Studies on detection of Candida antigen in the sera of mice inoculated orally with Candida albicans

Summary The authors succeeded in establishing a murine model of systemic candidiasis being disseminated from the primary gastrointestinal lesions caused by oral inoculation of Candida albicans. Using this model, an attempt was made for detecting the Candida antigen by enzyme-linked immunosorbent assay using avidin-biotin (AB-ELISA) from the serum of infected mice.Gastrointestinal candidiasis was formed in all of the 20 mice treated with the drugs (antibiotics, antineoplastic agents, hydrocortisone, etc.) and inoculated orally with C. albicans. Fourteen of these mice suffered from submucosal candidiasis, and C. albicans was cultured from the visceral organs in 12 of them. The assay by AB-ELISA was able to detect 1.0 ng/ml Candida mannan in the mouse serum. The Candida antigen was detected in the sera of 11 of the 14 mice with submucosal candidiasis. However, the antigen could not be detected in the sera of the 6 mice with intramucosal candidiasis.The assay by AB-ELISA is more sensitive and specific for the diagnosis of systemic candidiasis than other serological assays.     

Effects of muscimol and baclofen on levels of monoamines and their metabolites in the El mouse brain

We compared the changes in monoamines and their metabolites in the El mouse brain induced by GABA-A and GABA-B receptor agonists. Muscimol was used as a GABA-A receptor agonist, and baclofen as a GABA-B receptor agonist. Muscimol (3 mg/kg) significantly increased the DOPAC level in all parts of the mouse brain and the HVA level in the cortex, striatum, and midbrain. No significant change was observed in the dopamine (DA) level. These findings suggest that muscimol may accelerate both the synthesis and catabolism of DA. Baclofen (20 mg/kg) increased the DA level in the hippocampus and midbrain, and the DOPAC level in the hippocampus. Muscimol increased 5-HIAA levels and decreased 5-HT levels. This result suggests that 5-HT metabolism is accelerated by muscimol. No change in 5-HT or 5-HIAA levels was induced by baclofen. The GABA-A receptor system seems to have a potent effect not only on DA neurons, but on 5-HT neurons. However, the GABA-B receptor system appears to have almost no effect on 5-HT neurons, though it appears to have some effect on DA neurons.     

Repression of Asolectin-Dependent Activation of Partially Lipid Depleted ATPase Prepared from the Plasma Membrane-Enriched Fraction of Cucumber Roots due to Ca2+ Starvation

The ATPase activity of the plasma membrane-enriched fractionwas severely inhibited by withdrawal of Ca²⁺ from the mediumfor 5 days, although the root system appeared to be unaffectedto visual inspection. Partially lipid-depleted ATPases withsimilar ratios of phospholipid to protein were prepared fromthe plasma membrane-enriched fraction of cucumber roots culturedwith control medium and one lacking Ca²⁺, and their propertieswere compared. SDS disc polyacrylamide gel electrophoresis showedthat the polypeptide components were essentially similar betweencontrol and Ca²⁺-starved roots. Partially lipid-depleted ATPasereassociated with asolectin, the lecithin from soybean, showedtypical characteristics of plasma membrane type ATPase; pH optimumat 6.5, high specificity for ATP as substrate and strong inhibitionby vanadate but not nitrate. The activity of reassociated ATPaseobtained from the control roots was apparently higher than theactivity obtained from Ca²⁺-starved roots. The amount of asolectinrequired for maximum activation of the partially lipid-depletedATPase prepared from control roots was much lower than thatprepared from Ca²⁺-starved roots. Reassociation of partiallylipid-depleted ATPase with asolectin produced higher ATPaseactivity than that with individual phospholipids. The activationof partially lipid-depleted ATPase prepared from control rootswith asolectin was not inhibited by addition of a sample preparedfrom Ca²⁺-starved roots. Thus, a decrease in the functionalassociation of ATPase with phospholipids might be one of thephysiological injuries in root cell membranes of cucumber causedby Ca²⁺ starvation. ¹Permanent address: Department of Horticulture, College of Agriculture,Chonnam National University, Chonnam 500, Korea. (Received February 23, 1988; Accepted August 18, 1988)     

Repression of Proton extrusion from Intact Cucumber Roots and the Proton Transport Rate of Microsomal Membrane Vesicles of the Roots due to Ca2+ Starvation

Proton extrusion from cucumber roots decreased markedly duringCa²⁺ starvation in the presence of KC1. Vesicles with ATP-dependentproton transport activity were prepared from the microsomalmembrane fraction of control and Ca²⁺-starved roots. The protontransport rate of the vesicles from Ca²⁺-starved roots was repressedto less than half of the vesicles prepared from the controlroots. K⁺-Mg²⁺-ATPase activity associated with the vesiclesprepared from Ca²⁺-starved roots was approximately one-thirdof the activity associated with those prepared from controlroots. K_m values of the proton transport rate and K⁺-Mg²⁺-ATPasefor ATP were much higher in vesicles prepared from Ca²⁺-starvedroots. The repression of proton extrusion linked with K⁺ uptake inthe Ca²⁺-starved roots could be largely caused by the reducedproton pumping activity associated with microsomal membranesin the roots. (Received May 25, 1987; Accepted October 14, 1987)     

Increase in Proton-Transport Activity of Tonoplast Vesicles as an Adaptive Response of Barley Roots to NaCl Stress

H⁺-Transport activity of the vesicles prepared from barley rootswas studied at the early phase after application of NaCl stress.The activity reached maximal level at 3 days after the treatmentwith 200 mM NaCl which moderately reduced the growth. This activityincrease could be suppressed in the presence of cycloheximideand actinomycin D. The properties of the membrane vesicles associated with H⁺-transportactivity prepared from both control and NaCl-stressed rootssuggested that it was of tonoplast origin based on the followingfindings: optimal pH at 7.5, strong inhibition by nitrate butnot by vanadate, and stimulation by chloride. The density gradient centrifugation of vesicles with DextranT70 did not show any detectable difference in the distributionpatterns of H⁺-transport activities between control and NaClstressedroots. Furthermore, K_m values for ATP of the H⁺-transport activityof vesicles prepared from control and NaCl-stressed roots werethe same. Therefore, H⁺-transport activity with properties similarto those of the control roots was increased by NaCl stress.The results are discussed in terms of an adaptive mechanismof barley against salt stress. ¹Permanent address: Department of Horticulture, College of Agriculture,Chonnam National University, Chonnam 500, Korea. (Received April 18, 1988; Accepted July 20, 1988)     

Modulation of cerebral acetylcholine metabolism by the dorsal diencephalic conduction system in the rat

We investigated the effects of interruption of the impulse flow in the habenulopeduncular pathways by local infusion of tetrodotoxin on the acetylcholine and choline content in selected dopamine rich regions in the forebrain and midbrain in rats. The tetrodotoxin infusion caused a marked increase in acetylcholine content in the medial frontal cortex, striatum and ventral tegmental area+interpeduncular nucleus, but not in the limbic area or the substantia nigra, whereas choline content was reduced only in both the striatum and ventral tegmental area+interpeduncular nucleus. There was an increase in 3,4-dihydroxyphenylacetic acid content in the striatum after the manipulation. These findings suggest that the dorsal diencephalic conduction system may be involved in the integration of the activity of cholinergic neurons in the forebrain and midbrain regions and striatal dopanine neurons may play a role in the modulation of cholinergic neurons.     

Protective effect of cerulein on memory impairment induced by protein synthesis inhibitors in rats.

Previous studies have demonstrated that NMDA receptor antagonists and protein kinase C inhibitors induced marked memory impairment in rats, but that peripherally administered cerulein (CER) prevented these effects. In the present study, the effect of subcutaneously administered CER on amnesia induced by protein synthesis inhibitors was examined in passive and active avoidance responses and in the Morris water maze test. Intraperitoneal injection of the inhibitors produced marked memory impairment, but the effect was abolished by combined administration with CER. The effective dose of subcutaneously injected CER was, on a molar basis, three thousand- and six thousandfold less than the dose of anisomycin, and two hundred eighty- and three thousandfold less than the dose of puromycin in the passive and active avoidance response experiments, respectively. Similarly, in the Morris water maze test, behavioral disturbances produced by the protein synthesis inhibitors were abolished by CER. These results indicate the effectiveness of CER in preventing memory impairment induced by protein synthesis inhibitors.     

Morphological differentiation of endothelial cells co-cultured with astrocytes on type-I or type-IV collagen

Summary In this study bovine aortic endothelial cells were co-cultured with astrocytes from fetal Wistar Kyoto rats. Endothelial cells growing on type-I collagen, development. Although some cells appeared to be mature, horseradish peroxidase penetrated within 1 min of incubation through the intercellular junctions of these endothelial elements maintained on type-I collagen. In contrast, endothelial cells on type-IV collagen, co-cultured with astrocytes, were well developed; their intercellular junctions were well established, and plasmalemmal vesicles reduced in number. As a result, horseradish peroxidase was unable to penetrate through the endothelial cells grown on type-IV collagen and co-cultured with astrocytes because of the reduced extent of the junctional and vesicular transport. These findings reveal that (1) type-IV collagen is essential for the differentiation of endothelial cells, (2) endothelial cell-astrocyte interactions occur during co-culture, and (3) endothelial permeability depends on astrocyte-produced factors, in addition to type-IV collagen.     

Gene structure and expression of a tobacco endochitinase gene in suspension-cultured tobacco cells

We have isolated and characterized the genomic clone CHN50 corresponding to tobacco basic endochitinase (E.C.3.2.1.14). DNA sequence and blotting analysis reveal that the coding sequence of the gene present on CHN50 is identical to that of the cDNA clone pCHN50 and, moreover, the CHN50 gene has its origin in the progenitor of tobacco, Nicotiana sylvestris. Tobacco basic chitinases are encoded by a small gene family that consists of at least two members, the CHN50 gene and a closely related CHN17 gene which was characterized previously. By northern blot analysis, it is shown that the CHN50 gene is highly expressed in suspension-cultured tobacco cells and the mRNA accumulates at late logarithmic growth phase. To identify cis-DNA elements involved in the expression of the CHN50 gene in suspensioncultured cells, the chimeric gene consisting of 1.1 kb CHN50 5 upstream region fused to the coding sequence of -glucuronidase (GUS) was introduced by electroporation into protoplasts isolated from suspension-cultured tobacco cells. Transient GUS activity was found to be dependent on the growth phase of the cultured cells, from which protoplasts had been prepared. Functional analysis of 5 deletions suggests that the distal region between -788 and -345 contains sequences that potentiate the high-level expression in tobacco protoplasts and the region (-68 to -47) proximal to the TATA box functions as a putative silencer.     

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene inEscherichia coli

Summary The application of an inducible regulation system using the trytophanase operon promoter (TPase promoter; P_tna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of P_tna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, l-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-P_tnaTS was increased 500-fold compared to that of the cultured host strain. About 1 mol l-tryptophan/l reaction mixture was formed from indole and l-serine at 37° C for 3.5 h. Offprint requests to: H. Yukawa