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Biomedical subjects

R Kuhn

Publications and source records attributed to R Kuhn.

At least 163 records · Page 9Linked to original sources
PubMed1990

Free flow electrophoresis for the purification of proteins: I. Zone electrophoresis and isotachophoresis.

The principles and some applications of free flow zone electrophoresis and isotachophoresis are described. The influence of (i) carrier electrolyte conductivity on the migration velocity and (ii) band shape on zone electrophoresis was investigated. The technique was found convenient for studying the effect of pH on the mobility of proteins to create a mobility curve. The purification of alcohol dehydrogenase from a crude yeast extract revealed the separation power of zone electrophoresis for complex protein mixtures. Without additional steps, a purification factor of 5.4, with a recovery of 97% alcohol dehydrogenase, was achieved. Free flow isotachophoresis was applied to the purification of immunoglobulins from human serum. Disadvantages of this technique are the time-consuming development of an optimized separation system and the empirical search for suitable spacers. Also, reaching of the steady state becomes increasingly difficult as the number of sample components increases.

Alcohol Dehydrogenase↗
PubMed1990

Free-flow electrophoresis for the purification of proteins: II. Isoelectric focusing and field step electrophoresis.

Two modes of continuous isoelectric focusing are described. The development of a natural pH gradient, consisting of a mixture of three buffer solutions, and the focusing behavior of human serum albumin is investigated. The advantages of isoelectric focusing in an artificial pH gradient of three buffer solutions are demonstrated on the purification of alpha-amylase from an E. coli protein extract. Furthermore the principle of field step electrophoresis is presented. The most important factors influencing the efficiency: (i) residence time, (ii) conductivity of the sample and (iii) sample zone width, are discussed. The use of a larger sized device to allow simultaneous multiple injections of the sample demonstrates the feasibility of scaling-up field step electrophoresis. This approach permits a throughput of about 20 mL sample solution per minute.

Bacterial Proteins↗
PubMed1990

A conserved element in the leader mediates post-meiotic translation as well as cytoplasmic polyadenylation of a Drosophila spermatocyte mRNA.

We have previously shown that Mst87F (previously called mst(3)g1-9), a gene which is exclusively expressed in the male germ line of Drosophila melanogaster, is subject to negative translational control. While transcription of this gene takes place premeiotically, translation occurs only after the elongation of spermatids is complete. We report here the identification of a sequence element within the first 45 nucleotides of the leader which is crucial for this translational regulation. Sequence comparison with six other genes, which form a gene family with Mst87F, shows the conservation of a twelve nucleotide element within this leader segment. It is found in all genes at positions +28 to +39 of the leader. Deletion of this element or alteration of two nucleotides by in vitro mutagenesis both lead to the breakdown of the translational control mechanism. The poly(A) tail of the Mst87F mRNA becomes longer and heterogeneous in length when the mRNA is recruited for translation. We present evidence that the control for this additional polyadenylation also resides within the conserved element of the leader.

Animals↗
PubMed1990

Subclinical course of cholesterol ester storage disease (CESD) diagnosed in adulthood. Report on two cases with remarks on the nature of the liver storage process.

An extremely benign variant of cholesterol ester storage disease (CESD) was diagnosed in two female patients aged 43 and 56 years. In one of them the course was entirely subclinical until a stroke at the age of 47, most probably a complication of secondary hyperlipoproteinaemia. The diagnosis was made accidentally in vivo during extensive examination for concomitant monoclonal gammapathy. The other patient (aged 56), still displays a fairly stable course with minor dyspeptic symptoms. The clinical findings in both patients were confined to moderate well tolerated hepatomegaly, hyperlipoproteinaemia of IIb type and xanthelasmata. Acid lipase activity was markedly deficient in peripheral leukocytes and cultured fibroblasts. These cases represent a rare adult variant the existence of which should be borne in mind in the differential diagnosis of chronic liver disease in advanced age and of hyperlipoproteinaemic states. The diagnostic criteria for the routine clinicopathological steps are summarized with emphasis on a special lipopigment deposition pattern, encompassing inhibition and modification of lipofuscin generation in hepatocytes and an excess of ceroid production in both portal and intralobular histiocytes. The varied ultrastructural appearance of the lysosomal limiting membrane complex is described.

Acetylesterase↗
PubMed1990

A monoclonal antibody recognizing selectively rat granulocytes.

In the present study, a murine monoclonal antibody, RK-4 (IgG1) specific for rat granulocytes and their precursors in blood and bone marrow is described. Results obtained by immunocytological and flowcytometry analysis showed this mAb to recognize selectively a surface antigen on neutrophils, eosinophils and basophils. Analysis of cryosections of various lymphatic and non-lymphatic organs demonstrates that this mouse antibody does not cross-react with any other structures. By flow cytometric analysis we found peripheral blood leukocytes to consist of about 15-20% granulocytes. RK-4 is therefore a valuable tool for studying differentiation and function of granulocytes under various conditions.

Animals↗
PubMed1990

Inhibition of epoxide hydrolase by valproic acid in epileptic patients receiving carbamazepine.

The effect of valproic acid (VPA) on the disposition of carbamazepine-10,11-epoxide (epoxide) was studied in five epileptic patients on chronic carbamazepine (CBZ) therapy. The individual pharmacokinetic parameters influencing epoxide disposition were determined in the presence and absence of VPA. VPA significantly decreased the clearance of unbound epoxide (an in vivo index of epoxide hydrolase activity), but did not appear to affect epoxide formation. VPA also increased the free concentrations of both CBZ and epoxide.

Adolescent↗
PubMed1990

The gene encoding peripheral myelin protein zero is located on mouse chromosome 1.

We have used somatic cell hybrids to map the gene encoding protein zero (P0), the major structural protein of peripheral myelin. Analysis of Southern blots of DNA obtained from these hybrids allows us to unambiguously assign the P0 gene to mouse chromosome 1. This observation indicates that mutations in the P0 gene do not account for Trembler, a chromosome 11 mutation that specifically affects myelination in the peripheral nervous system.

Animals↗
PubMed1989

Application of free flow electrophoresis to the preparative purification of basic proteins from an E. coli cell extract.

The application of free flow electrophoresis (FFE) to the purification of a basic protein from a complex protein mixture was investigated. For this purpose lysozyme (E.C.3.2.1.17) from hen egg white, serving as a model for a basic protein, was added to a crude E. coli cell extract and reisolated. For three techniques of FFE (zone electrophoresis, isoelectric focusing and field step electrophoresis), suitable electrolyte systems were developed. The purity, purification factor, recovery and throughput were determined for the optimized experiments. A combination of field step electrophoresis and zone electrophoresis gave the best purification factor (9.5) and the highest recovery (95%). The purification factors achieved in zone electrophoresis and isoelectric focusing were comparable to each other and ranged from 3.5 to 4.75. In isoelectric focusing, 94% of the enzyme activity was recovered. Zone electrophoresis gave recoveries of 82% and 87%, respectively. Purities of more than 95% were achieved with all the techniques described. With the exception of zone electrophoresis, all the techniques effected a concentration of the enzyme during the separation. Zone electrophoresis and field step electrophoresis were very simple in application.

Bacterial Proteins↗
PubMed1989

Free flow electrophoresis as a method for the purification of enzymes from E. coli cell extract.

The application of the four techniques of free flow electrophoresis (zone electrophoresis, isotachophoresis, isoelectric focusing and field step electrophoresis) for the purification of proteins from a complex protein mixture was investigated. For this purpose alpha-amylase (EC 3.2.1.1) from Aspergillus oryzae was added and reisolated from E. coli cell extract. The chosen enzyme and the biological extract are models for many industrial separation problems. In optimized experiments purity, purification factor, yield, throughput and efficiency were calculated. The best results were obtained with field step electrophoresis in combination with zone electrophoresis. High purity (0.82 mg enzyme/mg total protein) and high throughput (111 mL sample/h) were achieved using this technique. Field step electrophoresis gave the best throughput (330 mL sample/h), but low purity (0.63 mg enzyme/mg total protein). This technique can also be used for a simple concentration of the sample. With zone electrophoresis a purity of more than 0.95 mg enzyme/mg total protein was obtained, which was the best of all techniques. However, the enzyme concentration was decreased due to dilution with buffer solution after the separation. Isotachophoresis was the most difficult technique, combined with a relatively low recovery of 31% of the enzyme activity. In a purification scheme, free flow electrophoresis is able to substitute one or even several chromatography steps with a negligible loss of biological activity.

Aspergillus oryzae↗
PubMed1989

SCIP: a glial POU domain gene regulated by cyclic AMP.

We have isolated cDNA clones encoding SCIP, a POU domain gene expressed by myelin-forming glial of the central and peripheral nervous systems. In purified Schwann cells cultured in the absence of neurons, expression of SCIP is suppressed. This suppression is relieved by cAMP, and induction of SCIP mRNA by this second messenger precedes cAMP induction of myelin-specific genes. Similarly, SCIP expression in vivo precedes full expression of myelin-specific genes in developing oligodendrocytes and Schwann cells. The sequence of the SCIP POU domain is identical to that of Tst-1, a recently identified member of a family of POU domain genes expressed by restricted subsets of neurons. Our results demonstrate that SCIP is also expressed by myelin-forming glia and suggest that it plays a central role in the progressive determination of these cells and their commitment to myelination.

Amino Acid Sequence↗
PubMed1988

Cis-acting regions sufficient for spermatocyte-specific transcriptional and spermatid-specific translational control of the Drosophila melanogaster gene mst(3)gl-9.

In Drosophila spermatogenesis transcription occurs only premeiotically while translation can be detected also in postmeiotic spermatids. To analyse the underlying processes mst(3)gl-9, a gene specifically expressed in the male germ cells of Drosophila melanogaster, was studied. The putative protein encoded by mst(3)gl-9 is mostly composed of repetitive Cys-Gly-Pro motifs. The transcriptional and translational control of expression of mst(3)gl-9 has been investigated by P-mediated transformation. Only 102 bp of 5' upstream sequences and the first 201 bp of the gene are sufficient to maintain the gene specific characteristics of expression, namely premeiotic transcription and postmeiotic translation separated by 3 days of development.

Amino Acid Sequence↗
PubMed1986

'Vital' forms of depression.

Vital depression must be diagnosed when the patient complains of fatigue, oppressive feelings of heaviness and narrowness as well as inhibition of thinking and acting and when such symptoms are more obvious in the morning than in the afternoon or evening. Besides there is an inability to experience pleasure and to pursue interests. Vital depression however is the target symptom for the indication of treatment with specific antidepressants. This applies not only to endogenous but also to reactive depressions and to various other psychiatric diseases at the bottom of which vital depressive symptoms can be found when investigated.

Adjustment Disorders↗