Hemagglutinating activity in extracts of mycelia from submerged mushroom cultures.

Extracts from mycelia of seven different mushrooms agglutinated erythrocytes of several species. More than one agglutinating factor was identified in the extracts of three different mycelia. Agglutination was partially inhibited nonspecifically by high concentrations of glucose, galactose, mannose, fucose, and rhamnose.     

The interaction of cholesterol with bilayers of phosphatidylethanolamine

The interaction of cholesterol with the glycerol backbone segments of phospholipids was studied in bilayers of phosphatidylethanolamine containing equimolar amounts of cholesterol. Glycerol selectively deuterated at various positions was supplied to the growth medium of Escherichia coli strain 131 GP which is defective in endogeneous glycerol synthesis. The procedure enables the stereospecific labeling of the three glycerol backbone segments of the membrane phospholipids. Phosphatidylethanolamine with wild-type fatty acid composition was purified from E. coli cells and deuterium magnetic resonance spectra were obtained either from dispersions of pure phosphatidylethanolamine or from equimolar mixtures of phosphatidylethanolamine with cholesterol. For comparative purposes 1,2-di[9,10-²H₂]elaidoyl-sn-glycero-3-phosphoethanolamine and [3-α-²H]cholesterol were synthesized in order to monitor the behavior of the fatty acyl chains and of the cholesterol molecule itself. For all deuterated segments the deuterium quadrupole splittings as well as the deuterium spin-lattice (T₁) relaxation times were measured as a function of temperature. The glycerol backbone was found to be a remarkably stable structural element of the phospholipid molecule. The quadrupole splittings of the backbone segments changed only by at most 2 kHz upon incorporation of 50 mol % cholesterol. This was in contrast to the fatty acyl chains where the same amount of cholesterol increased the quadrupole splitting by more than 20 kHz. The glycerol segments exhibited the shortest T₁ relaxation times of all CH₂ segments indicating that the glycerol backbone is the slowest motional moiety of the lipid molecule. Addition of cholesterol has no effect on the backbone motion but the fast reorientation rate of the trans-double bonds in 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine is increased dramatically.     

Expression of multiple Na+,K(+)-ATPase genes reveals a gradient of isoforms along the nonpigmented ciliary epithelium: functional implications in aqueous humor secretion.

The Na+,K(+)-ATPase alpha 1, alpha 2, and alpha 3 subunit isoforms have been shown to be differentially expressed in the nonpigmented (NPE) and pigmented (PE) cells of the ocular ciliary epithelium (CE) (Martin-Vasallo et al., J. Cell. Physiol., 141:243-252, 1989; Ghosh et al., J. Biol. Chem., 265:2935-2940, 1990). In this study we analyzed and compared the pattern of expression of the multiple Na+,K(+)-ATPase alpha (alpha 1, alpha 2, alpha 3) subunit genes with the pattern of expression of the Na+,K(+)-ATPase beta (beta 1, beta 2) subunit genes along the bovine CE. We have selected three regions in the CE, referred to as 1) the anterior region of the pars plicata, near the iris; 2) the middle region of the pars plicata; and 3) the posterior region of the pars plana, near the ora serrata. Using isoform-specific cDNA probes and antibodies for the Na+,K(+)-ATPase alpha 1, alpha 2, alpha 3, beta 1, and beta 2 subunits on Northern and Western blot analysis, we found that mRNA and polypeptides are expressed in all three CE regions with different abundance. The pattern of expression of alpha and beta isoforms detected along the NPE cell layers suggests a gradient of alpha 1, alpha 2, alpha 3, beta 1, and beta 2 mRNAs and polypeptides that correlates with decreasing Na+,K(+)-ATPase activity from the most anterior region at the pars plicata towards the posterior region at the ora serrata. We also found marked differences in the pattern of immunolocalization of Na+,K(+)-ATPase alpha 1, alpha 2, alpha 3, beta 1, and beta 2 subunit isoforms in different regions of the CE. In the anterior region, NPE cells stained intensely at the basal lateral membrane with specific monoclonal and polyclonal antibodies for each of the alpha (alpha 1, alpha 2, alpha 3) and beta (beta 1, beta 2) Na,K-ATPase isoforms. In the middle and posterior regions of the CE, NPE cells showed lower or absent levels of staining with alpha 1, alpha 2, alpha 3, and beta 1 antibodies, although staining with beta 2 was abundant. In contrast, PE cells throughout the CE were stained at the basal lateral membrane by antibodies to alpha 1 and beta 1, while no staining signals were detected with the rest of the antibodies (i.e. alpha 2, alpha 3, and beta 2). Our results support the conclusion that the three alpha and two beta isoforms of the Na+,K(+)-ATPase are differentially expressed in the two cell layers that make up the CE.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro fertilization of water buffalo follicular oocytes and their ability to cleave in vitro

Water buffalo (Murrah) oocytes were collected from ovaries obtained from a local slaughterhouse. They were classified according to the character of the cumulus cells under a stereomicroscope and then cultured in 25 mM Hepes buffered tissue culture medium-199 (TCM-199) supplemented with 5% estrous water buffalo serum in an atmosphere containing 5% CO(2) in air at 39 degrees C. After 20 to 24 hours of in vitro maturation, the oocytes were cultured at 38.5 degrees C in TCM-199 supplemented with 1% estrous water buffalo serum and in an atmosphere containing 5% CO(2) in air. Oocytes with compact and dense cumulus cells cleaved significantly further (P<0.01, 67.3%, 33 49 ) than those with fair, partially denuded oocytes with thin cumulus layers (27.5%, 25 91 ) or small remnants of cumulus cells and poor naked oocytes (3 100 ). A substantial variation in fertilization and developmental rates (16.0 to 43.8%) was observed among 4 different bulls. Late morulae were transferred nonsurgically into 14 buffalo recipients on Day 6 or 7 of their estrous cycle. One recipient was diagnosed to be pregnant by palpation per rectum on Day 60 and delivered a calf in October 1991.     

Activation of methionine by Escherichia coli methionyl-tRNA synthetase

In the present work, we have examined the function of three amino acid residues in the active site of Escherichia coli methionyl-tRNA synthetase (MetRS) in substrate binding and catalysis using site-directed mutagenesis. Conversion of Asp52 to Ala resulted in a 10,000-fold decrease in the rate of ATP-PPi exchange catalyzed by MetRS with little or no effect on the Km's for methionine or ATP or on the Km for the cognate tRNA in the aminoacylation reaction. Substitution of the side chain of Arg233 with that of Gln resulted in a 25-fold increase in the Km for methionine and a 2000-fold decrease in kcat for ATP-PPi exchange, with no change in the Km for ATP or tRNA. These results indicate that Asp52 and Arg233 play important roles in stabilization of the transition state for methionyl adenylate formation, possibly directly interacting with complementary charged groups (ammonium and carboxyl) on the bound amino acid. Primary sequence comparisons of class I aminoacyl-tRNA synthetases show that all but one member of this group of enzymes has an aspartic acid residue at the site corresponding to Asp52 in MetRS. The synthetases most closely related to MetRS (including those specific for Ile, Leu, and Val) also have a conserved arginine residue at the position corresponding to Arg233, suggesting that these conserved amino acids may play analogous roles in the activation reaction catalyzed by each of these enzymes. Trp305 is located in a pocket deep within the active site of MetRS that has been postulated to form the binding cleft for the methionine side chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterisation of a beta-glucosidase (cellobiase) from a mushroom Termitomyces clypeatus

A beta-glucosidase with cellobiase activity was purified to homogeneity from the culture filtrate of the mushroom Termtomyces clypeatus. The enzyme had optimum activity at pH 5.0 and temperature 65 degrees C and was stable up to 60 degrees C and within pH 2-10. Among the substrates tested, p-nitrophenyl-beta-D-glucopyranoside and cellobiose were hydrolysed best by the enzyme. Km and Vm values for these substrates were 0.5, 1.25 mM and 95, 91 mumol/min per mg, respectively. The enzyme had low activity towards gentiobiose, salicin and beta-methyl-D-glucoside. Glucose and cellobiose inhibited the beta-D-glucosidase (PNPGase) activity competitively with Ki of 1.7 and 1.9 mM, respectively. Molecular mass of the native enzyme was approximated to be 450 kDa by HPLC, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated a molecular mass of 110 kDa. The high molecular weight enzyme protein was present both intracellularly and extracellularly from the very early growth phase. The enzyme had a pI of 4.5 and appeared to be a glycoprotein.     

X-Ray radiography for studying diffusion characteristics of sucrose in plaster of paris matrix containing yeast cells

Summary X-Ray radiography was employed to monitor the diffusion of sucrose into plaster of Paris matrix containing 20% yeast cells. It was observed that the depth of penetration of tracer P_b detected by radiography matched with the substrate penetration detected by chemical test. However electron probe microanalysis (EPMA) did not yield any conclusive evidence regarding the movements of tracer P_b and substrate to the same extent.     

A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite.     

Acid pH-induced fusion of cells by herpes simplex virus glycoproteins gB an gD

Enveloped animal viruses enter host cells either by direct fusion at neutral pH or by endocytosis. Herpes simplex virus (HSV) is believed to fuse with the plasma membrane of cells at neutral pH, and the glycoproteins gB and gD have been implicated in virus entry and cell fusion. Using cloned gB or gD genes, we show that cells expressing HSV-1 glycoproteins gB or gD can undergo fusion to form polykaryons by exposure only to acidic pH. The low pH-induced cell fusion was blocked in the presence of monoclonal antibodies specific to the glycoproteins. Infection of cells expressing gB or gD glycoproteins with HSV-1 inhibited the low pH-induced cell fusion. The results suggest that although the glycoproteins gB and gD possess fusogenic activity at acidic pH, other HSV proteins may regulate it such that in the virus-infected cell, this fusion activity is blocked.