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B Grinde

Publications and source records attributed to B Grinde.

At least 19 recordsLinked to original sources
PubMed1992

Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins.

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected. The catalytic efficiency of the RSV protease on cognate substrates varied by as much as 30-fold. The least efficiently processed substrate, p2-p10, represents the cleavage site between the RSV p2 and p10 proteins. This peptide was inhibitory to the AMV as well as the HIV-1 and HIV-2 protease cleavage of other substrate peptides with Ki values in the 5-20 microM range. Molecular modeling of the RSV protease with the p2-p10 peptide docked in the substrate binding pocket and analysis of a series of single-amino acid-substituted p2-p10 peptide analogues suggested that this peptide is inhibitory because of the potential of a serine residue in the P1' position to interact with one of the catalytic aspartic acid residues. To open the binding pocket and allow rotational freedom for the serine in P1', there is a further requirement for either a glycine or a polar residue in P2' and/or a large amino acid residue in P3'. The amino acid residues in P1-P4 provide interactions for tight binding of the peptide in the substrate binding pocket.

Amino Acid Sequence↗
PubMed1992

Mutations that alter the activity of the Rous sarcoma virus protease.

Mutations designed by analysis of the Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV)-1 protease (PR) crystal structures were introduced into 1) the substrate binding pocket, 2) the substrate enclosing "flaps," and 3) surface loops of RSV PR. Each mutant PR was expressed in Escherichia coli. Changes in activity were detected by following cleavage of a truncated (NC-PR) precursor polypeptide in E. coli and cleavage of synthetic peptide substrates representing RSV and HIV-1 PR cleavage sites in vitro. Mutations in the substrate binding pocket exchanged amino acid residues located close to the substrate in the HIV-1 PR for structurally equivalent residues in the RSV PR. Changing histidine 65 to glycine (H65G) gave an inactive enzyme, while a double mutant R105P,G106V, as well as the triple mutant, H65G,R105P,G106V, produced enzymes which showed significant activity toward a substrate that represented a HIV-1 cleavage site. Mutating the catalytic aspartate (D37S) or an adjacent conserved alanine to threonine (A40T), produced inactive enzymes. In contrast, the substitution A40S was active, but showed a reduced rate of catalysis. Mutations in the flaps of conserved glycines (G69L, G70L) produced inactive PRs. Two extended RSV PR surface loops were shortened to the size found in HIV-1 PR and resulted in drastically reduced activity. These results have confirmed some of the basic predictions made from structural models but have also revealed unexpected roles and interactions in the protein.

Amino Acid Sequence↗
PubMed1992

Analysis of substrate interactions of the Rous sarcoma virus wild type and mutant proteases and human immunodeficiency virus-1 protease using a set of systematically altered peptide substrates.

In the preceding study, mutant Rous sarcoma virus (RSV) proteases are described in which three amino acids found in the human immunodeficiency virus-1 (HIV-1) protease (PR) were substituted into structurally comparable positions (Grinde, B., Cameron, C.E., Leis, J., Weber, I., Wlodawer, A., Burstein, H., Bizub, D., and Skalka, A. M. (1992) J. Biol. Chem. 267, 9481-9490). In this report, the activity of the wild type and these mutant PRs are compared using a set of RSV NC-PR peptide substrates with single amino acid substitutions in each of the P4 to P3' positions. With most substrates, the relative activities of the two active mutants followed that of the RSV PR. Substitutions in the P1 and P1' positions were an exception; in this case, the mutants behaved more like the HIV-1 PR. These results confirm predictions from structural analyses which indicate that residues 105 and 106 of the RSV PR are important in forming the S1 and S1' binding subsites. These results, further analyzed with the aid of computer modeling of the RSV PR with different substrates, provide an explanation for why only partial HIV-1 PR-like behavior was introduced into the above RSV PR mutants.

Amino Acid Sequence↗
PubMed1992

Mutations in the central polypurine tract of HIV-1 result in delayed replication.

The reverse transcription of HIV-1 generates a linear genomic DNA with a single-stranded gap. The gap is believed to be the result of plus-strand priming from a second, centrally located, polypurine tract (PPT). A mutant containing four amino-acid-neutral purine-to-pyrimidine changes within the central PPT did not replicate as fast as wild-type virus. Another mutant with the entire 15-bp PPT deleted was replication-deficient. The results indicate that plus-strand priming at the central PPT is important for viral replication, possibly by ensuring efficient DNA synthesis.

Base Sequence↗
PubMed1991

Genital human papilloma virus infection in Oslo studied by dot blot DNA hybridization and the polymerase chain reaction.

Samples from patients with genital condyloma acuminata or with cervical condylomas and/or dysplasia and from women without cytological/clinical evidence of cervical affection were examined by dot blot DNA hybridization or the polymerase chain reaction (PCR). The PCR was much more sensitive than dot blot, more than doubling the human papilloma virus (HPV) findings. HPV DNA, mainly HPV 6/11, was detected in 18 of 19 biopsies of condyloma acuminata, whereas HPV 16 was most frequently detected in the 21 cervices (76%) with condyloma and/or dysplasia. HPV 16 was detected in eight of 103 cervical smears with no signs of infection. The prevalence of HPV 16 in cervical samples was somewhat higher than expected. This suggests that, in Oslo, HPV 16 is a common HPV type in women with cytologically normal cervices. HPV 18 was relatively rare and was detected only in combination with other HPVs.

Adult↗
PubMed1991

Survival of HIV-1 activity after disinfection, temperature and pH changes, or drying.

A recently developed assay for measuring infectious HIV-1 particles was used to determine the stability of the virus under various storage conditions as well as the effect of commonly used disinfectants. At the optimum pH of 7.1 the half life of the virus ranged from approx. twenty-four hours at 37 degrees C to no significant loss over 6 months at -75 degrees C. Drying the virus on a glass surface or freezing caused a 5-12 fold and 4-5 fold decrease of activity, respectively. The dried preparations, however, were about as stable as when stored in a buffered solution. A solution of iodine and detergent (2% Jodopax) was the only disinfectant examined which removed all detectable HIV-1 activity. Isopropanol and ethanol were more potent than acetone; however, all three solvents left some viable particles after a 30 min treatment with 70% solutions.

1-Propanol↗
PubMed1991

The plus strand is discontinuous in a subpopulation of unintegrated HIV-1 DNA.

During reverse transcription the synthesis of plus strand viral DNA is initiated from an RNA polypurine primer immediately upstream of the U 3 region. The polypurine tract (PPT) sequence at this site is in HIV-1 also present in the middle of the genome. Here we demonstrate that a subpopulation of linear unintegrated HIV-1 DNA has a discontinuity in the plus strand within less than 50 bp from this central PPT, consistent with its utilization as a plus strand initiation site.

DNA Replication↗
PubMed1991

cDNA and amino acid sequences of rainbow trout (Oncorhynchus mykiss) lysozymes and their implications for the evolution of lysozyme and lactalbumin.

The complete 129-amino-acid sequences of two rainbow trout lysozymes (I and II) isolated from kidney were established using protein chemistry microtechniques. The two sequences differ only at position 86, I having aspartic acid and II having alanine. A cDNA clone coding for rainbow trout lysozyme was isolated from a cDNA library made from liver mRNA. Sequencing of the cloned cDNA insert, which was 1 kb in length, revealed a 432-bp open reading frame encoding an amino-terminal peptide of 15 amino acids and a mature enzyme of 129 amino acids identical in sequence to II. Forms I and II from kidney and liver were also analyzed using enzymatic amplification via PCR and direct sequencing; both organs contain mRNA encoding the two lysozymes. Evolutionary trees relating DNA sequences coding for lysozymes c and alpha-lactalbumins provide evidence that the gene duplication giving rise to conventional vertebrate lysozymes c and to lactalbumin preceded the divergence of fishes and tetrapods about 400 Myr ago. Evolutionary analysis also suggests that amino acid replacements may have accumulated more slowly on the lineage leading to fish lysozyme than on those leading to mammal and bird lysozymes.

Amino Acid Sequence↗
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.

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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↗