist Frederich Miescher first observed DNA in the late 1800s. But nearly a century passed from that discovery until researchers unraveled the structure of the DNA molecule and realized its central importance to biology.
For many years, scientists debated which molecule carried life's biological instructions. Most thought that DNA was too simple a molecule to play such a critical role. Instead, they argued that proteins were more likely to carry out this vital function because of their greater complexity and wider variety of forms.
The importance of DNA became clear in 1953 thanks to the work of James Watson, Francis Crick, Maurice Wilkins and Rosalind Franklin. By studying X-ray diffraction patterns and building models, the scientists figured out the double helix structure of DNA - a structure that enables it to carry biological information from one generation to the next.
Scientist use the term "double helix" to describe DNA's winding, two-stranded chemical structure. This shape - which looks much like a twisted ladder - gives DNA the power to pass along biological instructions with great precision.
To understand DNA's double helix from a chemical standpoint, picture the sides of the ladder as strands of alternating sugar and phosphate groups - strands that run in opposite directions. Each "rung" of the ladder is made up of two nitrogen bases, paired together by hydrogen bonds. Because of the highly specific nature of this type of chemical pairing, base A always pairs with base T, and likewise C with G. So, if you know the sequence of the bases on one strand of a DNA double helix, it is a simple matter to figure out the sequence of bases on the other strand.
DNA's unique structure enables the molecule to copy itself during cell division. When a cell prepares to divide, the DNA helix splits down the middle and becomes two single strands. These single strands serve as templates for building two new, double-stranded DNA molecules - each a replica of the original DNA molecule. In this process, an A base is added wherever there is a T, a C where there is a G, and so on until all of the bases once again have partners.
In addition, when proteins are being made, the double helix unwinds to allow a single strand of DNA to serve as a template. This template strand is then transcribed into mRNA, which is a molecule that conveys vital instructions to the cell's protein-making machinery.
Last Reviewed: October 1, 2008
Nov 22, 2008
Protocols of TUNEL
Fixing cells
1. Wash cells on slides.
2. Fix cells with 2-4% paraformaldehyde in PBS for 15 minutes.
3. Wash the cells in PBS three times for 5 minutes.
4. Permeabilize cells with 0.2% Trion X-100 in PBS for 5 minutes at room temperature.
5. Wash the cells in PBS three times for 5 minutes.
Fluorescein labeling
6. Equilibrate in the equilibration buffer (200 mM potassium or sodium cacodylate (pH 6.6), 25 mM Tris-HCl (pH6.6), 0.2 mM DTT, 0.25 mg/ml BSA, 2.5 mM cobalt chloride).
7. Make a labeling mix: 90 ml equilibration buffer, 10 ml nucleotide mix (50 mM fluorescein-12-dUTP, 100 mM dATP, 10 mM Tris-HCl (pH7.6), 1 mM EDTA), and 2 ml TdT enzyme (20 units).
8. Add 50-100 ml labeling mix to each slide, cover with a plastics cover slide. Incubate at 37 °C for 1 hour in a dark humidified chamber.
9. Wash the cells in 2X SSC three times for 10 minutes each in dark.
biotin labeling
6a. Equilibrate with 200 ml of 1X TdT buffer + 1 mM Cobalt Chloride for 5 minutes.
7a. Incubate in 100 ml of the labeling mix (1X TdT buffer, 1 mM Cobalt Chloride, 10 mM BrdUTP, 250 units TdT enzyme/ml) at 37 °C for 1 hour in a humidified chamber.
8a. Wash in PBS for 3 times, 5 minutes each.
9a. Add 200 ml FITC-avidin or Texas red-avidin (1:100) in 4X SSC/0.2% BSA.
10a. Incubate at 37 °C for 1 hour in a dark humidified chamber.
11a. Wash the cells in 4X SSC for 5 minutes in dark.
BrdU labeling
6b. Equilibrate with 200 ml of 1X TdT buffer (0.2M potassium or sodium cacodylate, 25mM TRIS-HCl, pH 6.6, 0.25mg/ml BSA) + 1 mM cobalt chloride for 5 minutes.
7b. Incubate in 100 ml of the labeling mix (1X TdT buffer, 1 mM cobalt chloride, 2.5 mM biotin-dATP, 250 units terminal transferase TdT enzyme/ml) at 37 °C for 1 hour in a humidified chamber.
8b. Rinse 3X in PBS.
9b. Add diluted anti-BrdU-FITC antibody and incubate at room temperature for 1 hour in the dark.
10b. Wash cells 3X in PBS in the dark.
Nuclei staining
12. Stain cells in 1 ml/ml propidium iodide or 10 mg/ml DAPI in PBS. Or mount directly in VECTASHIELD + DAPI. RNase may be included in the nuclei staining to eliminate staining for RNA.
TUNEL Protocol
TUNEL Staining Protocol for Apoptosis Detection - Enzyme Method
TUNEL assay
TUNEL procedure for bovine embryos
TUNEL Protocol (Hyde Lab.)
Springer Protocols: Full Text: TUNEL Assay: An Overview of Techniques
Cell Surface Labeling and ‘TUNEL’ Protocol
Springer Protocols: TUNEL and Immunofluorescence double staining.
TUNEL STAINING PROTOCOL
TUNEL Staining Protocol - Enzyme
TUNEL Staining Protocol - Fluorescence
TUNEL Labeling on paraffin section
Detection of Cell Death in Spider Embryos Using TUNEL.
TUNEL Protocol
Detection of TUNEL and GFP.
Analysis of TUNEL Staining by Flow Cytometry to Detect Apoptosis .
References:
TUNEL Apoptotic Cell Detection in Tissue Sections: Critical Evaluation and Improvement.
TUNEL Protocols. The three modalities of TUNEL we tested (laboratory protocol, ApopTag kit, and Boehringer Mannheim kit) yielded no significantly different ...www.jhc.org/cgi/content/full/46/3/327
Sensational Human Genome Discovery
In whose image was The Adam – the prototype of modern humans, Homo sapiens – created?
The Bible asserts that the Elohim said: “Let us fashion the Adam in our image and after our likeness.” But if one is to accept a tentative explanation for enigmatic genes that humans possess, offered when the deciphering of the human genome was announced in mid-February, the feat was decided upon by a group of bacteria!
“Humbling” was the prevalent adjective used by the scientific teams and the media to describe the principal finding – that the human genome contains not the anticipated 100,000 - 140,000 genes (the stretches of DNA that direct the production of amino-acids and proteins) but only some 30,000+ -- little more than double the 13,601 genes of a fruit fly and barely fifty percent more than the roundworm’s 19,098. What a comedown from the pinnacle of the genomic Tree of Life!
Moreover, there was hardly any uniqueness to the human genes. They are comparative to not the presumed 95 percent but to almost 99 percent of the chimpanzees, and 70 percent of the mouse. Human genes, with the same functions, were found to be identical to genes of other vertebrates, as well as invertebrates, plants, fungi, even yeast. The findings not only confirmed that there was one source of DNA for all life on Earth, but also enabled the scientists to trace the evolutionary process – how more complex organisms evolved, genetically, from simpler ones, adopting at each stage the genes of a lower life form to create a more complex higher life form – culminating with Homo sapiens.
The “Head-scratching” Discovery
It was here, in tracing the vertical evolutionary record contained in the human and the other analyzed genomes, that the scientists ran into an enigma. The “head-scratching discovery by the public consortium,” as Science termed it, was that the human genome contains 223 genes that do not have the required predecessors on the genomic evolutionary tree.
How did Man acquire such a bunch of enigmatic genes?
In the evolutionary progression from bacteria to invertebrates (such as the lineages of yeast, worms, flies or mustard weed – which have been deciphered) to vertebrates (mice, chimpanzees) and finally modern humans, these 223 genes are completely missing in the invertebrate phase. Therefore, the scientists can explain their presence in the human genome by a “rather recent” (in evolutionary time scales) “probable horizontal transfer from bacteria.”
In other words: At a relatively recent time as Evolution goes, modern humans acquired an extra 223 genes not through gradual evolution, not vertically on the Tree of Life, but horizontally, as a sideways insertion of genetic material from bacteria…
An Immense Difference
Now, at first glance it would seem that 223 genes is no big deal. In fact, while every single gene makes a great difference to every individual, 223 genes make an immense difference to a species such as ours.
The human genome is made up of about three billion neucleotides (the “letters” A-C-G-T which stand for the initials of the four nucleic acids that spell out all life on Earth); of them, just a little more than one percent are grouped into functioning genes (each gene consists of thousands of "letters"). The difference between one individual person and another amounts to about one “letter” in a thousand in the DNA “alphabet.” The difference between Man and Chimpanzee is less than one percent as genes go; and one percent of 30,000 genes is 300.
So, 223 genes is more than two thirds of the difference between me, you and a chimpanzee!
An analysis of the functions of these genes through the proteins that they spell out, conducted by the Public Consortium team and published in the journal Nature, shows that they include not only proteins involved in important physiological but also psychiatric functions. Moreover, they are responsible for important neurological enzymes that stem only from the mitochondrial portion of the DNA – the so-called “Eve” DNA that humankind inherited only through the mother-line, all the way back to a single “Eve.” That finding alone raises doubt regarding that the "bacterial insertion" explanation.
A Shaky Theory
How sure are the scientists that such important and complex genes, such an immense human advantage, was obtained by us --“rather recently”-- through the courtesy of infecting bacteria?
“It is a jump that does not follow current evolutionary theories,” said Steven Scherer, director of mapping of the
“We did not identify a strongly preferred bacterial source for the putative horizontally transferred genes,” states the report in Nature. The Public Consortium team, conducting a detailed search, found that some 113 genes (out of the 223) “are widespread among bacteria” – though they are entirely absent even in invertebrates. An analysis of the proteins which the enigmatic genes express showed that out of 35 identified, only ten had counterparts in vertebrates (ranging from cows to rodents to fish); 25 of the 35 were unique to humans.
“It is not clear whether the transfer was from bacteria to human or from human to bacteria,” Science quoted Robert Waterson, co-director of Washington University’s Genome Sequencing Center, as saying.
But if Man gave those genes to bacteria, where did Man acquire those genes to begin with?
The Role of the Anunnaki
Readers of my books must be smiling by now, for they know the answer.
They know that the biblical verses dealing with the fashioning of The Adam are condensed renderings of much much more detailed Sumerian and Akkadian texts, found inscribed on clay tablets, in which the role of the Elohim in Genesis is performed by the Anunnaki – “Those Who From Heaven to Earth Came.”
As detailed in my books, beginning with The 12th Planet (1976) and even more so in Genesis Revisited and The Cosmic Code, the Anunnaki came to Earth some 450,000 years ago from the planet Nibiru – a member of our own solar system whose great orbit brings it to our part of the heavens once every 3,600 years. They came here in need of gold, with which to protect their dwindling atmosphere. Exhausted and in need of help in mining the gold, their chief scientist Enki suggested that they use their genetic knowledge to create the needed Primitive Workers.
When the other leaders of the Anunnaki asked: How can you create a new being? He answered:
"The being that we need already exists;
all that we have to do is put our mark on it.”
The time was some 300,000 years ago.
What he had in mind was to upgrade genetically the existing hominids, who were already on Earth through Evolution, by adding some of the genes of the more advanced Anunnaki. That the Anunnaki, who could already travel in space 450,000 years ago, possessed the genomic science (whose threshold we have now reached) is clear not only from the actual texts but also from numerous depictions in which the double-helix of the DNA is rendered as Entwined Serpents (a symbol still used for medicine and healing) -- see illustration ‘A’ below.
When the leaders of the Anunnaki approved the project (as echoed in the biblical ”Let us fashion the Adam”), Enki with the help of Ninharsag, the Chief Medical Officer of the Anunnaki, embarked on a process of genetic engineering, by adding and combining genes of the Anunnaki with those of the already-existing hominids.
When, after much trial and error breathtakingly described and recorded in antiquity, a “perfect model” was attained, Ninharsag held him up and shouted: “My hands have made it!” An ancient artist depicted the scene on a cylinder seal (illustration ‘B’).
And that, I suggest, is how we had come to possess the unique extra genes. It was in the image of the Anunnaki, not of bacteria, that Adam and Eve were fashioned.
A Matter of Extreme Significance
Unless further scientific research can establish, beyond any doubt, that the only possible source of the extra genes are indeed bacteria, and unless it is then also determined that the infection
(“horizontal transfer”) went from bacteria to Man and not from Man to bacteria, the only other available solution will be that offered by the Sumerian texts millennia ago.
Until then, the enigmatic 223 alien genes will remain as an alternative – and as a corroboration by modern science of the Anunnaki and their genetic feats on Earth.
by Zecharia Sitchin
from ZechariaSitchin Website
Advances in DNA (Gene) -2
11: Arch Pharm Res. 2008 Nov;31(11):1483-1488. Epub 2008 Nov 21.
Compound K suppresses ultraviolet radiation-induced apoptosis by inducing DNA repair in human keratinocytes.
Cai BX, Luo D, Lin XF, Gao J.
Department of Dermatology, the First Affiliated
Ultraviolet (UV)-induced DNA damage is a crucial molecular trigger for sunburn cell formation and skin cancer. Nucleotide excision repair (NER) is the main mechanism in repairing UVB-induced DNA damage of mammalian cells. The purpose of this study is to investigate the functional role of ginsenoside compound K on HaCaT cells (a keratinocyte-derived permanent cell line) irradiated by UV. Hoechst 33258 staining were performed in analyzing UV-induced apoptosis on keratinocytes which were treated with compound K. ImmunoDotBlot assay was used in detecting cyclobutane pyrimidine dimers, the main DNA damage. Western blot analysis was applied for analyzing XPC and ERCC1, two of the NER proteins. Compound K inhibited UV-induced apoptosis of keratinocytes and caused a notable reduction in UV-specific DNA lesions which was due to induction of DNA repair. In agreement with this, compound K induced the expression of particular components of the NER complex, such as XPC and ERCC1. Our results demonstrate that compound K can protect cells from apoptosis induced by UV radiation by inducing DNA repair.
12: Arch Pharm Res. 2008 Nov;31(11):1413-1418. Epub 2008 Nov 21.
Topoisomerase I and II inhibitory constituents from the bark of Tilia amurensis.
Choi JY, Seo CS, Zheng MS, Lee CS, Son JK.
Two coumarins (1 and 6), one flavan-3-ol (2), one fatty acid (3), and two lignan glycosides (4 and 5) were isolated from the EtOAc and CH(2)Cl(2) extract of the bark of Tilia amurensis. Their chemical structures were identified by comparing their physicochemical and spectral data with those of published in literatures. Compounds 4, 5, and 6 were isolated from Tilia genus for the first time. Compounds 2 and 3 showed potent inhibitory activity against both DNA topoisomerase I (IC(50) values; 49 muM and 4 muM, respectively, with 18 muM of positive control compound, comptothecin) and DNA topoisomerase II (IC(50) values; 13 muM and 3 muM, respectively, with 50 muM of positive control compound, etoposide). However, all compounds did not showed cytotoxicity against the human colon adenocarcinoma cell line (HT-29), the human breast adenocarcinoma cell line (MCF-7), and human liver hepatoblastoma cell line (HepG-2).
13: Rev Port Pneumol. 2008 Nov/Dez;14(6):727-746.
HLA class I and II and TNF-alpha gene polymorphisms in sarcoidosis patients.
[Article in Portuguese, English]
Morais A, Alves H, Lima B, Delgado L, Gonçalves R, Tafulo S.
Serviço de Pneumologia do Hospital São João, Porto / Pulmonology Unit, Hospital São João,
Introduction: Several factors suggest a genetic predisposition to sarcoidosis, namely the recognition of race as a risk factor and the occurrence of familial clustering of cases. Several studies have reported an association of sarcoidosis and HLA class I and especially class II alleles in different populations. Aim: HLA class I, class II and TNF-alpha genotyping in a group of sarcoidosis patients and its relation with clinical presentation and outcome. Material and methods: A total of 104 sarcoidosis patients were included. Clinical presentation, functional, radiology, BAL findings and organ involvement were studied. HLA- A*, -B*, -C*, DRB1*, DQB1* and TNF-alpha were genotyped by molecular biology methods. DNA was extracted from peripheral blood and PCR-SSP and PCR-reverse hybridisation me- thods were used. Allele frequencies were compared with controls from the same region. The X2 test was used for discrete values and the Kruskal-Wallis test for continuous values. Results: When patients were compared with controls we noticed increased frequencies of B*08 (10.6% vs. 6.1%), O.R.=1.8, C.I.=[1.1;3.1], p=0.02; DRB1*12 (4.3% vs. 1.7%), O.R.=2.63, C.I.=[1.1;6.1], p=0.03. Patients with erythema nodosum have increased frequencies of the alleles DRB1*03 (28% vs. 9.3%), R.R.=2.39, C.I.=[1.5;3.8], pc=0.01 and DQB1*02 (38% vs. 18%), R.R.=2.1, C.I.=[1.3;3.3], pc=0.02. Allele DQB1*03 is decreased in patients with obstructive pattern R.R.=0.53, C.I.=[0.3;0.9], pc=0.05. Allele DRB1*15 is related to restrictive pattern and reduced diffusion capacity (21.1% vs. 6.6%), R.R.=2.46, C.I.=[1.35;4.48], p=0.01 and (18.1% vs. 3.8%), R.R.=1.87, pc= 0.05 respectively. The TNF-alpha A/A (high) genotype is significantly associated with erythema nodosum (p=0.04). Conclusions: These data add support to the genetic association of HLA class I and II with sarcoidosis in terms of susceptibility, type of presentation, severity and outcome. Moreover as previously described in other populations, the TNF-alpha A/A (high) genotype has a significant association with erythema nodosum. Rev Port Pneumol 2008; XIV (6): 727-746 Key-words: Sarcoidosis, genetics, HLA.
14: Lab Chip. 2008 Dec;8(12):2135-2145. Epub 2008 Oct 20.
Microchip DNA electrophoresis with automated whole-gel scanning detection.
Lo RC, Ugaz VM.
Artie McFerrin Department of Chemical Engineering,
Gel electrophoresis continues to play an important role in miniaturized bioanalytical systems, both as a stand alone technique and as a key component of integrated lab-on-a-chip diagnostics. Most implementations of microchip electrophoresis employ finish-line detection methods whereby fluorescently labeled analytes are observed as they migrate past a fixed detection point near the end of the separation channel. But tradeoffs may exist between the simultaneous goals of maximizing resolution (normally achieved by using longer separation channels) and maximizing the size range of analytes that can be studied (where shorter separation distances reduce the time required for the slowest analytes to reach the detector). Here we show how the miniaturized format can offer new opportunities to employ alternative detection schemes that can help address these issues by introducing an automated whole-gel scanning detection system that enables the progress of microchip-based gel electrophoresis of DNA to be continuously monitored along an entire microchannel. This permits flexibility to selectively observe smaller faster moving fragments during the early stages of the separation before they have experienced significant diffusive broadening, while allowing the larger slower moving fragments to be observed later in the run when they can be better resolved but without the need for them to travel the entire length of the separation channel. Whole-gel scanning also provides a continuous and detailed picture of the electrophoresis process as it unfolds, allowing fundamental physical parameters associated with DNA migration phenomena (e.g., mobility, diffusive broadening) to be rapidly and accurately measured in a single experiment. These capabilities are challenging to implement using finish-line methods, and make it possible to envision a platform capable of enabling separation performance to be rapidly screened in a wide range of gel matrix materials and operating conditions, even allowing separation and matrix characterization steps to be performed simultaneously in a single self-calibrating experiment.
15: Lab Chip. 2008 Dec;8(12):2091-2104. Epub 2008 Nov 5.
Development of a digital microfluidic platform for point of care testing.
Sista R, Hua Z, Thwar P, Sudarsan A, Srinivasan V, Eckhardt A, Pollack M, Pamula V.
Advanced Liquid Logic Inc.,
Point of care testing is playing an increasingly important role in improving the clinical outcome in health care management. The salient features of a point of care device are rapid results, integrated sample preparation and processing, small sample volumes, portability, multifunctionality and low cost. In this paper, we demonstrate some of these salient features utilizing an electrowetting-based Digital Microfluidic platform. We demonstrate the performance of magnetic bead-based immunoassays (cardiac troponin I) on a digital microfluidic cartridge in less than 8 minutes using whole blood samples. Using the same microfluidic cartridge, a 40-cycle real-time polymerase chain reaction was performed within 12 minutes by shuttling a droplet between two thermal zones. We further demonstrate, on the same cartridge, the capability to perform sample preparation for bacterial infectious disease pathogen, methicillin-resistant Staphylococcus aureus and for human genomic DNA using magnetic beads. In addition to rapid results and integrated sample preparation, electrowetting-based digital microfluidic instruments are highly portable because fluid pumping is performed electronically. All the digital microfluidic chips presented here were fabricated on printed circuit boards utilizing mass production techniques that keep the cost of the chip low. Due to the modularity and scalability afforded by digital microfluidics, multifunctional testing capability, such as combinations within and between immunoassays, DNA amplification, and enzymatic assays, can be brought to the point of care at a relatively low cost because a single chip can be configured in software for different assays required along the path of care.
16: Acta Biochim Pol. 2008 Nov 20. [Epub ahead of print]
Self-association of Chaetopterus variopedatus sperm histone H1-like. Relevance of arginine content and possible physiological role.
Salvati D, Conforti S, Conte M, Matassa DS, Fucci L, Piscopo M.
Department of Structural and Functional Biology,
Self-association of histones H1 from calf thymus and from sperm of the marine worm Chaetopterus variopedatus was studied on native and glutaraldehyde cross-linked molecules by PAGE and by salt-induced turbidity measurements. Multiple polymers were generated by native sperm histone H1-like after glutaraldehyde cross-linking while the same treatment on its lysine- or arginine-modified derivatives and on somatic histone H1 failed to induce polymerization. This result suggests the relevance of arginine content in the formation of histone H1-like polymers particularly because Chaetopterus variopedatus and calf thymus histones H1 have similar content of lysine but different K\R ratio (2 and 15, respectively). Salt-induced turbidity experiments confirmed the high tendency of sperm histone H1-like to form oligomers, particularly in the presence of phosphate ions. Native PAGE analysis in the presence of phosphate supported this hypothesis. The reported results suggest that phosphate ions connecting lysine and arginine side chain groups contribute to the interaction of sperm histone H1-like with DNA in chromatin and play a key role in organization and stabilization of the chromatin higher order structures.
17: Acta Biochim Pol. 2008 Nov 20. [Epub ahead of print]
TNFalpha-induced activation of NFkappaB protects against UV-induced apoptosis specifically in p53-proficient cells.
Szołtysek K, Pietranek K, Kalinowska-Herok M, Pietrowska M, Kimmel M, Widłak P.
The signaling pathways that depend on p53 or NFkappaB transcription factors are essential components of cellular responses to stress. In general, p53 is involved in either activation of cell cycle arrest or induction of apoptosis, while NFkappaB exerts mostly anti-apoptotic functions; both regulatory pathways apparently interfere with each other. Here we aimed to analyze the effects of NFkappaB activation on DNA damage-induced apoptosis, either p53-dependent or p53-independent, in a set of human cell lines. Four cell lines, HCT116 and RKO colon carcinoma, NCI-H1299 lung carcinoma and HL60 myeloblastoma, each of them in two congenic variants either containing or lacking transcriptionally competent p53, were used. Cells were incubated with TNFalpha cytokine to activate NFkappaB and then treated with ultraviolet or ionizing radiation to induce apoptosis, which was assessed by measurement of the sub-G1 cell fraction. We observed that treatment with TNFalpha resulted in a significant reduction in the frequency of apoptotic cells in UV-irradiated p53-proficient lines (with exception of the UV-resistant NCI-H1299 cells). This anti-apoptotic effect was lost when cells were pretreated with parthenolide, an inhibitor of NFkappaB activation. In marked contrast, TNFalpha-pretreatment of p53-deficient lines resulted in an increased frequency of apoptotic cells after UV irradiation (with exception of HL60 cells). Such anti- and pro-apoptotic influence of TNFalpha was less obvious in cells treated with ionizing radiation. The data clearly indicates functional interference of both signaling pathways upon the damage-induced apoptotic response, yet the observed effects are both cell type- and stimulus-specific.
18: Mol Vis. 2008;14:2076-2086. Epub 2008 Nov 17.
PCNA interacts with Prox1 and represses its transcriptional activity.
Chen X, Patel TP, Simirskii VI, Duncan MK.
Department of Biological Sciences,
PURPOSE: Prox1 is a transcription factor which can function either as a transcriptional activator, transcriptional repressor or a transcriptional corepressor. This paper seeks to better understand the role of protein-protein interactions in this multitude of functions. METHODS: We performed a yeast two-hybrid screen of an 11.5 day post coitum (dpc) mouse embryo cDNA library using the homeo-Prospero domain of Prox1 as bait. Computer modeling, cotransfection analysis and confocal immunolocalization were used to investigate the significance of one of the identified interactions. RESULTS: Proliferating cell nuclear antigen (PCNA) was identified as a Prox1 interacting protein. Prox1 interactions with PCNA require the PCNA interacting protein motif (PIP box), located in the Prospero domain of Prox1. Computer modeling of this interaction identified the apparent geometry of this interface which maintains the accessibility of Prox1 to DNA. Prox1 activated the chicken betaB1-crystallin promoter in cotransfection tests as previously reported, while PCNA squelched this transcriptional activation. CONCLUSIONS: Since PCNA is expressed in the lens epithelium where Prox1 levels are low, while chicken betaB1-crystallin expression activates in lens fibers where Prox1 expression is high and PCNA levels are low, these data suggest that Prox1-PCNA interactions may in part prevent the activation of betaB1-crystallin expression in the lens epithelium.
19: Mol Vis. 2008;14:2067-2075. Epub 2008 Nov 17.
Identification of five novel mutations in the long isoform of the USH2A gene in Chinese families with Usher syndrome type II.
Dai H, Zhang X, Zhao X, Deng T, Dong B, Wang J, Li Y.
Beijing Institute of Ophthalmology,
PURPOSE: Usher syndrome type II (USH2) is the most common form of Usher syndrome, an autosomal recessive disorder characterized by moderate to severe hearing loss, postpuberal onset of retinitis pigmentosa (RP), and normal vestibular function. Mutations in the USH2A gene have been shown to be responsible for most cases of USH2. To further elucidate the role of USH2A in USH2, mutation screening was undertaken in three Chinese families with USH2. METHODS: Three unrelated Chinese families, consisting of six patients and 10 unaffected relatives, were examined clinically, and 100 normal Chinese individuals served as controls. Genomic DNA was extracted from the venous blood of all participants. The coding region (exons 2-72), including the intron-exon boundary of USH2A, was amplified by polymerase chain reaction (PCR). The PCR products amplified from the three probands were analyzed using direct sequencing to screen sequence variants. Whenever substitutions were identified in a patient, restriction fragment length polymorphism analysis, or single strand conformation polymorphism analysis was performed on all available family members and the control group. RESULTS: Fundus examination revealed typical fundus features of RP, including narrowing of the vessels, bone-speckle pigmentation, and waxy optic discs. The ERG wave amplitudes of three probands were undetectable. Audiometric tests indicated moderate to severe sensorineural hearing impairment. Vestibular function was normal. Five novel mutations (one small insertion, one small deletion, one nonsense, one missense, and one splice site) were detected in three families after sequence analysis of USH2A. Of the five mutations, four were located in exons 22-72, specific to the long isoform of USH2A. CONCLUSIONS: The mutations found in our study broaden the spectrum of USH2A mutations. Our results further indicate that the long isoform of USH2A may harbor even more mutations of the USH2A gene.
20: PLoS Pathog. 2008 Nov;4(11):e1000213. Epub 2008 Nov 21.
Extracellular DNA Chelates Cations and Induces Antibiotic Resistance in Pseudomonas aeruginosa Biofilms.
Mulcahy H, Charron-Mazenod L, Lewenza S.
Department of Microbiology and Infectious Diseases,
Biofilms are surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, bacterial polysaccharides and proteins, which are up to 1000-fold more antibiotic resistant than planktonic cultures. To date, extracellular DNA has been shown to function as a structural support to maintain Pseudomonas aeruginosa biofilm architecture. Here we show that DNA is a multifaceted component of P. aeruginosa biofilms. At physiologically relevant concentrations, extracellular DNA has antimicrobial activity, causing cell lysis by chelating cations that stabilize lipopolysaccharide (LPS) and the outer membrane (
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