Cell surface proteins of Candida albicans: preparation of extracts and improved detection of proteins.

We have reexamined the detection of the components in a beta-mercaptoethanol and ammonium carbonate buffer extract of surface proteins of Candida albicans and the effects of postextraction manipulation of the extract on recovery of extract components. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), preferential staining of some moieties was observed when bands detected by a commercial silver staining method or a Coomassie Brilliant Blue (CBB) staining method were compared. Additional protein bands that were either not detected or poorly detected by a single method alone were readily observed by a combined silver-CBB staining method. This method also detected alterations in the profile of extracted proteins from organisms grown in the presence of galactose or hemoglobin rather than glucose. Two-dimensional electrophoresis (2-DE) gel analysis by double stain showed better detection of several acidic and basic protein spots. Less than 10% of the extract as determined by a dye-binding assay was lost following either or both lyophilization and dialysis. These manipulations of the extract did not change the protein profile following SDS-PAGE as determined by the combined staining or Western blot analysis of a 70 kDa protein. These observations suggest that soluble cell wall proteins are not unusually sensitive to procedures routinely used in protein purification. In addition, these studies suggest that a modified staining method that combines both silver stain and CBB stain provides improved detection of cell wall proteins compared to either method alone.

Variations in mRNA transcript levels of cell wall-associated genes of Saccharomyces cerevisiae following spheroplasting.

mRNA transcript levels of 38 genes from Saccharomyces cerevisiae were investigated during attempted spheroplast regeneration. Many of the genes selected are involved in cell wall biosynthesis. Spheroplasts did not regenerate into osmotically competent cells during the experiment. However, at a mRNA level, the quantities of transcripts were altered between the experimental and control populations. KRE11, EGT2 and MSS10 had their transcript levels increased by more than 10-fold during attempted spheroplast regeneration. A further six genes, FLO1, TIR1, SED1, HKR1, YGR189 and MUC1, showed transcript level increases of at least 5-fold. Five genes showed a change in transcript levels from an undetectable level to a detectable level: SKT5, KRE1, KRE5, SEC53 and DHS1. PMT2 showed a rapid decrease in mRNA levels followed by an increase to the basal level. Thus, cell stress genes, biosynthetic genes and some glycosylphosphatidylinositol-anchored cell wall proteins have their transcript levels increased in regenerating spheroplasts, but their transcription was not sufficient to initiate the replacement of the cell wall in liquid medium.

Enolase is present in the cell wall of Saccharomyces cerevisiae.

Non-covalently attached or soluble cell wall proteins of Saccharomyces cerevisiae were extracted using a high pH/2-mercaptoethanol procedure and were separated for peptide sequencing using 2D-PAGE. A partial N-terminal sequence of a major protein spot was obtained and showed high identity with enolase gene products. Western blotting with an anti-enolase antibody confirmed that enolase was present in the cell wall extract. Biotinylation of cells prior to protein extraction with a membrane impermeable biotinylating agent confirmed that the detection was not owing to cell lysis during extraction. Transmission immunoelectron microscopy showed enolase to be present in the cell wall. Enolase contains no known secretion signal that would localize it to the cell wall. Thus S. cerevisiae must have further mechanisms for targeting proteins to the cell wall.

A comparison of youthful inmates who have committed violent versus nonviolent crimes.

The incidence of violent crimes committed by youthful offenders in the United States is increasing. In this report, 150 inmates in a prison for youths (ages 14 to 24 years) who were treated by a psychiatrist were compared with 150 control subjects on several parameters including those who had committed violent crimes (V) versus those who had committed nonviolent crimes (NV). There were more similarities than differences between those who had committed V versus NV crimes. There was no statistically significant difference between the V and NV groups in most diagnostic categories, the Beck Hopelessness Scale, the IQ scale, the MMPI scores, job stability, and whether they were treated by a psychiatrist or not. There were, however, some significant differences. The V group was younger than the NV group; those with a diagnosis of paranoid schizophrenia disorder had committed V crimes, while those with a diagnosis of dysthymic disorder had committed NV crimes.

The C-terminus of yeast plasma membrane H+-ATPase is essential for the regulation of this enzyme by heat shock protein Hsp30, but not for stress activation.

Several stresses cause additional activation to the glucose-stimulated plasma membrane H+-ATPase activity of yeast, but it is not clear how this is achieved. We recently reported that cells lacking the integral plasma membrane heat shock protein Hsp30 display enhanced increases in plasma membrane H+-ATPase activity with either heat shock or weak organic acid stress (Piper, P.W., Ortiz-Calderon, C., Holyoak, C., Coote, P. and Cole, M. (1997) Cell Stress and Chaperones 2, 12-24), indicating that the stress induction of Hsp30 acts to reduce stress stimulation of the H+-ATPase. In this study it is shown that Hsp30 acts to reduce the Vmax of H+-ATPase in heat shocked cells. Its action is lost with deletion of the C-terminal 11 amino acids of the H+-ATPase, a deletion that does not abolish the stress stimulation of this enzyme. Mutation of the Thr-912 residue within the C-terminal domain of H+-ATPase, a potential site of phosphorylation by the Ca2+-calmodulin-dependent protein kinase, also abolishes any effect of Hsp30. Hsp30 may therefore be acting on a Thr-912 phosphorylated form of the H+-ATPase.

Description of an outpatient psychiatric population in a youthful offender's prison.

Prisons are receiving increased numbers of inmates with mental and emotional problems. This study describes some of the characteristics and treatment of such an outpatient population. It was determined that a typical patient is a white male, 19 years old, of average intelligence, with a sporadic work record and poor academic performance, who quits high school in his freshman year. He has a history of substance abuse and is likely to have a multidrug habit. He is likely to have had a traumatic childhood and had psychiatric treatment as a child or young adolescent, as well as having attended special classes in school and counseling for drug abuse. The great majority of patients were diagnosed as having either mood, adjustment, or psychotic disorders. All were treated with a psychotropic medication and case management and also with some type of accepted individual and/or group counseling. In this population, there is a high incidence of expression of aggression requiring medication and counseling with the patient's permission. Patients responded well to treatment, but usually requested to discontinue treatment when symptoms diminished. However, approximately half of them returned for medication when symptoms recurred.

Natural history of serous detachments of the retinal pigment epithelium.

Fifty eyes of 40 patients with serous (avascular) detachments of the retinal pigment epithelium were followed up for an average of 22 months. Of the 50 eyes, 14 (28%) developed bleeding beneath the retinal pigment epithelium and retina or subretinal neovascularization detected on fluorescein angiography. These changes did not occur in eyes of patients younger than age 56 years at diagnosis or in detachments less than 1 disk diameter in size, whereas 35.9% (14) of eyes in patients older than age 56 years developed vascular complications. Vascular complications were uncommon when the initial detachment did not involve the fovea. Visual acuity declined in only one (9.1%) of eyes in patients younger than age 56 years but in 20 (51.3%) of eyes in patients age 56 years or greater.

The prevalence of HLA-B7 in presumed ocular histoplasmosis in patients with peripheral atrophic scars.

Patients identified in the Walkersville, Maryland, epidemiologic study of presumed ocular histoplasmosis as having only peripheral atrophic scars showed no increase in frequency of HLA-B7 over a control population. Because this antigen is increased in patients with a history of active macular or peripapillary lesions, these findings indicate an inherent predisposition in some patients to develop posterior pole lesions after an ocular infection with Histoplasma capsulatum. The precise mechanisms by which the histocompatibility complex participates in the pathogenesis of these lesions is unknown, but our data are compatible with previous suggestions that alterations in the immune response of these individuals are in some way important in the development of disciform scarring in the posterior pole.

The prevalence of HLA-B7 in presumed ocular histoplasmosis.

Eighteen patients with the presumed ocular histoplasmosis syndrome were typed for HLA histocompatability antigens and 78% were found to have the B7 antigen. This data implies an underlying immunogenic predisposition to the development of the clinical eye findings and active posterior pole lesions in the presumed ocular histoplasmosis syndrome. It is likely that some feature of the immune system of these patients renders them more susceptible either to the initial infection or to a later activation of posterior pole lesions mediated through immunologic mechanisms.