Biomedical subjects

B Grinde

Publications and source records attributed to B Grinde.

At least 37 records · Page 2Linked to original sources

PubMed1996

Sensitive detection of human Caliciviridae by RT-PCR.

A semi-nested reverse transcriptase-polymerase chain reaction (RT-PCR) was developed for the detection of human Caliciviridae. The method was evaluated on faecal samples from patients with gastroenteritis sent to the Norwegian National Institute of Public Health for routine diagnosis by direct electron microscopy (EM). Of 166 samples, 49 were found to contain Caliciviridae by EM, while 7 samples contained other viruses. A total of 74 samples was positive by PCR, including all the samples with EM detectable Caliciviridae, while specimens containing other agents were negative. Phylogenetic analysis of RNA sequences from 14 Norwegian samples indicated that the viruses present in Norway are evenly distributed when compared to sequences of human Caliciviridae from other countries. The PCR primers should therefore be useful for samples from other regions. The phylogenetic analysis did not cluster viruses with a calici-like morphology, but mingled them with sequences from Norwalk-like viruses, indicating that the two morphological types do not represent separate genogroups.

Base Sequence↗

PubMed1996

Prevalence of human papillomavirus in cervical scrapes, as analyzed by PCR, in a population-based sample of women with and without cervical dysplasia.

HPV is suspected of being a major cause of cancer of the uterine cervix. To understand the risk of disease in the general population of women, it is important to estimate the prevalence of HPV infection in a random population-based sample of women without disease. In this study, a total of 231 randomly selected women without dysplasia (controls) were examined, and compared with 103 women with histologically confirmed CIN II-III (patients). The prevalence of HPV DNA in cervical scrapes was determined by general nested PCR, which was expected to detect any relevant HPV type commonly found in cervical samples. The nested positive samples were typed with type-specific PCR. In the general nested PCR, 15% of the controls were positive, compared to 91% of the patients. In the population-based sample, 2.2% had HPV types 6 and 11 and 10% had types 16, 18, 31, and 33. In both groups, HPV DNA was observed less frequently in women above than below the age of 30. The results are among the few population-based figures on the prevalence of HPV in women, and provide a baseline for understanding the risk of developing cancer of the uterine cervix, and determining the proportion of women to be included in intervention studies.

Adult↗

PubMed1995

Detection of all serotypes of human astrovirus by the polymerase chain reaction.

A reverse transcription (RT) and polymerase chain reaction (PCR) was designed for the detection of astroviruses based on a conserved nucleotide sequence in the 3'-end of the genome of the 7 known serotypes of human astrovirus. Thirty-eight samples found to contain astrovirus by electron microscopy (EM) were used for evaluation of the assay. The samples were dialyzed for 1 h to remove potential low molecular weight inhibitors of the RT-PCR. Immediately before RT, 1 microliters of the samples were incubated at 94 degrees C for 2 min to disrupt the viral particles. Thirty-six of the samples were positive by PCR, including samples of all 7 serotypes. The two samples that were negative, could hve been false positive by EM, or the viral RNA could have been degraded. All other viruses examined, including calici-, rota- and enteroviruses, were negative.

Base Sequence↗

PubMed1995

Sensitive detection of group A rotaviruses by immunomagnetic separation and reverse transcription-polymerase chain reaction.

An immunomagnetic separation (IMS) method was developed for concentrating rotaviruses from environmental samples, as well as a reverse transcription-polymerase chain reaction (RT-PCR) for the sensitive and specific detection of group A rotaviruses. Magnetic beads were coated with monoclonal antibodies directed against the group-specific, inner capsid protein (VP6) and subsequently used to capture and purify the virus with the help of a magnet. The genome was made available for RT by heat-disrupting the viral particles. A single 40-cycle PCR was as sensitive as a nested PCR, both detecting 0.005 PFU of the Wa strain, corresponding to approximately 5 particles as indicated by EM. The nested PCR was positive for all the group A strains tested, but negative for group C rotaviruses and other RNA viruses. The IMS-RT-PCR method functioned satisfactorily with virus seeded out in fresh water samples; with sea water, the IMS removed most, but not all, inhibiting activity.

Antigens, Viral↗

PubMed1994

[Gene therapy. Methods and applications].

Modern techniques in molecular biology and cell biology will probably make gene therapy, i.e. therapeutic transfer of genes to the patient's cells, available for treatment of many genetic diseases, cancer, cardiovascular diseases and infectious diseases. For genetic diseases the treatment will involve the transfer of a functional copy of the defect gene. The strategy for treatment of cancer may include the transfer of genes that induce the death of cancer cells via toxic molecules, and genes that enhance the immune response to tumour cells. After several years of preclinical studies, the National Institutes of Health in the USA has, up to February 1994, approved 56 protocols for clinical trials in human gene therapy. Most of the protocols include use of viruses to aid gene delivery. This paper briefly reviews the scientific basis for gene therapy, and discusses some clinical applications of somatic gene therapy in humans.

Genetic Diseases, Inborn↗

PubMed1994

Detection of hepatitis A virus in clinical and environmental samples by immunomagnetic separation and PCR.

Magnetic beads coated with antibodies against surface epitopes of the hepatitis A virus (HAV) captured efficiently viral particles from various types of samples. Contaminating substances, including particulate material, were removed by using a magnet to retain the beads during the washing procedure. A sensitive reverse transcriptase/nested polymerase chain reaction (RT-PCR) was developed to confirm the presence of viral particles on the beads. The above technique was shown to be useful for the detection of a laboratory strain of HAV added to polluted river water, sea water and fecal extracts.

Base Sequence↗

PubMed1993

Comparison of the substrate-binding pockets of the Rous sarcoma virus and human immunodeficiency virus type 1 proteases.

A steady state kinetic analysis of the avian myeloblastosis virus/Rous sarcoma virus (AMV/RSV) and human immunodeficiency virus Type 1 (HIV-1) retroviral proteases (PRs) was carried out using a series of 40 peptide substrates that are derivatives of the AMV/RSV nucleocapsid-PR cleavage site. These peptides contain single amino acid substitutions in each of the seven positions of the minimum length substrate required by the PR for specific and efficient cleavage. These peptide substrates are distinguished by the individual enzyme subsites of the AMV/RSV and HIV-1 PRs. The molecular basis for similarities and differences of the individual subsites for both proteases is discussed using steady state kinetic data and modeling based on crystal structures.

Amino Acid Sequence↗

PubMed1993

Sequence comparison and mutational analysis of elements that may be involved in the regulation of DNA synthesis in HIV-1.

The large number of sequenced clones of HIV-1 and related viruses made it possible to indicate conserved elements with potential regulatory or structural functions. Such analysis was combined with directed mutagenesis in order to investigate the importance of elements that may influence the initiation of plus-strand DNA synthesis. The main site for plus-strand initiation is a polypurine tract near the 3' end of the viral RNA (the 3' PPT). An exact copy of this PPT is located in the middle of the genome (the internal PPT). Upstream from the internal PPT there is an inverted repeat. Mutants designed to upset the internal PPT (i.e., purine to pyrimidine changes), as well as mutants designed to abolish the potential stem-loop formation (changes around the internal PPT or in the upstream inverted repeat) both resulted in viruses with a reduced ability to replicate. Upsetting the stem-loop formation was, however, less harmful than changing the polypurine nature of the PPT. Changing a conserved T on the 3' side of the PPT to a C did not affect the phenotype.

Base Sequence↗

PubMed1993

Detection of human astrovirus serotype 1 by the polymerase chain reaction.

Astroviruses are small, plus-strand RNA viruses associated with diarrhoea, mostly in children. The diagnostic method commonly used is electron microscopy. We have designed a nested polymerase chain reaction (PCR) based on the recently reported nucleotide sequence of the 3' end of the genome of a human astrovirus serotype 1, the most common form. The PCR was positive for the ten serotype 1 samples tested, while being negative for all other viruses tested, including astrovirus type 2, 3, 4 and 5, calicivirus, rotavirus and picornaviruses. Fecal extracts from patients with diarrhoea were analysed directly or after isolation of RNA, the former method being at least as sensitive. Titration of fecal extracts by PCR indicated the presence of up to 10(11) viral particles per ml in feces.

Base Sequence↗

PubMed1993

Evolution of mouse mammary tumor virus-related sequences in the human genome.

The human genome contains numerous copies of elements with sequence homology to the mouse mammary tumor virus (MMTV). We have been interested in whether these elements are still actively transposing in the germ lines, and investigated this question by looking at restriction fragment patterns. Genomic DNA from humans and selected animals were digested with three different restriction enzymes and hybridized with five probes representative of five subgroups of the human MMTV-related elements. Two polymorphisms as well as two cases of sexual dimorphism were discovered in human DNA. The latter indicate the presence of at least two elements of the MMTV family on the Y chromosome. DNA samples from non-primate mammals were all unreactive. Both chimpanzee and rhesus monkey had restriction patterns of roughly the same number of bands and total intensity as humans. The number of differences in patterns between humans, and between humans and chimpanzee or rhesus monkey, were compatible with being caused by point mutations. The data indicate that these elements were actively spreading in the genome some time before the split between apes and Old World monkeys, while being relatively inactive, at least as to transpositions in the germ line, in later ages.

Animals↗

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↗