The spacer region of XPG mediates recruitment to nucleotide excision repair complexes and determines substrate specificity

XPG has structural and catalytic roles in nucleotide excision repair (NER) and belongs to the FEN-1 family of structure-specific nucleases. XPG contains a stretch of over 600 amino acids termed the "spacer region" between the conserved N- and I-nuclease regions. Its role is unknown, and it is not similar to any known protein. To investigate its possible functions, we generated and analyzed several deletion mutants of XPG. The spacer region is not required for endonuclease activity, but amino acids 111-550 contribute to the substrate specificity of XPG, and they are required for interaction with TFIIH and for NER activity in vitro and in vivo. Deletion of residues 184-210 and 554-730 leads only to a partial defect in NER activity and a weakened interaction with TFIIH. XPGDelta184-210 and XPGDelta554-730 are not observed at sites of local UV damage in living cells by immunofluorescence, suggesting that the weakened interaction between XPG and TFIIH results in an NER reaction with altered kinetics. This study demonstrates that the N-terminal portion of the spacer region is particularly important for NER progression by mediating the XPG-TFIIH interaction and XPG substrate specificity.     

Heterochromatin characterization of four fish species of the family Loricariidae (Siluriformes)

The karyotypic structures and the composition and distribution of the heterochromatin in the karyotypes of four catfish species belonging to four Loricariidae subfamilies were analysed, namely: Neoplecostomus microps (Neoplecostominae) with 2n=54 chromosomes, Harttia loricariformis (Loricariinae) with 2n=56 chromosomes, Hypostomus affinis (Hypostominae) with 2n=66 chromosomes and Upsilodus sp. (Upsilodinae) with 2n=96 chromosomes. The amount and composition of heterochromatin was quite unequal among the studied species, being copious and mainly GC-rich in Upsilodus sp. and scarce and balanced in H. loricariformis. All of the H. affinis heterochromatin is GC-rich and related with nucleolar organizing regions. N. microps show low quantity of interstitial and GC-rich heterochromatin, one of them being related with NORs. Trends in the macrokaryotypic diversification as well as in the distribution pattern of the heterochromatin are discussed.     

Heterogeneity in the biosynthesis of mucin O-glycans from Trypanosoma cruzi tulahuen strain with the expression of novel galactofuranosyl-containing oligosaccharides

Sialoglycoprotein from Trypanosoma cruzi strains participates in important biological functions in which the O-linked glycans play a pivotal role, and their structural diversity may be related to the parasite's virulence pattern. To provide supporting evidence for this idea, we have determined the structure of novel linear and branched alpha-O-GlcNAc-linked oligosaccharides present on the mucins of the T. cruzi Tulahuen strain. The O-glycans were isolated as oligosaccharide alditols by reductive beta-elimination, purified, and characterized by nuclear magnetic resonance spectroscopy and methylation analysis. Two core families were synthesized by the parasite: the Galfbeta1-->4GlcNAc and Galpbeta1-->4GlcNAc. The Galfbeta1-->4GlcNAc core yields three series of O-chain structures. In the first, the Galf residue is nonsubstituted, while in the other series it is elongated by the activity of galactopyranosyl or galactofuranosyl transferases giving rise to Galp-beta-(1-->2)-Galf-beta-(1-->4) or Galf-beta-(1-->2)-Galf-beta-(1-->4) substructures not previously observed. The three series can arise by further galactopyranosylation of the GlcNAc O-6 arm. Sialylation was the only observed elaboration of the Galpbeta1-->4GlcNAc core family. Thus the determination of the structures of the O-glycans from T. cruzi Tulahuen mucins confirms the strain specificity of the glycosylation and predicts a relationship between it and parasite pathogenicity and the epidemiology of Chagas' disease.     

The ribs of Eupatorieae (Asteraceae): of wide taxonomic value or reliable characters only among certain groups?

Rib numbers have traditionally been used in identification keys of Eupatorieae. Some species, however, demonstrate variations in this character much greater than those registered in the literature, making the use of this trait controversial. The large variations seen among some species of Eupatorieae examined in this work were found to be related to the fact that rib number is more closely associated with the pressure exerted by neighboring flowers on the capitulum than with genetic factors. According to our results, ribs are not a reliable taxonomic character for broad use in Eupatorieae, being especially inappropriate for Chromolaena stachyophylla, Praxelis pauciflora, and Vittetia orbiculata. The association between the occurrence of vascular bundles and ribs is also discussed, and the variability seen in some Eupatorieae is analyzed. Our data indicate that the formation of the ribs on the cypselae is determined more by the position occupied by the flower on the inflorescence than by the occurrence of vascular bundles.     

Pre-treatment of central venous catheters with the cathelicidin BMAP-28 enhances the efficacy of antistaphylococcal agents in the treatment of experimental catheter-related infection

An in vitro antibiotic susceptibility assay for Staphylococcus aureus biofilms developed on 96-well polystyrene tissue culture plates was performed to elucidate the activity of the 27 residues cathelicidin peptide BMAP-28, quinupristin/dalfopristin (Q/D), linezolid, and vancomycin. Efficacy studies were performed in a rat model of staphylococcal CVC infection. Silastic catheters were implanted into the superior cava. Twenty-four hours after implantation the catheters were filled with BMAP-28. Thirty minutes later rats were challenged via the CVC with 1.0x10(6) CFU of S. aureus strain Smith diffuse. Administration of antibiotics into the CVC at a concentration equal to the MBC observed using adherent cells, or at a much higher concentration (1024 microg/mL) began 24 h later. The inhibition activities of all antibiotics against adherent bacteria were at least two-four-fold lower that against freely growing cells. When antibiotics were used in BMAP-28 pre-treated wells, they showed higher activities. The in vivo studies showed that when CVCs were pre-treated with BMAP-28 or with a high dose of antibiotics, biofilm bacterial load was reduced from 10(7) to 10(3) CFU/mL and bacteremia reduced from 10(3) to 10(1) CFU/mL. When CVCs were treated with both BMAP-28 and antibiotics, biofilm bacterial load was further decreased to 10(1) CFU/mL and bacteremia was not detected. These results suggest that CVC pre-treated with BMAP-28 represents an attractive choice for the treatment of device-related infections caused by staphylococci.