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Biomedical subjects

B Grinde

Publications and source records attributed to B Grinde.

At least 55 records · Page 3Linked to original sources
PubMed1990

Modified oligopeptides designed to interact with the HIV-1 proteinase inhibit viral replication.

The human immunodeficiency virus 1 (HIV-1) codes for a proteinase that cuts viral proteins at specific sites. We have tested 13 modified oligopeptides related to these cleavage sites to see if they inhibit viral replication. To indicate whether a decrease in replication could be due to a general inhibition of cell metabolism, we also measured the effect of the peptides on cellular protein synthesis. Three of the peptides tested (Ac-Gln-Asn-Sta-Val-NH2, Ac-Gln-Asn-Sta-Val-Val-NH2, and Ac-Glu-Asn-Sta-Ile-NH2) inhibited HIV-1 replication at concentrations that did not inhibit protein synthesis. Ac-Gln-Asn-Sta-Val-NH2 was the most potent, causing an approximately 40% decrease in viral replication, measured as the synthesis of HIV-1 antigens and the formation of infectious particles.

Amino Acid Sequence↗
PubMed1990

An assay for quantifying infectious HIV particles.

A method for assessing the number of infectious particles in preparations of HIV has been developed. Virus was mixed with cells to allow binding of virus. The cells were then cast in an agar gel to block any further transfer of virus between the cells. After 4 days of incubation the cells initially infected with HIV expressed viral antigens. The percentage of infected cells was then determined by indirect immunofluorescence. The method was developed for HIV, but is presumably suitable for any virus that can replicate in cells not attached to a surface.

Agar↗
PubMed1990

Virus quantification by immunofluorescence of cells grown in agar.

We have developed a method for assessing the number of infectious viral particles by measuring what we call fluorescence initiating units (FIV). The present work has been done with HIV, but the methods should be applicable to other viruses as well. Briefly described, cells are mixed with virus and then cast in an agar gel to block further transfer of virus. After a period of incubation sufficient to allow infected cells to express virus antigens, the percentage of infected cells is determined by indirect immunofluorescence.

Cell Line↗
PubMed1989

A lysozyme isolated from rainbow trout acts on mastitis pathogens.

The antibacterial effects of two lysozymes purified from rainbow trout kidney (type I and II) were tested on eight bacterial strains isolated from cases of clinical mastitis (staphylococci, streptococci and coliforms). Three other lytic agents were included in the experiments as controls: hen egg-white lysozyme, lysostaphin and mutanolysin. Proliferating bacteria were incubated with the various lytic agents, either in hearts infusion broth or in milk. The type II rainbow trout lysozyme decreased the number of live bacteria (colony forming units) of all the strains tested, but was most efficient against staphylococci. The other two lysozymes had little effect.

Animals↗
PubMed1989

Unhealthy genes.

Explore the source record for details and available documents.

Genetic Diseases, Inborn↗
PubMed1989

The proteinase inhibitor pepstatin A inhibits formation of reverse transcriptase in H9 cells infected with human immunodeficiency virus 1.

Retroviruses depend on a virus-encoded proteinase. As this enzyme is an interesting target for antiviral therapy, we examined the effect of various low-molecular-weight proteinase inhibitors, as well as a few oligopeptides related to the proteolytic cleavage sites, on the replication of HIV-1 in H9 cells. The increase in reverse transcriptase activity during incubation was assumed to reflect viral replication. Cellular DNA synthesis was measured to quantitate the adverse effects of the inhibitors on the cells. Only one of the substances tested, pepstatin A, had an appreciable selective effect on viral replication. Substances that decreased DNA synthesis generally caused an equally large decrease in reverse transcriptase activity.

CD4-Positive T-Lymphocytes↗
PubMed1989

Detecting inhibition of coxsackievirus replication by measuring DNA synthesis--effect of proteinase inhibitors.

The replication of picornaviruses can be monitored by microscopic examination of the cytopathogenic effect. We found that measuring cellular DNA synthesis gave a more objective and reliable estimate of the viral effect on the cells. Furthermore, by simultaneously measuring DNA synthesis in uninfected cells, we obtained a sensitive indication of whether the agents tested influenced cellular activity. The method was employed to investigate the effect of various proteinase inhibitors on the replication of coxsackie-B3 virus in HEp2 cells. Certain inhibitors of metallo-proteinases had a limited, but consistent, protective effect against viral activity.

Anti-Bacterial Agents↗
PubMed1988

Purification and characterization of two lysozymes from rainbow trout (Salmo gairdneri).

1. Two different lysozymes, designated I and II, were purified from the kidney of rainbow trout. The enzymes had isoelectric points of approximately 9.5 and 9.65, and differed in their binding characteristics to a cation exchanger. Lysozyme II had the highest specific activity against Micrococcus luteus. 2. By sodium dodecyl sulphate gel electrophoresis, a molecular mass of 14.4 kDa was established for the two lysozymes. 3. For both type I and II enzymes, optimum pH under the present conditions was 5.5 and optimum temperature (at pH 6.2) around 45 degrees C. 4. N-terminal amino acid sequence determination indicated that the two trout lysozymes were c-type lysozymes. 5. The fish lysozymes had a much higher rate of diffusion in agar than did the other lysozymes tested (possibly due to less interaction with agarose). This implies that a foreign lysozyme, such as hen egg white lysozyme, should not be used as a standard when assaying lysozyme activity with the lysoplate technique.

Amino Acid Sequence↗
PubMed1987

Methionine is a regulator of starvation-induced proteolysis in Tetrahymena.

The ciliate Tetrahymena thermophila responds to starvation by drastically increasing the rate of proteolysis. The response was reversed by resuspending the cells in a defined growth medium. Among the components of this medium only amino acids were active in inhibiting proteolysis. One amino acid, methionine, accounted for at least 75% of the effect of the complete medium, strongly indicating that in Tetrahymena methionine is the main regulator of step-down proteolysis, a process generally connected with autophagy in eukaryotic cells. The fact that one amino acid has such a drastic effect should make the system well suited for further investigations of the regulation of this process.

Amino Acids↗
PubMed1986

Proteolytic response to nutritional step-down in Tetrahymena.

The ciliate Tetrahymena thermophila is usually grown in a medium containing proteose peptone and yeast extract as organic nutrients. When the ciliate is transferred to step-down conditions, i.e., an inorganic medium, it is shown that the cells respond by rapidly and drastically increasing their rate of protein degradation. A method for measuring the response to step-down conditions is presented, and the response is characterized. The types of proteinases involved are indicated by the use of specific inhibitors. It is concluded that Tetrahymena reacts in much the same way as mammalian cells, and provides a suitable system for investigating the regulation of protein degradation.

Aminooxyacetic Acid↗
PubMed1986

Perforated hepatocytes as a system for studying intracellular proteolysis.

Isolated rat hepatocytes were treated either by electroshock or enterotoxin in order to perforate the plasma membrane. Under appropriate conditions perforated cells could be incubated for a couple of hours without further disruption. The rate of proteolysis was higher in the perforated cells than in homogenized cells. ATP, a thiol reagent and Ca2+/Mg2+ stimulated proteolysis. By using inhibitors, it was shown that the proteolysis was partly lysosomal, and that all four classes of endoproteinases were involved.

Acid Phosphatase↗
PubMed1985

Autophagy and lysosomal proteolysis in the liver.

Autophagy is defined as any process whereby cellular macromolecules destined for degradation gain access to the lysosomes. A review is presented on the physiological significance, mechanisms and regulation of autophagy in hepatocytes, concentrating on the issue of regulation. The article ends by discussing techniques available for future research.

Amino Acids↗
PubMed1984

Effect of amino acid metabolites on lysosomal protein degradation. A regulatory role for kynurenine?

Possible derivatives of the amino acids tryptophan, tyrosine and histidine were examined as to their effect on protein metabolism in isolated rat hepatocytes. One of the substances tested, kynurenine (a main product of the catabolism of tryptophan), might be a physiological regulator of the lysosomal degradation of endogenous protein, because of the following. (a) Kynurenine decreased the lysosomal (i.e. methylamine-sensitive) pathway of degradation to a much greater extent than its parent amino acid, without interfering with the non-lysosomal pathway. (b) Kynurenine did not appreciably reduce the (lysosomal) degradation of the endocytosed protein asialo-fetuin, or the rate of protein synthesis, indicating a specificity of action. (c) Electron micrographs revealed a reduction in secondary lysosomes due to kynurenine.

Amino Acids↗
PubMed1984

Inhibition of autophagy by benzyl alcohol.

Benzyl alcohol caused a rather complete and selective inhibition of the methylamine sensitive (i.e., the putative lysosomal) pathway of protein degradation in isolated rat hepatocytes. The effect was found to be entirely reversible within 30 min of removing the agent. A morphometric examination of electron micrographs revealed that the inhibition of lysosomal protein degradation coincided with a block in the formation of autophagic vacuoles. The number of acidic vacuoles (i.e., vacuoles induced to swell by adding methylamine) was not drastically reduced.

Animals↗
PubMed1983

Role of Ca2+ for protein turnover in isolated rat hepatocytes.

Experiments with bivalent-cation chelators (EGTA and EDTA), a Ca2+ ionophore (A23187) and a Ca2+-channel blocker (verapamil) indicate that Ca2+ is required for the lysosomal degradation of endogenous protein in hepatocytes. A distinction is made between lysosomal and non-lysosomal degradation by using the lysosomotropic agent methylamine. As Ca2+ does not appear to be required for the lysosomal degradation of endocytosed asialo-fetuin, the Ca2+-dependence for the degradation of endogenous protein is probably connected with the formation of autophagic vacuoles or the fusion of autophagic vacuoles with lysosomes. EGTA and EDTA had a slight inhibitory effect on the non-lysosomal degradation. This effect could be due to the activity of non-lysosomal Ca2+-dependent thiol proteinases. Together with previous experiments with thiol-proteinase inhibitors, the present experiments indicate that these proteinases have a very limited impact on the bulk protein degradation in the isolated hepatocytes.

Animals↗
PubMed1983

Inhibition of hepatic protein degradation by synthetic analogues of chymostatin.

Analogues of the microbial proteinase inhibitor chymostatin have been synthesized. The two most promising analogues were tested on protein turnover in isolated rat hepatocytes. Their effect is much similar to the effect of chymostatin, but the analogues are even more powerful inhibitors, probably due to an increased effect on lysosomal thiol proteinases. The analogues blocked most of the lysosomal (i.e. methylamine-sensitive) degradation of endogenous protein and caused a 50% inhibition of the non-lysosomal degradation; the effect occurred rapidly and was reversed upon washing the cells. One of the analogues, Z-Arg-Leu-Phe(H), is the most potent inhibitor of hepatic protein degradation so far found.

Animals↗
PubMed1983

The thiol proteinase inhibitors, Z-Phe-PheCHN2 and Z-Phe-AlaCHN2, inhibit lysosomal protein degradation in isolated rat hepatocytes.

The effects on protein metabolism of Z-Phe-PheCHN2 and Z-Phe-AlaCHN2 were examined in isolated rat hepatocytes. The two thiol proteinase inhibitors caused a drastic reduction in the degradation of both endogenous and endocytosed (asialo-fetuin) protein. The inhibition was not additive to that of the lysosomotropic base methylamine, indicating that Z-Phe-PheCHN2 and Z-Phe-AlaCHN2 only affect lysosomal degradation. At high concentrations (0.1-1 mM) both inhibitors reduced protein synthesis strongly. This finding indicates non-specific/toxic effects, which may limit the usefulness of the inhibitors.

Animals↗