Expression and activity of polymorphisms in the 67-kDa protein of the NADPH oxidase system

The NADPH oxidase system plays a central role in the antimicrobial activity of phagocytes. This system is initiated by the translocation of cytosolic proteins p67phox, p47phox and p40phox to be in close contact with membrane flavocytochrome b558. This event begins the electron transfer from cytosolic NADPH to molecular oxygen to produce superoxide anions. Herein, a functional analysis is presented of p67phox polymorphisms identified from healthy humans. Mutations were generated in the p67phox cDNA by site-directed mutagenesis and then transiently expressed in COS7 cells that also expressed gp91phox, p22phox, and p47phox from stable transgenes. The changes Va1166lle, Pro329Ser and His389Gln correspond to possible polymorphisms identified in healthy individuals revealed a functional activity similar to COSphox cells transiently transfected with WT p67phox; therefore, these modifications are not associated with genetic deficiencies in NADPH oxidase. In conclusion, the COSphox system represents an easily transfectable model for analysis of NADPH oxidase function in intact cells. The analysis of mutant derivatives of p67phox provides insight into molecular mechanisms by which this subunit regulates the NADPH oxidase.     

Characterization of the c-Myb-responsive region and regulation of the human type I collagen alpha 2 chain gene by c-Myb

We have characterized the role of c-Myb and B-Myb in the regulation of human type I collagen alpha2 chain gene expression in fibroblastic cells. We have identified four Myb-binding sites (MBSs) in the promoter. Transactivation assays on wild type and mutant promoter-reporter constructs demonstrated that c-Myb, but not B-Myb, can transactivate the human type I collagen alpha 2 chain gene promoter via the MBS-containing region. Electrophoretic mobility shift assay experiments showed that c-Myb specifically binds to each of the four MBS; however, the mutagenesis of site MBS-4 completely inhibited transactivation by c-Myb, at least in the full-length promoter. In agreement with these results, c-myb(-/-) mouse embryo fibroblasts (MEFs) showed a selective lack of expression of type I collagen alpha 2 chain gene but maintained the expression of fibronectin and type III collagen. Furthermore, transforming growth factor-beta induced type I collagen alpha 2 chain gene expression in c-myb(-/-) MEFs, implying that the transforming growth factor-beta signaling pathway is maintained and that the absence of COL1A2 gene expression in c-myb(-/-) MEFs is a direct consequence of the lack of c-Myb. The demonstration of the importance of c-Myb in the regulation of the type I collagen alpha 2 chain gene suggests that uncontrolled expression of c-Myb could be an underlying mechanism in the pathogenesis of several fibrotic disorders.     

Observations on the life history and descriptions of coccidia (Apicomplexa) from the western chorus frog,Pseudacris triseriata triseriata,from eastern Nebraska

Two hundred and twenty-four anurans of 6 species (47 adults and 16 tadpoles of Rana blairi, 35 R. catesbeiana, 31 Hyla chrysoscelis, 30 adults and 46 tadpoles of Pseudacris triseriata triseriata, 11 Bufo woodhousii, and 8 Acris crepitans) from Pawnee Lake, Lancaster County, Nebraska, were surveyed for coccidian parasites during March 2001 to May 2002. Of these, 23 of 30 (77%) adults and 4 of 46 (9%) tadpoles of P. t. triseriata shed oocysts of Isospora cogginsi n. sp. Oocysts of I. cogginsi were ovoid, 19.3 x 15.1 (18-23 x 11-20) microm, with a thin, smooth, colorless, single-layered wall, with no micropyle or oocyst residuum. Sporocysts were ovoid, 13.3 x 9.9 (11-15 x 9-13) microm, with a thin, colorless, smooth wall, and Stieda body absent. Sporocyst residuum was present, 5.5 x 5.3 (4-7 x 4-7) microm, consisting of numerous granules. Histological examination of frogs and tadpoles infected with the new species revealed endogenous stages including mature meronts, developing microgamonts, mature microgametes, mature macrogamonts, and young unsporulated oocysts located in the cytoplasm of the epithelial cells of the small intestine. Concurrently, 2 adult P. t. triseriata shed oocysts of Eimeria streckeri. Oocysts of E. streckeri were spherical, 15.7 x 15.4 (14-17 x 14-19) microm, with a thin, smooth, single-layered, colorless wall with an oocyst residuum composed of numerous granules surrounding a large vacuolated area, with a previously undescribed globularlike body present within the vacuole, and no micropyle. Sporocysts were ovoid, 9.1 x 6.1 (7-10 x 5-7) microm, with a thin, colorless, smooth wall with a Stieda body and sporocyst residuum. Our results are the first to document infection of adult and tadpole stages of frogs of the same species with the same species of coccidian, indicating that adult frogs may contaminate breeding ponds with oocysts during their breeding season and infect tadpoles directly by the ingestion of sporulated oocysts.     

A parallel optimization approach for controlling allele diversity in conservation schemes

We propose a novel method to control allelic diversity in conservation schemes based on an optimization problem, characterized by a convex program subject to integer linear constraints. Departing from previous studies considering similar problems, we implement a parallel simulated annealing algorithm to minimize the number of alleles lost across generations. The proposed algorithm shows excellent timing and minimization performances. Execution time decreases linearly with the number of processors used, providing similar results in all cases.     

Cinnamic acid amides from Chenopodium album: effects on seeds germination and plant growth

Seven cinnamic acid amides have been isolated from Chenopodium album. The structures have been attributed by means of their spectral data. One of them, N-trans-4-O-methylferuloyl 4′-O-methyldopamine, is described for the first time. Their effects on germination and growth of dicotyledons Lactuca sativa L. (lettuce) and Lycopersicon esculentum L. (tomato) and of monocotyledon Allium cepa L. (onion) as standard target species have been studied in the range concentration 10⁻⁴-10⁻⁷ M.     

Feasibility of cytology-based cervical cancer screening in rural Cameroon

OBJECTIVE: To determine the prevalence of cervical cancer and its precursors in a rural population in Cameroon and to evaluate the feasibility of a cytology-based screening program in such areas. STUDY DESIGN: A prospective study was conducted in the rural town of Bafang. Following an advocacy campaign, 750 women were recruited. After a clinical examination, all women had a Pap smear with the Cervex Brush. Each sample had two preparations, conventional and liquid based. The conventional smears were interpreted in Bafang. Cytologically abnormal cases, those with clinical inflammation and/or macroscopic cervical lesions, had a colposcopic examination and directed biopsy. HSIL and colposcopically abnormal cases were treated with large loop excision of the transformation zone (LLETZ). The liquid-based preparations and histopathology were performed in Geneva and the results sent to Cameroon for patient follow-up. RESULTS: Mean age and parity of the women screened were 43.7 years and 7.8, respectively. The conventional smears showed 3.6% cervical abnormalities: 2% (15/740) ASCUS/LSIL and 1.6% (12/740) HSIL. The liquid-based preparations showed 12.6% (91/722) cervical abnormalities: 10.1% (73/722) ASCUS/LSIL and 2.5% (18/722) HSIL. Fifty percent of samples in both preparations showed evidence of inflammation. Histology was performed on 64 colposcopically directed punch biopsies and LLETZ specimens. The histologic diagnoses agreed with the cytologic findings in 60% (14/23) of conventional smears and 85% (12/14) of liquid-based preparations. CONCLUSION: There is a high rate of cervical intraepithelial neoplasia in the unscreened rural population of Cameroon. The situation is complicated by a high rate of cervical infection. A population-based cytologic screening program for cervical cancer would not be feasible in rural Cameroon because of high cost, low quality and limited technical facilities. Rural Africa requires an algorithm using a simple, low-cost technique of mass screening and an improved cytology service only to triage selected patients.     

TrwD, the hexameric traffic ATPase encoded by plasmid R388, induces membrane destabilization and hemifusion of lipid vesicles

TrwD, a hexameric ATP hydrolase encoded by plasmid R388, is a member of the PulE/VirB11 protein superfamily of traffic ATPases. It is essential for plasmid conjugation, particularly for expression of the conjugative W pilus. In the present study, we analyzed the effects that TrwD produced on unilamellar vesicles consisting of cardiolipin and phosphatidylcholine in equimolar amounts. TrwD induced dose-dependent vesicle aggregation and intervesicular mixing of the lipids located in the outer monolayers in the presence of calcium. It also induced extensive leakage of the vesicular aqueous contents. A point mutant of TrwD with a mutation in the P loop of the nucleotide-binding region (K203Q) that lacks both ATPase activity and the ability to support conjugation showed the same behavior as native TrwD in all of these processes, which were independent of the presence of ATP. Structure prediction methods revealed a close similarity to Helicobacter pylori protein HP0525, another member of the PulE/VirB11 family, whose crystal structure is known. The interpretation of our data in the light of this structure is that TrwD interacts with the lipid bilayer through hydrophobic regions in its N-terminal domain, which leads to a certain degree of membrane destabilization. TrwD appears to be a part of the conjugation machinery that interacts with the membranous systems in order to facilitate DNA transfer in bacteria.     

Susceptibility to proteolysis of triosephosphate isomerase from two pathogenic parasites: characterization of an enzyme with an intact and a nicked monomer

The susceptibility to subtilisin of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM) was studied. Their amino sequence and 3D structure are markedly similar. In 36 h of incubation at a molar ratio of 4 TIM per subtilisin, TcTIM underwent extensive hydrolysis, loss of activity, and large structural alterations. Under the same conditions, only about 50% of the monomers of TbTIM were cleaved in two sites. The higher sensitivity of TcTIM to subtilisin is probably due to a higher intrinsic flexibility. We isolated and characterized TbTIM that had been exposed to subtilisin. It exhibited the molecular mass of the dimer, albeit it was formed by one intact and one nicked monomer. Its k(cat) with glyceraldehyde 3-phosphate was half that of native TbTIM, with no change in K(m). The intrinsic fluorescence of nicked TbTIM was red-shifted by 5 nm. The association between subunits was not affected. The TbTIM data suggest that there are structural differences in the two monomers or that alterations of one subunit change the characteristics of the other subunit. In comparison to the action of subtilisin on TIMs from other species, the trypanosomal enzymes appear to be unique.