Role of Influenza A virus protein NS1 in regulating host nuclear body ND10 complex formation and its involvement in establishment of viral pathogenesis.

Influenza viral infection is a seasonal infection which causes widespread acute respiratory issues among humans globally. This virus changes its surface receptor composition to escape the recognition process by the host's immune cells. Therefore, the present study focussed to identify some other important viral proteins which have a significant role in establishment of infection and having apparent conserved structural composition. This could facilitate the permanent vaccine development process or help in designing a drug against IAV (influenza A virus) infection which will eliminate the seasonal flu shot vaccination process. The NS1 (Non-structural protein 1) protein of IAV maintains a conserved structural motif. Earlier studies have shown its significant role in infection establishment. However, the mechanism by which viruses escape the host's ND10 antiviral action remains elusive. The present study clearly showed that IAV infection and NS1 transfection in A549 cells degraded the main component of the ND10 anti-viral complex, PML and therefore, inhibited the formation of Daxx-sp100-p53-PML complex (ND10) at the mid phase of infection/transfection. PML degradation activated the stress axis which increased cellular ROS (reactive oxygen species) levels as well as mitochondrial dysfunction. Additionally, IAV/NS1 increased cellular stress and p53 accumulation at the late phase of infection. These collectively activated apoptotic pathway in the host cells. Along with the inactivation of several interferon proteins, IAV was found to decrease p-IKKε. A549 cells transfected with pcDNA3.1-NS1 showed a similar effect in the interferon axis and IKKε. Moreover, NS1 induced the disintegration of the host's ND10 complex through the changes in the SUMOylation pattern of the PML nuclear body. These findings suggest the possible mechanism of how NS1 helps IAV to establish infection in the host cells. However, it demands further detailed study before targeting NS1 to develop permanent vaccines or novel drugs against IAV in future.

Cross-cultural adaptation of Nepalese literacy and stigma of suicide scales (LOSS-SF-Nep and SOSS-SF-Nep) among Nepalese medical and nursing students.

INTRODUCTION: Nepal is a country in South-east Asia with high suicide. There is ongoing trend of emerging research on suicide from Nepal but there is lack of validated scale in measuring literacy or stigma. In the view of poor media reporting and large treatment gap, this study was conducted. All previous validation studies were done in non-Hindu populations. METHODS: A cross-sectional study was planned where the short forms of Nepalese literacy of suicide scale (LOSS-SF-Nep) and Stigma of Suicide Scale (SOSS-SF-Nep) were validated using standard procedure at a medical college in southern Nepal. Medical and nursing students of all batches were approached offline after successful pretesting. The psychometric properties of the scales were tested, and the statuses of literacy and stigma were assessed. Patient Health Questionnaire-9 and General Anxiety Disorder Scale-7 were used for revealing depression and generalized anxiety. RESULTS: Three hundred and nineteen Nepalese students participated and most of them were males, belonged to nuclear family, upper-middle socioeconomic status and represented 46 out of 77 districts of Nepal. The mean score of LOSS-SF-Nep was 6.36 ± 1.92 and literacy ranged from 37.9% to 89.7%. The deeper exposure to suicidal patients was associated with better literacy. Factor analysis of SOSS-SF-Nep revealed three subscales: stigmatization, isolation/depression, and normalization/glorification and had acceptable psychometric properties. Gender, occupation of head of the family, region and years of education, using mental health services, and depression were associated with variable literacy or stigma. CONCLUSION: Literacy and stigma scales were validated in Nepali, and SOSS factor structures were revealed with modified descriptors. The literacy and stigma levels in medical students were calculated for the first time in Nepal and Hindu majority population.

Enhanced inflammasome-mediated inflammation and impaired autophagy in peripheral blood mononuclear cells is associated with non-alcoholic fatty liver disease severity.

AIMS: Identification of the progress of non-alcoholic fatty liver disease (NAFLD) is crucial for their effective treatment. Circulating peripheral blood mononuclear cells (PBMC) could be a surrogate monitor instead of complicated and expensive biopsies. Changes in immuno-metabolic status in NAFLD patients may be reflected by an expression of different PBMC-specific molecular markers. It was hypothesized that impaired autophagy with enhanced inflammasome activation is a critical molecular event in PBMC that could contribute to systemic inflammation associated with NAFLD progression. MAIN METHODS: A cross-sectional study with a sample size of 50 subjects were undertaken from a governmental facility in Kolkata, India. Major anthropometric, biochemical, and dietary parameters were recorded. Cellular and serum samples of NAFLD patients were analyzed for oxidative stress, inflammation, inflammasome activation, and autophagic flux by western blot, flow cytometry, immunocytochemistry. KEY FINDINGS: Baseline anthropometric and clinical parameters were found associated with NAFLD severity. Elevated systemic inflammation was reflected by higher proinflammatory markers like iNOS, Cox-2, IL-6, TNF-α, IL-1ß, hsCRP in the serum of NAFLD subjects (p < 0.05). ROS-induced NLRP3 inflammasomes marker proteins were upregulated (p < 0.05) in PBMC along with NAFLD severity. Expression of autophagic markers such as LC3B, Beclin-1 and its regulator pAMPKα were found diminished (p < 0.05) with a concomitant rise of p62. Colocalization of NLRP3 with LC3B proteins in PBMC was found diminished along NAFLD severity. SIGNIFICANCE: Present data provide mechanistic evidence of impaired autophagy and intracellular ROS triggered inflammasome activation in PBMC, which could potentially exacerbate NAFLD severity.

Evaluation of spike protein antigens for SARS-CoV-2 serology.

BACKGROUND: Spike protein domains are being used in various serology-based assays to detect prior exposure to SARS-CoV-2 virus. However, there has been limited comparison of antibody titers against various spike protein antigens among COVID-19 infected patients. METHODS: We compared four spike proteins (RBD, S1, S2 and a stabilized spike trimer (ST)) representing commonly used antigens for their reactivity to human IgG antibodies using indirect ELISA in serum from COVID-19 patients and pre-2020 samples. ST ELISA was also compared against the EUROIMMUN IgG ELISA test. Further, we estimated time appropriate IgG and IgA seropositivity rates in COVID-19 patients using a panel of sera samples collected longitudinally from the day of onset of symptoms (DOS). RESULTS: Among the four spike antigens tested, the ST demonstrated the highest sensitivity (86.2 %; 95 % CI: 77.8-91.7 %), while all four antigens showed high specificity to COVID-19 sera (94.7-96.8 %). 13.8 % (13/94) of the samples did not show seroconversion in any of the four antigen-based assays. In a double-blinded head-to-head comparison, ST based IgG ELISA displayed a better sensitivity (87.5 %, 95 % CI: 76.4-93.8 %) than the EUROIMMUN IgG ELISA (67.9 %, 95 % CI: 54.8-78.6 %). Further, in ST-based assays, we found 48 % and 50 % seroconversion in the first six days (from DOS) for IgG and IgA antibodies, respectively, which increased to 84 % (IgG) and 85 % (IgA) for samples collected ≥22 days from DOS. CONCLUSIONS: Comparison of spike antigens demonstrates that spike trimer protein is a superior option as an ELISA antigen for COVID-19 serology.

Smokeless tobacco induces toxicity and apoptosis in neuronal cells: a mechanistic evaluation.

Smokeless tobacco (SLT) or chewing tobacco has been a highly addictive practice in India across ages, posing major threat to the systemic health and possibly neurodegeneration. Earlier studies showed components of SLT could be harmful to neuronal health. However, mechanism of SLT in neurodegeneration remained unexplored. This study investigated the detrimental role of SLT on differentiated neuronal cell lines, PC12 and SH-SY5Y by using graded doses of water soluble lyophilised SLT. Reduced cell viability, compromised mitochondrial structure and functions were observed when neuronal cell lines were treated with SLT (6 mg/mL) for 24 h. There was reduction of oxidative phosphorylation and aerobic glycolysis as determined by diminution of ATP production (2.5X) and basal respiration (1.9X). Mitochondrial membrane potential was dropped by 3.5 times. Bid, a pro-apoptotic Bcl-2 family protein, has imperative role in regulating mitochondrial outer membrane permeabilization and subsequent cytochrome c release leading to apoptosis. This article for the first time indicated the involvement of Bid in SLT mediated neurotoxicity and possibly neurodegeneration. SLT treatment enhanced expression of cleaved-Bid in time dependent manner. The involvement of Bid was further confirmed by using Bid specific shRNA which reversed the effects of SLT and conferred significant protection from apoptosis up to 72 h. Thus, our results clearly indicated that SLT induced neuronal cell death occurred via production of ROS, alteration of mitochondrial morphology, membrane potential and oxidative phosphorylation, inactivation of survival pathway and activation of apoptotic markers mediated by Bid. Therefore, Bid could be a potential future therapeutic target for SLT induced neurodegeneration.

Ferulic acid enhances the radiation sensitivity of lung and liver carcinoma cells by collapsing redox homeostasis: mechanistic involvement of Akt/p38 MAPK signalling pathway.

The major drawback of anticancer therapy is the development of resistance against drugs and radiation at the later phase of treatment which may lead to recurrences of the disease. Therefore, strategy was taken to enhance radiation sensitivity of lung (A549) and liver (HepG2) carcinoma cells by treatment with ferulic acid (FA) prior to irradiation. FA pre-treatment initially decreased reactive oxygen species (ROS) level in carcinoma cells which induced reductive stress and cytostasis. To overcome this stress, cellular mechanism increased the Keap1 level to down-regulate nuclear localisation of Nrf2 and its dependent antioxidant system. The antioxidant system reached the lowest level after 3 and 6 h of FA treatment in A549 and HepG2 cells respectively. As endogenous ROS were still being generated at same rate, ROS level was clearly higher than control which changed the reductive stress to oxidative stress. Exposure to γ-radiation in this condition further increased ROS level and caused radio-sensitisation of carcinoma cells. Combination of irradiation (IR) and FA activated mitochondrial apoptotic pathway and concomitantly inhibited the cell cycle progression and survival pathway over the IR group. Moreover, the combination treatment showed significant tumour regression, caspase 3 activation and nuclear fragmentation in tumour tissue compared to radiation alone. In contrast, FA pre-treatment protected peripheral blood mononuclear cells (PBMC) and normal lung fibroblast WI38 cells from radiation damage. Together, combination treatment offers effective strategy of killing cancer cells and demonstrates its potential for increasing the efficacy of radio-therapy.

An association study of severity of intellectual disability with peripheral biomarkers of disabled children in a rehabilitation home, Kolkata, India.

The current investigation has identified the biomarkers associated with severity of disability and correlation among plethora of systemic, cellular and molecular parameters of intellectual disability (ID) in a rehabilitation home. The background of study lies with the recent clinical evidences which identified complications in ID. Various indicators from blood and peripheral system serve as potential surrogates for disability related changes in brain functions. ID subjects (Male, age 10 ± 5 yrs, N = 45) were classified as mild, moderate and severe according to the severity of disability using standard psychometric analysis. Clinical parameters including stress biomarkers, neurotransmitters, RBC morphology, expressions of inflammatory proteins and neurotrophic factor were estimated from PBMC, RBC and serum. The lipid peroxidation of PBMC and RBC membranes, levels of serum glutamate, serotonin, homocysteine, ROS, lactate and LDH-A expression increased significantly with severity of ID whereas changes in RBC membrane ß-actin, serum BDNF, TNF-α and IL-6 was found non-significant. Structural abnormalities of RBC were more in severely disabled children compared to mildly affected ones. The oxidative stress remained a crucial factor with severity of disability. This is confirmed not only by RBC alterations but also with other cellular dysregulations. The present article extends unique insights of how severity of disability is correlated with various clinical, cellular and molecular markers of blood. This unique study primarily focuses on the strong predictors of severity of disability and their associations via brain-blood axis.

Alteration of murine duodenal morphology and redox signalling events by reactive oxygen species generated after whole body γ-irradiation and its prevention by ferulic acid.

Radiation-induced gastrointestinal syndrome occurs due to the clonogenic loss of crypt cells and villi depopulation, resulting in disruption of the mucosal barrier, bacterial invasion, inflammation, and sepsis. In this study, we investigated the role of ferulic acid (FA) against ionising radiation-induced duodenal injury and subsequent alterations in redox signalling events in wild type male Swiss albino mice. Mice were administered with FA at a dose of 50 mg/kg body weight for 5 consecutive days prior to exposure of 2.5, 5 and 10 Gy doses of γ-radiation. Histopathological and electron microscopic images revealed marked duodenal injuries in a dose-dependent manner. FA prevented radiation induced damage and loss of cryptic stem cells and the shortening of duodenal villus length. FA pretreatment further suppressed NF-κB-dependent activation of inflammatory pathways and augmented Nrf2 nuclear translocation with higher expression of Mn-SOD and heme-oxygenase one (HO1) activity to combat with radiation induced duodenal stress. The colocalisation of NF-κB and Nrf2 transcription factors in the nuclei of the duodenum indicated their interaction in radiation and the FA combination group. Moreover, FA treatment inhibited phosphatidyl serine (PS) externalisation, and loss of mitochondrial membrane potential in duodenal cells. Animals exposed to 10-Gy irradiation exhibited over activation of p53, p21, caspase 3, poly ADP ribose polymerase (PARP) and DNA double-strand break which were ameliorated by FA treatment. Therefore, this article first uncovers the modulatory effect of FA on radiation-induced ROS/NF-κB/Nrf2/p53-caspase 3-PARP axis in the duodenum and establishing biological function of FA in protecting duodenum from radiation damage with a detailed mechanistic approach.

Radiosensitizing effect of ellagic acid on growth of Hepatocellular carcinoma cells: an in vitro study.

Failure of treatment for cancer in clinic by radio/chemotherapy is generally attributed to tumour resistance. Therefore, it is important to develop strategies to increase the cytotoxicity of tumour cells by radiation in combination with unique tumour selective cytotoxic agents. We evaluated the potential of ellagic acid (EA) as an enhancer of oxidative stress in cancer cells. HepG2 cells were treated with EA (10 µM) for 12 h prior to exposure of single 7.5 Gy dose of irradiation. Treatment of HepG2 cells with EA and gamma radiation showed increased reactive oxygen species generation, up regulation of p53 protein expression, decreased survival markers level like p-Akt, p-NF-kB and p-STAT3 which were significantly higher after radiation treatment alone. We also found that combination treatment increased G2/M phase cell population, decreased IL-6, COX-2 and TNF-α expression and caused a loss in mitochondrial membrane potential with decreased level of angiogenesis marker MMP-9. Over expression of Bax and activation of caspase 3 indicated the apoptosis of the cells. The results provided a strong unique strategy to kill cancer cells HepG2, using less radiation dose along with effective pro-oxidant dose of EA.

Hypoxia-Induced Let-7d Has a Role in Pericyte Differentiation.

The microvascular pericyte is an important regulatory cell that maintains tissue homeostasis. One of the mechanisms by which pericytes maintain tissue homeostasis is through the induction of endogenous adaptative changes to stress signals. These adaptations include migration, differentiation and induction of angiogenesis. We have investigated pericyte responses to hypoxic stress (1 % O2) and have reported that pericytes adapt to hypoxia, in part, through changes in endogenous and released microRNAs (miRNAs). Of those miRNAs, Let-7d plays an important role. We exposed pericytes to hypoxia with and without basic fibroblast growth factor (bFGF) in stem cell medium. The expression of Let-7d in pericyte-derived neurospheres was determined. Evidence of differentiation was determined by immunocytochemistry. Hypoxia enhanced pericyte spheres were positive for Let-7d. The transcription factor Sox2, a marker of cell differentiation, was also induced in pericytic spheres. Taken together, our results suggest that pericyte expression of Let-7d in response to hypoxia and bFGF is involved in pericyte differentiation. Thus, for the first time, we propose a pathway for induction of pericyte differentiation. Modulation of this pathway in pericytes may be an important target in tissue repair.

Backbone and stereospecific (13)C methyl Ile (δ1), Leu and Val side-chain chemical shift assignments of Crc.

Carbon catabolite repression (CCR) allows bacteria to selectively assimilate a preferred compound among a mixture of several potential carbon sources, thus boosting growth and economizing the cost of adaptability to variable nutrients in the environment. The RNA-binding catabolite repression control (Crc) protein acts as a global post-transcriptional regulator of CCR in Pseudomonas species. Crc triggers repression by inhibiting the expression of genes involved in transport and catabolism of non-preferred substrates, thus indirectly favoring assimilation of preferred one. We report here a nearly complete backbone and stereospecific (13)C methyl side-chain chemical shift assignments of Ile (δ1), Leu and Val of Crc (~ 31 kDa) from Pseudomonas syringae Lz4W.

NADPH oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system, associated with demyelination and neurodegeneration. The mechanisms of tissue injury are poorly understood, but recent data suggest that mitochondrial injury may play an important role in this process. Mitochondrial injury can be triggered by reactive oxygen and nitric oxide species, and we recently provided evidence for oxidative damage of oligodendrocytes and dystrophic axons in early stages of active multiple sclerosis lesions. In this study, we identified potential sources of reactive oxygen and nitrogen species through gene expression in carefully staged and dissected lesion areas and by immunohistochemical analysis of protein expression. Genome-wide microarrays confirmed mitochondrial injury in active multiple sclerosis lesions, which may serve as an important source of reactive oxygen species. In addition, we found differences in the gene expression levels of various nicotinamide adenine dinucleotide phosphate oxidase subunits between initial multiple sclerosis lesions and control white matter. These results were confirmed at the protein level by means of immunohistochemistry, showing upregulation of the subunits gp91phox, p22phox, p47phox, nicotinamide adenine dinucleotide phosphate oxidase 1 and nicotinamide adenine dinucleotide phosphate oxidase organizer 1 in activated microglia in classical active as well as slowly expanding lesions. The subunits gp91phox and p22phox were constitutively expressed in microglia and were upregulated in the initial lesion. In contrast, p47phox, nicotinamide adenine dinucleotide phosphate oxidase 1 and nicotinamide adenine dinucleotide phosphate oxidase organizer 1 expression were more restricted to the zone of initial damage or to lesions from patients with acute or early relapsing/remitting multiple sclerosis. Double labelling showed co-expression of the nicotinamide adenine dinucleotide phosphate oxidase subunits in activated microglia and infiltrated macrophages, suggesting the assembly of functional complexes. Our data suggest that the inflammation-associated oxidative burst in activated microglia and macrophages plays an important role in demyelination and free radical-mediated tissue injury in the pathogenesis of multiple sclerosis.

Pathogenic T cell responses against aquaporin 4.

Inflammatory lesions in the central nervous system of patients with neuromyelitis optica are characterized by infiltration of T cells and deposition of aquaporin-4-specific antibodies and complement on astrocytes at the glia limitans. Although the contribution of aquaporin-4-specific autoantibodies to the disease process has been recently elucidated, a potential role of aquaporin-4-specific T cells in lesion formation is unresolved. To address this issue, we raised aquaporin-4-specific T cell lines in Lewis rats and characterized their pathogenic potential in the presence and absence of aquaporin-4-specific autoantibodies of neuromyelitis optica patients. We show that aquaporin-4-specific T cells induce brain inflammation with particular targeting of the astrocytic glia limitans and permit the entry of pathogenic anti-aquaporin-4-specific antibodies to induce NMO-like lesions in spinal cord and brain. In addition, transfer of aquaporin-4-specific T cells provoked mild (subclinical) myositis and interstitial nephritis. We further show that the expression of the conformational epitope, recognized by NMO patient-derived aquaporin-4-specific antibodies is induced in kidney cells by the pro-inflammatory cytokine gamma-interferon. Our data provide further support for the view that NMO lesions may be induced by a complex interplay of T cell mediated and humoral immune responses against aquaporin-4.

Inflammation induced by innate immunity in the central nervous system leads to primary astrocyte dysfunction followed by demyelination.

Primary loss and dysfunction of astrocytes may trigger demyelination, as seen in neuromyelitis optica, an inflammatory disease of the central nervous system. In most patients affected by this disease, injury to astrocytes is initiated by the action of autoantibodies targeting aquaporin 4 (AQP-4), a water channel on astrocytes. We show here that damage of astrocytes and subsequent demyelination can also occur in the absence of autoantibody-mediated mechanisms. Following injection of lipopolysaccharide into the white matter initial microglia activation is followed by a functional disturbance of astrocytes, mainly reflected by retraction of astrocytic foot processes at the glia limitans and loss of AQP-4 and connexins, which are involved in the formation of gap junctions between astrocytes and oligodendrocytes. Demyelination and oligodendrocyte degeneration in this model follows astrocyte pathology. Similar structural abnormalities were also seen in a subset of active lesions in multiple sclerosis. Our studies suggest that astrocyte injury may be an important early step in the cascade of lesion formation in brain inflammation.

Extracellular matrix in multiple sclerosis lesions: Fibrillar collagens, biglycan and decorin are upregulated and associated with infiltrating immune cells.

Extracellular matrix (ECM) proteins can modify immune reactions, e.g. by sequestering or displaying growth factors and by interacting with immune and glial cells. Here we quantified by quantitative polymerase chain reaction (qPCR) expression of 50 ECM components and 34 ECM degrading enzymes in multiple sclerosis (MS) active and inactive white matter lesions. COL1A1, COL3A1, COL5A1 and COL5A2 chains were induced strongly in active lesions and even more in inactive lesions. These chains interact to form collagen types I, III and V, which are fibrillar collagens. Biglycan and decorin, which can decorate fibrillar collagens, were also induced strongly. The fibrillar collagens, biglycan and decorin were largely found between the endothelium and astrocytic glia limitans in the perivascular space where they formed a meshwork which was closely associated with infiltrating immune cells. In active lesions collagen V was also seen in the heavily infiltrated parenchyma. Fibrillar collagens I and III inhibited in vitro human monocyte production of CCL2 (MCP-1), an inflammatory chemokine involved in recruitment of immune cells. Together, ECM changes in lesions with different activities were quantified and proteins forming a perivascular fibrosis were identified. Induced fibrillar collagens may contribute to limiting enlargement of MS lesions by inhibiting the production of CCL2 by monocytes.

REM sleep deprivation-induced noradrenaline stimulates neuronal and inhibits glial Na-K ATPase in rat brain: in vivo and in vitro studies.

Increased noradrenaline, induced by rapid eye movement (REM) sleep deprivation, stimulates Na-K ATPase activity in the rat brain. The brain contains neurons as well as glia and both possess Na-K ATPase, however, it was not known if REM sleep deprivation affects the enzyme in both types of cells identically. Rats were REM sleep deprived by the flowerpot method and free moving, large platform and recovery controls were carried out. Na-K ATPase activity was measured in membranes prepared from whole brain as well as from neuronal and glial fractions separated from REM sleep-deprived and control rats. The effects of noradrenaline (NA) in different fractions were studied with or without in vivo i.p. treatment of prazosin, an alpha1-adrenpceptor antagonist, as well as in vitro membranes prepared from neurons and glia separated from normal rat brain. Further, to confirm the findings, membranes were prepared from neuro2a and C6 cell lines treated with NA in the presence and absence of prazosin and Na-K ATPase activity was estimated. The results showed that neuron and neuro2a as well as glia and C6 possess comparable Na-K ATPase activity. After REM sleep deprivation the neuronal Na-K ATPase activity increased, while the glial enzyme activity decreased and these changes were mediated by NA acting on alpha1-adrenoceptor; comparable results were obtained by treating the neuro2a and C6 cell lines with NA. The opposite actions of NA on neuronal and glial Na-K ATPase activity probably help maintain neuronal homeostasis.

Involvement of Leishmania donovani major surface glycoprotein gp63 in promastigote multiplication.

The major surface glycoprotein gp63 of the kinetoplastid protozoal parasite Leishmania is implicated as a ligand mediating uptake of the parasite into, and survival within, the host macrophage. By expressing gp63 antisense RNA from an episomal vector in L. donovani promastigotes, gp63-deficient transfectants were obtained. Reduction of the gp63 level resulted in increased generation times, altered cell morphology, accumulation of cells in the G2/M phase of the cell cycle, and increased numbers of binucleate cells with one or two kinetoplasts. Growth was stimulated, and the number of binucleate cells reduced, by addition to the culture of a bacterially expressed fusion protein containing the fibronectin-like SRYD motif and the zinc-binding (metalloprotease) domain of gp63. These observations support an additional role of gp63 in promastigote multiplication; the fibronectin-like properties of gp63 may be important in this process

Nuclear entry of poliovirus protease-polymerase precursor 3CD: implications for host cell transcription shut-off.

Host cell transcription mediated by all three RNA polymerases is rapidly inhibited after infection of mammalian cells with poliovirus (PV). Both genetic and biochemical studies have shown that the virus-encoded protease 3C cleaves the TATA-binding protein and other transcription factors at glutamine-glycine sites and is directly responsible for host cell transcription shut-off. PV replicates in the cytoplasm of infected cells. To shut-off host cell transcription, 3C or a precursor of 3C must enter the nucleus of infected cells. Although the 3C protease itself lacks a nuclear localization signal (NLS), amino acid sequence examination of 3D identified a potential single basic type NLS, KKKRD, spanning amino acids 125-129 within this polypeptide. Thus, a plausible scenario is that 3C enters the nucleus in the form of its precursor, 3CD, which then generates 3C by auto-proteolysis ultimately leading to cleavage of transcription factors in the nucleus. Using transient transfection of enhanced green fluorescent protein (EGFP) fusion polypeptides, we demonstrate here that both 3CD and 3D are capable of entering the nucleus in PV-infected cells. However, both polypeptides remain in the cytoplasm in uninfected HeLa cells. Mutagenesis of the NLS sequence in 3D prevents nuclear entry of 3D and 3CD in PV-infected cells. We also demonstrate that 3CD can be detected in the nuclear fraction from PV-infected HeLa cells as early as 2 h postinfection. Significant amount of 3CD is found associated with the nuclear fraction by 3-4 h of infection. Taken together, these results suggest that both the 3D NLS and PV infection are required for the entry of 3CD into the nucleus and that this may constitute a means by which viral protease 3C is delivered into the nucleus leading to host cell transcription shut-off.

Cell cycle regulation of hepatitis C and encephalomyocarditis virus internal ribosome entry site-mediated translation in human embryonic kidney 293 cells.

We have established stably transformed human embryonic kidney cell lines (HEK293) containing bicistronic constructs to study regulation of viral internal ribosome entry site (IRES)-mediated translation in vivo. These cells produce Renilla luciferase (Rluc) in a cap-dependent manner, while Firefly luciferase (Luc) synthesis is mediated by IRES elements. Using these cell lines, we demonstrate here that IRES-mediated translation directed by both hepatitis C (HCV) and encephalomyocarditis (EMCV) virus varies with the cell cycle. Experiments involving arrest of the cell lines at different phases of the cell cycle, release of synchronized cells from cell cycle arrest, as well as direct sorting of the cells based on position in the cell cycle have shown that the activity of the HCV and EMCV IRES elements is lowest during the G2/M phase in HEK293 cells. These results suggest that cellular trans-acting factors either stimulate viral IRES-mediated translation during G1 and S phases or repress translation during the G2/M phase in HEK293 cells.

Effect of textile waste water on the spermatogenesis of male albino rats.

Textile waste water released from dyeing and printing industries situated in Sanganer, Jaipur (India), brought about inhibition of spermatogenesis in male rats. Water analysis showed the presence of heavy metals at more than permissible limits. Oral administration of waste water to the rats at the dose level of 26.6 ml kg(-1) body wt. significantly reduced the weights of testes, epididymides and seminal vesicle. Treated animals showed a notable depression of various stages of spermatogenesis. The production of spermatids was inhibited by 70.8% in waste-water-treated rats. The populations of spermatogonia, preleptotene spermatocytes and secondary spermatocytes were decreased by 67.2, 71.1 and 73.2%, respectively. The total number of Sertoli cells was affected after waste water treatment. Reduced sperm count and motility resulted in treated groups. A significant fall in the content of various biochemical parameters of reproductive tissues was observed after water treatment.