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Biomedical subjects

R Kramer

Publications and source records attributed to R Kramer.

At least 73 records · Page 4Linked to original sources
PubMed1987

Some aspects of cytochrome P450-dependent denitrosation of N-nitrosamines.

The present paper deals with three aspects of cytochrome P450-dependent denitrosation of N-nitrosamines. (1) Nitrate was found in addition to nitrite as a metabolic product of the denitrosation reaction when N-nitrosamines were incubated with a microsomal system. This could also be shown when nitric oxide was added to the microsomes. (2) In order to determine the amount of denitrosation in vivo, the nitroso group of N-nitroso-N-methylaniline was labelled with the 15N isotope and administered to rats; then, the concentrations of 15N-nitrate and 15-N-nitrite in the urine were quantified by measuring the reaction of nitrate and benzene to nitrobenzene. It is estimated from these data that about 33% of the applied dose of 15N-nitroso-N-methylaniline is denitrosated in vivo. (3) Although N-nitrosodiphenylamine (NDPhA) has been classified as a noncarcinogen, recent long-term and short-term studies have cast some doubt. In order to evaluate the mechanism by which NDPhA exerts its possible genetoxic effects, its metabolism was studied in vitro, and NDPhA and its metabolites were tested for induction of DNA single-strand breaks in rat hepatocytes and in Chinese hamster V79 cells. One metabolite was identified as diphenylamine; others were suspected to be the 4-hydroxylated derivative and its corresponding quinoneimine. NDPhA caused DNA damage in rat hepatocytes but not in V79 cells. Diphenylamine also gave negative results in V79 cells, but its putative metabolite, diphenylhydroxylamine, induced a significant increase in DNA single-strand breaks.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Secretion of somatostatin by Saccharomyces cerevisiae. Correct processing of an alpha-factor-somatostatin hybrid.

Somatostatin is a 14-amino acid peptide hormone that is proteolytically processed from its precursor, prosomatostatin, by a paired-basic-specific protease localized in the Golgi apparatus and secretory vesicles. Yeast (Saccharomyces cerevisiae MAT alpha) synthesize an analogous peptide hormone precursor, pro-alpha-factor, that contains tandem repeats of alpha factor (13 amino acids) flanked by spacers that include paired basic residues. To investigate the role of these two pro regions in mediating intracellular transport and processing, cloned genes specific for preprosomatostatin and prepro-alpha-factor were used to generate recombinants encoding hybrids between the alpha-factor pro region (amino-terminal) and somatostatin (carboxyl-terminal). These recombinants were inserted into yeast expression vectors under control of either the native alpha-factor promoter or the inducible yeast PHO5 (acid phosphatase) promoter. Yeast transformed with these plasmids expressed the hybrid messenger RNAs constitutively (alpha-factor promoter) or when induced in phosphate-deficient medium (PHO5 promoter). Radioimmunoassay of culture media revealed the secretion of up to 200 ng of immunoreactive somatostatin/10(7) cells. Metabolic labeling with [35S]cysteine, followed by immunoprecipitation with anti-somatostatin antibodies revealed two forms of hybrid precursor intracellularly, one of Mr 25,000, containing core carbohydrates, and a second of Mr 11,000, which was unglycosylated. Translation of mRNA extracted from these transformants in the wheat germ cell-free system revealed that the Mr 11,000 form was the primary translation product, whereas the Mr 25,000 species could be generated in vitro by inclusion of mammalian rough microsomes. The secreted immunoreactive material was shown to be authentic somatostatin by high pressure liquid chromatography analysis and protein sequencing. These results demonstrate that the yeast processing enzymes recognize these chimeric precursors, resulting in the secretion of the mature peptide hormone.

Chromatography, High Pressure Liquid↗
PubMed1986

Characterization of the gene for the microbody (glycosomal) triosephosphate isomerase of Trypanosoma brucei.

To determine how microbody enzymes enter microbodies, we are studying the genes for glycosomal (microbody) enzymes in Trypanosoma brucei. Here we present our results for triosephosphate isomerase (TIM), which is found exclusively in the glycosome. We found a single TIM gene without introns, having one major polyadenylated transcript of 1500 nucleotides with a long untranslated tail of approximately 600 nucleotides. By a novel method, suitable for low abundance transcripts, we demonstrate that TIM mRNA contains the 35-nucleotide leader sequence (mini-exon) also found on several other trypanosome mRNAs. The TIM gene and a DNA segment of at least 6 kbp upstream of the gene are transcribed at an equal rate in isolated nuclei, suggesting that the gene is part of a much larger transcription unit. The predicted protein is of the same size as TIMs from other organisms and shares approximately 50% amino acid homology with other eukaryote TIMs, somewhat less with prokaryote TIMs. Trypanosome TIM is the most basic of all TIMs sequenced thus far. This is, in part, due to the presence of two clusters of positively charged residues in the molecule which may act as a signal for entry into glycosomes.

Amino Acid Sequence↗
PubMed1986

The fos gene product undergoes extensive post-translational modification in eukaryotic but not in prokaryotic cells.

To investigate the properties of the fos oncogene, we have constructed bacterial and yeast vectors which express the entire fos-coded protein (Fos) and two C-terminal deletion products. In Escherichia coli, Fos proteins were expressed from the phage lambda pL promotor under the control of the temperature-sensitive lambda repressor. In vitro transcription/translation studies indicate that these vectors produce Fos proteins of the expected sizes. However, in vivo, Fos protein accumulation is observed only in hosts with the Lon- phenotype. In Saccharomyces cerevisiae, the fos gene was expressed from the PHO5 promoter which is induced under low-phosphate conditions. In contrast to the situation in E. coli, in which the heterologous proteins appeared as single major products when subjected to sodium dodecyl sulfate - polyacrylamide gel electrophoresis, the Fos proteins in S. cerevisiae displayed extensive Mr heterogeneity. Pulse-chase analyses indicated that this heterogeneity was a consequence of extensive post-translational modification. These modifications occurred to an equivalent extent on the products coded by the fos gene with C-terminal deletions and thus appear not to be controlled by the missing domain.

Animals↗
PubMed1986

Corticosteroid responsive immune thrombocytopenia in Hodgkin's disease.

A 72-year-old female is described in whom immune thrombocytopenia (ITP) and fever were the presenting features of stage IIB Hodgkin's disease (HD) of the nodular sclerosis type. Thrombocytopenia resolved following steroid therapy, and after having received MOPP chemotherapy the patient is in sustained remission and with normal platelet counts 18 months later. Review of the 35 cases published to date with ITP in HD shows that of 20 patients treated with corticosteroids alone, 14 failed to respond, 2 had a partial response and only 4 patients had a good response. In contrast, of 29 splenectomised patients only 7 failed to respond, 2 had a partial response, and 20 had a good response. The state of activity of HD was closely correlated with the response of thrombocytopenia to treatment by either corticosteroids or splenectomy: only 7 of 17 patients with active HD achieved a resolution of thrombocytopenia as compared to 15 of 17 patients with HD in remission. Thus, control of thrombocytopenia is easier in patients in remission than in patients with active HD, and splenectomy is more efficient in controlling thrombocytopenia than corticosteroids.

Adolescent↗
PubMed1985

HTLV-III env gene products synthesized in E. coli are recognized by antibodies present in the sera of AIDS patients.

The envelope gene of HTLV-III, the retrovirus directly linked to AIDS, encodes a protein of 856 amino acids. Our sequence analysis of the cloned HTLV-III (HXB-3) env gene and its comparison with other isolates reveal significant divergence, especially in the external portion of this protein. A large segment of the env gene (1800 bp) was inserted into the expression vector pEV-vrf3, and a corresponding 68 kd protein, which encompasses both the extracellular and the membrane-associated regions of the native protein, was produced in E. coli. Several smaller polypeptides, which appear to be internal initiation products, were also produced. All 50 AIDS patient sera obtained from different locations in the United States specifically recognized the bacterially synthesized envelope proteins, as judged by Western blots. This suggests that these proteins will be useful for the diagnosis of HTLV-III infection and possibly as a vaccine against AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Molecular cloning and regulated expression of the human c-myc gene in Escherichia coli and Saccharomyces cerevisiae: comparison of the protein products.

mRNA from human HL-60 cells was used to prepare a cDNA library, from which two full-length clones that encompass the complete c-myc coding region were isolated. One clone, pM1-11, contains all three exons of human c-myc. The second clone, pM4-10, represents a relatively rare transcript that initiated in the first intron and includes the coding exons 2 and 3. The cDNA insert in pM1-11 was used to express the human c-myc protein in both prokaryotic and eukaryotic cells. Insertion of the coding sequences in exons 2 and 3 into the appropriate expression vectors yielded detectable c-myc protein in Escherichia coli lacking the Lon protease and in Saccharomyces cerevisiae upon induction. The protein produced in E. coli has an apparent size of 60 kDa and appears to be unmodified, as it is identical in size to the protein synthesized in an in vitro system. In contrast, yeast cells synthesize two myc proteins, of 60 kDa and 62 kDa. The difference in apparent molecular mass between the two proteins appears to be due, in part, to phosphorylation. Subcellular fractionation of yeast cells showed that the c-myc phosphoprotein is located predominantly in the nuclear fraction.

Base Sequence↗
PubMed1985

[Local anesthetic effects of morphine and naloxone].

The effects of both morphine (Mo) and naloxone (Nal), topically applied to the desheathed saphenous nerve, were studied in Sprague-Dawley rats during 22 min of superfusion at various concentrations (Mo: 0.086, 0.28, 0.86, 2.8, 8.6 mM/l; Nal: 0.1, 0.33, 1.0 mM/l) by recording single C-fibres. C-fibres were electrically stimulated and the latencies of action potentials were measured. Both drugs impaired nerve conduction in a dose-dependent, fully reversible manner. At high concentrations, conduction was blocked in 5 of 11 (Mo 2.8 mM/l) and 3 of 8 (Nal 1 mM/l) fibres. Mo (8.6 mM/l) instantly blocked all C-fibres investigated. Subsequent washing out of the drug resulted in complete recovery. The findings indicate that Mo and Nal also show local anaesthetic properties; a non-opiate receptor-specific mechanism is postulated. In spite of their blocking action, at present neither Mo nor Nal seems to provide distinct advantages over other agents that have been used successfully to induce reversible nerve blockade at peripheral sites. Due to the rather high dosage necessary (1-2 mg/ml), well-known side-effects of these opiates must also be taken into consideration.

Action Potentials↗
PubMed1984

Identification of subpopulations of human urinary plasminogen activators.

Enzymes functioning as plasminogen activators in commercial urokinase preparations and individual human urine concentrates were subjected to affinity chromatography on columns of lentil lectin-sepharose, ricin-sepharose, wheat germ agglutinin-sepharose, lotus lectin-sepharose and concanavalin A-sepharose. Chromatography of the enzymes from both sources yielded similar results for all lectins except lentil lectin. Urokinase from several commercial sources was approximately 50% adherent to lentil lectin-sepharose while only 5-10% of the urinary plasminogen activators from individuals was adherent to this lectin. SDS-PAGE followed by zymography indicated that the observed differences between commercial and individual samples could be due to the presence in urine concentrates of subpopulations of plasminogen activators which were absent from the commercial samples.

Adult↗
PubMed1982

Double-blind multicenter comparison of cyclacillin and amoxicillin for the treatment of acute otitis media.

A multicenter double-blind study of 363 pediatric outpatients with acute otitis media demonstrated that cyclacillin, administered in three divided doses (three times a day) for ten days, is as effective as and better tolerated than a similar regimen of amoxicillin. The bacteriologic cure rate for patients with middle-ear cultures was 98% in both treatment groups. Remission of signs and symptoms was significantly faster in the cyclacillin group (3.1 days) than in the amoxicillin group (4.3 days) (P less than 0.05). Of the 179 children treated with cyclacillin, three (1.7%) had drug-related diarrhea in contrast to 18 of the 184 (9.8%) children treated with amoxicillin (P less than 0.01). Cyclacillin produces a rapid resolution of symptoms and a low incidence of diarrhea.

Adolescent↗
PubMed1982

[Quality criteria of phlebography examination methods. Technically conditioned possibilities of error prediction (author's transl)].

The article describes the technical and anatomical limitations of information supplied by phlebography. Injection of contrast medium into the v. mediana hallucis enables optimal contrast medium distribution. The more proximal the site of injection, the greater the uncertainty of achieving continuous filling of the deep veins with contrast medium. Obstructions during compression (ulcer, oedema) may prove an obstacle to the assessment of insufficient v. perforantes due to overfilling of superficial veins with contrast medium. Even without any abnormal finding it is not possible to stain muscle veins and the v. femoris profunda in about 50% of the examinations. Muscular traction can result in deformation of mainly the v. poplitea and hence results in discontinuous filling with contrast medium if the musculature is not properly relaxed. The veins of the thigh and pelvis must be stained with high contrast in a continuous manner, since otherwise occlusions and collaterals are overlooked in an otherwise normal distal phlebogram. Valve motility can be assessed without additional examination measures directly after ascending phlebography during recumbency of the patient.

Contrast Media↗
PubMed1982

[Criteria for diagnostic and therapeutic methods of vascular embolization (kidneys, spleen, liver) (author's transl)].

Basing on the findings in patients with extensive carcinomas of the kidneys, the article reports on the various possibilities of application of occlusion spirals (Gianturco's method), of injectable occlusion materials and a combinations of both methods. In the authors' own experience, the spiral technique has mostly proved successful in achieving embolization of the arteries of the kidney 24 hours before surgery. Both the spiral technique as well as the use of injectable emblizates and a combination of both are used in the treatment of macrohaematurias in non-operable kidney tumours. The article reports on the technical conditions, indication and methodical limitations of embolization of the splenic artery, basing on three actual cases. The conditions and technique of embolization of branches of the hepatic artery are discussed by means of the findings obtained in two patients with liver metastases of an insulinoma.

Adult↗