Comparison of fungal viability assays using Candida albicans yeast cells undergoing prolonged incubation in the absence of nutrients.

Staining methods for determining fungal viability are usually assessed by comparisons with enumeration of colony-forming units (CFU) on solid media. The purpose of the present study was to compare viability as assessed by the acridine orange (AO) and MTT methods with the numbers of CFUs obtained for Candida albicans yeast cells undergoing prolonged incubation in distilled water. In initial assessments of the assays using various proportions of control and heat-killed C. albicans, the AO and MTT methods consistently indicated significantly higher values for viability than did CFU determinations. Experiments using organisms cultured overnight revealed that approximately 95% of the cells were capable of dividing at least once in a microscopic proliferation assay, whereas only 69% were capable of forming colonies. Parallel assays comparing AO uptake and MTT reduction gave excellent agreement with the microscopic proliferation assay, but not with CFU determinations. Using organisms undergoing prolonged incubations in distilled water, much lower viabilities were obtained with the CFU method at 7 and 10 days than with the microscopic proliferation assay or the two staining methods. These results indicate that the AO and MTT assays correlate well with the ability of C. albicans to divide at least once, but may not accurately indicate the percentage of organisms actually able to form colonies.

Expression of genes encoding peroxisomal proteins in Saccharomyces cerevisiae is regulated by different circuits of transcriptional control.

In Saccharomyces cerevisiae induction of the FOX3 gene, encoding peroxisomal 3-oxoacyl-CoA thiolase, by growth on oleate as sole carbon source, is exerted via the cis-acting DNA element designated oleate response element (ORE) (Einerhand et al. (1991) Eur. J. Biochem. 200, 113-122). The transcription factor(s) binding to this upstream activation site (UAS) are still unknown, however. Induction of another peroxisomal enzyme, citrate synthase (CIT2) is dependent on the products of two genes called RTG1 and RTG2 (Liao and Butow (1993) Cell 72, 61-71). In the present study we have investigated whether RTG1 controls other genes coding for peroxisomal proteins, and whether such control takes place via the ORE. A number of genes coding for a variety of peroxisomal proteins such as: thiolase and catalase (peroxisomal matrix proteins), PAS3p (a peroxisomal membrane protein) and PAS10p (a protein involved in the import of peroxisomal proteins) were studied in their response to RTG1. Although the RTG1 and 2 products proved to be required for the increase in number and volume of peroxisomes upon induction by oleate, the single promoter output of the chosen set of genes remained practically unchanged in a rtg1 mutant strain. In addition gel retardation experiments indicated that RTG1 does not bind to the ORE. The behavior of genes coding for the various proteins also varied during repression, derepression and induction, indicating that probably a number of proteins are involved in tuning the output of each gene to cellular demand.

The upstream region of the FOX3 gene encoding peroxisomal 3-oxoacyl-coenzyme A thiolase in Saccharomyces cerevisiae contains ABF1- and replication protein A-binding sites that participate in its regulation by glucose repression.

Expression of the FOX3 gene, which encodes yeast peroxisomal 3-oxoacyl-coenzyme A thiolase, can be induced by oleate and repressed by glucose. Previously, we have shown that induction was mediated by an oleate response element. Just upstream of this element a negatively acting control region that mediated glucose repression was found. In order to study this negative control region, we carried out DNA-binding assays and analyzed phenotypes of mutations in this region and in the trans-acting factor CAR80, which is identical to UME6. DNA-binding assays showed that two multifunctional yeast proteins, ABF1 and RP-A, interacted with the negative control element independently of the transcriptional activity of the FOX3 gene. ABF1 and RP-A, the latter being identical to BUF, were able to bind to DNA independently of one another but also simultaneously. The phenotypes of mutations in either DNA-binding sites of ABF1, RP-A, or both, which affected the DNA binding of these factors in vitro, indicated that these sites and the proteins that interact with them participate in glucose repression. The involvement of the RP-A site in glucose repression was further supported by our observation that the CAR80 gene product, which is required for repression mediated by the RP-A site, was essential for maintenance of glucose repression. In addition to the RP-A site in the FOX3 promoter, similar sequences were observed in other genes involved in peroxisomal function. RP-A proved to bind to all of these sequences, albeit with various affinities. From these results it is concluded that the ABF1 and RP-A sites are being required in concert to mediate glucose repression of the FOX3 gene. In addition, coordinated regulation of expression of genes involved in peroxisomal function in response to glucose is mediated by proteins associated with the RP-A site, probably RP-A and CAR80.

Evaluation of the antibacterial effects of intracanal Nd:YAG laser irradiation.

Fifty canals of extracted single-rooted teeth were prepared to a size 50 master apical file, sterilized in ethylene oxide, and inoculated with a known quantity of Bacillus stearothermophilus spores. Five groups of 10 canals each were used. The control group received no treatment. The four treatment groups were exposed to pulsed Nd:YAG laser radiation or 0.5% NaOCl alone and in combination. The root canals were flushed with sterile distilled water to recover spores, and serial dilutions were incubated on blood agar and the number of colony-forming units recovered was determined. Analysis of the data indicated a 2-log reduction in colony-forming units among the four treatment groups as compared with the controls; however, no significant differences were observed among the treatment groups. In none of the treatment groups were the root canals sterilized.

Characterization of a transcriptional control element involved in proliferation of peroxisomes in yeast in response to oleate.

Oleate induces the transcription of genes involved in peroxisome biogenesis and stimulates the proliferation of these organelles in Saccharomyces cerevisiae. Previously, we have reported the identification of a region containing a positive regulatory element in the 5' flanking region of the FOX3 gene encoding the peroxisomal enzyme 3-oxoacyl-CoA thiolase. This region contains a 23-bp imperfect inverted-repeat sequence. Full induction, in response to oleate, is mediated by the intact dyad. However, one half-site of the inverted repeat is also able to mediate induction of transcription in response to oleate, albeit to a small extent. Furthermore, the weak binding of protein to each part of the inverted repeat proved to be correlated with the weak activation of transcription, in support of oleate. A DNase-I footprint covered the entire dyad and DNA band-shift experiments indicated that one or more trans-acting factors bind to the imperfect palindrome. The binding of protein to this element seems to be correlated with transcriptional activation, since mutations in both halves of the inverted dyad affected both transcriptional activation and protein binding in vitro. Similar oleate-responsive elements are commonly found in the 5' flanking regions of genes encoding proteins involved in peroxisome biogenesis and the factor(s) binding to oleate-responsive element(s) could therefore be involved in coordination of the expression of oleate-inducible genes and the proliferation of peroxisomes.

Decreased number of vasopressin immunoreactive neurons in the medial amygdala and locus coeruleus of the aged rat.

The earlier described age-related decreases in vasopressin innervation of extra-hypothalamic rat brain regions were found to coincide with a decrease in vasopressin expression in the cells where these fibers originated. A significant age-related decrease in the number of vasopressin-immunoreactive cell bodies was found in the medial amygdala and locus coeruleus of senescent Brown-Norway (BN/BiRij) rats (33 months) when compared to middle-aged (19 months) and young (3 months) rats. In addition, total testosterone plasma levels were significantly reduced in middle-aged and old rats as compared to young animals and the number of vasopressin-immunoreactive cells in both the medial amygdala and locus coeruleus correlated significantly with the decreased testosterone levels in a similar way as found earlier for vasopressin terminals.

[Immediate and late results following endarterectomy of the carotid bifurcation]. / Sofort- und Spätergebnisse nach Endarteriektomie der Karotisgabel.

In a retrospective study we analysed two groups each consisting of 100 consecutive patients of similar age and sex distribution who underwent surgery for carotid disease with an intervening period of 5 years (group A 1980/82, group B 1986/87) between the collectives. Against a background of changing indications, tactics and techniques the aim of the study was to detect any differences between the two groups. Group A had a higher proportion of coronary and peripheral vascular disease. The states of cerebral ischemia I, II and III were distributed equally, but state IV was seen more frequently in group B (p less than 0.05). The number of shunt/without shunt operations in group A was 97/2, in group B 10/84 (p less than 0.005). The external carotid artery was deobliterated in 58/81 cases group A versus group B (p less than 0.005). We closed the artery by direct suture in 8/31 (p less than 0.005), by autologous venous patch in 53/26 (p less than 0.005) and by Dacron patches in 39/41 patients. In group A the operative mortality was zero and in group B 1 patient died; one patient in group B developed sudden occlusion (with TIA) postoperatively. Transient intra-/postoperative neurological deficits occurred in 1/2, permanent in 4/2 patients (n.s.). 54/25 patients have died up to 31/08/91. Coronary heart disease was the main cause of late complications and deaths in group A (p less than 0.025). Statistically, there was no dependence of neurological deficits on group, sex, age or intraoperative management. Only patients with preoperative PRINDS hat a higher postoperative neurological deficit rate than the others.

Impaired function of immune reactivity to Listeria monocytogenes in diet-fed mice.

Mice fed a diet high in cholesterol, lard, and sucrose were shown to exhibit an impairment of specific immunity to Listeria monocytogenes. Whereas titers of L. monocytogenes in livers of normal mice decreased rapidly after 6 days of infection, L. monocytogenes persisted in livers of diet-fed mice. Adoptive transfer experiments indicated that L. monocytogenes-immune spleen cells are generated in diet-fed mice. However, the function of immune spleen cells from donors of either nutritional status was impaired in diet-fed recipients. The results indicate that the site(s) of impairment of specific immunity to L. monocytogenes in diet-fed mice occurs at a stage beyond the generation of immune T-cells.

Antibiotic susceptibility of black-pigmented Bacteroides isolates from the human oral cavity.

The minimal inhibitory concentrations of penicillin and six other antibiotics were determined for 66 oral black-pigmented Bacteroides isolates by using the National Committee for Clinical Laboratory Standards proposed standard agar dilution technique. These results plus iodometric determination of beta-lactamase activity showed that oral isolates of black-pigmented Bacteroides are remaining relatively susceptible to commonly used antibiotics.

Inhibition of host resistance by nutritional hypercholesteremia.

Previous experiments showed that nutritionally induced hypercholesteremia in mice caused an increase in susceptibility to coxsackievirus B, with a marked suppression of cellular infiltrates in infected tissues and an increased mortality. The present studies demonstrated that a hypercholesteremic diet was associated with an inhibition in host resistance as measured by susceptibility to Listeria monocytogenes infection and the growth of two transplanted syngeneic murine tumors. Moreover, the ability of Corynebacterium parvum to induce regression of a transplanted methylcholanthrene-induced fibrosarcoma was inhibited in hypercholesteremic hosts, as was the histiocytic infiltration normally accompanying C. parvum inoculation. In contrast, the peritoneal macrophages from C. parvum-treated hypercholesteremic mice were indistinguishable from similarly treated macrophages from normal mice with respect to their in vitro tumoricidal activity and the presence of a cell surface antigen associated with activated macrophages. Hypercholesteremia was also associated with a decreased antibody response to sheep erythrocytes in vivo, but dit not appear to exert a detrimental effect on B- or T-cell blastogenesis when tested in vitro. The findings that the hypercholesteremic diet was associated with an impairment in the host immune response and increased susceptibility to viral, bacterial, and tumor cell challenge are discussed with respect to virus-lipid interactions in the pathogenesis of atherogenesis and diabetes mellitus.

Suppression of aortic elastic tissue autofluorescence for the detection of viral antigen.

Suppression of aortic elastic tissue autofluorescence was achieved by employing a modification of Verhoeff's elastic tissue staining procedure. Consquently, coxsackievirus B antigen present in the aortic media was detected by conventional fluorescent antibody staining.