Mouse Hepatitis Virus Infection Upregulates Genes Involved in Innate Immune Responses

Neurotropic recombinant strain of Mouse Hepatitis Virus, RSA59, induces meningo-encephalitis, myelitis and demyelination following intracranial inoculation. RSA59 induced neuropathology is partially caused by activation of CNS resident microglia, as demonstrated by changes in cellular morphology and increased expression of a microglia/macrophage specific calcium ion binding factor, Iba1. Affymetrix Microarray analysis for mRNA expression data reveals expression of inflammatory mediators that are known to be released by activated microglia. Microglia-specific cell surface molecules, including CD11b, CD74, CD52 and CD68, are significantly upregulated in contrast to CD4, CD8 and CD19. Protein analysis of spinal cord extracts taken from mice 6 days post-inoculation, the time of peak inflammation, reveals robust expression of IFN-γ, IL-12 and mKC. Data suggest that activated microglia and inflammatory mediators contribute to a local CNS microenvironment that regulates viral replication and IFN-γ production during the acute phase of infection, which in turn can cause phagolysosome maturation and phagocytosis of the myelin sheath, leading to demyelination.     

Interaction between Nbp35 and Cfd1 Proteins of Cytosolic Fe-S Cluster Assembly Reveals a Stable Complex Formation in Entamoeba histolytica

Iron-Sulfur (Fe-S) proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1) protein and Nucleotide binding protein 35 (Nbp35). In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151) of Nbp35 and (G5-V6, M34-D39 and G46-A52) of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD) simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins without any apparent assistance of mitosomes.     

Mouse-Hamster Chimeric Prion Protein (PrP) Devoid of N-Terminal Residues 23-88 Restores Susceptibility to 22L Prions,but Not to RML Prions in PrP-Knockout Mice

Prion infection induces conformational conversion of the normal prion protein PrP^C, into the pathogenic isoform PrP^Sc, in prion diseases. It has been shown that PrP-knockout (Prnp^0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/Prnp^0/0 mice, neither developed the disease nor accumulated MHM2^ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice developed the disease with abundant accumulation of MHM2^ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2^ScΔ23-88, and that the co-expressing wild-type PrP^C can stimulate the conversion of MHM2Δ23-88 to MHM2^ScΔ23-88 in trans. In the present study, we confirmed that Tg(MHM2Δ23-88)/Prnp^0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/Prnp^0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2^ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2^ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice. However, wild-type PrP^Sc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice, compared with RML- and 22L-inoculated Prnp^0/+ mice. These results show that MHM2Δ23-88 itself can convert into MHM2^ScΔ23-88 without the help of the trans-acting PrP^C, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrP^C stimulates the conversion of MHM2Δ23-88 into MHM2^ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrP^C into PrP^Sc.     

Thermodynamic properties of strongly coupled plasma in the presence of an external magnetic field

Thermodynamic properties of a Yukawa system consisting of dust particles in plasma are studied in the presence of an external magnetic field. It is assumed that dust particles interact with each other by a modified potential in the presence of a magnetic field. A molecular dynamics code is developed to calculate this internal energy for the entire system. Based on the values of the internal energy given by the code, the Helmholtz free energy and pressure are calculated for the system.     

Membrane Topology of Human Presenilin-1 in SK-N-SH Cells Determined by Fluorescence Correlation Spectroscopy and Fluorescent Energy Transfer

Presenilin-1 (PS1) protein acts as passive ER Ca²⁺ leak channels that facilitate passive Ca²⁺ leak across ER membrane. Mutations in the gene encoding PS1 protein cause neurodegeneration in the brains of patients with familial Alzheimer’s disease (FAD). FADPS1 mutations abrogate the function of ER Ca²⁺ leak channel activity in human neuroblastoma SK-N-SH cells in vitro (Das et al., J Neurochem 122(3):487–500, 2012) and in mouse embryonic fibroblasts. Consequently, genetic deletion or mutations of the PS1 gene cause calcium (Ca²⁺) signaling abnormalities leading to neurodegeneration in FAD patients. By analogy with other known ion channels it has been proposed that the functional PS1 channels in ER may be multimers of several PS1 subunits. To test this hypothesis, we conjugated the human PS1 protein with an NH₂-terminal YFP-tag and a COOH-terminal CFP-tag. As expected YFP–PS1, and PS1–CFP were found to be expressed on the plasma membranes by TIRF microscopy, and both these fusion proteins increased ER Ca²⁺ leak channel activity similar to PS1 (WT) in SK-N-SH cells, as determined by functional calcium imaging. PS1–CFP was either expressed alone or together with YFP–PS1 into SK-N-SH cell line and the interaction between YFP–PS1 and PS1–CFP was determined by Förster resonance energy transfer analysis. Our results suggest interaction between YFP–PS1 and PS1–CFP confirming the presence of a dimeric or multimeric form of PS1 in SK-N-SH cells. Lateral diffusion of PS1–CFP and YFP–PS1 in the plasma membrane of SK-N-SH cells was measured in the absence or in the presence of glycerol by fluorescence correlation spectroscopy to show that both COOH-terminal and NH₂-terminal of human PS1 are located on the cytoplasmic side of the plasma membrane. Therefore, we conclude that both COOH-terminal and NH₂-terminal of human PS1 may also be oriented on the cytosolic side of ER membrane.     

Application of response surface method to evaluate the cytotoxic potency of Ulva fasciata Delile,a marine macro alga

Bioprospecting of marine natural products has recently produced a substantial number of drug candidates. Ulva fasciata Delile, belonging to the family Ulvaceae, is a green marine macro alga that grows profusely on the coastal seashore of South India. In the present study, we investigated the in vitro cytotoxic potential of a methanolic extract of U.fasciata Delile (MEUF) using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay against human colon carcinoma (HT-29), human hepatocyte carcinoma (Hep-G2), and human breast carcinoma (MCF-7) cell lines. Response surface methodology (RSM) was applied using central-composite experimental design (CCD) to obtain optimum combined effect of concentration and cancer cells with highest cytotoxicity. The effect of concentration, cancer cell lines as independent variables on absorbance (OD), percent cell survival and percent cell inhibition as dependent variables was investigated. Maximum cytotoxic activity of MEUF was established for Hep-G2 with lowest OD or percent cell survival; highest percent cell inhibition with significant difference (p > 0.05) was compared to HT-29 and MCF-7.     

Seasonal variation in expression pattern of genes under HSP70

Heat shock protein 70 (HSP70) is one of the most abundant and best characterized heat shock protein family that consists of highly conserved stress proteins, expressed in response to stress, and plays crucial roles in environmental stress tolerance and adaptation. The present study was conducted to identify major types of genes under the HSP70 family and to quantify their expression pattern in heat- and cold-adapted Indian goats (Capra hircus) with respect to different seasons. Five HSP70 gene homologues to HSPA8, HSPA6, HSPA1A, HSPA1L, and HSPA2 were identified by gene-specific primers. The cDNA sequences showed high similarity to other mammals, and proteins have an estimated molecular weight of around 70 kDa. The expression of HSP70 genes was observed during summer and winter. During summer, the higher expression of HSPA8, HSPA6, and HSPA1A was observed, whereas the expression levels of HSPA1L and HSPA2 were found to be lower. It was also observed that the expression of HSPA1A and HSPA8 was higher during winter in both heat- and cold-adapted goats but downregulates in case of other HSPs. Therefore, both heat and cold stress induced the overexpression of HSP70 genes. An interesting finding that emerged from the study is the higher expression of HSP70 genes in cold-adapted goats during summer and in heat-adapted goats during winter. Altogether, the results indicate that the expression pattern of HSP70 genes is species- and breed-specific, most likely due to variations in thermal tolerance and adaptation to different climatic conditions.     

A Short Sequence Motif in the 5′ Leader of the HIV-1 Genome Modulates Extended RNA Dimer Formation and Virus Replication

The 5′ leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication.     

Functional Cross-talk between Distant Domains of Chikungunya Virus Non-structural Protein 2 Is Decisive for Its RNA-modulating Activity

Chikungunya virus (CHIKV) non-structural protein 2 (nsP2) is a multifunctional protein that is considered a master regulator of the viral life cycle and a main viral factor responsible for cytopathic effects and subversion of antiviral defense. The C-terminal part of nsP2 possesses protease activity, whereas the N-terminal part exhibits NTPase and RNA triphosphatase activity and is proposed to have helicase activity. Bioinformatics analysis classified CHIKV nsP2 into helicase superfamily 1. However, the biochemical significance of a coexistence of two functionally unrelated modules in this single protein remains unknown. In this study, recombinant nsP2 demonstrated unwinding of double-stranded RNA in a 5′–3′ directionally biased manner and RNA strand annealing activity. Comparative analysis of NTPase and helicase activities of wild type nsP2 with enzymatic capabilities of different truncated or N-terminally extended variants of nsP2 revealed that the C-terminal part of the protein is indispensable for helicase functionality and presumably provides a platform for RNA binding, whereas the N-terminal-most region is apparently involved in obtaining a conformation of nsP2 that allows for its maximal enzymatic activities. The establishment of the protocols for the production of biochemically active CHIKV nsP2 and optimization of the parameters for helicase and NTPase assays are expected to provide the starting point for a further search of possibilities for therapeutic interventions to suppress alphaviral infections.