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Biomedical subjects

A Becker

Publications and source records attributed to A Becker.

At least 55 records · Page 3Linked to original sources
PubMed1994

Mutational analysis of human endothelin receptors ETA and ETB identification of regions involved in the selectivity for endothelin 3 or cyclo-(D-Trp-D-Asp-Pro-D-Val-Leu).

Two endothelin(ET)-receptor subtypes have been identified in mammals. They differ in their affinity towards the ET isopeptides with ETA displaying an ET-1-selective profile and ETB a non-selective one. To identify the regions responsible for the differential selectivity, chimeric forms were engineered by sequentially exchanging extracellular regions together with their flanking transmembrane domains. Two sets of reciprocal receptor mutants were thereby generated and analysed by expression in COS-7 cells. The recombinant receptor chimeras were characterised by direct and competitive radioligand-binding analysis. COS-7 cells transfected with vectors for the mutant receptors exhibited specific saturable [3-125I]iodotyrosyl ET-1 (125I-ET-1) binding, with affinities comparable to those of the wild-type receptors (apparent Ki approximately 1-6 x 10(-9) M). An average of 10(5)-10(6) binding sites/cell was calculated for the wild-type and mutant forms. In competition experiments using 125I-ET-1 and unlabeled ET-3, an ETB-selective agonist, we detected a clear switch from an ET-1-selective profile to a non-isopeptide-selective profile in ETA chimeras where the second extracellular loop and the flanking transmembrane domains IV and V, or the third extracellular loop and the flanking transmembrane domains VI and VII, had been exchanged for the corresponding parts of ETB. The opposite effect, namely a switch from a non-isopeptide-selective to an ET-1-selective binding, was observed for the mirror ETB chimeras where the symmetrical exchange had been operated. Using 125I-ET-1 and the ETA-specific antagonist cyclo-(D-Trp-D-Asp-Pro-D-Val-Leu) (BQ123), we were able to map the main determinants responsible for this selectivity to the N-terminal moiety of this receptor. Therefore, the ability for the interaction with ET-3 or BQ123 is governed by two different regions of the ET receptors.

Amino Acid Sequence↗
PubMed1994

Purification of human big endothelin 1 derived through cleavage with collagenase and dipeptidylpeptidase IV from a fusion protein expressed in Escherichia coli.

The cDNA coding for human big endothelin 1 (bigET-1), preceded by an optimized collagenase recognition sequence and followed by a stop codon, was fused in frame to the C-terminal region of alkaline phosphatase (AP). The fusion protein (AP-bigET), expressed in Escherichia coli K12 upon the lowering of organic phosphate concentrations, consisted of alkaline phosphatase (1-447), the collagenase cleavage site (Gly-Pro-Ala)4, and glycylprolyl-bigET-1. AP-bigET accumulated intracellularly in the form of inclusion bodies that were extensively washed and finally extracted by 8 M urea to yield highly enriched AP-bigET. Upon digestion of the fusion protein with collagenase, two disulfide conformeres of glycylprolyl-bigET-1 (bigET-1A and bigET-1B) could be purified by reverse-phase FPLC. Upon treatment with dipeptidylpeptidase IV to remove the N-terminal glycylprolyl-dipeptide, the later-eluting form of bigET-1 (bigET-1B) coeluted with authentic human bigET-1 on reverse-phase HPLC. BigET-1A and bigET-1B were formed at a ratio of 1:3. After reduction and S-pyridylethylation, both conformers coeluted with authentic but reduced bigET-1. Their amino acid sequences were identical. Both forms were converted by digestion with pepsin to the respective ET-1 conformeres (ET-1A and ET-1B) that were purified. In vasoconstriction assays, ET-1B but not ET-1A, at 10(-8) M, evoked a maximal response indistinguishable from that of authentic ET-1.

Alkaline Phosphatase↗
PubMed1994

The influence of diazepam on learning processes impaired by pentylenetetrazol kindling.

Repeated administration of pentylenetetrazol (PTZ) induces kindling and impairs shuttle-box learning. The available literature suggesting a close connection between seizure frequency and mental deficits in human epileptics allows us to hypothesize that seizure inhibition prevents the progressive mental retardation associated with kindling. In order to investigate the effect of motor seizure inhibition on mental impairment we administered diazepam (DZP) doses of 0.5 and 2.5 mg/kg, respectively 60 min prior to the 10 convulsant injections. After completion of kindling the learning performance of the rats was tested in the shuttle-box. PTZ kindling resulted in diminished shuttle-box learning. In control rats treated with DZP no significant changes in their learning ability occurred. Although DZP was found to suppress kindling development effectively a worsened shuttle-box learning could be observed in all PTZ groups treated with DZP.

Animals↗
PubMed1994

Effects of the peptide BCH-325 upon the efficacy of common antiepileptic drugs.

It was shown that BCH-325 (Pro-D-Phe-Pro-Gly), a des-tyrosine derivative of beta-casomorphin, exhibits anticonvulsive effects. In the present study, we combined the peptide with clinically used antiepileptic drugs (phenytoine, carbamazepine, diazepam, phenobarbital, and dipropyl acetate) to investigate possible interactions and, moreover, to draw conclusions concerning the mode of action of BCH-325. There were no interactions with phenytoine and carbamazepine, which might be interpreted to mean that BCH-325 exerted no action on sodium channels. The efficacy of the combinations with the other drugs (diazepam, phenobarbital, dipropyl acetate) was increased. The data suggest that the anticonvulsant effect of BCH-325 might occur by interference with elements of GABA/benzodiazepinergic neurotransmission.

Amino Acid Sequence↗
PubMed1994

Purification, cloning, and expression of a human enzyme with acyl coenzyme A: cholesterol acyltransferase activity, which is identical to liver carboxylesterase.

An enzyme with acyl coenzyme A:cholesterol acyltransferase (ACAT) activity was isolated from porcine liver, and sequences derived from trypsinized peptides indicated homology to liver carboxylesterase. By use of degenerate primers, human cDNA clones were identified, which were identical to human liver carboxylesterase. Expression of the full-length cDNA in Chinese hamster ovary (CHO) cells led to an approximately threefold increase in cellular ACAT activity. This was accompanied by an approximately 20-fold increase of cellular cholesteryl ester content. By light and electron microscopy, recombinant CHO cells contained numerous lipid droplets that were not present in control CHO cells. Expression of an antisense cDNA in HepG2 cells reduced cellular ACAT activity by 35% compared with control. To further investigate the role of the enzyme in cellular cholesterol homeostasis, regulation of the mRNA was investigated in 7-day cultured human mononuclear phagocytes (MNPs). When these cells were incubated in lipoprotein-deficient serum for 18 hours, the mRNA for ACAT/carboxylesterase was almost not detectable on Northern blots, whereas after incubation with acetylated low-density lipoproteins, a strong hybridization signal was obtained. This is evidence that the mRNA of ACAT/carboxylesterase is induced by cholesterol loading. It is concluded from the data presented that ACAT/carboxylesterase is relevant for cellular cholesterol esterification in vivo. The regulation in MNPs indicates that the enzyme is also involved in foam cell formation during early atherogenesis.

Amino Acid Sequence↗
PubMed1994

[Cytogenetic studies of Chilean children with acute lymphoblastic leukemia].

Acute lymphoblastic leukemia (ALL) is the most frequent childhood cancer. The leukemic cells of ALL patients show several well defined numeric and structural chromosomal abnormalities which are universally known for its prognostic implications. We studied a group of 44 children with ALL, to investigate the incidence of chromosome aberrations in ALL, its lymphocyte lineage and some clinical feature associations, and the finding of non previously described aberrations. A high proportion of patients (79.5%) showed chromosomal abnormalities. Most of them had a pseudodiploid karyotype (46 chromosomes), characterized mainly by a translocation. In relation to chromosome number, 27% of them were hyperdiploid with more than 50; 9% hyperdiploid between 47-50 and 7% hypodiploid (less than 46). Among structural aberrations found, were the following recurrent translocations: t (1; 19), t (4; 11), t (9; 22) in 6.8%, 9.1% and 2.3% of cases respectively, all related to an early B immunophenotype. Other translocations found, compromised regions 7q22, 9p21 - 24. Two new translocations in ALL were found: t (1; 5)(q23; q33), apparently balanced, and t (13; 21)(q14; q22), unbalanced. Other recurrent structural changes found were: deletion (6q), (7q), (9p), (11q), (12p), inversion (3q), isochromosome (7q), maker chromosomes and double minutes. The distribution of chromosome abnormalities in this group of patients was in agreement with previous reports from other investigators.

Adolescent↗
PubMed1994

[Anemia associated with acute post streptococcal glomerulonephritis].

Thirty-three children with post-streptococcal acute glomerulonephritis, age mean: 8.3 years (range: 6 - 12) were studied prospectively. Mean initial hematocrit (Hct) was 31.6% with 90% showing Hct under the normal lower limit for this age group. Reticulocyte index (RI) was < 0.5 in half of the cases. Serum iron concentration, total iron binding capacity (TIBC) and percentage of transferrin saturation were normal for this age group although 75% of the children had increased serum ferritin levels. At the time of discharge, Hct increased to 35.1% but 44% still had anemia. Hct increased spontaneously for 105 days stabilizing at 38%. Based on Hct changes, 3 groups were defined: Group I (3 individuals): normal upon discharge; Group II (19): partial recovery at discharge, slow recovery stabilizing after 105 days; Group III (11): lower Hct, slower recovery but with RI significantly higher than group II (0.96 vs 0.45 p < 0.01). Our data suggest that although hemodilution is present in all, it may be considered the solely factor only in 3 cases (Group I). In group II, evidence of bone marrow depression was indicated by the low RI. On the other hand, the intense anemia that could not be justified only by hemodilution and marrow depression in group III, suggests other pathogenic factors.

Acute Disease↗
PubMed1993

Stable expression of human endothelin receptors ETA and ETB by transfected baby hamster kidney cells.

The cDNAs for human endothelin receptors ETA and ETB were subcloned into the eukaryotic expression vector pMPSV/CMV and transfected, in parallel with plasmids carrying resistances for hygromycin B and puromycin, into baby hamster kidney (BHK) cells. Cell clones constitutively expressing high levels of either receptor were obtained through the combined selective pressure of both antibiotics. This was further confirmed by Northern blot analysis using ETA- or ETB-specific oligonucleotide probes. The calculated KD for endothelin (ET)-1 binding to ETA and ETB were 2.2 x 10(-10) M and 5.3 x 10(-10) M, respectively. Competitive binding experiments using the different ET forms showed the expected isopeptide-selective and non-isopeptide-selective profiles for ETA and ETB, respectively. BQ 123, a specific ETA antagonist, competed with ET-1 for binding to ETA but not to ETB.

Animals↗
PubMed1993

Characterisation of the ATP-dependent taurocholate-carrier protein (gp110) of the hepatocyte canalicular membrane.

The canalicular domain-specific glycoprotein gp110, which recently has been shown to function as an ATP-dependent taurocholate transporter, has been purified 1800-fold from rat liver plasma membranes. gp110 has been characterised as an integral plasma membrane protein with M(r) of 100,000-115,000 and pI of 2.5-3.5 and possesses a highly glycosylated and negatively charged extra-cellular domain. The broad range of M(r) and pI values results from the existence of numerous glycoforms composed of sialylated N-glycans. After deglycosylation, the polypeptide has M(r) 48,000 and pI 5.0. In primary cultures of rat hepatocytes, gp110 is synthesised with M(r) 110,000, while in the presence of tunicamycin the non-glycosylated form has M(r) 48,000. In the presence of 1-deoxymannojirimycin, two forms of M(r) 83,000 and M(r) 91,000 were found, which were converted by endo-beta-N-acetylglucosaminidase H into a single 52,000-M(r) band, indicating the existence of two basic glycoforms at the oligomannosyl stage of biosynthesis. gp110 was phosphorylated at serine residues in primary cultures of hepatocytes. The sequences of ten internal peptides of gp110 were identical to the sequence of the high-M(r) form of ecto-ATPase, but ecto-ATPase activity from plasma-membrane extracts was not depleted by anti-(gp110) serum. In contrast, Fab fragments of these antibodies inhibit the aggregation of freshly isolated hepatocytes.

ATP-Binding Cassette Transporters↗
PubMed1993

Expression of human endothelin receptor ETB by Escherichia coli transformants.

Membrane fractions from Escherichia coli transformed with an expression vector for the human endothelin receptor ETB were found to exhibit specific and saturable binding sites for labeled ET-1. Binding properties were similar to those observed in eukaryotic cells: KD was 110 pM and the three endothelin isoforms were equipotent in competition experiments. The importance of the cysteines located in each of the three extracellular loops was assessed by replacing them individually with alanine residues. Impaired binding was observed in each case but the effect was far more pronounced for the cysteine mutants of the first and second loop pointing to their critical importance for active ETB expression.

Base Sequence↗
PubMed1993

Bibrotoxin, a novel member of the endothelin/sarafotoxin peptide family, from the venom of the burrowing asp Atractaspis bibroni.

A new member of the endothelin/sarafotoxin family of vasoconstrictor peptides, bibrotoxin (BTX), was isolated from the venom of the burrowing asp Atractaspis bibroni by reversed-phase FPLC. The amino acid sequence of BTX differs from SRTX-b in the substitution Ala4 instead of Lys4, which suggests that it represents the peptide isoform of Atractaspis bibroni corresponding to SRTX-b. BTX competed for [125I]ET-1 binding to human ETB-type receptor with a Ki of 3.2 x 10(-9) M compared to 4.2 x 10(-9) M for SRTX-b. In rat thorax aorta BTX induced vasoconstrictions with a threshold concentration of 3 x 10(-8) M compared to 1 x 10(-9) for ET-1.

Amino Acid Sequence↗
PubMed1993

Analysis of the Rhizobium meliloti exoH/exoK/exoL fragment: ExoK shows homology to excreted endo-beta-1,3-1,4-glucanases and ExoH resembles membrane proteins.

Nucleotide sequencing of a 4.15 kb DNA fragment from megaplasmid 2 of Rhizobium meliloti 2011 revealed the location of the genes exoH, exoK and exoL. The putative proteins encoded by these genes have molecular weights of 41, 30, and 44 kDa, respectively. The hydrophobicity profile of the ExoH amino acid sequence resembles that of transmembrane proteins. The predicted exoL gene product does not contain hydrophobic regions, indicating a cytoplasmic localization. The exoK gene product is characterized by a putative signal peptide and exhibits significant homology to endo-beta-1,3-1,4-glucanases of bacilli and Clostridium thermocellum. R. meliloti exoK mutants induced pink nodules and synthesized a reduced amount of exopolysaccharide (EPS). Colonies of this mutant showed a delay in the appearance of the Calcofluor white fluorescence. In addition, the formation of the characteristic halo was strongly delayed. R. meliloti exoL and exoH mutants induced pseudonodules. The exoH, but not the exoL mutant, synthesized an EPS that could be precipitated by cetyl pyridinium chloride (CPC) and also by ethanol. Plasmid integration mutagenesis revealed promoter regions preceding exoH, exoK and exoL.

Amino Acid Sequence↗
PubMed1993

Identification and analysis of the Rhizobium meliloti exoAMONP genes involved in exopolysaccharide biosynthesis and mapping of promoters located on the exoHKLAMONP fragment.

Sequence analysis of a 7.494 kb DNA fragment from megaplasmid 2 of Rhizobium meliloti 2011 involved in exopolysaccharide I (EPS I) biosynthesis revealed the presence of five exo genes designated exoA, exoM, exoN, exoO, and exoP. ExoN was found to show strong homology to a UDP-glucose pyrophosphorylase from Acetobacter xylinum, whereas ExoO displayed weak homologies to the NodC proteins from R. meliloti and R. loti. Surprisingly, different mutations in exoP resulted in divergent phenotypes. One exoP mutant was able to establish an effective symbiosis with alfalfa, although no EPS I polymer could be detected. In contrast, other exoP mutations prevented the formation of an effective symbiosis. The transcriptional organization of the exoA-exoP gene region has been analysed in conjunction with the exoH, exoK and exoL genes. Using exo-lacZ transcription fusions in association with plasmid integration mutagenesis a strong promoter was identified upstream of exoH, which is able to direct transcription of the whole exoHKLAMONP gene cluster. A much weaker promoter upstream of exoL was found to be involved in the transcription of the exoLAMONP genes. In addition, weak promoters were identified upstream of exoK, exoA, exoN and exoP.

Amino Acid Sequence↗
PubMed1993

Effect of antihypertensive drugs on glomerular hyperfiltration and renal haemodynamics. Comparison of captopril with nifedipine, metoprolol and celiprolol.

Glomerular hyperfiltration and hypertension may contribute to the progression of chronic renal insufficiency regardless of the underlying disease. Protein restriction and antihypertensive treatment are used to slow the decline in renal function. However, little is known about the interaction of protein loading and antihypertensive treatment on glomerular haemodynamics in humans. This paper compares the renal haemodynamic effects of beta-adrenoceptor blockers with those of the calcium channel antagonist nifedipine and the ACE inhibitor captopril on resting glomerular filtration and during glomerular hyperfiltration. In two separate studies the effects of nifedipine, captopril, metoprolol, and celiprolol on renal haemodynamics have been investigated. In two groups of healthy volunteers (n = 13) inulin and PAH clearances were measured, first under fasting conditions and afterwards during aminoacid infusion. In fasting subjects nifedipine and metoprolol induced glomerular hyperfiltration, while celiprolol and captopril did not significantly affect GFR. Without premedication, and also after nifedipine, metoprolol and celiprolol, the aminoacid infusion significantly increased the GFR. After premedication with captopril, however, aminoacid-induced hyperfiltration was prevented. In fasting subjects captopril, celiprolol and metoprolol elevated PAH clearance. With our without premedication aminoacid infusion increased renal plasma flow compared to baseline on the control day. We conclude that in healthy subjects, acute administration of antihypertensive drugs results in different renal haemodynamic responses. In contrast to captopril and celiprolol, nifedipine and metoprolol induce glomerular hyperfiltration like protein loading. Thus, they may counteract the renal haemodynamic effects of protein restriction. Celiprolol behaves similarly to captopril, since it increases renal perfusion without inducing glomerular hyperfiltration, a pattern which might reflect lower glomerular pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Antihypertensive Agents↗
PubMed1993

Resorbed lateral incisors adjacent to impacted canines have normal crown size.

The importance of detecting maxillary canine impaction is that it may compromise dental health, particularly since a small but significant proportion of impacted canines is associated with the resorption of the roots of neighboring teeth. It has been shown that the existence of small and peg-shaped lateral incisors is highly correlated with maxillary canine palatal impaction. To date, however, it has not been established whether the root resorption that occurs has a similar correlation or a predilection for one or other types of lateral incisor crown structure. Accordingly, a group of patients with buccally or palatally impacted canines, in which root resorption of the lateral incisor could be diagnosed roentgenographically, was examined and compared with a group of cases with palatally impacted canines, none of which showed root resorption, which served as controls. The mesiodistal crown dimension of the lateral incisor in the experimental group was found to be normal in all patients except three (13%), which was significantly different from the distribution of lateral incisor size in the control group (p < 0.001). In the majority of the cases, aggressive root resorption of the lateral incisor root had occurred. We would speculate that in these cases, the normal-sized and early developing lateral incisor root obstructs the deviated eruption path of the canine and consequently stands a considerably greater chance of being damaged by resorption.

Adolescent↗