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Y Bromberg

Publications and source records attributed to Y Bromberg.

At least 19 recordsLinked to original sources
PubMed2006

The neuroprotective adenosine-activated signal transduction pathway involves activation of phospholipase C.

We have demonstrated before that exposure of neuronal cultures to poisoning by iodoacetic acid (IAA) followed by "reperfusion" (IAA-R insult), results in severe cytotoxicity, which could be markedly attenuated by prior activation of the adenosine A1 receptors. We also have demonstrated that adenosine activates a signal transduction pathway (STP), which involves activation of PKC epsilon and opening of KATP channels. Here, we provide proof for the involvement also of phospholipase C (PLC) in the neuronal protective adenosine-activated STP. R-PIA, a specific A1 adenosine receptor agonist, was found to enhance neuronal PLC activity and protect against the IAA-R insult. The PLC inhibitor U73122, abrogated both R-PIA-induced effects. These results demonstrate that activation of PLC is a vital step in the neuronal protective adenosine-induced STP.

Adenosine↗
PubMed2004

Clinical and biochemical manifestations and molecular characterization of the mutation HPRT Jerusalem.

A novel point mutation (I137T) was identified in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) encoding gene, in a patient with partial deficiency of the enzyme. The mutation, ATT to ACT (substitution of isoleucine to threonine), occurred at codon 137, which is within the region encoding the binding site for 5-phosphoribosyl-1-pyrophosphate (PRPP). The mutation caused decreased affinity for PRPP, manifested clinically as a Lesch-Nyhan variant (excessive purine production and delayed acquisition of language skills). The partial HPRT deficiency could be detected only by measuring HPRT activity in intact fibroblasts (uptake of hypoxanthine into nucleotides).

Binding Sites↗
PubMed2000

Kelley-Seegmiller syndrome due to a unique variant of hypoxanthine-guanine phosphoribosyltransferase: reduced affinity for 5-phosphoribosyl-1-pyrophosphate manifested only at low, physiological substrate concentrations.

A male child, who presented at the age of 3.5 years with acute renal failure, was diagnosed as having partial deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT; EC 2.4.2.8). The underlying HPRT mutation was unique in that the specific activity of HPRT in erythrocyte and in fibroblast lysates was normal, but the rate of uptake of hypoxanthine into nucleotides of intact cultured fibroblasts was markedly reduced (23% of normal). The low functioning of HPRT in the intact fibroblasts was associated with decreased utilization of endogenously generated hypoxanthine and with decreased utilization of the cosubstrate 5-phosphoribosyl-1-pyrophosphate (PRPP). The non-utilized hypoxanthine was excreted into the incubation medium. The accumulation of PRPP was indicated by the 2.3-fold increase in the rate of uptake of adenine into intact cell nucleotides and by the 7. 5-fold enhancement of the rate of de novo purine synthesis. Kinetic studies of HPRT activity in fibroblast lysates revealed reduced affinity of the enzyme for PRPP (apparent K(m) 500 microM in comparison to 25 microM in control lysates), manifested in low activity at low (physiological), but not at high PRPP concentrations. The apparent K(m) for hypoxanthine was normal (23 microM in comparison to 14.2 microM in control lysates). With allopurinol treatment, our patient has had no problems since presentation, and is developing normally at 5 years of age.

Acute Kidney Injury↗
PubMed1999

EHD1--an EH-domain-containing protein with a specific expression pattern.

A cDNA that is a member of the eps15 homology (EH)-domain-containing family and is expressed differentially in testis was isolated from mouse and human. The corresponding genes map to the centromeric region of mouse chromosome 19 and to the region of conserved synteny on human chromosome 11q13. Northern analysis revealed two RNA species in mouse. In addition to the high levels in testis, expression was noted in kidney, heart, intestine, and brain. In human, three RNA species were evident. The smaller one was predominant in testis, while the largest species was evident in other tissues as well. The predicted protein sequence has an EH domain at its C-terminus, including an EF, a Ca2+ binding motif, and a central coiled-coil structure, as well as a nucleotide binding consensus site at its N-terminus. As such, it is a member of the EH-domain-containing protein family and was designated EHD1 (EH domain-containing 1). In cells in tissue culture, we localized EHD1 as a green fluorescent protein fusion protein, in transferrin-containing, endocytic vesicles. Immunostaining of different adult mouse organs revealed major expression of EHD1 in germ cells in meiosis, in the testes, in adipocytes, and in specific retinal layers. Results of in situ hybridization to whole embryos and immunohistochemical analyses indicated that EHD1 expression was already noted at day 9.5 in the limb buds and pharyngeal arches and at day 10.5 in sclerotomes, at various elements of the branchial apparatus (mandible and hyoid), and in the occipital region. At day 15.5 EHD1 expression peaked in cartilage, preceding hypertrophy and ossification, and at day 17.5 there was no expression in the bones. The EHD1 gene is highly conserved between nematode, Drosophila, mouse, and human. Its predicted protein structure and cellular localization point to the possibility that EHD1 participates in ligand-induced endocytosis.

Amino Acid Sequence↗
PubMed1996

Adenine nucleotide metabolism in primary rat neuronal cultures.

The metabolism of adenine nucleotides (AdRN) has been studied previously in whole brains, brain slices and brain extracts, containing mixed populations of neurons and glia. The availability of primary neuronal cultures enables us to study these pathways in almost pure neuronal preparations. The aim of the present study was to characterize the relative importance of the pathways of AdRN metabolism in the neurons. The metabolic fate of (8-14C) adenine and of AdRN prelabeled with (8-14C)adenine were studied in immature and mature primary rat neuronal cultures. Specific inhibitors were used to clarify the various metabolic fluxes, which were evaluated based on the time-related changes in the distribution of label (the cellular nucleotide content did not change during incubation). The turnover rate of AdRN was found to reflect mainly conversion of label to acid insoluble derivatives (AID) and partly degradation to hypoxanthine. The turnover was faster in the immature neurons. The combined addition of 2'-deoxycoformycin (2'-dCF) and of 5'-amino-5'-deoxyadenosine, inhibiting adenosine metabolism, resulted in both cultures in enhanced loss of label from AdRN, mainly to adenosine and adenine. This finding indicates the activity of the futile cycle AMP-->adenosine-->AMP. In both cultures, in the presence of these inhibitors, the ratio (hypoxanthine + inosine)/(adenine + adenosine) was 1.1, indicating that the fluxes through AMP deamination and AMP dephosphorylation are about equal. Addition of L-alanosine, inhibiting the conversion of IMP to AMP, resulted in both cultures, but especially in the mature neurons, in enhanced loss of label from AdRN to hypoxanthine and inosine. This finding indicates the functioning of the adenine nucleotide cycle (AMP-->IMP-->adenylosuccinic acid-->AMP). Under conditions of enhanced degradation of ATP (induced by iodoacetate and antimycin A), addition of 2'-dCF resulted in the immature cultures in lowering the ratio (hypoxanthine + inosine + IMP)/(adenine + adenosine) to 0.62, indicating a shift in favor of AMP dephosphorylation.

Adenine Nucleotides↗
PubMed1995

Metabolism of guanine and guanine nucleotides in primary rat cardiomyocyte cultures.

The metabolic fate of labeled guanine and of prelabeled guanine nucleotides (GuRN) was studied in cultured rat cardiomyocytes. Special attention was given to guanine salvage in comparison to degradation; to the contribution of GuRN to adenine nucleotides (AdRN); to the fluxes from GMP to IMP and from IMP to GMP; and to the degradation pathways of GuRN. In accordance with the 3- to 4-fold higher activity of guanine deaminase (guanase), in comparison to that of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the rate of guanine deamination to xanthine exceeded that of guanine incorporation into nucleotides (at 4 microM) by 13.2-fold. The label from guanine incorporated into nucleotides was found mainly (81%) in GuRN, but also in IMP and AdRN. The prelabeled GuRN lost 43% of the label in 4 h, reflecting mainly degradation to xanthine (and uric acid) and synthesis of nucleic acids. Blocking nucleoside degradation was associated with a marked accumulation of label in guanosine and inosine (guanosine/inosine labeling ratio is 1.25). The results indicate that in the myocardium guanine is a poor substrate for salvage synthesis of GuRN and that its contribution to the homeostasis of adenine nucleotides is negligible; that GMP degradation to xanthine proceeds through both guanosine and IMP; and that the cardiomyocytes contain the activity of GMP reductase and of the enzymes converting IMP to GMP.

Animals↗
PubMed1995

Developmental changes in purine nucleotide metabolism in cultured rat astroglia.

The present study was conducted in order to clarify the role of the glia in brain purine metabolism. This, in connection with the clarification of the etiology of the neurological manifestations associated with some of the inborn errors of purine metabolism in man. Purine nucleotide content, the capacity for de novo and salvage purine synthesis and the activity of several enzymes of purine nucleotide degradation, were assayed in primary cultures of rat astroglia in relation to culture age. The capacity of the intact cells to produce purine nucleotides de novo exhibited a marked decrease with the culture age, but the activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), catalyzing salvage nucleotide synthesis, increased. Aging was also associated with a marked increase in the activity of the degradation enzymes AMP deaminase, purine nucleoside phosphorylase (PNP) and guanine deaminase (guanase). The activity of adenosine deaminase and of AMP-5'-nucleotidase, increased markedly during the first 17 days in culture, but decreased thereafter. The results indicate that purine nucleotide metabolism in the cultured astroglia is changing with aging to allow the cells to maintain their nucleotide pool by reutilization of preformed hypoxanthine, rather than by de-novo production of new purines. Aging is also associated with increased capacity for operation of the adenine nucleotide cycle, contributing to the homeostasis of adenine nucleotides and to the energy charge of the cells. In principle, the age-related alterations in purine metabolism in the astroglia resemble those occurring in the maturating neurons, except for the capacity to produce purines de novo, which exhibited inverse trends in the two tissues. However, in comparison to the neurons, the cultured astroglia possess the capacity for a more intensive metabolism of purine nucleotides.

Animals↗
PubMed1994

The GDP-bound form of the small G protein Rac1 p21 is a potent activator of the superoxide-forming NADPH oxidase of macrophages.

Phagocytes produce superoxide by the assembly of a multicomponent complex that utilizes NADPH for the reduction of molecular oxygen (NADPH oxidase). The components participating in the assembly are a membrane-bound flavocytochrome and three cytosolic proteins, one of which was shown to be a dimer of the small GTP-binding protein (G protein) Rac1 p21 or Rac2 p21 with GDP dissociation inhibitor for Rho (Rho GDI). We determined the identity and quantity of the nucleotide bound to Rac1 p21 by high performance anion exchange chromatography of extracts prepared from highly purified Rac1 p21-Rho GDI, isolated from guinea pig macrophage cytosol. Rac1 p21 contained only GDP at a ratio of close to 1 mol of GDP per mol of G protein. The GDP-bound form of Rac1 p21 complexed to Rho GDI functioned as a potent activator of NADPH oxidase in a cell-free system that contained no free GTP or ATP. We propose that the GDP-bound form of Rac1 p21 might be the physiological activator of NADPH oxidase in macrophages, following its dissociation from Rho GDI, and that nucleotide exchange or conversion to GTP is not necessarily involved.

Amino Acid Sequence↗
PubMed1994

Effects of differentiation-inducing agents on purine nucleotide metabolism in an ovarian cancer cell line.

The effects of the differentiation-inducing agents sodium butyrate (NaOBt), dimethylsulfoxide (DMSO) and mycophenolic acid (MA), on purine nucleotide metabolism, was studied in an ovarian carcinoma cell line (GZL-8). Exposure to these agents inhibited cell proliferation, but did not affect cell viability. Three hours following exposure, NaOBt and DMSO moderately decelerated purine synthesis de novo, but MA accelerated it three-fold, this being associated with a two-fold increase in the excretion of hypoxanthine and xanthine into the incubation medium. NaOBt and DMSO did not affect the cellular nucleotide content, but MA caused a 73% decrease in GTP content and about a 50% increase in the cellular content of UTP. The following alterations in cellular enzyme activity were observed 72 h following exposure: NaOBt decreased the activity of hypoxanthine-guanine phosphoribosyltransferase and increased the activity of IMP and of AMP 5'-nucleotidases, DMSO increased the activity of IMP 5'-nucleotidase, and MA increased the activity of the two nucleotidases. The results suggest that, in the carcinoma cell line studied, the differentiation process induced by NaOBt and DMSO may be associated with a general shift in the direction of purine metabolism from anabolism to catabolism, whereas that induced by MA is associated with a specific decrease in the production of GTP.

Butyrates↗
PubMed1993

Characterization of the alterations in purine nucleotide metabolism in hypoxanthine-guanine phosphoribosyltransferase-deficient rat neuroma cell line.

A rat neuroma cell line (B103 4C), deficient of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), was utilized as a model tissue in search for the biochemical basis of the Lesch-Nyhan syndrome (LNS). The HGPRT-deficient neurons exhibited the following properties: an almost complete absence of uptake of guanine and of hypoxanthine into intact cell nucleotides (0.92% and 0.69% of normal, respectively); a significant increase in the availability of 5'-phosphoribosyl-1-pyrophosphate; a three- to fourfold acceleration of the rate of de novo nucleotide synthesis; a normal excretion of xanthine, but 15-fold increase in the excretion of hypoxanthine into the culture media; a normal cellular purine nucleotide content, including the absence of 5-amino-4-imidazole carboxamide nucleotides (Z-nucleotides), but enhanced turnover of adenine nucleotides (loss of 86% of the radioactivity of the prelabeled pool in 24 h, in comparison to 73% in the normal line), and an elevated UTP content. The results suggest that, under physiological conditions, guanine salvage does not occur in the normal neurons, but that hypoxanthine salvage is of great importance in the homeostasis of the adenine nucleotide pool. The finding of the normal profile of purine nucleotides in the HGPRT-deficient neurons indicates that the lack of hypoxanthine salvage is adequately compensated by the enhanced de novo nucleotide synthesis. These results did not furnish evidence in support of the possibility that GTP or ATP depletion, or Z-nucleotide accumulation, occurs in HGPRT-deficient neurons and that these are etiological factors causing the neurological abnormalities in LNS.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗
PubMed1992

Metabolic fate of hypoxanthine and inosine in cultured cardiomyocytes.

The metabolic fate of labeled hypoxanthine and inosine, degradation products of adenine nucleotides, was studied in cultured beating cardiomyocytes, in order to assess the physiological significance of their contribution to salvage nucleotide synthesis in the heart. Inosine and hypoxanthine were found to be incorporated into nucleotides by a similar rate, but in the presence of 8-aminoguanosine, a potent inhibitor of purine nucleoside phosphorylase (EC 2.4.2.1), the rate of inosine incorporation into nucleotides was markedly reduced (by 75%), indicating that inosine incorporation to IMP (inosinic acid) occurs following its degradation to hypoxanthine. The proportion of hypoxanthine converted to IMP by hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) is markedly greater than that degraded to xanthine and uric acid by xanthine oxidase (EC 1.3.2.3). However, close to 50% of the IMP formed was degraded to inosine by IMP 5'-nucleotidase (EC 3.1.3.5). The results demonstrate the activity of the following futile cycle in the cardiomyocytes: hypoxanthine----IMP----inosine----hypoxanthine. The rational for the activity of this energy consuming cycle is yet unclear.

Cells, Cultured↗
PubMed1990

Developmental changes in the activity of enzymes of purine metabolism in rat neuronal cells in culture and in whole brain.

The activities (Vmax) of several enzymes of purine nucleotide metabolism were assayed in premature and mature primary rat neuronal cultures and in whole rat brains. In the neuronal cultures, representing 90% pure neurons, maturation (up to 14 days in culture) resulted in an increase in the activities of guanine deaminase (guanase), purine-nucleoside phosphorylase (PNP), IMP 5'-nucleotidase, adenine phosphoribosyltransferase (APRT), and AMP deaminase, but in no change in the activities of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenosine deaminase, adenosine kinase, and AMP 5'-nucleotidase. In whole brains in vivo, maturation (from 18 days of gestation to 14 days post partum) was associated with an increase in the activities of guanase, PNP, IMP 5'-nucleotidase, AMP deaminase, and HGPRT, a decrease in the activities of adenosine deaminase and IMP dehydrogenase, and no change in the activities of APRT, AMP 5'-nucleotidase, and adenosine kinase. The profound changes in purine metabolism, which occur with maturation of the neuronal cells in primary cultures in vitro and in whole brains in vivo, create an advantage for AMP degradation by deamination, rather than by dephosphorylation, and for guanine degradation to xanthine over its reutilization for synthesis of GMP. The physiological meaning of the maturational increase in these two ammonia-producing enzymes in the brain is not yet clear. The striking similarity in the alterations of enzyme activities in the two systems indicates that the primary culture system may serve as an appropriate model for the study of purine metabolism in brain.

Adenosine Monophosphate↗
PubMed1989

Prenatal diagnosis of Lesch-Nyhan syndrome: experience with three fetuses at risk.

Our experience with the prenatal detection of the Lesch-Nyhan syndrome (LNS; hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency) in three fetuses at risk is reported. Enzyme activities were measured in cultured amniocytes in two pregnancies, and in tissues and cultures obtained from chorionic villus sampling (CVS) in a third pregnancy. In all tissues the specific activities of HGPRT and adenine phosphoribosyltransferase (APRT) were determined and APRT/HGPRT ratios were calculated. In addition to the enzyme assays, the rate of purine synthesis de novo was assessed in the two amniocyte cultures, and the rate of [14C]hypoxanthine incorporation into nucleotides and sensitivity to azaguanine were measured in one of the amniocyte cultures. We report the diagnosis of normal fetuses by study of amniocytes in two pregnancies and of LNS using CVS in one pregnancy. In all three cases the diagnosis was confirmed.

Adenine Phosphoribosyltransferase↗
PubMed1987

Activation of the superoxide forming NADPH oxidase in a cell-free system by sodium dodecyl sulfate. Characterization of the membrane-associated component.

Sodium dodecyl sulfate (SDS) was shown to elicit NADPH-dependent superoxide (O2-) production by a cell-free system derived from sonically disrupted resting guinea pig macrophages (Bromberg, Y., and Pick, E. (1985) J. Biol. Chem. 260, 13539-13545). O2- production was absolutely dependent on the cooperation between a membrane-associated component, sedimenting with the 48,000 X g pellet and a cytosolic factor, nonsedimentable at 265,000 X g. The present report describes the solubilization and characterization of the membrane-associated component of the SDS-activable O2(-)-forming NADPH oxidase (operationally termed pi). Treatment of the 48,000 X g pellet with 30 mM octyl glucoside resulted in complete transfer of pi to the soluble fraction. The solubilized pellet produced an average of 0.92 mumol of O2-/mg of protein/min upon reduction of octyl glucoside content below the critical micellar concentration and in the presence of cytosol, 100 microM SDS, and 0.2 mM NADPH. The activity of solubilized pellet-cytosol combinations was also expressed as NADPH-dependent, azide-resistant oxygen consumption and hydrogen peroxide production. pi was inactivated by the sulfhydryl reagent p-chloromercuribenzoate. Solubilized pellet contained spectroscopically detectable cytochrome b559 (225.6 +/- 15.0 pmol/mg mg protein). Both pi and cytochrome b559 were bound by Cibacron Blue Sepharose and could be eluted by a gradient of octyl glucoside (0-30 mM) in the presence of 1 M KCl. On high performance gel filtration on Superose 12, both pi and cytochrome b559 eluted in the excluded volume; when 25 mM octyl glucoside was present in the elution buffer, pi was partially dissociated from cytochrome b559. Sequential purification of pi on Blue Sepharose followed by gel filtration on Superose 12 in the presence of 25 mM octyl glucoside lead to complete resolution of pi from cytochrome b559 (pi was found in the Mr = 28,000 - 11,000 range while the bulk of cytochrome b559 eluted in the Mr = 113,000 - 71,000 range). We propose that pi is distinct from cytochrome b559 and represents a membrane-associated component in an amphiphile-activated electron transport chain from NADPH to oxygen.

Animals↗
PubMed1985

Activation of NADPH-dependent superoxide production in a cell-free system by sodium dodecyl sulfate.

Sodium dodecyl sulfate (SDS) elicits the production of superoxide (O2-) by a cell-free system represented by sonically disrupted guinea pig peritoneal macrophages. O2- generation requires NADPH and a heat-sensitive cellular component, is proportional to the amount of macrophage protein, and exhibits a pH optimum of 6.5-7. The kinetic parameters of the SDS-stimulated enzyme are: Km (+/- S.E.) = 0.0367 +/- 0.003 mM NADPH and Vmax (+/- S.E.) = 73.46 +/- 9.09 nmol O2-/mg of protein/min. O2- production is dependent on the cooperation between a particulate subcellular component sedimentable at 48,000 X g and a cytosolic factor present in the 48,000 X g supernatant. The activity of both components is destroyed by heating at 80 degrees C. Pretreatment of intact macrophages with phorbol myristate acetate results in the partial removal of the requirement for cytosolic factor; SDS is now capable of activating the isolated 48,000 X g pellet. Among a large number of anionic, cationic, and nonionic detergents tested, only the anionic detergents SDS and sodium dodecyl sulfonate are capable of eliciting O2- production in the cell-free system, SDS being the more potent stimulant. It is proposed that the structural requirements that make these compounds capable of activating the O2- forming NADPH oxidase in a cell-free system are the presence of an anionic polar head and a long hydrophobic alkyl tail. We suggest that sodium salts of long chain unsaturated fatty acids that were found by us to be capable of stimulating O2- production in a cell-free system (Bromberg, Y., and Pick, E. (1984) Cell. Immunol. 88, 213-221) owe their activity to the fact that they function as anionic detergents.

Animals↗