Conformation of the second disulfide loop in human transforming growth factor-alpha studied by two-dimensional NMR spectroscopy

The determination of the conformation of a cyclic heptadecapeptide derived from the second loop of human transforming growth factor-α, [Ala²¹]-hTGF-α-(16–32), is described. Two-dimensional homonuclear Hartmann–Hahn and rotating-frame cross-relaxation spectroscopy in H₂O were used to obtain the complete proton resonance assignments and the necessary distance constraints between nonbonded hydrogen atoms to derive a conformation without involving any energy minimization. The result is an ellipsoidal-shaped structure with a turn at Gly19 and a bend formed by residues 26–29, Gln-Glu-Asp-Lys. Comparison is made with the second loop of human epidermal growth factor and the results are discussed in terms of receptor binding and biological activity.     

The sucrose fuel cell: Efficient biomass conversion using a microbial catalyst

Sucrose was used as a fuel in a thionine-mediated microbial fuel cell containingProteus vulgaris serving as the biocatalyst in the anode compartment. The measured yields show that under suitable conditions the substrate may be oxidised quantitatively to electricity and carbon dioxide.     

Turnover of proteoglycans in skin fibroblast cultures derived from patients with mucopolysaccharidoses

The turnover of proteoglycans in the extracellular matrix was studied in fibroblasts cultures derived from patients with mucopolysaccharidosis (MPS) and healthy donors. The cells were labelled with 35S-sulfate and 14C-glucosamine and it was found that in MPS-fibroblasts the rate of extracellular matrix turnover was hardly affected, where as the intracellular turnover was severely inhibited. Similar results were obtained with normal fibroblasts treated with 20 mM ammonia. Autoradiography revealed that MPS fibroblasts have a preferential accumulation of 35S-sulfate labelled material in the nuclear area, indicating that the nucleus may also be affected in MPS pathology. It is suggested that, although lysosomal enzymes are an important factor in intracellular proteoglycan turnover, they do not play a crucial role in the turnover of extracellular matrix proteoglycans.     

Structure and function in the herpes simplex virus 1 RNA-binding protein U(s)11: mapping of the domain required for ribosomal and nucleolar association and RNA binding in vitro.

The herpes simplex virus 1 US11 protein is an RNA-binding regulatory protein that specifically and stably associates with 60S ribosomal subunits and nucleoli and is incorporated into virions. We report that US11/ beta-galactosidase fusion protein expressed in bacteria bound to rRNA from the 60S subunit and not the 40S subunit. This binding reflects the specificity of ribosomal subunit association. Analyses of deletion mutants of the US11 gene showed that specific RNA binding activity, nucleolar localization, and association with 60S ribosomal subunits were found to map to the amino acid sequences of the carboxyl terminus of US11 protein, suggesting that these activities all reflect specific binding of US11 to large subunit rRNA. The carboxyl-terminal half of the protein consists of a regular tripeptide repeat of the sequence RXP and constitutes a completely novel RNA-binding domain. All of the mutant US11 proteins could be incorporated into virus particles, suggesting that the signal for virion incorporation either is at the amino-terminal four amino acids or is redundant in the protein.     

Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity.

The scrapie amyloid (prion) protein (PrP27-30) is the protease-resistant core of a larger precursor (PrPSc) and a component of the infectious scrapie agent; the potential to form amyloid is a result of posttranslational event or conformational abnormality. The conformation, heat stability, and solvent-induced conformational transitions of PrP27-30 were studied in the solid state in films by CD spectroscopy and correlated with the infectivity of rehydrated and equilibrated films. The exposure of PrP27-30 in films to 60 degrees C, 100 degrees C, and 132 degrees C for 30 min did not change the beta-sheet secondary structure; the infectivity slightly diminished at 132 degrees C and correlated with a decreased solubility of PrP27-30 in sodium dodecyl sulfate (SDS), probably due to cross-linking. Exposing PrP27-30 films to formic acid (FA), trifluoroacetic acid (TFA), trifluoroethanol (TFE), hexafluoro-2-propanol (HFIP), and SDS transformed the amide CD band, diminished the mean residue ellipticity of aromatic bands, and inactivated scrapie infectivity. The convex constraint algorithm (CAA) deconvolution of the CD spectra of the solvent-exposed and rehydrated solid state PrP27-30 identified five common spectral components. The loss of infectivity quantitatively correlated with a decreasing proportion of native, beta-pleated sheet-like secondary structure component, an increasing amount of alpha-helical component, and an increasingly disordered tertiary structure. The results demonstrate the unusual thermal stability of the beta-sheet secondary structure of PrP27-30 protein in the solid state. The conformational perturbations of PrP27-30 parallel the changes in infectivity and suggest that the beta-sheet structure plays a key role in the physical stability of scrapie amyloid and in the ability to propagate and replicate scrapie.     

Characterization of a BHK(TK-) cell clone resistant to postattachment entry by herpes simplex virus types 1 and 2.

BHK(TK-) cells selected for resistance to polyethylene glycol-mediated fusion give rise to clones that are resistant to herpes simplex virus (HSV) infection. We have characterized one such clone, designated 95-19, and found that it is resistant to entry of HSV type 1 (HSV-1), HSV-2, and the related alphaherpesvirus pseudorabies virus (PRV). Single-step growth experiments show no detectable replication of multiple strains of HSV-1 and HSV-2 on 95-19 cells. Three lines of evidence suggest that these cells are resistant to postattachment entry. (i) Measurements of binding of radiolabeled virus show that heparin-sensitive binding of HSV-1 and HSV-2 to 95-19 cells is identical to binding to BHK(TK-) cells, suggesting that the block to replication occurs after attachment to heparan sulfate proteoglycan. (ii) 95-19 cells exposed to HSV-1 or HSV-2 at high multiplicity show no detectable immediate-early (IE) mRNA expression. (iii) Exposure of attached virus and cells to polyethylene glycol results in partial recovery of both IE gene expression and virus yield in single-step growth. The degrees of recovery of single-step yield and IE gene expression are similar, suggesting that the only block to single-step replication is at the point of virus entry and that these cells are deficient in some cellular factor required for efficient postattachment entry of free virus. 95-19 cells are also highly resistant to entry by cell-to-cell spread, suggesting that the same cellular factor participates in both types of entry.     

The all-D-configuration segment containing the IKVAV sequence of laminin A chain has similar activities to the all-L-peptide in vitro and in vivo.

Laminin is a basement membrane glycoprotein that has diverse biological activities. A sequence on the A chain containing IKVAV (Ile-Lys-Val-Ala-Val) has been shown to promote neurite outgrowth, cell adhesion, and tumor growth and metastasis. Here we have determined the structural requirements of this synthetic peptide for biological activity. Twelve-amino acid-long all-L- (LAM-L) and all-D-peptide (LAM-D) segments as well as an alternating D- and L-amino acid-containing peptide (LAM-DL), which included the IKVAV sequence (residues 2097-2108), were synthesized. Circular dichroism spectral analysis revealed a mirror image conformation of LAM-D and LAM-L with mainly beta-sheet and to a minor extent alpha-helical structure. LAM-DL did not exhibit any significant ordered conformational features. LAM-D and LAM-L showed similar cell attachment activities for rat pheochromocytoma cells (PC12), whereas LAM-DL was inactive. A peptide analog with randomized IKVAV sequence (LAM-RM) was also inactive. A similar trend was observed in competition experiments of the four peptides with laminin in analogous cell attachment assays. In in vivo experiments, both LAM-D and LAM-L were capable of increasing tumor growth when subcutaneously injected into mice with murine melanoma cells B16F10. Results indicate that the conformational status of the IKVAV domain is a contributing factor in determining the biological activity but that there is no strict requirement for a specific chirality. There is a likely sequence specificity to the IKVAV region.     

Isolation and identification of 4-hydroxy- and 4-oxoretinoic acid. In vitro metabolites of all-trans-retinoic acid in hamster trachea and liver.

Incubation of [3H]retinoic acid in the presence of hamster liver 10000g supernatant produces several metabolites that are more polar than the parent compound. Two of these metabolites are identical with synthetic all-trans-4-hydroxyretinoic acid and all-trans-4-oxoretinoic acid both in ultraviolet absorption and mass spectral characteristics and in migration rates on two different reverse-phase high-pressure liquid chromatographic systems. The metabolites produced in a cell-free liver incubation reaction also migrate on a high-pressure liquid chromatography column together with metabolites isolated from a tracheal organ culture system. Both the metabolites and the synthetic standards show less biological activity than the parent all-trans-retinoic acid in a tracheal organ culture assay.