Antibiotics and anaerobes of gut origin

Hundreds of bacterial species make up human gut flora. Of these, 99% are anaerobic bacteria. Although anaerobes are part of the normal commensal flora, they can become opportunistic pathogens, causing serious, sometimes fatal infections if they escape from the colonic milieu. Most often, this escape occurs as a result of perforation, surgery, diverticulitis or cancer. Infections involving anaerobic bacteria are often difficult to treat because antibiotic resistance is increasing among the genera, mediated primarily through horizontal transfer of a plethora of mobile DNA transfer factors. Some of these transfer factors can also be transmitted to aerobic bacteria. It is becoming increasingly clear that antibiotic resistance trends have to be carefully monitored, and the transfer factors and mechanisms of transfer understood at a molecular level to avoid negative clinical outcomes when infections involve anaerobic bacteria.     

Transmembrane domain 5 of the LdNT1.1 nucleoside transporter is an amphipathic helix that forms part of the nucleoside translocation pathway

Transporters of the equilibrative nucleoside transporter (ENT) family promote the uptake of nucleosides, nucleobases, and a variety of therapeutic drugs in eukaryotes from protozoa to mammals. Despite its importance, the translocation pathway that mediates the internalization of these substrates has not been identified yet in any of the ENT carriers. Previous genetic studies on the LdNT1.1 nucleoside transporter from Leishmania donovani defined two amino acid residues in predicted transmembrane domains (TMD) 5 and 7 that may line this translocation pathway. The role of TMD5 in forming a portion of the aqueous channel was investigated using the substituted-cysteine accessibility method. A series of 22 cysteine substitution mutants spanning predicted TMD5 were created from a fully functional, cysteine-less, parental LdNT1.1. Cysteine replacement at six positions (M(176)C, T(186)C, S(187)C, Q(190)C, V(193)C, and K(194)C) produced permeases that were inhibited by incubation with sulfhydryl-specific methanethiosulfonate reagents, denoting their solvent accessibility to the translocation pathway. Adenosine was able to block this thiol modification, implying that access to the domain becomes restricted as a consequence of the substrate binding. Strikingly, the Q(190)C substitution interacted differentially with the substrates adenosine and uridine, suggesting that binding of adenosine but not uridine might directly occlude this position. When superimposed on a helical model, all six mutants clustered along one face of the amphipathic alpha-helix predicted for TMD5, strongly suggesting its involvement in the translocation pathway through LdNT1.1.     

Crystallographic studies on structural features that determine the enzymatic specificity and potency of human angiogenin: Thr44, Thr80, and residues 38-41

Human angiogenin (Ang) is a potent inducer of blood vessel formation and is a member of the pancreatic ribonuclease superfamily. Its enzymatic activity is unusually weak and biased toward cleavage after cytidine nucleotides. As part of an ongoing investigation into the structural basis of Ang's characteristic activity, we have determined the crystal structures of three Ang variants having novel activity. (i) The structure of T44D-Ang indicates that Asp44 can participate directly in pyrimidine binding and that the intrinsic hydrogen-bonding capability of this residue largely governs the pyrimidine specificity of this variant. Unexpectedly, the mutation also causes the most extensive disruption of the C-terminus seen in any Ang variant thus far. This allows the side chain of Arg101 to penetrate the B(1) site, raising the possibility that it participates in substrate binding as occurs in ribonuclease 4. (ii) The structure of T80A-Ang supports the view that Thr80 plays little role in maintaining the obstructive conformation of the C-terminus and that its participation in a hydrogen bond with Thr44 selectively weakens the interaction between Thr44 and N3 of cytosine. (iii) ARH-II is an angiogenin/RNase A chimera in which residues 38-41 of Ang are replaced with the corresponding residues (38-42) of RNase A. Its structure suggests that the guest segment influences catalysis by subtle means, possibly by reducing the pK(a) of the catalytic lysine. The loss of angiogenic activity is not attributable to disruption of known cell-binding or nuclear translocation sites but may be a consequence of the chimera's enhanced ribonucleolytic activity.     

Identification of domain boundaries within the N-termini of TAP1 and TAP2 and their importance in tapasin binding and tapasin-mediated increase in peptide loading of MHC class I

Before exit from the endoplasmic reticulum (ER), MHC class I molecules transiently associate with the transporter associated with antigen processing (TAP1/TAP2) in an interaction that is bridged by tapasin. TAP1 and TAP2 belong to the ATP-binding cassette (ABC) transporter family, and are necessary and sufficient for peptide translocation across the ER membrane during loading of MHC class I molecules. Most ABC transporters comprise a transmembrane region with six membrane-spanning helices. TAP1 and TAP2, however, contain additional N-terminal sequences whose functions may be linked to interactions with tapasin and MHC class I molecules. Upon expression and purification of human TAP1/TAP2 complexes from insect cells, proteolytic fragments were identified that result from cleavage at residues 131 and 88 of TAP1 and TAP2, respectively. N-Terminally truncated TAP variants lacking these segments retained the ability to bind peptide and nucleotide substrates at a level comparable to that of wild-type TAP. The truncated constructs were also capable of peptide translocation in vitro, although with reduced efficiency. In an insect cell-based assay that reconstituted the class I loading pathway, the truncated TAP variants promoted HLA-B*2705 processing to similar levels as wild-type TAP. However, correlating with the observed reduction in tapasin binding, the tapasin-mediated increase in processing of HLA-B*2705 and HLA-B*4402 was lower for the truncated TAP constructs relative to the wild type. Together, these studies indicate that N-terminal domains of TAP1 and TAP2 are important for tapasin binding and for optimal peptide loading onto MHC class I molecules.     

Synthesis and biological evaluation of new 4beta-anilino- and 4beta-imido-substituted podophyllotoxin congeners

A series of C-4-anilino- and C-4-imido-substituted new podophyllotoxin congeners have been designed, synthesized, and evaluated for their cytotoxicity and DNA topoisomerase-II (topo-II) inhibition potential. Some of these compounds have exhibited promising in vitro anticancer and topo-II inhibition activity.     

Differential zinc and DNA binding by partial peptides of human protamine HP2

The Zn(II) binding by partial peptides of human protamine HP2: HP2_1–15; HP2_1–25, HP2_26–40, HP2_37–47, and HP2_43–57 was studied by circular dichroism (CD). Precipitation of a 20mer DNA by these partial peptides and the effects of Zn(II) thereon were investigated using polyacrylamide gel electrophoresis (GE). The results of this study suggest that reduced HP2 (thiol groups intact) can bind Zn(II) at various parts of the molecule. In the absence of DNA, the primary Zn(II) binding site in reduced HP2 is located in the 37–47 sequence (involving Cys37, His39, His43, and Cys47), while in the presence of DNA, the strongest Zn(II) binding is provided by sequences 12–22 (by His12, Cys13, His19, and His22) and 43–57 (His43, Cys47, Cys53, and His57). In its oxidized form, HP2 can bind zinc through His residues of the 7–22 sequence. Zn(II) markedly enhances DNA binding by all partial peptides. These findings suggest that Zn(II) ions may be a regulatory factor for sperm chromatin condensation processes.     

Generation and analysis of expressed sequence tags from the salt-tolerant mangrove species Avicennia marina (Forsk) Vierh

Salinization poses an increasingly serious problem in coastal and agricultural areas with negative effects on plant productivity and yield. Avicennia marina is a pantropical mangrove species that can survive in highly saline conditions. As a first step towards the characterization of genes that contribute to combating salinity stress, the construction of a cDNA library of A. marina genes is reported here. Random expressed sequence tag (EST) sequencing of 1,841 clones produced 1,602 quality reads. These clones were classified into functional categories, and blast comparisons revealed that 113 clones were homologous to genes earlier implicated in stress responses, of which the dehydrins are the most predominant in this category. Of the ESTs analyzed, 30% showed homology to previously uncharacterized genes in the public plant databases. Of these 30%, 52 clones were selected for reverse Northern analysis: 26 were shown to be up-regulated and five shown to be down-regulated. The results obtained by reverse Northern analysis were confirmed by Northern analysis for three clones.