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
Nov 22, 2008
Protocols of TUNEL
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 (
Advances in DNA (Gene) -1
1: Inflamm Bowel Dis. 2008 Nov 20. [Epub ahead of print]
Twin studies reveal specific imbalances in the mucosa-associated microbiota of patients with ileal Crohn's disease.
Willing B, Halfvarson J, Dicksved J, Rosenquist M, Järnerot G, Engstrand L, Tysk C, Jansson JK.
Department of Microbiology,
Background: Large interindividual variation in the composition of the intestinal microbiota between unrelated individuals has made it challenging to identify specific aspects of dysbiosis that lead to Crohn's disease (CD).Methods: To reduce variations in exposure during establishment of the gut flora and the influence of genotype, we studied the mucosa-associated microbiota of monozygotic twin pairs that were discordant (n = 6) or concordant (n = 4) for CD. DNA was extracted from biopsies collected from 5 locations between the ileum and rectum. Bacterial 16S ribosomal RNA genes were amplified and community composition assessed by terminal-restriction fragment length polymorphism, cloning and sequencing, and quantitative real-time polymerase chain reaction (PCR).Results: The microbial compositions at all biopsy locations for each individual were similar, regardless of disease state, but there were differences between individuals. In particular, individuals with predominantly ileal CD had a dramatically lower abundance (P < style="">
2: Genes Chromosomes Cancer. 2008 Nov 20. [Epub ahead of print]
Identification of a potential "hotspot" DNA region in the RUNX1 gene targeted by mitoxantrone in therapy-related acute myeloid leukemia with t(16;21) translocation.
Ottone T, Hasan SK, Montefusco E, Curzi P, Mays AN, Chessa L, Ferrari A, Conte E, Noguera NI, Lavorgna S, Ammatuna E, Divona M, Bovetti K, Amadori S, Grimwade D, Lo-Coco F.
Dipartimento di Biopatologia e Diagnostica per Immagini, University "Tor Vergata,"
The translocation t(16;21) involving RUNX1 (AML1) and resulting in the RUNX1-CBFA2T3 fusion is a rare but recurrent abnormality mostly found in therapy-related acute myeloid leukemia (t-AML) associated with agents targeting topoisomerase II (topo II). We characterized, at the genomic level, the t(16;21) translocation in a patient who developed t-AML after treatment of multiple sclerosis with mitoxantrone (MTZ). Long template nested PCR of genomic DNA followed by direct sequencing enabled the localization of RUNX1 and CBFA2T3 (ETO2) breakpoints in introns 5 and 3, respectively. Sequencing of the cDNA with specific primers showed the presence of the expected RUNX1-CBFA2T3 fusion transcript in leukemic cells. The RUNX1 intron 5 breakpoint was located at nucleotide position 24,785. This region contained an ATGCCCCAG nucleotide sequence showing approximately 90% homology to a "hotspot" DNA region ATGCCCTAG present in intron 6 of PML previously identified in therapy-related acute promyelocytic leukemia cases arising following treatment with MTZ. This study suggests a wider distribution in the human genome, and particularly at genes involved in chromosome translocations observed in t-AML, of DNA regions (hotspot) targeted by specific topo II drugs. (c) 2008 Wiley-Liss, Inc.
3: Hum Mutat. 2008 Nov 19. [Epub ahead of print]
Genomic deletions of OFD1 account for 23% of oral-facial-digital type 1 syndrome after negative DNA sequencing.
Thauvin-Robinet C, Franco B, Saugier-Veber P, Aral B, Gigot N, Donzel A, Van Maldergem L, Bieth E, Layet V, Mathieu M, Teebi A, Lespinasse J, Callier P, Mugneret F, Masurel-Paulet A, Gautier E, Huet F, Teyssier JR, Tosi M, Frébourg T, Faivre L.
Centre de Génétique, Hôpital d'Enfants, CHU Dijon, France.
Oral-facial-digital type I syndrome (OFDI) is characterised by an X-linked dominant mode of inheritance with lethality in males. Clinical features include facial dysmorphism with oral, dental and distal abnormalities, polycystic kidney disease and central nervous system malformations. Considerable allelic heterogeneity has been reported within the OFD1 gene, but DNA bi-directional sequencing of the exons and intron-exon boundaries of the OFD1 gene remains negative in more than 20% of cases. We hypothesized that genomic rearrangements could account for the majority of the remaining undiagnosed cases. Thus, we took advantage of two independent available series of patients with OFDI syndrome and negative DNA bi-directional sequencing of the exons and intron-exon boundaries of the OFD1 gene from two different European labs: 13/36 cases from the French lab; 13/95 from the Italian lab. All patients were screened by a semiquantitative fluorescent multiplex method (QFMPSF) and relative quantification by real-time PCR (qPCR). Six OFD1 genomic deletions (exon 5, exons 1-8, exons 1-14, exons 10-11, exons 13-23 and exon 17) were identified, accounting for 5% of OFDI patients and for 23% of patients with negative mutation screening by DNA sequencing. The association of DNA direct sequencing, QFMPSF and qPCR detects OFD1 alteration in up to 85% of patients with a phenotype suggestive of OFDI syndrome. Given the average percentage of large genomic rearrangements (5%), we suggest that dosage methods should be performed in addition to DNA direct sequencing analysis to exclude the involvement of the OFD1 transcript when there are genetic counselling issues. (c) 2008 Wiley-Liss, Inc.
4: Nat Prod Res. 2008 Nov;22(16):1441-1450.
Apoptotic cell death through inhibition of protein kinase CKII activity by 3,4-dihydroxybenzaldehyde purified from Xanthium strumarium.
Lee BH, Yoon SH, Kim YS, Kim SK, Moon BJ, Bae YS.
Department of Biochemistry,
The CKII inhibitory compound was purified from the fruit of Xanthium strumarium by organic solvent extraction and silica gel chromatography. The inhibitory compound was identified as 3,4-dihydroxybenzaldehyde by analysis with FT-IR, FAB-Mass, EI-Mass, (1)H-NMR and (13)C-NMR. 3,4-dihydroxybenzaldehyde inhibited the phosphotransferase activity of CKII with IC(50) of about 783 microM. Steady-state studies revealed that the inhibitor acts as a competitive inhibitor with respect to the substrate ATP. A value of 138.6 microM was obtained for the apparent K(i). Concentration of 300 microM 3,4-dihydroxybenzaldehyde caused 50% growth inhibition of human cancer cell U937. 3,4-dihydroxybenzaldehyde-induced cell death was characterised with the cleavage of poly(ADP-ribose) polymerase and procaspase-3. Furthermore, the inhibitor induced the fragmentation of DNA into multiples of 180 bp, indicating that it triggered apoptosis. This induction of apoptosis by 3,4-dihydroxybenzaldehyde was also confirmed by using flow cytometry analysis. Since CKII is involved in cell proliferation and oncogenesis, these results suggest that 3,4-dihydroxybenzaldehyde may function by inhibiting oncogenic disease, at least in part, through the inhibition of CKII activity.
5: Scand J Infect Dis. 2008 Nov 20:1-7. [Epub ahead of print]
A nosocomial outbreak of Candida parapsilosis in southern
Brillowska-Dabrowska A, Schon T, Pannanusorn S, Lonnbro N, Bernhoff L, Bonnedal J, Haggstrom J, Wistedt A, Fernandez V, Arendrup MC.
Unit of Mycology and Parasitology, Statens Serum Institute,
In a haematology ward, Candida parapsilosis was found in blood cultures from 4 patients within a month. As C. parapsilosis is known to have a restricted genetic diversity, a combined methodological approach was adopted to establish a possible epidemiological relationship among the isolates (n = 9). Multilocus sequence typing and random amplified polymorphic DNA analysis suggested a clonal origin of the isolates. The clonal origin was confirmed by microsatellite analysis, a method that displayed the highest discriminatory level and readily differentiated cluster isolates from 2 epidemiologically unrelated strains of C. parapsilosis. The use of novel methods of genotyping such as microsatellite analysis will facilitate epidemiological investigations of potential clonal outbreaks of fungaemia.
6: J Comput Aided Mol Des. 2008 Nov 21. [Epub ahead of print]
Binding of the Zn(2+) ion to ferric uptake regulation protein from E. coli and the competition with Fe(2+) binding: a molecular modeling study of the effect on DNA binding and conformational changes of Fur.
Jabour S, Hamed MY.
Computational Science Program, Chemistry Department,
The three dimensional structure of Ferric uptake regulation protein dimer from E. coli, determined by molecular modeling, was docked on a DNA fragment (iron box) and Zn(2+) ions were added in two steps. The first step involved the binding of one Zn(2+) ion to what is known as the zinc site which consists of the residues Cys 92, Cys 95, Asp 137, Asp141, Arg139, Glu 140, His 145 and His 143 with an average metal-Nitrogen distance of 2.5 A and metal-oxygen distance of 3.1-3.2 A. The second Zn(2+) ion is bound to the iron activating site formed from the residues Ile 50, His 71, Asn 72, Gly 97, Asp 105 and Ala 109. The binding of the second Zn(2+) ion strengthened the binding of the first ion as indicated by the shortening of the zinc-residue distances. Fe(2+), when added to the complex consisting of 2Zn(2+)/Fur dimer/DNA, replaced the Zn(2+) ion in the zinc site and when a second Fe(2+) was added, it replaced the second zinc ion in the iron activating site. The binding of both zinc and iron ions induced a similar change in Fur conformations, but shifted residues closer to DNA in a different manner. This is discussed along with a possible role for the Zn(2+) ion in the Fur dimer binding of DNA in its repressor activity.
7: J Neuroimmune Pharmacol. 2008 Nov 21. [Epub ahead of print]
TNF Alpha Production in Morphine-Treated Human Neural Cells Is NF-kappaB-Dependent.
Sawaya BE, Deshmane SL, Mukerjee R, Fan S, Khalili K.
Department of Neuroscience, Center for Neurovirology, Temple University School of Medicine, 1900N 12th Street, Philadelphia, PA, 19122, USA, sawaya@temple.edu.
The cytokine tumor necrosis factor alpha (TNFalpha) is a key factor in several inflammatory diseases and its levels increase in response to a variety of internal or external stimuli. The regulation of the TNFalpha promoter is mediated by several transcription factors including the nuclear factor kappa B protein (NF-kappaB). This study examines the role of NF-kappaB in the regulation of TNFalpha production by morphine in microglia. Using reverse transcriptase polymerase chain reaction, we demonstrated the presence of morphine receptors in these cells. We next demonstrated the ability of morphine to promote TNFalpha production and secretion by these cells using a cytokine array assay. Transient transfection experiments led to the identification of the region located between nucleotides -751 and -615 within the TNFalpha promoter as being responsive to morphine treatment. The DNA sequence of this region contains a motif indicative of a potential NF-kappaB binding site. The use of a small interfering RNA directed against p65, a subunit of NF-kappaB, demonstrated that TNFalpha induction by morphine is NF-kappaB-dependent. All of the effects of morphine were reversed by the morphine inhibitor, naloxone. These data provide important insights into the effects of morphine on microglia.
8: Neurochem Res. 2008 Nov 21. [Epub ahead of print]
Neuronal Mitochondrial Toxicity of Malondialdehyde: Inhibitory Effects on Respiratory Function and Enzyme Activities in Rat Brain Mitochondria.
Long J, Liu C, Sun L, Gao H, Liu J.
Institute for Brain Aging and Dementia,
Malondialdehyde (MDA) is a product of oxidative damage to lipids, amino acids and DNA, and accumulates with aging and diseases. MDA can possibly react with amines so as to modify proteins and inactivate enzymes; it can also modify nucleosides so as to cause mutagenicity. Brain mitochondrial dysfunction is a major contributor to aging and neurodegenerative diseases. We hypothesize that MDA accumulated during aging targets mitochondrial enzymes so as to cause further mitochondrial dysfunction and additional contributions to aging and neurodegeneration. Herein, we investigated the neuronal mitochondrial toxic effects of MDA on mitochondrial respiration and activities of enzymes (mitochondrial complexes I-V, alpha-ketoglutarate dehydrogenase (KGDH) and pyruvate dehydrogenase (PDH)), in isolated rat brain mitochondria. MDA depressed mitochondrial membrane potential, and also showed a dose-dependent inhibition of mitochondrial complex I- and complex II-linked respiration. Complex I and II, and PDH activities were depressed by MDA at >/=0.2 mumol/mg; KGDH and complex V were inhibited by >/=0.4 and >/=1.6 mumol MDA/mg, respectively. However, MDA did not have any toxic effects on complex III and IV activities over the range 0-2 mumol/mg. MDA significantly elevated mitochondrial reactive oxygen species (ROS) and protein carbonyls at 0.2 and 0.002 mumol/mg, respectively. As for the antioxidant defense system, a high dose of MDA slightly decreased mitochondrial GSH and superoxide dismutase. These results demonstrate that MDA causes neuronal mitochondrial dysfunction by directly promoting generation of ROS and modifying mitochondrial proteins. The results suggest that MDA-induced neuronal mitochondrial toxicity may be an important contributing factor to brain aging and neurodegenerative diseases.
9: Mod Rheumatol. 2008 Nov 22. [Epub ahead of print]
A1330V polymorphism of low-density lipoprotein receptor-related protein 5 gene and self-reported incident fractures in Japanese female patients with rheumatoid arthritis.
Furuya T, Urano T, Ikari K, Kotake S, Inoue S, Hara M, Momohara S, Kamatani N, Yamanaka H.
We attempted to determine whether the A1330V polymorphism of the low-density lipoprotein receptor-related protein 5 (LRP5) gene is associated with a risk of self-reported incident fractures and hypercholesterolemia in Japanese patients with rheumatoid arthritis (RA). DNA samples, laboratory data, and clinical data were obtained from 563 female RA patients who participated in the Institute of Rheumatology Rheumatoid Arthritis (IORRA) observational cohort study. A1330V genotyping was performed using a custom TaqMan assay. Multiple logistic regression analyses showed that any incident fracture was significantly associated with older age (P = 0.000000036), high Japanese Health Assessment Questionnaire (J-HAQ) score (P = 0.016), and high daily prednisolone dose (P = 0.031), but not with the A1330V polymorphism, while serum total cholesterol levels >/=220 mg/100 mL were independently correlated with baseline older age (P = 0.00011), low J-HAQ score (P = 0.0098), high body mass index (P = 0.024), 1330VV genotype (P = 0.027), and high daily prednisolone dose (P = 0.031). Our results suggest that this LPR5 polymorphism does not appear to be a clinically useful marker for the prediction of fracture risk in Japanese female RA patients, although it is associated with increased serum total cholesterol levels.
10: World J Surg. 2008 Nov 21. [Epub ahead of print]
Nonviral Delivery for Genomic Therapy of Cancer.
Templeton NS.
Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, M/S 390, Houston, TX, 77030, USA, NANCYT@bcm.tmc.edu.
We have developed improved liposomal nanoparticles that efficiently condense nucleic acids, proteins, viruses, drugs, and mixtures of these agents on the interior of bilamellar invaginated structures (BIVs) produced by a novel extrusion procedure. The liposomal complexes have extended half-life in the circulation, serum stability, and broad biodistribution; are targetable to specific organs and cell types; can penetrate through tight barriers in several organs; are fusogenic with cell membranes and avoid endosomes; are optimized for nucleic acid:lipid ratio and colloidal suspension in vivo; can be size fractionated to produce totally homogeneous populations of complexes prior to injection; are nontoxic, nonimmunogenic, and can be repeatedly administered; and they are stable in liquid suspensions and freeze-dried formulations. We can add specific ligands either by ionic interactions or by covalent attachments to the surface of these nucleic acid-liposome complexes to accomplish targeted delivery to specific cell surface receptors. Furthermore, the charge on the surface of these complexes can be modified to avoid uptake by nontarget cells using our novel technology called "reversible masking." We have also achieved high-dose systemic delivery of these complexes without toxicity in vivo by further purification of plasmid DNA. At present, these complexes are injected intravenously into patients in clinical trials to treat lung cancer and will be used in upcoming trials to treat breast, pancreatic, and head and neck cancers. Notably, BIV complexes are being injected intravenously into patients with non-small-cell lung carcinoma who have failed to respond to chemotherapy. These patients are living longer and have demonstrated objective responses, including tumor regression.
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