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Hsc20 is a 20 kDa J-protein that regulates the ATPase activity and peptide-binding specificity of Hsc66, an hsp70-class molecular chaperone. We report herein the crystal structure of Hsc20 from Escherichia coli determined to a resolution of 1.8 A using a combination of single isomorphous replacement (SIR) and multi-wavelength anomalous diffraction (MAD). The overall structure of Hsc20 consists of two distinct domains, an N-terminal J-domain containing residues 1-75 connected by a short loop to a C-terminal domain containing residues 84-171. The structure of the J-domain, involved in interactions with Hsc66, resembles the alpha-topology of J-domain fragments of Escherichia coli DnaJ and human Hdj1 previously determined by solution NMR methods. The C-terminal domain, implicated in binding and targeting proteins to Hsc66, consists of a three-helix bundle in which two helices comprise an anti-parallel coiled-coil. The two domains make contact through an extensive hydrophobic interface ( approximately 650 A(2)) suggesting that their relative orientations are fixed. Thus, Hsc20, in addition to its role in the regulation of the ATPase activity of Hsc66, may also function as a rigid scaffold to facilitate positioning of the protein substrates targeted to Hsc66., (Copyright 2000 Academic Press.)

Hsc20 is a 20-kDa auxiliary protein that functions with the molecular chaperone Hsc66 in Escherichia coli. Crystals of Hsc20 suitable for X-ray diffraction analysis were grown using the hanging drop vapor diffusion technique in polyethylene glycol 400 containing dioxane as an additive to slow growth. The crystals are monoclinic and belong to the space group C2 with unit cell dimensions a = 125.4 A, b = 71.9 A, c = 68.8 A, and beta = 97.0 degrees. The crystals diffract to a minimum d-spacing of approximately 2.5 A resolution, and a native data set was collected to 2.7 A. The results of a self-rotation function analysis revealed threefold symmetry, suggesting three molecules of Hsc20 in the asymmetric unit and, hence, 12 molecules in the unit cell; this corresponds to a Vm value of 2.6 A3/Da and a solvent content of approximately 53% in the crystals. Structure determination by isomorphous replacement is in progress.

Much of our understanding of P450 reaction mechanisms derives from studies on P450cam, a bacterial camphor hydroxylase. P450cam has served as the model for understanding detailed structure/function relationships in mammalian P450 enzymes, which have not proved amenable to x-ray crystallographic techniques. To expand and improve the P450 model, we solved the structure of P450eryF, a cytochrome P450 involved in erythromycin biosynthesis. The overall structure of P450eryF is similar to that of P450cam, but differs in the exact positioning of several alpha-helices, which results in the enlargement of the substrate-binding pocket. P450eryF also differs from P450cam in having alanine in place of the highly conserved threonine residue in the active site. To assess the role of this alanine residue, two mutant forms of P450eryF and a substrate analog were examined. Our findings suggest that P450eryF has evolved an active site that utilizes the substrate to assist in catalysis. In addition, the enlarged substrate binding pocket of P450eryF enables P450eryF to bind certain steroid compounds and azole-based steroid hydroxylase inhibitors. Crystals have been obtained for P450eryF complexed with the antifungal drug ketoconazole, and the high-resolution structure has been determined.

A highly conserved threonine in the active site of cytochromes P450 has been proposed to participate in O2 binding and cleavage. Cytochrome P450eryF is unusual in having alanine in place of this threonine and an ordered active site water molecule (Wat 519) which is hydrogen bonded to the substrate 5-hydroxyl group and is in position to operate as an acid catalyst required for cleaving dioxygen. To asses the role of this alanine residue and Wat 519 in catalysis, two mutant forms of P450eryF (Ala --> Ser,Ala --> Thr) and a substrate analogue lacking a 5-hydroxyl group were examined using kinetic, spectral and crystallographic techniques. In each case decreased catalytic activity was correlated with a loss or repositioning of Wat 519. These findings suggest that P450eryF utilizes the substrate to assist in the acid-catalysed dioxygen bond cleavage reaction.

Differential scanning calorimetry was used to evaluate the effects of polymerizing conditions and the addition of pigments for a heat-activated maxillofacial silicone (MDX 4-4515). Unpigmented specimens were examined in the starting condition and after polymerizing at 100 degrees C for 1 hour, 4 hours, and 20 hours, and specimens of four different pigmented silicones processed 1 hour at 100 degrees C were also included. The DSC scanning was performed from -150 degrees C to 200 degrees C at 10 degrees C per minute, and a strong melting-recrystallization peak at approximately -40 degrees C was found for all eight specimen groups (N = 5). Modulated differential scanning calorimetry resolved this peak into separate melting and recrystallization peaks, and confirmed the existence of a very weak glass transition peak in this silicone at approximately -125 degrees C. The mechanical properties of the MDX 4-4515 silicone appear to be dominated by melting and recrystallization, rather than the glass transition, and differences may exist in the viscoelastic and creep behavior for the unpigmented and pigmented materials.

Cytochrome P450eryF catalyzes the 6S-hydroxylation of 6-deoxyerythronolide B, the initial reaction in a multistep pathway to convert 6-deoxyerythronolide B into the antibiotic, erythromycin. The overall structure of P450eryF is similar to that of P450cam but differs in the exact positioning of several alpha-helices. The largest difference occurs in the B' helix and results in the enlargement of the substrate-binding pocket of P450eryF. The substrate is positioned with the macrolide ring perpendicular to the haem plane and contacts seven hydrophobic residues and three solvent molecules. The substrate participates in a network of hydrogen bonds that may provide a proton shuttle pathway in the oxygen cleavage reaction.

Cytochrome P450eryF was overexpressed in Escherichia coli and purified in high yield. Crystals of the protein in the presence of the substrate, 6-deoxyerythronolide B, have been obtained by the hanging drop vapor diffusion method, using polyethylene glycol 4000 as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 54.16 A, b = 79.67 A, and c = 99.48 A and one molecule per asymmetric unit. A complete native data set has been collected to a resolution of 2.1 A, and anomalous dispersion difference Patterson maps have revealed the location of the single heme iron atom.

Electro-oculographic recordings of caloric-induced nystagmus were conducted in a number of normal and disordered subjects. On the basis of the results, implementation of a clinical measuring system is considered; this is based on the interbeat interval, selected in view of its favourable sensitivity and stability. Two stages of processing are involved; first, the determination of the interbeat intervals, second the analysis. The first stage has been implemented off-line on a general purpose computer and also, in on-line mode, by special purpose electronics. The second stage requires computer analysis. We have used the rank magnitude sequences of interbeat intervals as the basis of the analysis.

Plasmids have an important role in the pathogenicity of certain bacterial species, and Escherichia coli provides the most complete example of the relationship involved. Enterotoxigenic strains of E. coli, in addition to producing heat-stable and/or heat-labile enterotoxins, may also produce a haemolysin and fimbriate cell surface antigens which facilitate the adherence of the bacterial cell to the mucosa of the small bowel. Numerous studies have shown that these properties are plasmid-mediated and that the plasmids act in concert to confer on the host bacterium the ability to produce enteric disease in man and in animals. Moreover, studies with invasive strains of E. coli have shown that the Col V plasmid, which codes for the synthesis of colicin V, significantly enhances the pathogenicity of its host bacterium. Although the relationship between Col V plasmids and virulence is unclear, reports indicate that Col V-containing strains of E. coli are better able to survive in the alimentary tract and that colicine V itself inhibits macrophage function. It is probable that bacterial virulence is a complex phenomenon involving both chromosomal and plasmid genes. We describe here a virulence plasmid which mediates tissue invasiveness in human pathogenic strains of Yersinia enterocolitica.

The shell thickness and mass of eggs of the dipper (Cinclus cinclus) collected on streams of different pH in Wales and Scotland were measured. The aluminium, phosphorus and calcium content of their invertebrate prey were also measured. In a regression analysis, significant at p<0.05, stream pH accounted for up to 7% of the variance in shell thickness, with shells decreasing by 2.5% of the overall mean with each unit of pH decline. In the Welsh sample, differences in shell thickness due to pH were small compared with differences between years across all sites. In data pooled between Scotland and Wales, pH accounted for 17% of the variance in egg mass, but a greater proportion (25%) in Scottish eggs alone. Aluminium concentrations in invertebrates showed no relationship with stream pH, but calcium levels in two insect orders increased significantly with pH. Calcium rich prey, such as Gammarus, were found only in circumneutral streams. The importance of calcium in the diet of dippers before and during egg formation is discussed. No evidence that aluminium in prey adversely affected dipper eggs was found.

The in vivo disintegration of luting cements was determined at 6- and 12-month intervals in two test series of 20 participants each. Four cements were inserted in the wells located in the mesial and distal surfaces of cast crowns. Glass ionomer, silicophosphate, polycarboxylate, and zinc phosphate cements prepared with recommended powder/liquid ratios are discussed, and are ranked with respect to disintegration at 6 and 12 months.