Metastases suppressor NME2 associates with telomere ends and telomerase and reduces telomerase activity within cells

Analysis of chromatin-immunoprecipitation followed by sequencing (ChIP-seq) usually disregards sequence reads that do not map within binding positions (peaks). Using an unbiased approach, we analysed all reads, both that mapped and ones that were not included as part of peaks. ChIP-seq experiments were performed in human lung adenocarcinoma and fibrosarcoma cells for the metastasis suppressor non-metastatic 2 (NME2). Surprisingly, we identified sequence reads that uniquely represented human telomere ends in both cases. In vivo presence of NME2 at telomere ends was validated using independent methods and as further evidence we found intranuclear association of NME2 and the telomere repeat binding factor 2. Most remarkably, results demonstrate that NME2 associates with telomerase and reduces telomerase activity in vitro and in vivo, and sustained NME2 expression resulted in reduced telomere length in aggressive human cancer cells. Anti-metastatic function of NME2 has been demonstrated in human cancers, however, mechanisms are poorly understood. Together, findings reported here suggest a novel role for NME2 as a telomere binding protein that can alter telomerase function and telomere length. This presents an opportunity to investigate telomere-related interactions in metastasis suppression.     

Integrin α9β1-mediated cell migration in glioblastoma via SSAT and Kir4.2 potassium channel pathway

The α9β1 integrin accelerates cell migration through binding of the α9 cytoplasmic domain to SSAT, which catalyzes the catabolism of higher order polyamines, spermidine and spermine, to the lower order polyamine, putrescine. SSAT levels were downregulated at both the mRNA and protein levels by shRNA-mediated simultaneous knockdown of MMP-9 and uPAR/cathepsin B. In addition, we noted a prominent reduction in the expression of SSAT with MMP-9 and uPAR/cathepsin B knockdown in the tumor regions of 5310 injected nude mice brains. Further, SSAT knockdown in glioma xenograft cells significantly reduced their migration potential. Interestingly, MMP-9, uPAR and cathepsin B overexpression in these xenograft cells significantly elevated SSAT mRNA and protein levels. The migratory potential of MMP-9/uPAR/cathepsin B-overexpressed 4910 and 5310 cells was not affected by either glybenclamide (Kir 6.x inhibitor) or tertiapin-Q (Kir 1.1 and 3.x inhibitor) but instead was significantly inhibited by either barium or Kir4.2 siRNA treatments. Co-localization of α9 integrin with Kir4.2 was observed in both 4910 and 5310 xenograft cells. However, MMP-9 and uPAR/cathepsin B knockdown in these cells prominently reduced the co-localization of α9 with Kir4.2. Taken together, our results clearly demonstrate that α9β1 integrin-mediated cell migration utilizes SSAT and the Kir4.2 potassium channel pathway, and inhibition of the migratory potential of these glioma xenograft cells by simultaneous knockdown of MMP-9 and uPAR/cathepsin B could be attributed to the reduced SSAT levels and co-localization of α9 integrin with Kir4.2 inward rectifier potassium channels.     

Slow amyloid nucleation via α-helix-rich oligomeric intermediates in short polyglutamine-containing huntingtin fragments

The 17-amino-acid N-terminal segment (htt(NT)) that leads into the polyglutamine (polyQ) segment in the Huntington's disease protein huntingtin (htt) dramatically increases aggregation rates and changes the aggregation mechanism, compared to a simple polyQ peptide of similar length. With polyQ segments near or above the pathological repeat length threshold of about 37, aggregation of htt N-terminal fragments is so rapid that it is difficult to tease out mechanistic details. We describe here the use of very short polyQ repeat lengths in htt N-terminal fragments to slow this disease-associated aggregation. Although all of these peptides, in addition to htt(NT) itself, form α-helix-rich oligomeric intermediates, only peptides with Q(N) of eight or longer mature into amyloid-like aggregates, doing so by a slow increase in β-structure. Concentration-dependent circular dichroism and analytical ultracentrifugation suggest that the htt(NT) sequence, with or without added glutamine residues, exists in solution as an equilibrium between disordered monomer and α-helical tetramer. Higher order, α-helix rich oligomers appear to be built up via these tetramers. However, only htt(NT)Q(N) peptides with N=8 or more undergo conversion into polyQ β-sheet aggregates. These final amyloid-like aggregates not only feature the expected high β-sheet content but also retain an element of solvent-exposed α-helix. The α-helix-rich oligomeric intermediates appear to be both on- and off-pathway, with some oligomers serving as the pool from within which nuclei emerge, while those that fail to undergo amyloid nucleation serve as a reservoir for release of monomers to support fibril elongation. Based on a regular pattern of multimers observed in analytical ultracentrifugation, and a concentration dependence of α-helix formation in CD spectroscopy, it is likely that these oligomers assemble via a four-helix assembly unit. PolyQ expansion in these peptides appears to enhance the rates of both oligomer formation and nucleation from within the oligomer population, by structural mechanisms that remain unclear.     

Deletion of Specific Immune-Modulatory Genes from Modified Vaccinia Virus Ankara-Based HIV Vaccines Engenders Improved Immunogenicity in Rhesus Macaques

Modified vaccinia virus Ankara (MVA) is a safe, attenuated orthopoxvirus that is being developed as a vaccine vector but has demonstrated limited immunogenicity in several early-phase clinical trials. Our objective was to rationally improve the immunogenicity of MVA-based HIV/AIDS vaccines via the targeted deletion of specific poxvirus immune-modulatory genes. Vaccines expressing codon-optimized HIV subtype C consensus Env and Gag antigens were generated from MVA vector backbones that (i) harbor simultaneous deletions of four viral immune-modulatory genes, encoding an interleukin-18 (IL-18) binding protein, an IL-1β receptor, a dominant negative Toll/IL-1 signaling adapter, and CC-chemokine binding protein (MVAΔ4-HIV); (ii) harbor a deletion of an additional (fifth) viral gene, encoding uracil-DNA glycosylase (MVAΔ5-HIV); or (iii) represent the parental MVA backbone as a control (MVA-HIV). We performed head-to-head comparisons of the cellular and humoral immune responses that were elicited by these vectors during homologous prime-boost immunization regimens utilizing either high-dose (2 × 10⁸ PFU) or low-dose (1 × 10⁷ PFU) intramuscular immunization of rhesus macaques. At all time points, a majority of the HIV-specific T cell responses, elicited by all vectors, were directed against Env, rather than Gag, determinants, as previously observed with other vector systems. Both modified vectors elicited up to 6-fold-higher frequencies of HIV-specific CD8 and CD4 T cell responses and up to 25-fold-higher titers of Env (gp120)-specific binding (nonneutralizing) antibody responses that were relatively transient in nature. While the correlates of protection against HIV infection remain incompletely defined, our results indicate that the rational deletion of specific genes from MVA vectors can positively alter their cellular and humoral immunogenicity profiles in nonhuman primates.     

Association analysis of p16 (CDKN2A) and RB1 polymorphisms with susceptibility to cervical cancer in Indian population

The potent tumor suppressors P16 and RB1 are the key regulators of cell cycle machinery in eukaryotes. Polymorphisms in these genes play an important role in the outcome of various diseases including cancer. In the present study, we evaluated the association of p16 and RB1 polymorphisms with cervical cancer susceptibility in Indian population. We screened 150 histologically confirmed cervical cancer cases along with equal number of healthy controls with normal cervical cytology. PCR-RFLP method was employed for genotyping of SNPs in p16 C540G (rs11515), C580T (rs3088440) in the 3′-UTR of exon 3 and RB1 A153104G (rs4151580) located in the intron 18 and confirmed by direct sequencing. Both patients and controls were screened for HPV infection. In this case–control study 84.67% (127/150) of cases were found to be positive for HPV DNA sequence. Women carrying p16 C540G carrier genotypes 540 (CG/GG) may have protective effect for the development of cervical cancer (P = 0.0001, OR = 0.31, 95% CI = 0.17–0.56). And SNP at C580T of p16 gene was found to be negatively associated with the risk of cervical cancer (P = 0.0004, OR = 0.04, 95% CI = 0.002–0.63). p16 (540C/580T) has emerged as a major risk haplotype (P = 0.033, OR = 1.47, 95% CI = 1.05–2.07) whereas p16 (540G/580T) as a chief protective haplotype (P = 0.014, OR = 0.39, 95% CI = 0.18–0.83) for the development of cervical cancer among Indian women. Contrary to this, SNP at A153104G of RB1 gene showed statistically significant association (P = 0.035, OR = 1.69, 95% CI = 1.06–2.68) with increased susceptibility for the development of cervical cancer. Our results suggest that single nucleotide polymorphisms in p16, RB1 genes may affect the susceptibility to cervical cancer collectively.     

Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice

Biliary pancreatitis is the most common etiology for acute pancreatitis, yet its pathophysiological mechanism remains unclear. Ca(2+) signals generated within the pancreatic acinar cell initiate the early phase of pancreatitis, and bile acids can elicit anomalous acinar cell intracellular Ca(2+) release. We previously demonstrated that Ca(2+) released via the intracellular Ca(2+) channel, the ryanodine receptor (RyR), contributes to the aberrant Ca(2+) signal. In this study, we examined whether RyR inhibition protects against pathological Ca(2+) signals, acinar cell injury, and pancreatitis from bile acid exposure. The bile acid tauro-lithocholic acid-3-sulfate (TLCS) induced intracellular Ca(2+) oscillations at 50 μM and a peak-plateau signal at 500 μM, and only the latter induced acinar cell injury, as determined by lactate dehydrogenase (LDH) leakage. Pretreatment with the RyR inhibitors dantrolene or ryanodine converted the peak-plateau signal to a mostly oscillatory pattern (P < 0.05). They also reduced acinar cell LDH leakage, basolateral blebbing, and propidium iodide uptake (P < 0.05). In vivo, a single dose of dantrolene (5 mg/kg), given either 1 h before or 2 h after intraductal TLCS infusion, reduced the severity of pancreatitis down to the level of the control (P < 0.05). These results suggest that the severity of biliary pancreatitis may be ameliorated by the clinical use of RyR inhibitors.     

Pulvinus: an ideal explant for plant regeneration in <Emphasis Type="Italic">Caesalpinia bonduc</Emphasis> (L.) Roxb., an important ethnomedicinal woody climber

This study demonstrates the morphogenic potential of pulvinus, an important organ situated at the base of the petiole or rachis of leguminous plants. Plant regeneration via pulvinus-derived calli of Caesalpinia bonduc has been achieved. Organogenic calli have been derived from the explant 45 days after culture on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with 6-benzylaminopurine (BA). Optimum callus induction (100%) occurred when the pulvini were cultured on MS medium fortified with 6 mg l⁻¹ 2,4-D and 1 mg l⁻¹ BA. The highest shoot induction was obtained when the calli were transferred to MS medium supplemented with 5 mg l⁻¹ BA and 1 mg l⁻¹ indole-3-acetic acid (IAA). On this medium, 87% cultures responded with an average number of 4.2 shoots per culture. The maximum root induction from the regenerated shoots was observed on half strength MS medium containing 6 mg l⁻¹ indole-3-butyric acid (IBA). Here 100% shoots rooted with a mean number of 6.3 roots per shoot. The regenerated plantlets were acclimatized and subsequently showed normal growth. This efficient protocol will be helpful for propagating elite clones on a mass scale and could be utilized for genetic transformation study.     

Characterization and in vitro immunomodulatory screening of fructo-oligosaccharides of Asparagus racemosus Willd

Asparagus racemosus Linn. (Fam. Liliaceae) is an ethno-pharmacologically acclaimed Ayurvedic medicinal plant. In the present study, aqueous extract of A. racemosus (ARC) was fractionated and screened for the polysaccharide fraction (ARP). The characterization was done by enzymatic, Size Exclusion, gas chromatography with flame ionization detector (GC-FID), high pressure anion exchange chromatography (HPAEC) and thin layer chromatographic analyses. Phyto-chemical evaluation confirmed the presence of 26.7% of 2 → 1 linked fructo-oligosaccharides (FOS). They have a degree of polymerization (DP) of nearly 7-8. Cytotoxicity evaluation on P388 cell lines was consistent with low cytotoxicity of the extracts. In vitro Natural Killer (NK) cell activity was evaluated using human peripheral blood mononuclear cells (PBMC) isolated from whole blood on a ficoll-hypaque density gradient. K562 a myeloid leukemia cell line, were used as target cells. ARC, tested over the range 0.2-50 μg/ml, showed a dose-related stimulation of NK cell activity with a peak increase of 16.9 ± 4.4% at 5.6 μg/ml. However, ARP demonstrated a higher stimulatory activity of 51.8 ± 1.2% at 25 μg/ml. The results indicate that the FOS from A. racemosus potentiates the NK cell activity and this could be an important mechanism underpinning the ‘Rasayana’ properties of this plant.     

Fatty Acid Biosynthesis in Pseudomonas aeruginosa Is Initiated by the FabY Class of β-Ketoacyl Acyl Carrier Protein Synthases

The prototypical type II fatty acid synthesis (FAS) pathway in bacteria utilizes two distinct classes of β-ketoacyl synthase (KAS) domains to assemble long-chain fatty acids, the KASIII domain for initiation and the KASI/II domain for elongation. The central role of FAS in bacterial viability and virulence has stimulated significant effort toward developing KAS inhibitors, particularly against the KASIII domain of the β-acetoacetyl-acyl carrier protein (ACP) synthase FabH. Herein, we show that the opportunistic pathogen Pseudomonas aeruginosa does not utilize a FabH ortholog but rather a new class of divergent KAS I/II enzymes to initiate the FAS pathway. When a P. aeruginosa cosmid library was used to rescue growth in a fabH downregulated strain of Escherichia coli, a single unannotated open reading frame, PA5174, complemented fabH depletion. While deletion of all four KASIII domain-encoding genes in the same P. aeruginosa strain resulted in a wild-type growth phenotype, deletion of PA5174 alone specifically attenuated growth due to a defect in de novo FAS. Siderophore secretion and quorum-sensing signaling, particularly in the rhl and Pseudomonas quinolone signal (PQS) systems, was significantly muted in the absence of PA5174. The defect could be repaired by intergeneric complementation with E. coli fabH. Characterization of recombinant PA5174 confirmed a preference for short-chain acyl coenzyme A (acyl-CoA) substrates, supporting the identification of PA5174 as the predominant enzyme catalyzing the condensation of acetyl coenzyme A with malonyl-ACP in P. aeruginosa. The identification of the functional role for PA5174 in FAS defines the new FabY class of β-ketoacyl synthase KASI/II domain condensation enzymes.