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The lysosomal α-mannosidases are class II mannosidases that belong to glycoside hydrolase family 38 and play an important role in the degradation of asparagine-linked carbohydrates of glycoproteins. Based on peptide similarity to human and bovine lysosomal mannosidase (LM), recombinant α-mannosidase from Drosophila melanogaster (dLM408) was cloned and heterologously expressed in Pichia pastoris. The recombinant form of dLM408 designed for structural analysis lacks the transmembrane domain and was crystallized using standard vapour-diffusion and counter-diffusion techniques. The crystals grew as flat plates and as tetragonal bipyramids, respectively. The plate-shaped crystals exhibited the symmetry of space group P2(1)2(1)2(1) and diffracted to a minimum d-spacing of 3.5 Å.

Permeabilization of yeast and other fungal cells by osmotic shock enabled the in situ assays of intracellular plasma membrane-bound enzymes, such as beta-1,3-glucan synthase, chitin synthase, and Na(+)/K(+) ATPase as well as the soluble, cytoplasmic enzymes, such as lactate dehydrogenase and alpha-glucosidase. The permeabilization was accomplished by rapid changes in osmolarity of the washing buffer at 0 degrees C whereby 0.5-3.5 M glycerol, sorbitol, and/or mannitol and/or 1 M KCl could be used as the osmolytes. No appreciable leakage of intracellular proteins occurred during the permeabilization procedure. The described procedure caused practically complete cell permeabilization while avoiding treatments with organic solvents, detergents, and other xenobiotics currently used for the permeabilization of microbial cells.

Transfer of exponentially growing cells of the yeast Saccharomyces cerevisiae to hyperosmotic growth medium containing 0.7-1 M KCl, 1 M mannitol, and/or 1 M glycerol caused cessation of yeast growth for about 2 h; thereafter, growth resumed at almost the original rate. During this time, formation of fluorescent patches on the inner surface of cell walls stained with Primulin or Calcofluor white was observed. The fluorescent patches also formed in solutions of KCl or when synthesis of the cell wall was blocked with cycloheximide and/or 2-deoxyglucose. The patches gradually disappeared as the cells resumed growth, and the new buds had smooth cell walls. Electron microscopy of freeze-etched replicas of osmotically stressed cells revealed deep plasma membrane invaginations filled from the periplasmic side with an amorphous cell wall material that appeared to correspond to the fluorescent patches on the cell surface. The rate of incorporation of D-[U-14C]glucose from the growth medium into the individual cell wall polysaccharides during osmotic shock followed the growth kinetics. No differences in cell wall composition between osmotically stressed yeast and control cells were found. Hyperosmotic shock caused changes in cytoskeletal elements, as demonstrated by the disappearance of microtubules and actin microfilaments. After 2-3 h in hyperosmotic medium, both microtubules and microfilaments regenerated to their original polarized forms and the actin patches resumed their positions at the apices of growing buds. The response of S. cerevisiae strains with mutations in the osmosensing pathway genes hog1 and pbs2 to hyperosmotic shock was similar to that of the wild-type strain. We conclude that, besides causing a temporary disassembling of the cytoskeleton, hyperosmotic shock induces a change in the organization of the cell wall, apparently resulting from the displacement of periplasmic and cell wall matrix material into invaginations of the plasma membrane created by the plasmolysis.

The synthesis of endoglucanase by young induced mycelia of Trichoderma reesei QM 9414 incubated in the presence of 1 mM sophorose (a potent cellulase inducer) was stimulated or repressed by additions of dibutyryl cyclic AMP (dBcAMP), depending on the concentration. At low concentrations (10(-6) and 10(-5) M), dBcAMP stimulated the formation of endoglucanase; higher concentrations of dBcAMP (10(-3) and 10(-2) M) repressed the synthesis of endoglucanase. Addition of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) at 1 and 10 microM concentrations to young induced mycelia caused an increase in intracellular cAMP and stimulated the production of endoglucanase. Neither exogenous dBcAMP nor IBMX was capable of inducing endoglucanase synthesis by itself, and neither was able to relieve catabolite repression of endoglucanase synthesis caused by glucose. All of the monosaccharides tested caused a more or less transient increase in intracellular cAMP. However, the effect of these treatments on endoglucanase synthesis was varied. The phosphorylable hexoses, both metabolizable and nonmetabolizable, increased the intracellular level of glucose-6-phosphate or its analogs and repressed endoglucanase synthesis. Nonphosphorylable sugars, such as 6-deoxyglucose, xylose, L-fucose, and (or) L-sorbose, did not influence the glucose-6-phosphate level and stimulated endoglucanase production to varying degrees. It is concluded that both cAMP and glucose-6-phosphate are involved in regulating cellulase synthesis in T. reesei. However, these factors seem to act in opposing directions.