Cytoplasmic Translocation of Polypyrimidine Tract-Binding Protein and Its Binding to Viral RNA during Japanese Encephalitis Virus Infection Inhibits Virus Replication

Japanese encephalitis virus (JEV) has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5′- and 3′-non-coding regions (NCRs). The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB) interacts in vitro with both the 5′-NCR of the positive-sense genomic RNA - 5NCR(+), and its complementary sequence in the negative-sense replication intermediate RNA - 3NCR(-). The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(-) RNA with viral RNA-dependent RNA polymerase (NS5 protein), an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA)-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.     

Cytogenetic Analysis of Bone Marrow and Peripheral Blood Samples Stored in Fixative for Several Years

We have developed a method which improves the spreading of chromosomes and permits banding analysis of cytogenetic samples of bone marrow and unstimulated peripheral blood which have been stored in fixative for up to 15 years. Metaphase cells had been harvested as usual and stored in fixative (acetic acid:methanol 1:3) at -15 C. The procedure includes 4-5 changes of fixative (acetic acid:ethanol 1:1). Next, cells are dropped onto a chilled, wet slide. The back of the slide is then rinsed with 70% ethanol and dried by ignition. C-, G-, Q-, or R-banding patterns can now be obtained with these specimens. The procedure is useful for reinvestigation of cytogenetic samples that were obtained prior to the development of banding techniques.     

Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: a consequence of the formation of "native-like conformation".

The influence of n-propanol on the overall -helical conformation of -globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of -helical conformation is induced into these proteins atpH 6.0 and 4°C in the presence of relatively low concentrations of n-propanol. The induction of -helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of -globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The -helical conformation induced into - and -globin by n-propanol decreased as the temperature is raised from 4 to 24°C. In contrast, the -helical conformation of both - and -chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced -helical conformation of globins and the -helical conformation of - and -chains. A cross-correlation of the n-propanol induced increase in the fluorescence of -globin with the corresponding increase in the -helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the -helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a native-like structure. The induction of -helical conformation into these proteins in the presence of n-propanol and the consequent generation of native-like conformation is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an -helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume -helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high -helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of -helical proteins. Consistent with this concept, the induction of -helical conformation into shorter polypeptide fragments of 30 residues, (e.g., _1-30, which exists in an -helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.     

Subtilisin modification of monodeamidated ribonuclease-A

Limited proteolysis of RNAase-Aa₁ (monodeamidated ribonuclease-A) by subtilisin results in the formation of an active RNAase-S type of derivative, namely RNAase-Aa₁S. RNAase-Aa₁S was chromatographically distinct from RNAase-S, but exhibited very nearly the same enzymic activity, antigenic conformation and susceptibility to trypsin as did RNAase-S. Fractionation of RNAase-Aa₁S by trichloroacetic acid yielded RNAase-Aa₁S-protein and RNAase-Aa₁S-peptide, both of which are inactive by themselves, but regenerate active RNAase-Aa₁S′ when mixed together. RNAase-Aa₁S-peptide was identical with RNAase-S-peptide, whereas the protein part was distinct from that of RNAase-S-protein. Titration of RNAase-Aa₁S-protein with S-peptide exhibited slight but noticeably weaker binding of the peptide to the deamidated S-protein as compared with that of native protein. Unlike the subtilisin digestion of RNAase-A, which gives nearly 100% conversion into RNAase-S, the digestion of RNAase-Aa₁ gives only a 50% conversion. The resistance of RNAase-Aa₁ to further subtilisin modification after 50% conversion is apparently due to the interaction of RNAase-Aa₁ with its subtilisin-modified product. RNAase-S was also found to undergo activity and structural changes in acidic solutions, similar to those of RNAase-A. The initial reaction product (RNAase-Sa₁) isolated by chromatography was not homogeneous. Unlike the acid treatment of RNAase-A, which affected only the S-protein part, the acid treatment of RNAase-S affected both the S-protein and the S-peptide region of the molecule.     

Age-related qualitative and quantitative changes in tRNA population of rat skeletal muscle

Total tRNA was purified from skeletal muscle of young, adult and old female albino rats. Age-dependent variation of total tRNA was the same with respect to tRNA content and biological activity as measured by amino acid acceptor capacity. The tRNA content was more in young rats and showed a gradual decrease in the adult and old rats. The relative abundancy of eleven aminoacyl-tRNAs were checked at each age and during aging. Arginyl, glutamyl and tyrosyl-tRNAs do not show any quantitative or qualitative change with age.     

Molecular evolution and mosaicism of leptospiral outer membrane proteins involves horizontal DNA transfer

Leptospires belong to a genus of parasitic bacterial spirochetes that have adapted to a broad range of mammalian hosts. Mechanisms of leptospiral molecular evolution were explored by sequence analysis of four genes shared by 38 strains belonging to the core group of pathogenic Leptospira species: L. interrogans, L. kirschneri, L. noguchii, L. borgpetersenii, L. santarosai, and L. weilii. The 16S rRNA and lipL32 genes were highly conserved, and the lipL41 and ompL1 genes were significantly more variable. Synonymous substitutions are distributed throughout the ompL1 gene, whereas nonsynonymous substitutions are clustered in four variable regions encoding surface loops. While phylogenetic trees for the 16S, lipL32, and lipL41 genes were relatively stable, 8 of 38 (20%) ompL1 sequences had mosaic compositions consistent with horizontal transfer of DNA between related bacterial species. A novel Bayesian multiple change point model was used to identify the most likely sites of recombination and to determine the phylogenetic relatedness of the segments of the mosaic ompL1 genes. Segments of the mosaic ompL1 genes encoding two of the surface-exposed loops were likely acquired by horizontal transfer from a peregrine allele of unknown ancestry. Identification of the most likely sites of recombination with the Bayesian multiple change point model, an approach which has not previously been applied to prokaryotic gene sequence analysis, serves as a model for future studies of recombination in molecular evolution of genes.     

Microvascular PO2 during extreme hemodilution with hemoglobin site specifically PEGylated at Cys-93(beta) in hamster window chamber

The oxygen transport capacity of nonhypertensive polyethylene glycol (PEG)-conjugated hemoglobin solutions were investigated in the hamster chamber window model. Microvascular measurements were made to determine oxygen delivery in conditions of extreme hemodilution [hematocrit (Hct) 11%]. Two isovolemic hemodilution steps were performed with a 6% Dextran 70 (70-kDa molecular mass) plasma expander until Hct was 35% of control. Isovolemic blood volume exchange was continued using two surface-modified PEGylated hemoglobins (P5K2, P(50) = 8.6, and P10K2, P(50) = 8.3; P(50) is the hemoglobin Po(2) corresponding to its 50% oxygen saturation) until Hct was 11%. P5K2 and P10K2 are PEG-conjugated hemoglobins that maintain most of the hemoglobin allosteric properties and have a cooperativity index of n = 2.2. The effects of these molecular solutions were compared with those obtained in a previous study using MP4, a PEG-modified hemoglobin whose P(50) was 5.4 and cooperativity was 1.2 (Tsai et al., Am J Physiol Heart Circ Physiol 285: H1411-H1419, 2003). Tissue oxygen levels were higher after P5K2 (7.0 +/- 2.5 mmHg) and P10K2 (6.3 +/- 2.3 mmHg) versus MP4 (1.7 +/- 0.5 mmHg) or the nonoxygen carrier Dextran 70 (1.3 +/- 1.2 mmHg). Microvascular oxygen delivery was higher after P5K2 and P10K2 (2.22 and 2.34 ml O(2)/dl blood) compared with MP4 (1.41 ml O(2)/dl blood) or Dextran 70 (0.90 ml O(2)/dl blood); however, all these values were lower than control (7.42 ml O(2)/dl blood). The total hemoglobin in blood was similar in all cases; therefore, the improvement in tissue Po(2) and oxygen delivery appears to be due to the increased cooperativity of the new molecules.     

Large Dense-Core Vesicles in Rat Adrenal After Reserpine: Levels of mRNAs of Soluble and Membrane-Bound: Constituents in Chromaffin and Ganglion Cells Indicate a Biosynthesis of Vesicles with Higher Secretory Quanta

Abstract: Rats were injected with a large dose of reserpine known to stimulate the adrenal medulla. Various times after drug treatment the mRNA levels of several constituents of large dense-core vesicles were determined by northern blot analysis and in situ hybridization. The latter method allowed detection of changes in mRNA levels not only in chromaffin cells, but also in the ganglion cells found in adrenal medulla. Levels of the mRNAs of secretory components of large dense-core vesicles (chromogranins A and B., secretogranin II, VGF, and neuropeptide Y) increased in chromaffin cells by 215–857% after 1–3 days of drug treatment. For partly membrane-bound components (dopamine β-hydroxylase, prohormone convertase 2, carboxypeptidase H., and peptidylglycine α-amidating monooxygenase) the changes ranged from 182 to 315%, whereas for glycoprotein III and for intrinsic membrane proteins (cytochrome b ₆₆₁ and vesicle monoamine transporter 2) no change occurred. In ganglion cells the mRNAs that could be detected for VGF, neuropeptide Y., secretogranin II, carboxypeptidase H., and vesicle monoamine transporter 1 showed an analogous pattern of change, with significant increases for the secretory proteins and no change for the membrane components. From these and previous results we suggest the following concept: Long-lasting stimulation of chromaffin cells or neurons does not induce the biosynthesis of a larger number of vesicles but rather leads to the formation of vesicles containing higher secretory quanta of chromogranins and neuropeptides. Key Words : ChromograninSecretogranin II—Monoamine transporter—Prohormone convertase 2—Carboxypeptidase H—Cytochrome b ₆₆₁-Clusterin.     

Host Range of an Insecticidal Crystal Protein of Bacillus thuringiensis subsp. kurstaki Produced in Escherichia coli

A crylA(c)-like gene of Bacillus thuringiensis subsp. kurstaki strain HD1 was over-expressed in Escherichia coli from a multicopy plasmid. Biological toxicity tests conducted on the larvae of three lepidopteran insects showed that the host range of transgenic E. coli HB 101 (pRT 200) was a subset of the host range of B. thuringiensis kurstaki HD1. Both were toxic to the larvae of Helicoverpa armigera (Gram pod borer) and Bombyx mori (Silkworm). However. though the sporecrystal formulation of HDl was toxic to the larvae of Phthorimaea operculella (Potato tuber moth). the transgenic E. coli was not. Product of St toxin gene other than crylA(c) present In HD1 may be responsible for Its toxicity to the larvae of P. opercuiella.     

The extrinsic proteins of photosystem II: update

Planta - Recent investigations have provided important new insights into the structures and functions of the extrinsic proteins of Photosystem II. This review is an update of the last major review...