Biomedical subjects

A Negro

Publications and source records attributed to A Negro.

At least 55 records · Page 3Linked to original sources

PubMed1993

Aromatic diamidines are reversible inhibitors of porcine kidney diamine oxidase.

The inhibitory ability of aromatic diamidines has been studied on porcine kidney diamine oxidase. The reversibility of drug-protein interactions has been tested by means of exhaustive dialysis experiments, showing in all cases a reversible binding pattern. Ki values obtained by means of Lineweaver-Burk plots were: stilbamidine 12 microM, 2-OH-stilbamide 8.5 microM, phenamidine 4 microM, propamidine 8 microM, dibromopropamidine 4.9 microM and amicarbalide 12 microM.

Acquired Immunodeficiency Syndrome↗

PubMed1993

Brain-derived neurotrophic factor selectively rescues mesencephalic dopaminergic neurons from 2,4,5-trihydroxyphenylalanine-induced injury.

Brain-derived neurotrophic factor (BDNF) supports the survival of sensory neurons as well as retinal ganglion cells, basal forebrain cholinergic neurons, and mesencephalic dopaminergic neurons in vitro. Here we examined the ability of BDNF to confer protection on cultured dopaminergic neurons against the neurotoxic effects of 6-hydroxyDOPA (TOPA or 2,4,5-trihydroxyphenylalanine), a metabolite of the dopamine pathway suggested to participate in the pathology of Parkinson's disease. Cells prepared from embryonic day 14-15 rat mesencephalon were maintained with 10-50 ng/ml BDNF for 7 days prior to addition of TOPA (10-30 microM) for 24 hr. In BDNF-treated cultures, the extensive loss (> 90%) of tyrosine hydroxylase immunopositive cells was virtually (< 10%) eliminated, while the equally drastic loss (> 90%) of the overall cell population was limited to only a 25-30% recovery. Furthermore, the monosialoganglioside GM1 (1-10 microM), although inactive alone, acted synergistically with subthreshold amounts of BDNF to rescue tyrosine hydroxylase-positive cells against TOPA neurotoxicity. These results add impetus to exploring the therapeutic potential of gangliosides and BDNF in Parkinson's disease.

Animals↗

PubMed1993

Nerve growth factor antibodies recognize neurotrophin-3.

The immunological properties of the neurotrophins NGF, BDNF, and NT-3 were compared using polyclonal and monoclonal antibodies against the beta subunit of mouse NGF. Affinity-purified anti-NGF IgG consistently recognized NGF and NT-3 on Western blots, and inhibited the trophic activity of NGF and NT-3 but not BDNF. In contrast, anti-NGF monoclonal antibodies did not block the trophic activity of either NT-3 or BDNF. These results are consistent with the greater structural overlap between NGF and NT-3 than between NGF and BDNF.

Animals↗

PubMed1993

4',6-Diamidino 2-phenylindole is a new reversible inhibitor of diamine oxidase and S-adenosyl-L-methionine decarboxylase from mammalian tissues.

1. 4',6-Diamidino-2-phenylindole is a powerful reversible inhibitor of porcine kidney diamine oxidase and partially purified rabbit liver S-adenosyl-L-methionine decarboxylase. 2. This diamidine has shown to be a competitive inhibitor of porcine kidney diamine oxidase with a Ki value of 13 microM. 3. A similar inhibitory pattern has been found on rat liver S-adenosyl-L-methionine decarboxylase with an estimated Ki of 21 microM.

Adenosylmethionine Decarboxylase↗

PubMed1993

Pharmacokinetics of triclabendazole in rabbits.

1. Pharmacokinetic profiles of triclabendazole (TCBZ) following intravenous (i.v.) and oral administration of the drug in rabbits were carried out. 2. In normal rabbits, TCBZ was metabolized rapidly to its sulphoxide (TCBZ-SO) and sulphone (TCBZ-SO2) derivatives following administration, with undetectable concentrations of unchanged TCBZ in the plasma of the treated animals at any time (detection limit, 10 ng/ml). 3. The disposition kinetics of this drug in rabbits can be described by a two-compartment open model. 4. Mean peak concentrations in plasma of TCBZ-SO and TCBZ-SO2 of 12.41 micrograms/ml and 9.5 micrograms/ml occurred 7.5 and 9.5 hr after oral administration, respectively. 5. Both metabolites were eliminated slowly from plasma with elimination half-lives of 16.86 hr for the sulphoxide and 13 hr for the sulphone. 6. The area under the plasma concentration versus time curve (AUC) was 240 mg hr/l for the sulphoxide, higher than that found for the sulphone, 185 g hr/l.

Administration, Oral↗

PubMed1993

Production and characterization of monoclonal antibodies to human ciliary neurotrophic factor with defined epitope recognition.

Ten mouse hybridoma lines producing monoclonal antibodies (Mabs) against recombinant human ciliary neurotrophic factor (rhCNTF) have been obtained. Two monoclonal antibodies belonging to the IgG1 class were selected and characterized. Their specificity was established by ELISA and Western blotting. Epitopes recognized by the two Mabs were investigated with ELISA and Western blotting by using rhCNTF mutants, rhCNTF fragments and synthetic peptides mimicking different portions of the CNTF molecule. The carboxy-terminal part of the CNTF and particularly the sequence between aa 150 and 159 appeared to constitute the immunodominant group. The fact that certain amino acid sequences of CNTF are conserved among species was utilized to examine the crossreactivity patterns of the two Mabs with rat sciatic nerve CNTF by Western blotting and immunohistochemistry. These antibodies will be useful for studying the distribution of CNTF in the nervous system and in developing an enzyme-linked immunosorbent assay for the quantitative determinations of CNTF in various neuropathologies.

Amino Acid Sequence↗

PubMed1993

Neurotrophin-3 upregulates NGF receptors in a central nervous system glial cell line.

The low-affinity nerve growth factor (NGF) receptor (LNGFR) binds the neurotrophins NGF, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) with similar affinities. Here we report on the ability of NT-3 to regulate the expression of the LNGFR in C6 glioma cells. LNGFR-like immunoreactivity (LNGFR-IR) was examined in C6 cells treated for 16 h with NT-3 and exposed to the antibody 192-IgG followed by immunoglobulins conjugated with colloidal gold by means of ultrastructural morphometric analysis. Untreated C6 cells exhibited some positive LNGFR-IR, while C6 cells treated with NT-3 displayed significantly increased (2.3 fold) LNGFR-IR. The increase in LNGFR protein was accompanied by a greater quantity of LNGFR mRNA in NT-3-treated cells. Thus, LNGFR can be upregulated by the structurally related neurotrophin NT-3.

Animals↗

PubMed1992

Synthesis and purification of biologically active rat brain-derived neurotrophic factor from Escherichia coli.

The cDNA for rat brain-derived neurotrophic factor was cloned as the prepro and mature sequences into two independent expression vectors under control of the T7 promoter. When these vectors were transfected into Escherichia coli the prepro and mature forms of brain-derived neurotrophic factor accounted for about 20% and 25% of total E. coli proteins, and displayed molecular sizes of 26 kDa and 15 kDa, respectively. Mature brain-derived neurotrophic factor was extracted from E. coli inclusion bodies, refolded in the presence of CuCl2 and purified. The resulting protein had an ED50 of 3 ng/ml in supporting survival of cultured embryonic dorsal root ganglion neurons.

Animals↗

PubMed1992

High-performance liquid chromatographic methods for the determination of the penems SCH 29482 and FCE 22101 in human serum and urine.

High-performance liquid chromatographic methods have been developed for the determination of two 6-(1-hydroxyethyl)penems, SCH 29482 (I) and FCE 22101 (II), in serum and urine. Serum samples were combined with an equal volume of methanol to remove proteins and, after centrifugation, an aliquot of the supernatant was analysed by ion-pair chromatography on a reversed-phase C18 column with hexadecyltrimethylammonium bromide as the ion-pairing agent. The compounds were detected by their ultraviolet absorbance at 305 nm for II and 322 nm for I. Urine samples were diluted, filtered and analysed by the same chromatographic procedure. At concentrations of 1-500 micrograms/ml of each compound, the within- and between-day precisions were 1.8-3.6 and 2.6-5.1%, respectively. The detection limit was 0.2 micrograms/ml for I and 0.3 micrograms/ml for II.

Anti-Bacterial Agents↗

PubMed1992

Determination of luxabendazole in biological fluids by high-performance liquid chromatography.

Luxabendazole, a new benzimidazole, is a highly potent broad-spectrum anthelmintic. A high-performance liquid chromatographic method has been developed for its determination in serum and urine samples. In order to optimize the clean-up of samples we compared two procedures: C18 Sep-Pak cartridges and ultrafiltration through a cellulose membrane with a 30,000 relative molecular mass cut-off. In order to obtain the most suitable mobile phase, we studied the influence of pH and acetonitrile content on the capacity factor (k'). Chromatographic separation and quantification were performed on a reversed-phase column packed with 5-microns Nucleosil C18. The mobile phase was acetonitrile-0.05 M phosphate buffer (pH 7.0), (40:60, v/v). The column effluent was monitored by ultraviolet-visible spectrophotometry at 290 nm. The method shows good recovery, precision and accuracy. The lower limit of detection for luxabendazole is 15 ng/ml in serum samples and 25 ng/ml in urine samples.

Animals↗

PubMed1992

Reversed-phase ion-pair high-performance liquid chromatographic determination of triclabendazole metabolites in serum and urine.

An ion-pair high-performance liquid chromatographic method was developed for measuring the concentrations of triclabendazole metabolites (sulphoxide and sulphone) in plasma and urine samples. The diluted biological fluids are ultrafiltered before chromatography through a 30,000 relative molecular mass cut-off filter and then injected into a C18 column. They are then isocratically eluted with a mobile phase consisting of 0.05 M phosphate buffer (pH 7.0)-acetonitrile (55:45, v/v) with addition of 1.0 mmol/l sodium decanesulphonate and monitored by ultraviolet-visible spectrophotometry at 312 nm. Recoveries over the range 0.01-9.0 micrograms/ml for triclabendazole sulphoxide and sulphone are, respectively, 91.7% and 91.6% in serum and 90.3% and 90.2% in urine. For both metabolites, the limit of detection is 10 ng/ml in both urine and serum.

Animals↗

PubMed1992

Synthesis of the biologically active beta-subunit of human nerve growth factor in Escherichia coli.

The gene (NGFB) encoding the beta subunit of mature human nerve growth factor (hNGFB) was subcloned into the pJLA503 expression vector under the control of bacteriophage promoters PR and PL, and expressed in Escherichia coli. The recombinant protein represented approximately 3% of the total cellular protein. Biologically active hNGFB was solubilized (0.2% total NGFB) and purified by cation-exchange chromatography and it yielded two bands on polyacrylamide-gel electrophoresis under nonreducing conditions, corresponding to the monomeric (14 kDa) and homodimeric (26.5 kDa) forms of the molecule. Both hNGFB forms were immunopositive on Western blots with rabbit anti-NGFB antibodies; however, following additional purification, only the species corresponding to the hNGFB homodimer was biologically active on cultured chicken dorsal root ganglion neurons. These results demonstrate the feasibility of synthesizing the biologically active form of hNGFB in E. coli.

Base Sequence↗

PubMed1992

Interleukin-1 beta regulates proenkephalin gene expression in astrocytes cultured from rat cortex.

Glial cells execute essential functions in central nervous system (CNS) development and are also believed to play important roles during gliosis in response to trauma or disease. These developmental and pathological states have also been associated with elevated expression of opioid genes. Because levels of the cytokine interleukin-1 beta (IL-1 beta) increase following CNS lesions, we examined the possible influence of IL-1 beta on the expression of opioid genes in astrocytes cultured from rat cortex. Proenkephalin mRNA expression was stimulated by IL-1 beta in a time- and concentration-dependent manner, being maximal with 5 U/ml IL-1 beta at 4 h. Although the beta-adrenergic agonist isoproterenol was also active, interferon, glutamate, and carbachol were not. Unlike isoproterenol, the actions of IL-1 beta were not associated with a cyclic adenosine monophosphate (AMP)-dependent pathway. Interleukin-1 beta also regulated a proenkephalin-chloramphenicol acetyltransferase fusion gene transiently transfected into astrocytes, with a dose-response similar to that active in proenkephalin mRNA. These effects of IL-1 beta were region-specific, not being observed with either cerebellar or hippocampal astrocytes; however, isoproterenol was active in the latter cell populations. Proenkephalin mRNA in cortical astrocytes was stimulated following a temperature stress. These results suggest that enhanced proenkephalin gene expression in astrocytes by IL-1 beta may be important in neuroimmune interactions and in trauma-induced CNS injury or stress.

Animals↗

PubMed1992

Nerve growth factor (NGF) receptors in a central nervous system glial cell line: upregulation by NGF and brain-derived neurotrophic factor.

The neurotrophic proteins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are related in their primary amino acid structures. In this study we investigated the extent to which the low-affinity NGF receptor (LNGFR) in C6 glioma cells can discriminate between the neurotrophins NGF and BDNF. LNGFR-immunoreactivity (IR) was studied in C6 cells treated for 16 hr with NGF (50 ng/ml) or BDNF (10 ng/ml), using immunogold labelling and electron microscopic morphometric analysis. The cells were exposed to the anti-NGFR antibody 192-IgG, followed by immunoglobulin conjugated with colloidal gold. Untreated C6 cells exhibited some surface gold label (positive LNGFR-IR). Cells treated with NGF or BDNF displayed significantly increased LNGFR-IR on all surfaces in terms of gold labeling, which was more pronounced in NGF-treated cells. LNGFR-IR was also localized in coated endocytotic vesicles, in smooth endoplasmic reticulum, and in secondary multivesicular lysosomes in neurotrophin-treated and untreated cells. The increase in LNGFR protein was further substantiated by a correspondingly higher content of LNGFR mRNA detected after 15 hr of either NGF or BDNF treatment. These results suggest that the LNGFR in glial cells can be upregulated by the structurally related neurotrophins NGF and BDNF.

Animals↗

PubMed1992

Recombinant human ciliary neurotrophic factor alters the threshold of hippocampal pyramidal neuron sensitivity to excitotoxin damage: synergistic effects of monosialogangliosides.

Ciliary neurotrophic factor (CNTF) is a multifunctional protein which not only promotes neuronal survival in vitro and in vivo but also controls cell division of neuronal precursors, transmitter differentiation, and glial cell differentiation. Recent studies have indicated that neurotrophic factors can alter hippocampal neuronal threshold to excitotoxin sensitivity. To examine such a role for CNTF, cultures of rat embryonic hippocampal neurons were maintained with recombinant human CNTF for different times, prior to exposure to a toxic dose of glutamate at 5 days in vitro for a further 24 hr. The cytotoxic action of 200 microM glutamate (approximately 40% of pyramidal neurons remaining after 24 hr) was reduced in a concentration-dependent manner in cultures receiving a prior exposure to CNTF within the first 3 days of cell plating: 30 ng/ml CNTF permitted about 75% of the initial number of pyramidal neurons to survive. Presentation of CNTF less than 48 hr before glutamate challenge was ineffective at up to 100 ng/ml. When pyramidal neurons were cultured with a subthreshold concentration (2 ng/ml) of CNTF together with 10 microM of the monosialoganglioside GM1 (or its inner ester form) in the same paradigm, the resulting neuronal survival was similar to that seen with 30 ng/ml CNTF in the face of a glutamate challenge. Such low doses of either CNTF or ganglioside alone were ineffective. The ability of trophic factors to influence the threshold of neuronal sensitivity to excitatory amino acid injury suggests that these proteins could play an important role in the reparative capacity of acutely traumatized central neurons and in neurodegenerative diseases linked to an excitotoxic mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗

PubMed1992

Photochemical stroke and brain-derived neurotrophic factor (BDNF) mRNA expression.

In situ hybridization and Northern blotting were used to study the expression of brain-derived neurotrophic factor (BDNF) mRNA in the rat brain following photochemical stroke. A focal thrombotic lesion of the sensorimotor cortex was produced by intravenously injecting the light-sensitive dye rose bengal and exposing the skull to a controlled beam of light. Four hours after the light exposure the level of BDNF mRNA was increased in the hippocampus and cortex ipsilateral and perifocal to the lesion. The stroke-induced BDNF mRNA increase was prevented by the non-competitive glutamate receptor blocker dizocilpine (MK-801). The results indicate that the activation of N-methyl-D-aspartate (NMDA)-sensitive glutamate receptors is involved in the stroke-triggered stimulation of BDNF mRNA increase.

Animals↗

PubMed1991

Cloning and expression of human ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) is a survival factor for avian ciliary ganglion neurons and a variety of other neuronal cell types in vitro. We report here the cloning of the entire genomic sequence encoding human CNTF and its primary structure. Biologically active CNTF has been expressed in Chinese hamster ovary cells from a human genomic DNA clone. Human CNTF has no significant sequence similarity to any previously reported protein, although approximately 84% similarity exists compared with rat and rabbit CNTF. The lack of both an N-terminal signal sequence and consensus sequences for glycosylation or hydrophobic regions, and the fact that active CNTF is expressed but not released into the culture medium of transfected cells, argue in favour of human CNTF as a cytosolic protein. These data provide a basis for understanding the role of CNTF in nervous system physiology and pathology.

Amino Acid Sequence↗

PubMed1991

Synthesis, purification, and characterization of human ciliary neuronotrophic factor from E. coli.

The cDNA for human ciliary neuronotrophic factor (CNTF) has been cloned into an expression vector under the control of the T7 promoter. The BL21 strain of E. coli was transformed with this vector. Human CNTF accounted for about 30% of the total bacterial protein after induction with isopropyl-B-D-thiogalactopyranoside. This human CNTF was purified to homogeneity from inclusion bodies by a combination of ion exchange chromatography and reverse-phase high performance liquid chromatography. The amino-terminal amino acid sequence of the purified protein was identical to the deduced amino acid sequence; however, the methionyl residue has been removed. On SDS-PAGE gels, human CNTF displayed a molecular weight of about 24 kDa, in accord with its deduced molecular mass; a pI of 5.8 indicates the acidic nature of the molecule. A proposed structure for human CNTF includes major alpha helical regions. The ED50 of purified human CNTF was approximately 30 pM, using cultured embryonic day 10 chicken dorsal root ganglion neurons; no activity was observed with neurons from embryonic day 8 ganglia. Polyclonal antibodies prepared against both a synthetic peptide of CNTF and the entire human CNTF protein recognized a single 24 kDa band on Western blots, corresponding to human CNTF. However, only the antibodies against intact CNTF blocked its biological activity. This represents the first molecular expression and purification of human CNTF.

Base Sequence↗