Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp.

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Primary structure of pilin protein from Bacteroides nodosus strain 216: comparison with the corresponding protein from strain 198

The amino acid sequence of pilin protein from Bacteroides nodosus strain 216 was determined. The protein had a calculated molecular weight of 15962 and contained the same number of amino acid residues (151) as the pilin from the previously sequenced strain 198. The sequence of the first 44 residues was common to both strains, including the unusual amino-terminal amino acid, N-methylphenylalanine. Of the remaining 107 residues, 37% of them differed between the two strains. Comparison of hydrophilicity profiles constructed from the sequence data indicated that a conserved region around residues 71-72 was probably the site of an antigenic determinant.     

Pathophysiologic alterations in Biomphalaria glabrata infected with Angiostrongylus costaricensis

Hemolymph glucose, alkaline phosphatase, lactic dehydrogenase, and creatine phosphokinase in Biomphalaria glabrata infected with Angiostrongylus costaricensis were significantly higher on day 27 postinfection (PI) than in uninfected snails. Hemolymph total calcium from infected snails was less on days 6, 12, and 27 PI than that from controls. Total hemolymph protein was similar for controls and infected animals during the entire study. Throughout the study the mean number of amoebocytes/mm³ hemolymph from infected snails was significantly less than that for controls. Mean total wet weights of digestive gland and foot muscle from infected and uninfected snails was similar throughout the study. Mean μg glycogen/mg wet weight of digestive gland from infected snails was significantly greater on days 24, 27, and 28 PI than that from controls. Mean μg glycogen/mg wet weight of foot muscle from infected snails was significantly reduced between days 12 and 28 PI from that of uninfected snails. It is suggested that hemolymph glucose and digestive gland glycogen in infected snails are augmented by glycogen breakdown in the foot muscle of parasitized animals. Elevations in hemolymph enzymes are due to tissue destruction by larvae emerging from the foot muscle of infected snails. Parasite-induced derangements in shell metabolism underlie observed changes in hemolymph calcium in infected snails.     

Unusual stability of the Methanospirillum hungatei sheath.

The proteinaceous sheath of Methanospirillum hungatei was isolated by lysing cells in 50 mM dithiothreitol, separating the sheath from other cellular material by discontinuous sucrose density centrifugation, and removing the "cell spacers" with dilute NaOH. The isolated sheath material consisted of hollow tubes which had a highly ordered surface array. The stability of the sheath to treatment with denaturants and to enzymatic digestion was examined by a turbidimetric assay in conjunction with electron microscopy and optical or electron diffraction. The sheath was resistant to a range of proteases and also was not digested by peptidoglycan-degrading enzymes, a lipase, a cellulase, a glucosidase, or Rhozyme (a mixture of galactosidases, acetylglucosaminidase, acetylgalactosaminidase, fucosidase, and mannosidases). In addition to being unaffected by common salts, thiol-reducing agents, and EDTA, the layer was resistant to powerful denaturants such as 6 M urea, 6 M guanidinium hydrochloride, 10 M LiSCN, cyanogen bromide, sodium periodate, and 1% sodium dodecyl sulfate. Strong bases, boiling 3 N HCl, and performic acid did attack the sheath; in these cases, the array was systematically disassembled in a progressive manner, which was followed by electron microscopy. The layer was slightly modified by N-bromosuccinimide in urea, but the array remained intact. The stability of the sheath was remarkable, not only as compared to other bacterial surface arrays, but also as compared to proteins generally, and possibly indicated the presence of covalent cross-links between protein subunits.     

Macrophage-induced cytostasis: kinetic analysis of bromodeoxyuridine-pulsed cells

The effect of tumoricidal macrophages on the cell cycle progression of six different cell lines was studied using an anti-bromodeoxyuridine (BrdUrd) monoclonal antibody to follow the traverse of BrdUrd-labeled cells. Exponentially growing cultured mammalian cells, from six different cell lines, were prepulsed with BrdUrd before exposure to tumoricidal macrophages. The cultured cells were then analyzed as a function of time for DNA content (by propidium iodide staining) and for BrdUrd incorporation (using a fluoresceinisothiocyanate [FITC]-conjugated anti-BrdUrd monoclonal antibody). The position of the cells in cycle and the progression of the BrdUrd-labeled cohort was followed using flow cytometry. The cell lines examined were: Colon 26, BALB/c-3T3, ST3T3 (a spontaneously transformed, tumorigenic clone of 3T3), WCHE5 (a clone of whole Chinese hamster embryo cells), RIF (a radiation-induced fibrosarcoma), and A101D (a human melanoma). The bivariate distributions showed that for all six cell lines the BrdUrd-labeled cohort in the control cultures progressed around the cell cycle during the first 12 h of culture, as the cells exponentially increased. In contrast, when each cell line was incubated with tumoricidal macrophages, the BrdUrd-labeled cohort did not progress through cell cycle but remained in S phase throughout the 12-h culture period. There was also no evidence for progression of cells out of G1. The data show that cells were arrested in every phase of cell cycle. This study suggests that cytostasis, as manifested by the termination of progression in all phases of the cell cycle, is a universal phenomenon induced by tumoricidal macrophages.     

Competitive antagonists of bradykinin

The first sequence-related competitive inhibitors of the classic kinin in vitro (rat uterus guinea pig ileum) and in vivo (rat blood pressure) assays have been developed. Replacement of the proline residue at position 7 of bradykinin (BK) with a D-phenylalanine residue is the key modification which converts BK agonists into antagonists. [D-Phe7]-BK exhibits moderate (pA2 = 5.0) inhibition of BK activity on the guinea pig ileum but possesses weak BK-like myotropic activity on the isolated rat uterus and 2-4% of BK depressor potency in the rat blood pressure assay. The additional replacement of the phenylalanine residues at positions 5 and 8 of [D-Phe7]-BK with the isosteric beta-(2-thienyl)-alanine residue produces a potent antagonist of BK activity on the uterus (pA2 = 6.4), ileum (pA2 = 6.3), and in the rat blood pressure assay. The antagonism of BK action on smooth muscle is specific for kinins (BK, kallidin, Met-Lys-BK), but neither inhibitor antagonizes the smooth muscle activity of angiotensin or substance P. Inhibition is competitive and fully reversible.     

Subcellular localization and glycoprotein nature of the invertase from the fission yeast Schizosaccharomyces pombe

The subcellular localization of the enzyme invertase in Schizosaccharomyces pombe cells, both repressed and derepressed for synthesis of the enzyme, was studied. Most of the invertase was found to be located outside the plasma membrane and only a small percentage was found to be associated to membranes. A substantial portion of the external enzyme remained firmly bound to cell-wall material.All of the invertase recovered in soluble form from cellular extracts reacted with concanavalin A and with the lectin from Bandeiraea simplicifolia seeds, indicating the presence in the enzyme of a carbohydrate moiety which probably contains terminal mannosyl (or structurally related) and galactosyl residues.The possibility of the presence of two different forms of invertase in S. pombe was considered. An intracellular, soluble form of invertase, devoid of carbohydrate, similar to the small invertase of the budding yeast Saccharomyces cerevisiae, was not found in S. pombe. However, the Michaelis constant for sucrose of the enzyme present in repressed cells was smaller than that of the invertase synthesized under derepressing conditions, although this difference could also be the result of a different pattern of glycosylation of the invertase synthesized under different growth conditions.     

N2-(1-carboxyethyl)methionine. A ''pseudo-opine'' in octopine-type crown-gall tumours.

A novel methionine-containing plasmid-determined compound, N2-(1-carboxyethyl)methionine (NCEM) has been identified in crown-gall tumours induced by octopine-type strains of Agrobacterium tumefaciens. NCEM is probably synthesized by octopine synthase. Cell-free preparations from octopine-type strains of A. tumefaciens can degrade NCEM; however, the bacterium cannot transport the compound into the cell, although these strains can take up and degrade the octopine family of opines.