Experimental Germ Tube Induction in Candida albicans: An Evaluation of the Effect of Sodium Bicarbonate on Morphogenesis and Comparison with Pooled Human Serum

Objective. The potential of NaHCO3 versus human serum to induce germ tube formation in Candida albicans was investigated. Specimens. A total of 100 isolates were obtained from oral swabs of patients presenting with thrush. Approval for the study was granted by the Joint Research Ethics Committee (JREC/23/08). Method. Confirmed C. albicans isolates by routine methods were tested for germ tube induction using 5 different concentrations of Tris-maleate buffered NaHCO3 and Tris-maleate buffer control. Standard control strains included were C. albicans (ATCC 10231) and C. krusei (ATCC 6258). Microculture was done in 20 μL inoculums on microscope slides for 3 hours at 37°C. The rate of germ tube formation at 10-minute intervals was determined on 100 isolates using the optimum 20 mM Tris-maleate buffered NaHCO3 concentration. Parallel germ tube formation using human serum was done in test tubes. Results. The optimum concentration of NaHCO3 in Tris-maleate buffer for germ tube induction was 20 mM for 67% of isolates. Only 21% of isolates formed germ tubes in Tris-maleate buffer control. There was no significant difference in induction between human serum and Tris-maleate buffered NaHCO3. Conclusion. Tris-maleate buffered NaHCO3 induced germ tube formation in C. albicans isolates at rates similar to human serum.

Phagocytosis and hydrogen peroxide production of Eosinophils in the rats spontaneously infected with Mycoplasma pulmonis

The ability of hydrogen peroxide (H2O2) production was cytochemically compared in eosinophils (EP) between specific pathogen free (SPF) and spontaneously Mycoplasma pulmonis-infected male rats (Wistar strain).Peritoneal cells including EP (PEP) were harvested with a cold Hanks' balanced salt solution containing 0.1% glucose (HBSSG). Simultaneously, blood granulocytes with eosinophils (BEP) were isolated from the same animals by a Ficoll-Hypaque method. Cells were incubated with latex particles in HBSSG for 60-90 min. Thereafter, cells were washed and transferred into a cerium (Ce) medium consisting of 0.1 M tris - maleate buffer, pH 7.5, 1mM CeCl3, 10 mM sodium azide, 0.1. glucose and 0.25 M sucrose. The incubation was carried out for 20 min at 37°C with occasional stirring. Some cells were incubated for 60 min at 37 °C in a DAB medium containing 0.1% 3.3’-diaminobenzidine 4HCl with particles in 0.1 M phosphate buffer, pH 7.4. All cells were then fixed with glutaraldehyde and osmium tetroxide before processing for electron microscopy, x-ray microanalysis of the subcellular electron-dense reaction deposits was performed under a Tracor-Northern EDX attached to a JEM-1200EX STEM system.

Production and partial characterization of elastase of Bacillus subtilis isolated from the cervices of human females

Conditions are described for the production of extracellular elastase by Bacillus subtilis. The yield of enzyme was maximum in shake–cultures grown in Syncase medium at 37 °C and was stable in culture supernatants. The enzyme, purified by ammonium sulphate precipitation and Sephadex G-75 gel filtration, showed a molecular weight of 25 000 and activity between pH 6.0 and 9.5, with an optimum of 9.0 in Tris–maleate buffer. Elastinolytic activity was maximum in glycine–NaOH buffer and minimum in phosphate buffer. Enzyme activity was adversely affected by temperature ≥ 40 °C.

Partial purification and characterization of an extracellular proteinase from Aeromonas hydrophila strain A4

SummaryAn extracellular metalloproteinase from Aeromonas hydrophila strain A4, isolated from milk, was purified by a factor of 300 by chromatogrpahy on DEAE-cellulose and Sephadex G-150. The enzyme had a mol. wt of 43000 and contained 2 g atom Ca/mol. It was active over a pH range 4·8–9·5 and had optimum activity on casein at pH 7·0 with Km = 0·17 mM. It was strongly inactivated by metal chelators and the apoenzyme was fully reactivated with Ca2+, Mn2+ or Co2+. Heavy metal ions such as Ag+, Hg2+, Fe2+, Zn2+, Cd2+, Ni2+ and Cu2+ totally or partly inactivated the enzymic activity at 5 mM concentration. The enzyme was not inactivated by diisopropylfluorophosphate, soyabean trypsin inhibitor or sulphydryl group reagents. It was optimally active at 45 °C; above 50 °C activity declined rapidly, but significant activity persisted at 4 °C. It was heat labile in phosphate or Tris-maleate buffer but exogenous Ca2+ afforded protection.

Subcellular localization of H2O2 production in human neutrophils stimulated with particles and an effect of cytochalasin-B on the cells

The ultrastructural localization of H2O2 production in suspended polymorphonuclear leukocytes (PMN) stimulated with particles was studied using CeCl3 technique. PMN stimulated with opsonized zymosan or polystylene latex with or without IgG were incubated in 0.1 M Tris- maleate buffer with 1 mM CeCl3 and 10 mM aminotriazole. Cells were then fixed and embedded in a resin for electron microscopy. The reaction product of cerium perhydroxide was observed on the phagosomal membranes and on the areas of the plasma membrane engulfing the particles. Catalase or ferricytochrome-c decreased the deposits. p-Benzoquinone (O2- scavenger) inhibited the formation of the deposits, but KCN or NaN3 enhanced it. Pretreatment with p-diazobenzenesulfonic acid inhibited the reaction. In some PMN pretreated with cytochalasin-B, cellular aggregation was observed. The H2O2 production in these cells were observed on the membrane adherent to the particles and on the contact surface of the membrane of adjoining PMN. The plasma membrane was damaged and the electron-dense product was diffused into the cytoplasm. These results clearly show that H2O2 production is initiated at the area of the plasma membrane adherent to the particles and that H2O2 is released before the completion of phagocytosis.

Subcellular localization of H2O2 production in human neutrophils stimulated with particles and an effect of cytochalasin-B on the cells

The ultrastructural localization of H2O2 production in suspended polymorphonuclear leukocytes (PMN) stimulated with particles was studied using CeCl3 technique. PMN stimulated with opsonized zymosan or polystylene latex with or without IgG were incubated in 0.1 M Tris- maleate buffer with 1 mM CeCl3 and 10 mM aminotriazole. Cells were then fixed and embedded in a resin for electron microscopy. The reaction product of cerium perhydroxide was observed on the phagosomal membranes and on the areas of the plasma membrane engulfing the particles. Catalase or ferricytochrome-c decreased the deposits. p-Benzoquinone (O2- scavenger) inhibited the formation of the deposits, but KCN or NaN3 enhanced it. Pretreatment with p-diazobenzenesulfonic acid inhibited the reaction. In some PMN pretreated with cytochalasin-B, cellular aggregation was observed. The H2O2 production in these cells were observed on the membrane adherent to the particles and on the contact surface of the membrane of adjoining PMN. The plasma membrane was damaged and the electron-dense product was diffused into the cytoplasm. These results clearly show that H2O2 production is initiated at the area of the plasma membrane adherent to the particles and that H2O2 is released before the completion of phagocytosis.

The assay and partial characterization of macromolecular heparin depolymerase activity in rat small intestine

Homogenates of rat small intestine can depolymerize macromolecular rat skin heparin (RS heparin) to products similar in size to commercial heparin [Horner (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 3469–3473]. This activity is attributed to an enzyme provisionally named ‘macromolecular heparin depolymerase’. An assay for macromolecular heparin depolymerase activity in rat small intestine has been developed, based on the action of the enzyme on 35S-labelled macromolecular RS heparin. The depolymerized products are separated into two peaks by gel chromatography through columns of Bio-Gel A-15m. The amount of label in the second peak, expressed as a percentage of the total radioactivity, is the index of enzyme activity. The pH optimum was found to be 6.0 and the temperature optimum 45 degrees C. The enzyme was shown to be most stable in 50mM-Tris/maleate buffer containing 1 mM-EDTA. Macromolecular heparin depolymerase activity measured as a function of time and substrate concentration produced curves typical of an enzymic reaction. Evidence was obtained demonstrating that the activity did not originate from bacteria in the intestine. Macromolecular heparin depolymerase activity was increased by dilution and storage at 7 degrees C for 24 h. This suggests that homogenates of rat small intestine contain an unstable inhibitor of the enzyme.

Iodination of Calsequestrin in the Sarcoplasmic Reticulum of Rabbit Skeletal Muscle: A Re-Examination

The exposed proteins of sarcoplasmic reticulum (SR) vesicles from skeletal muscle were iodinated with the use of Sepharose 4B-bound lactoperoxidase, so that the location of the proteins in the membrane could be determined. It was found that the pattern of protein labelling could be modified simply by changing the constituents of the incubation media. This implies that the position or configuration of a particular protein in the membrane can be altered by the local environment. When the reaction was performed in the presence of 25 mM tris-maleate, pH 7 �0, alone, the Ca2+ pump ATPase (molecular weight 105000) and calsequestrin (47000) were both heavily labelled, suggesting they are at least partially exposed on the outer surface of the membrane. By contrast the high affinity calcium-binding protein (55000) was not labelled. However, when the vesicles were iodinated under conditions that were suitable for ATPase activity and Ca2+ accumulation, namely in the presence of 25 mM tris-maleate, pH 7 �0, 5 mM ATP, 5 mM Mg2+ and 0�05 mM Ca2+, a different pattern of labelling was obtained. No labelling of calsequestrin was observed whereas the extent of labelling of the Ca2+ pump ATPase remained about the same. The inclusion of anyone of the additives mentioned was effective in inhibiting the iodination of calsequestrin in the SR vesicle. When added alone, Ca2+ was more effective than Mg2+ in preventing labelling of calsequestrin. Half-maximal inhibition was observed at concentrations of approximately 0�05 mM Ca 2+ and 0�2-0�3 mM Mg2+ . Although much reduced, significant labelling of calsequestrin was observed even in the presence of 5 mM ATP. Investigations with partially purified calsequestrin revealed that the iodination of calsequestrin was the same in both the presence and absence of 1 mM Ca2 +. Therefore the reduction in label observed in intact SR vesicles probably represents a change in the location of calsequestrin within the membrane, rather than inhibition by Ca2+ of the iodination sites of the protein itself.