Comparative genome analysis of potential regulatory elements in the ABCG5-ABCG8 gene cluster

The excretion of sterols from the liver and intestine is regulated by the ABCG5 and ABCG8 transporters. To identify potential regulatory elements, 152 kb of the human ABCG5-ABCG8 gene cluster was sequenced and comparative genome analysis was performed. The two genes are oriented in a head-to-head configuration and are separated by a 374-bp intergenic region, which is highly conserved among several species. Using a reporter construct, the intergenic region was found to act as a bidirectional promoter. A conserved GATA site in the intergenic region was shown by site-directed mutagenesis to act as a repressor for the ABCG5 promoter. The intergenic region was also shown to be partially responsive to treatment by LXR agonists. In summary, several potential regulatory elements were found for the ABCG5 and ABCG8 genes, and the intergenic region was found to act as a bidirectional promoter.     

Structure-based design of potent histatin analogues

Conformational studies of human salivary peptide, histatin 3 (Hst3), were performed by nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy in a membrane-mimicking environment. The structural information that was obtained was used in the design of peptide analogues with improved antifungal activity. In the presence of increasing concentrations of L-alpha-dimyristoylphosphatidylcholine (L-alpha-DMPC) lipid vesicles, a dramatic increase in a minimum at 198 nm is observed in the CD spectra of Hst3. The NMR data of Hst3 in the presence of L-alpha-DMPC lipid vesicles reveal the proximity of residues Y(10) and S(20), indicating the existence of a more compact structure. Peptide analogues were designed on the basis of this observation, which incorporated a disulfide bond to stabilize an extended loop in this region of the sequence. One of these, peptide 4, was 100 times more potent than Hst5 against Saccharomyces cerevisiae cells. Conformational analysis of peptide 4 revealed a looped structure with charged residues protruding on the outside surface, while a combination of aromatic residues and histidines are packed into an internal core.     

Nucleotide variation,haplotype structure,and association with end-stage renal disease of the human interleukin-1 gene cluster

A dense gene-based SNP map was constructed across a 360-kb region containing the interleukin-1 gene cluster (IL1A, IL1B, and IL1RN), focusing on IL1RN. In total, 95 polymorphisms were confirmed or identified primarily by direct sequencing. Polymorphisms were precisely mapped to completed BAC and genomic sequences spanning this region. The polymorphisms were typed in 443 case-control subjects from Caucasian and African American groups. Consecutive pair-wise marker linkage disequilibrium was not strictly correlated with distance and ranged from D'=0.0079 to 1.000 and D'=0.0521 to 1.0000 in Caucasians and African Americans, respectively. Single markers and haplotypes in IL1 cluster genes were evaluated for association with end-stage renal disease (ESRD). Eleven SNPs show some evidence of association with ESRD, with the strongest associations in two IL1A variants, one SNP, rs1516792-3, in intron 5 (p=0.0015) and a 4-bp insertion/deletion within the 3'UTR, rs16347-2 (p=0.0024), among African Americans with non-T2DM-associated ESRD.     

Acute toxicity of lead, steel, and an iron-tungsten-nickel shot to mallard ducks (Anas platyrhynchos)

Twenty mallards (Anas platyrhynchos) of both sexes were dosed by oral gavage with Heavi-Shot (H-S; Environ-Metal, Inc., Sweet Home, Oregon, USA) pellets, 20 with steel shot, and 10 with lead (Pb) pellets, all of equal size. All pellets were fired from a shotgun into an absorbent material, retrieved, and weighed prior to introduction into the ducks. Birds were fed whole kernel corn and grit and observed for signs of toxicity for 30 days following dosing. Hevi-Shot pellets lost an average of 6.2% of their mass and steel shot pellets lost 57% of their mass in the birds' gizzards. Almost all (90%) of the Pb shot dosed birds died before the end of the study, while no mortality was observed in the steel or H-S dosed groups. Even though total food consumption differed between the H-S and steel shot groups, mean bird weight change was not different. There were no significant morphologic or histopathologic abnormalities of the liver and kidney in the H-S and steel shot groups. Results indicated that mallards dosed orally with eight No. 4 H-S pellets were not adversely affected over a 30-day period, and that H-S provides another environmentally safe nontoxic shot for use in waterfowl hunting.     

Enzymatic function of loop movement in enolase: preparation and some properties of H159N,H159A,H159F,and N207A enolases

The hypothesis that His159 in yeast enolase moves on a polypeptide loop to protonate the phosphoryl of 2-phosphoglycerate to initiate its conversion to phosphoenolpyruvate was tested by preparing H159N, H159A, and H159F enolases. These have 0.07%–0.25% of the native activity under standard assay conditions and the pH dependence of maximum velocities of H159A and H159N mutants is markedly altered. Activation by Mg²⁺ is biphasic, with the smaller Mg²⁺ activation constant closer to that of the catalytic Mg²⁺ binding site of native enolase and the larger in the mM range in which native enolase is inhibited. A third Mg²⁺ may bind to the phosphoryl, functionally replacing proton donation by His159. N207A enolase lacks an intersubunit interaction that stabilizes the closed loop(s) conformation when 2-phosphoglycerate binds. It has 21% of the native activity, also exhibits biphasic Mg²⁺ activation, and its reaction with the aldehyde analogue of the substrate is more strongly inhibited than is its normal enzymatic reaction. Polypeptide loop(s) closure may keep a proton from His159 interacting with the substrate phosphoryl oxygen long enough to stabilize a carbanion intermediate.     

The H159A mutant of yeast enolase 1 has significant activity

The function of His159 in the enolase mechanism is disputed. Recently, Vinarov and Nowak (Biochemistry (1999) 38, 12138-12149) prepared the H159A mutant of yeast enolase 1 and expressed this in Escherichia coli. They reported minimal (ca. 0.01% of the native value) activity, though the protein appeared to be correctly folded, according to its CD spectrum, tryptophan fluorescence, and binding of metal ion and substrate. We prepared H159A enolase using a multicopy plasmid and expressed the enzyme in yeast. Our preparations of H159A enolase have 0.2-0.4% of the native activity under standard assay conditions and are further activated by Mg(2+) concentrations above 1 mM to 1-1.5% of the native activity. Native enolase 1 (and enolase 2) are inhibited by such Mg(2+) concentrations. It is possible that His159 is necessary for correct folding of the enzyme and that expression in E. coli leads to largely misfolded protein.     

Binding of multivalent carbohydrates to concanavalin A and Dioclea grandiflora lectin. Thermodynamic analysis of the "multivalency effect"

Binding of a series of synthetic multivalent carbohydrate analogs to the Man/Glc-specific lectins concanavalin A and Dioclea grandiflora lectin was investigated by isothermal titration microcalorimetry. Dimeric analogs possessing terminal alpha-D-mannopyranoside residues, and di-, tri-, and tetrameric analogs possessing terminal 3, 6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside residues, which is the core trimannoside of asparagine-linked carbohydrates, were selected in order to compare the effects of low and high affinity analogs, respectively. Experimental conditions were found that prevented precipitation of the carbohydrate-lectin cross-linked complexes during the isothermal titration microcalorimetry experiments. The results show that the value of n, the number of binding sites on each monomer of the lectins, is inversely proportional to the number of binding epitopes (valency) of each carbohydrate. Hence, n values close to 1.0, 0.50, and 0.25 were observed for the binding of mono-, di-, and tetravalent sugars, respectively, to the two lectins. Importantly, differences in the functional valency of a triantennary analog for concanavalin A and D. grandiflora lectin are observed. The enthalpy of binding, DeltaH, is observed to be directly proportional to the number of binding epitopes in the higher affinity analogs. For example, DeltaH of a tetravalent trimannoside analog is nearly four times greater than that of the corresponding monovalent analog. Increases in K(a) values of the multivalent carbohydrates relative to monovalent analogs, known as the "multivalency effect," are shown to be due to more positive entropy (TDeltaS) contributions to binding of the former sugars. A general thermodynamic model for distinguishing binding of multivalent ligands to a single receptor with multiple, equal subsites versus binding to separate receptor molecules is given.     

Intravital Imaging of a Massive Lymphocyte Response in the Cortical Dura of Mice after Peripheral Infection by Trypanosomes

Peripheral infection by Trypanosoma brucei, the protozoan responsible for sleeping sickness, activates lymphocytes, and, at later stages, causes meningoencephalitis. We have videoed the cortical meninges and superficial parenchyma of C56BL/6 reporter mice infected with T.b.brucei. By use of a two-photon microscope to image through the thinned skull, the integrity of the tissues was maintained. We observed a 47-fold increase in CD2+ T cells in the meninges by 12 days post infection (dpi). CD11c+ dendritic cells also increased, and extravascular trypanosomes, made visible either by expression of a fluorescent protein, or by intravenous injection of furamidine, appeared. The likelihood that invasion will spread from the meninges to the parenchyma will depend strongly on whether the trypanosomes are below the arachnoid membrane, or above it, in the dura. Making use of optical signals from the skull bone, blood vessels and dural cells, we conclude that up to 40 dpi, the extravascular trypanosomes were essentially confined to the dura, as were the great majority of the T cells. Inhibition of T cell activation by intraperitoneal injection of abatacept reduced the numbers of meningeal T cells at 12 dpi and their mean speed fell from 11.64 ± 0.34 μm/min (mean ± SEM) to 5.2 ± 1.2 μm/min (p = 0.007). The T cells occasionally made contact lasting tens of minutes with dendritic cells, indicative of antigen presentation. The population and motility of the trypanosomes tended to decline after about 30 dpi. We suggest that the lymphocyte infiltration of the meninges may later contribute to encephalitis, but have no evidence that the dural trypanosomes invade the parenchyma.     

HDL: structure, function and metabolism.

The major advances in our knowledge of the structure, function and metabolism of the plasma lipoproteins have occurred as a result of the rapid increase in our knowledge of the structure and function of the apolipoproteins, lipoproteins, and the heterogeneity of the individual classes of lipoproteins. Over the last decade, there has been a tremendous increase in our knowledge of the structure and molecular properties of ApoA-I and ApoA-II which has permitted an analysis of the functions of these apolipoproteins in lipid and lipoprotein metabolism and the initiation of kinetic studies of HDL metabolism. The elucidation of the structures of the ApoA-I and ApoA-II genes has permitted the determination of genetic defects resulting in decreased levels of HDL and premature cardiovascular disease, as well as the identification of new diseases (e.g. hereditary systemic amyloidosis). The future focus of research on HDL will be the analysis of the individual lipoprotein particles within HDL which have different physiological functions and important roles in reverse cholesterol transport. An improved understanding of the role of HDL in the transport of cellular cholesterol to the liver and the exchange of cholesterol between plasma lipoproteins will provide critical information on cholesterol metabolism in normal subjects and permit the elucidation of the molecular defects of new genetic diseases which may be associated with the development of premature cardiovascular disease.