New microsatellite multiplex PCR for Candida albicans strain typing reveals microevolutionary changes

Five new microsatellite loci were described and characterized for use as molecular markers for the identification and genetic differentiation of Candida albicans strains. Following the typing of 72 unrelated clinical isolates, the analysis revealed that they were all polymorphic, presenting from 5 to 30 alleles and 8 to 46 different genotypes. The discriminatory power obtained by combining the information generated by three microsatellites used in a multiplex PCR amplification strategy was 0.99, the highest ever reported. The multiplex PCR was later used to test a total of 114 C. albicans strains, including multiple isolates from the same patient collected from different body locations and along episodes of vulvovaginal infections. Three different scenarios for strain relatedness were identified: (i) different isolates that were revealed to be the same strain, (ii) isolates that were the same strain but that apparently underwent a process of microevolution, and (iii) isolates that corresponded to different strains. Analysis of the microevolutionary changes between isolates from recurrent infections indicated that the genotype alterations observed could be the result of events that lead to the loss of heterozygosity (LOH). In one case of recurrent infection, LOH was observed at the CAI locus, and this could have been related to exposure to fluconazole, since such strains were exposed to this antifungal during treatment. The analysis of microsatellites by a multiplex PCR strategy was found to be a highly efficient tool for the rapid and accurate differentiation of C. albicans strains and adequate for the identification of fine microevolutionary events that could be related to strain microevolution in response to environmental stress conditions.     

Identification of N-acetylneuraminic acid and its 9-O-acetylated derivative on the cell surface of Cryptococcus neoformans: influence on fungal phagocytosis.

Sialic acids from sialoglycoconjugates present at the cell surface of Cryptococcus neoformans yeast forms were analyzed by high-performance thin-layer chromatography, binding of influenza A and C virus strains, enzymatic treatment, and flow cytofluorimetry with fluorescein isothiocyanate-labeled lectins. C. neoformans yeast forms grown in a chemically defined medium contain N-acetylneuraminic acid and its 9-O-acetylated derivative. A density of 3 x 10(6) residues of sialic acid per cell was found in C. neoformans. Sialic acids in cryptococcal cells are glycosidically linked to galactopyranosyl units as inferred from the increased reactivity of neuraminidase-treated yeasts with peanut agglutinin. N-Acetylneuraminic acids are alpha-2,6 and alpha-2,3 linked, as indicated by using virus strains M1/5 and M1/5 HS8, respectively, as agglutination probes. The alpha-2,6 linkage markedly predominated. These findings were essentially confirmed by the interaction of cryptococcal cells with the lectins Sambucus nigra agglutinin and Maackia amurensis agglutinin. We also investigated whether the sialyl residues present in C. neoformans are involved in the fungal interaction with a cationic solid-phase substrate and with mouse resident macrophages. Adhesion of yeast cells to poly-L-lysine was mediated, in part, by sialic acid residues, since the number of adherent cells was markedly reduced after treatment with bacterial neuraminidase. The enzymatic removal of sialic acids also made C. neoformans yeast cells more susceptible to endocytosis by macrophages. The results show that sialic acids are components of the cryptococcal cell surface that contribute to its negative charge and protect yeast forms against phagocytosis.     

Involvement of rat cytochrome 1A1 in the biotransformation of kaempferol to quercetin: relevance to the genotoxicity of kaempferol

Kaempferol is a flavonoid widely distributed in edible plantsand has been shown to be genotoxic to V79 cells in the absenceof external metabolizing systems. The presence of an externalmetabolizing system, such as rat liver homogenates (S9 mix),leads to an increase in its genotoxicity, which is attributedto its biotransformation to the more genotoxic flavonoid quercetin,via the cytochrome P450 (CYP) mono-oxygenase system. In thepresent work we investigated the mechanisms of the genotoxicityof kaempferol further. Special attention has been given to therole of CYP in the genotoxicity of this flavonoid. We studiedthe induction of mutations in Salmonella typhimurium TA98 inthe presence and in the absence of S9 mix and the inductionof chromosomal aberrations (CAs) and micronuclei (MN) by kaempferolin V79 cells in the presence and in the absence of S9 mix. Toevaluate the role of different CYP in the biotransformationof kaempferol we studied the induction of CAs and MN in V79cells genetically engineered for the expression of rat CYP 1A1,1A2 and 2B1. In addition we performed CYP inhibition studiesusing the above-mentioned indicators and high performance liquidchromatography (HPLC) analysis. The results obtained in thiswork suggest that rat CYP 1A1 is, among the cytochromes studied,the one that plays the major role in the transformation of kaempferolinto quercetin. The relevance of these findings to the humansituation is discussed. ⁴To whom correspondence should be addressed     

Analysis of CDKN1A polymorphisms: markers of cancer susceptibility?

The CDKN1A (TP21) gene encodes a 21-kD protein that is a critical downstream mediator of wild-type TP53 and an important regulator of the cell cycle. Failure in the function of this gene would be expected to result in abnormal cell proliferation and transformation. Tumor-associated mutations of the coding region of the TP21 are rare. On the other hand, some TP21 polymorphisms have been identified and characterized by single base substitutions. In the present study, we investigated the potential role of TP21 gene polymorphisms in skin, head, and neck tumorigenesis. A total of 261 samples were examined by polymerase chain reaction single-strand conformational analysis, and one mutation at codon 31 and four polymorphisms in exons 2 (codon 55) and 3 [nucleotide (nt)590] and in promoter region (nt2298) were identified. In conclusion, this investigation confirmed the rarity of mutations in this gene, arguing against a role for TP21 mutations in skin, head, and neck cancers. Also, our results show significant differences in nt2298 allele frequencies between normal individuals and skin malignant tumors (P < 0.05). The results suggest that this polymorphism affects TP21 transactivator binding and may be important during the pathogenesis of skin cancer.     

Segmental jejunal lipomatosis--a rare cause of intestinal obstruction

A rare case of a segmental small intestinal (jejunal) lipomatosis is described. A 33-year-old male was admitted with a clinical diagnosis of an acute intestinal obstruction. A plain erect abdominal x-ray showed multiple air-fluid levels. On an exploratory laparotomy, a jejunojejunal intussusception was found secondary to a segmental submucosal lipomatosis. This was treated by a segmental resection and anastomosis, which resulted in a complete cure. Here we present this case with a review of the relevant literature.     

Structural and functional analysis of a new desmin variant causing desmin‐related myopathy

Desmin‐related myopathy is a familial or sporadic disease characterized by skeletal muscle weakness and cardiomyopathy as well as the presence of intracytoplasmic aggregates of desmin‐reactive material in the muscle cells. Previously, two kinds of deletions and eight missense mutations have been identified in the desmin gene and proven to be responsible for the disorder. The present study was conducted to determine structural and functional defects in a pathogenic desmin variant that caused a disabling disorder in an isolated case presenting with distal and proximal limb muscle weakness and cardiomyopathy. We identified a novel heterozygous Q389P desmin mutation located at the C‐terminal part of the rod domain as the causative mutation in this case. Transfection of desmin cDNA containing the patient’s mutation into C2.7, MCF7, and SW13 cells demonstrated that the Q389P mutant is incapable of constructing a functional intermediate filament network and has a dominant negative effect on filament formation. We conclude that Q389P mutation is the molecular event leading to the development of desmin‐related myopathy. Hum Mutat 18:388–396, 2001. © 2001 Wiley‐Liss, Inc.     

Lipoperoxidation products and thiol antioxidants in chromium exposed workers

Hexavalent chromium is an established carcinogenic agent, which is not directly reactive with DNA. Its genotoxicity involves a reduction step, producing reactive oxygen species and radicals, and also lower valence forms which form stable complexes with intracellular macromolecules. The trivalent form of chromium may directly react with the genetic material and has also been shown to generate oxidative damage in vitro. To further evaluate the importance of in vivo oxidative DNA damage in the toxicity of each valence form, we conducted a comparative study on hexavalent and trivalent chromium-exposed workers (manual metal arc stainless steel welders and leather tanning workers), focusing on the total oxidative status by quantifying the level of lipoperoxidation products in urine. Thiol antioxidants are important in response to oxidative stress, and therefore, the concentration of glutathione and cysteine in peripheral blood lymphocytes was also determined. Chromium exposure was evaluated by quantifying total chromium in plasma and urine. Both groups had a significant increase in lipid peroxidation products expressed as malondialdehyde (MDA) in urine (tanners 1.42 +/- 0.61 micromol/g creatinine, welders 1.67 +/- 1.13 micromol/g creatinine versus controls 0.81 +/- 0.26 micromol/g creatinine, P < 0.005 in both cases) but only welders had a significant decrease in glutathione concentration in lymphocytes. There was a positive correlation between chromium in plasma and urinary MDA in welders, but not in tanners. This work is part of a larger study of which major results have been published previously including cytogenetics and DNA-protein cross-links in workers exposed to the two different forms of chromium. These results are compared with the results of oxidative damage from this study.     

Pulmonary and mediastinal "sarcoidosis" following surgical resection of cancer

Previous reports indicate that enlarged hilar and mediastinal lymph nodes caused by sarcoid-like reactions may develop after curative resection of cancer, and their presence does not necessarily denote neoplastic recurrence. Reports further suggest that coexisting pulmonary infiltrates in this setting may be related to sarcoidosis. In this study, we describe two patients who had resected lung and gastric cancer and who later developed pulmonary interstitial infiltrate, concurrent with progressive mediastinal lymphadenopathy initially thought to be caused by intrathoracic dissemination of their cancer. These changes were shown by open lung biopsy to be a benign, granulomatous reaction interpreted as sarcoidosis. Thus, it is important to recognize this clinical pattern when pulmonary infiltrates develop after complete treatment of cancer in an otherwise relapse-free patient and to encourage lung or lymph node biopsy in these particular settings in order to confirm a sarcoid-like reaction, thereby avoiding unnecessary chemotherapy for presumed tumor recurrence.     

Induction of an adaptive response to quercetin, mitomycin C and hydrogen peroxide by low doses of quercetin in V79 Chinese hamster cells

The adaptive response is a phenomenon by which cells exposedto low, non-cytotoxic doses of a genotoxicant become significantlyresistant to a subsequent higher dose of the same or anothergenotoxic agent. Induction of the adaptive response has beenmainly studied using ionizing radiation and alkylating agentsas genotoxic agents. However, other mutagenic agents may warrantfurther study, since the adaptive response as a whole may bean important general biological mechanism to maintain geneticintegrity and thus could prevent carcinogenic initiation ofcells. The exposure to mutagenic agents present, or formed,in the diet is considered an important factor in the etiologyof human tumors and a considerable number of these agents havenot yet been identified or characterized. Flavonoids are a largegroup of polyphenolic quinoids found in a wide variety of ediblefruits and vegetables and a few, such as quercetin, presentgenotoxic activity in vitro. The mechanisms of mutagenicityof quercetin involve the production of oxygen radicals throughan autoxidation process dependent on pH value and the presenceof oxygen. Although there are few doubts regarding the mutagenicityof quercetin invitro, carcinogenicity of this flavonoid is stillcontroversial. In view of these conflicting results and theradiomimetic nature of the mutagenicity of flavonoids, we addressedthe question of cell exposure to quercetin at the low levelspresent in the diet leading to adaptation to further exposureto mutagens or carcinogens. The work reported here concernsinduction of an adaptive response by low doses of quercetinto challenging doses of quercetin and other compounds, namelyhydrogen peroxide and mitomycin C, using induction of chromosomalaberrations in V79 cells as the end point. ⁴To whom correspondence should be addressed: Tel: +351 1 3610290; Fax: +351 1 3622018; Email: jose.rueff{at}gene.unl.mailpac.pt