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Y Berman

Publications and source records attributed to Y Berman.

12 recordsLinked to original sources
PubMed2000

Defective prodynorphin processing in mice lacking prohormone convertase PC2.

Prodynorphin, a multifunctional precursor of several important opioid peptides, is expressed widely in the CNS. It is processed at specific single and paired basic sites to generate various biologically active products. Among the prohormone convertases (PCs), PC1 and PC2 are expressed widely in neuroendocrine tissues and have been proposed to be the major convertases involved in the biosynthesis of hormonal and neural peptides. In this study we have examined the physiological involvement of PC2 in the generation of dynorphin (Dyn) peptides in mice lacking active PC2 as a result of gene disruption. Enzymological and immunological assays were used to confirm the absence of active PC2 in these mice. The processing profiles of Dyn peptides extracted from brains of these mice reveal a complete lack of Dyn A-8 and a substantial reduction in the levels of Dyn A-17 and Dyn B-13. Thus, PC2 appears to be involved in monobasic processing, leading to the generation of Dyn A-8, Dyn A-17, and Dyn B-13 from prodynorphin under physiological conditions. Brains of heterozygous mice exhibit only half the PC2 activity of wild-type mice; however, the levels of Dyn peptides in these mice are similar to those of wild-type mice, suggesting that a 50% reduction in PC2 activity is not sufficient to significantly reduce prodynorphin processing. The disruption of the PC2 gene does not lead to compensatory up-regulation in the levels of other convertases with similar substrate specificity because we find no significant changes in the levels of PC1, PC5/PC6, or furin in these mice as compared with wild-type mice. Taken together, these results support a critical role for PC2 in the generation of Dyn peptides.

Animals↗
PubMed1999

Specificity of the dynorphin-processing endoprotease: comparison with prohormone convertases.

The cleavage specificity of a monobasic processing dynorphin converting endoprotease is examined with a series of quench fluorescent peptide substrates and compared with the cleavage specificity of prohormone convertases. A dynorphin B-29-derived peptide, Abz-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-Arg-Ser-Glneddnp (where Abz is o-aminobenzoyl and eddnp is ethylenediamine 2,4-dinitrophenyl), that contains both dibasic and monobasic cleavage sites is efficiently cleaved by the dynorphin converting enzyme and not cleaved by two propeptide processing enzymes, furin and prohormone convertase 1. A shorter prorenin-related peptide, Dnp-Arg-Met-Ala-Arg-Leu-Thr-Leu-eddnp, that contains a monobasic cleavage site is cleaved by the dynorphin converting enzyme and prohormone convertase 1 and not by furin. Substitution of the P1' position by Ala moderately affects cleavage by the dynorphin-processing enzyme and prohormone convertase 1. It is interesting that this substitution results in efficient cleavage by furin. The site of cleavage, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry, is N-terminal to the Arg at the P1 position for the dynorphin converting enzyme and C-terminal to the Arg at the P1 position for furin and prohormone convertase 1. Peptides with additional basic residues at the P2 and at P4 positions also serve as substrates for the dynorphin converting enzyme. This enzyme cleaves shorter peptide substrates with significantly lower efficiency as compared with the longer peptide substrates, suggesting that the dynorphin converting enzyme prefers longer peptides that contain monobasic processing sites as substrates. Taken together, these results suggest that the cleavage specificity of the dynorphin converting enzyme is distinct but related to the cleavage specificity of the prohormone convertases and that multiple enzymes could be involved in the processing of peptide hormones and neuropeptides at monobasic and dibasic sites.

Aspartic Acid Endopeptidases↗
PubMed1999

Dynorphin A processing enzyme: tissue distribution, isolation, and characterization.

Limited proteolysis of the dynorphin precursor (prodynorphin) at dibasic and monobasic processing sites results in the generation of bioactive dynorphins. In the brain and neurointermediate lobe of the pituitary, prodynorphin is processed to produce alpha and beta neo endorphins, dynorphins (Dyn) A-17 and Dyn A-8, Dyn B-13, and leucine-enkephalin. The formation of Dyn A-8 from Dyn A-17 requires a monobasic cleavage between Ile and Arg. We have identified an enzymatic activity capable of processing at this monobasic site in the rat brain and neurointermediate lobe of the bovine pituitary; this enzyme is designated "dynorphin A-17 processing enzyme." In the rat brain and neurointermediate lobe, a majority of the Dyn A processing enzyme activity is membrane-associated and can be released by treatment with 1% Triton X-100. This enzyme has been purified to apparent homogeneity from the membrane extract of the neurointermediate lobe using preparative iso-electrofocussing in a granulated gel pH 3.5 to 10, FPLC using anion exchange chromatography, and non-denaturing electrophoresis. The Dyn A processing enzyme exhibits a pI of about 5.8 and a molecular mass of about 65 kDa under reducing conditions. The Dyn A processing enzyme is a metalloprotease and has a neutral pH optimum. It exhibits substantial sensitivity to metal chelating agents and thiol agents suggesting that this enzyme is a thiol-sensitive metalloprotease. Specific inhibitors of other metallopeptidases such as enkephalinase [EC 3.4.24.11], the enkephalin generating neutral endopeptidase [EC 3.4.24.15], or NRD convertase do not inhibit the Dyn A processing enzyme activity. In contrast, specific inhibitors of angiotensin converting enzyme inhibit the activity. The purified enzyme is able to process a number of neuropeptides at both monobasic and dibasic sites. These characteristics are consistent with a role for the Dyn A processing enzyme in the processing of Dyn A-17 and other neuropeptides in the brain.

Amino Acid Sequence↗
PubMed1998

Bax cleavage is mediated by calpain during drug-induced apoptosis.

The anti-apoptotic molecule Bcl-2 is located in the mitochondrial and endoplasmic reticulum membranes as well as the nuclear envelope. Although its location has not been as rigorously defined, the pro-apoptotic molecule Bax appears to be mainly a cytosolic protein which translocates to the mitochondria upon induction of apoptosis. Here we identify a protease activity in mitochondria-enriched membrane fractions from HL-60 cells capable of cleaving Bax which is absent from the cytosolic fraction. Bax protease activity is blocked in vitro by cysteine protease inhibitors including E-64 which distinguishes it from all known caspases and granzyme B, both of which are involved in apoptosis. Protease activity is also blocked by inhibitors against the calcium-activated neutral cysteine endopeptidase calpain. Partial purification of the Bax protease activity from HL-60 cell membrane fractions by column chromatography revealed that a calpain-like activity was the protease responsible for Bax cleavage. In addition, purified calpain enzymes cleaved Bax in a calcium-dependent manner. Pretreatment of HL-60 cells with the specific calpain inhibitor calpeptin effectively blocked both drug-induced Bax cleavage and calpain activation, but not PARP cleavage or cell death. These results suggest that calpains and caspases are activated during drug-induced apoptosis and that calpains, along with caspases, may be involved in modulating cell death by acting selectively on cellular substrates.

Alanine↗
PubMed1998

Instability of the amyloidogenic cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type.

A cystatin C variant with L68Q substitution and a truncation of 10 NH2-terminal residues is the major constituent of the amyloid deposited in the cerebral vasculature of patients with the Icelandic form of hereditary cerebral hemorrhage with amyloidosis (HCHWA-I). Variant and wild type cystatin C production, processing, secretion, and clearance were studied in human cell lines stably overexpressing the cystatin C genes. Immunoblot and mass spectrometry analyses demonstrated monomeric cystatin C in cell homogenates and culture media. While cystatin C formed concentration-dependent dimers, the HCHWA-I variant dimerized at lower concentrations than the wild type protein. Amino-terminal sequence analysis revealed that the variant and normal proteins produced and secreted are the full-length cystatin C. Pulse-chase experiments demonstrated similar levels of normal and variant cystatin C production and secretion. However, the secreted variant cystatin C exhibited an increased susceptibility to a serine protease in conditioned media and in human cerebrospinal fluid, explaining its depletion from the cerebrospinal fluid of HCHWA-I patients. Thus, the amino acid substitution may induce unstable cystatin C with intact inhibitory activity and predisposition to self-aggregation and amyloid fibril formation.

Amino Acid Sequence↗
PubMed1997

Effects of an intrahypothalamic injection of antisense oligonucleotides for preproenkephalin mRNA in female rats: evidence for opioid involvement in lordosis reflex.

Previous studies in female rats have shown that estrogen increases preproenkephalin (PPE) mRNA levels in the ventrolateral part of the ventromedial nucleus of the hypothalamus (VMHVL), an area implicated in the modulation of sexual behavior. In order to assess the physiological role of hypothalamic opioid expression in lordosis reflex 16-mer oligodeoxynucleotide (ODN) directed towards the PPE mRNA were acutely microinjected above the VMH of estradiol-primed ovariectomized rats. Estradiol-induced lordosis behavior was observed in response to a stud male 2 days thereafter. Antisense (without or with 4 mismatches) ODN injections near the VMHVL resulted in a significant reduction in lordosis quotient compared to control (reverse sense) ODN treatment or to antisense ODN injections targeted anterior or posterior to the VMHVL. In contrast, locomotor activity of these animals in the open-field test was not affected by ODN treatments. Enkephalin immunoreactive levels were determined by radioimmunoassay in the preoptic area, a major terminal field of the VMHVL. Estradiol-induced enkephalin levels were greatly reduced in antisense-treated groups. Using the in situ hybridization technique, PPE mRNA levels in the VMHVL were also determined. A 1.5-2-fold increase in PPE mRNA levels was observed in estradiol-treated rats compared to ovariectomized rats as previously described. This increase in PPE mRNA levels was not affected by ODN treatment, suggesting that the reduction of enkephalin expression was mainly due to physical blockade of PPE mRNA translation and not to its degradation. Taken together, these data further support the behavioral role of PPE expressing VMHVL neurons. They also highlight the in vivo potency of acute administration of antisense phosphorothioate ODNs in blocking neuronal target gene expression.

Amino Acid Substitution↗
PubMed1997

Chronic food restriction and streptozotocin-induced diabetes differentially alter prodynorphin mRNA levels in rat brain regions.

It was previously reported that chronic food restriction and streptozotocin-induced diabetes lead to brain region-specific changes in levels of Prodyn-derived peptides. These changes parallel behavioral adaptations that are reversed by opioid antagonists. In the present study, effects of food restriction and diabetes on Prodyn gene expression were measured in rat brain regions using a quantitative solution hybridization mRNA assay. Picogram amounts of Prodyn mRNA were determined in extracts of five brain regions. The highest density of Prodyn mRNA was observed in extracts of nucleus accumbens (4.68 pg/microg total RNA), bed nucleus of the stria terminalis (4.18 pg/microg), and in caudate nucleus (3.51 pg/microg). Lower levels were observed in the lateral hypothalamus (1.87 pg/microg) and central nucleus of the amygdala (1.22 pg/microg). Food restriction and diabetes both markedly increased the levels of Prodyn mRNA in the central amygdala (163% and 93%, respectively). Levels in the lateral hypothalamus were also increased (35% and 29%, respectively), though only the food-restriction effect was statistically significant. Neither treatment altered prodynorphin mRNA levels in the caudate nucleus, nucleus accumbens or bed nucleus of the stria terminalis. These results suggest that dynorphin neurons in central amygdala and lateral hypothalamus may be involved in behavioral or physiological adaptations to sustained metabolic need.

Animals↗
PubMed1995

Curve-shift analysis of self-stimulation in food-restricted rats: relationship between daily meal, plasma corticosterone and reward sensitization.

Chronic food restriction lowers the threshold for lateral hypothalamic electrical self-stimulation (LHSS). This effect has previously been interpreted to reflect a sensitization of reward. In the present study a curve-shift method was used to explicitly differentiate effects of food restriction on brain stimulation rewarding efficacy and performance. Food restriction consistently shifted rate-frequency curves to the left, lowering the M-50 and Theta-0 parameters of rewarding efficacy. Asymptotic rates of reinforcement and slopes of rate-frequency functions were unaffected, confirming that food restriction does not facilitate LHSS by enhancing performance. In this and previous studies, LHSS in food-restricted rats was measured in the period immediately preceding the daily meal when hunger (i.e., period since last meal) and plasma corticosterone are at peak levels. In the light of evidence that corticosterone may regulate sensitivity of the mesolimbic dopamine pathway and account for the sensitizing effect of stress on psychomotor effects of opiates and stimulants, LHSS and corticosterone were measured in the immediate pre-and post-meal periods. While all food-restricted rats displayed elevated corticosterone levels in the pre-meal period and generally displayed a decline to control levels in the post-meal period, the sensitization of reward was not reversed in the post-meal period. These results indicate that chronic food restriction produces a sensitization of reward that does not depend upon the acute state of hunger that precedes the daily meal and does not vary with dynamic changes in plasma corticosterone level.

Animals↗
PubMed1995

Effects of streptozotocin-induced diabetes on prodynorphin-derived peptides in rat brain regions.

Pharmacological studies suggest that diabetes produces changes in the brain opioid system, affecting several behavioral functions including analgesia, feeding and self-stimulation. Previous investigations of opioid receptor binding have failed to explain the unusual opioid pharmacology of the diabetic animal. In the present study, the effects of streptozotocin-induced diabetes on levels of three immunoreactive (ir)-prodynorphin-derived peptides, ir-dynorphin A1-17 (A1-17), ir-dynorphin A1-8 (A1-8) and ir-dynorphin B1-13 (B1-13), were determined in eleven brain regions known to be involved in appetite, taste and reward. Diabetes was found to increase levels of A1-17 in the ventromedial and dorsomedial hypothalamic nuclei (+60% and +25%, respectively) and levels of A1-8 in the dorsomedial and lateral hypothalamus (+45% and +35%, respectively). The possible significance of these results is discussed in relation to (i) diabetic hyperphagia, (ii) medial hypothalamic transduction of circulating insulin levels, and (iii) the potentiation of reward by metabolic need states.

Animals↗
PubMed1994

Effects of chronic food restriction on prodynorphin-derived peptides in rat brain regions.

Chronic food restriction produces a variety of physiological and behavioral adaptations including a potentiation of the reinforcing effect of food, drugs and lateral hypothalamic electrical stimulation. Previous work in this laboratory has revealed that the lowering of self-stimulation threshold by food restriction is reduced by mu- and kappa-selective opioid antagonists. In the present study, the effect of chronic food restriction on levels of three prodynorphin-derived peptides, namely dynorphin A1-17 (A1-17), dynorphin A1-8 (A1-8) and dynorphin B1-13 (B1-13) were measured in eleven brain regions known to be involved in appetite, taste and reward. Food restriction increased levels of A1-17 in dorsal medial (+19.6%), ventral medial (+24.2%) and medial preoptic (+82.9%) hypothalamic areas. Levels of A1-17 decreased in the central nucleus of the amygdala (-35.1%). Food restriction increased levels of A1-8 in nucleus accumbens (+34.4%), bed nucleus of the stria terminalis (+24.5%) and lateral hypothalamus (+41.9%). Food restriction had no effect on levels of B1-13. A1-17 is highly kappa-preferring and the brain regions in which levels increased all have a high ratio of kappa: mu and delta receptors. A1-8 is less discriminating among opioid receptor types and the brain regions in which levels increased have a low ratio of kappa: mu and delta receptors. The present results suggest that food restriction alters posttranslational processing within the dynorphin A domain of the prodynorphin precursor, possibly leading to a change in the balance between kappa and non-kappa opioid receptor stimulation in specific brain regions.

Animals↗