Biomedical subjects

A Lapidot

Publications and source records attributed to A Lapidot.

At least 19 recordsLinked to original sources

PubMed2001

Neomycin B-arginine conjugate, a novel HIV-1 Tat antagonist: synthesis and anti-HIV activities.

HIV-1 transactivating protein Tat is essential for virus replication and progression of HIV disease. HIV-1 Tat stimulates transactivation by binding to HIV-1 transactivator responsive element (TAR) RNA, and while secreted extracellularly, it acts as an immunosuppressor, an activator of quiescent T-cells for productive HIV-1 infection, and by binding to CXC chemokine receptor type 4 (CXCR4) as a chemokine analogue. Here we present a novel HIV-1 Tat antagonist, a neomycin B-hexaarginine conjugate (NeoR), which inhibits Tat transactivation and antagonizes Tat extracellular activities, such as increased viral production, induction of CXCR4 expression, suppression of CD3-activated proliferation of lymphocytes, and upregulation of the CD8 receptor. Moreover, Tat inhibits binding of fluoresceine isothiocyanate (FITC)-labeled NeoR to human peripheral blood mononuclear cells (PBMC), indicating that Tat and NeoR bind to the same cellular target. This is further substantiated by the finding that NeoR competes with the binding of monoclonal Abs to CXCR4. Furthermore, NeoR suppresses HIV-1 binding to cells. Importantly, NeoR accumulates in the cell nuclei and inhibits the replication of M- and T-tropic HIV-1 laboratory isolates (EC(50) = 0.8-5.3 microM). A putative model structure for the TAR-NeoR complex, which complies with available experimental data, is presented. We conclude that NeoR is a multitarget HIV-1 inhibitor; the structure, and molecular modeling and dynamics, suggest its binding to TAR RNA. NeoR inhibits HIV-1 binding to cells, partially by blocking the CXCR4 HIV-1 coreceptor, and it antagonizes Tat functions. NeoR is therefore an attractive lead compound, capable of interfering with different stages of HIV infection and AIDS pathogenesis.

Amino Acid Sequence↗

PubMed2001

Effect of endogenous beta-hydroxybutyrate on glucose metabolism in the diabetic rabbit brain: a (13)C-magnetic resonance spectroscopy study of [U-(13)C]glucose metabolites.

The neurological consequences of diabetes mellitus have recently been receiving greater attention in both clinical and experimental settings. The deleterious effect of hyperglycemia and altered oxidative substrate availability on the diabetic brain is the subject of many studies. The aim of the present study was to examine the effect of the altered metabolic environment, namely, hyperglycemia and hyperketonemia, on glucose metabolism in the diabetic brain. More specifically, we examined the effect of diabetes on the glucose flux via the pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) pathways and subsequent metabolism in the tricarboxylic acid cycles in neurons and glia. To this end, [U-(13)C]glucose was infused into the circulation of alloxan-induced diabetic young adult rabbits, and the [(13)C]glucose metabolites were subsequently studied in brain extracts by (13)C-NMR. Significantly elevated brain glucose levels were found. In the hyperketonemic rabbits, elevated cerebral levels of beta-hydroxybutyrate (beta-HBA) were found. Alterations in the labeling patterns of glutamine in the hyperketonemic group lead to the conclusion that the elevated beta-HBA levels inhibit glucose metabolism, mostly in glia. This results in accumulation of glucose in the diabetic brain. In addition, altered levels of glutamine, glutamate, and GABA were also attributed to the effect of beta-HBA on brain metabolism. The possible role of these metabolic perturbations in causing neurological damage remains to be investigated.

3-Hydroxybutyric Acid↗

PubMed2001

Energy fuel utilization by fetal versus young rabbit brain: a 13C MRS isotopomer analysis of [U-(13)C]glucose metabolites.

The principle substrate for brain metabolism is glucose, which provides both energy and the carbon skeletons of glutamate and glutamine, via the TCA cycle. The existence of two distinct cerebral metabolic compartments, neurons and glia, involved in glutamate and glutamine synthesis, respectively, is a widely accepted concept. In previous work, the relative glucose flux via pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) in adult rabbit brain, using 13C NMR isotopomer analysis of glutamate and glutamine, was quantified. In this work, manifestation of cerebral compartmentation in the near-term fetal rabbit was investigated, using the above approach. Following infusion of [U-13C]glucose into maternal circulation (1 mg/kg per min) for 60-70 min, fetal brains were excised and brain extracts were studied by 13C NMR. The labelling patterns of fetal cerebral metabolites differed from those observed in the young adult brain. The most significant differences were found for glutamine labelling patterns. We suggested that these differences are a result of increased utilization of non-labeled fuels, mainly beta-hydroxybutyrate (beta-HBA) in the glia, the site of glutamine synthesis. In addition, we have shown that acute exposure to elevated beta-HBA levels leads to increased uptake, but not utilization, into the fetal rabbit brain; no increase in uptake is observed in the adult brain. We have also demonstrated that during short-term starvation, although no changes are detected in plasma and cerebral glucose levels in the fetal and young adult brain, amino acid levels and energy metabolism are altered in the young adult brain.

3-Hydroxybutyric Acid↗

PubMed2001

Women's dreams reported during first pregnancy.

This study analysed women's dreams reported during first pregnancy, a subject matter located at the crossroads of the psychology of dreams and the psychology of pregnancy. In the comparison of dreams reported by first-time pregnant women, to those reported by controls, we hypothesized that pregnant women's dreams would: (i) include more pregnancy-related content; (ii) display a higher degree of anxiety; and (iii) rate higher on a primary-process thinking (PPT) scale. As predicted, it was found that pregnancy-related contents significantly occupied pregnant women's dreams, a fact that might be attributed to an attempt to process and master the experience. Contrary to our expectations, it was found that anxiety and PPT were not significantly higher among pregnant women. An attempt to account for these findings raised methodological, as well as theoretical issues, consequently leading to a re-examination of the original hypotheses. Thus, it was claimed that the linkage of pregnancy to increased anxiety and PPT is grossly unbalanced.

Adult↗

PubMed2001

The "rhinogram".

Explore the source record for details and available documents.

Forms and Records Control↗

PubMed2000

Anti-human immunodeficiency virus activity of novel aminoglycoside-arginine conjugates at early stages of infection.

Conjugates of L-arginine with aminoglycosides have already been described as potent in vitro inhibitors of the HIV-1 Tat-trans-activation responsive element interaction. The polycationic nature of these agents leads us to suggest that they may be active against HIV-1 replication by inhibiting earlier stages of the virus life cycle. We have found that R4K and R3G, kanamycin A, and gentamicin C, conjugated with arginine, inhibited HIV-1 NL4-3 replication at EC50 values of 15 and 30 microM for R3G and R4K, respectively, without a detectable tonic effect on MT-4 cells at concentrations higher than 4000 and about 1000 microM, respectively. Both compounds inhibited the binding of a monoclonal antibody (12G5) directed to CXCR4 as well as the intracellular Ca2+ signal induced by the chemokine SDF-1alpha on CXCR4+ cells, suggesting that aminoglycoside-arginine conjugates interact with CXCR4, the coreceptor used by T-tropic, X4 strains of HIV-1. On the other hand, CB4K, a conjugate of kanamycin A with gamma-guanidinobutyric acid, structurally similar to R4K, failed to display any anti-HIV activity of CXCR4 antagonist activity. An HIV-1 strain that was made resistant to the known CXCR4 antagonist AMD3100 was cross-resistant to both R4K and R3G. However, unlike SDF-1alpha and R4K, R3G inhibited the binding of HIV-1 to MT-4 cells. Aminoglycoside-arginine conjugates inhibit HIV replication by interrupting the early phase of the virus life cycle, namely virus binding to CD4 cells and interaction with CXCR4. R3G and R4K may serve as prototypes of novel anti-HIV agents and should be further studied.

Anti-HIV Agents↗

PubMed2000

Aminoglycoside-arginine conjugates that bind TAR RNA: synthesis, characterization, and antiviral activity.

Regulation of HIV gene expression is crucially dependent on binding of the trans-activator protein, Tat, to the trans-activation response RNA element, TAR, found at the 5' end of all HIV-1 transcripts. Tat-TAR interaction is mediated by a short arginine-rich domain of the protein. Disruption of this interaction could, in theory, create a state of complete viral latency. A new class of small-molecule peptidomimetic TAR RNA binders, conjugates of aminoglycosides and arginine, was recently designed [Litovchick, A., Evdokimov, A. G., and Lapidot, A. (1999) FEBS Lett. 445, 73-79]. Two of these compounds, the tri-arginine derivative of gentamicin C (R3G) and the tetra-arginine derivative of kanamycin A (R4K), bind efficiently and specifically to TAR RNA. These compounds display negligible toxicity while being transported and accumulated in cell nuclei. Here we present a detailed synthesis and chemical characterization of the aminoglycoside-arginine conjugates R3G and R4K as well as GB4K, the tetra-gamma-guanidinobutyric derivative of kanamycin A. Their binding sites on TAR RNA were assigned by RNase A, uranyl nitrate, and lead acetate footprinting. The conjugates interact with TAR RNA in the widened major groove, formed by the UCU bulge and the neighboring base pairs of the upper stem portion of TAR, the binding site of Tat protein, and Tat-derived peptides (e.g., R52). Our results suggest an additional binding site of R4K and R3G compounds, in the lower stem-bulge region of TAR. The antiviral activity of the conjugates in cultured equine dermal fibroblasts infected with equine infectious anemia virus, used as a model system of HIV-infected cells, is also presented.

Animals↗

PubMed1999

Low temperature cycled PCR protocol for Klenow fragment of DNA polymerase I in the presence of proline.

A method for performing cycled PCR at low temperatures, using the thermolabile Klenow fragment of DNA polymerase I, is reported. Application of proline as a buffer additive in the range of 3.0-5.5 M remarkably increases the thermal stability of the polymerase and decreases the denaturation temperature of DNAtemplate. This method might be applicable to a broad spectrum of thermolabile DNA polymerases in cycled PCR and other methods of DNA amplification.

DNA Polymerase I↗

PubMed1999

Effect of tetrahydropyrimidine derivatives on protein-nucleic acids interaction. Type II restriction endonucleases as a model system.

2-Methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyri- midine (THP(A) or hydroxyectoine) and 2-methyl,4-carboxy-3,4,5, 6-tetrahydropyrimidine (THP(B) or ectoine) are now recognized as ubiquitous bacterial osmoprotectants. To evaluate the impact of tetrahydropyrimidine derivatives (THPs) on protein-DNA interaction and on restriction-modification systems, we have examined their effect on the cleavage of plasmid DNA by 10 type II restriction endonucleases. THP(A) completely arrested the cleavage of plasmid and bacteriophage lambda DNA by EcoRI endonuclease at 0.4 mM and the oligonucleotide (d(CGCGAATTCGCG))2 at about 4.0 mM. THP(B) was 10-fold less effective than THP(A), whereas for betaine and proline, a notable inhibition was observed only at 100 mM. Similar effects of THP(A) were observed for all tested restriction endonucleases, except for SmaI and PvuII, which were inhibited only partially at 50 mM THP(A). No effect of THP(A) on the activity of DNase I, RNase A, and Taq DNA polymerase was noticed. Gel-shift assays showed that THP(A) inhibited the EcoRI-(d(CGCGAATTCGCG))2 complex formation, whereas facilitated diffusion of EcoRI along the DNA was not affected. Methylation of the carboxy group significantly decreased the activity of THPs, suggesting that their zwitterionic character is essential for the inhibition effect. Possible mechanisms of inhibition, the role of THPs in the modulation of the protein-DNA interaction, and the in vivo relevance of the observed phenomena are discussed.

DNA Restriction Enzymes↗

PubMed1999

Arginine-aminoglycoside conjugates that bind to HIV transactivation responsive element RNA in vitro.

HIV gene expression is crucially dependent on binding of the viral Tat protein to the transactivation RNA response element. A number of synthetic Tat-transactivation responsive element interaction inhibitors of peptide/peptoid nature were described as potential antiviral drug prototypes. We present a new class of peptidomimetic inhibitors, conjugates of L-arginine with aminoglycosides. Using a gel-shift assay and affinity chromatography on an L-arginine column we found that these compounds bind specifically to the transactivation responsive element RNA in vitro with Kd values in the range of 20-400 nM, which is comparable to the Kd of native Tat bound to the transactivation responsive element (10-12 nM). Confocal microscopy studies demonstrated that fluorescein-labelled conjugate penetrates into live cells. High affinity to the transactivation responsive element, low toxicity, and relative simplicity of synthesis make these compounds attractive candidates for antiviral drug design.

Aminoglycosides↗

PubMed1997

Quantitation of metabolic compartmentation in hyperammonemic brain by natural abundance 13C-NMR detection of 13C-15N coupling patterns and isotopic shifts.

In the present study, the removal of cerebral ammonia by glutamine synthetase (GS) and by reductive amination of 2-oxoglutarate by glutamate dehydrogenase in the presence of an amino donor group, was determined in hyperammonemic rabbit brains. The 15N enrichments of brain metabolite alpha-amino and amide positions of glutamine, glutamate, and alanine were determined by the indirect detection of 15N-labeled compounds of the 13C-15N spin coupling patterns of natural abundance 13C-NMR spectra. The 13C-NMR spectra of brain extracts were obtained from rabbits infused with 15NH4Cl with or without intraperitoneal infusion of the GS inhibitor, L-methionine DL-sulfoximine, in a reasonable acquisition time period. When 15NH4Cl was infused, [5-15N]glutamine and [2-15N]glutamine concentrations reached 5.2 mumol/100 mg protein and 3.6 mumol/100 mg protein, respectively, which indicates the relatively high activity of reductive amination of 2-oxoglutarate in the glutamate dehydrogenase reaction. The low concentration of [2-15N]glutamate, which is about 30% of that of [2-15N]glutamine obtained in this study, suggests that very little glutamine serves as a precursor of neuronal glutamate. When GS was inhibited by L-methionine DL-sulfoximine, a flux of 15NH4+ via the residual activity of GS was accompanied by an apparent increase of [2-15N]glutamate and [15N]alanine concentrations (2.9 mumol/100 mg protein and 1.8 mumol/100 mg protein, respectively). These findings and those obtained from 13C-13C isotopomer analysis (Lapidot and Gopher, 1994b) suggest that astrocytic 2-oxoglutarate is partially utilized (together with an amino group donor) as a precursor for neuronal glutamate in the hyperammonemic brain when GS is inhibited. This process can partly replace GS activity in metabolizing ammonia in the hyperammonemic rabbit brain.

Amino Acids↗

PubMed1996

Overexpression and single-step purification of a thermostable xylanase from Bacillus stearothermophilus T-6.

Xylanase T-6 is a thermostable alkaline-tolerant enzyme that is produced by Bacillus stearothermophilus T-6. Xylanase T-6 was found to bleach pulp effectively at pH 9 and 65 degrees C and was used successfully on an industrial-scale mill trial. To facilitate the future characterization of the protein via X-ray analysis and protein engineering, it was necessary to overexpress the enzyme in Escherichia coli. The xylanase gene was cloned into T-7 polymerase expression vectors and its expression was optimized. The enzyme was found to constitute over 70% of the cell protein and it was efficiently purified from the host proteins by a single heating step. Over 2 g soluble and active enzyme per 1 culture were achieved.

Amino Acid Sequence↗

PubMed1996

Induction of synthesis of tetrahydropyrimidine derivatives in Streptomyces strains and their effect on Escherichia coli in response to osmotic and heat stress.

The metabolic responses of a number of Streptomyces strains to osmotic and heat stress were studied by 13C nuclear magnetic resonance spectroscopy. During cell growth in a chemically defined medium supplemented with 0.5 M NaCl, tetrahydropyrimidine derivatives (THPs), 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine [THP(A)] and, to a lesser extent, 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)], were found to accumulate in a significant amount in all bacteria examined. In addition, when the growth temperature was shifted from 30 to 39 degrees C, the intracellular concentration of THP(A) increased significantly. Moreover, exogenously provided THP(A) or THP(B) or both reversed inhibition of Escherichia coli growth caused by osmotic stress and increased temperature. Although the ability of Streptomyces strains to tolerate high concentrations of NaCl is well known, very little is known about the osmoregulatory strategy in Streptomyces strains. Similarly, the mechanism by which compatible solutes accumulate in a variety of microorganisms is not understood. Our findings suggest the possibility of a novel mechanism of protection of DNA against salt and heat stresses involving the THPs.

Biological Transport↗

PubMed1996

Dynamic parameters of maternal amino acid metabolism and fetal growth.

This study presents kinetic parameters of glycine metabolism during pregnancy and the influence of fuel availability on fetal growth. The kinetic studies were done on patients with gestational diabetes mellitus (diet treated) and pre-gestational diabetes mellitus (diet and insulin treated) that was accompanied by increased fetal growth and pregnancy-induced hypertension and, during the third trimester of pregnancy, by intrauterine growth retardation. Gas chromatography-mass spectrometry was used to determine the 15N enrichment of plasma glycine, and to calculate the pool sizes, turnover rate constants, fluxes and metabolic clearance rates. Glycine pool sizes in pre-gestational diabetes were significantly larger than those in normal, hypertensive and gestational diabetes pregnancies. Glycine turnover rate constants and metabolic clearance rates were not significantly different between the normal pregnant women, the hypertensive, and the two diabetic groups of pregnant women. Glycine fluxes were significantly higher in the pre-gestational diabetic pregnant women than in those with gestational diabetes, hypertension, and normal pregnancy. Pre-gestational diabetic pregnant women delivered fetuses with higher birthweights than the other three groups. Fetal birthweight of the hypertensive women was significantly lower than among the normal and diabetic women. Stable isotope methodology using labeled amino acids provides a powerful tool for clinical studies of maternal protein metabolism and its relationship to fetal growth.

Birth Weight↗

PubMed1995

Tetrahydropyrimidine derivatives inhibit binding of a Tat-like, arginine-containing peptide, to HIV TAR RNA in vitro.

The ability of a small molecule, 2-methyl,4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyrimidine (THP(A)), which accumulates intracellularly in various streptomyces, to inhibit the interaction of Tat peptide (R52) with TAR RNA is presented. Using gel-shift assay, we found that the inhibition constant Ki of THP(A) is 50-100 nM, which is in the range of the binding constants of Tat peptide and protein. THP(A) is approximately 10(6) times more tightly bound than the free L-arginine. The high binding affinity may be attributed to the special delocalized positive charge on the NCN group and the hydroxyl group at the 5 position of this molecule. A model for THP(A)-TAR interaction, analogous to the arginine guanidinum group-TAR interaction, is presented. The relatively high uptake of THP(A) by mammalian cells warrants in vivo Tat/TAR inhibition studies.

Bacterial Proteins↗

PubMed1995

Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum.

The major cellulose-binding domain (CBD) from the cellulosome of Clostridium thermocellum YS was cloned and overexpressed in Escherichia coli. The expressed protein was purified efficiently by a modification of a novel procedure termed affinity digestion. The properties of the purified polypeptide were compared with those of a related CBD derived from a cellulosome-like complex of a similar (but mesophilic) clostridial species, Clostridium cellulovorans. The binding properties of the two proteins with their common substrate were found to be very similar. Despite the similarity in the amino acid sequences of the two CBDs, polyclonal antibodies raised against the CBD from C. thermocellum failed to interact with the protein from C. cellulovorans. Chemical modification of the single cysteine of the CBD had little effect on the binding to cellulose. Biotinylation of this cysteine allowed the efficient binding of avidin to cellulose, and the resultant matrix is appropriate for use as a universal affinity system.

Affinity Labels↗

PubMed1994

Cerebral metabolic compartmentation. Estimation of glucose flux via pyruvate carboxylase/pyruvate dehydrogenase by 13C NMR isotopomer analysis of D-[U-13C]glucose metabolites.

A method is presented for determining the compartmentation of amino acid metabolism in the brain. 13C NMR spectroscopy, and more specifically, homonuclear 13C-13C spin coupling patterns of 13C-labeled amino acids were used to measure the relative flux of label from D-[U-13C]glucose through the anaplerotic pathway versus the oxidative pathway. Glucose flux through the pyruvate carboxylase pathway was quantitated following primed dose constant infusion of D-[U-13C]glucose to young rabbits at a rate of 1 mg/kg body weight per min. We demonstrate, for the first time, that multiplet spectra of three adjacent 13C isotopomer in 1,2,3-13C3 in glutamine and glutamate, which are derived from [1,2,3-13C3]pyruvate, present different isotopomer populations in glutamine in comparison to that in glutamate. This is due to two different metabolic compartments characterized by the presence or absence of glutamine synthetase activity and two different tricarboxylic acid cycles, one preferentially mediated by pyruvate carboxylase and the other by pyruvate dehydrogenase. Our results indicate that the anaplerotic pathway accounts for 34% of glutamine synthesis and only 16% of glutamate and gamma-aminobutyric acid syntheses in metabolic and isotopic steady state conditions. These results support the concept, and provide a quantitative measure, that glutamine and/or tricarboxylic acid cycle intermediates are supplied by astrocytes to neurons to replenish the neurotransmitter pool of gamma-aminobutyric acid and glutamate.

Animals↗

PubMed1994

Cloning and DNA sequence of the gene coding for Bacillus stearothermophilus T-6 xylanase.

Bacillus stearothermophilus T-6 produces an extracellular thermostable xylanase. Affinity-purified polyclonal serum raised against the enzyme was used to screen a genomic library of B. stearothermophilus T-6 constructed in lambda-EMBL3. Two positive phages were isolated, both containing similar 13-kb inserts, and their lysates exhibited xylanase activity. A 3,696-bp SalI-BamHI fragment containing the xylanase gene was subcloned in Escherichia coli and subsequently sequenced. The open reading frame of xylanase T-6 consists of 1,236 bp. On the basis of sequence similarity, two possible -10 and -35 regions, a ribosome-binding site at the 5' end of the gene and a potential transcriptional termination motif at the 3' end of the gene, were identified. From the previously known N-terminal amino acid sequence of xylanase T-6 and the possible ribosome-binding site, a putative 28-amino-acid signal peptide was deduced. The mature xylanase T-6 consists of 379 amino acids with a calculated molecular weight and pI of 43,808 and 6.88, respectively. Multiple alignment of beta-glycanase amino acid sequences revealed highly conserved regions. Northern (RNA) blot analysis indicated that the xylanase T-6 transcript is about 1.4 kb and that the induction of this enzyme synthesis by xylose is on the transcriptional level.

Amino Acid Sequence↗