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S J Chanock

Publications and source records attributed to S J Chanock.

67 records · Page 4Linked to original sources
PubMed1995

PCR and single-strand conformational polymorphism for recognition of medically important opportunistic fungi.

The application of PCR technology to molecular diagnostics holds great promise for the early identification of medically important pathogens. PCR has been shown to be useful for the detection of the presence of fungal DNA in both laboratory and clinical samples. Considerable interest has been focused on the utility of selecting universal primers, those that recognize constant regions among most, if not all, medically important fungi. Once an amplicon, or piece of amplified DNA determined by the unique pair of oligonucleotide primers, has been generated, several different methods may be used to distinguish between genera and between species. The two major approaches have utilized differences in restriction enzyme digestion patterns or hybridization with specific probe. We report the application of single-strand conformational polymorphism (SSCP) as a technique to delineate the differences between fungal species and/or genera. Minor sequence variations in small single-stranded DNA cause subtle changes in conformation, allowing these strands to be separated on polyacrylamide gels by SSCP. We used a 197-bp fragment amplified from the 18S rRNA gene, common to all medically important fungi. After amplification, the fragments were denatured and run on an acrylamide-glycerol gel at room temperature or 4 degrees C for 4.5 or 4 h, respectively. Under room temperature conditions, the SSCP patterns for Candida albicans, Candida tropicalis, and Candida parapsilosis were identical and all strains within each species demonstrated the same pattern. These patterns differed markedly from those of the genus Aspergillus. The SSCP patterns of major and minor bands at room temperature permitted distinction between strains of Aspergillus fumigatus and Aspergillus flavus. There also was consistency of the SSCP banding pattern among different strains of the same Aspergillus species. The SSCP patterns for other medically important opportunistic fungi, such as Cryptococcus neoformans, Pseudallescheria boydii, and Rhizopus arrhizus, were sufficiently unique to permit distinction from those of C. albicans and A. fumigatus. We conclude that the technique of PCR-SSCP provides a novel method by which to recognize and distinguish medically important opportunistic fungi and which has potential applications to molecular diagnosis, taxonomic classification, molecular epidemiology, and elucidation of mechanisms of antifungal drug resistance.

Aspergillus↗
PubMed1995

The phosphorylation targets of p47phox, a subunit of the respiratory burst oxidase. Functions of the individual target serines as evaluated by site-directed mutagenesis.

The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to O2- at the expense of NADPH. Dormant in resting cells, the oxidase is activated by exposing the cells to appropriate stimuli. During activation, p47phox, a cytosolic oxidase subunit, becomes extensively phosphorylated on a number of serines located between S303 and S379. To determine whether this phosphorylation is necessary for oxidase activation, we examined phorbol-elicited oxidase activity in EBV-transformed B lymphoblasts deficient in p47phox after transfection with plasmids expressing various S-->A mutants of p47phox. The mutant containing S-->A mutations involving all serines between S303 and S379 [S(303-379)A] was not phosphorylated, did not translocate to plasma membrane during activation and was almost devoid of function. As to individual serines, S379 was of special interest because (a) p47 phox S379 was phosphorylated in phorbol-activated lymphoblasts expressing wild-type p47phox, and (b) p47phox S379A failed to translocate to the membrane, and was as functionless as p47phox S(303-379)A; other single S-->A mutations had little effect on oxidase activity. These findings suggest that the phosphorylation of S379 may be important for oxidase activation in whole cells.

Amino Acid Sequence↗
PubMed1994

Isolation of a complex of respiratory burst oxidase components from resting neutrophil cytosol.

The respiratory burst oxidase of neutrophils is a multicomponent enzyme, dormant in resting cells, that catalyzes the reduction of oxygen to O2- at the expense of NADPH. In the resting neutrophil, some of the components of the oxidase, including proteins p47phox and p67phox, are in the cytosol, while the rest are in a fraction that usually copurifies with plasma membrane. Recent evidence has suggested that at least some of the cytosolic oxidase components exist as a complex. We have now purified such a complex from the cytoplasm of resting neutrophils using an affinity column prepared with an antibody that recognizes the C-terminal decapeptide of p47phox. Immunoblotting showed that the complex contained both p47phox and p67phox. When supplemented with recombinant p67phox, the complex displayed considerable activity in a cell-free oxidase-activating system, and even without added p67phox, the complex could more than double O2- production in an oxidase-activating system supplemented with suboptimal amounts of cytosol. Isolation of the complex was blocked by preincubating the affinity column with CFSTKRKLASAV, the peptide against which the antibody was raised. On gel filtration, the complex migrated with a molecular weight of 240-300K, similar to that observed with whole neutrophil cytosol. The p47phox/p67phox ratio in the gel-filtered complex was approximately 1 to 1. These results indicate that in resting neutrophil cytosol, p47phox and p67phox exist as a complex.

Amino Acid Sequence↗
PubMed1993

Gene targeting of X chromosome-linked chronic granulomatous disease locus in a human myeloid leukemia cell line and rescue by expression of recombinant gp91phox.

The X chromosome-linked chronic granulomatous disease (X-CGD) locus, which encodes the gp91phox subunit of the phagocyte respiratory-burst oxidase cytochrome b, was disrupted by homologous recombination in the PLB-985 human myeloid cell line to develop an in vitro model of X-CGD. Superoxide formation was absent in targeted cells after differentiation to granulocytes but was rescued by stable transfection and expression of wild-type gp91phox cDNA. The targeted cell line should be useful in experiments aimed at defining functional regions within gp91phox by expression of mutant gp91phox cDNAs, complementing studies of naturally occurring mutations in X-CGD. In addition, the mutant line provides a model system in which to establish an experimental basis for the treatment of X-CGD patients with gene replacement therapy. Rescued clones containing even modest amounts of recombinant gp91phox had respiratory-burst activity comparable to the wild-type PLB-985 line, suggesting that functional correction of X-CGD neutrophils may not require high-level expression of gp91phox.

Blotting, Northern↗
PubMed1992

O2- production by B lymphocytes lacking the respiratory burst oxidase subunit p47phox after transfection with an expression vector containing a p47phox cDNA.

The respiratory burst oxidase of phagocytes and B lymphocytes is a complicated enzyme that catalyzes the one-electron reduction of oxygen by NADPH. It is responsible for the O2- production that occurs when these cells are exposed to phorbol 12-myristate 13-acetate or other appropriate stimuli. The activity of this enzyme is greatly decreased or absent in patients with chronic granulomatous disease, an inherited disorder characterized by a severe defect in host defense against bacteria and fungi. In every chronic granulomatous disease patient studied to date, an abnormality has been found in a gene encoding one of four components of the respiratory burst oxidase: the membrane protein p22phox or gp91phox, or the cytosolic protein p47phox or p67phox. We report here that O2- production was partly restored to phorbol 12-myristate 13-acetate-stimulated Epstein-Barr virus-transformed B lymphocytes from a patient with p47phox-deficient chronic granulomatous disease by transfection with an expression plasmid containing a p47phox cDNA inserted in the sense direction. No detectable O2- was produced by untransfected p47phox-deficient lymphocytes or by p47phox-deficient lymphocytes transfected with an antisense plasmid. The finding that O2- can be produced by p47phox-deficient B lymphocytes after the transfer of a p47phox cDNA into the deficient cells suggests that this system could be useful for studying the function of mutant p47phox proteins in whole cells.

Amino Acid Sequence↗
PubMed1989

Pediatric infection with the human immunodeficiency virus: issues for the otorhinolaryngologist.

Pediatric HIV infection is increasing at a rapid rate. It has many features that distinguish it from the adult forms of the disease. An understanding of the epidemiology and pathophysiology of HIV infection in children is necessary for the clinician to accurately diagnose and effectively treat infected children. Particular emphasis is placed on the infectious and neoplastic conditions of the head and neck region in infected children.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Genetic reassortment of mammalian reoviruses in mice.

Reassortments between type 1 (Lang) and type 3 (Dearing) reoviruses were isolated from suckling mice infected perorally with an inoculum containing both type 1 and type 3 viruses. A total of five distinct reassortants (designated as E1 through E5) were isolated from animals during the course of the experiment. Two reassortants (E1 and E2) represented the majority of the reassortants isolated. The majority of genes of types E1 and E2 were derived from type 1 (Lang). However, E1 had an M2 gene and an S1 gene derived from type 3 (Dearing), while E2 had M2 and S2 genes derived from type 3 (Dearing). Thus, nonrandom reassortment between mammalian reoviruses can be demonstrated in vivo.

Animals↗
PubMedDate not supplied

New evidence that Candida albicans possesses additional ATP-binding cassette MDR-like genes: implications for antifungal azole resistance.

Emergence of resistance of Candida albicans to antifungal triazoles is increasingly recognized as an important cause of refractory mucosal candidiasis in HIV-infected patients. Recently, CDR1, which is thought to be analogous to the human MDR-1 P-glycoprotein, has been cloned in C. albicans. It has been proposed that its expression is partially responsible for fluconazole resistance in C. albicans. This gene is characterized by the presence of an ATP binding cassette (ABC) region and is distinct from the BENr gene which does not encode such a functional domain. As the molecular basis for fluconazole resistance appears to be multifactorial, we considered that there may be other ATP binding cassette-containing MDR genes that may potentially contribute to antifungal azole resistance in C. albicans. We therefore sought to identify potential target sequences that may be derived from candidate genes that share homology with the ATP binding cassette region of the human MDR-1 P-glycoprotein. Degenerate oligonucleotide primers based on the known sequence from the ATP binding cassette region of the human MDR-1 P-glycoprotein were used to amplify PCR products within the range of 100 bp in length from C. albicans isolates (3 fluconazole-susceptible and 3 fluconazole-resistant). Sequence analysis of individually subcloned PCR products, derived from the six isolates revealed 34 sequences in total. The results of our study identified 14 clones (with at least one per isolate) with a high degree of homology to the ATP binding cassette of the human MDR-1 P-glycoprotein. The BLAST search did not disclose homology of these new sequences to the C. albicans CDR1 gene, suggesting that C. albicans may possess more than one MDR-like gene. We conclude that C. albicans may possess one or more additional genes encoding ATP binding cassette MDR-like proteins that are distinct from CDR 1 and which could participate in the development of fluconazole resistance.

AIDS-Related Opportunistic Infections↗