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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