Yeast mannans inhibit binding and phagocytosis of zymosan by mouse peritoneal macrophages.

We have examined the effects of various mannans, glycoproteins, oligosaccharides, monosaccharides, and sugar phosphates on the binding and phagocytosis of yeast cell walls (zymosan) by mouse peritoneal macrophages. A phosphonomannan (PO(4):mannose ratio = 1:8:6) from kloeckera brevis was the most potent inhibitor tested; it inhibited binding and phagocytosis by 50 percent at concentrations of approximately 3-5 mug/ml and 10 mug/ml, respectively. Removal of the phosphate from this mannan by mild acid and alkaline phosphatase treatment did not appreciably reduce its capacity to inhibit zymosan phagocytosis. The mannan from saccharomyces cerevisiae mutant LB301 inhibits phagocytosis by 50 percent at 0.3 mg/ml, and a neutral exocellular glucomannan from pichia pinus inhibited phagocytosis by 50 percent at 1 mg/ml. Cell wall mannans from wild type S. cervisiae X2180, its mnn2 mutant which contains mannan with predominantly 1(arrow)6- linked mannose residues, yeast exocellular mannans and O-phosphonomannans were less efficient inhibitors requiring concentrations of 1-5 mg/ml to achieve 50 percent reduction in phagocytosis. Horseradish peroxidase, which contains high-mannose type oligosaccharides, was also inhibitory. Mannan is a specific inhibitor of zymosan binding and phagocytosis. The binding and ingestion of zymosan but not of IgG- or complement-coated erythrocytes can be obliterated by plating macrophages on substrates coated with poly-L-lysin (PLL)-mannan. Zymosan uptake was completely abolished by trypsin treatment of the macrophages and reduced by 50-60 percent in the presence of 10 mM EGTA. Pretreatment of the macrophages with chloroquine inhibited zymosan binding and ingestion. These results support the proposal that the macrophage mannose/N-acetylglucosamine receptor (P. Stahl, J.S. Rodman, M.J. Miller, and P.H. Schlesinger, 1978, Proc. Natl. Acad. Sci. U.S.A. 75:1399-1403, mediates the phagocytosis of zymosan particles.

Phenylalanine transfer RNA: molecular dynamics simulation.

Yeast phenylalanine transfer RNA was subjected to a 12-picosecond molecular dynamics simulation. The principal features of the x-ray crystallographic analysis are reproduced, and the amplitudes of atomic displacements appear to be determined by the degree of exposure of the atoms. An analysis of the hydrogen bonds shows a correlation between the average length of a bond and the fluctuation in that length and reveals a rocking motion of bases in Watson-Crick guanine X cytosine base pairs. The in-plane motions of the bases are generally of larger amplitude than the out-of-plane motions, and there are correlations in the motions of adjacent bases.

On the relations between the elemental surface composition of yeasts and bacteria and their charge and hydrophobicity.

The elemental surface composition of eleven microorganisms was determined by X-ray photoelectron spectroscopy. Bacteria could be distinguished from yeasts by higher nitrogen and phosphate concentrations. Overall physico-chemical properties, electrical charge and hydrophobicity, were also investigated: the former by electrophoretic mobility measurements, the latter by contact angle and by hydrophobic interaction chromatography. Phosphate plays the major role in determining the surface electrostatic charge. A correlation is observed between the N/P atomic concentration ratio and the electrostatic charge. In bacteria, hydrophobicity is directly related to concentration of carbon in hydrocarbon form and inversely related to oxygen concentration or to the N/P ratio. For yeasts, a positive correlation is found between hydrophobicity and the N/P ratio, pointing at the role of proteins in determining the hydrophobicity.

Purification of an acidic alpha-D-mannosidase from Aspergillus saitoi and specific cleavage of 1,2-alpha-D-mannosidic linkage in yeast mannan.

An acidic alpha-D-mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) has been isolated from culture filtrate of Aspergillus saitoi. The extracellular alpha-mannosidase was homogeneous in polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 51 000 and the isoelectric point pH 4.5. The purified enzyme has a pH optimum of 5.0, a Km of 0.45 mM with baker's yeast mannan and has no activity towards p-nitrophenyl-alpha-D-mannoside. The mode of action of the enzyme has been studied with baker's yeast mannan and saké yeast mannan. The enzyme cleaves specifically the 1,2-alpha-linked side chain, producing free mannose.

Sequence homology of nuclear and mitochondrial DNAs of different yeasts.

1. Both nuclear and mtDNA of four different yeasts show approximately 10% homology as measured by DNA-DNA filter hybridization. These homologous sequences are mainly attributable to the ribosomal cistrons. 2. Melting curve analysis shows that the heterologous mitochondrial DNA-DNA hybrids contain several times more mismatching than the nuclear DNA-DNA hybrids. 3. DNA-rRNA hybridization shows that the sequences of the ribosomal cistrons in both the nuclear and the mitochondrial genome have been conserved during evolution. 4. However, melting curve analysis of the DNA-RNA hybrids shows that the sequence of the nuclear ribosomal cistrons have undergone considerable fewer nucleotide substitutions than their mitochondrial counterparts. 5. The results suggest that the mitochondrial ribosomal cistrons have evolved more rapidly than the nuclear cistrons. This is discussed in the light of theories on the rat of molecular evolutin.

Binding of yeast tRNAPhe anticodon arm to Escherichia coli 30 S ribosomes.

A 15-nucleotide fragment of RNA having the sequence of the anticodon arm of yeast tRNAPhe was constructed using T4 RNA ligase. The stoichiometry and binding constant of this oligomer to poly(U)-programmed 30 S ribosomes was found to be identical to that of deacylated tRNAPhe. The anticodon arm and tRNAPhe also compete for the same binding site on the ribosome. These data indicate that the interaction of tRNAPhe with poly(U)-programmed 30 S ribosomes is primarily a result of contacts in the anticodon arm region and not with other parts of the transfer RNA. Since similar oligomers which cannot form a stable helical stem do not bind ribosomes, a clear requirement for the entire anticodon arm structure is demonstrated.

Codon-induced transfer RNA association. A property of transfer RNA involved in its adaptor function?

It is shown by equilibrium sedimentation that the binding of cognate codons to tRNAPhe (yeast), tRNAPhe (Escherichia coli), tRNALys, tRNAfMet and of the wobble codon UUU to tRNAPhe (yeast) induces dimerization of codon transfer RNA complexes. Analysis of the sedimentation profiles with a quantitative evaluation of the coupling between sedimentation and association equilibrium provides dimerization constants in the range from 1 X 10(4) to 6 X 10(4) M-1. These results on various tRNAs from different organisms suggest that the codon-induced tRNA association is a general phenomenon. Probably the codon-induced tRNA association facilitates the aminoacyl transfer reaction.

Expression of a foreign eukaryotic gene in Saccharomyces cerevisiae: beta-galactosidase from Kluyveromyces lactis.

Three recombinant DNA vectors carrying the beta-galactosidase structural gene, LAC4, from the yeast Kluyveromyces lactis were constructed and transformed into Saccharomyces cerevisiae. All transformants expressed the beta-galactosidase activity of LAC4. However, the level of enzyme activity varied, being highest in cells transformed with vectors which are maintained as multicopy plasmids and lowest in cells transformed with a vector which integrates into chromosomes. Enzyme levels probably reflect gene dosage. LAC4 is very stable when integrated into a chromosome, but unstable when carried on a plasmid. Therefore, stability is a property of the recombinant vector rather than of LAC4, LAC4-coded beta-galactosidase synthesized in either S. cerevisiae or in K. lactis is the same as judged by two-dimensional polyacrylamide gel electrophoresis. However S. cerevisiae transformed with LAC4 cannot grow on lactose, probably because lactose does not enter the cell.

On the sequence homology of the ribosomal proteins, Escherichia coli S11, yeast rp59 and Chinese hamster S14.

A strong sequence homology was found among the ribosomal proteins of the different species, S11 of Escherichia coli, rp59 of Saccharomyces cerevisiae and S14 of Chinese hamster ovary cell. The significance of this series of highly conserved proteins is discussed.

A simple method for extraction of DNA from fungi and yeasts with anhydrous hydrogen fluoride.

A novel method is described for the extraction of DNAs from fungi and yeasts. Anhydrous hydrogen fluoride (HF) selectively cleaves their cell walls under mild conditions (for 5 min at 0 degrees C), enabling the effective extraction of DNAs from organisms with a cell wall. A possible mechanism for this method concerning the selective cleavage of O-glycosidic linkages in cell walls has been described previously [(1977) Anal. Biochem. 82,289-309]. The extracted DNA is intact: in fact, the yeast DNA is directly applicable for restriction analysis and transformation of Escherichia coli.

The redox-cycling assay is not suited for the detection of pyrroloquinoline quinone in biological samples.

Based on the results of the so-called redox-cycling assay it has been claimed that various common foods and beverages as well as mammalian body fluids and tissues contain substantial quantities (microM) of free PQQ [M. Paz et al. (1989) in: PQQ and Quinoproteins (J.A. Jongejan and J.A. Duine, eds.) Kluwer Academic Publishers, Dordrecht, pp. 131-143 and J. Killgore et al. (1989) Science 245, 850-852]. However, by investigating samples from such sources with a biological assay of nM sensitivity, we could not confirm these claims. Analysis of the samples with procedures that proved adequate for the detection of PQQ adducts and conjugates gave equally negative results. To account for the positive response in the redox-cycling assay, as opposed to the negative results obtained by other methods, a search was made for those substances in these samples that caused the false-positive reactions. It was found that a number of commonly occurring biochemicals like ascorbic and dehydroascorbic acid, riboflavin and to a lesser extent pyridoxal phosphate, gave a positive response in the redox-cycling assay. The amounts of these interfering substances that were determined in the samples by independent methods could well explain the response. In separate experiments it was found that the effect of PQQ added to biological samples was obscured over an appreciable range of concentrations. For these reasons it must be concluded that the redox-cycling assay is not suited for the detection of PQQ in these samples. Any claims that are based on the results of this method should be disregarded.

Presence of the methylester of 5-carboxymethyl uridine in the wobble position of the anticodon of tRNAIII Arg from brewer's yeast.

The methylester of 5-carboxymethyluridine (mcm5U), its degradation product 5-carboxymethyluridine (cm5U) and the corresponding nucleotide (cm5Up) were isolated from brewer's yeast tRNAIII Arg or from the dodecanucleotide containing the anticodon. Their chromatographic and electrophoretic properties and their UV absorbing spectra were identical to that of the corresponding synthetic compounds. The gas chromatographic behavior and the mass spectrum of mcm5U obtained from tRNAIII Arg and of a synthetic sample were also identical ; the rare occurence of a thermal reciprocal bimolecular methyl-hydrogen transfer in the mass spectrometer ion source was observed. A mild alkaline treatment of tRNAIII Arg leads to the saponification of mcm5U into cm5U (within the tRNA), which can be again esterified in the presence of a yeast homogenate and (methyl-14C) S adenosylmethionine. The radioactivity was found in the mcm5U located in the wobble position of the anticodon of tRNAIII Arg. The presence of this odd nucleotide in that position could possibly restrict the codon-anticodon interaction of tRNAIII Arg.

The distribution of pyrophosphatidic acid in nature.

The occurrence of a novel phospholipid, pyrophosphatidic acid, in the lipid extracts of yeasts (23 species), bacteria (E. coli), algae (chlorella), mammalia (human, rabbit, guinea pig, and mouse), insect (cockroach), fish (carp), mollusc (clam), and spermatophyta (spinach) was investigated. Pyrophosphatidic acid was found exclusively in the lipid extracts of several kinds of yeast species, but not in other normal living species (animals, plants, and microorganisms) so far investigated. All of the yeast species containing this lipid belong to the asporogenous yeasts (Cryptococcus neoformans CBS-132, Cryptococcus laurentii Z 6-5, Rhodotorula glutinis H 3-9-1, Rhodotorula rubra AY-2, Kloeckera apiculata KK-3, and Trichosporon cutaneum KC 4-3), and ballistosporogenous yeast (Sporobolomyces salmonicolor WF 174). In contrast, no detectable amount of pyrophosphatidic acid was found in the cellular lipids of ascosporogenous yeasts.

Distribution of immunoreactive glia maturation factor-like molecule in organs and tissues.

Using the monoclonal antibody G2-09 raised against bovine glia maturation factor (GMF), we screened various rat organs and tissues for GMF-like immunoreactivity. In the adult animal, with the exception of the heart, GMF was found exclusively in the nervous system, with the cerebellum exhibiting higher specific activity than other brain regions. The nature of the immunoactivity in the heart is presently unclear. None of the body fluids collected from humans, including serum and cerebrospinal fluid, possessed detectable GMF immunoactivity. A phylogenetic comparison revealed the presence of GMF in the brain of al vertebrates studied, from fish to primates. GMF was absent from bacteria and yeast. An ontogenetic study on rats showed the highest GMF level in the fetal brain, with a gradual but steady decrease after birth. However, a substantial amount of GMF persisted even in older animals. GMF was localized in astrocytes and Bergmann glia in the rat brain, using immunostaining at the light microscopic level.

Intensity and anisotropy decays of the Wye base of yeast tRNA(Phe) as measured by frequency-domain fluorometry.

The intensity and anisotropy decays of Wye base fluorescence from yeast tRNA(Phe) were determined by frequency-domain fluorometry. The intensity decay is at least a double exponential in the presence and absence of Mg2+, but the multi-exponential character of the decay is more pronounced in the absence of Mg2+. The anisotropy decay displays components due to overall tRNA rotational diffusion and to local torsional motions. The amplitude of the local motion is decreased 2-fold by the presence of Mg2+. The results are broadly consistent with a more homogeneous environment for the Wye base in the presence of Mg2+.

Methylation and acetolysis of extracellular D-mannans from yeast.

Methylation-fragmentation analyses were conducted on a series of extra-cellular, yeast alpha-D-linked mannans representing six different structural types. D-Mannans of low degree of branching were produced by Hansenula capsulata strains and by species related to H. holstii, The former consisted primarily of (1 leads to 2)- and (1 leads to 6)-linked D-mannosyl residues; the latter, of (1 leads to 2)- and (1 leads to 3)-linked D-mannosyl residues. Although the remaining structural types were highly branched, each gave distinct methylation-patterns indicative of (1 leads to 6)-linked backbones to which are appended non-(1 leads to 6)-linked side-chains. Acetolysis studies were correlated with the methylation analyses, and the correlation demonstrated that each branched polymer possesses side chains of heterogeneous length.

Lipid composition of 30 species of yeast.

The detailed composition of cellular lipid of more than 23 species of yeast has been determined quantitatively by thinchrography on quartz rods, a method previously used for estimating cellular lipids of seven species of yeast. That data was fortified by neutral and phospholipid quantitations on 30 species of yeast cells. Most of the test organisms contained 7-15% total lipid and 3-6% total phospholipid per dry cell weight, except for the extremely high accumulation of triglycerides in two species of Lipomyces. Qualitatively, 30 species of yeast cells contained similar neutral lipid constituents (triglyceride, sterol ester, free fatty acid, and free sterol) and polar lipid components (phosphatidyl choline, phosphatidyl ehtanolamine, phosphatidyl serine, phosphatidyl inositol, cardiolipin, and ceramide monohexoside) without minor constituents. Based on the quantitative composition of neutral lipids, the 30 species of yeast were divided into two groups , the triglyceride predominant group and the sterol derivative group. These groupings were fairly well overlapped from the standpoint of the distribution characteristics of fatty acid. The relative polar lipid compositions also grossly resembled each other. Only one exception of polar lipid composition in yeast cells was found in Rhodotorula rubra species which contained phosphatidyl ethanolamine as the most abundant phospholipid. Fatty acid distribution patterns in yeast cells consistently coincided with other reports concerning fatty acid composition of yeast cells. Correlation of lipid composition and classification of yeasts are suggested and discussed.

[Essay of proteins after aldehyde treatment in biological objects (author's transl)]. / Proteinbestimmung nach Aldehydbehandlung biologischer Objekte.

Following fixation with formalin or glutaraldehyde, the protein content of bovine serum albumin, lysozyme, non-disintegrated yeast cells and liver nuclei of mice is determined by means of a brom-phenol blue staining procedure and the widely used Lowry-technique. The bromphenol blue technique permits determinations of soluble as well as particulate proteins or protein mixtures fixed with up to 6% formalin or 2% glutaraldehyde. Recovery rates differ no more than 20% as related to unfixed controls. Using the bromphenol blue method it is not necessary to separate aldehyde or any interfering material by additional steps prior to determination. Factors for correcting protein content of biological material after aldehyde treatment are available. In this way, comparative biochemical as well as cyto- and histochemical investigations of enzymatic activities after aldehyde fixation are possible. Some advantages of the bromphenol blue technique with special reference to the analysis of particulate and/or aldehyde-fixed specimens are discussed.

Cell wall of pathogenic yeasts and implications for antimycotic therapy.

Yeast cell wall is a complex, multilayered structure where amorphous, granular and fibrillar components interact with each other to confer both the specific cell shape and osmotic protection against lysis. Thus it is widely recognized that as is the case with bacteria, yeast cell wall is a major potential target for selective chemotherapeutic drugs. Despite intensive research, very few such drugs have been discovered and none has found substantial application in human diseases to date. Among the different cell wall components, beta-glucan and chitin are the fibrillar materials playing a fundamental role in the overall rigidity and resistance of the wall. Inhibition of the metabolism of these polymers, therefore, should promptly lead to lysis. This indeed occurs and aculeacin, echinocandin and polyoxins are examples of agents producing such an action. Particular attention should be focused on chitin synthesis. Although quantitatively a minor cell wall component, chitin is important in the mechanism of dimorphic transition, especially in Candida albicans, a major human opportunistic pathogen. This transition is associated with increased invasiveness and general virulence of the fungus. Yeast cell wall may also limit the effect of antifungals which owe their action to disturbance of the cytoplasmic membrane or of cell metabolism. Indeed, the cell wall may hinder access to the cell interior both under growing conditions and, particularly, during cell ageing in the stationary phase, when important structural changes occur in the cell wall due to unbalanced wall growth (phenotypic drug resistance).