ABSTRACT
Major histocompatabilty (MHC) proteins rely heavily on peptide backbone recognition for ligation. Nonetheless, modifications to the polyamide backbone of a tetrapeptide ligand can be made without abrogating binding.
Subject(s)
Histocompatibility Antigens Class II/drug effects , Histocompatibility Antigens Class II/metabolism , Peptides/chemistry , Peptides/pharmacology , Binding Sites , HLA-DR1 Antigen/drug effects , HLA-DR1 Antigen/metabolism , Hemagglutinins/chemistry , Inhibitory Concentration 50 , Lactams/chemistry , Ligands , Molecular Mimicry , Molecular Structure , Phosphoproteins/drug effects , Phosphoproteins/metabolism , Structure-Activity Relationship , Transcription Factors/drug effects , Transcription Factors/metabolismABSTRACT
Tetrapeptide derived major histocompatability (MHC) II ligands have been developed that contain no unadulterated peptide bonds. These are the 'least peptidic' ligands for any MHC protein yet reported.
Subject(s)
Histocompatibility Antigens Class II/chemistry , Histocompatibility Antigens Class II/metabolism , Peptides/chemistry , Drug Design , Histocompatibility Antigens Class II/drug effects , Inhibitory Concentration 50 , Lactams/chemistry , Ligands , Molecular Mimicry , Structure-Activity RelationshipABSTRACT
The discovery and physico-chemical characterization of three novel and minor virginiamycin M1 analogs as potent gastrin antagonists from a culture of a strain of Streptomyces olivaceus are described. These analogs are L-156,586, L-156,587 and L-156,588. They are, respectively, 15-dihydro-13,14-anhydro-, 13,14-anhydro- and 13-desoxy-analogs of virginiamycin M1. We also chemically converted virginiamycin M1 (via L-156,587) to L-156,586 and its unnatural epimer, L-156,906. These analogs are competitive and selective antagonists of gastrin and brain cholecystokinin binding at nanomolar concentrations. These are the most potent gastrin/brain cholecystokinin antagonists from natural products. The same compounds showed poor Gram-positive antibiotic activity versus virginiamycin M1. Structurally related Gram-positive antibiotics, griseoviridin and madumycin I, were inactive in gastrin and brain cholecystokinin binding at up to 100 microM.
Subject(s)
Cholecystokinin/antagonists & inhibitors , Gastrins/antagonists & inhibitors , Streptomyces/metabolism , Virginiamycin/analogs & derivatives , Animals , Bacteria/drug effects , Binding, Competitive , Brain/metabolism , Chromatography, High Pressure Liquid , Fermentation , Gastric Mucosa/metabolism , Guinea Pigs , Liver/metabolism , Magnetic Resonance Spectroscopy , Male , Pancreas/metabolism , Rats , Receptors, Cholecystokinin/metabolism , Streptomyces/classification , Virginiamycin/biosynthesis , Virginiamycin/chemistry , Virginiamycin/metabolism , Virginiamycin/pharmacologySubject(s)
Complement C5a/antagonists & inhibitors , Peptides, Cyclic/biosynthesis , Streptomyces/metabolism , Chromatography, Gel , Fermentation , Humans , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Conformation , Molecular Structure , Peptides, Cyclic/chemistry , Peptides, Cyclic/isolation & purification , X-Ray DiffractionABSTRACT
The aims of these studies are (1) to determine whether, and by what mechanism(s), underexpression of M1 and/or NS1 protein restricts replication and cytopathogenicity in mouse brain cells of human influenza viruses which are closely related to the neurovirulent WSN variant but not selected for the neurovirulent phenotype; (2) to learn, ultimately, whether similarly restricted replication in natural infections might be enough to cause direct or indirect, immunologically mediated, neuropathology. On the basis of immunostaining, we have suggested that, in "aged" mouse embryo brain (MEB) cell cultures infected with A/PR/8/34 (PR8) or A/WS/33 (WS), M1 protein expression is restricted mainly in mature astrocytes (the dominant cell type in such cultures), but not in mature oligodendrocytes or neurons. Here we show that amounts of radiolabeled M1 protein in lysates of MEB cultures infected with PR8, WS, or WSN differ in proportion to previously reported single-cycle yields of trypsin-activated infectious virions. Low or undetectable cell-associated M1 does not reflect accelerated degradation, but tends to be accompanied by increased M2 protein (a product of spliced mRNA7). Radiolabeled NS1 is reduced, NS2 relatively increased, in "aged" MEB cultures infected at low m.o.i. with PR8, at high m.o.i. with WS as well, but not with WSN. In contrast, actively dividing and differentiating astrocyte-enriched or "young" MEB cultures tend to produce greatly increased amounts of NS2 even though NS1 may be at "normal" levels, both relative to those in similarly infected CEF cultures. We show, in extension of comparative studies by others on permissive and abortive FPV-infected cell systems, that virus strain-, cell type-, and perhaps differentiation-dependent variations in efficiency of mRNA 7 and 8 transcription and/or splicing are primary factors controlling variable expression of M and NS proteins in mouse brain cell cultures.
Subject(s)
Brain/microbiology , Influenza A Virus, H1N1 Subtype , Influenza A virus/physiology , RNA Splicing , RNA, Viral/genetics , Viral Proteins/genetics , Animals , Astrocytes/microbiology , Autoradiography , Brain/cytology , Capsid/analysis , Capsid/biosynthesis , Capsid/genetics , Cells, Cultured , Densitometry , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Viral , Humans , Influenza A virus/genetics , Mice , Precipitin Tests , RNA Processing, Post-Transcriptional , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/metabolism , Transcription, Genetic , Viral Core Proteins/analysis , Viral Core Proteins/biosynthesis , Viral Core Proteins/genetics , Viral Matrix Proteins/analysis , Viral Matrix Proteins/biosynthesis , Viral Matrix Proteins/genetics , Viral Nonstructural Proteins , Viral Proteins/analysis , Viral Proteins/biosynthesis , Virus ReplicationABSTRACT
The in vitro and in vivo oxytocin/arginine vasopressin (OT/AVP) antagonist properties of two cyclic hexapeptides derived from a newly discovered natural product (L-156,373) of Streptomyces silvensis are described. In radioligand binding assays, L-156,373 [cyclo(L-Pro-D-Phe-N-OH-L-Ile-D-piperazyl-L-piperazyl-N-Me-D -Phe)] exhibited moderate affinity for rat uterine OT receptors (Ki, 150 nM), with some selectivity (approximately 20-fold) vs. liver AVP-V1 and kidney AVP-V2 receptors. Dehydroxylation of N-hydroxyisoleucine and oxidation of the piperazic acid residues of L-156-373 produced an interesting derivative, L-365,209. These structural modifications increased OT receptor affinity and selectivity by 20- and 2.5-5-fold, respectively. In the isolated rat uterus, L-365,209 was a potent (apparent dissociation constant, 1.7 nM) and competitive OT antagonist. L-365,209 also blocked the effects of AVP at both AVP-V1 (phosphatidylinositol turnover in rat hepatocytes) and AVP-V2 (adenylate cyclase in rat kidney medulla) receptors, but only at low micromolar concentrations. L-365,209, given iv to anesthetized rats, antagonized the action of exogenous OT on the uterus (ID50, 460 micrograms/kg) with a relatively long duration of action. L-365,209 represents a unique class of compounds that provides an entirely new approach for the design of antagonists for these neurohypophyseal hormones.
Subject(s)
Oxytocin/antagonists & inhibitors , Peptides/pharmacology , Receptors, Vasopressin , Streptomyces/analysis , Animals , Arginine Vasopressin/antagonists & inhibitors , Female , In Vitro Techniques , Liver/cytology , Liver/metabolism , Peptides/metabolism , Peptides, Cyclic/metabolism , Rats , Rats, Inbred Strains , Receptors, Angiotensin/metabolism , Receptors, Oxytocin , Uterus/metabolismABSTRACT
The responses of mouse embryo brain (MEB) cell cultures and of Madin-Darby canine kidney cells and chicken embryo fibroblasts to infection with A/PR/8/34 (PR8), A/WS/33 (WS), or the neurovirulent WSN variant were compared in terms of (i) single-cycle yields of hemagglutinating and associated neuraminidase (NA) activities and plaque-forming particles, the latter with or without trypsin activation [PFU(TR++) or PFU(TR--), respectively], and (ii) expression of nucleoprotein (NP), M1, and NS1 protein, determined for specific cell types by immunostaining, for whole culture lysates by Western blot analysis of NP and M1. Primary MEB cultures grown in serum-enriched medium were infected after 6 days (young), when none of the cells reacted specifically and exclusively with any of the nerve cell marker antibodies used, or after greater than or equal to 21 days (aged), when astrocytes (the predominant cell type), neurons, and oligodendrocytes were morphologically and immunologically mature. Secondary astrocyte-enriched cultures were used when they contained 90 to 99% of their cells as astrocytes at an early stage of differentiation. By all criteria, young MEB cultures were only marginally less permissive for each of the three viruses than were chicken embryo fibroblasts or Madin-Darby canine kidney cells. Aged MEB cultures, by comparison, produced undiminished NP, hemagglutinin, and neuraminidase, but yields of PFU(TR++) and expression of M1 protein (relative to NP) were reduced for all three viruses, most for PR8 and least for WSN; relative reduction of NS1 protein was demonstrable only in PR8-infected aged cultures. Immunostaining revealed low levels of M1 and NS1 expression only in astrocytes, not in oligodendrocytes and neurons. In PR8-infected mature astrocytes, NP accumulated in the nucleus; it persisted in some cells for at least 8 weeks after infection. The presence of NP did not seem to interfere with cell division. Secondary MEB cultures containing 90 to 99% immature astrocytes were less restricted than were aged primary cultures. Thus, it appears that reduced permissivity of nerve cell cultures, as measured in this study, is most closely correlated with advancing differentiation and maturity of astroglial cells. Assembled virions, including those that score as PFU(TR++) in restricted cultures (e.g., PR8-infected aged MEB), may be mainly products of mature oligodendrocytes and neurons.
Subject(s)
Brain/microbiology , Influenza A virus/growth & development , Animals , Blotting, Western , Brain/cytology , Cell Differentiation , Cells, Cultured , Dogs , Fibronectins/analysis , Hemagglutinins, Viral/analysis , Immunoenzyme Techniques , Influenza A virus/immunology , Influenza A virus/pathogenicity , Mice , Mitosis , Neuraminidase/metabolism , Neurons/cytology , Neurons/microbiology , Nucleoproteins/immunology , Viral Matrix Proteins/metabolism , Virus ReplicationABSTRACT
Difficidin and oxydifficidin, two novel macrocyclic polyene lactone phosphate esters were discovered in fermentation broths of each of two strains of Bacillus subtilis: ATCC 39320 and ATCC 39374. Difficidin and oxydifficidin each showed a broad spectrum of activity against aerobic and anaerobic bacteria. Many of the susceptible aerobes and anaerobes were human pathogens resistant to one or more antibiotics. Difficidin and oxydifficidin when administered intraperitoneally protected mice against an otherwise lethal bacteremia caused by Klebsiella pneumoniae (ED50 in mg/kg of 1.31 and 15.6 respectively). Neither difficidin nor oxydifficidin were effective when administered via the subcutaneous route.