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Biomedical subjects

B Harrach

Publications and source records attributed to B Harrach.

49 records · Page 3Linked to original sources
PubMed1988

Bovine herpesvirus 1: rapid diagnosis of infection by direct filter hybridization.

Direct filter hybridization (DFH) was applied as a simple method of nucleic acid hybridization to diagnose bovine herpesvirus 1 (BHV-1) infection without previous purification of nucleic acids from the specimens. The DNA of BHV-1 was cleaved with the restriction endonuclease Pst I and randomly cloned into pKH47 plasmids. The clones were labelled with 32P or biotin and selected on uninfected and infected cells for the highest specific activity to detect BHV-1 infection. Two clones, which detected about 10 infected cells, were selected for the diagnosis of BHV-1 in cattle. On specimens collected during experimental and natural disease, the DFH showed to be in concordance with the standard method of virus isolation. This simple hybridization technique proved to be a sensitive and rapid alternative to virus isolation. Specific diagnosis of BHV-1 infection can be made even in simply equipped laboratories within 10 h.

Animals↗
PubMed1987

Interaction of small dermatan sulfate proteoglycan from fibroblasts with fibronectin.

Immunogold labeling was used to localize the core protein of small dermatan sulfate proteoglycan (DS-PG) on the surface of cultured human fibroblasts. At 4 degrees C, DS-PG core protein was uniformly distributed over the cell surface. At 37 degrees C, gold particles either became rearranged in form of clusters or remained associated with fibrils. Double-label immunocytochemistry indicated the co-distribution of DS-PG core protein and fibronectin in the fibrils. In an enzyme-linked immunosorbent assay, binding of DS-PG from fibroblast secretions and of its core protein to fibronectin occurred at pH 7.4 and at physiological ionic strength. Larger amounts of core protein than of intact proteoglycan could be bound. Fibronectin peptides containing either the heparin-binding domain near the COOH-terminal end or the heparin-binding NH2 terminus were the only fragments interacting with DS-PG and core protein. Competition and replacement experiments with heparin and dermatan sulfate suggested the existence of adjacent binding sites for heparin and DS-PG core protein. It is hypothesized that heparan sulfate proteoglycans and DS-PG may competitively interact with fibronectin.

Cell Membrane↗
PubMed1985

Macrocyclic trichothecene toxins produced by Stachybotrys atra strains isolated in Middle Europe.

A total of 17 strains of Stachybotrys atra isolated in Hungary and Czechoslovakia were cultured on Sabouraud agar, and the toxins produced by them were chemically analyzed by gas-liquid chromatography, high-pressure liquid chromatography, and mass spectroscopy. Furthermore, brine shrimp (Artemia salina) bioassay was used for the determination of toxicity of the compounds examined. Macrocyclic trichothecenes (satratoxins H and G, roridin E, and verrucarin J as well as two other unidentified macrocyclic trichothecenes) were found in all of the cultures tested. The identities of satratoxins H and G, roridin E, and verrucarin J were qualitatively determined by high-pressure liquid chromatography and gas-liquid chromatography. The ratio of satratoxins H and G and roridin E was found to be similar in each of the strains tested, but the amount of verrucarin J found was different in each of them. One of the unidentified macrocyclic trichothecenes was equivalent to the compound isolated by Harrach et al. (Harrach et al., Appl. Environ. Microbiol. 41:1428-1433, 1981). The other one proved to be a newly isolated macrocyclic trichothecene toxin. Stachybotryotoxicosis, one of the oldest mycotoxicoses known, and a serious problem in Middle Europe (Gy. Danko, Magy. Allatorv. Lapja 31:226-232, 1976), is believed to be caused by macrocyclic trichothecene toxins produced by Stachybotrys atra (R. M. Eppley, in Rodricks et al., ed., Mycotoxins in Human and Animal Health, p. 285-293, 1977). Forty years ago, the death of animals in the Soviet Union was associated with this fungus (C. U. Ruhliada, in Proceedings of the All-Union Sci. and Tech. Conf., p. 47-51, 1980).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Isolation of satratoxins from the bedding straw of a sheep flock with fatal stachybotryotoxicosis.

During a period of several weeks, more than 100 sheep died at a Hungarian farm. The animals exhibited fleece loosing, and hemorrhaging was the most important autopsy finding. Pasteurella haemolytica was cultured from various organs. The bedding straw was abundantly covered with Stachybotrys atra, and removal of the straw stopped the disease. Methanol extraction of the bedding straw followed by solvent partitioning, column chromatography, preparative thin-layer chromatography, and high-pressure liquid chromatography led to the isolation of satratoxins G and H, which were characterized by thin-layer chromatography, high-pressure liquid chromatography, and mass spectroscopy. This is the first isolation and characterization of toxins from a field sample of material responsible for an outbreak of stachybotryotoxicosis.

Animals↗
PubMed1981

Macrocyclic trichothecene toxins produced by a strain of Stachybotrys atra from Hungary.

A strain of Stachybotrys atra isolated from a field case of stachybotryotoxicosis in Hungary was cultured in Hungary. All of the compounds toxic to brine shrimp were separated from the culture extract by solvent partition, column chromatography, and preparative thin-layer chromatography. Two of the toxic compounds were identified as verrucarin J and satratoxin H by comparison with pure standards resolved by high-pressure liquid chromatography and characterized by mass spectrometry. Two other toxic components were identified as roriden E and satratoxin G on the basis of their mass spectra. The fifth toxic compound was identified as a macrocyclic trichothecene based on the following findings: a positive 4-(p-nitrobenzyl)pyridine color reaction, hydrolysis resulting in verrucarol verified by combined gas chromatography-mass spectrometry, and a characteristic trichothecene proton-nuclear magnetic resonance spectrum. This macrocyclic trichothecene has a molecular ion (528) identical to satratoxin H, and its mass spectrum is similar; however, its Rf value on Silica Gel G differs.

Chemical Phenomena↗
PubMedDate not supplied

Polyclonal antibodies against formaldehyde-modified apolipoprotein A-I. An approach to circumventing fixation-induced loss of antigenicity in immunocytochemistry.

A central requirement for the subcellular localization of synthesized and/or endocytozed lipoproteins is the availability of antibodies against the antigen to be localized. The preparation of cells for electron microscopy, in particular the fixation and embedding routine, influences the antigenicity, often resulting in a markedly reduced labeling intensity. To ameliorate fixation-induced changes in antigenicity, we produced antibodies against pre-fixed human apolipoprotein (apo) A-I. Purified apo A-I was fixed with 4% formaldehyde and was used to raise polyclonal antibodies in rabbits. The antiserum was purified by protein A-Sepharose followed by affinity chromatography with the fixed antigen coupled to vinylsulfone-activated agarose. The specificity of the antibodies was ascertained by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis against different fixed and unfixed lipoproteins. Nonspecific binding to unfixed or to fixed apolipoproteins was not observed. Thus, the antibodies reacted specifically with apo A-I and recognized the fixed as well as the unfixed protein. In ELISA, the reaction of the antibodies was markedly enhanced with the fixed antigen, indicating that the antibodies were directed against epitopes characteristically modified by the fixation. The efficacy of the antibodies for light and electron microscopy was tested on HepG2 cells and on human liver cells which are known to synthesize apo A-I. When HepG2 cells were exposed to anti-apo A-I antibodies followed by a secondary fluorescein isothiocyante-labeled antibody, fluorescence was found intracellularly in distinct regions. Electron microscopy revealed that the endoplasmic reticulum, and in particular the trans elements of the Golgi complexes, were the main compartments stained for apo A-I both in HepG2 cells, as shown by the immunoperoxidase technique, and in human hepatocytes, as shown by the protein A-gold technique on ultrathin cryosections. Furthermore, we were able to localize apo A-I in foam cells of the human aortic plaque by postembedding immunolabeling techniques. These findings demonstrate the potential of antibodies to fixed lipoproteins in impaired localization at the light microscopic and electron microscopic levels.

Animals↗
PubMedDate not supplied

Display of low density lipoprotein receptors is clustered, not dispersed, in fibroblast and hepatocyte plasma membranes.

Although the principal details of low density lipoprotein (LDL) uptake by receptor-mediated endocytosis and its subsequent intracellular fate have been thoroughly investigated, an aspect of this mechanism that continues to provoke controversy concerns the manner of display of LDL receptors upon their initial insertion at the cell surface. While our studies based on electron microscopy of platinum/carbon replicas of gold-labeled cells have previously suggested a clustered display pattern, others have concluded, before and since, that LDL receptors are inserted individually at random widely dispersed sites in the plasma membrane. In this article, we present a series of experiments designed to discriminate between these competing hypotheses. In addition to the use of LDL-colloidal gold complexes, visualized electron microscopically, on cells subjected to a variety of experimental procedures, these experiments include the application of anti-apolipoprotein B-100 antibodies, anti-LDL-receptor antibodies, and direct visualization of native (unlabeled) LDL molecules at the cell surface. All results point to a loose-cluster arrangement, not one involving widely dispersed individual units, as the initial display pattern of newly inserted LDL receptors. A comparison of LDL and beta-very low density lipoprotein receptor distribution in fibroblasts and hepatocytes suggests that this cluster pattern is a characteristic of the LDL (apolipoprotein B/E) receptor across cell types, but that the closely related apolipoprotein E receptor differs in that it is inserted individually in a highly dispersed state, in common with a variety of other receptor types.

Animals↗