A combined approach of 16S rRNA and a functional marker gene, soxB to reveal the diversity of sulphur-oxidising bacteria in thermal springs.

With the advent of new molecular tools, new taxa of sulphur-oxidising bacteria (SOB) in diverse environments are being discovered. However, there is a significant gap of knowledge about the ecology and diversity of SOB in thermal springs. Here, the species diversity and phylogenetic affiliations of SOB were investigated using 16S rRNA and functional gene marker, soxB in thermal springs of Thane district of Maharashtra, India. Most SOB detected by 16S rDNA sequences belong to different operational taxonomic units (OTU's): Firmicutes, α-, ß-, γ-Proteobacteria and Actinobacteria with the dominance of first class. However, the soxB gene clone library sequences had shown affiliation with the ß-, γ- and α-Proteobacteria. ß-Proteobacteria-related sequences were dominant, with 53.3% clones belonging to genus Hydrogenophaga. The thiosulphate oxidation assay carried out for different isolates having distinct identity showed the mean sulphate-sulphur production from 117.86 ± 0.50 to 218.82 ± 2.56 mg SO4-S l-1 after 9 days of incubation. Also, sulphur oxidation by the genus Nitratireductor, Caldimonas, Geobacillus, Paenibacillus, Brevibacillus, Tristrella and Chelatococcus has been reported for the first time that reveals ecological widening over which thiotrophs are distributed.

Anti-inflammatory effect of dikaempferol rhamnopyranoside, a diflavonoid from Eugenia jambolana Lam. Leaves.

Traditionally, the Indian Blackberry or locally called Jamun, Eugenia jambolana Lam. (Syn.: Syzygium cumini), is well known for its pharmacological potential, particularly anti-inflammatory. Here, we studied kaempferol-7-O-α-L-rhamnopyranoside]-4'-O-4'- [kaempferol-7-O-α-L-rhamnopyranoside (EJ-01) isolated from the E. jambolana leaves for possible anti-inflammatory activity. EJ-01 (3, 10 and 30 mg/kg, p.o.) was assessed for anti-inflammatory activity using carrageenan-induced paw edema model in mice by determining edema volume, myeloperoxidase (MPO), nitrite plus nitrate (NOx) and cytokine levels in paw edema tissue. EJ-01 significantly attenuated the edema, MPO levels, tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1ß) levels in the edema of paw at the 5th hour after carrageenan injection at all doses. EJ-01 (30 mg/kg) decreased the nitric oxide (NO) levels of the edema of paw at the 5th hour after carrageenan injection. The anti-inflammatory mechanisms of EJ-01 might be related to the decrease in the level of edema paw by reduced activities of NO and MPO. It probably exerts anti-inflammatory effects through the suppression of TNF-α and IL-1ß. Therefore, we conclude that EJ-01 could be positively exploited for itspotential benefits against inflammatory diseases and support the pharmacological basis of E. jambolana as traditional herbal medicine for the treatment of inflammatory diseases.

Organization of the noncontiguous promoter components of adenovirus VAI RNA gene is strikingly similar to that of eucaryotic tRNA genes.

The intragenic transcriptional control region (internal promoter) of the adenovirus type 2 VAI RNA gene was mutated by deletion, insertion, and substitution of DNA sequences at the plasmid level. The mutant plasmids were assayed for in vitro transcriptional activity by using HeLa cell extracts. The mutant clones with substitution or insertion of DNA sequences or both between nucleotides +18 and +53 of the VAI RNA gene were all transcriptionally active, although to various extents. Substitution of unrelated DNA sequences up to +26 or between +54 and +61 abolished the transcriptional activity completely. Based on these results, the intragenic promoter sequences of the VAI RNA gene can be subdivided into two components: element A, +10 to +18; and element B, +54 to +69. The distance between the A and B components could be enlarged from its normal 35 base pairs to 75 base pairs without destroying the transcriptional activity. However, a deletion of 4 or 6 base pairs in the DNA segment separating the A and B components (segment C) reduced the transcriptional activity of the genes to less than 2% of that of the wild type. When the VAI RNA gene with its element A or B was substituted for the corresponding element A or B of the Xenopus laevis tRNAMet gene, the hybrid genes transcribed close to the level of the wild-type VAI RNA gene and about 10- to 20-fold more efficiently than the tRNAMet gene. Thus, the organization of DNA sequences in the internal promoter of the VAI RNA gene appears to be very similar to that of eucaryotic tRNA genes. This similarity suggests an evolutionary relationship of the VAI RNA gene to tRNA genes.

Structural requirements of adenovirus VAI RNA for its translation enhancement function.

Recently, by genetic and biochemical approaches, it has been shown that adenovirus VAI RNA is required for efficient translation of viral mRNAs at late times after infection. To understand the nucleotide sequences and the domains of the VAI RNA that are responsible for the role of VAI RNA in enhancement of translation, a mutational analysis of the VAI gene was undertaken. Deletion, substitution, and insertion mutations covering most of the nucleotide sequences of VAI RNA were introduced into the VAI gene at the plasmid level. These mutant genes were then reintroduced into the virus, and growth properties of the mutant viruses were studied. The majority of the mutants retained normal or nearly normal levels of biological function. Mutations in the region between +43 and +53 and between +107 and the 3' end of the gene resulted in a considerable loss of activity. These mutants, however, grew significantly better than did an adenovirus type 5 mutant lacking both functional VAI and VAII genes, indicating that they retain a portion of their activity. Because no one mutation was able to completely abolish the function, we suggest that the VAI RNA may have multiple functional sites for its translation modulation function. These multiple sites may be short oligonucleotide sequences that may interact with cellular or viral components or both during translation.

Selective and opposing actions of progesterone receptor isoforms in human endometrial stromal cells.

Transcriptional regulation by progesterone is mediated primarily through the two progesterone receptor (PR) isoforms, PR-A and PR-B. Primary human endometrial stromal cell cultures, in which endogenous PR expression was lost, were infected with adenovirus expressing PR-A, PR-B, or both. Global gene expression analysis was conducted on vehicle and 30 nM progesterone (P4) treated cells following 12 h treatment. Interestingly, many genes regulated by PR-B alone or upon PR-A and PR-B co-expression, did not overlap with each other or with the PR-A expression group. Although many genes known to be progestin regulated in the uterus in vivo were also regulated in this study, markedly little overlap with published P4 regulated genes from human breast cancer cells was observed. Progesterone dose response curves were generated for several genes demonstrating gene selective potency and efficacy for each PR isoform. Furthermore, the PR isoforms opposed each other in regulation of tissue factor, with PR-B increasing and PR-A decreasing both mRNA and protein levels. Our data provide a view of global gene expression by PR isoforms in human endometrial cells and a comparison with other cell types. The specific genes and regulation patterns found provide groundwork to revealing the mechanism of PR isoform selectivity, and perhaps ultimately to the tissue selective properties these receptors appear to exhibit.

A re-look at the duration of human pregnancy.

INTRODUCTION: The duration of human pregnancy is arbitrarily taken as 280 days (40 weeks). Foetuses are considered to be at high risk once pregnancy goes beyond the expected date of confinement. This study was carried out with the aim of determining the mean gestation age of low-risk pregnancies that went into spontaneous labour and the incidence of adverse outcomes in relation to gestation. METHODS: Low-risk singleton pregnancies admitted in spontaneous labour at a single community hospital in the Udupi district of Karnataka in South India, from December 2002 to December 2003, were analysed for mean gestational age at the onset of spontaneous labour and rates of perinatal complications by gestational age. RESULTS: Among the 1,094 women who went into spontaneous labour, the mean gestational age was 272.1 +/- 9 days. A significantly increased incidence of meconium-stained amniotic fluid beyond 39 weeks of gestation was observed. 783 of 1,094 women (80 percent) had delivered during the period of 261-280 days of pregnancy (period of one standard deviation around the mean gestational age at delivery). There was significant increase in perinatal morbidity indicators and mortality rates once the pregnancy carried beyond 280 days. CONCLUSION: Mean gestational age at the onset of labour for women native to the area of study was 272 days (standard deviation 9 days). Pregnancies beyond a duration of 280 days showed significantly increased perinatal morbidity. It is suggested that there is a need for determining the length of gestation and to compile gestation-wise incidence of meconium-stained amniotic fluid as an indicator of foetal maturity or the undisclosed risk factor, in addition to other neonatal morbidity indicators for different populations.

Adenovirus vectors for gene expression.

Adenoviruses possess a combination of features that make them highly suitable as vectors for expression of heterologous genes. Non-conditional and non-defective adeno-vectors have been constructed to obtain high level expression of a number of foreign genes and some of them have been shown in animal models to exhibit excellent promise as vaccine candidates.

Grave's disease associated with immunoglobulin A nephropathy: A rare association.

Immunoglobulin A (Ig A) nephropathy is the most common form of primary glomerulonephritis. The association of Ig A nephropathy with Grave's disease has not been reported so far. We report a case of 20-year-old female with Grave's disease who presented with edema, facial puffiness, and decreased urine output. She was found to be hypertensive with renal failure and nephrotic range proteinuria. Renal biopsy revealed features of Ig A nephropathy. The patient was treated with oral corticosteroids (1 mg/kg/day). To our knowledge, this is the first case showing association of Grave's disease with Ig A nephropathy.

Results of treatment of distal extra: articular femur fractures with locking plates using minimally invasive approach--experience with 25 consecutive geriatric patients.

PURPOSE: Fractures involving the femur in older adults are reasonably common. The aim of this study was to evaluate the results of MIPO technique using locking plates in geriatric patients for distal extra-articular femur fractures. METHODS: About 25 consecutive patients with distal extra-articular femur fractures aged 60 years and above were treated using locking plates and minimally invasive technique. Patients were studied prospectively over a period of 3 years. Parameters studied included patient demographics, fracture type, time taken for the surgery, time to union and any complications. RESULTS: Mean age of patients was 66.5 years. Nineteen (76%) patients were females. Most of fractures in our study were type 33A2 fractures (64%). Average time to full weight bearing was 14.32 weeks, and fractures united at an average of 16.88 weeks. There were two (8%) patients with superficial infection, two (8%) with implant tenderness. One (4%) patient developed knee stiffness. Five (20%) patients had extension lag of average 5°. One (4%) patient sustained a peri-implant fracture at 2 months. None of the patients developed non-union or delayed union. According to criteria laid by Schatzker's and Lambert, excellent results were achieved in 22 (88%) patients. CONCLUSIONS: Outcome of minimally invasive fixation of distal extra-articular femur fractures with locking plates in patients of age 60 years and above seems to be good with high union rate despite high prevalence of osteoporosis and comminution.

A novel human estrogen receptor beta: identification and functional analysis of additional N-terminal amino acids.

A novel human estrogen receptor beta (hERbeta) was cloned from human testis mRNA, ovary and thymus cDNA utilizing PCR and 5' RACE methods. The 5' end of hERbeta contained an additional open reading frame, in-frame and upstream of the published clones. hERbeta encodes a protein of 530 amino acids with an approximate molecular weight of 63 kDa and is larger than the previously reported rat, mouse and human protein. To determine the functional role of additional N-terminal amino acids, we compared the transcription functions of receptor lacking (hERbetaT) and receptor containing (hERbetaL) this N-terminal extension. hERbetaL is more active than hERbetaT in transactivating ERE-based reporter genes. hERbetaL, but not hERbetaT, attenuated cytokine mediated NFkappaB activation. Taken together, the additional N-terminal amino acids appear to play a role in modulating estrogen responsive gene expression in vitro.

Emphysematous pyelonephritis: Outcome with conservative management.

Emphysematous pyelonephritis is a life-threatening condition characterized by necrotising gas forming infection of the renal parenchyma. We describe eight patients seen over a period of 2 years, 62.5% males and 37.5% females with age range between 21 and 65 years. About 75% patients had diabetes mellitus. Six patients were managed conservatively. One patient required nephrectomy with percutaneous drainage and one patient died without surgical intervention.

Adenovirus mutants with DNA sequence perturbations in the intragenic promoter of VAI RNA gene allow the enhanced transcription of VAII RNA gene in HeLa cells.

Ad2 VAI gene strongly competes for transcription with VAII gene in vitro. It has been suggested that this competition may be a basis for the large excess of VAI gene transcription in virus infected cells at late times. We have studied the effect of the DNA sequence perturbations of the intragenic promoter of the VAI gene on transcription of VAII gene at the level of viral chromosome. Several Ad5 mutants with mutations in the promoter of VAI gene were constructed and transcription of their VAI and VAII genes were analyzed in the infected cells. It was found that transcription of VAII gene increased dramatically when either Box A or Box B promoter sequences of VAI gene were mutated or when the entire VAI gene was replaced by a DNA segment with an unrelated DNA sequence. Thus, at late times, active transcription of VAI gene appears to partially repress transcription of VAII gene. Those mutants which synthesized large quantities of VAII RNA only grew more slowly yielding a titer which was 1/10 of that of their parent but 5 to 6 fold higher than that of an Ad5 mutant lacking both VAI and VAII genes.

Two small RNAs encoded by Epstein-Barr virus can functionally substitute for the virus-associated RNAs in the lytic growth of adenovirus 5.

Adenovirus (Ad) serotypes 2 and 5 synthesize large amounts of two low molecular weight RNAs designated virus-associated (VA) 1 and 2. Recently, genetic and biochemical approaches have been used to show that Ad2 VA1 RNA is required for efficient translation of viral mRNAs produced at late times after infection. Primate cells harboring the Epstein-Barr virus genome (EBV) synthesize large amounts of two low molecular weight RNAs: EBER1 and EBER2. Striking similarities of gene organization have been noted between the genes coding for Ad5 VA RNAs and the EBV EBERs [Rosa, M. D., Gottlieb, E., Lerner, M. R. & Steitz, J. A. (1981) Mol. Cell. Biol. 1, 785-796]. To examine whether the EBRs can functionally substitute for the VA RNAs for the lytic growth of Ad5, we have constructed an Ad5 substitution mutant in which the two VA RNA genes have been deleted and replaced by an EBV DNA segment coding for the two EBERs. The resulting Ad5 mutant synthesizes large amounts of the EBERs and is viable. Thus, two small RNAs of EBV origin, having primary and secondary structures different from those of the VA RNAs, can functionally substitute for the VA RNAs in the lytic growth of Ad5. These results are discussed in the context of mechanism of function of the VA RNAs.

Construction and analysis of additional adenovirus substitution mutants confirm the complementation of VAI RNA function by two small RNAs encoded by Epstein-Barr virus.

Adenovirus VAI RNA is essential for the efficient initiation of translation of viral mRNAs at late times after infection. Recently, by constructing an adenovirus type 5 substitution mutant, we showed that the Epstein-Barr virus encoded two small RNAs complemented for the VAI RNA function in the adenovirus type 5 lytic growth (Bhat and Thimmappaya, Proc. Natl. Acad. Sci. USA 80:4789-4793, 1983). This observation was based on our inability to propagate an adenovirus type 5 mutant lacking functional VAI and VAII genes. Subsequently, it was found that this mutant was viable and able to grow to a low titer. Therefore, we examined the complementation of the VAI RNA function by the Epstein-Barr virus-encoded RNAs by constructing additional adenovirus type 5 substitution mutants containing multiple copies of the Epstein-Barr virus-encoded RNA genes in nonessential early transcriptional region III. The new substitution mutants synthesized viral polypeptides at late times at levels comparable to those observed in wild type-infected cells. Our results convincingly demonstrated that the two Epstein-Barr virus-encoded RNAs can efficiently complement for the VAI RNA-mediated translational defect in adenovirus-infected cells.

Efficient expression of small RNA polymerase III genes from a novel simian virus 40 vector and their effect on viral gene expression.

In the past, simian virus 40 (SV40) has been used as a cloning vehicle to clone foreign genes by substituting portions of the viral genome vital for viral replication. Propagation of these defective viruses required a helper virus and the recombinant viruses obtained could be grown only as a mixture. In this study, we describe a novel nondefective SV40 vector to clone small RNA polymerase III genes. Two small RNA polymerase III genes, an amber suppressor human serine tRNA gene and the adenovirus (Ad) VAI RNA gene, were cloned in the intron region of the large-T antigen gene of SV40 after deleting DNA sequences coding for the small-t polypeptide. The recombinant viruses grew to wild type levels and showed no growth defects. When CV-1p cells were infected with these viruses, the cloned RNA polymerase III genes were expressed at high levels at late times. Interestingly, large amounts VAI RNA in CV-1p cells infected with SV40-VA recombinant virus, did not enhance translation of viral mRNAs significantly but did lead to a 3 to 4 fold increase in the steady state levels of large-T mRNA suggesting a novel function for VAI RNA in SV40 infected monkey cells. Furthermore, VAI mutants which fail to function in Ad infected human cells also failed to enhance the levels of large-T mRNAs in monkey cells infected with SV40. The simple SV40 vector described here may be useful to study the structure and function of small RNA polymerase III genes in the context of a eucaryotic chromosome. In addition, the nondefective recombinant SV40 which expresses the suppressor tRNA gene at high levels may provide a useful helper system to propagate animal viruses with amber mutations in essential genes.

Molecular characterization of a new variant of hepatitis B virus in a persistently infected homosexual man.

Based on the diversity of nucleotide sequences of cloned hepatitis B virus DNA genomes, we have predicted possible replication of genetic variants of human hepatitis B virus. This prediction is exemplified by studies of a chronic carrier of HBsAg/adw2, who lacked anti-HBc but carried exceedingly high levels of hepatitis B virus DNA in serum. Molecular characterization of a number of clones revealed a restriction map that deviated significantly from the typical pattern of the adw2 subtype, especially around the EcoRI site commonly used as a reference point. Mutations appearing consistently in the precore and core regions included (a) mutation in the precore region resulting in a termination codon after the initiation codon, (b) mutation of the core initiation codon and (c) an inframe insert of 36 nucleotides in the precore region with a new initiation site for the core protein. The 36-nucleotide insertion resulted in a new core protein with 12 extra amino acids at its amino-terminal end. A few scattered point mutations were clustered in the amino-terminal half of the core gene. Although the core protein of this hepatitis B virus variant carried immunologically detectable HBcAg, the absence of a humoral immune response to HBcAg could have been caused by previous infection with human immunodeficiency virus. This naturally occurring human hepatitis B virus variant replicated efficiently without expressing the precore region, confirming previous observations made of the artificial mutants of duck hepatitis B virus.

Regulated processing of hepatitis C virus core protein is linked to subcellular localization.

Posttranslational processing and subcellular localization of the HCV core protein are critical steps involved in the assembly of hepatitis C virus (HCV). In this study, both of these events were investigated by in vitro translation and transient COS-1 cell transfection of core protein expression constructs. Mutations at amino acid residues 173 to 174 and 191 to 192 disrupted processing events at the two putative cleavage sites in the C-terminal hydrophobic region of the core protein, indicating that these residues are implicated in the pathway of core protein maturation. As a result, two forms of core protein, C173 and C191, were detected by immunoblotting. Indirect immunofluorescence experiments showed that core proteins C173 and C191, when produced from HCV full-length protein or various polyprotein precursors, displayed a cytoplasmic localization. The C173 species, however, was translocated to the nucleus when expressed in the absence of C191. These findings indicate that preferential cleavage may occur during core protein maturation and that the association of the C191 with the C173 species may contribute to the distinct subcellular distribution of core protein. This may provide a possible mechanism for the control of the diverse biological functions of core protein during HCV replication and assembly.

The hepatitis C virus NS2 protein generated by NS2-3 autocleavage is required for NS5A phosphorylation.

Hepatitis C virus (HCV) is dependent on NS2-3 autocleavage and NS5A phosphorylation for its life cycle. We demonstrate that NS5A, when released from the NS2-5 polyprotein of the BK virus strain, is phosphorylated as two distinct forms, pp56 and pp58. Deletion analysis indicates that the appearance of pp58 requires NS2 in cis, while pp56 is NS2 independent. Disruption of NS2-3 autoproteolysis by directed mutagenesis results in loss of pp58. Expression of a construct producing NS2-3 is sufficient to restore pp58 in trans. These data indicate that generation of functional NS2 via autocleavage of the NS2-3 precursor and NS5A phosphorylation are consecutive processes, suggesting coordinate regulation during virus propagation in vivo.

Suppression of the translation defect phenotype specific for a virus-associated RNA-deficient adenovirus mutant in monkey cells by simian virus 40.

Human cells infected with adenovirus type 2 (Ad2) or Ad5 require VAI RNA for efficient translation of viral mRNAs at late times after infection. The Ad5 mutant dl-sub720 synthesized neither virus-associated I (VAI) nor VAII RNAs, and infection of human cells with this mutant resulted in reduced virion polypeptide synthesis. Infection of monkey cells with this mutant also resulted in drastic reduction of polypeptide synthesis compared with wild-type (WT) adenovirus infections. Steady-state levels of hexon-specific mRNA were found to be comparable in WT- and mutant-infected monkey cells. The in vitro translation experiments showed that double-mutant- and WT-infected cells contained comparable levels of translatable hexon mRNA (and other adenovirus late mRNAs), suggesting that the severe inhibition of hexon protein synthesis in the VA mutant involves a translation block. Preinfection of monkey cells with simian virus 40 fully restored the efficient translation of this mRNA in the VA mutant infections to the level observed in WT-infected cultures. These results raise the possibility that simian virus 40 may encode or induce factors that suppress the translation block that occurs during adenovirus infections in the absence of the VA RNAs.