Role of group A p21-activated kinases in activation of extracellular-regulated kinase by growth factors

The canonical extracellular-regulated kinase (ERK) signaling cascade, consisting of the Ras-Raf-Mek-ERK module, is critically important to many cellular functions. Although the general mechanism of activation of the ERK cascade is well established, additional noncanonical components greatly influence the activity of this pathway. Here, we focus on the group A p21-activated kinases (Paks), which have previously been implicated in regulating both c-Raf and Mek1 activity, by phosphorylating these proteins at Ser(338) and Ser(298), respectively. In NIH-3T3 cells, expression of an inhibitor of all three group A Paks reduced activation of ERK in response to platelet-derived growth factor (PDGF) but not to epidermal growth factor (EGF). Similar results were obtained in HeLa cells using small interference RNA-mediated simultaneous knockdown of both Pak1 and Pak2 to reduce group A Pak function. Inhibition of Pak kinase activity dramatically decreased phosphorylation of Mek1 at Ser(298) in response to either PDGF or EGF, but this inhibition did not prevent Mek1 activation by EGF, suggesting that although Pak can phosphorylate Mek1 at Ser(298), this event is not required for Mek1 activation by growth factors. Inhibition of Pak reduced the Ser(338) phosphorylation of c-Raf in response to both PDGF and EGF; however, in the case of EGF, the reduction in Ser(338) phosphorylation was not accompanied by a significant decrease in c-Raf activity. These findings suggest that Paks are required for the phosphorylation of c-Raf at Ser(338) in response to either growth factor, but that the mechanisms by which EGF and PDGF activate c-Raf are fundamentally different.     

Immune Activation in the Female Genital Tract: Expression Profiles of Soluble Proteins in Women at High Risk for HIV Infection

Soluble cervicovaginal biomarkers of inflammation, immune activation and risk of HIV acquisition are needed to reliably assess the safety of new biomedical prevention strategies including vaccines and microbicides. However, a fuller understanding of expression profiles in women at high risk for HIV infection is crucial to the effective use of these potential biomarkers in Phase 3 trial settings. We have measured 45 soluble proteins and peptides in cervicovaginal lavage samples from 100 HIV negative women at high risk for HIV infection. Women were followed over one menstrual cycle to investigate modulation by hormonal contraception, menstrual cycle phase, recent sexual exposure and intravaginal practices. Women using injectable DMPA had increased concentration of several soluble proteins of the innate and adaptive immune system, including IL-1α, IL-1β, IL-2, MIP-1β, IP-10, IL-8, TGF-β, HBD4, IgA, IgG1, and IgG2. Women using combined oral contraceptives had a similar signature. There were differences in concentrations among samples from post-ovulation compared to pre-ovulation, notably increased immunoglobulins. Increased prostate-specific antigen, indicative of recent sexual exposure, was correlated with increased IL-6, MCP-1, and SLPI, and decreased GM-CSF and HBD3. The identified signature profiles may prove critical in evaluating the potential safety and impact on risk of HIV acquisition of different biomedical intervention strategies.     

Dosimetric parameters of the new design 103Pd brachytherapy source based on Monte Carlo study

In this study version 5 of the MCNP photon transport simulation was used to calculate the dosimetric parameters for new palladium brachytherapy source design following AAPM Task Group No. 43U1 report. The internal source components include four resin beads of 0.6 mm diameters with ¹⁰³Pd uniformly absorbed inside and one cylindrical copper marker with 1.5 mm length. The resin beads and marker are then encapsulated within 0.8 mm in diameter and 4.5 mm long cylindrical capsule of titanium. The dose rate constant, Λ, line and point-source radial dose function, g_L(r) and g_P(r), and the anisotropy function, F(r,θ) of the IR01-¹⁰³Pd seed have been calculated at distances from 0.25 to 5 cm. All the results are in good agreement with previously published thermoluminescence-dosimeter measured values [3] for the source. The dosimetric parameters calculated in this work showed that in dosimetry point of view, the IR01-¹⁰³Pd seed is suitable for use in brachytherapy of prostate cancer.     

Human telomeres that carry an integrated copy of human herpesvirus 6 are often short and unstable,facilitating release of the viral genome from the chromosome

Linear chromosomes are stabilized by telomeres, but the presence of short dysfunctional telomeres triggers cellular senescence in human somatic tissues, thus contributing to ageing. Approximately 1% of the population inherits a chromosomally integrated copy of human herpesvirus 6 (CI-HHV-6), but the consequences of integration for the virus and for the telomere with the insertion are unknown. Here we show that the telomere on the distal end of the integrated virus is frequently the shortest measured in somatic cells but not the germline. The telomere carrying the CI-HHV-6 is also prone to truncations that result in the formation of a short telomere at a novel location within the viral genome. We detected extra-chromosomal circular HHV-6 molecules, some surprisingly comprising the entire viral genome with a single fully reconstituted direct repeat region (DR) with both terminal cleavage and packaging elements (PAC1 and PAC2). Truncated CI-HHV-6 and extra-chromosomal circular molecules are likely reciprocal products that arise through excision of a telomere-loop (t-loop) formed within the CI-HHV-6 genome. In summary, we show that the CI-HHV-6 genome disrupts stability of the associated telomere and this facilitates the release of viral sequences as circular molecules, some of which have the potential to become fully functioning viruses.     

A highly sensitive and specific multiplex PCR assay for simultaneous detection of Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus

Aims: To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human. Methods and Results: Species‐specific PCR primers were designed based on toxR gene for V. cholerae and V. parahaemolyticus, and vvhA gene for V. vulnificus. The multiplex PCR was validated with 488 Vibrio strains including 322 V. cholerae, 12 V. vulnificus, and 82 V. parahaemolyticus, 20 other Vibrio species and 17 other bacterial species associated with human diseases. It could detect the three target bacteria without any ambiguity even among closely related species. It showed good efficiency in detection of co‐existing target species in the same sample. The detection limit of all the target species was ten cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient for simultaneous detection of these potentially pathogenic Vibrio species in clinical and environmental samples. Significance and Impact of the Study: This simple, rapid and cost‐effective method can be applicable in a prediction system to prevent disease outbreak by these Vibrio species and can be considered as an effective tool for both epidemiologist and ecologist.     

Dual functions of Nbs1 in the repair of DNA breaks and proliferation ensure proper V(D)J recombination and T-cell development

Immunodeficiency and lymphoid malignancy are hallmarks of the human disease Nijmegen breakage syndrome (NBS; OMIM 251260), which is caused by NBS1 mutations. Although NBS1 has been shown to bind to the T-cell receptor alpha (TCRα) locus, its role in TCRβ rearrangement is unclear. Hypomorphic mutations of Nbs1 in mice and patients result in relatively mild T-cell deficiencies, raising the question of whether the truncated Nbs1 protein might have clouded a certain function of NBS1 in T-cell development. Here we show that the deletion of the entire Nbs1 protein in T-cell precursors (Nbs1(T-del)) results in severe lymphopenia and a hindrance to the double-negative 3 (DN3)-to-DN4 transition in early T-cell development, due to abnormal TCRβ coding and signal joints as well as the functions of Nbs1 in T-cell expansion. Chromatin immunoprecipitation (ChIP) analysis of the TCR loci reveals that Nbs1 depletion compromises the loading of Mre11/Rad50 to V(D)J-generated DNA double-strand breaks (DSBs) and thereby affects resection of DNA termini and chromatin conformation of the postcleavage complex. Although a p53 deficiency relieves the DN3→DN4 transition block, neither a p53 deficiency nor ectopic expression of TCRαβ rescues the major T-cell loss in Nbs1(T-del) mice. All together, these results demonstrate that Nbs1's functions in both repair of V(D)J-generated DSBs and proliferation are essential for T-cell development.     

Acylated 5,7,2',6'-oxygenated flavone glycosides from Andrographis alata

Five acylated 5,7,2',6'-oxygenated flavone glycosides along with the known 5,2',6'-trihydroxy-7-methoxyflavone-2'-O-beta-d-glucopyranoside have been isolated from the whole plant of Andrographis alata. The structures of the compounds were established from spectral (mainly 1D and 2D NMR) and chemical studies.     

Frequent Intra-Subtype Recombination among HIV-1 Circulating in Tanzania

The study estimated the prevalence of HIV-1 intra-subtype recombinant variants among female bar and hotel workers in Tanzania. While intra-subtype recombination occurs in HIV-1, it is generally underestimated. HIV-1 env gp120 V1-C5 quasispecies from 45 subjects were generated by single-genome amplification and sequencing (median (IQR) of 38 (28–50) sequences per subject). Recombination analysis was performed using seven methods implemented within the recombination detection program version 3, RDP3. HIV-1 sequences were considered recombinant if recombination signals were detected by at least three methods with p-values of ≤0.05 after Bonferroni correction for multiple comparisons. HIV-1 in 38 (84%) subjects showed evidence for intra-subtype recombination including 22 with HIV-1 subtype A1, 13 with HIV-1 subtype C, and 3 with HIV-1 subtype D. The distribution of intra-patient recombination breakpoints suggested ongoing recombination and showed selective enrichment of recombinant variants in 23 (60%) subjects. The number of subjects with evidence of intra-subtype recombination increased from 29 (69%) to 36 (82%) over one year of follow-up, although the increase did not reach statistical significance. Adjustment for intra-subtype recombination is important for the analysis of multiplicity of HIV infection. This is the first report of high prevalence of intra-subtype recombination in the HIV/AIDS epidemic in Tanzania, a region where multiple HIV-1 subtypes co-circulate. HIV-1 intra-subtype recombination increases viral diversity and presents additional challenges for HIV-1 vaccine design.     

The Membrane-Associated Transient Receptor Potential Vanilloid Channel Is the Central Heat Shock Receptor Controlling the Cellular Heat Shock Response in Epithelial Cells

The heat shock response (HSR) is a highly conserved molecular response to various types of stresses, including heat shock, during which heat-shock proteins (Hsps) are produced to prevent and repair damages in labile proteins and membranes. In cells, protein unfolding in the cytoplasm is thought to directly enable the activation of the heat shock factor 1 (HSF-1), however, recent work supports the activation of the HSR via an increase in the fluidity of specific membrane domains, leading to activation of heat-shock genes. Our findings support the existence of a plasma membrane-dependent mechanism of HSF-1 activation in animal cells, which is initiated by a membrane-associated transient receptor potential vanilloid receptor (TRPV). We found in various non-cancerous and cancerous mammalian epithelial cells that the TRPV1 agonists, capsaicin and resiniferatoxin (RTX), upregulated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70 and Hsp90 respectively, while the TRPV1 antagonists, capsazepine and AMG-9810, attenuated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70, Hsp90, respectively. Capsaicin was also shown to activate HSF-1. These findings suggest that heat-sensing and signaling in mammalian cells is dependent on TRPV channels in the plasma membrane. Thus, TRPV channels may be important drug targets to inhibit or restore the cellular stress response in diseases with defective cellular proteins, such as cancer, inflammation and aging.