Changes in external trehalase activity during human serum-induced dimorphic transition in Candida albicans

Yeast-like cells (blastoconidia) of Candida albicans growing exponentially on a glucose-containing medium (YPD) exhibited low external trehalase activity and stored a negligible amount of intracellular trehalose. The addition of human serum at 37 degrees C to exponential cultures promoted a high degree of germ-tube formation with no significant changes in trehalase activity or trehalose content. In contrast, stationary cells accumulated a large amount of trehalose, while external trehalase remained at a low and practically constant level. However, resting cultures were unable to enter the dimorphic program, except when they were supplemented with fresh YPD and serum together. Only under these conditions was trehalase activated and trehalose hydrolyzed. Specific inhibition of external trehalase by validoxylamine A caused a certain delay in, and a lower level of, germ-tube formation, but did not totally block the dimorphic conversion. These results suggest that external trehalase is not involved in the serum-induced morphological transition in C. albicans.     

Trehalase activity in genetically diabetic mice (serum, kidney, and liver).

Trehalase activity was determined in serum, liver, and kidney in alloxan treated Swiss mice and in homozygous (Ob/Ob, Db/Db) and heterozygous (Ob/+, Db/m+) diabetic mice. Both alloxan and genetic diabetic mice exhibited a large increase in serum and liver trehalase activity with no change in kidney trehalase activity. The heterozygotes (Ob/+, Db/m+) showed only a slight increase of enzyme activity. Further quantitative differences were noticed between the genetic and alloxan diabetic animals. The liver enzyme activity increased from 10- to more than 20-fold in the liver of the homozygous Ob/Ob and Db/Db strains and only 3-fold (not significant compared to controls) in the alloxan treated animals. The above results suggest a regulatory relationship between the genes coding for trehalase and the enzymes of glucose metabolism activity involved in the development of the metabolic anomalies of diabetes. The structural gene for trehalase may well have survived elimination of selective pressure during phylogenesis and remained part of a co-regulated group of glucose metabolising enzymes. This could explain its sensitivity to mutations affecting glucose metabolism and its sensitivity to insulin directed regulatory mechanisms.     

Synthesis, accumulation and hydrolysis of trehalose during growth of peanut rhizobia in hyperosmotic media

We examined and compared the activities of synthetic and hydrolytic enzymes involved in trehalose metabolism, in three peanut rhizobia strains grown in control, hypersaline, and non-ionic hyperosmotic media. Results indicated that the effects of hyperosmolarity on the synthesis and the degradation of the disaccharide were diverse. In the salt-tolerant slow-growing strain Bradyrhizobium sp. ATCC 10317, we observed increased synthesis and accumulation of trehalose under hyperosmolarity imposed by either NaCl or PEG-8000. In the other two peanut rhizobia strains, the disaccharide level did not change under hypersalinity. In the salt-sensitive slow-growing strain Bradyrhizobium sp. USDA 3187, intracellular trehalose diminished in late stationary phase-cells grown with PEG, this reduction was accompanied by both an increased activity of synthetic enzymes and a decreased activity of trehalase. In the salt-tolerant fast-growing strain Rhizobium sp. TAL 1000, we also observed a reduction of intracellular trehalose under PEG-mediated growth, this decrease was early and transiently accompanied by an enhancement of trehalase activity, afterwards, the activity of synthetic enzymes augmented.     

The immunogenic and co-operative effects of lymphocyte nuclear material from antigen-primed mice

When nuclei from the lymphocytes of mice immunized with rabbit erythrocytes are injected into unprimed animals the number of cells forming antibody to rabbit erythrocytes increases to a level approaching that of the immunized donor animals. The specificity of the response induced by nuclei is equally as good as that induced by native antigen. When `T' and `B' mice are immunized with rabbit erythrocytes a poor response is observed but when the nuclei from primed `T' animals are used to immunize `B' mice the number of antibody-producing cells is very much increased. In view of current theories about cellular co-operation it is pointed out that this co-operative effect of `T' lymphocyte nuclei is independent of both cell membrane and additional antigen.     

Induction of an Autologous Immune-complex Glomerulonephritis in the Rat by Intravenous Injection of Heterologous Anti-rat Kidney Tubular Antibody: I. Production of Chronic Progressive Immune-complex Glomerulonephritis

An autoimmune kidney disease morphologically and functionally similar to the Heymann autoimmune nephrosis can be produced in rats by the injection of BSA followed by heterologous anti-rat kidney tubular antibody. Animals injected with the anti-kidney tubular antibody only, developed a milder form of the typical renal lesion without proteinuria. Control animals injected with BSA or normal rabbit serum alone and BSA and normal rabbit serum together did not develop a progressive type of kidney disease.Gamma-globulin eluted from the developed lesions of the heterologous antibody induced glomerulonephritis was autologous IgG which reacted with the periluminal zone of the proximal tubules on normal rat kidney sections in a fluorescent antibody test. Gamma-globulin eluted from kidneys of homologous renal antigen induced autologous immune complex (AIC) nephritis reacted with normal rat kidney sections in a similar manner.It is suggested that heterologous anti-tubular antibody reaching the proximal convoluted tubules is reabsorbed and releases the nephritogenic antigen with subsequent formation of autoantibody to it. The continuous release of the nephritogenic antigen and the development of the chronic progressive autologous immune-complex glomerulonephritis is maintained by autoantibody produced as the result of the ongoing autoimmune processes.     

Dissemination of beads coated with trehalose 6,6'-dimycolate: a possible role for coagulation in the dissemination process

When spread as a monolayer on the surface of hydrophobic beads and injected into mice, the mycobacterial glycolipid, trehalose 6,6'-dimycolate, reproduces the biologic effects traditionally associated with virulent mycobacteria, including acute inflammation, granuloma formation, and immune adjuvancy. Repeated intraperitoneal injection of glycolipid-coated beads into young C57Bl/6 mice elicits a granulomatous peritonitis, with concomitant dissemination of beads from the peritoneum to distant organs. Glycolipid-coated beads which disseminate from the peritoneum to other sites elicit neither acute inflammation nor granulomata. The coagulation system may be involved in the dissemination of glycolipid-coated beads as evidenced by the following: fibrinogen is a necessary cofactor of the trehalose dimycolate monolayer; diffuse peritoneal and pulmonary hemorrhage accompanies bead dissemination; peritoneal exudate collected shortly after intraperitoneal injection of glycolipid-coated beads is enriched in coagulant activity; coagulability of blood from trehalose dimycolate-treated animals is reduced; and anticoagulation inhibits the inflammatory response to glycolipid-coated beads. In this report, the dissemination of trehalose dimycolate-coated beads is characterized, and a role for the coagulation system in this process is proposed.     

Immune-complex nephritis in the rabbit produced by injections of rat renal tubular fraction 3 antigen

Rabbits used for the production of anti-rat kidney tubular fraction 3 antibody developed a form of immune-complex glomerulonephritis which was characterized by the deposition of immune complexes in the glomerular basement membrane and in the mesangium. The deposits were composed of the injected rat kidney antigen and rabbit antibody to the injected antigen. Eluted gamma-globulin obtained from the diseased rabbit kidneys reacted only with the brush-border region of the proximal convoluted tubules of normal rat kidney sections but not with normal rabbit kidney sections, in an indirect fluorescent antibody test. The developing kidney disease does not appear to have an autoimmune component.     

Immune-complex nephritis in the rabbit produced by injections of rat renal tubular fraction 3 antigen.

Rabbits used for the production of anti-rat kidney tubular fraction 3 antibody developed a form of immune-complex glomerulonephritis which was characterized by the deposition of immune complexes in the glomerular basement membrane and in the mesangium. The deposits were composed of the injected rat kidney antigen and rabbit antibody to the injected antigen. Eluted gamma-globulin obtained from the diseased rabbit kidneys reacted only with the brush-border region of the proximal convoluted tubules of normal rat kidney sections but not with normal rabbit kidney sections, in an indirect fluorescent antibody test. The developing kidney disease does not appear to have an autoimmune component.     

Identification of Candida glabrata by a 30-second trehalase test

Rapid (30-s) trehalase tests done with material from colonies of 482 yeasts suspended in a drop of trehalose solution on a commercially supplied glucose test strip were positive for 225 (99.1%) of 227 Candida glabrata isolates grown on either of two differential media, Candida ID medium or CandiSelect medium. The test was positive for only 3 (1.2%) and 12 (4.7%) of 255 isolates of other medically important yeast species grown on the same two media, respectively. A rapid maltase test done with a subset of 255 yeast isolates was negative for all but 1 of 64 trehalase-positive C. glabrata isolates, raising the specificity of the rapid testing for C. glabrata to 98.4 to 100%, depending on the isolation medium used. Rapid trehalase and maltase tests done independently in two laboratories with 217 yeast isolates showed sensitivities of 96.0 to 98.0% and specificities of 98.2 to 99.4% for identification of C. glabrata from colonies grown on Candida ID medium. The specificity was much lower because of frequent false-positive trehalose test results when the source of colonies was Sabouraud agar formulated with 4% glucose. We conclude that direct recognition of C. albicans as blue colonies on Candida ID isolation medium coupled with the performance of the 30-s trehalase and maltase tests for C. glabrata among the white colonies on this medium will allow the rapid presumptive identification of the two yeast species most commonly encountered in clinical samples.     

Passive Heymann-like nephritis in the rabbit.

Rabbits injected with a guinea-pig anti-rabbit kidney fraction 3 antiserum developed immune complex deposition in the glomerulus which disappeared by the sixth day. The distribution of the glomerular deposits was similar to those of passive Heymann nephritis in the rat. A glomerular fixed antigen was not demonstrated in normal rabbit kidneys after perfusion with the guinea-pig anti-tubular antiserum. When tubular antigen was injected intraperitoneally in saline it could be detected in the glomeruli and after injection of the anti-tubular antiserum the deposits stained with increased intensity. Addition of a small amount of anti-GBM antiserum to the anti-tubular antiserum enhanced the size and number of glomerular deposits and increased their survival time. It is concluded that there is no pre-existing tubular antigen in the rabbit glomerulus and that the injected heterologous anti-tubular antiserum releases antigen from an extraglomerular source, probably the proximal tubules, and this released antigen becomes trapped in the glomerulus.     

Passive Heymann-like nephritis in the rabbit

Rabbits injected with a guinea-pig anti-rabbit kidney fraction 3 antiserum developed immune complex deposition in the glomerulus which disappeared by the sixth day. The distribution of the glomerular deposits was similar to those of passive Heymann nephritis in the rat. A glomerular fixed antigen was not demonstrated in normal rabbit kidneys after perfusion with the guinea-pig anti-tubular antiserum. When tubular antigen was injected intraperitoneally in saline it could be detected in the glomeruli and after injection of the anti-tubular antiserum the deposits stained with increased intensity. Addition of a small amount of anti-GBM antiserum to the anti-tubular antiserum enhanced the size and number of glomerular deposits and increased their survival time. It is concluded that there is no pre-existing tubular antigen in the rabbit glomerulus and that the injected heterologous anti-tubular antiserum releases antigen from an extraglomerular source, probably the proximal tubules, and this released antigen becomes trapped in the glomerulus.     

Increased orbital volume after periodic intrabulbar injections of silicone in growing rabbits

The intent of this experiment was to determine whether with an increase in ocular mass there would be an increase in orbital volume. Eighteen, four-week old, 220 to 420 gm Dutch rabbits were used. In 12 animals silicone was injected into one eye but not the opposite one. No silicone was injected into the eyes of six untreated rabbits. In a ten week period the total amount of ten silicone injections into each treated eye ranged from 1.2 to 1.6 ml. After each rabbit was euthanatized, elastic rubber base imprints were made of both cleaned orbits to determine the volumes. In the untreated animals the differences between the right and left orbital volumes ranged from — 0.1 to 0.2 ml (— 2.4 to 4.7%). The mean of the differences was not statistically significant. When the eyes were injected with silicone, the differences in orbital volumes between the injected and non-injected sides ranged from 0.6 to 0 ml (14.6 to 0%). The mean of the differences was 0.3 ml which is statistically significant. It was concluded that periodic intrabulbar injections of silicone in the growing rabbit significantly increased orbital volume.     

Studies of the pathogenetic role of humoral antibodies reacting with subcellular organ antigens: I. Active immunization of rabbits with rat-organ antigens

Rat organ homogenates were fractionated by differential centrifugation and the fractions were incorporated into incomplete Freund's adjuvant and administered intramuscularly to rabbits. Gel-diffusion precipitin and complement-fixation tests showed the development of antibodies which reacted with rat, and in lower titres with rabbit organ fractions. The rabbit antisera to liver and kidney fractions reacted with preparations of both these organs, and antisera to liver fractions (nuclei, mitochondria and supernate) reacted with all three of these preparations. Hepatic lesions were observed in those animals which developed antibodies shown capable of reacting in vitro with liver tissue constituents. These changes were most pronounced in rabbits immunized with liver mitochondria, in which inflammatory changes and fibrosis were related to the biliary ductules, and in those immunized with liver nuclear or kidney mitochondrial fractions, in which the changes appeared mainly around the larger interlobular hepatic ducts. Changes occurred also in the kidneys, but were observed also in control animals injected with normal rat serum.     

Characterization and regulation of the trehalose synthesis pathway and its importance in the pathogenicity of Cryptococcus neoformans

The disaccharide trehalose has been found to play diverse roles, from energy source to stress protectant, and this sugar is found in organisms as diverse as bacteria, fungi, plants, and invertebrates but not in mammals. Recent studies in the pathobiology of Cryptococcus neoformans identified the presence of a functioning trehalose pathway during infection and suggested its importance for C. neoformans survival in the host. Therefore, in C. neoformans we created null mutants of the trehalose-6-phosphate (T6P) synthase (TPS1), trehalose-6-phophate phosphatase (TPS2), and neutral trehalase (NTH1) genes. We found that both TPS1 and TPS2 are required for high-temperature (37 degrees C) growth and glycolysis but that the block at TPS2 results in the apparent toxic accumulation of T6P, which makes this enzyme a fungicidal target. Sorbitol suppresses the growth defect in the tps1 and tps2 mutants at 37 degrees C, which supports the hypothesis that these sugars (trehalose and sorbitol) act primarily as stress protectants for proteins and membranes during exposure to high temperatures in C. neoformans. The essential nature of this pathway for disease was confirmed when a tps1 mutant strain was found to be avirulent in both rabbits and mice. Furthermore, in the system of the invertebrate C. elegans, in which high in vivo temperature is no longer an environmental factor, attenuation in virulence was still noted with the tps1 mutant, and this supports the hypothesis that the trehalose pathway in C. neoformans is involved in more host survival mechanisms than simply high-temperature stresses and glycolysis. These studies in C. neoformans and previous studies in other pathogenic fungi support the view of the trehalose pathway as a selective fungicidal target for use in antifungal development.     

Passive immune complex glomerulonephritis in mice: models for various lesions found in human disease. I. High avidity complexes and mesangiopathic glomerulonephritis.

Intravenous injection of mice with soluble complexes of highly avid rabbit antibody to egg albumin, prepared by dissolution of equivalence precipitates in large quantities of antigen, resulted in a purely mesangial localization of the complexes. When animals received three injections of complexes per day for 1 day it was noted that precipitates dissolved in 80 times the equivalence amount of antigen produced slight mesangial changes. When such complexes were injected for 2 or 3 days, outright mesangiopathic glomerulonephritis was observed in an increasing proportion of the animals. Equivalent amounts of antigen alone did not produce lesions.     

Quantitative assessment of the effects of platelet depletion in the autologous phase of nephrotoxic serum nephritis.

The effects of platelet depletion with antibody have been studied in two models of the autologous phase of nephrotoxic nephritis in the rabbit. In the 'telescoped' model (animals pre-immunized to sheep IgG injected with sheep nephrotoxic antibody), platelet depletion did not alter intraglomerular fibrin deposition or evidence of glomerular damage, but did significantly reduce proteinuria during the first 3 days of the 5 day experiment. In the 'passive' model (animals injected with hyperimmune rabbit antiserum to sheep IgG 48 hr after sheep nephrotoxic antibody and killed 3 hr later), platelet depletion was associated with significantly fewer intraglomerular polymorphonuclear leucocytes (PMN), but again did not alter intraglomerular fibrin deposition. The results indicate that platelets are involved in the initiation of glomerular PMN localization in the autologous phase, but that fibrin-induced glomerular injury is platelet-independent.     

Effects of Plagiorchis elegans (Trematoda: Plagiorchiidae) infection on the carbohydrate metabolism of fourth instar Aedes aegypti (Diptera: Culicidae)

Infection of fourth-instar Aedes aegypti (L.) with the entomopathogenic digenean Plagiorchis elegans (Rudolphi) alters the carbohydrate metabolism of the insect. Within 24 h of cercarial penetration, total body extracts of infected fourth instars exhibited decreased trehalase activity, increased trehalose-6-phosphatase activity, and a concomitant accumulation of trehalose when compared with uninfected larvae. The amounts of glucose, glycogen and lipids, and the activity of glycogen phosphorylase a were similar in extracts of infected and control larvae. The predominant fatty acids, in both control and infected larvae, were C 18:0, C 18:1, and C 18:3. There were no significant differences in the types or proportions of fatty acids found in control and infected larvae. Parasitic infection is discussed in terms of impaired trehalose metabolism.     

Preparation of a Relatively Acid-Stable Antigen (CEA) by Disruption of its Specific Precipitate with Antibody

The carcinoembryonic antigen (CEA) of intestinal tract cancers is presently purified by extraction from a homogen-ate of such tissues with 1.0M perchloric acid (PCA), followed by two steps of chromatography and one of electrophoresis. In this study, rabbit antisera prepared against such purified CEA, and absorbed with normal human plasma, liver, kidney, spleen and brain, gave a conventional precipitin curve with a water homogenate or with a dialyzed PCA extract of intestinal tract cancers. The immune precipitate was almost completely soluble in PCA. Following dialysis of this solution against cold water, a new precipitate composed mainly of denatured antibody appeared. The remaining solution contained purified antigen. Recovery of this antigen (CEA-R), as judged by radioimmunoassay, appeared optimal when 3.0M PCA was used for disruption of the immune precipitate, and presence of 1.8M urea was helpful. When injected into rabbits, CEA-R was not as strongly antigenic as CEA. Comparison by immunodiffusion and immunoelectrophoresis against their specific rabbit anti-sera indicated that CEA and CEA-R share some specificities but are not identical.     

Preparation of a Relatively Acid-Stable Antigen (CEA) by Disruption of its Specific Precipitate with Antibody

The carcinoembryonic antigen (CEA) of intestinal tract cancers is presently purified by extraction from a homogen-ate of such tissues with 1.0M perchloric acid (PCA), followed by two steps of chromatography and one of electrophoresis. In this study, rabbit antisera prepared against such purified CEA, and absorbed with normal human plasma, liver, kidney, spleen and brain, gave a conventional precipitin curve with a water homogenate or with a dialyzed PCA extract of intestinal tract cancers. The immune precipitate was almost completely soluble in PCA. Following dialysis of this solution against cold water, a new precipitate composed mainly of denatured antibody appeared. The remaining solution contained purified antigen. Recovery of this antigen (CEA-R), as judged by radioimmunoassay, appeared optimal when 3.0M PCA was used for disruption of the immune precipitate, and presence of 1.8M urea was helpful. When injected into rabbits, CEA-R was not as strongly antigenic as CEA. Comparison by immunodiffusion and immunoelectrophoresis against their specific rabbit anti-sera indicated that CEA and CEA-R share some specificities but are not identical.     

Induction of an autologous immune complex glomerulonephritis in the rat by intravenous injection of heterologous anti-rat kidney tubular antibody. III. Long-term effects.

The long-term effects of a single intravenous injection of anti-rat kidney fraction 3 antibody were studied in rats. At the end of the experiment at one year the kidneys had deposition of immune complexes in the glomeruli staining for autologous IgG and C-3. The number and size of the deposits did not appear to increase during the experiment although proteinuria eventually developed in most test animals. Control rats injected with normal rabbit serum had no immune complexes in their kidneys.