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Biomedical subjects

N Srivastava

Publications and source records attributed to N Srivastava.

At least 55 records · Page 3Linked to original sources
PubMed1999

Molecular bases of low production rates of apolipoprotein B-100 and truncated apoB-82 in a mutant HepG2 cell line generated by targeted modification of the apolipoprotein B gene.

In subjects with familial hypobetalipoproteinemia heterozygous for truncated forms of apolipoprotein B, both apoB-100 and the truncated forms are produced at lower than expected rates. We studied the mechanism of low levels of apoB in a cell model produced by targeted modification of the apob gene of HepG2 cells. One of the three alleles of apob was found to be targeted. The targeted cells expressed apoB-100 and B-82. The media of mutant cells contained 56% of the levels of apoB-100 present in the media of wild-type (WT) HepG2 cells. ApoB-82 was present at 11% of the apoB-100 levels in mutant cell media. An 85-kD protein (apoB-15) representing the N-terminal fragment of apoB was also secreted, but only in the mutant cell media. We examined the mechanism of low levels of apoB-82. Cellular apoB-82 mRNA was 11% of apoB-100 mRNA, lower than the 33% expected, but consistent with relative levels of apoB-82 in the media. ApoB mRNA transcription in WT and the mutant cells did not differ, while the levels of apoB-82 mRNA in nuclei and polysomes were 46% and 12% of the levels of apoB-100 mRNA, respectively, suggesting that the lower levels of apoB-82 mRNA were due to altered message stability. In a pulse/chase experiment with [35S] methionine, at zero time of chase, the amounts of apoB-100 in mutant cells was 66% that of WT levels, consistent with the modification of one allele. The fractions of newly synthesized apoB-100 secreted into the media at 2 h were 10% in the mutant cells and 19% in the WT cells, suggesting greater presecretory degradation of apoB-100 in the mutant cells. Thus, low levels of mutant apoB-82 mRNA gave rise to the low levels of apoB-82, while low levels of apoB-100 were due to low rates of secretion.

Alleles↗
PubMed1999

Effect of endodontic smear layer and various solvents on the calcium ion diffusion through radicular dentin--an in vitro study.

External root resorption and ankylosis remains the major cause of failure of replanted teeth. This study was conducted to explore the different ways to increase the pH of periradicular area in order to overcome the problem of root resorption and ankylosis. 60 freshly extracted permanent anteriors were used after removing the crown at CEJ. After biomechanical preparation Ca (OH)2 was injected and assays were done using EDTA, Citric and tannic acid Assays were repeated. Calcium diffusion and pH in the root exterior was measured using spectrophotometer. Results showed that dentin is permeable to calcium & hydroxyl ions and placement of Ca (OH)2 in the canal resulted in its increased recovery and alkaline pH periradicularly. Smear layer removal did not result in significant increase in Ca++ recovery or alkaline pH however combination of EDTA & NaOCl was found best than the other two.

Calcium↗
PubMed1998

Effects of ethanol on development of locus coeruleus brain stem transplants in oculo.

In this investigation, we studied the effects of ethanol (EtOH) on the development of noradrenergic (NE) neurons of the locus coeruleus. Fetal brainstem tissue from embryonic days 15-17 was grafted into the anterior chamber of the eye of adult rats. Two different experimental groups were exposed to 16% EtOH in the drinking water during different developmental windows. The first group received EtOH 24 h after transplantation and during the whole experimental period of 7 weeks (continuous EtOH), and the second group only during the last 5 weeks of the experimental period (delayed EtOH). The control group received water ad libitum. After 7 weeks, all the animals were sacrificed and morphological evaluations were performed. Immunohistochemical analysis showed that axon bundle formation and NE fiber outgrowth into the host iris was significantly reduced in the continuous EtOH-treated group compared to controls. We also studied the morphology of TH-positive neurons and processes in the intraocular transplants. A significant decrease in TH-positive staining intensity was observed in the continuous EtOH-treated group compared to controls. Moreover, we found a significant decrease in cell size and neuronal survival in both EtOH-treated groups compared to controls. The present results suggest that chronic EtOH exposure during development leads to an altered axonal outgrowth and decreased cell sizes and number of NE neurons in intraocular brain stem grafts. Furthermore, we found that NE neurons are more sensitive to EtOH exposure during the last prenatal days and the first postnatal week of development, compared to a later developmental period.

Animals↗
PubMed1997

Estrogen up-regulates apolipoprotein E (ApoE) gene expression by increasing ApoE mRNA in the translating pool via the estrogen receptor alpha-mediated pathway.

The antiatherogenic property of estrogens is mediated via at least two mechanisms: first by affecting plasma lipoprotein profiles, and second by affecting the components of the vessel wall. Raising plasma apolipoprotein E (apoE) in mice protects them against diet-induced atherosclerosis (Shimano, H., Yamada, N., Katsuki, M., Gotoda, T., Harada, K., Murase, T., Fukuzawa, C., Takaku, F., and Yazaka, Y. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1750-1754). It is possible that estrogen may be antiatherogenic at least in part by increasing plasma apoE levels. Therefore, we studied the regulation of apoE by estrogen. A survey of 15 inbred strains of mice showed that some mouse strains responded to injections or subcutaneously implanted pellets of estradiol by raising their apoB and apoE levels and some did not. We performed detailed studies in two "responder" strains, C57L and C57BL, and two "non-responder" strains, C3H and BALBc. Responders increased their plasma apoE levels 2.5-fold. Non-responders' levels were altered +/-10%. In the responders the distribution of apoE among the plasma lipoproteins shifted from high density lipoprotein toward the apoB-containing lipoprotein fractions. In nonresponders the shift was toward high density lipoprotein. Hepatic apoE mRNA levels and relative rates of apoE mRNA transcription were unchanged in all strains, suggesting that apoE regulation occurred at posttranscriptional loci. Therefore, we measured apoE synthesis in fresh liver slices and on isolated hepatic polysomes. Two-fold increases were noted but only in responders accompanied by selective 1.5-fold increases in polysomal apoE mRNA levels. Similar increases in apoE synthesis were also observed in castrated C57BL mice given either physiological or pharmacological replacement doses of estradiol, but not testosterone, suggesting that the effect of estradiol was specific on the distribution of apoE mRNA in the translationally active polysomal pool. Next, we examined whether the effects of estrogen on apoE translation were mediated by estrogen receptors (ER). ER-alpha knock-out mice and their wild-type littermates were administered estradiol. As expected, apoE levels and hepatic apoE synthesis increased more than 2-fold in the wild-type littermates, but only 20% increases in the plasma apoE and hepatic synthesis were observed in the ER knock-out mice. Hepatic apoE mRNA levels did not change in either the wild-type or the ER knock-out mice. Thus, estradiol up-regulates apoE gene expression by increasing levels of apoE mRNA in the polysomal translating pool. Furthermore, the increased polysomal recruitment of apoE mRNA is largely mediated by estrogen receptors.

Animals↗
PubMed1997

Morphological alterations in the peripheral and central nervous systems of mice lacking glial cell line-derived neurotrophic factor (GDNF): immunohistochemical studies.

Glial cell line-derived neurotrophic factor (GDNF) is a member of the TGF-beta superfamily of growth factors with neurotrophic activity on midbrain dopaminergic neurons and on developing and mature motoneurons of the brainstem and spinal cord. To investigate the extent of GDNF dependency of central and peripheral nervous structures during development, we have performed an immunohistochemical analysis of sections from the whole head including brain, peripheral ganglia, developing teeth and tongue, as well as intestines, in mutant mice lacking a part of the third exon that encodes the GDNF protein. As described previously, these null-mutated mice lack most of the enteric nerve plexus and are subject to agenesis or severe dysgenesis of the kidneys. In the present communication, we examined the development of vibrissae and incisor and molar teeth, as well as the innervation of these structures, and found no differences between null-mutated and control mice. A decrease in the immunohistochemical labeling intensity with tyrosine hydroxylase was observed in the superior cervical ganglion (SCG), as well as in the pontine nucleus locus coeruleus, and the sympathetic innervation of blood vessels and glands in the head was significantly decreased. None of the brain nuclei studied exhibited any significant decreases in the total number of neurons, but the packing density of neurons in the nucleus locus coeruleus was decreased. These data indicate that GDNF might be one neurotrophic factor that contributes to the development of central and peripheral noradrenergic neurons.

Animals↗
PubMed1997

Collateral sprouting of central noradrenergic neurons during aging: histochemical and neurochemical studies in intraocular triple transplants.

The sprouting capacity of aged noradrenergic neurons of the brain-stem nucleus locus coeruleus (LC) was examined using intraocular transplants of fetal tissues. Fetal hippocampal tissue (E18) and LC tissue (E15) were transplanted together as a double transplant into the anterior chamber of the eye of young adult Fischer 344 rats. The double transplants were allowed to mature for 14-18 months, after which an additional fetal hippocampal transplant was placed next to the LC graft. The triple transplants were monitored for overall growth and vascularization for an additional 2-6 months. Immunohistochemical examinations showed that both young (2-6 months old) and aged (16-24 months old) hippocampal cografts contained a plexus of thin varicose tyrosine hydroxylase (TH)-immunoreactive fibers extending throughout the grafted hippocampal tissues. However, the aged hippocampal grafts contained a denser uniform plexus of TH-positive fibers compared to the young transplants. Immunohistochemistry with synapsin antibodies demonstrated that both the young and the aged hippocampal transplants contained much higher densities of synaptic elements than the LC grafts. In vivo electrochemical measurements of potassium-evoked overflow of norepinephrine (NE) in the grafts showed that similar amounts of NE overflow were detected in both the young and the aged hippocampal grafts. HPLC-EC measurements of NE levels in the grafts revealed that there were similar amounts of NE in the young and the aged grafts, and the grafts did not contain serotonin or dopamine. In summary, the findings of the present study show that aged LC neurons are capable of undergoing collateral sprouting producing a functional NE neuronal system when introduced to an appropriate young target.

Animals↗
PubMed1997

Biochemical contents, their variation and changes in free amino acids during seed germination in Terminalia arjuna.

The leaves, twigs, stem and bark of T. arjuna were analysed for their protein, phenol, tannin, nitrate, oxalate in addition to vitamin C, anthocyanin and chlorophyll in the leaves. The variation of some of these parameters in the leaves with season and leaf position was also studied. The time course changes in amino acids and protein during seed germination in T. arjuna, showed initial decrease in protein followed by increase at subsequent stages. The seeds contain high level of serine (21.7%) and glutamic acid (22.6%) the later decreased as the germination progressed. After 30 days seeds showed higher amounts of serine (26.0%), valine (2.8%), proline (10.6%), methionine (3.4%), histidine (5.6%) and lysine (7.4%) while threonine, glutamic acid, tyrosine and arginine were in lower amounts than that of initial stage at 0 day.

Amino Acids↗
PubMed1996

Pharmacokinetics of the conventional and microemulsion formulations of cyclosporine in pancreas-kidney transplant recipients with gastroparesis.

Cyclosporine (CsA) is an immunosuppressive drug requiring dose individualization and regular control due to its highly variable pharmacokinetics. Since gastroparesis may influence the absorption of CsA, a randomized cross-over study was performed to assess the pharmacokinetics and tolerability of a novel microemulsion CsA formulation in comparison with the standard CsA dosage form in six stable pancreas-kidney transplant recipients with scintigraphically proven gastroparesis. The absorption of CsA was investigated during three 2-hr study days during each treatment period, and a full pharmacokinetic profile was done for each formulation. No adverse events or differences in tolerability/safety parameters between the treatments were found. The average AUC (0-->2 hr) was 150% higher after the novel formulation. The coefficient of variation in AUC (0-->2 hr) for both formulations was comparable (37% after the microemulsion and 40% after the standard formulation). The median time at which blood CsA levels exceeded the preceding trough level by 20% was 30 min (range: 30 -> 718 min) after the standard formulation. With approximately the same average dose, the AUCss tau after the microemulsion was 81% higher than the standard formulation, while predose and 12-hr trough levels were similar. The average maximal CsA plasma level after the microemulsion was 396 ng/ml (95% CI: 71-722 ng/ml) higher than after the standard formulation. The median time at which the highest blood levels were observed was 90 min (range: 150 -> 718 min) after the standard formulation. The time profiles of the CsA metabolites followed those of the parent compound. The microemulsion resulted in a higher systemic exposure to CsA than the standard formulation in pancreas-kidney transplant patients with diabetic gastroparesis, but substantial variability in blood concentrations remained.

Adult↗
PubMed1996

Role of GABAA receptors in the GABA attenuation of ethanol neurotoxicity.

We have previously reported that GABA reverses the neuronotoxic effects of ethanol in neuroblast-enriched cultures derived from 3-day-old whole chick embryo (E3WE). In the present study, we examined the effects of GABA agonists and antagonists on morphological growth patterns and on cholinergic neuronal phenotypic expression, using choline acetyltransferase (ChAT) activity as a marker. E3WE neuroblast-enriched cultures showed positive immunoreactivity for neurofilament and as previously reported, control cultures exhibited the characteristic pattern of outgrowth of neurites of varying thickness radiating from the aggregates. In contrast, cultures grown in ethanol consisted of neuronal aggregates lacking fasciculation but having a complex network of individual thin neurites. Both GABA and GABAA agonist muscimol enhanced neuritic fasciculation and arborization in control and ethanol-treated cultures, and this growth enhancement was inhibited by GABAA antagonist bicuculline. No effects were noted with GABAB agonist baclofen. GABA increased ChAT activity in E3WE control cultures, as previously reported. A similar effect was seen with GABAA agonist muscimol, but not with GABAB agonist baclofen. However, the GABA effect was not apparent in the presence of GABAB antagonist phaclofen. Thus, it appears that the cholinotrophic effects of GABA are mediated by both GABAA and GABAB receptors. In ethanol-treated cultures the already-reported ChAT decline was reversed by GABA and muscimol, but not by baclofen. Moreover, the GABA effect in ethanol-treated cultures was not antagonized by GABAB antagonist phaclofen, suggesting that the GABA effect was mediated by a GABAA receptor. We conclude from these findings that the cholinotrophic effects of GABA are mediated by GABAA and GABAB receptors, while the rescuing effects of GABA in the ethanol-treated cultures are mediated via GABAA receptors.

Animals↗
PubMed1996

A proton nuclear magnetic resonance investigation of the conformation of daunomycin.

The 500 MHz proton NMR spectra of 4.95 mM daunomycin in D2O have been investigated in the temperature range 277-350 K. Down field shifts of approximately 0.15 to 0.20 ppm in 1H, 2H and 3H protons with increasing temperature indicate that daunomycin exists in aggregated form at 277 K which is stabilized by stacking of aromatic rings. The 7H, 10axH and 10eqH protons show a change in chemical shift of 0.12 to 0.16 ppm, while other ring A/sugar protons shift by 0.0 to 0.08 ppm due to self association. The drug exists in monomer state at about 350 K. The conformational features have been ascertained from NOESY spectra at 297 K. The 7H proton is found to be strongly coupled to 8axH (J = 5 Hz) as compared to 8eqH (J = 2 Hz), while the 4'H-5'H connectivity is not observed in the COSY spectra. Besides the NOESY cross peaks between the spin-spin coupled protons, several intramolecular NOEs are seen. The 8axH and 8eqH are equally distant from 5'H proton. The distances of 3'H and 4'H daunosamine sugar protons from the ring A protons--7H, 8eqH, 9COCH3--are in the range 2.9-3.1 A, giving moderate cross peaks in NOESY spectra. The observed results imply the existence of predominantly 9H8 half chair conformation of ring A in aqueous solution, which is marginally different from that obtained by X-ray crystal analysis.

Antibiotics, Antineoplastic↗
PubMed1996

A new apolipoprotein B truncation (apo B-43.7) in familial hypobetalipoproteinemia: genetic and metabolic studies.

We describe a new truncation of apolipoprotein (apo) B in a white kindred with familial hypobetalipoproteinemia (FHBL). Apo B-43.7, found in a daughter and her father, was due to a C --> T change in base position 6162 of the apo B gene converting the arginine (residue 1986) codon CGA to a stop codon TGA. Both subjects were heterozygotes, and both apo B-43.7- and apo B-100-containing particles were present in plasma. On density gradient ultracentrifugation (DGUC), approximately 30% to 40% of apo B-43.7 floated with very-low-density lipoprotein (VLDL)/intermediate-density lipoprotein (IDL)-density particles and 60% to 70% floated with high-density lipoprotein (HDL)-density particles. To assess the metabolism of apo B, 13C-leucine was infused and its rates of appearance in and disappearance from apo B-43.7- and apo B-100-containing particles were quantified by multicompartmental kinetic analysis. Apo B-100 entered plasma via VLDL with a production rate of 30 mg x kg-1 x d-1. Fractional catabolic rates (FCRs) for apo B-100 VLDL, IDL, and low-density lipoprotein (LDL) were 20.0, 16.0, and 0.46 pools x d-1, respectively. The production rate of apo B-43.7 was 9.6 mg x kg-1 x d-1, and FCRs for apo B-43.7 VLDL- and HDL-like particles were 12.0 and 1.8 pools x d-1, respectively. Approximately 30% of apo B-43.7 in HDL-density particles was derived from VLDL apo B-43.7, and about 70% appeared to enter the plasma as HDLs. The relatively low production rate of apo B-43.7 is compatible with previous reports that apo B truncations are produced at lower rates than their apo B-100 counterparts.

Adult↗
PubMed1996

Expression, purification and properties of recombinant E. coli ribonuclease III.

Cloned RNase III gene in a T7 RNA polymerase promoter system was expressed in Escherichia coli cells lacking endogenous RNase III, and the over-expressed recombinant RNase III was purified to homogeneity using ion exchange, exclusion and affinity column chromatography. The overexpressed RNase III was found to separate with the membrane fraction after sonication, which was solubilized, fractionated with (NH)2SO4 and the active fractions used for further purification. The properties of the purified recombinant RNase III were studied using the synthetic RNA substrate, 3[H]poly[A].poly[U], and the natural substrates, 7S and p10Sa RNAs, and compared with the partially purified RNase III from wild-type E. coli cells. The recombinant RNase III showed maximal activity at 37 degrees C and at a pH range of 6.9 to 7.4, which was similar to the RNase III purified from the wild-type cells. Recombinant RNase III efficiently hydrolyzed 3[H].poly[A].poly[U] in the presence of Mg2+. However, the recombinant RNase III cleaved natural RNA substrates efficiently and accurately in the presence of Mn2+. A concentration of Mn2+ ranging from 150 to 300 microM was found to be optimal; concentrations higher than 0.5 mM were inhibitory. Other divalent cations did not support RNase III activity. Monovalent cations, Na+, K+ and NH4+ at 20 mM were equally effective in stimulating RNase III activity although they were not absolutely required for the activity. The thermal stability of the recombinant RNase III was examined at two temperatures, 37 degrees and 50 degrees C. Incubation of RNase III at 37 degrees C for 30 min did not affect activity, but it lost almost 50% of its activity when incubated at 50 degrees C for 30 min. Thus, the recombinant RNase III prefers Mn2+ for the cleavage of natural substrates and exhibits several properties similar to the wild-type RNase III.

Chromatography↗
PubMed1996

Nitric oxide is necessary for a switch from stationary to locomoting phenotype in epithelial cells.

The restitution of epithelial integrity is accomplished in part by cell migration. Studying this process, we have found that nitric oxide (NO) release migrating epithelial BSC-1 cells displayed a biphasic response to the inflicted wounds; an initial transient release of NO is followed by a delayed sustained elevation. Whereas the constitutive endothelial NO synthase (NOS) did not show any spatial or temporal changes associated with wounding, the inducible NOS became expressed 3 h after wounding and showed higher abundance at the edges of epithelial wounds. L-Arginine (L-Arg) or NO donor, S-nitroso-N-acetyl-DL-penicillamine, exerted motogenic effect in epithelial and endothelial cells. Inhibition of NOS with NG-nitro-L-arginine methyl ester (L-NAME) or a selective knockout of inducible NOS with antisense oligodeoxynucleotides reduced the rate of spontaneous or epidermal growth factor (EGF)-induced BSC-1 cell migration. Migrating cells showed the polarized expression of NOS, suggesting a head-to-rear NO gradient. Several growth factors (EGF, insulin-like growth factor I, hepatocyte growth factor, and fibroblast growth factor) were motogenic for BSC-1 cells, but this effect was abrogated by pretreatment with L-NAME. We conclude that endogenous NO production is a prerequisite for BSC-1 cell migration. A vectorial NO release may be essential for the spatially and temporally coordinated reciprocal phenomena that occur at the leading and trailing edge of locomoting epithelial cells. Although the exact mode of NO action remains uncertain, it is conceivable that the production of NO serves as a cellular switch from the stationary to the locomoting epithelial phenotype.

Animals↗
PubMed1996

Analysis and potential of excretory-secretory antigens of Setaria cervi for immunodiagnosis of human filariasis.

The excretory-secretory (E-LS) products released by the adult Setaria cervi, a bovine filarial parasite, were used to raise polyvalent hyperimmune serum in rabbits. Analysis of E-S products, using anti-E-S serum showed the presence of 10-14 immunogenic proteins, the rabbit anti-E-S serum showed reciprocal antibody titres in the range of 100,000-250,000 by enzyme linked immunosorbent assay. The anti-E-S antibodies could detect circulating antigen in filarial patients sera by Counter immunoelectrophoresis.

Animals↗
PubMed1996

Apolipoprotein E gene expression in various tissues of mouse and regulation by estrogen.

Apolipoprotein (apo) E is associated with several classes of lipoproteins and serves as a ligand for the receptor mediated uptake of cholesterol-rich particles by hepatocytes and peripheral tissues. Variant forms of apo E is also associated with dyslipidemia and late-onset of Alzheimer's Disease (AD). We report here expression of apoE in various mouse tissues, and regulation of apoE in liver, kidney, brain and testes by supraphysiological doses of estrogen. ApoE mRNA was quantified by RNase protection assay and translatable apoE mRNA by in vitro translation. As an internal control the levels of beta-actin mRNA were also quantified. Highest levels of apoE were expressed in liver (220-280 pg/mu g RNA) with negligible levels in small intestine. Brain expressed highest levels of total (35-40 pg/mu g RNA) and translatable apoE mRNA next only to liver. Other tissues that expressed relatively higher levels of apoE were adrenals, testes and ovary. ApoE was also found to be expressed in heart, lung, kidney and spleen. Regulation of apoE gene expression by estrogen (3 mu g 17beta-estradiol/ g body weight/ day for 5 consecutive days) was studied in liver, kidney, brain and testes of 4 mouse strains. Hepatic apoE mRNA did not change significantly in any of the mouse strains following estradiol administration. Of note was significant increases in the levels of brain apoE mRNA in the strain C3H. These studies demonstrate that estrogen regulates apoE gene expression in a tissue-specific manner in mice, and increases in apoE mRNA in the brain by estrogen may have implications in late-onset of Alzheimer's Disease.

Animals↗
PubMed1996

The multifaceted roles of the RNA processing enzyme ribonuclease III.

Ribonuclease III was initially characterized as an endoribonuclease specific for double stranded RNA. Subsequently RNase III was found to be involved in the processing and maturation of ribosomal and tRNAs. Recent studies demonstrate that RNase III also participates in the processing of small stable RNAs. A number of other biological processes in which RNase III participates are: (a), conversion of polycistronic transcript of the bacteriophage T7 early region into discrete monocistronic mRNAs, (b), controlling expression of a variety of genes by processing of gene transcripts, (c), autoregulation of its own gene and (d), regulation of mRNA stability and stimulation of translation. No single processing enzyme displays such a wide variety of roles in RNA metabolism and gene expression as RNA processing enzyme ribonuclease III. This review provides an account of the various roles of RNase III in regulating gene expression and RNA metabolism.

Bacteriophage T7↗