Immunofluorescence colocalization of the 90-kDa heat-shock protein and microtubules in interphase and mitotic mammalian cells

A mouse monoclonal antibody (AC88) that was raised against the 88-kDa heat-shock protein of the water mold, Achlya ambisexualis, and that cross-reacts with the 90-kDa mammalian heat-shock protein (hsp90), and an antibody against tubulin were used to localize hsp90 and microtubules, respectively, in the same cultured rat endothelial and PtK1 epithelial cells by indirect immunofluorescence. AC88 and tubulin antibodies labeled the same structures in cells at all stages of the cell cycle, regardless of whether cells were permeabilized before or after fixation. Labeling of cell structures by both AC88 and anti-tubulin antibodies was identically affected by treating cells with colcemid. Double labeling with AC88 and anti-tubulin antibodies in interphase and mitotic cells is consistent with the conclusion that all microtubules are labeled and that no subclass of microtubules is preferentially labeled. Fluorescent labeling by AC88 was prevented by preabsorption of the antibody with purified rat hsp90 but was unaffected by preabsorption with purified 6S tubulin dimer. In contrast to AC88, fluorescent labeling by an anti-tubulin antibody was prevented by preabsorption with tubulin dimer but was unaffected by preabsorption with rat hsp90. Western-blot analysis demonstrated no cross-reactivity of AC88 for tubulin and no cross-reactivity of the anti-tubulin antibody for hsp90. A polyclonal antiserum fraction from a rabbit immunized with the 89-kDa heat-shock protein from chicken also labeled the mitotic apparatus in dividing cells and, somewhat less distinctly, fibrous structures in interphase cells. Labeling by hsp89 anti-serum was prevented by absorption with hsp90. AC88 also labeled microtubules in cultured mouse (L929 and 3T3), rat (endothelium and TRST), hamster (CHO) and primate (BSC, COS-1 and HeLa) cell lines. The demonstration of colocalization of hsp90 with microtubules should provide a valuable clue to eventual understanding of the cellular function of this ubiquitous, conserved and abundant stress-response protein.     

Aggregate-formation by Clostridium butyricum

Summary Clostridium butyricum was grown in a glucose-limited chemostat culture at a dilution rate of 0.1 h^–1 at pH 6.0. With 0.9% w/v input glucose in the medium the cells were found to grow in suspension and glucose was fermented completely to acetate and butyrate. An increase in the input concentration of glucose resulted in increased concentrations of end-products, but not all extra glucose was consumed. It could be demonstrated that this was due to a lowering of the maximal growth rate by elevated levels of butyric acid. However, prolonged growth in the presence of high glucose concentrations led to an increase in biomass. This was caused by the selection of a variant that was less sensitive to butyrate. This variant was able to form aggregates in an anaerobic gas-lift reactor at high dilution rates. Inoculation of these aggregates in a conventional chemostat culture with high glucose input resulted in an aggregated culture that remained stable for at least 6 months, and in which all glucose was consumed. Whether the organisms grew in suspension or in aggregates was found to be determined by the concentration of butyrate. The isolation of aggregate-forming variants from chemostat cultures leads to a very simple and new type of immobilization technique.Offprint requests to: G. R. Zoutberg     

Continuous production of gibberellic acid in a fixed-bed reactor by immobilized mycelia of Gibberella fujikuroi in calcium alginate beads

Summary The continuous production of gibberellic acid with immobilized mycelia of Gibberella fujikuroi was maintained over a hundred days in a tubular fixed-bed reactor. Free mycelium at the beginning of the storage phase was harvested from G. fujikuroi shake-flask culture and was immobilized by ionotropic gelation in calcium alginate beads.The continuous recycle production system consisted of a fixed-bed reactor, a container in which the culture medium was heated, stirred and aerated, and valves for sample withdrawal or reactant addition during the first 1320 h (55 days). A two-phase continuous extractor was then added for the last 960 hours (40 days). Free and immobilized mycelium shake-flask cultures with the same strain used in the continuous culture system were also realized to compare growth, maintenance and production parameters. The results show about the same gibberellic acid productivity in both free and immobilized mycelium shakeflask cultures: 0.384 and 0.408 mgGA₃·gBiomass^-1 ·day^-1, respectively, whereas in the continuous system the gibberellic acid production is about twice as large for a similar biomass: 0.768 mgGA₃·gBiomass^-1·day^-1. Several factors affecting the overall productivity of the immobilized systems were found to be: the quality and the quantity of mycelia in the biocatalyst beads and the immobilization conditions.     

Evidence that vascular actions of PHI are mediated by a VIP-preferring receptor

Vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) are homologous neuropeptides which share vasodilatory properties. This paper addresses the question of whether PHI exerts its vascular action via a receptor distinct from that for VIP. Radioligand binding experiments were done using [Tyr(125I)10]VIP, [Tyr(125I)22]porcine PHI, [Tyr(125I)10]rat PHI and arterial preparations from rat, bovine and porcine species. The radioiodination of rat PHI by the lactoperoxidase-glucose oxidase method and analysis of the structure of the major radiolabeled derivatives were described. All the receptor binding experiments identified a VIP-preferring receptor irrespective of which radioligand or arterial preparation was utilized. VIP and PHI peptides demonstrated cross-desensitization in studies of relaxation of porcine coronary arterial strips in vitro. The present results favor the conclusion that the vascular actions of the PHI peptides are best explained by binding to a VIP-preferring receptor.     

Structure and biological features of the temperate phage of Bacillus thuringiensis var. galleriae 69/9, sensitive to chloroform

A new temperate bacteriophage designated Px1 has been isolated from the culture of Bacillus thuringiensis var. galleriae 69/6 producing enthobacterin. The bacteriophage belongs to morphological group B1 in accordance with the classification by D. Reanney and H. Ackerman. The bacteriophage head has an isometric multifaceted form with 40 nm diameter. The length of its noncontractile transversely lined tail is 130 nm. High sensitivity to chloroform is peculiar of the phage. The lytical specter of the phage Px1 has been studied. The phage is shown to be capable of efficient transduction of plasmids between the bacteria of Bacillus cereus group.     

Isolation of recombinant vaccine strains of poliovirus from patients with poliomyelitis

Oligonucleotide maps of some poliovirus type 2 strains isolated from polio cases, while being clearly related to that of the Sabin vaccine type 2 strain, exhibited, nevertheless, marked differences from the reference (vaccine) map. Several large oligonucleotides derived from 4 such strains were subjected to enzymatic sequencing. The results strongly suggest that all of them were intertypic recombinants between the Sabin strains. The 5'-parts of the genomes of these strains were derived from the type 2 vaccine whereas the 3'-parts were of type 1 (in 2 strains) or type 3 (in other 2 strains) origin.     

Expert systems in medicine

The emergence of the artificial intelligence (AI) in computer technology and its application in the medical field enables the researchers to carry out such intelligent activities like image processing, medical reasoning systems, clinical decision supporting and natural language understanding, etc. A gastroenterological expert system application is briefly demonstrated in this paper. Similar expert systems can be seen to be useful in the research of gastrointestinal cytoprotection, including the plan of different compounds with cytoprotective effect, experimental and clinical medical research.     

Antibiotics formed by Actinomadura fulvescens

Novel antibiotics with in vitro activity against gram-positive bacteria were isolated from Actinomadura fulvescens INA 3321 and INA 3852. Conditions for biosynthesis and isolation of antibiotics 3321 and 3852, as well as their physicochemical and biological properties were studied. Chromatographic analysis of the antibiotics revealed that each of them contained two biologically active components. The components were separated with preparative chromatography. Physicochemical properties of the components showed that antibiotics 3321 and 3852 were similar. UV and IR spectroscopy suggested that antibiotics 3321 and 3852 were original compounds not described earlier.