Isolation and identification of 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3, a new metabolite of 1,25-dihydroxyvitamin D3 produced in rat kidney

A new metabolite of vitamin D3 was produced in vitro by perfusing rat kidneys with 1,25-dihydroxyvitamin D3 (4 X 10(-6) M). It was isolated and purified from the lipid extract of the kidney perfusate by high-performance liquid chromatography. By means of ultraviolet absorption spectrophotometry, mass spectrometry, chemical derivatization, and chemical synthesis, the new metabolite was identified as 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3. Along with the new metabolite, three other previously identified metabolites, namely, 1,24,25-trihydroxyvitamin D3, 1,25-dihydroxy-24-oxovitamin D3, and 1,23,25-trihydroxy-24-oxovitamin D3, were also isolated. The new metabolite was also formed when 1,23,25-trihydroxy-24-oxovitamin D3 was used as the substrate. Thus, the new metabolite fits into the following metabolic pathway: 1,25-dihydroxyvitamin D3----1,24(R),25-trihydroxyvitamin D3----1,25-dihydroxy-24-oxovitamin D3----1,23,25-trihydroxy-24-oxovitamin D3----1,23-dihydroxy-24,25,26,27-tetranorvitamin D3. Further, we used 1 alpha,25-dihydroxy[1 beta-3H]vitamin D3 in the kidney perfusion system and demonstrated 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 as the major further metabolite of 1,25-dihydroxyvitamin D3, circulating in the final perfusate when kidneys were perfused with 1,25-dihydroxyvitamin D3 (6 X 10(-10) M) for 4 h. The biological activity of 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 (C-3 alcohol) and its metabolic relationship to 1-hydroxy-23-carboxy-24,25,26,27-tetranorvitamin D3 (calcitroic acid or C-23 acid), the other previously identified side-chain cleavage metabolite of 1,25-dihydroxyvitamin D3, are unknown and are presently undergoing investigation.     

Perinuclear localization of slow troponin C m RNA in muscle cells is controlled by a cis-element located at its 3' untranslated region

The process of mRNA localization within a specific cytoplasmic region is an integral aspect of the regulation of gene expression. Furthermore, colocalization of mRNAs and their respective translation products may facilitate the proper assembly of multi-subunit complexes like the thick and thin filaments of muscle. This postulate was tested by investigating the cytoplasmic localization of three mRNAs-the alpha-actin, slow troponin C (sTnC), and slow troponin I (sTnI), which encode different poly-peptide partners of the thin filament. Using in situ hybridization we showed that all three thin filament mRNAs are localized in the perinuclear cytoplasm of cultured C2C12 muscle cells. Their localization differs from that of the nonmuscle beta-actin mRNA, which is localized in the peripheral region of both proliferating nondifferentiated myoblasts and the differentiated myocytes. Analysis of the localization signal of the sTnC mRNA showed that a 40-nucleotide-long region of the sTnC mRNA 3' UTR is sufficient to confer the perinuclear localization on a heterologous reporter beta-Gal mRNA. This localization signal showed tissue specificity and worked only in the differentiated myocytes, but not in the proliferating myoblasts or in HeLa cells. The predicted secondary structure of the localization signal suggests the presence of multiple stem and loop structures in this region of the 3' UTR. Mutations within the stem region of the localization signal, which abolish the base pairing in this region, significantly reduced its perinuclear mRNA localization activity. Using UV-induced photo-cross-linking of RNA and proteins we found that a myotube-specific 42-kDa polypeptide binds to the localization signal.     

Circadian clocks: neural and peripheral pacemakers that impact upon the cell division cycle

Circadian clocks are pervasive entities that allow organisms to maintain rhythms of approximately 24h, independently of external cues, thereby adapting them to the solar cycle. Recent studies have shown that molecular circadian clocks are important for the proper orchestration of the cell division cycle. For the first time, this provides a framework to understand the interactions between these two evolutionarily linked timers. Here we review the current model of the circadian clock and the molecular methods that can be used to investigate its function. We then map out links to the cell cycle at the cellular level. Furthermore, we review recent progress that has linked dysfunction of the clockwork with the pathogenesis of cancer. Disruption of circadian timing (as occurs in jet-lag, shift work and dementia) thus has far reaching consequences for normal regulation of cell division. The implications of this for the health of a "24-h society" are apparent.     

A Genetic Strategy to Demonstrate the Occurrence of Spontaneous Mutations in Nondividing Cells within Colonies of Escherichia Coli

A genetic strategy was designed to examine the occurrence of mutations in stationary-phase populations. In this strategy, a parental population of cells is able to survive under both permissive and restrictive conditions whereas mutants at a particular target locus exhibit a conditional-lethal phenotype. Thus, by growing the population to stationary phase under restrictive conditions and then shifting it to permissive conditions, mutations that had arisen in stationary phase can be studied without confounding effects caused by the occurrence of similar mutations during growth of the population. In two different applications of this strategy, we have studied the reversion to Lac(+) in stationary phase of several Lac(-) mutations in Escherichia coli. Our results indicate that a variety of spontaneous point mutations and deletions, particularly those that are sensitive to the mechanisms of replication slippage (for their generation) and methyl-directed mismatch repair (for their correction), can arise in nondividing populations of cells within a colony. The frequency of their occurrence was also elevated in mutS strains, which are defective in such mismatch repair. These data have relevance to the ongoing debate on adaptive or directed mutations in bacteria.     

Seasonal variation in the biomass, quantity and quality of agar from Gelidiella acerosa (Forsskal) Feldmann et Hamel (Gelidiales, Rhodophyta) from the Gulf of Mannar Marine Biosphere Reserve, India

The Gulf of Mannar on the southeast coast of India, which has recently been declared as biosphere reserve, is the main harvesting place of Gelidiella acerosa for the Indian agar industry. The biomass harvested from natural stocks was estimated to be over 10 000 tonnes dry weight (DW) in the last two decades and is a serious cause of concern on long term sustainability of this valuable resource from this region. The present study reports the seasonal variation in the biomass and agar yield and quality from G. acerosa in four different habitats such as Rameswaram (rocky intertidal), Ervadi (lower intertidal), Sethukarai (subtidal) and Krusadai Island (lagoon reef area). A maximum biomass of 260 ± 26 g DW m^?2 was recorded from Ervadi, whereas Sethukarai showed the highest percentage cover (69.83 ± 4.83%) and density (208.20 ± 30.16 plants m^?2). Biomass, percentage cover and density were lowest in Rameswaram and significantly lower (P < 0.001) than the other three stations. A single peak in biomass was observed with autumn maxima in Ervadi and Rameswaram and southwest monsoon maxima in Sethukarai and Krusaidai Island. Length of G. acerosa was maximum (9.65 ± 0.25 cm) in Ervadi and the branch index value was maximum (24.70 ± 4.01 br^?1 g^?1 DW) for Rameswaram. Agar yield (37.24 ± 7.59%) and gel strength (448.66 ± 6.50 g/cm²) were higher in Sethukarai plants. The agar yields attained a peak in the northeast monsoon and decreased in summer, whereas a reverse trend was observed for gel strength. Evidently, agar yield showed a strong negative correlation with gel strength (P < 0.0001) and gelling (P < 0.0001) and melting temperatures (P < 0.01). The single annual peak in biomass observed in the present study, as compared with two peaks reported earlier, are attributed to the continuous overharvesting that would have had an effect on the overall annual biomass production. The findings of this study reveal that a single harvest during the autumn months (January to March) could yield optimum biomass with moderate agar and that would be the best for sustainable usage and conservation of this resource from this region.     

Optimization of L-asparaginase production from novel Enterobacter sp., by submerged fermentation using response surface methodology

The culture conditions and nutritional rations influencing the production of extra cellular antileukemic enzyme by novel Enterobacter aerogenes KCTC2190/MTCC111 were optimized in shake-flask culture. Process variables like pH, temperature, incubation time, carbon and nitrogen sources, inducer concentration, and inoculum size were taken into account. In the present study, finest enzyme activity achieved by traditional one variable at a time method was 7.6 IU/mL which was a 2.6-fold increase compared to the initial value. Further, the L-asparaginase production was optimized using response surface methodology, and validated experimental result at optimized process variables gave 18.35 IU/mL of L-asparaginase activity, which is 2.4-times higher than the traditional optimization approach. The study explored the E. aerogenes MTCC111 as a potent and potential bacterial source for high yield of antileukemic drug.     

Biodegradation of toxic organic compounds using a newly isolated <Emphasis Type="Italic">Bacillus</Emphasis> sp. CYR2

The objective of this study was to isolate a new bacterium and investigate its ability for degradation of various toxic organic compounds. Based on 16S rRNA gene sequence and phylogenetic analysis, the isolated strain was identified as Bacillus sp. CYR2. Degradation of various toxic compounds and growth of CYR2 strain were evaluated with 2 and 4% inoculum sizes. All the experiments were conducted for 6 days, flasks were incubated at 30oC under 180 rpm. Among the 2 and 4% inoculum sizes, bacteria showed highest growth and toxic compounds degradation at 4% inoculum size. Especially, compared to 2% inoculum size, growth of the strain CYR2 at 4% inoculum size was increased by 15.1 folds with 4-secondarybutylphenol, 9.1 folds with phenol, and 5.4 folds with 4-tertiary-butylphenol. Strain CYR2 at 4% inoculum size showed highest removal of phenol (84 ± 5%), followed by 4-tertiary-butylphenol (66 ± 3%), 4-secondary-butylphenol (63 ± 5%) and 4-nonylphenol (57 ± 6%). Compared with 2% inoculum size, degradation ability of strain CYR2 with 4% inoculum size was enhanced by 3.45 times with 4-tertiary-octylphenol, and 2.53 times with 4-tertiarybutylphenol. Our results indicated that the newly isolated Bacillus sp. CYR2 can be used for in situ bioremediation of phenol and alkylphenols contaminated water.     

Design,synthesis and in vitro cytotoxicity studies of novel β-carbolinium bromides

A series of novel β-carbolinium bromides has been synthesized from easily accessible β-carbolines and 1-aryl-2-bromoethanones. The newly synthesized compounds were evaluated for their in vitro anticancer activity. Among the synthesized derivatives, compounds 16l, 16o and 16s exhibited potent anticancer activity with IC₅₀ values of <10 μM against tested cancer cell lines. The most potent analogue 16l was broadly active against all the tested cancer cell lines (IC₅₀ = 3.16–7.93 μM). In order to test the mechanism of cell death, we exposed castration resistant prostate cancer cell line (C4-2) to compounds 16l and 16s, which resulted in increased levels of cleaved PARP1 and AO/EB staining, indicating that β-carbolinium salts induce apoptosis in these cells. Additionally, the most potent β-carbolines 16l and 16s were found to inhibit tubulin polymerization.     

Novel potentially antifungal hybrids of 5-flucytosine and fluconazole: Design,synthesis and bioactive evaluation

A series of novel potentially antifungal hybrids of 5-flucytosine and fluconazole were designed, synthesized and characterized by ¹H NMR, ¹³C NMR, IR and HRMS spectra. Bioactive assay manifested that some prepared compounds showed moderate to good antifungal activities in comparison with fluconazole and 5-flucytosine. Remarkably, the 3,4-dichlorobenzyl hybrid 7h could inhibit the growth of C. albicans ATCC 90023 and clinical resistant strain C. albicans with MIC values of 0.008 and 0.02?mM, respectively. The active molecule 7h could not only rapidly kill C. albicans but also efficiently permeate membrane of C. albicans. Molecular docking study revealed that compound 7h could interact with the active site of CACYP51 through hydrogen bond. Quantum chemical studies were also performed to explain the high antifungal activity. Further preliminary mechanism research suggested that molecule 7h could intercalate into calf thymus DNA to form a steady supramolecular complex, which might block DNA replication to exert the powerful bioactivities.     

Macromolecular assemblages. Theory and simulation

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