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Biomedical subjects

A B Reddy

Publications and source records attributed to A B Reddy.

At least 19 recordsLinked to original sources

Differential resynchronisation of circadian clock gene expression within the suprachiasmatic nuclei of mice subjected to experimental jet lag.

Disruption of the circadian timing system arising from travel between time zones ("jet lag") and rotational shift work impairs mental and physical performance and severely compromises long-term health. Circadian disruption is more severe during adaptation to advances in local time, because the circadian clock takes much longer to phase advance than delay. The recent identification of mammalian circadian clock genes now makes it possible to examine time zone adjustments from the perspective of molecular events within the suprachiasmatic nucleus (SCN), the principal circadian oscillator. Current models of the clockwork posit interlocked transcriptional/post-translational feedback loops based on the light-sensitive Period (Per) genes and the Cryptochrome (Cry) genes, which are indirectly regulated by light. We show that circadian cycles of mPer expression in the mouse SCN react rapidly to an advance in the lighting schedule, whereas rhythmic mCry1 expression advances more slowly, in parallel to the gradual resetting of the activity-rest cycle. In contrast, during a delay in local time the mPer and mCry cycles react rapidly, completing the 6 hr shift together by the second cycle, in parallel with the activity-rest cycle. These results reveal the potential for dissociation of mPer and mCry expression within the central oscillator during circadian resetting and a differential molecular response of the clock during advance and delay resetting. They highlight the indirect photic regulation of mCry1 as a potentially rate-limiting factor in behavioral adjustment to time zone transitions.

Analysis of Variance↗

Seasonal regulation of food intake and body weight in the male Siberian hamster: studies of hypothalamic orexin (hypocretin), neuropeptide Y (NPY) and pro-opiomelanocortin (POMC).

The aim of these experiments was to investigate the relationship between hypothalamic expression of orexin (also called hypocretin), neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) mRNA and seasonal cycles of body weight and food intake in the Siberian hamster. Adult males were transferred from long days of 16 h light and 8 h dark to short days of 8 h light and 16 h dark, a procedure known to induce major reductions in food intake and body weight in this species. After 8 weeks of exposure to short days, while body weight was declining, hypothalamic NPY mRNA levels as assessed by in situ hybridization were slightly lower (P < 0.05) than in age-matched controls exposed to long days. After 12 weeks with short days, when body weight would be expected to have reached its seasonal nadir, POMC mRNA levels were lower (P < 0.05) than in hamsters under long days. At no stage did orexin mRNA levels in hamsters under short days differ significantly from levels in those under long days. To investigate further the role of these peptide systems in seasonal changes in body weight and food intake, two provocative tests were carried out. Firstly, a 48-h fast induced a significant increase (P < 0.025) in hypothalamic NPY mRNA levels in both long- and short-day conditions, but did not change hypothalamic POMC or orexin mRNA levels. Secondly, systemic (intraperitoneal) treatment with recombinant murine leptin (5 mg/kg body weight) significantly decreased (P < 0.01) food intake over a 6-h post-treatment period in both long- and short-day conditions. However, this acute leptin treatment did not induce significant changes in hypothalamic orexin, NPY or POMC mRNA abundance. The increase in NPY expression in both long- and short-day conditions following food restriction and the suppression of food intake by leptin in both conditions suggests that acute homeostatic mechanisms operate in both long-day (obese) and short-day (nonobese) conditions. The lack of major changes in orexin, NPY and POMC in such different metabolic states suggest that other central systems must play a greater role in generating these states. Such findings are consistent with the 'sliding set-point' hypothesis, that is, seasonal cycles in food intake and fat metabolism are brought about by as yet unknown central mechanisms that chronically alter the level ('set point') around which homeostasis occurs, rather than resulting from changes in the potency of the acute feedback mechanisms themselves.

Animals↗

Negative regulation by dexamethasone of fluvastatin-inducible CYP2B expression in primary cultures of rat hepatocytes: role of CYP3A.

Fluvastatin (Fluva), a synthetic inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, induces CYP2B1/2 in rat liver and primary cultured rat hepatocytes. However, the overall profile of CYP induction, which includes induction of CYP4A, suggests that Fluva is not a typical "phenobarbital (PB)-like" inducer. Several treatments affecting diverse cell signaling pathways have been reported to modify PB-inducible CYP2B expression in primary cultured rat hepatocytes. We examined the effects of selected treatments on the ability of Fluva to induce CYP2B1/2 mRNA. Only dexamethasone (Dex) produced effects on Fluva-inducible CYP2B1/2 mRNA expression that differed from those produced on PB-inducible CYP2B1/2 mRNA expression. Dex concentrations up to 10(-7) M of potentiated PB (10(-4) M)-mediated CYP2B1/2 mRNA induction, while higher Dex concentrations produced a progressive reduction in PB-induced CYP2B1/2 mRNA levels. By contrast, Dex concentrations up to 10(-8) M had no effect on Fluva (3 x 10(-5) M)-induced CYP2B1/2 mRNA levels, while Dex concentrations of 10(-7) M and higher markedly suppressed Fluva-mediated CYP2B1/2 mRNA induction. The concentrations of several glucocorticoids that produced suppression of Fluva-induced CYP2B1/2 mRNA levels were the same concentrations that induced CYP3A mRNA. Treatment with pregnenolone 16 alpha-carbonitrile also produced a concentration-dependent suppression of Fluva-induced CYP2B1/2 mRNA levels. Dex-mediated suppression of Fluva-induced CYP2B1/2 mRNA was concentration-dependently reversed when hepatocytes were cotreated with troleandomycin, a selective CYP3A inhibitor. The amounts of Fluva detected in culture medium and cells were reduced significantly when hepatocytes were incubated with Dex. However, Dex-mediated suppression of Fluva-induced CYP2B1/2 mRNA expression was not overcome when hepatocytes were incubated with Fluva concentrations greater than 3 x 10(-5) M, suggesting that mechanisms other than CYP3A-catalyzed metabolism may contribute to Dex-mediated suppression of Fluva-induced CYP2B1/2 expression.

Animals↗

Comparative pharmacodynamics of CYP2B induction by DDT, DDE, and DDD in male rat liver and cultured rat hepatocytes.

In this study the pharmacodynamics were characterized of rat hepatic cytochrome P-450 2B (CYP2B) induction by the pesticide DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane] and its metabolites DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene], which is bioretained, and DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)ethane], which is metabolized further and therefore less prone to bioaccumulate. DDT, DDE, and DDD were each found to be pure phenobarbital-type cytochrome P-450 inducers in the male F344/NCr rat, causing induction of hepatic CYP2B and CYP3A, but not CYP1A. The ED50 values for CYP2B induction (benzyloxyresorufin O-dealkylation) by DDT, DDE, and DDD were, respectively, 103, 88, and > or = 620 ppm in diet (14 d of exposure). The efficacies (Emax values) for induction of benzyloxyresorufin O-dealkylation by DDT, DDE, and DDD were 24-, 22-, and > or = 1-fold, respectively, compared to control values. The potencies of the three congeners for CYP2B induction appeared also to be similar, with EC50 values (based on total serum DDT equivalents) of 1.5, 1.8, and > or = 0.51 microM, respectively. The EC50 values based on DDT equivalents in hepatic tissue were 15, 16, and > or = 5.9 micromol/kg liver tissue, respectively. In primary cultures of adult rat hepatocytes, DDT, DDE, and DDD each displayed ability to induce total cellular RNA coding for CYP2B (ED50 values of 0.98, 0.83, and > or = 2.7 microM, respectively). These results suggest that DDT, DDE, and DDD each possess a high degree of intrinsic CYP2B-inducing ability for rat liver, despite marked differences in bioretention among the congeners.

Animals↗

Regulation of rat hepatic cytochrome P450 expression by sterol biosynthesis inhibition: inhibitors of squalene synthase are potent inducers of CYP2B expression in primary cultured rat hepatocytes and rat liver.

The effects of treatment with squalestatin 1, a potent inhibitor of squalene synthase, the first committed enzyme of sterol biosynthesis, were examined on cytochrome P450 expression in primary cultured rat hepatocytes and rat liver. Incubation of cultured hepatocytes with squalestatin 1 caused marked accumulations (maximal elevations that were approximately 25-100% of phenobarbital-elicited increases) of CYP2B mRNA and immunoreactive protein but not of CYP1A, CYP3A, or CYP4A. Squalestatin 1 treatment increased CYP2B and 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA content in hepatocyte cultures with comparable potencies (ED50 = 5.0 and 18 nM, respectively), and significantly induced CYP2B (mRNA, immunoreactive protein, and pentoxyresorufin O-dealkylase activity) in the livers of treated rats, producing maximal increases at a dose of 25 mg/kg/day that were approximately 32-87% of phenobarbital-induced increases. Squalestatin 1 treatment induced both CYP2B1 and CYP2B2 and activated reporter gene expression in cultured hepatocytes transiently transfected with a plasmid containing approximately 2.4 kb of CYP2B1 gene 5'-flanking region or containing a previously described phenobarbital-responsive region. Coincubation of cultured hepatocytes with 25-hydroxycholesterol suppressed squalestatin 1-mediated CYP2B and 3-hydroxy-3-methylglutaryl coenzyme A mRNA induction with approximately the same potency. Treatment of cultures with SQ-34919, a structurally distinct squalene synthase inhibitor, produced the same selective CYP2B mRNA induction as did squalestatin 1. These results suggest that inhibition of hepatic sterol synthesis activates processes that culminate in increased CYP2B gene transcription.

Animals↗

Regulation of cytochrome P450 expression by inhibitors of hydroxymethylglutaryl-coenzyme A reductase in primary cultured rat hepatocytes and in rat liver.

It was previously demonstrated that treatment of primary cultured rat hepatocytes with lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, induced the mRNAs for several cytochromes P450 (P450s), including CYP2B1/2, CYP3A1/2, and CYP4A. In this study, we have compared the effects of lovastatin with those of three additional HMG-CoA reductase inhibitors (simvastatin, pravastatin, and the structurally dissimilar drug fluvastatin) on P450 expression in primary cultured rat hepatocytes, and we have also characterized the effects of in vivo treatment with fluvastatin on P450 expression in rat liver. Treatment of cultured hepatocytes with lovastatin, simvastatin, or fluvastatin increased CYP2B1/2, CYP3A1/2, and CYP4A mRNA and immunoreactive protein levels over the dose range (3 x 10(-6) to 3 x 10(-5) M) required to increase the amount of HMG-CoA reductase mRNA. The increases in CYP2B1/2 levels produced by 3 x 10(-5) M fluvastatin treatment were larger than those produced by lovastatin or simvastatin treatment or by treatment with 10(-4) M phenobarbital. In contrast, treatment of cultured hepatocytes with 3 x 10(-5) M lovastatin, simvastatin, or fluvastatin increased CYP3A1/2 and CYP4A mRNA and immunoreactive protein to lower levels than those produced by treatment with 10(-5) M dexamethasone or 10(-4) M ciprofibrate. Treatment of cultured hepatocytes with pravastatin had little or no effect on the amount of any of the P450s examined, although this drug induced HMG-CoA reductase mRNA as effectively as did fluvastatin. Incubation of hepatocytes with 10(-4) M fluvastatin increased CYP1A1 mRNA to 67% of the level induced by treatment with 10(-5) M beta-naphthoflavone. Doses of 50 or 100 mg/ kg/day fluvastatin administered for 3 days to rats increased the hepatic levels of CYP2B1/2 and CYP4A mRNA and immunoreactive protein, although to much lower levels than those produced by treatment with phenobarbital or ciprofibrate, respectively. Treatment of rats with fluvastatin had no effect on hepatic levels of CYP3A1/2 mRNA or immunoreactive protein. However, treatment with 50 mg/kg/day fluvastatin induced CYP1A1 mRNA and protein. The effects of fluvastatin treatment on P450 expression seen in primary cultured rat hepatocytes thus largely recapitulated the effects seen in vivo. The differences in effects among the HMG-CoA reductase inhibitors suggest that simple inhibition of HMG-CoA reductase cannot explain all of the effects of these drugs on P450 expression.

Animals↗

Isolation and characterization of complementary DNA to proliferating cell nucleolar antigen P40.

Proliferating cell nucleolar antigen P40 is a late G1-specific protein, which was found in a variety of human tumors (A. Chatterjee, J. W. Freeman, and H. Busch. Cancer Res., 47: 1123-1129, 1987). Two overlapping complementary DNA clones for antigen P40 were isolated by immunoscreening a lambda gt11 human expression library. The complete nucleotide sequence of the clones was determined. The complementary DNAs encode the Mr 30,000 portion of the COOH-terminal portion of the protein. The mRNA for P40 was 2.8 kilobases long and was expressed maximally in G1 cells in cell cycle. A series of deletion mutants of the expressed peptide was constructed and the deletion mutants were expressed in Escherichia coli. Using these mutants, the epitope region of P40 recognized by a P40-specific monoclonal antibody was identified. The hydropathy plot based on the protein sequence revealed that this region of the protein is largely hydrophilic. This protein is unique and differs in sequence from other proliferating cell nuclear/nucleolar antigen proteins of similar molecular weight such as protein B23 and cyclin.

Antibodies, Monoclonal↗

Characterization of mycobacteriophage I8 and its unrelatedness to mycobacteriophages I1, I3 and I5.

Homology among the genomes of mycobacteriophages I1, I3, I5 and I8 has been studied. Based on restriction endonuclease cleavage patterns, dot blot hybridization and Southern blot hybridization analysis, the DNAs of phages I1, I3 and I5 have been shown to be homologous and indistinguishable, but entirely different from phage I8. Unlike the others, the I8 genome does not harbour any single-strand interruptions. The DNA is 43 kb in length with limited cyclic permutations and has a G + C content of 54%. The presence of 5-methylcytosine in I8 DNA was indicated from the restriction patterns of MspI and HpaII. The number of sites and fragment sizes for several restriction enzymes on I8 DNA has been determined. Phage I8 has a replication cycle of 300 min, with a latent period of 180 min, a rise period of 120 min and a burst size of 100. The viability of phage I8 is significantly reduced by treatment with organic solvents.

DNA, Viral↗

Presence of random single-strand gaps in mycobacteriophage I3 DNA.

The genomic double-stranded DNA of mycobacteriophage I3, when denatured with alkali, heat, formamide or dimethylsulfoxide, breaks down to heterogeneous-sized single-strand (ss) fragments smaller than the expected intact unit genome length suggesting the presence of random ss interruptions on both the strands. The occurrence of the interruptions at random is also demonstrated by two-dimensional gel electrophoresis of the restriction fragments of I3 DNA. These interruptions have no adverse effect on the phage infectivity or DNA transfectivity. Studies with nuclease BAL 31 and end-labeling analysis confirm the presence of random interruptions. Detailed analysis using T4 DNA ligase, nuclease S1 and DNA polymerase I Klenow fragment revealed that the interruptions are in the form of small gaps rather than single phosphodiester bond breaks. The average length of the gap is about 10 nucleotides long and there are 13 to 14 such gaps per DNA molecule.

DNA, Single-Stranded↗

Soleus muscle adenosine-5'-triphosphate (ATP) levels in Mastomys natalensis with Dipetalonema viteae infections: effect of diethylcarbamazine.

Adenosine-5'-triphosphate (ATP) levels in the soleus muscles of Mastomys natalensis during the patent phase of Dipetalonema viteae infection were studied. Decreased ATP levels were found in the infected animals as compared with the uninfected controls. Diethylcarbamazine citrate produced an 'anaphylactic reaction' and enhanced microfilaraemia in the infected animals, but did not cause any lowering of the soleus muscle ATP in the infected as well as in the uninfected animals.

Adenosine Triphosphate↗

Microfilarial periodicity in Mastomys natalensis.

The microfilarial level in the peripheral blood of Mastomys natalensis infected with the filarial parasites, Litomosoides carinii, Brugia pahangi, B. malayi and Dipetalonema viteae was monitored at two-hour intervals for 24 hours. The microfilariae of B. pahangi and B. malayi exhibited nocturnal and diurnal subperiodicity, respectively; no such periodicity was seen in L. carinii and D. viteae infections. The level of B. pahangi and D. viteae microfilariae in peripheral blood was significantly increased when the host was anaesthetized with diethylether or pentothal sodium. Ether-induced anaesthesia had no effect on the level of B. malayi microfilariae but pentothal was most effective. The peripheral blood count of L. carinii microfilariae tended to decrease in the anaesthetized animals but the reduction was not statistically significant.

Anesthetics↗

Nasoenteral feeding tube insertion via fiberoptic endoscope for enteral hyperalimentation.

Total parenteral nutrition can maintain good nutritional status in selected patients. However, it can be accompanied by serious complications. It is generally agreed that enteral alimentation is more economical and safer. Gut should be used for nutritional replenishment whenever feasible. However, large-bore nasogastric feeding tubes can cause problems. Even fine-bore nasogastric tubes can cause aspiration pneumonia in obtunded and debilitated patients. In some patients it is clearly desirable to have the tip of the feeding tube in the distal duodenum or proximal jejunum. Previously described methods for placement of nasoenteral tubes may be unsatisfactory. We describe a safe, simple, and reliable method for endoscopic insertion of fine-bore nasoenteral feeding tube. We have used this method on 15 patients without complication.

Aged↗