The role of glucose in the pH regulation of germ-tube formation in Candida albicans

It has been reported that Candida albicans can form germ-tubes only in the narrow pH range of 6-8, and that by changing only the pH one can regulate germ-tube formation. We found that the pH minimum for germ-tube formation could be dramatically lowered by eliminating the glucose present in many induction solutions. Lee's medium lacking glucose, ethanol, N-acetyl-D-glucosamine, and proline induced germ-tubes at pH values as low as 3 under most conditions. The presence of as little as 1 mM-glucose in these induction solutions was sufficient to cause the cells to grow either as yeasts with multiple buds or as pseudohyphae when the pH was 3.7. However, when C. albicans was grown in any of the above induction solutions (with the exception of ethanol), containing 200 mM-glucose buffered at pH 5.8, not only were germ-tubes formed, but their rate of formation and length were also increased. Preincubation of the cells in a solution buffered at pH 3.7 and containing 200 mM-glucose, before exposure to induction solutions lacking glucose at pH 3.7 or at pH 5.8, did not inhibit germ-tube formation. Likewise, addition of glucose after 45 min exposure to an induction solution was without effect. Theophylline and dibutyryl cAMP did not counteract the action of glucose. Other sugars which suppressed germ-tube formation at low pH were fructose, galactose, mannose, xylose, gluconic acid and the nonmetabolizable sugar 3-O-methylglucose. These results indicate that pH does not directly regulate dimorphism in C. albicans, and that glucose or its metabolites may play an important role.     

Demonstration of up-regulated IL 2 receptor expression on an in vitro cloned BCL1 subline

We have established BCL1 CL-3 cells capable of responding to B15-TRF and interleukin 2 (IL 2). This clone has both high affinity and low affinity receptors for IL 2 (IL 2R), but IL 2 by itself did not stimulate either proliferation or immunoglobulin (Ig) secretion. B15-TRF, which possesses both growth and differentiation activity, causes an increase in size of CL-3 cells and renders CL-3 cells responsive to IL 2, including an increased expression of IL 2R (eight-fold to 10-fold) and the differentiation of CL-3 cells into Ig secretion (60 to 80% of cultured cells). CL-3 cells pretreated with B15-TRF for 12 hr become competent to respond to IL 2 by up-regulation of IL 2R within 12 hr. In contrast CL-3 cells pretreated with IL 2 for 12 hr required 24 hr B15-TRF stimulation to result in IL 2R up-regulation. Thus the ordered action of B15-TRF and IL 2 is the most effective operational pathway for the up-regulation of IL 2R. This IL 2-mediated IL 2R up-regulation and induction of Ig synthesis depends upon the concentration of IL 2 in the culture. Both responses seem to be caused by IL 2 molecules bound to high affinity IL 2R. However, the possibility of involvement of low affinity IL 2R can not be vigorously excluded. In fact the level of IL 2 required for a response is far higher than that needed for activated T cell proliferation. This cloned BCL1 subline promises to be a useful tool for studying the regulation and mechanisms of B cell responses.     

Inhibition of cell proliferation by putative metabolites and non-degradable analogs of methotrexate-gamma-dimyristoylphosphatidylethanolamine

Previous investigations have shown that untargeted liposomes, in which methotrexate is anchored to the lipid bilayers as methotrexate-gamma-dimyristoylphosphatidylethanolamine (methotrexate-gamma-DMPE), can inhibit in vitro cell proliferation. To test the possibility that this inhibition may involve extracellular metabolism of methotrexate-gamma-DMPE, we have degraded it chemically (dilute alkali) or enzymatically (phospholipase A2, phospholipase C, phospholipase C plus phosphatase), and assayed the products using human lymphoblastoid T cells or a subline that has a defective methotrexate transport system. Neither methotrexate-gamma-(1-myristoyl)-glycerophosphorylethanolamine, methotrexate-gamma-glycerophosphorylethanolamine, methotrexate-gamma-phosphorylethanolamine, nor methotrexate-gamma-ethanolamine resemble methotrexate-gamma-DMPE sensitized liposomes or the free derivative in their ability to block tritiated deoxyuridine incorporation into DNA. When added extracellularly, these putative metabolites manifest a higher ID50 concentration and/or, unlike the liposomes or unincorporated methotrexate-gamma-DMPE, utilize the methotrexate transport system to enter cells. Additionally, we have synthesized methotrexate-gamma-dihexadecylphosphatidylethanolamine and methotrexate-gamma-hexadecylphosphorylethanolamine, analogs of methotrexate-gamma-DMPE that cannot be hydrolyzed by phospholipases A2, C and D; liposomes prepared with these derivatives are markedly less potent cytotoxic agents than methotrexate-gamma-DMPE sensitized liposomes. All together, these results are consistent with the conclusion that methotrexate-gamma-DMPE must undergo intracellular metabolism to exert optimal inhibition; they also bear on possible mechanisms by which methotrexate-gamma-DMPE may enter cells.     

Distribution and fine structure of ependymal cells possessing intracellular cysts in the aqueductal wall of the rat brain

Summary The wall of the cerebral aqueduct was examined in 20 male rats at the light- and electron-microscopic levels. Disorder in ciliary orientation was occasionally seen in ordinary ependymal cells. Ependymal cells possessing intracellular cysts of 5 to 30 urn in diameter were observed within and beneath the aqueductal ependyma in all animals examined. Light-microscopic reconstruction from serial, 10-m thick frontal sections revealed an extensive distribution of cystic ependymal cells (CECs), especially along the ependymal ridges in the rostral half of the aqueduct, and along the dorsal region of the aqueductal lining in the caudal half. Both cystic and surface membranes of CECs bore microvilli and cilia. Ectopic ependymal cells (EECs) characterized by densely packed microvilli, well-developed intermediate junctions and cilia, but without cysts, were situated in the subependymal region adjacent to a CEC or another EEC. The ependymal ridges were long, narrow and sporadically stratified ependymal linings extending rostrocaudally and bilaterally along the aqueductal surface. Tanycyte-like cells filled the surface region of the ridge, and CECs and EECs were frequently seen in the core. Intraventricularly injected microperoxidase was detected among densely packed microvilli but not in the cystic lumina of CECs, indicating that EECs and CECs are distinct entities.     

Changes in cerebral level of monoamines by Japanese encephalitis virus infection

The changes in monoamine levels of different brain regions following Japanese encephalitis virus (JEV) intraperitoneal inoculation were examined in experimentally JEV-infected mice. In addition, virus distribution was studied using infectivity assay and immuno-histochemistry of viral antigen. 1) The level of monoamines in brain tissues was not affected by 48 hours after viral inoculation, but marked effects were elicited at 96 hours after the inoculation. The cerebral concentration of 5-hydroxyindole-3-acetic acid (5 HIAA) was increased, while that of dopamine (DA) showed a decrease. Especially these alteration were observed in the cerebral cortex, but not in the cerebellum. 2) The viral growth in the brain was observed at 48 hours after the inoculation. The growth in the cerebellum, however, was found to be lower than those in other cerebral regions. 3) The viral antigen was detected in the cerebral cortex, hippocampus, mesencephalon and diencephalon in addition to the substantia nigra and striatum. From these results, it is presumed that clinical manifestation of JEV infection may involve the changes in the metabolism of neurotransmitter, especially those of DA and serotonin in the brain.     

Effect of calcium overload on the phosphoinositide breakdown in the rat left ventricular papillary muscle

We investigated the effect of Ca²⁺ overload on the phospholipase C-catalyzed hydrolysis of phosphoinositides in the rat left ventricular papillary muscle. Ca²⁺ overload on the papillary muscle was induced by treatment with 0.3 mM ouabain in Ca²⁺-containing medium following either Ca²⁺-containing or Ca²⁺-free superfusion. The phosphoinositide breakdown was evaluated by determining accumulations of [³H]inositol phosphates ([³H]IPs) in the tissues prelabeled with [³H]inositol. Ca²⁺ repletion following Ca²⁺-free superfusion resulted in a rapid but small increase in resting tension that was not followed by contracture, nor was it associated with a significant increase in [³H]IPs accumulations. Treatment with ouabain following Ca²⁺-containing superfusion increased resting tension after a lag period of several minutes and produced contracture associated with an increase in [³H]IPs accumulations. The ouabain induced increases in resting tension, and accumulations of [³H]IPs were significantly potentiated by prior Ca²⁺-free superfusion instead of Ca²⁺-containing superfusion. There was a significant positive correlation between increases in resting tension and the phosphoinositide breakdown. The increased resting tension and the accumulations of [³H]IPs were not antagonized by treatments with prazosin plus atropine or indomethacin, but were abolished by superfusion with Ca²⁺-free buffer solution. Although the enhanced phospholipase C-catalyzed hydrolysis of phosphoinositides appears to be a consequence rather than a cause of increased intracellular Ca²⁺, such a biochemical change may provoke a positive feedback mechanism to develop the muscle contracture through the putative intracellular messenger action of inositol triphosphate and diacylglycerol.Abbreviations [³H]IPs [³H]Inositol Phosphates - IP Inositol Phosphate - IP₂ Inositol Bisphosphate - IP₃ Inositol Trisphosphate - PI Phosphatidylinositol - PI-4-P Phosphatidylinositol-4-phosphate - PI-4,5-P₂ Phosphatidylinositol 4,5-bisphosphate - PRZ Prazosin - ATR Atropine - INDO Indomethacin - min Minutes     

Two distinct O-methyltransferases in aflatoxin biosynthesis

The substances belonging to the sterigmatocystin group bear a close structural relationship to aflatoxins. When demethylsterigmatocystin (DMST) was fed to Aspergillus parasiticus NIAH-26, which endogenously produces neither aflatoxins nor precursors in YES medium, aflatoxins B1 and G1 were produced. When dihydrodemethylsterigmatocystin (DHDMST) was fed to this mutant, aflatoxins B2 and G2 were produced. Results of the cell-free experiment with S-adenosyl-[methyl-3H]methionine showed that first the C-6-OH groups of DMST and DHDMST are methylated to produce sterigmatocystin and dihydrosterigmatocystin (O-methyltransferase I) and then the C-7-OH groups are methylated to produce O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin (DHOMST) (O-methyltransferase II). However, no methyltransferase activity was observed when either OMST, DHOMST, 5,6-dimethoxysterigmatocystin, 5-methoxysterigmatocystin, or sterigmatin was incubated with the cell extract. Treatment of the cell extract with N-ethylmaleimide inhibited O-methyltransferase I activity but not that of O-methyltransferase II. Furthermore, these O-methyltransferases were different in their protein molecules and were involved in both the reactions from DMST to OMST and DHDMST to DHOMST. The reactions described in this paper were not observed when the same mold had been cultured in YEP medium.     

Functional alterations induced by fusarium T-2 toxin in Madin-Darby bovine Kidney (MDBK) and primary fetal bovine Kidney (PFBK) cultures

We recently reported that primary fetal bovine Kidney (PFBK) cells were consistently more sensitive to the cytotoxic effects of fusarium T-2 toxin than Madin-Darby bovine kidney (MDBK) cells in culture. The present report examined the influence of T-2 on selected biochemical parameters of these two culture types. T-2 toxin inhibited incorporation of labeled thymidine, uridine, and leucine in both culture types; at lower concentrations of the toxin, PFBK cells were affected to a greater extent than MDBK cells. T-2 toxin inhibited both the transport of thymidine as well as thymidine incorporation into macromolecules in MDBK cells during initial periods, but did not affect uridine incorporation. The cellular enzymes, K⁺- dependent phosphatase and succinic dehydrogenase were inhibited in MDBK but not in PFBK cultures; acid phosphatase was not influenced in either culture types. In a cell-free system none of the above enzymes were affected by T-2 until the toxin concentration exceeded 10^?5M.     

Changes in polyamine-oxidizing capacity of peroxisomes under various physiological conditions in rats

Rat liver peroxisomal polyamine oxidase activity was determined under various physiological conditions by using the peroxidase method with phenol and 4-aminoantipyrine. N1-Acetylpolyamines such as N1-acetylspermine and N1-acetylspermidine were better substrates than the free polyamines. The polyamine oxidase activity in rat peroxisomes increased significantly when cell proliferation was high. The activity began to appear in fetal liver at the 16th approximately 18th day of pregnancy and peaked in neonatal liver on the first day (approx. 1.7-times higher than in adult liver). In regenerating rat liver, only polyamine oxidase activity among the peroxisomal enzymes tested was increased considerably 12 h after partial hepatectomy (approx. 2.8-fold over the control liver). Finally, the enzyme activity was significantly increased by administration of clofibrate, a peroxisome proliferator, which also causes hepatomegaly. In all cases, the increase in polyamine oxidase activity was not more than 3-fold. Since the level of polyamine oxidase activity in the normal liver is more than adequate in relation to the level of the substrates, the slight but significant increase under conditions of cell proliferation may have a role in modulating levels of polyamines in the proliferating liver tissue.     

Origin of changes in electrical impedance during the growth and fermentation process of yeast in batch culture

The electrical impedance of the culture medium shows complex changes during the growth and fermentation process of yeast, and this prevents its possible application for the monitoring of certain yeast activities. Clarification of the mechanism of such changes is thus essential for practical use. As a first step toward this aim, the impedance, yeast concentration, and pH of a batch culture medium were measured using special cells with two compartments and also the usual type of cell with one compartment. In the special cells, the yeast was cultured in one compartment only. Conducting ions and nonconducting substances diffused through an intermediate porous membrane sandwiched between the two compartments. The impedances of the two compartments were measured simultaneously by the four-electrode method. The main mechanism responsible for increasing the impedance was the conducting ions produced by the yeast extract added as a nutrient to the culture broth by certain nonconducting substances during the process of growth. The increase in the yeast concentration was also a minor factor increasing the impedance. These increases surpassed the impedance decrease caused by the increase of H(+) ions produced by some accumulated acidic substances, and the impedance thus increased.