Absence of DICER in Monocytes and Its Regulation by HIV-1

MicroRNAs (miRNAs) are a class of small RNA molecules that function to control gene expression and restrict viral replication in host cells. The production of miRNAs is believed to be dependent upon the DICER enzyme. Available evidence suggests that in T lymphocytes, HIV-1 can both suppress and co-opt the host''s miRNA pathway for its own benefit. In this study, we examined the state of miRNA production in monocytes and macrophages as well as the consequences of viral infection upon the production of miRNA. Monocytes in general express low amounts of miRNA-related proteins, and DICER in particular could not be detected until after monocytes were differentiated into macrophages. In the case where HIV-1 was present prior to differentiation, the expression of DICER was suppressed. MicroRNA chip results for RNA isolated from transfected and treated cells indicated that a drop in miRNA production coincided with DICER protein suppression in macrophages. We found that the expression of DICER in monocytes is restricted by miR-106a, but HIV-1 suppressed DICER expression via the viral gene Vpr. Additionally, analysis of miRNA expression in monocytes and macrophages revealed evidence that some miRNAs can be processed by both DICER and PIWIL4. Results presented here have implications for both the pathology of viral infections in macrophages and the biogenesis of miRNAs. First, HIV-1 suppresses the expression and function of DICER in macrophages via a previously unknown mechanism. Second, the presence of miRNAs in monocytes lacking DICER indicates that some miRNAs can be generated by proteins other than DICER.     

A simple clustering algorithm can be accurate enough for use in calculations of pKs in macromolecules

Structure and function of macromolecules depend critically on the ionization states of their acidic and basic groups. Most current structure-based theoretical methods that predict pK of ionizable groups in macromolecules include, as one of the key steps, a computation of the partition sum (Boltzmann average) over all possible protonation microstates. As the number of these microstates depends exponentially on the number of ionizable groups present in the molecule, direct computation of the sum is not realistically feasible for many typical proteins that may have tens or even hundreds of ionizable groups. We have tested a simple and robust approximate algorithm for computing these partition sums for macromolecules. The method subdivides the interacting sites into independent clusters, based upon the strength of site-site electrostatic interaction. The resulting partition function is factorizable into computationally manageable components. Two variants of the approach are presented and validated on a representative test set of 602 proteins, by comparing the pK(1/2) values computed by the proposed method with those obtained by the standard Monte Carlo approach used as a reference. With 95% confidence, the relative error introduced by the more accurate of the two methods is less than 0.25 pK units. The algorithms are one to two orders of magnitude faster than the Monte Carlo method, with the typical settings. A graphical representation is introduced that visualizes the clusters of strong site-site interactions in the context of the three-dimensional (3D) structure of the macromolecule, facilitating identification of functionally important clusters of ionizable groups; the approach is exemplified on two proteins, bacteriorhodopsin and myoglobin.     

Adaphostin and other anticancer drugs quench the fluorescence of mitochondrial potential probes

Fluorescent dyes are widely used to monitor changes in mitochondrial transmembrane potential (DeltaPsim). When MitoTracker Red CMXRos, tetramethylrhodamine methyl ester (TMRM), and 3,3'dihexyloxacarbocyanine iodide (DiOC6(3)) were utilized to examine the effects of the experimental anticancer drug adaphostin on intact cells or isolated mitochondria, decreased fluorescence was observed. In contrast, measurement of tetraphenylphosphonium uptake by the mitochondria using an ion-selective microelectrode failed to show any effect of adaphostin on DeltaPsim. Instead, further experiments demonstrated that adaphostin quenches the fluorescence of the mitochondrial dyes. Structure-activity analysis revealed that the adamantyl and p-aminobenzoic acid moieties of adaphostin are critical for this quenching. Anticancer drugs containing comparable structural motifs, including mitoxantrone, aminoflavone, and amsacrine, also quenched the mitochondrial probes. These results indicate the need for caution when mitochondrial dyes are utilized to examine the effects of xenobiotics on DeltaPsim and suggest that some previously reported direct effects of anticancer drugs on mitochondria might need re-evaluation.     

Effect of Triphala on oxidative stress and on cell-mediated immune response against noise stress in rats

Stress is one of the basic factors in the etiology of number of diseases. The present study was aimed to investigate the effect of Triphala (Terminalia chebula, Terminalia belerica and Emblica officinalis) on noise-stress induced alterations in the antioxidant status and on the cell-mediated immune response in Wistar strain male albino rats. Noise-stress employed in this study was 100 dB for 4 h/d/15 days and Triphala was used at a dose of 1 g/kg/b.w/48 days. Eight different groups of rats namely, non-immunized: control, Triphala, noise-stress, Triphala with noise-stress, and corresponding immunized groups were used. Sheep red blood cells (5×10⁹ cells/ml) were used to immunize the animals. Biochemical indicators of oxidative stress namely lipid peroxidation, antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), ascorbic acid in plasma and tissues (thymus and spleen) and SOD, GPx and corticosterone level in plasma were estimated. Cell-mediated immune response namely foot pad thickness (FPT) and leukocyte migration inhibition (LMI) test were performed only in immunized groups. Results showed that noise-stress significantly increased the lipid peroxidation and corticosterone level with concomitant depletion of antioxidants in plasma and tissues of both non-immunized and immunized rats. Noise-stress significantly suppressed the cell-mediated immune response by decreased FPT with an enhanced LMI test. The supplementation with Triphala prevents the noise-stress induced changes in the antioxidant as well as cell-mediated immune response in rats. This study concludes that Triphala restores the noise-stress induced changes may be due to its antioxidant properties.     

Structure/function mapping of amino acids in the N-terminal zinc finger of the human immunodeficiency virus type 1 nucleocapsid protein: residues responsible for nucleic acid helix destabilizing activity

The nucleocapsid protein (NC) of HIV-1 is 55 amino acids in length and possesses two CCHC-type zinc fingers. Finger one (N-terminal) contributes significantly more to helix destabilizing activity than finger two (C-terminal). Five amino acids differ between the two zinc fingers. To determine at the amino acid level the reason for the apparent distinction between the fingers, each different residue in finger one was incrementally replaced by the one at the corresponding location in finger two. Mutants were analyzed in annealing assays with unstructured and structured substrates. Three groupings emerged: (1) those similar to wild-type levels (N17K, A25M), (2) those with diminished activity (I24Q, N27D), and (3) mutant F16W, which had substantially greater helix destabilizing activity than that of the wild type. Unlike I24Q and the other mutants, N27D was defective in DNA binding. Only I24Q and N27D showed reduced strand transfer in in vitro assays. Double and triple mutants F16W/I24Q, F16W/N27D, and F16W/I24Q/N27D all showed defects in DNA binding, strand transfer, and helix destabilization, suggesting that the I24Q and N27D mutations have a dominant negative effect and abolish the positive influence of F16W. Results show that amino acid differences at positions 24 and 27 contribute significantly to finger one's helix destabilizing activity.     

Hyperhomocysteinemia decreases intestinal motility leading to constipation

Elevated levels of plasma homocysteine (Hcy) called hyperhomocysteinemia (HHcy) have been implicated in inflammation and remodeling in intestinal vasculature, and HHcy is also known to aggravate the pathogenesis of inflammatory bowel disease (IBD). Interestingly, colon is the pivotal site that regulates Hcy levels in the plasma. We hypothesize that HHcy decreases intestinal motility through matrix metalloproteinase-9 (MMP-9)-induced intestinal remodeling leading to constipation. To verify this hypothesis, we used C57BL/6J or wild-type (WT), cystathionine β-synthase (CBS(+/-)), MMP-9(-/-), and MMP-9(-/-) + Hcy mice. Intestinal motility was assessed by barium meal studies and daily feces output. Plasma Hcy levels were measured by HPLC. Expression of ICAM-1, inducible nitric oxide synthase, MMP-9, and tissue inhibitors of MMPs was studied by Western blot and immunohistochemistry. Reactive oxygen species (ROS) including super oxide were measured by the Invitrogen molecular probe method. Tissue nitric oxide levels were assessed by a commercially available kit. Plasma Hcy levels in the treated MMP-9 group mice were comparable to CBS(+/-) mice. Barium meal studies suggest that intestinal motility is significantly decreased in CBS(+/-) mice compared with other groups. Fecal output-to-body weight ratio was significantly reduced in CBS(+/-) mice compared with other groups. There was significant upregulation of MMP-9, iNOS, and ICAM-1 expression in the colon from CBS(+/-) mice compared with WT mice. Levels of ROS, superoxide, and inducible nitric oxide were elevated in the CBS(+/-) mice compared with other groups. Results suggest that HHcy decreases intestinal motility due to MMP-9-induced intestinal remodeling leading to constipation.     

Deep brain stimulation amplitude alters posture shift velocity in Parkinson’s disease

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is now widely used to alleviate symptoms of Parkinson’s disease (PD). The specific aim of this study was to identify posture control measures that may be used to improve selection of DBS parameters in the clinic and this was carried out by changing the DBS stimulation amplitude. A dynamic posture shift paradigm was used to assess posture control in 4 PD STN-DBS subjects. Each subject was tested at 4 stimulation amplitude settings. Movements of the center of pressure and the position of the pelvis were monitored and several quantitative indices were calculated. The presence of any statistically significant changes in several normalized indices due to reduced/no stimulation was tested using the one-sample t test. The peak velocity and the average movement velocity during the initial and mid phases of movement towards the target posture were substantially reduced. These results may be explained in terms of increased akinesia and bradykinesia due to altered stimulation conditions. Thus, the dynamic posture shift paradigm may be an effective tool to quantitatively characterize the effects of DBS on posture control and should be further investigated as a tool for selection of DBS parameters in the clinic.     

AVPR1A and SLC6A4 polymorphisms in choral singers and non-musicians: a gene association study

Amateur choral singing is a common pastime and worthy of study, possibly conferring benefits to health and social behaviour. Participants might be expected to possess musical ability and share some behavioural characteristics. Polymorphisms in genes concerned with serotonergic neurotransmission are associated with both behaviour and musical aptitude. Those investigated previously include the variable number tandem repeats RS1, RS3 and AVR in the AVPR1A (arginine vasopressin receptor 1a) gene and STin2 in the SLC6A4 (solute carrier family 6 [neurotransmitter transporter, serotonin], member 4) gene, as well as the SLC6A4 promoter region polymorphism, 5-HTTLPR. We conducted a genetic association study on 523 participants to establish whether alleles at these polymorphisms occur more commonly in choral singers than in those not regularly participating in organised musical activity (non-musicians). We also analysed tagging single nucleotide polymorphisms (SNPs) for AVPR1A and SLC6A4 to determine whether other variants in these genes were associated with singer/non-musician status. At the STin2 polymorphism, overall association with singer/non-musician status was evident at P = 0.006. The 9-repeat (P = 0.04) and 12-repeat (P = 0.04) alleles were more common in singers and the 10-repeat allele less so (P = 0.009). Odds ratios were 0.73 (95% CI 0.57-0.94) for the 10-repeat allele and 2.47 (95% CI 0.88-6.94) for the rarer 9-repeat allele. No overall association was detected at P<0.05 between any other polymorphism and singer/non-musician status. Our null findings with respect to RS3, RS1 and AVR, polymorphisms associated with musical ability by other authors, suggest that choir membership may depend partly on factors other than musical ability. In a related musical project involving one participating choir, a new 40-part unaccompanied choral work, "Allele", was composed and broadcast on national radio. In the piece, each singer's part incorporated their personal RS3 genotype.     

Role of Direct Repeat and Stem-Loop Motifs in mtDNA Deletions: Cause or Coincidence?

Deletion mutations within mitochondrial DNA (mtDNA) have been implicated in degenerative and aging related conditions, such as sarcopenia and neuro-degeneration. While the precise molecular mechanism of deletion formation in mtDNA is still not completely understood, genome motifs such as direct repeat (DR) and stem-loop (SL) have been observed in the neighborhood of deletion breakpoints and thus have been postulated to take part in mutagenesis. In this study, we have analyzed the mitochondrial genomes from four different mammals: human, rhesus monkey, mouse and rat, and compared them to randomly generated sequences to further elucidate the role of direct repeat and stem-loop motifs in aging associated mtDNA deletions. Our analysis revealed that in the four species, DR and SL structures are abundant and that their distributions in mtDNA are not statistically different from randomized sequences. However, the average distance between the reported age associated mtDNA breakpoints and their respective nearest DR motifs is significantly shorter than what is expected of random chance in human (p<10(-4)) and rhesus monkey (p = 0.0034), but not in mouse (p = 0.0719) and rat (p = 0.0437), indicating the existence of species specific difference in the relationship between DR motifs and deletion breakpoints. In addition, the frequencies of large DRs (>10 bp) tend to decrease with increasing lifespan among the four mammals studied here, further suggesting an evolutionary selection against stable mtDNA misalignments associated with long DRs in long-living animals. In contrast to the results on DR, the probability of finding SL motifs near a deletion breakpoint does not differ from random in any of the four mtDNA sequences considered. Taken together, the findings in this study give support for the importance of stable mtDNA misalignments, aided by long DRs, as a major mechanism of deletion formation in long-living, but not in short-living mammals.     

Rapid diagnosis of leptospirosis in patients with different clinical manifestations by 16S rRNA gene based nested PCR

Leptospirosis, a zoonosis of global importance and it is underreported in India and more than 50,000 severe cases are reported each year. Here we present the evaluation of 16S rRNA based nested PCR assay for the rapid identification of human leptospires using serum and urine samples. The study includes 261 suspected cases for leptospirosis with different clinical manifestations. 16S rRNA based nested PCR assay was compared and evaluated against the conventional serological methods such as MAT and ELISA. The technique enabled amplification of a 289 bp product with notable percentage of positivity in all sample groups including 94.8 in pediatric cases, 93 in pregnant women, 94.2 in renal failure, 87.8 in jaundice and 94.6 in common febrile cases. The sensitivity and specificity was 94.4% and 100%, respectively. The technique proved to be prompt and effective for the diagnosis of leptospiral infection at the acute phase of the disease. PCR based approach detects leptospiral DNA from the clinical samples both at the acute and leptospiruria phase on comparison with its counter parts where detection is made possible only after 7 days or 7–30 days post-infection. In this regard PCR based diagnosis of leptospirosis should be made available for clinicians for the early diagnosis and prompt treatment of the disease.