Biomedical subjects

R L Krogsrud

Publications and source records attributed to R L Krogsrud.

9 recordsLinked to original sources

PubMed1994

A potent peptidomimetic inhibitor of HSV ribonucleotide reductase with antiviral activity in vivo.

Herpes simplex viruses (HSV) types 1 and 2 encode their own ribonucleotide reductases (RNRs) (EC 1.17.4.1) to convert ribonucleoside diphosphates into the corresponding deoxyribonucleotides. Like other iron-dependent RNRs, the viral enzyme is formed by the reversible association of two distinct homodimeric subunits. The carboxy terminus of the RNR small subunit (R2) is critical for subunit association and synthetic peptides containing these amino-acid sequences selectively inhibit the viral enzyme by preventing subunit association. Increasing evidence indicates that the HSV RNR is important for virulence and reactivation from latency. Previously, we reported on the design of HSV RNR inhibitors with enhanced inhibitory potency in vitro. We now report on BILD 1263, which to our knowledge is the first HSV RNR subunit-association inhibitor with antiviral activity in vivo. This compound suppresses the replication of HSV-1, HSV-2 and acyclovir-resistant HSV strains in cell culture, and also strongly potentiates the antiviral activity of acyclovir. Most importantly, its anti-herpetic activity is shown in a murine ocular model of HSV-1-induced keratitis, providing an example of potent nonsubstrate-based antiviral agents that prevent protein-protein interactions. The unique antiviral properties of BILD 1263 may lead to the design of new strategies to treat herpesvirus infections in humans.

Amino Acid Sequence↗

PubMed1993

A solid-phase assay for the binding of peptidic subunit association inhibitors to the herpes simplex virus ribonucleotide reductase large subunit.

The herpes simplex virus (HSV) ribonucleotide reductase is comprised of two nonidentical homodimeric subunits whose association is essential for enzymatic activity. In order to evaluate the affinity of a series of peptidic inhibitors with the enzyme's large subunit (R1), we have developed a sensitive solid-phase binding assay. The assay entails the use of a nonneutralizing antibody directed against the R1 subunit of the enzyme to immobilize either the native holoenzyme from HSV-1-infected cells or a recombinantly expressed HSV-2 R1 subunit. In either case, the radioiodinated peptidic inhibitor 125I-desamino-Tyr-(N-methyl)-Val-Ile-Asp-(gamma-N,N-diethyl)-Asp-Leu demonstrated specific, saturable binding to the HSV R1 that could be competed by the nonapeptide Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu corresponding to the C-terminal sequence of the HSV ribonucleotide reductase small subunit (R2) or by recombinant HSV R2, but not by C-terminally truncated HSV R2 or murine R2. Our results provide direct evidence that inhibitors based on the carboxy-terminal amino acid sequence of HSV R2 compete with intact HSV R2 for a common binding site on HSV R1. The utility and sensitivity of this binding assay were further demonstrated by the ability to detect and discriminate ribonucleotide reductase inhibitors in the low nanomolar range.

Amino Acid Sequence↗

PubMed1987

Presence of alpha-melanocyte-stimulating hormone in bovine pituitary intraglandular colloid of intermediate lobe origin.

Alpha-melanocyte-stimulating hormone (alpha-MSH), one of several peptide hormones originating in the intermediate lobe of the pituitary as proopiomelanocortin, was discovered in bovine pituitary intraglandular colloid by using radioimmunoassay. The quantity of alpha-MSH varied from 5 to 368 micrograms/mg protein in the three pools. The importance of this finding is discussed in light of the possibility that the colloid is a transport medium for alpha-MSH and other intermediate lobe hormones.

Animals↗

PubMed1987

Isolation and characterization of monoclonal antibodies to proline dehydrogenase from Escherichia coli K-12.

The PutA protein of Escherichia coli K-12 serves as both proline dehydrogenase and the repressor controlling the expression of genes putP and putA. Thirty-eight hybridoma cell lines were isolated using mice immunized with proline dehydrogenase purified from a bacterial membrane extract. The monoclonal antibodies secreted by those cells showed varying affinities for proline dehydrogenase by enzyme-linked immunosorbent assay (ELISA). Nine antibodies labelled the PutA protein in Western blots after sodium dodecyl sulfate--polyacrylamide gel electrophoresis and two of the five tested also labelled the undenatured PutA protein. Three antibodies bound proteins present in a peripheral membrane protein fraction from both putA+ bacteria and a putA::Tn5 mutant strain. Urea denaturation eliminated the proline:2,6-dichloroindophenol (DCIP) oxidoreductase activity, but did not alter the immunoreactivity of the PutA protein. Tween 20, which caused 1.8-fold increases in Km (proline) and Vmax for proline:DCIP oxidoreductase, increased the avidity of the antibody from hybridoma line 2.1C10.3 fivefold. The antibodies from hybridoma lines 2.1C10.2, 1.2C10.3, and 1.1B07.1 were shown by electron microscopy of immunogold-labelled preparations or by ELISA to bind the membrane-associated PutA protein, whereas those from hybridoma lines 2.1A08.2 and 1.4C09.1 failed to recognize that antigen form. These antibodies will serve as probes of the relationships among protein domain, conformation, and function for the PutA protein.

Antibodies, Monoclonal↗

PubMed1986

Proline transport and osmotic stress response in Escherichia coli K-12.

Proline is accumulated in Escherichia coli via two active transport systems, proline porter I (PPI) and PPII. In our experiments, PPI was insensitive to catabolite repression and was reduced in activity twofold when bacteria were subjected to amino acid-limited growth. PPII, which has a lower affinity for proline than PPI, was induced by tryptophan-limited growth. PPII activity was elevated in bacteria that were subjected to osmotic stress during growth or the transport measurement. Neither PPI nor uptake of serine or glutamine was affected by osmotic stress. Mutation proU205, which was similar in genetic map location and phenotype to other proU mutations isolated in E. coli and Salmonella typhimurium, influenced the sensitivity of the bacteria to the toxic proline analogs azetidine-2-carboxylate and 3,4-dehydroproline, the proline requirements of auxotrophs, and the osmoprotective effect of proline. This mutation did not influence proline uptake via PPI or PPII. A very low uptake activity (6% of the PPII activity) observed in osmotically stressed bacteria lacking PPI and PPII was not observed when the proU205 lesion was introduced.

Amino Acid Transport Systems, Neutral↗

PubMed1979

Characterization of stem cells and progenitors of hemopoiesis by cell sorting.

Cell sorting has been used as a method for characterizing hemopoietic stem cells and progenitors. Fluorescent antibody-surface labels and changes in fluorescence polarization induced by in vitro stimulation with potential hemopoietic regulators were used. As detected by significant enrichment of CFU-S (pluripotent stem cells) in fluorescence-activated cell sorting, some CFU-S bear 'unique antigens' recognized by rabbit anti-human brain sera, human anti-human sperm sera, and 129 anti-F9 serum, but not A . TH anti-A . TL (Ia) ascites. Significant changes in fluorescence polarization induced by in vitro stimulation of mouse bone marrow with potential hemopoietic regulators were also observed; further, progenitors of human T-lymphocyte colonies were observed to exhibit a significantly decreased mean polarization value after short-term stimulation with PHA-LCM (phytohemagglutinin-stimulated leukocyte conditioned medium).

Animals↗

PubMed1979

Hemopoietic stem cells bear sperm-associated antigens.

A fluorescence-activated cell sorter has been used to sort out enriched populations of murine hemopoietic stem cells from mouse bone marrow, based on fluorescence of cells labeled with various antisera. Significant positive enrichment for stem cells was obtained with rabbit anti-human brain, human anti-human sperm and mouse anti-mouse F9 sera but not with mouse anti-mouse Ia serum. These results support the hypothesis that the hemopoietic stem cell exhibits 'unique' surface antigens cross-reactive with sperm, brain and embryonic antigens.

Animals↗

PubMed1977

Age-related changes in T cell function.

A comparison was made of the abilities of carrier (BGG)-primed T cell populations from young (4-month old), middle-aged (14- and 19-month old) and old (31- and 34-month old) mice to collaborate with hapten (DNP)-primed B cells from young mice in a cell-transfer system. The plaque-forming cell responses to 2,4-dinitrophenol (DNP) were measured by a modification of the Jerne plaque assay. The DNP-specific antibody-forming cell responses of old T cell/young B cell combinations were significantly lower than those of young T cell/young B cell combinations, both in the number of T cells needed for peak response and in the size of that response. These data indicate that the primed T cell populations of old mice are deficient by a factor of 6 in their ability to initiate B cell proliferation and differentiation into antibody-forming cells.

Aging↗

PubMed1977

Serologic identification of hemopoietic progenitor cell antigens common to mouse and man.

An immunologic approach to the identification of human hemopoietic stem cell antigens has yielded two types of antisera. Both recognize antigens on hemopoietic progenitors which are identical or cross-reactive with determinants on sperm, brain, and, across the species barrier, with murine hemopoietic stem cells (CFU-S). The first antiserum was the result of an autoimmune reaction in a post-vasectomy patient. The second was produced in rabbits after injection of human brain cells.

Animals↗