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 amphotericin B with fluconazole in the treatment of acute AIDS-associated cryptococcal meningitis. The NIAID Mycoses Study Group and the AIDS Clinical Trials Group.

BACKGROUND. Intravenous amphotericin B, with or without flucytosine, is usually standard therapy for cryptococcal meningitis in patients with the acquired immunodeficiency syndrome (AIDS). Fluconazole, an oral triazole agent, represents a promising new approach to the treatment of cryptococcal disease. METHODS. In a randomized multicenter trial, we compared intravenous amphotericin B with oral fluconazole as primary therapy for AIDS-associated acute cryptococcal meningitis. Eligible patients, in all of whom the diagnosis had been confirmed by culture, were randomly assigned in a 2:1 ratio to receive either fluconazole (200 mg per day) or amphotericin B. Treatment was considered successful if the patient had had two consecutive negative cerebrospinal fluid cultures by the end of the 10-week treatment period. RESULTS. Of the 194 eligible patients, 131 received fluconazole and 63 received amphotericin B (mean daily dose, 0.4 mg per kilogram of body weight in patients with successful treatment and 0.5 mg per kilogram in patients with treatment failure; P = 0.34). Treatment was successful in 25 of the 63 amphotericin B recipients (40 percent; 95 percent confidence interval, 26 percent to 53 percent) and in 44 of the 131 fluconazole recipients (34 percent; 95 percent confidence interval, 25 percent to 42 percent) (P = 0.40). There was no significant difference between the groups in overall mortality due to cryptococcosis (amphotericin vs. fluconazole, 9 of 63 [14 percent] vs. 24 of 131 [18 percent]; P = 0.48); however, mortality during the first two weeks of therapy was higher in the fluconazole group (15 percent vs. 8 percent; P = 0.25). The median length of time to the first negative cerebrospinal fluid culture was 42 days (95 percent confidence interval, 28 to 71) in the amphotericin B group and 64 days (95 percent confidence interval, 53 to 67) in the fluconazole group (P = 0.25). Multivariate analyses identified abnormal mental status (lethargy, somnolence, or obtundation) as the most important predictive factor of a high risk of death during therapy (P less than 0.0001). CONCLUSIONS. Fluconazole is an effective alternative to amphotericin B as primary treatment of cryptococcal meningitis in patients with AIDS. Single-drug therapy with either drug is most effective in patients who are at low risk for treatment failure. The optimal therapy for patients at high risk remains to be determined.     

Positional cloning of the gene for X-linked retinitis pigmentosa 3: homology with the guanine-nucleotide-exchange factor RCC1

The gene for retinitis pigmentosa 3 (RP3), the most frequent form of X- linked RP (XLRP), has been mapped previously to a chromosome interval of less than 1000 kbp between the DXS1110 marker and the OTC locus at Xp21.1-p11.4. Employing a novel technique, YAC Representation Hybridization (YRH)', we have recently identified a small XLRP associated microdeletion in this interval, as well as several putative exons including the 3' end of a gene that was truncated by the deletion. cDNA library screening and sequencing of a cosmid centromeric to the deletion has now enabled us to identify numerous additional exons and to detect several point mutations in patients with XLRP. The predicted gene product shows homology to RCC1, the guanine-nucleotide- exchange factor (GEF) of the Ras-like GTPase Ran. Our findings suggest that we have cloned the long-sought RP3 gene, and that it may encode the GEF of a retina-specific GTP-binding protein.      

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Enhanced oxidative burst in immunologically activated but not elicited polymorphonuclear leukocytes correlates with fungicidal activity.

Polymorphonuclear neutrophils (PMN) induced locally in immune mice by intraperitoneal injection of antigen exhibit enhanced fungicidal activity compared with PMN elicited with thioglycolate. The mechanism of the differences in these PMN populations was studied. Sublethal infection was used to produce immunity to Blastomyces dermatitidis. A correlation was sought between the ability of PMN to kill, or not kill, B. dermatitidis and the production of the oxidative burst, as measured by luminol-enhanced chemiluminescence (CL). Although elicited PMN cocultured with Candida albicans produced a burst of CL and were candidacidal, killing did not occur when PMN were cocultured with B. dermatitidis. Lack of killing of B. dermatitidis by elicited PMN correlated with lack of stimulation of a brisk oxidative burst. In contrast to elicited PMN, PMN induced by B. dermatitidis antigen responded to this fungus with a burst of CL and a significant reduction of inoculum CFU (80%). Furthermore, these PMN when cocultured with C. albicans produced an enhanced burst of CL, and killing was enhanced compared with that by elicited PMN, e.g., 86 versus 58%. The CL burst and killing of B. dermatitidis by antigen-induced PMN was abrogated in the presence of catalase, implying a critical role for hydrogen peroxide. Partial but significant depression of CL and killing in the presence of dimethyl sulfoxide, a hydroxyl radical scavenger, identified hydroxyl radical, or its metabolites, as a toxic product(s) responsible for a significant fraction of fungicidal activity. These results indicate that the metabolic activity and microbicidal activity of PMN can be altered (enhanced) at the site of an immunological reaction and thus could constitute an important factor in resistance.     

A locus for posterior polymorphous corneal dystrophy (PPCD3) maps to chromosome 10

Posterior polymorphous corneal dystrophy (PPCD) is an autosomal dominant disorder characterized by corneal endothelial abnormalities, which can lead to blindness due to loss of corneal transparency and sometimes glaucoma. We mapped a new locus responsible for PPCD in a family in which we excluded the previously reported PPCD locus on 20q11, and the region containing COL8A2 on chromosome 1. Results of a 317-marker genome scan provided significant evidence of linkage of PPCD to markers on chromosome 10, with single-point LOD scores of 2.63, 1.63, and 3.19 for markers D10S208 (at (circumflex)theta = 0.03), D10S1780 (at (circumflex)theta = 0.00), and D10S578 (at (circumflex)theta = 0.06). A maximum multi-point LOD score of 4.35 was found at marker D10S1780. Affected family members shared a haplotype in an 8.55 cM critical interval that was bounded by markers D10S213 and D10S578. Our finding of another PPCD locus, PPCD3, on chromosome 10 indicates that PPCD is genetically heterogeneous. Guttae, a common corneal finding sometimes observed along with PPCD, were found among both affected and unaffected members of the proband's sib ship, but were absent in the younger generations of the family. Evaluation of phenotypic differences between family members sharing the same affected haplotype raises questions about whether differences in disease severity, including differences in response to surgical interventions, could be due to genetic background or other factors independent of the PPCD3 locus.     

Identification of a common immunodominant protein in culture filtrates of three Nocardia species and use in etiologic diagnosis of mycetoma.

Diagnosis of systemic nocardiosis has been hampered by difficulty in the rapid isolation and identification of the organism and by the frequent need for invasive biopsies. We show that a 55,000-Mr protein previously found in Nocardia asteroides is also present in N. brasiliensis and N. caviae culture filtrates. We also used the antigen in a dot blot assay to identify patients infected with N. brasiliensis. Sera from patients with mycetoma secondary to N. brasiliensis (n = 6) or Actinomadura madurae (n = 2) and 10 hospitalized controls were tested. Six (100%) of the sera from the patients with nocardial mycetoma had positive dot blots to purified antigen, whereas all 10 of the control sera were negative. Serum from one of two patients infected with A. madurae also reacted with the antigen. Thus, the 55-kilodalton protein appears to be produced by three medically important species of Nocardia and is useful when used in an immunoblot assay for identifying patients infected with these organisms.     

Charles Lasègue and his 'Considerations on Sciatica'

The eponym Lasègue sign has been applied to the increase in sciatic pain caused by flexing the extended lower extremity on the abdomen. The sign was never put into writing by Lasègue but by his pupils. He did not describe the test in the usual reference, "Considerations on Sciatica," in 1864. That article has to do with his analysis of then-current theories of sciatica and his own clinical observations. Sciatica was divided into a benign and a serious form, and two examples of each were described. Emphasis was laid on the constant, fixed sciatic pain, as contrasted with the irregular, largely nocturnal, episodes of lancinating pain. Atrophy of leg muscles was not to be explained on the basis of disuse but by a disorder of the nerve, which also was responsible for the typical neuralgia, unlike that of any other part of the body except possibly neuralgia of the brachial plexus. Treatments currently available (cupping, vesicants, and injections of atropine solution) were unavailing. The steps are unknown by which Lasègue came to modify his 1864 views that any sort of flexion or extension of the lower extremity did not exacerbate the pain; in the 1881 thesis of his pupil, Forst, that straight-leg raising sign is described and illustrated and ascribed to his teacher, Professor Lasègue.     

A novel mis-sense mutation (G1381A) in the G6PD gene identified in a Chinese man

Objective　To detect new mutations among 29 glucose-6-phosphate dehydrogenase (G6PD) deficient individuals from Yunnan province. Methods　The nitroblue tetrazolium (NBT) method was used to screen G6PD deficient individuals. Mutation was identified by single strand conformation polymorphism (SSCP), amplification created restriction site (ACRS), amplification refractory mutation system (ARMS) and DNA sequencing. Results　Among 29 cases, 18 cases of G1388A, 1 case of C1004A, and 1 case of G1381A were identified. Nine cases remained to be defined. The G1381A mutation is a novel mis-sense mutation, with a substitution of threonine for alanine (A461T). The resultant G6PD had reduced enzymatic activity. In addition, G1381A caused a restriction site of Stu I to disappear, providing a rapid method for the detection of this mutation. Conclusion　A novel mis-sense mutation G1381A was found. This mutation results in a substitution of threonine for alanine, producing enzyme with reduced activity. The loss of the Stu I restriction site offers a rapid method for the detection of this mutation.