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Biomedical subjects

O Reiner

Publications and source records attributed to O Reiner.

46 records · Page 3Linked to original sources
PubMed1988

Differential expression of the human glucocerebrosidase-coding gene.

Gaucher disease is an inborn error of sphingolipid metabolism. It is due to decreased enzymatic activity of glucocerebrosidase (GCase) which causes accumulation of glucocerebrosides, mainly in cells of the reticulo-endothelial system. The disorder is clinically heterogenous and can include central nervous system signs. However, the manifestations of the disease in most cases are restricted to a limited number of cell types and organs. This could be explained by highly differential expression of the human gcs gene. To test this notion, the level of GCase-specific mRNA was determined in different human cell lines by hybridizing Northern blots to a human GCase-specific cDNA probe or by using the RNase protection method. It was found that epithelial cells exhibit high levels of GCase mRNA while skin fibroblasts and promyelocytes show intermediate steady-state levels of this RNA. Macrophages have low steady-state levels of GCase mRNA and in B-cells it is hardly detectable. Moreover, when B-cells or skin fibroblasts were transfected with a vector harbouring the bacterial cat gene coupled to the human gcs gene promoter, the levels of CAT expressed in each cell type were directly correlated to the amount of endogenous GCase RNA. Comparison of the GCase mRNA levels in Gaucher-versus non-Gaucher-derived cells revealed that in Gaucher cells this RNA is always more abundant than in the corresponding non-Gaucher counterparts, suggesting the involvement of a feed-back mechanism sensitive to the levels of actual enzymatic activity.

Blotting, Northern↗
PubMed1988

Structural analysis of the human glucocerebrosidase genes.

Two different genomic clones containing the entire coding sequence of human glucocerebrosidase were isolated from a fetal liver library using a cDNA probe previously cloned by us. These clones correspond to two human glucocerebrosidase genes, designated 6-1 and 10-2. Clone 6-1 contains sequences homologous to the cDNA we cloned previously. The promoter regions of the genes were identified by S1 analysis and sequenced. They contain TATA- and CAAT-like boxes, but lack a GGCGGG motif. When coupled to the bacterial gene coding for chloramphenicol acetyl transferase (CAT) and transfected to Gaucher skin fibroblast lines, both promoter fragments enhanced CAT activity. The promoter of gene 6-1 was eight times more efficient than the promoter of gene 10-2. Northern blot analysis revealed three human glucocerebrosidase RNA species of 6, 2.6, and 2.2 kb in size. The 6-kb transcript is probably a nuclear transcript whereas the 2.6-kb and 2.2-kb transcripts are cytoplasmic species which emerge from polyadenylation at different sites.

Base Sequence↗
PubMed1987

Efficient in vitro and in vivo expression of human glucocerebrosidase cDNA.

A human glucocerebrosidase cDNA clone was isolated from a human chronic myelogenous leukemia (line K562) cDNA library using a 36-nucleotide-long synthetic probe (GC-36). The 2.4-kb cDNA contains 184 bp of 5' nontranslated sequences, the complete coding region, and 546 bp of 3' nontranslated sequences followed by 100 bp of poly(A). A primer extension experiment indicated that the cDNA is at least 51 bp shorter than the mRNA at the 5' end. In normal human placenta as well as in fibroblasts from Gaucher's disease patients, a major mRNA species of 2.6 kb hybridizes with the cDNA probe. The amounts of the glucocerebrosidase mRNA in normal placenta and Gaucher's cells are comparable. The cDNA was linked to the SP6 promoter and transcribed in vitro. The resultant RNA, when translated in a cell-free system, yielded a polypeptide of 55 kD, which is the size expected from the coding sequence. The cDNA was inserted into an SV40 shuttle vector, under the transcription control of the SV40 early promoter. COS-M6 cells were transfected with this construct and the biological activity of the cDNA was assayed by monitoring the increase in glucocerebrosidase activity, using 4-methyl umbiliferyl glucopyranoside as a substrate. There was a two- to three-fold increase in enzymatic activity in the transfected cells, compared to nontransfected cells. These results prove the authenticity of the glucocerebrosidase cDNA and provide the basis for experiments to understand the nature of the genetic alterations responsible for Gaucher's disease.

Cloning, Molecular↗
PubMed1978

The role of the electrogenic sodium pump in the potassium relaxation of the rabbit ear artery.

1. The mechanism of the potassium-induced relaxation of contracted vascular smooth muscle has been studied in helical strips of rabbit ear artery by recording isometric tension and membrane potential. The arteries were stimulated with a standard concentration of 2 x10(-8) M noradrenaline producing about 20% of the maximal contractile response to noradrenaline. 2. At K+ concentrations between 1.2 and 20.0 mM, a positive correlation was found between [K+]0 and the contractile response to noradrenaline, i.e. increasing [K+]0 enhanced the effect of noradrenaline. However, when noradrenaline was added after a 10 min exposure to reduced [K+]0 (1.2 mM) and [K+]0 was then increased to 5.9 mM after the tension had reached a plateau, a relaxation of about 50% occurred instead of the expected increase in tension. The relaxation was preceded by a membrane hyperpolarization of about 8 mV. 3. A biphasic change of both membrane potential and tension resulted when, after exposure of arterial strips to 1.2 mM K+ and stimulation with noradrenaline, [K+]0 was increased to 20.0 mM: hyperpolarization and relaxation developed faster than at 5.9 mM K+, but after 1 min the hyperpolarization changed to a depolarization followed by an increase in tension. 4. Ca2+-free solution and D 600 (an inhibitor of Ca2+ transmembrane flux) diminished, but did not abolish, the tension response to noradrenaline; in the presence of D 600, the hyperpolarization upon increasing [K+]0 after a previous exposure to 1.2 mM K+ was not affected, but a relaxation no longer occurred. 5. It is concluded that a) electrogenic ion transport causes the hyperpolarization that occurs when [K+]0 is increased after a period of exposure to low [K+]0; b) the hyperpolarization leads to relaxation by blocking the influx of extracellular Ca2+, but does not affect that component of the contractile response which is due to the release of intracellular Ca2+.

Animals↗
PubMed1976

Action of prostaglandin, PGF2alpha, on the uterus of the pregnant rat.

The effects of prostaglandin F2alpha (PG) have been studied on the transmembrane potentials and contractions in isolated myometrial strips from pregnant rats. The results showed that: 1. The sensitivity of the myometrium to exogenous PG increases from day 19 to day 22 of gestation. 2. The electrical response to PG, at maximally effective doses (10-6 to 10-5 M) consists of a slow depolarization which upon reaching threshold initiates spike discharge. 3. These actions are most pronounced at term (day 22) and are due to a direction of PG on the myometrial cells. 4. D-600 (a methoxy derivative of verapamil) abolishes spike discharge and the phasic contractions induced by PG but has no effect on the slow depolarization and the accompanying increase in tonic tension. 5. The slow depolarization is dependent upon the presence of sodium in the external environment and is unaffected by the removal of calcium. 6. The spikes (and phasic contractions) are dependent upon the presence of calcium in the external environment.

Animals↗
PubMed1975

Action of D-600 on spontaneous and electrically stimulated activity of the parturient rat uterus.

The effects of D-600, the methoxy derivative of verapamil, on the parturient rat uterus have been studied. At concentrations of 10(-8) to 10(-7) M, D-600 abolishes spontaneous contractions and reduces the force of electrically evoked contractions. The first action is associated with a reduction in amplitude of the slow waves (oscillations of membrane potential which underlie rhythmic bursts of spikes initiating contractions). The second is associated with a reduction in frequency of spikes within the bursts accompanying the electrically stimulated contractions. This reduction results from a decrease in the slope of the prepotential in cells in pacemaker areas of the muscle. D-600 also decreased the height and rising velocity of the conducted action potentials, but these actions cannot account entirely for the reduction in contractile force.

Action Potentials↗
PubMedDate not supplied

The unfolding story of two lissencephaly genes and brain development.

Formation of our highly structured human brain involves a cascade of events, including differentiation, fate determination, and migration of neural precursors. In humans, unlike many other organisms, the cerebral cortex is the largest component of the brain. As in other mammals, the human cerebral cortex is located on the surface of the telencephalon and generally consists of six layers that are formed in an orderly fashion. During neuronal development, newly born neurons, moving in a radial direction, must migrate through previously formed layers to reach their proper cortical position. This is one of several neuronal migration routes that takes place in the developing brain; other modes of migration are tangential. Abnormal neuronal migration may in turn result in abnormal development of the cortical layers and deleterious consequences, such as Lissencephaly. Lissencephaly, a severe brain malformation, can be caused by mutations in one of two known genes: LIS1 and doublecortin (DCX). Recent in vitro and in vivo studies, report on possible functions for these gene products.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗