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Biomedical subjects

A S Levine

Publications and source records attributed to A S Levine.

At least 19 recordsLinked to original sources
PubMed1992

Escherichia coli umuDC mutants: DNA sequence alterations and UmuD cleavage.

The products of the chromosomally encoded umuDC genes are directly required for mutagenesis in Escherichia coli. Strains with either umuD or umuC mutations are rendered phenotypically non-mutable. To ascertain the molecular basis of this non-mutability, we determined the DNA sequence alterations of seven chromosomal umuDC mutants. Six mutants (umuD1, umuD44, umuD77, umuC36, umuC25, and umuC104) were found to be single base-pair substitutions that resulted in missense mutations. The Tn5 transposon insertion mutation (umuC122) resulted in a missense mutation followed immediately by a termination codon, producing a truncated UmuC protein lacking 102 carboxyl-terminal amino acids. All of the mutations were found to reside in regions of the UmuD and UmuC proteins that share high homology with analogous proteins. Chemiluminescent immunoassays revealed that the umuD1, umuD44, and umuD77 mutations all resulted in a non-cleavable UmuD protein. Because UmuD cleavage is a prerequisite for mutagenesis, the lack of UmuD processing appears to be the molecular basis for the non-mutable phenotype in these strains. These studies re-emphasize the critical nature of the RecA-mediated cleavage of UmuD for inducible mutagenesis and provide insights into the functional domains of the UmuC protein.

Bacterial Proteins↗
PubMed1992

SV40 small t deletion mutants preferentially transform mononuclear phagocytes and B lymphocytes in vivo.

Hamsters injected intracardiacally with wild-type SV40 developed diverse tumors, with no one type predominating. However, hamsters injected with small t deletion mutants of SV40 uniformly developed true histiocytic or B-cell lymphomas, both of which arise from rapidly dividing cell populations, with very few tumors of other types. Our results are consistent with in vitro studies which suggest a requirement for the small t function in SV40 transformation of nondividing cells.

Animals↗
PubMed1992

Effects of methadone on free feeding in satiated rats.

A variety of opioids and opiates are known to increase short-term food intake. In the present study, we evaluated the effects of methadone on free feeding in satiated rats. We assessed the effect of methadone (0, 1.5, 3.0, 5.0, and 10.0 mg/kg) on food intake 1, 2, 4, and 6 h after injection for 3 consecutive days. Two hours after methadone administration, food intake was inversely related to dose, but after 6 h a direct relationship between dose and feeding was obtained. Food intake increased with repeated methadone administration. In Experiment 2, methadone (5.0 mg/kg) was injected and food was made available 0, 1, 2, or 3 h later. Maximal food intake occurred in the third and fourth hours following methadone administration. As in Experiment 1, food intake increased with repeated methadone administration. Increases in food intake following repeated methadone administration may have been due to the development of tolerance to effects of methadone that may interfere with feeding, such as sedation. In Experiment 3, methadone was administered daily or every fifth day, assuming that spacing injections would retard tolerance development. Repeated daily methadone administration was associated with increased food intake earlier in the session, whereas increases in food intake following spaced methadone administration occurred later in the session. These data indicate that methadone increases short-term feeding in satiated rats. This is in contrast to the reported decrease in food-reinforced behavior noted in operant studies. This contrast may be due to sedating or other disabling effects of methadone.

Animals↗
PubMed1992

Effects of neuropeptide Y on food-reinforced behavior in satiated rats.

The effect of NPY on behavior and food intake of food-satiated rats was examined under three different food availability conditions. Food was available during times when rats normally do not eat under either a fixed-ratio or fixed-interval reinforcement schedule, or it was freely available in the bottom of the cage (FF). Forty responses were required for each 45-mg food pellet under the ratio schedule (FR 40) and for the first response to occur 15 s after the previous reinforcement under the interval schedule (FI 15"). NPY (5 micrograms) significantly increased food intake under all conditions and increased food-reinforced responses under the FR and FI schedules. NPY's effect on food intake was greatest when food was freely available and least for rats working under the schedule requiring the most effort (FR 40). Food intake peaked after 3 days under repeated daily administration of NPY. Under free food access and under the fixed-interval schedule, eating and/or responding occurred almost immediately following the onset of the initial 4-h session under NPY. However, during the first session following NPY administration under the FR, rats emitted few responses during the first 2 h of the session. The onset of robust responding under the FR schedule began earlier with each successive daily administration of NPY. These data show NPY substantially increases food-maintained behavior and is a potent inducer of food intake even under conditions where considerable effort is required to obtain food. Further, the conditions under which food is made available can dramatically alter NPY's effect on the temporal pattern of food-maintained responding, feeding, and latency to eat.

Animals↗
PubMed1992

HeLa cell single-stranded DNA-binding protein increases the accuracy of DNA synthesis by DNA polymerase alpha in vitro.

To determine whether cellular replication factors can influence the fidelity of DNA replication, the effect of HeLa cell single-stranded DNA-binding protein (SSB) on the accuracy of DNA replication by HeLa cell DNA polymerase alpha has been examined. An in vitro gap-filling assay, in which the single-stranded gap contains the supF target gene, was used to measure mutagenesis. Addition of SSB to the in vitro DNA synthesis reaction increased the accuracy of DNA polymerase alpha by 2- to 8-fold. Analysis of the products of DNA synthesis indicated that SSB reduces pausing by the polymerase at specific sites in the single-stranded supF template. Sequence analysis of the types of errors resulting from synthesis in the absence or presence of SSB reveals that, while the errors are primarily base substitutions under both conditions, SSB reduces the number of errors found at 3 hotspots in the supF gene. Thus, a cellular replication factor (SSB) can influence the fidelity of a mammalian DNA polymerase in vitro, suggesting that the high accuracy of cellular DNA replication may be determined in part by the interaction between replication factors, DNA polymerase and the DNA template in the replication complex.

Bacterial Proteins↗
PubMed1992

Hypothalamic neuropeptide Y regulation of feeding and energy metabolism.

Neuropeptide Y is an important regulator of energy intake and expenditure. The central portion of this regulatory system appears to reside in the arcuate nucleus/paraventricular nucleus of the hypothalamus. The effects of neuropeptide Y on energy metabolism include increased food intake, decreased thermogenesis and increased white fat storage.

Animals↗
PubMed1992

The enhanced mutagenic potential of the MucAB proteins correlates with the highly efficient processing of the MucA protein.

Inducible mutagenesis in Escherichia coli requires the direct action of the chromosomally encoded UmuDC proteins or functional homologs found on certain naturally occurring plasmids. Although structurally similar, the five umu-like operons that have been characterized at the molecular level vary in their ability to enhance cellular and phage mutagenesis; of these operons, the mucAB genes from the N-group plasmid pKM101 are the most efficient at promoting mutagenesis. During the mutagenic process, UmuD is posttranslationally processed to an active form, UmuD'. To explain the more potent mutagenic efficiency of mucAB compared with that of umuDC it has been suggested that unlike UmuD, intact MucA is functional for mutagenesis. To examine this possibility, we have overproduced and purified the MucA protein. Although functionally similar to UmuD, MucA was cleaved much more rapidly both in vitro and in vivo than UmuD. In vivo, restoration of mutagenesis functions to normally nonmutable recA430, recA433, recA435, or recA730 delta(umuDC)595::cat strains by either MucA+ or mutant MucA protein correlated with the appearance of the cleavage product, MucA'. These results suggest that most of the differences in mutagenic phenotype exhibited by MucAB and UmuDC correlate with the efficiency of posttranslational processing of MucA and UmuD rather than an inherent activity of the unprocessed proteins.

Bacterial Proteins↗
PubMed1992

Simian virus 40 (SV40) small t antigen inhibits SV40 DNA replication in vitro.

We describe a biochemical function of simian virus 40 small t antigen, the inhibition of simian virus 40 large T antigen-mediated viral DNA replication in an in vitro replication system. Our results suggest that in this system, small t antigen prevents protein phosphatase 2A-mediated activation of large T antigen.

Antigens, Polyomavirus Transforming↗
PubMed1991

Central administration of the opioid antagonist, LY255582, decreases short- and long-term food intake in rats.

A variety of opioid antagonists have been reported to decrease short-term food intake, but few appear to reduce long-term intake. In the present study we evaluated the effect of a relatively new class of opioid antagonists, 3,4-dimethyl-4-phenylpiperidines, on short-term and long-term food intake after central administration. We also evaluated their affinities for the mu and kappa opioid receptor sites in synaptosomal membranes derived from rat whole brain tissue (minus cerebellum) and guinea-pig cortex, respectively. The affinities for the mu receptor sites were LY255582 greater than LY217273 greater than LY256897 greater than naloxone greater than LY227444. The affinities for the kappa receptor sites were LY255582 greater than LY256897 = LY217273 greater than LY227444. LY255582 reduced food intake for up to 24 h after a single intraventricular injection. Doses as low as 1 microgram of LY255582 decreased food intake for up to 4 h. All other drugs were much less powerful. Naloxone and LY256897 only decreased food intake after injection of the 100 microgram dose. LY227444 and LY217273 failed to decrease intake at all doses tested. LY255582 (100 micrograms) decreased food intake over a 7 day period when injected intraventricularly once per day. The body weight of the rats also decreased during the 7 day period. Upon cessation of drug administration body weights and food intake approached control levels. Thus, LY255582 appears to be a very potent and long-acting anorectic agent which may be useful in the treatment of obesity. The mu and kappa binding profile of the phenylpiperidines does not seem to clearly correlate with their anorectic activity.

Animals↗
PubMed1991

Purification of an ultraviolet-inducible, damage-specific DNA-binding protein from primate cells.

A UV-inducible, damage-specific DNA-binding (DDB) protein with high affinity for double-stranded UV-irradiated DNA has been identified recently in monkey kidney (CV-1) cells (Hirschfeld, S., Levine, A. S., Ozato, K., and Protić, M. (1990) Mol. Cell. Biol. 10, 2041-2048). We have now purified the DDB protein from extracts of CV-1 cells using hydroxylapatite, phosphocellulose, Mono S, and DNA-affinity column chromatography. The DDB activity, either from mock-treated or UV-induced cells, is heterodisperse in column chromatography, and separation of three forms of the protein was obtained on a phosphocellulose column. Analysis of purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that greater than 90% of all three forms is a protein of approximately 126 kDa. The size of the native DDB protein was deduced from gel filtration and native polyacrylamide gel electrophoresis to be approximately 210 kDa, which suggests that the native DDB protein in solution is a homodimer. Preparations of partially purified DDB protein from UV-treated cells have enhanced levels of DDB activity and the protein when compared with similar preparations from mock-treated cells. This damage-recognition protein, alone or in conjunction with other subunits, may be of general importance for the initial recognition of DNA damage in mammals.

Animals↗
PubMed1991

Beta-funaltrexamine (beta-FNA) decreases deprivation and opioid-induced feeding.

We studied the effect of the mu antagonist, beta-funaltrexamine (beta-FNA) on deprivation and opioid-induced feeding. Intracerebroventricular pre-treatment of 20 h deprived rats with 0.1, 1, 10 and 20 nmol of beta-FNA decreased feeding by 24%, 50%, 50% and 38% during the first hour. Central administration of beta-FNA (0.1, 1 and 10 nmol) also decreased feeding induced by the mu opioid agonist, DAMGO by 57%, 60% and 71%. Feeding induced by the delta agonist, DSLET, was decreased by pre-treatment with beta-FNA; but only during the 1-2 h time points, a time when relatively little food was ingested. Intraventricular injection of beta-FNA failed to alter feeding stimulated by the kappa opioid agonist, U-50,488H. These data further substantiate a role for the opioid receptor in deprivation and opioid-induced feeding.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1991

The discriminative stimulus effects of neuropeptide Y.

Neuropeptide Y (NPY), an endogenous peptide which strongly induces food intake, is demonstrated to have discriminative stimulus properties when administered intracerebroventricularly. Rats rapidly learned to press the appropriate lever during training. NPY discrimination was dose-dependent. NPY's discriminative stimulus properties were compared to those of two doses of Peptide YY (PYY) and 24 and 48 h of food deprivation, conditions which also increase feeding. Both doses of PYY generalized to NPY, supporting previous findings that PYY has effects similar to NPY. Although food deprivation increases feeding in a manner similar to NPY, food deprivation did not result in NPY-appropriate responding.

Animals↗
PubMed1991

Induction and cleavage of Salmonella typhimurium UmuD protein.

SOS mutagenesis in prokaryotes is dependent upon the inducible activity of the chromosomally encoded UmuDC proteins, or homologous proteins such as MucAB or ImpCAB which are found on naturally occurring plasmids. Relative to Escherichia coli, however, Salmonella typhimurium is much less responsive to the mutagenic effects of DNA-damaging agents, despite the fact that it possesses both chromosomally and plasmid encoded umu-like operons. In E. coli, activation of the UmuD mutagenesis protein to UmuD' via RecA-mediated proteolysis is a critical step in the mutation fixation pathway. We have used a polyclonal antiserum raised against the E. coli UmuD and UmuD' proteins to show that S. typhimurium expresses cross-reacting material only after treatment with the DNA-damaging agent mitomycin C. The S. typhimurium umuDC operon, therefore, appears to be regulated by mechanisms similar to the E. coli umuDC operon. After induction, the S. typhimurium UmuD protein was processed to UmuD' in both S. typhimurium and E. coli. However, the S. typhimurium UmuD protein appears to be cleaved more efficiently than the E. coli UmuD protein under similar conditions. The data suggest that conversion of UmuD to the mutagenically active UmuD' is not the rate-limiting factor accounting for the weakly mutable phenotype of S. typhimurium.

Bacterial Proteins↗
PubMed1991

The effect of cholecystokinin-octapeptide on food intake and consummatory behavior in lactating rats.

Rats are less sensitive to the satiating effect of CCK-8 during some reproductive states such as estrus and proestrus, and in ovariectomized rats following the administration of estradiol and progesterone. The sensitivity of rats to CCK-8's effect on food intake decreases as lactation progresses. During lactation, prolactin and progesterone levels are elevated. Implantation of ectopic pituitaries increases prolactin levels in males and females as well as progesterone levels in females. To evaluate whether or not prolactin elevation modifies CCK's effect on feeding, we studied the effect of CCK-8 on food intake during the early dark cycle in male and female rats implanted with ectopic pituitaries. As previously demonstrated, prolactin levels were elevated in both male and female pituitary-implanted rats and progesterone levels were elevated in the female rats. CCK-8 inhibited food intake in sham-operated male rats, but did not reliably decrease early dark cycle food intake in sham-operated or pituitary-implanted female rats or pituitary-implanted male rats. Thus an elevation in prolactin levels does not appear to modify the effect of CCK-8 on food intake in female rats. We also evaluated the effect of CCK on consummatory and maternal behavior in lactating rats. CCK-8 altered the meal patterns of lactating rats primarily by decreasing the rate of food consumption and increasing the latency to the first meal. The latency to the first meal of rats receiving CCK was increased during early and mid-lactation and the PW period, but not during late lactation compared to that of the saline-injected rats. CCK-8 did not modulate any of the maternal behaviors studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Diazepam-induced feeding in captive gray wolves (Canis lupus).

Diazepam doses of 0.2, 0.4, and 0.8 mg/kg induced feeding in sated gray wolves in a dose-dependent manner (p less than 0.001). Neither 0.8 mg/kg of the benzodiazepine antagonist, beta-CCP (p = 0.36), nor 0.8 mg/kg of the benzodiazepine inverse agonist, beta-CCE (p = 0.85), decreased the diazepam-induced hyperphagia. Five of 6 naive wolves (p = 0.003) ate dry dog food within 15.4 +/- 1.9 min of being given 0.4 mg/kg diazepam and freely chose dog food after the single diazepam administration.

Animals↗
PubMed1991

Effects of intracerebroventricular injection of neuropeptide Y on energy metabolism.

Our objective was to find out if central injection of neuropeptide Y (NPY) would alter brown fat thermogenesis and white fat lipoprotein lipase activity. The following three groups of Sprague-Dawley rats received five injections over 24 h into the right lateral ventricle: 1) NPY (5 micrograms/injection) and ad libitum food; 2) NPY (5 micrograms/injection) and food restricted to control intake; 3) saline injection and ad libitum food. The NPY ad libitum-fed group consumed more food than the saline controls or NPY food-restricted animals. Brown fat thermogenic activity, assessed by GDP binding, was decreased relative to saline controls in both NPY-treated groups. White fat lipoprotein lipase activity was greatly increased in both NPY treatment groups compared with saline controls. The NPY effects on brown and white fat were not explained by measures of serum insulin, glucagon, glucose, or other metabolites. In a follow-up experiment, we asked whether food was necessary for expression of the NPY effects. Brown fat mitochondrial GDP binding indicated NPY effect even when no food was ingested. We conclude that intracerebroventricular administration of NPY promotes white fat lipid storage and decreases brown fat thermogenesis in addition to its known effect of stimulating food intake.

Adipose Tissue↗
PubMed1991

Food deprivation-induced vs. drug-induced feeding: a behavioral evaluation.

Several neuroactive substances including neuropeptide Y (NPY), muscimol, and norepinephrine (NE) stimulate feeding in satiated rats. In the present study, we observed the behavioral patterns of rats stimulated to eat by food deprivation or by intracerebroventricular (icv) injection of orexigenic agents to explore the hypothesis that such agents produce a behavioral state resembling hunger. Animals that were food deprived for 24 h spent the majority of their time eating (35%), drinking (5%), resting (44%), and moving (13%) when food was available. If food was removed and substituted with a chewable substrate (plastic tube), they chewed on tubes for a brief period (5%) but spent most of their time moving (14%) or resting (77%). In the absence of food or tubes, they briefly moved about the cage (4%) and spent almost all of their time resting (94%). The patterns observed with the orexigenic drugs were different, particularly in the absence of food. NPY-injected rats were more active than deprived rats, spending 22% of their time moving in the presence of food, 47% in the presence of tubes, and 37% in the absence of food or tubes. Rats injected with muscimol demonstrated a marked increase in the time spent chewing and eating. These rats spent 67% of their time eating in the presence of food and chewed 25% of the time in the absence of either food or tubes. NE-injected rats also chewed when tubes were present (17%) or when no food or tubes were present (10%). Lag sequential analysis further documented differences in behavioral patterns amongst the various treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗