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Biomedical subjects

A Berkovich

Publications and source records attributed to A Berkovich.

At least 19 recordsLinked to original sources
PubMed1998

Molecular mechanisms of antioxidant enzyme expression in lung during exposure to and recovery from hyperoxia.

Manganese superoxide dismutase (MnSOD) activity falls approximately 50% in lung during 48 h of exposure of adult rats to > 95% O2 (L. B. Clerch and D. Massaro. J. Clin. Invest. 91: 499-508, 1993). We now show that hyperoxia also decreased MnSOD activity in lungs of adult baboons, making the phenomenon potentially more important to humans. In rats, a decrease in lung MnSOD activity during an initial 48 h of exposure to > 95% O2 and its increase during an immediately subsequent 24 h in air were due to decreases and increases, respectively, in MnSOD specific activity and synthesis rate; the latter was due to altered translational efficiency. The concentration in the lung of copper-zinc superoxide dismutase mRNA, catalase mRNA, and glutathione peroxidase mRNA, unchanged during the initial 48 h of exposure to O2, rose approximately twofold during reexposure to O2 after 24 h in air. The demonstration that the fall in MnSOD activity is translationally and posttranslationally regulated during the initial exposure to hyperoxia suggests that gene transfer to increase MnSOD activity in hyperoxic lungs may also require therapy that maintains translational efficiency and MnSOD specific activity.

Animals↗
PubMed1996

Pertussis toxin alters the concentration and turnover of manganese superoxide dismutase in rat lung.

Treatment of rats with pertussis toxin (PTX) decreases the activity of manganese superoxide dismutase (Mn-SOD) in the lung and results in oxygen toxicity in air (L. B. Clerch, G. Neithardt, U. Spencer, J. A. Melendez, G. D. Massaro, and D. Massaro. J. Clin. Invest. 93: 2482-2489, 1994). To examine aspects of the mechanism of the PTX-induced fall in Mn-SOD activity, we injected rats with PTX (50 micrograms/kg), killed the rats 72 h later, and measured the activity, concentration, specific activity, and turnover of Mn-SOD in the lung. Treatment with PTX caused an approximately 50% fall in Mn-SOD activity and Mn-SOD concentration but no change in Mn-SOD specific activity. PTX also caused an increase in Mn-SOD mRNA concentration, a fall in Mn-SOD synthesis, and an increase in the half-life of Mn-SOD and general proteins. We conclude the PTX-induced low concentration of Mn-SOD is due to a decrease of translational efficiency. We suggest that, under normoxic conditions, signal transduction via heterotrimeric guanine nucleotide binding proteins regulates the expression of Mn-SOD at the level of translation and Mn-SOD degradation.

Animals↗
PubMed1995

Inhibitory effect of ODN, a naturally occurring processing product of diazepam binding inhibitor, on secretagogues-induced insulin secretion.

Diazepam binding inhibitor (DBI1-86) is a peptide that is present in large amounts in the intestine and pancreas and which inhibits glucose-stimulated insulin release from both perfused pancreas and isolated islets in low nanomolar concentrations. Here, DBI33-50 (also known as ODN, octadecaneuropeptide), one of the naturally occurring processing products of DBI1-86, and certain synthetic modified derivatives, have been shown to inhibit glucose and glibenclamide-stimulated insulin secretion from isolated rat islets and glibenclamide-stimulated insulin secretion from hamster-insulinoma (HIT-T15) beta-cell line. DBI17-50 (TTN; triakontatetraneuropeptide), another prominent processing product of DBI, had no effect. The 50% inhibitory concentration (IC50) for the effect of ODN on insulin secretion induced by 8.3 of 16.7 mM glucose was approximately the same: 5 to 6 nM. Moreover, ODN inhibited insulin release induced by 0.01 or 1 microM glibenclamide with a similar IC50 (8 to 10 nM) in both isolated pancreatic islets and in HIT-T15 beta-cells. At concentration up to 1 microM, ODN had no effect on insulin secretion induced by PACAP (pituitary adenylate cyclase polypeptide), BAYK 8644 (methyl-(1,4-dihydro-2,6-dimethyl-3-nitro-4,2-trifluoromethylphenyl) pyridine-5-carboxylate), and only marginally it affected IBMX-(isobutylmethylxanthine) induced insulin secretion. This indicates that ODN does not act directly on ATP-regulated K+ channels, voltage dependent Ca2+ channels or cAMP production. In contrast, ODN inhibited insulin secretion induced by sodium nitroprussiate in a manner that is independent from the presence of extracellular Ca2+. These results suggest that ODN or ODN-like peptide fragments of DBI, may inhibit glucose or glibenclamide-induced insulin secretion via a signaling pathway that regulate the cytoplasmic free Ca2+ concentration.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Topology of two DBI receptors in human lymphocytes.

High-affinity binding sites for the isoquinoline carboxamide PK 11195 and 4'-chlorodiazepam (4'CD) in human lymphocytes are recognized by two putative endogenous ligands: diazepam binding inhibitor (DBI) and protoporphyrin IX. We have now demonstrated that several synthetic DBI peptides--analogues to naturally processed human DBI (H-DBI) fragments--differ from protoporphyrin IX in the manner in which they displace [3H]PK 11195 and [3H]4'CD from binding sites associated with intact and cell-free lymphocyte preparations. In particular, the peptide fragments DBI37-80 and DBI37-70 displaced [3H]PK 11195 and [3H]4'CD with higher affinity from their binding sites on intact lymphocytes (Ki approximately 3-5 microM) than from the sites in the cell-free preparation (Ki approximately 20 microM). In contrast, protoporphyrin IX displaced [3H]PK 11195 and [3H]4'CD with higher affinity in the cell-free preparation (Ki - 0.4 microM) than in intact lymphocytes (Ki > 50 microM). Because DBI peptide fragments and protoporphyrin IX do not readily penetrate the plasma membrane of lymphocytes, our results suggest the existence of binding sites located both on the external face of the plasma membrane and intracellularly. The plasma membrane binding sites, recognized essentially only by DBI fragments, are termed here plasma membrane DBI receptors (PDRs). In contrast, the intracellular binding sites, recognized by both DBI fragments and protoporphyrin IX, are presumably located on mitochondria and are termed mitochondrial DBI receptors (MDRs). Immunohistochemical electronmicroscopic studies with antibodies to the synthetic peptide fragments 62-76 of the rat MDR support the hypothesis that PDRs are expressed on lymphocyte plasma membranes.

Amino Acid Sequence↗
PubMed1991

The role of diazepam binding inhibitor and its processing products at mitochondrial benzodiazepine receptors: regulation of steroid biosynthesis.

The rate-limiting step in the biosynthesis of steroids is the transport of the substrate cholesterol from the outer to the inner mitochondrial membrane, where cholesterol is metabolized to pregnenolone. This transport is markedly stimulated by the action of hormones, such as adrenocorticotropic hormone (ACTH) and luteinizing hormone (LH) for adrenocortical and testicular Leydig cells, respectively. Recently, it was demonstrated that the peripheral-type or mitochondrial benzodiazepine receptor, abundant in steroidogenic tissues, is involved in the regulation of steroid biosynthesis. In search for an endogenous ligand for mitochondrial benzodiazepine receptors, regulating steroidogenesis, the effects of Diazepam Binding Inhibitor (DBI) were studied. The model systems used were the Y-1 adrenocortical and the MA-10 Leydig cell lines, previously shown to be valid steroidogenic models. Both cell lines contain significant levels of immunoreactive DBI. Purified DBI from rat brain, at high nanomolar concentrations, increased formation of pregnenolone, when added to mitochondrial preparations of both cell types; but at concentrations of DBI above 1 microM, a decrease in the stimulation was observed. Flunitrazepam, a benzodiazepine which binds to mitochondrial benzodiazepine receptors, with high nanomolar affinity, inhibited the stimulatory action of DBI on the formation of mitochondrial pregnenolone, indicating that DBI exerts its stimulatory effects through an action on mitochondrial benzodiazepine receptors. In order to determine the biologically active amino acid sequence in the DBI molecule, various fragments of DBI were synthesized and tested; also, peptides structurally unrelated to DBI were tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1991

Diazepam binding inhibitor and its processing products stimulate mitochondrial steroid biosynthesis via an interaction with mitochondrial benzodiazepine receptors.

A recognition site for benzodiazepines structurally different from that linked to various gamma-aminobutyric acid A (GABAA) receptor subtypes is located on the outer mitochondrial membranes of steroidogenic cells. This protein has been signified to be important in the regulation of steroid biosynthesis. Because of its location it is designated herein as the mitochondrial benzodiazepine receptor (MBR). A putative endogenous ligand for MBR is the peptide diazepam binding inhibitor (DBI), previously shown to displace drugs from MBR and to be expressed and stored in steroidogenic cells rich in MBR. The two model systems used to study steroidogenic regulation by DBI were the Y-1 adrenocortical and MA-10 Leydig cell lines previously shown to be applicable in studies of mitochondrial steroidogenesis. Both cell lines contain DBI as well as DBI processing products, including the DBI fragments that on reverse phase HPLC coelute with the naturally occurring triakontatetraneuropeptide [TTN; DBI-(17-50)] and octadecaneuropeptide [DBI-(33-50)]. When DBI purified from rat brain was added to mitochondria prepared from Y-1 and MA-10 cell lines, it increased the rates of pregnenolone formation in a dose-related manner. In both cell lines, maximal stimulation (3-fold) of mitochondrial steroidogenesis was obtained with 0.33 microM DBI, with an EC50 of approximately 0.1 microM. However, DBI concentrations higher than 1 microM caused a smaller increase in pregnenolone formation. Flunitrazepam, a benzodiazepine that binds with high nanomolar affinity to MBR, was recently shown to act as an antagonist of ACTH and LH/hCG-induced steroidogenesis and was found in the present studies to inhibit DBI-stimulated mitochondrial steroidogenesis. During the incubation with mitochondria, DBI was partially processed to different peptide fragments, including octadecaneuropeptide and TTN. To determine whether DBI processing products influence mitochondrial steroid biosynthesis, several DBI fragments and other peptides structurally unrelated to DBI were tested. Among these, only TTN stimulated mitochondrial steroid synthesis in a dose-dependent manner similar to DBI.

Adrenal Cortex↗
PubMed1990

Effect of N-methylation on the modulation by synthetic peptides of the activity of the complement-factor-B-derived serine proteinase CVFBb.

Although they share the active-site catalytic triad of less-specific enzymes such as trypsin and chymotrypsin, the serine proteinases of the complement and coagulation cascades each cleave a highly restricted set of substrates. Peptides with sequences similar to that at which C3 is cleaved by the alternative-pathway complement proteinase CVFBb were synthesized by solid-phase methodology and examined for their effects on the activity of this enzyme as measured by three different types of assays. It was found that a peptide methylated at the scissile bond was a far more effective inhibitor of the cleavage of the protein substrate C5 and of the lysis of guinea-pig erythrocytes by the alternative pathway than was the equivalent unmethylated peptide. Whereas the unmethylated peptide inhibited cleavage of the peptide substrate, the methylated peptide actually stimulated cleavage in this assay. This stimulation was found to be due to a 2.8-fold increase in kcat; the dissociation constant for the substrate was not altered significantly. One model consistent with this behaviour is that the binding of the activator peptide in the extended substrate-recognition region stabilizes a catalytically more active conformation of the active site. A small peptide substrate may have access to such an activated active site, whereas the larger substrate, C5, may be excluded from the site. These results demonstrate that the observed effect of a given compound on activity of an enzyme with an extended substrate-recognition region may depend upon the substrate.

Amino Acid Sequence↗
PubMed1990

DBI (diazepam binding inhibitor): the precursor of a family of endogenous modulators of GABAA receptor function. History, perspectives, and clinical implications.

Biochemical, electrophysiological, and lately, molecular biological techniques have shown that GABAA receptors are heterogeneous supramolecular complexes and can be divided into at least three major subgroups: GABAA1, GABAA2, and GABAA3. They differ mainly in the structural and functional properties of the allosteric modulatory center associated with each one of them. This paper will review the present state of research based on the evidence that DBI (diazepam binding inhibitor) and its natural processing products can selectively modulate GABAergic transmission at different GABAA receptor subtypes. Furthermore, the possibility that the DBI family of peptides represents a novel and meaningful neurochemical correlate for neuropsychiatric pathology, sustained by an alteration of GABAergic transmission, will be discussed.

Animals↗
PubMed1990

A study of diazepam binding inhibitor (DBI) processing products in human cerebrospinal fluid and in postmortem human brain.

Diazepam binding inhibitor (DBI) is a neuropeptide of 11 kDa molecular size and is unevenly distributed in human and rat brain. It appears to function as a negative allosteric modulator of GABAA receptors. In the present paper, using antibodies directed against several synthetic peptides, which correspond to selective regions of human DBI (DBI 51-70, DBI 37-50, DBI 81-101), it is shown that DBI is processed into at least 6 peptide fragments in both postmortem human brain and in cerebrospinal fluid (CSF). One of these fragments was identified as the synthetic DBI 51-70 fragment (an eikosaneuropeptide, ENP) by combined chromatographic procedures. Immunoblotting analysis of the other fragments, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), revealed an apparent molecular size, ranging from 3-4 kDa for four of them and a larger molecular form of 8 kDa. On the basis of the immunological properties, a tentative amino acid sequence was deduced.

Acetates↗
PubMed1990

A natural processing product of rat diazepam binding inhibitor, triakontatetraneuropeptide (diazepam binding inhibitor 17-50) contains an alpha-helix, which allows discrimination between benzodiazepine binding site subtypes.

Synthetic peptides related to triakontatetraneuropeptide (TTN) [17TQPTDEEMLFIYSHFKQATVGDVNTDRPGLLDLK50; diazepam binding inhibitor (DBI) 17-50], a natural brain processing product of rat DBI, were analyzed for their physicochemical and ligand-receptor interaction characteristics. The ability of TTN and TTN-related fragments to displace [3H]flumazenil (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazol[1,5a] [1,4]-benzodiazepine-3-carboxylate) or [3H]Ro 5-4864 [7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1, 4-benzodiazepine-2-one] from their respective benzodiazepine (BZ) binding site subtypes was tested in intact cerebellar culture neurons or in homogenates of cultured astrocytes. These studies indicate that the C-terminal region of TTN, which is also present in DBI 22-50, eicosapentaneuropeptide (DBI 26-50), and octadecaneuropeptide (ODN) (DBI 33-50), but not in DBI 19-41, is essential for interaction with the BZ recognition sites. When the C-terminal lysine of ODN is blocked with an NH2 group, the ability of ODN to interact with the binding of [3H]flumazenil is lost. A comparison analysis of the binding data with the secondary structure characteristics of the peptides demonstrated that TTN (DBI 17-50) and DBI 22-50, which have hydrophobic portions and marked tendencies to produce alpha-helicity, specifically displace (apparent Ki, 5-6 microM) [3H] Ro 5-4864 from astroglial cell binding sites. Peptides (ODN, eicosapentaneuropeptide, OND-NH2) with very low tendencies to form alpha-helices and with virtually no hydrophobic structure were not able to displace Ro 5-4864 at concentrations of up to 100 microM. In contrast, ODN was a good displacer of [3H]flumazenil from intact neurons, with an apparent IC50 of 5 microM. These data suggest that the alpha-helical portion of TTN may be important for BZ receptor recognition and BZ receptor subtype discrimination.

Amino Acid Sequence↗
PubMed1989

Isolation and characterization of a rat brain triakontatetraneuropeptide, a posttranslational product of diazepam binding inhibitor: specific action at the Ro 5-4864 recognition site.

This report describes the purification and characterization from rat brain of triakontatetraneuropeptide (TTN, DBI 17-50), a major biologically active processing product of diazepam binding inhibitor (DBI). Brain TTN was purified by immunoaffinity chromatography with polyclonal octadecaneuropeptide, DBI 33-50) antibodies coupled to CNBr-Sepharose 4B followed by two reverse-phase HPLC steps. The amino acid sequence of the purified peptide is: Thr-Gln-Pro-Thr-Asp-Glu-Glu-Met-Leu-Phe-Ile-Tyr-Ser-His-Phe-Lys-Gln-Ala-Thr-Val - Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys. Synthetic TTN injected intracerebroventricularly into rats induces a proconflict activity (IC50 0.8 nmol/rat) that is prevented by the specific "peripheral" benzodiazepine (BZ) receptor antagonist isoquinoline carboxamide, PK 11195, but not by the "central" BZ receptor antagonist imidazobenzodiazepine, flumazenil. TTN displaces [3H]Ro 5-4864 from synaptic membranes of olfactory bulb with a Ki of approximately 5 microM. TTN also enhances picrotoxinin inhibition of gamma-aminobutyric acid (GABA)-stimulated [3H]flunitrazepam binding. These data suggest that TTN, a natural DBI processing product acting at "Ro 5-4864 preferring" BZ binding site subtypes, might function as a putative neuromodulator of specific GABAA receptor-mediated effects.

Amino Acid Sequence↗
PubMed1986

The nucleotide sequence of the mRNA encoding the fusion protein of measles virus (Edmonston strain): a comparison of fusion proteins from several different paramyxoviruses.

Membrane fusion is the primary cytopathic effect observed in cells infected with measles virus. The viral protein responsible for this process has previously been defined as the fusion (F) protein. Fusion is activated by the proteolytic cleavage of a precursor molecule (F0) to yield two disulfide-linked polypeptides (F1 and F2). In this paper the mRNA for the membrane fusion protein has been cloned and the resulting cDNAs were sequenced. A mRNA composed of 2377 nucleotides was found to contain one open reading frame which could potentially code for a protein of 550 amino acids. This corresponding gene product was identified as the fusion protein through use of antibodies directed against a synthetic peptide which was constructed from the deduced amino acid sequence. A long and rather G-C rich 5' terminus was found on the mRNA and this noncoding region may play some role in regulation of protein synthesis at the translational level. Protein sequence data derived from the cDNA clones revealed a highly conserved F1 amino terminus which is characteristic of most paramyxoviruses. Very little amino acid homology (except for the conservation of the F1 terminus and 9 cysteines) was evident when the sequence was compared to other paramyxovirus fusion proteins. However an overall hydrophobic nature was characteristic of all the F proteins and hydrophobicity plots for the fusion proteins of 4 different paramyxoviruses were very similar. Computer analysis was also employed to analyze the secondary structure of the measles virus F protein. Large stretches of alpha helix were characteristic of the regions which purportedly interact with membranes. The functional domains of the F protein and their similarity to those of the influenza hemagglutinin protein are discussed in this communication. We concluded that the distribution of hydrophobic regions capable of spanning biological membranes determines the fusogenic nature of the F protein.

Amino Acid Sequence↗