Activation of gene expression of the gamma-aminobutyric acid rather than the glutamatergic system in the preoptic area during the preovulatory gonadotropin surge of the rat.

There is increasing evidence that in the rat prior to and during the preovulatory LH surge, release rates of GABA in the preoptic area (POA) are decreased while no such changes occurred in the mediobasal hypothalamus (MBH). In addition, GnRH release appears to be facilitated by an increased preoptic excitation of glutamate (GLU). To investigate whether such changes of secretory activity of intrahypothalamic GABA or GLU neurons are associated with altered gene expression of biosynthetic enzymes or transporter proteins characteristic for either neuronal system, we determined mRNA levels of the two forms of the GABA-synthesizing enzyme glutamate decarboxylase (GAD65 and GAD67), the glutamate-synthesizing enzyme glutaminase (GLS), the GABA transporter type 1 (GAT-1) and the glutamate-aspartate transporter type 1 (GLAST). Competitive RT-PCRs using mutant cRNAs as internal standards were conducted with mRNA extracted from microdissected tissue of POA and MBH from diestrous, proestrous, and estrous rats. Proestrous animals were subgrouped according to their endocrine status as follows: 'prior to', on the 'ascending' or on the 'descending' limb of the LH peak, and 'after the LH surge (post)'. During the preovulatory LH surge, mRNA concentrations of GAD67 and GAT-1 in the POA were significantly increased compared to those observed on diestrous (2.8-fold for GAD67 and 2.5-fold for GAT-1, p < 0.01), while in the MBH the amount of both mRNAs remained constant. The expression levels of GAD65, GLS and GLAST were without any changes in the POA as well as in the MBH. These findings support the hypothesis that in rats induction of the preovulatory LH surge is controlled at the level of GnRH perikarya, and suggest that altered activities of intrapreoptic GABA neurons at both transcriptional and secretory levels are pivotal for the preovulatory activation of GnRH neurons.

Influence of oxidative stress induced by cysteamine upon the induction and development of thermotolerance in Chinese hamster ovary cells.

Chinese hamster ovary cells exposed to the sulfhydryl compound cysteamine combined with heat treatment at 44 degrees C developed thermotolerance within 8 h. After initial treatment either with 15 min cysteamine (0.4 mM) at 37 degrees C immediately followed by 15 min heat at 44 degrees C or with 15 min cysteamine (0.4 mM) at 44 degrees C, the magnitude of thermotolerance developed was identical. The D0 of the subsequent 44 degrees C heat survival curves increased by factors of 8.9 and 7.9, respectively. The kinetics of thermotolerance induction and the time to reach the maximum of thermotolerance expression after combined cysteamine treatment at 44 degrees C for 15 min was found to be comparable to the effects of 44 degrees C treatment alone for 30 min. The synergistic effect of cysteamine with the conditioning heat treatment at 44 degrees C was blocked by catalase (50 micrograms/ml). Following initial treatment with cysteamine at 37 degrees C, cells became thermotolerant within 2 h. The D0 of the survival curves for 44 degrees C heat treatments increased with duration (t1 = min, 37 degrees C) of the cysteamine (0.4 mM) exposure; e.g., the D0 increased by factors of 1.5, 1.6, 2.2, and 2.6 for t1 = 30, 60, 90, and 120 min. The induction of thermotolerance by cysteamine at 37 degrees C was completely blocked by the addition of catalase (50 micrograms/ml), present during the initial period of drug treatment. Combined cysteamine and heat treatment at 44 degrees C, but also cysteamine exposure at 37 degrees C, enhanced synthesis of heat shock proteins. The data suggest that oxidative stress by cysteamine can be synergistic with the conditioning heat treatment at 44 degrees C which induces thermotolerance. At 37 degrees C, cysteamine itself induces thermotolerance and the enhanced synthesis of heat shock proteins under these conditions.

Characterization of a secretory proteinase of Candida parapsilosis and evidence for the absence of the enzyme during infection in vitro.

The opportunistic yeastlike fungi of the genus Candida comprise three species which are proteolytic in vitro. Among them, C. albicans and C. tropicalis are of foremost medical importance. However, a strict correlation between extracellular proteolytic activity and virulence is opposed by the low virulence of the third proteolytic species, C. parapsilosis. We purified the secretory acid proteinase of C. parapsilosis (clinical isolate 265). The enzyme is a carboxyl proteinase (EC 3.4.23) like all other secretory Candida proteinases handled so far. Proteinase 265 is distinguished by a lower molecular weight (approximately 33,000); it has increased hydrophobicity, which accounts for inhibition of the enzyme by hemin, and required the presence of nonionic detergent in the initial steps of purification. The enzyme already undergoes alkaline denaturation at neutrality. Its activity is thus confined to the acid microenvironment of the fungal cell wall. Within this range, the enzyme may degrade immunoglobulins like immunoglobulin A1 (IgA1), IgA2, and secretory IgA. No indication was found for glycosylation of proteinase 265 and the related enzyme of C. albicans CBS 2730. However, the comparable proteinase of C. tropicalis 293 was identified as a manno protein. Antiserum against proteinase 265 cross-reacted strongly with corresponding enzymes from other Candida species. Antisera against proteinases of C. albicans and C. tropicalis reacted only weakly with proteinase 265. Thus, secretory Candida proteinases are likely to possess common and species-specific antigenic sites. In contrast to C. albicans, infection of phagocytes by C. parapsilosis 265 was not accompanied by secretion of fungal proteinase. This lack of induction of the enzyme under conditions of infection may account for the low virulence of most isolates of C. parapsilosis.

Identification and partial characterization of two proteinases from the cell envelope of Candida albicans blastospores.

As we have communicated previously, the fungal opportunist Candida albicans produces an angiotensin-1 liberating proteinase and a serine proteinase that acts as a converter of the coagulation factor X in vitro (25). Both enzymes are attached to delipidated cell fragments of fungal blastospores. They copurify upon ion exchange chromatography and gel filtration. The enzymes could be separated by hydrophobic chromatography on Phenylsepharose. According to their substrate and inhibition pattern, the enzymes have been classified as an aspartyl proteinase and a chymotrypsin-like proteinase. Their partial characterization includes estimates of the molecular weight and isoelectric points.

Secretion of acid proteinases by different species of the genus Candida.

Strains of several Candida species that are isolated from humans aside of C. albicans have been screened for secretion of acid proteinases. A third of the strains of C. tropicalis was strongly proteolytic, other strains of this species were moderately or virtually nonproteolytic; the variety of activities resembled pattern found among strains of C. albicans (32). Strains of C. parapsilosis produced 24% of the proteolytic activity of strongly proteolytic strains of C. tropicalis. The proteolytic activity of C. pseudotropicalis, C. krusei, and C. glabrata accounted for 7.5, 2.0, and 0.8% respectively. Four strains of C. guilliermondii developed strain-specific proteolysis after prolonged cultivation only. Five proteinases from strains of C. tropicalis cross reacted immunologically as did five proteinases from C. albicans. Interspecific cross reaction, though, was not observed when certain sera from rabbits were employed.

Detection of Candida proteinase by enzyme immunoassay and interaction of the enzyme with alpha-2-macroglobulin.

Double antibody immunoassay of secretory aspartic proteinases from 2 strains of the yeast Candida albicans was performed in microtest plates with rabbit antibody and peroxidase conjugated rabbit antibody. The test detects approximately 0.1 ng of proteinase and is 2 orders of magnitude more sensitive than direct measurement of enzymic activity with hemoglobin as a substrate. The immunoassay was affected both by alkaline denaturation, which is a common feature of aspartic proteinases, and by the presence of the proteinase scavenger alpha-2-macroglobulin. Conditions are described that allow for comparison of proteinases from different strains of yeast and minimize the interference of macroglobulin.