DNA-protein cross-linking via guanine oxidation: dependence upon protein and photosensitizer

DNA-protein cross-links form when guanine undergoes a 1-electron oxidation in a flash-quench experiment, and the importance of reactive oxygen species, protein, and photosensitizer is examined here. In these experiments, a strong oxidant produced by oxidative quenching of a DNA-bound photosensitizer generates an oxidized guanine base that reacts with protein to form the covalent adduct. These cross-links are cleaved by hot piperidine and are not the result of reactive oxygen species, since neither a hydroxyl radical scavenger (mannitol) nor oxygen affects the yield of DNA-histone cross-linking, as determined via a chloroform extraction assay. The cross-linking yield depends on protein, decreasing as histone > cytochrome c > bovine serum albumin. The yield does not depend on the cytochrome oxidation state, suggesting that reduction of the guanine radical by ferrocytochrome c does not compete effectively with cross-linking. The photosensitizer strongly influences the cross-linking yield, which decreases in the order Ru(phen)(2)dppz(2+) [phen = 1,10-phenanthroline; dppz = dipyridophenazine] > Ru(bpy)(3)(2+) [bpy = 2,2'-bipyridine] > acridine orange > ethidium, in accordance with measured oxidation potentials. A long-lived transient absorption signal for ethidium dication in poly(dG-dC) confirms that guanine oxidation is inefficient for this photosensitizer. From a polyacrylamide sequencing gel of a (32)P-labeled 40-mer, all of these photosensitizers are shown to damage guanines preferentially at the 5' G of 5'-GG-3' steps, consistent with a 1-electron oxidation. Additional examination of ethidium shows that it can generate cross-links between histone and plasmid DNA (pUC19) and that the yield depends on the quencher. Altogether, these results illustrate the versatility of the flash-quench technique as a way to generate physiologically relevant DNA-protein adducts via the oxidation of guanine and expand the scope of such cross-linking reactions to include proteins that may associate only transiently with DNA.     

Helicobacter pylori in familial clusters based on antibody profile

Studies have shown a high prevalence of Helicobacter pylori infection in close communities and that intrafamilial spread during early childhood may be a route of transmission. A total of 72 household members from 21 families were enrolled in this study. Sera from individuals showed 50/72 (69.4%) seropositive for IgG against H. pylori by ELISA. Western blots showed diversity in the protein profiles with molecular masses ranging from approximately 8 to 130 kDa. Cohen's kappa statistical analysis of the blot patterns showed that nine families demonstrated similar profiles (100%), while 4 other families showed varying similarities (17-50%). The results support the hypothesis of intrafamilial transmission of H. pylori. Furthermore, serological studies can be used as an effective approach to determine the familial status in relation to H. pylori infection.     

Description of Pintomyia limafalcaoae and Pintomyia antioquiensis,two new species of phlebotomine sand fly (Diptera,Psychodidae) from the Colombian Andes

Two new species of phlebotomine sand fly from Colombian Andes are described, belonging to the subgenus Pifanomyia of the genus Pintomyia. P. (P.) limafalcaoae sp. nov. for which both sexes are described, is assigned to the series pia while P. (P.) antioquiensis sp. nov., known only from the male, is included in the series verrucarum. The subgenus Pifanomyia is characterized and identification keys presented for the two new species.     

Plasmodium falciparum: in vitro activity of sulfadoxine and dapsone in field isolates from Kenya: point mutations in dihydropteroate synthase may not be the only determinants in sulfa resistance

We have determined the relationship between point mutations in the gene that encodes the sulfa target, dihydropteroate synthase (DHPS) and the chemosensitivity profile to sulfadoxine and dapsone in 67 isolates from Kilifi, Kenya. We assessed the presence of mutations at codons 436, 437, 540, 581, and 613 of dhps. The results showed that the dhps genotype had a strong influence on the sensitivity to sulfadoxine and dapsone, but that the correlation was far from perfect. Eleven isolates carried a wild-type dhps allele, but were resistant to sulfadoxine (IC(50) values >10 microg/ml), and 4/28 isolates were classed as sensitive to sulfadoxine (IC(50) values <10 microg/ml), but carried a triple mutant (436/437/613) allele of dhps. These data show that in low folate medium in vitro, the dhps genotype alone did not account completely for sulfadoxine or dapsone resistance; other factors such as the utilisation of exogenous folate must also be considered.     

Characterisation of a monoclonal antibody to carp IL-1beta and the development of a sensitive capture ELISA

A carp IL-1beta gene was identified from a subtraction hybridisation technology based cDNA library from activated carp leucocytes. This gene was cloned into pQE vector carrying 6xHis tag and the protein was expressed. Recombinant IL-1beta was used to produce hybridomas specific for carp IL-1beta. Monoclonal antibodies were purified by affinity column and a sandwich ELISA for IL-1beta was developed with a detection limit of 10 ng of the recombinant protein. Using the capture ELISA, the presence of native IL-1beta in culture supernatant of PHA-stimulated leucocytes from carp was identified, which was confirmed by SDS-PAGE and Western blot. Since IL-1beta is known to stimulate proliferation of T & B cells and macrophages, its ability to stimulate proliferation of carp leucocytes was studied using tritiated thymidine. The recombinant protein was found to significantly stimulate proliferation of head kidney and spleen cells from carp.     

Structural studies of the interaction between ubiquitin family proteins and proteasome subunit S5a

The 26S proteasome is essential for the proteolysis of proteins that have been covalently modified by the attachment of polyubiquitinated chains. Although the 20S core particle performs the degradation, the 19S regulatory cap complex is responsible for recognition of polyubiquitinated substrates. We have focused on how the S5a component of the 19S complex interacts with different ubiquitin-like (ubl) modules, to advance our understanding of how polyubiquitinated proteins are targeted to the proteasome. To achieve this, we have determined the solution structure of the ubl domain of hPLIC-2 and obtained a structural model of hHR23a by using NMR spectroscopy and homology modeling. We have also compared the S5a binding properties of ubiquitin, SUMO-1, and the ubl domains of hPLIC-2 and hHR23a and have identified the residues on their respective S5a contact surfaces. We provide evidence that the S5a-binding surface on the ubl domain of hPLIC-2 is required for its interaction with the proteasome. This study provides structural insights into protein recognition by the proteasome, and illustrates how the protein surface of a commonly utilized fold has highly evolved for various biological roles.     

DNA fingerprinting of human isolates of Salmonella enterica serotype Paratyphi B in Malaysia

AIMS: DNA fingerprinting of Salmonella enterica serotype Paratyphi B isolated in Malaysia during 1982-83, 1992 and 1996-2002 was carried out by pulsed-field gel electrophoresis (PFGE), antimicrobial susceptibility tests and D-tartrate utilization tests to assess the extent of genetic diversity of these isolates in Malaysia. METHODS AND RESULTS: Eighty-six human isolates and one food isolate of Salm. Paratyphi B were analysed by PFGE, antimicrobial susceptibility tests and D-tartrate utilization tests. Sixty-five strains were D-tartrate-negative (dT-) while 22 strains were D-tartrate-positive (dT+). Thirty-seven per cent of the Salm. Paratyphi B strains were resistant to one or more antimicrobial agents. PFGE analysis clearly distinguished the dT- and dT+ strains into two clusters based on the unweighted pair group average method (UPGMA). Twenty-two XbaI-pulsotypes were observed among the 65 dT- strains while 17 XbaI-pulsotypes were observed among the 22 isolates of Salm. Paratyphi B dT+. CONCLUSIONS: The present study showed that PFGE was very discriminative with 33.7% of the strains yielding distinct fingerprints. Paratyphoid fever in Malaysia is probably caused by one predominant, endemic clone of Salm. Paratyphi B dT- with various subtypes. There was no association between the pulsotypes and the severity of the disease indicating that the severity of the disease is probably multifactorial. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the present study verify the usefulness of PFGE in characterizing strains of Salm. Paratyphi B. This is the first report on the application of PFGE on a large collection of Salm. Paratyphi B in Malaysia.     

Redescription of Nyssomyia intermedia (Lutz & Neiva, 1912) and Nyssomyia neivai (Pinto, 1926) (Diptera: Psychodidae)

The phlebotomine sand flies Nyssomyia intermedia and Nyssomyia neivai are the probable vectors of American tegumentary leishmaniasis in the Southern and Southeastern regions of Brazil. These species form a complex, being difficult to separate between either females or males of the two members based on recognized morphological characteristics. Both N. intermedia and N. neivai are redescribed here in the search for characters that facilitate their correct identification. It was possible to differentiate females by means of spermathecal characteristics. Males could be separated with confidence by the tips of the genital filaments, which have the form of a deep spoon, the angle of the concavity being well accentuated in N. intermedia and much shallower in N. neivai.     

Stress memory in plants: a negative regulation of stomatal response and transient induction of rd22 gene to light in abscisic acid-entrained Arabidopsis plants

All organisms, including plants, perceive environmental stress, and they use this information to modify their behavior or development. Here, we demonstrate that Arabidopsis plants have memory functions related to repeated exposure to stressful concentrations of the phytohormone abscisic acid (ABA), which acts as a chemical signal. Repeated exposure of plants to ABA (40 micro m for 2 h) impaired light-induced stomatal opening or inhibited the response to a light stimulus after ABA-entrainment under both dark/light cycle and continuous light. Moreover, there were transient expressions of the rd22 gene during the same periods under both the growing conditions. Such acquired information in ABA-entrained plants produced a long-term sensitization. When the time of light application was changed, a transient induction of the rd22 gene in plants after ABA-entrainment indicated that these were light-associated responses. These transient effects were also observed in kin1, rab18, and rd29B. The transient expression of AtNCED3, causing the accumulation of endogenous ABA, indicated a possible regulation by ABA-dependent pathways in ABA-entrained plants. An ABA immunoassay supported this hypothesis: ABA-entrained plants showed a transient increase in endogenous ABA level from 220 to 250 pmol g-1 fresh mass at 1-2 h of the training period, whereas ABA-deficient (aba2) mutants did not. Taking into account these results, we propose that plants have the ability to memorize stressful environmental experiences, and discuss the molecular events in ABA-entrained plants.