Search PubMed⌕ Search

Biomedical subjects

A Klein

Publications and source records attributed to A Klein.

At least 163 records · Page 9Linked to original sources
PubMed1996

The occurrence of protein kinase C theta and lambda isoforms in retina of different species.

The localization and immunochemical identification of the novel protein kinase C theta (nPKC theta) and the atypical protein kinase C lambda (aPKC lambda) isoforms in retinas of different species were analyzed by immunohistochemistry and SDS-PAGE/Western blotting. nPKC theta immunoreactivity is associated with bipolar cells of mammalian (rabbit, rat and guinea pig) retinas but not the non-mammalian goldfish retina which has a lower concentration of nPKC theta. However, SDS-PAGE and Western blotting data indicate the antigen recognized by the nPKC theta monoclonal antibody in the retina is of a lower molecular weight than that expected for nPKC theta. This would suggest nPKC theta is more susceptible to degradation/breakdown than other PKC isoforms found in the retina or that the nPKC theta antibody may be recognizing an unknown retinal antigen. A comparison of nPKC theta and cPKC alpha immunoreactivities in bipolar cells shows unique distributions exist for the two isoforms. nPKC theta is present in the developing retina at an earlier stage than cPKC alpha. The typical 'transport' of cPKC alpha toward axonal terminals by phorbol-12,13-dibutyrate does not occur for nPKC theta yet both are translocated from the cytosolic to membrane compartments. The inner plexiform layer and the inner nuclear layer (putative horizontal cells) of all species examined (rabbit, rat, guinea pig and goldfish) exhibited positive immunoreactivity for aPKC lambda as confirmed by SDS-PAGE/Western blotting.

Animals↗
PubMed1996

The calcium channel agonist BAY k 8644 reduces ethanol intake and preference in alcohol-preferring AA rats.

Applying a 12-h limited access, two-bottle choice procedure, antialcohol effects of the 1,4-dihydropyridine (DHP) L-type calcium (Ca2+) channel agonist BAY k 8644 were investigated in alcohol-preferring AA rats. In this Wistar line, selectively bred for a high 10% v/v ethanol (EtOH) preference in a free choice situation, effects on EtOH preference and intake, as well as on food and total fluid intake were evaluated for racemic BAY k 8644 (0.1-1 mg/kg IP; 0.25-2 mg/kg PO), its agonistic (-)-enantiomer (0.1-1 mg/kg IP and PO) and its antagonistic (+)-enantiomer (10-50 mg/kg IP and PO). Irrespective of route of application, BAY k 8644 was found to be effective in reducing both EtOH intake and preference (minimal effective dose: 0.5 mg/kg; maximum effect: approximately 60% of baseline levels). The (+)-enantiomer, acting as a low-potency Ca2+ channel antagonist, also reduced EtOH intake and preference, but the effects were not very selective as food intake was also substantially reduced. Moreover, the effects were only obtained at relatively high doses (50 mg/kg). The essential enantiomer involved in the antialcohol effects of BAY k 8644 seems to be the (-)-enantiomer, acting as a strong Ca2+ channel agonist. This latter compound was potent (minimal effective dose: 0.3 mg/kg), very effective in reducing EtOH intake (maximum effect: 29% of baseline level) and preference (26% of baseline) and apparently more selective. Although slightly decreasing over days, effects of (-)-BAY k 8644 on EtOH intake and preference were shown to remain after repeated treatment (10 successive days, 0.3 mg/kg IP). Interestingly, the acute antialcohol effects of (-)-BAY k 8644 (0.3-1 mg/kg IP) could not be antagonized with the DHP L-type Ca2+ channel antagonists nimodipine (0.01-1 mg/kg IP) and (-)-nimodipine (1-30 mg/kg IP). The present results suggest that a mechanism of action other than L-type Ca2+ channel agonism is involved in the antialcohol effects of (+/-)- and (-)-BAY k 8644. Alternatively, it is possible that the previously described antialcohol effects of DHP Ca2+ channel antagonists are not related to antagonistic activity at Ca2+ channels. Finally, it cannot be excluded that a mechanism unrelated to Ca2+ channels is responsible for the antialcohol effects of both DHP Ca2+ channel agonists and antagonists.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1996

Development of quantitative Doppler indices for uteroplacental and fetal blood flow during the third trimester.

The aim of our study was to describe the development of uteroplacental and fetal blood flow during the third trimester. Doppler examination was carried out on 393 uncomplicated pregnancies with uncomplicated term delivery. Using a pulsed color Doppler, we calculated the maximum systolic, mean and maximum end-diastolic velocity after correcting the angle of insonation. Patients under tocolysis or other medication influencing blood flow parameters were excluded from this cross-sectional study. Summarizing the results gained by Doppler ultrasound investigation of the uteroplacental and fetal blood vessels, we created quantiles as quantitative Doppler indices for the maximum systolic, mean (TAMX = time averaged maximum velocity) and maximum end-diastolic velocity. The following conclusions could be drawn: (1) resistance to the blood flow in the maternal portion of the placenta does not change during the third trimester; (2) resistance to the blood flow on the fetal side of the placenta decreases up to week 42 of gestation; (3) cerebral vascular resistance decreases constantly up to gestational week 42; and (4) vascular resistance to the blood flow of the kidney decreases only slightly during the third trimester. This study offers clinically important values for quantitative Doppler flow velocimetry for the first time. We hope that our findings improve the usefulness of Doppler ultrasound as a diagnostic tool in obstetrical management.

Blood Flow Velocity↗
PubMed1996

Assessing myocardial perfusion with Albunex during coronary artery bypass surgery: technical considerations and safety of aortic root injections.

OBJECTIVE: To test the safety and report on limiting technical considerations, including optimal dosing of Albunex (Molecular Biosystems, Inc, Mallinckrodt Medical, St. Louis, MO) for myocardial opacification after intra-aortic root injections during cardiac surgery. DESIGN: This was a prospective randomized study with a control group who did not receive Albunex and a group who received intra-aortic root injections of Albunex. SETTING: Multicenter (two) independent university hospitals. PARTICIPANTS: 32 patients scheduled for elective coronary artery bypass surgery were evaluated after individual informed consent was obtained. INTERVENTIONS: 2 to 8 mL of Albunex were injected before and after coronary revascularization. MEASUREMENTS AND MAIN RESULTS: Quality of enhancement in each of four regions of the left ventricle was assessed from a short-axis mid-papillary ultrasound image by three experienced observers blinded to dose. Electrocardiogram (ECG), creatine phosphokinase (CPK) (MB fraction), and hemodynamics were evaluated at baseline and throughout the study period for up to 72 hours. No differences were noted between groups with respect to preoperative and postoperative CPK enzymes (CPK-MB fraction), ECG changes, hemodynamics, requirements for separation from CPB, need for postoperative inotropes, time to extubation, and time to discharge from the intensive care unit. The average total dose of Albunex injected was 19 mL +/- 4 (0.25 mL/kg). A single dose of 4.2 +/- 1.2 mL (0.05 mL/kg) appeared to offer optimal enhancement of contrast effect for myocardial perfusion assessment. CONCLUSION: Albunex is safe and easy to use for myocardial opacification when administered via an antegrade cardioplegia catheter into the aortic root during CPB.

Aged↗
PubMed1996

Isolation and characterization of insertional mutations in flagellin genes in the archaeon Methanococcus voltae.

Methanococcus voltae is a flagellated member of the Domain Archaea that has four flagellin genes arranged in two transcriptional units. One transcriptional unit encodes only flaA while the second is a multi-cistronic unit encoding three flagellin genes (flaB1, flaB2, and flaB3) as well as at least seven other open reading frames downstream. The polymerase chain reaction was used to amplify an internal fragment of the flaA gene which was subsequently cloned into an insertion vector developed for M. voltae. Transformation of protoplasts with this vector led to the isolation of mutant strains that had insertions in flaA or flaB2. Mutant strains carrying insertions in flaA had flagelia that were similar to wild-type cells in both number and appearance when viewed using the electron microscope. In addition, some of these mutant strains had profiles identical to the wild type in immunoblots developed with antisera raised against the 31 kDa flagellin of M. voltae. All flaA mutant strains and the wild-type cells showed immuno-cross-reactive bands at 33 and 31 kDa (corresponding to purified flagellins) as well as at 18 kDa. Some flaA mutant strains also showed an immuno-cross-reactive band at 27 kDa which probably represents a truncated flagellin produced by the insertion vector. However, both types of flaA mutant strains were less motile than the wild type in semi-swarm plate experiments. The mutant strain with an insertion in flaB2 was non-flagellated when examined by electron microscopy and it was non-motile in semi-swarm plate experiments. It represents the first structural mutant strain isolated in a methanogen. This mutant strain lacked the 33, 31, and 18 kDa immuno-cross-reactive bands observed in the wild type and flaA mutant strains, and instead had a novel band at 20 kDa. This band may represent an unmodified flagellin which still has an attached leader peptide. If so, then one of the downstream genes in the multi-cistronic transcriptional unit may encode a leader peptidase for the flagellin system.

Archaeal Proteins↗
PubMed1996

Antibabesial effect of the immunomodulator AS101 in mice: role of increased production of nitric oxide.

The immunomodulator AS101 has been shown to induce cell proliferation and to increase the secretion of a variety of cytokines. In the present study we evaluated the effect of AS101 on the pathogenicity of B. rodhaini infected mice. In order to clarify its mechanism of action we studied the ability of AS101 to activate neutrophils and macrophages, both of which inhibit parasite growth. More specifically, we studied the ability of AS101 to induce secretion of nitric oxide (NO). We found that AS101 protects mice from babesiosis in a time and dose dependent manner. At 10 and 20 micrograms/injection, two weeks prior to parasites, AS101 significantly increased the number of neutrophils and more than doubled the survival rate of infected mice. Similarly, at these concentrations when injected one month, or at 20 micrograms, injected 24 h before parasites. AS101 mitigated the course of infection and reduced by half the peak of parasitaemia. At 0.1 microgram/ml AS101 induced the secretion of significantly higher levels of NO in vitro than control. This was abrogated by adding the NO synthase inhibitor. NG-monomethyl-L-arginine. In vivo the antiparasitic protection of AS101 was abrogated by another NO synthase inhibitor, aminoguanidine. We found that AS101, partly by elevating levels of NO, can significantly mitigate the course of infection and thus increase survival, and may therefore be proven as an effective antiparasitic drug.

Adjuvants, Immunologic↗
PubMed1996

The SH3 domain of the Saccharomyces cerevisiae peroxisomal membrane protein Pex13p functions as a docking site for Pex5p, a mobile receptor for the import PTS1-containing proteins.

We identified a Saccharomyces cerevisiae peroxisomal membrane protein, Pex13p, that is essential for protein import. A point mutation in the COOH-terminal Src homology 3 (SH3) domain of Pex13p inactivated the protein but did not affect its membrane targeting. A two-hybrid screen with the SH3 domain of Pex13p identified Pex5p, a receptor for proteins with a type I peroxisomal targeting signal (PTS1), as its ligand. Pex13p SH3 interacted specifically with Pex5p in vitro. We determined, furthermore, that Pex5p was mainly present in the cytosol and only a small fraction was associated with peroxisomes. We therefore propose that Pex13p is a component of the peroxisomal protein import machinery onto which the mobile Pex5p receptor docks for the delivery of the selected PTS1 protein.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1996

Uptake and incorporation of an epitope-tagged sialic acid donor into intact rat liver Golgi compartments. Functional localization of sialyltransferase overlaps with beta-galactosyltransferase but not with sialic acid O-acetyltransferase.

The transfer of sialic acids (Sia) from CMP-sialic acid (CMP-Sia) to N-linked sugar chains is thought to occur as a final step in their biosynthesis in the trans portion of the Golgi apparatus. In some cell types such Sia residues can have O-acetyl groups added to them. We demonstrate here that rat hepatocytes express 9-O-acetylated Sias mainly at the plasma membranes of both apical (bile canalicular) and basolateral (sinusoidal) domains. Golgi fractions also contain 9-O-acetylated Sias on similar N-linked glycoproteins, indicating that O-acetylation may take place in the Golgi. We show here that CMP-Sia-FITC (with a fluorescein group attached to the Sia) is taken up by isolated intact Golgi compartments. In these preparations, Sia-FITC is transferred to endogenous glycoprotein acceptors and can be immunochemically detected in situ. Addition of unlabeled UDP-Gal enhances Sia-FITC incorporation, indicating a substantial overlap of beta-galactosyltransferase and sialyltransferase machineries. Moreover, the same glycoproteins that incorporate Sia-FITC also accept [3H]galactose from the donor UDP-[3H]Gal. In contrast, we demonstrate with three different approaches (double-labeling, immunoelectron microscopy, and addition of a diffusible exogenous acceptor) that sialyltransferase and O-acetyltransferase machineries are much more separated from one another. Thus, 9-O-acetylation occurs after the last point of Sia addition in the trans-Golgi network. Indeed, we show that 9-O-acetylated sialoglycoproteins are preferentially segregated into a subset of vesicular carriers that concentrate membrane-bound, but not secretory, proteins.

Acetylation↗
PubMed1996

Targeted disruption of the mouse sphingolipid activator protein gene: a complex phenotype, including severe leukodystrophy and wide-spread storage of multiple sphingolipids.

The four established or putative sphingolipid activator proteins derive from a large precursor protein encoded by a single gene. In addition to generating the four sphingolipid activator proteins, the precursor protein is suspected of having functions of its own, as, for example, a lipid binding/transport protein or a neurotrophic factor. The gene also appears to encode the Sertoli cell major sulfated glycoprotein. Sequence similarities have been noted with many other proteins of diverse functions. One patient and a fetus in a single family with a complete defect of this gene due to a mutation in the initiation codon exhibited complex pathological and biochemical abnormalities. Mutant mice homozygous for an inactivated gene of the sphingolipid activator protein precursor exhibit two distinct clinical phenotypes-neonatally fatal and later-onset. The latter develop rapidly progressive neurological signs around 20 days and die by 35-38 days. At 30 days, severe hypomyelination and periodic acid-Schiff-positive materials throughout the nervous system and in abnormal cells in the liver and spleen are the main pathology. Most prominently lactosylceramide, and additionally ceramide, glucosylceramide, galactosylceramide, sulfatide, and globotriaosylceramide are abnormally increased in the brain, liver, kidney, and their catabolism abnormally slow in cultured fibroblasts. Brain gangliosides are generally increased, particularly the monosialogangliosides. The clinical, pathological and biochemical phenotype closely resembles that of the human disease. This model not only allows further clarification of the physiological functions of the four individual sphingolipid activator proteins but also should be useful to explore putative functions of the precursor protein.

Animals↗
PubMed1996

Analysis of a splice-site mutation in the sap-precursor gene of a patient with metachromatic leukodystrophy.

Sphingolipid activator proteins (SAPs) are small, nonenzymatic glycoproteins required for the lysosomal degradation of various sphingolipids with a short oligosaccharide chain by their exohydrolases. Four of the five known activator proteins (sap-A-sap-D), also called "saposins," are derived from a common precursor by proteolytic processing. sap-B stimulates hydrolysis of sulfatides by arylsulfatase A in vivo. Its recessively inherited deficiency results in a metabolic disorder similar to classical metachromatic leukodystrophy, which is caused by a defect of arylsulfatase A. Here we report on a patient with sap-B deficiency. Reverse-transcription-PCR studies on the patient's mRNA revealed the occurrence of two distinct mutant species: one with an in-frame deletion of the first 21 bases of exon 6, the other with a complete in-frame deletion of this exon. The patient was homozygous for the underlying mutation, which was found to be a G-->T transversion within the acceptor splice site between intron e and exon 6, abolishing normal RNA splicing. Allele-specific oligonucleotide hybridization revealed that the parents and both grandfathers of the patient were carriers of this mutation. In order to analyze the fate of the mutant precursor proteins, both abnormal cDNAs were stably expressed in baby hamster kidney cells. Pulse-chase experiments showed that the deletion of 21 bp had no effect on the transport and the maturation of the encoded precursor. All sap forms except sap-B were detectable by immunochemical methods. The cDNA bearing a complete deletion of exon 6 encoded a shortened precursor of only 60 kD, and no mature SAPs were detectable. The carbohydrate chains of this polypeptide were of the high-mannose and hybrid type, indicating no transport of the mutant precursor beyond early Golgi apparatus. An endoplasmic-reticulum localization of this polypeptide was supported by indirect immunofluorescence analysis.

Animals↗
PubMed1996

Neurokinetic and myokinetic effects of the peptide xenin on the motility of the small and large intestine of guinea pig.

The effects of xenin 25, a peptide of the neurotensin/xenopsin family, were examined on the motility of the guinea pig jejunum and colon in vitro. In the jejunum, xenin induced a biphasic response: first a small relaxation and then a large contraction. In the colon, xenin induced relaxation. The tenia coli was contracted. Deletion or amidation of the C-terminal leucine inactivated xenin. A peptide sequence of 16 C-terminal amino acids was necessary to elicit a full response in the jejunum, whereas in the colon, the potencies of all fragments of xenin 25, including the 6 C-terminal amino acid sequence (xenin 6), were not significantly different from that of xenin 25. In the jejunum, contraction was abolished or reduced by tetrodotoxin, by atropine, by met-enkephalin, by antagonists to the tachykinin receptors NK1 and NK2 and by the P2-purinoceptor antagonist suramin. L-NNA, phentolamine, methysergide, hexamethonium and apamin had no effect. In the colon, all actions of xenin were tetrodotoxin-resistant; the potency of xenin to relax was reduced by apamin and suramin. The actions of xenin on small and large bowel were attenuated by the neurotensin receptor antagonist SR 48692. The xenin analog neurotensin was significantly less potent than xenin 25 in contracting the jejunum and more potent than xenin 25 in relaxing the colon. We conclude that xenin exerts excitatory effects on a neuronal subtype receptor in the jejunum, with participation of muscarinic, purinergic and tachykinin-related mechanisms. Xenin exerts relaxing effects on the colon by interaction with a myokinetic subtype receptor involving Ca(++)-dependent K+ channels and the P2-purinoceptor.

Animals↗