Cloning and Sequencing of a Candida albicans Catalase Gene and Effects of Disruption of This Gene

Catalase plays a key role as an antioxidant, protecting aerobic organisms from the toxic effects of hydrogen peroxide, and in some cases has been postulated to be a virulence factor. To help elucidate the function of catalase in Candida albicans, a single C. albicans-derived catalase gene, designated CAT1, was isolated and cloned. Degenerate PCR primers based on highly conserved areas of other fungal catalase genes were used to amplify a 411-bp product from genomic DNA of C. albicans ATCC 10261. By using this product as a probe, catalase clones were isolated from genomic libraries of C. albicans. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 487 amino acid residues. Construction of a CAT1-deficient mutant was achieved by using the Ura-blaster technique for sequential disruption of multiple alleles by integrative transformation using URA3 as a selectable marker. Resulting mutants exhibited normal morphology and comparable growth rates of both yeast and mycelial forms. Enzymatic analysis revealed an abundance of catalase in the wild-type strain but decreasing catalase activity in heterozygous mutants and no detectable catalase in a homozygous null mutant. In vitro assays showed the mutant strains to be more sensitive to damage by both neutrophils and concentrations of exogenous peroxide that were sublethal for the parental strain. Compared to the parental strain, the homozygous null mutant strain was far less virulent for mice in an intravenous infection model of disseminated candidiasis. Definitive linkage of CAT1 with virulence would require restoration of activity by reintroduction of the gene into mutants. However, initial results in mice, taken together with the enhanced susceptibility of catalase-deficient hyphae to damage by human neutrophils, suggest that catalase may enhance the pathogenicity of C. albicans.     

Comparison of assays to detect cytomegalovirus shedding in the semen of HIV-infected men

We sought to determine the optimal assays for cytomegalovirus (CMV) shedding in semen. Over a 2-month period, 149 HIV-1-infected men who have sex with men each provided up to three semen specimens. Specimens were tested for CMV by culture, rapid assay (shell vial) and polymerase chain reaction (PCR). By culture, 30% of seminal plasma and 28% of seminal cell specimens grew CMV. By rapid assay, results were 38 and 33%, respectively. By PCR, 56% of seminal cell specimens demonstrated CMV: 20% in a single semen specimen; 33% in two specimens; and 34% in all three specimens. Overall, 69% of men had CMV detected by PCR in at least one seminal cell specimen. By quantitative PCR, 14% had ten, 14% had 100, 16% had 1000, and 12% had 10 000 copies in 6.25 μl of semen analyzed. Adjusting for initial CD4+ cell count, men with CMV shedding demonstrated by PCR at the first visit were approximately four times as likely to shed CMV at a subsequent visit (RR 4.28, CI: 2.30–7.95). CMV shedding was associated with decreased CD4+ cell counts in peripheral blood (P=0.05). It is concluded that the PCR assay provided the greatest sensitivity among the three detection methods.     

Health beliefs and attitudes toward the prevention of osteoporosis in older women

OBJECTIVE: A pilot study to determine health belief factors associated with osteoporosis prevention behaviors in peri-and postmenopausal women. DESIGN: We administered a survey to a convenience sample of 60 women aged 40-95 years old in an urban family practice center and an associated retirement community. The self-reported questionnaire addressed demographics, osteoporosis risk factors, current preventive behaviors for osteoporosis, and health beliefs. RESULTS: The majority of women (89%) believed that osteoporosis is a serious condition, but only 29% perceived a personal susceptibility. Women were less concerned about osteoporosis when compared with cancer, cardiovascular disease, and neurologic disorders. Only 40% of women were taking active measures to prevent osteoporosis. There was no significant relationship between active osteoporosis prevention behaviors and five health belief factors (motivation, barrier, active participant in health care, frustration, and benefit) (p >or= 0.43). However, active behaviors to prevent osteoporosis were found to correlate with the single item "I am worried about developing osteoporosis" (p = 0.03). Most women surveyed would be willing to exercise and take calcium and a multivitamin to prevent osteoporosis. CONCLUSION: Few women are taking active measures to prevent osteoporosis despite their belief that it is a serious condition. Our data suggest that most women do not perceive a personal susceptibility to the disease. Only women who reported actively worrying about developing osteoporosis were more likely to be engaged in significant osteoporosis preventive behaviors.     

The effect of membrane fixed charges on diffusion potentials and streaming potentials

1. Electrical potential differences (p.d.''s) have been measured across an in vitro preparation of rabbit gall-bladder.2. When the gall-bladder separates identical bathing solutions, the p.d. is always zero, regardless of the composition of the bathing solution. Hence the gall-bladder is symmetrical: i.e. the mucosal and serosal cell membranes have the same relative permeability coefficients.3. Osmotic water flow causes streaming potentials of up to 20 mV, of a sign indicating greater permeability to cations than to anions.4. At constant osmolarity, streaming potentials increase slightly with NaCl concentration. Streaming potentials decrease considerably with changes in osmolarity resulting from changes in NaCl concentration.5. Diffusion potentials resulting from electrolyte concentration gradients are fitted well by the constant-field equation with the relative permeability coefficients P_Na = 1·00, P_Cl = 0·33, P_K = 2·3. These permeability coefficients are independent of osmolarity and of salt concentration.6. Relative to 0·25 mM-Ca, 5 mM-Ca reduces streaming potentials by 40%, NaCl diffusion potentials by 62%, and potassium diffusion potentials by 43%.7. The aqueous channels through which water and electrolytes traverse the cell membranes of the gall-bladder contain negative fixed charges, which are blocked by Ca. The physiological significance of the charges may be to reduce chloride permeability and thereby to increase the effectiveness of the gall-bladder in concentrating bile.8. The effect of pH, and analogy with surface charges of other cells, suggest that the charges are organic acids of low pK_a.     

Homeostasis of memory T cells

Summary: The pool of memory T cells is regulated by homeostatic mechanisms to persist for prolonged periods at a relatively steady overall size. Recent work has shown that two members of the common γ chain (γc) family of cytokines, interleukin‐7 (IL‐7) and IL‐15, govern homeostasis of memory T cells. These two cytokines work in conjunction to support memory T‐cell survival and intermittent background proliferation. Normal animals contain significant numbers of spontaneously arising memory‐phenotype (MP) cells, though whether these cells are representative of true antigen‐specific memory T cells is unclear. Nevertheless, it appears that the two types of memory cells do not display identical homeostatic requirements. For antigen‐specific memory CD8⁺ T cells, IL‐7 is primarily important for survival while IL‐15 is crucial for their background proliferation. For memory CD4⁺ T cells, IL‐7 has an important role, whereas the influence of IL‐15 is still unclear.     

Induction of CTL response by a minimal epitope vaccine in HLA A*0201/DR1 transgenic mice: dependence on HLA class II restricted T(H) response

CTL play a pivotal role in the immune response during viral infections. In this study, the HLA class II restricted T(H) requirement for optimal in vivo induction of HLA class I restricted CTL responses has been investigated. Towards this goal, transgenic mice expressing both HLA class I (A*0201 or A2.1) and class II (DRB1*0101 or DR1) molecules have been derived. Immunization of these mice with an HLA A*0201-restricted and CMV-specific CTL epitope (pp65(495-503)), and either of three different tetanus toxin-derived MHC class II-binding T(H) epitopes, resulted in a vigorous CTL response. CTL specific for the pp65(495-503) epitope were dramatically enhanced in mice expressing both the HLA-DR1 and HLA-A*0201 transgenes. Notably, preinjection of three TT peptides (TT(639-652), TT(830-843), and TT(947-967)) increased the capability of HLA A*0201/DR1 Tg mice to respond to subsequent immunization with the T(H) + CTL peptide mixture. These results indicate that the use of HLA A*0201/DR1 Tg mice constitute a versatile model system (in lieu of immunizing humans) for the study of both HLA class I and class II restricted T-cell responses. These studies provide a rational model for the design and assessment of new minimal-epitope vaccines based on their in vivo induction of a pathogen-specific CTL response.     

Characterization of anti-DNA B cells that escape negative selection

One of the challenges in the study of autoimmunity is to understand which autoreactive cells are subject to regulation and what mechanisms of regulation are operative. In mice transgenic for the R4A-gamma2b heavy chain of an anti-double stranded (ds) DNA antibody, the gamma2b heavy chain can pair with the full spectrum of endogenous light chains to produce a multitude of antibodies, including anti-dsDNA antibodies of different affinities and fine specificities. We have previously demonstrated the existence of two populations of anti-DNA B cells in non-autoimmune hosts: a high-affinity population which is rendered anergic in vivo, and a second high-affinity population which is deleted. We have now identified a third population of dsDNA-binding B cells. These cells produce germ-line-encoded antibodies with an apparent affinity for dsDNA that is 1 to 4 logs lower than the apparent affinities of antibodies made by anergic or deleted B cells, and represent a non-tolerized population which escapes regulation. Based on its characterization, we can define a molecular threshold for tolerance induction, and can speculate on the fate of these B cells when they are recruited to an immune response and undergo somatic mutation to become high-affinity anti-DNA B cells.     

Predominant type 1 CMV-specific memory T-helper response in humans: evidence for gender differences in cytokine secretion

Cell-mediated memory immune responses to viral antigens are important for protection against viruses causing persistent or acute infections. This study compared the cytokine profile of memory T-helper cells specific for cytomegalovirus (CMV) in healthy CMV-seropositive men and women. The cytokine response reflected T(H)1 bias, with dominant secretion of interferon (IFN)-gamma along with moderate levels of tumor necrosis factor-alpha, interleukin (IL)-10, and IL-2. Analyzed by gender, women had higher and significant spontaneous release of IFN-gamma and CMV-specific IL-2 secretion. Similar analysis with herpes simplex virus-1 showed a trend toward higher cytokine responsiveness in women, but the differences were not statistically significant. In contrast, men had statistically significant higher influenza virus-specific tumor necrosis factor-alpha secretion. IL-4 and IL-5, both T(H)2 cytokines, were low for all three viruses. The results show a predominant T(H)1 antiviral cytokine T-help memory response with significant differences linked to gender. Such differences may have an impact in the design of immunization strategies against CMV.