The two-stranded alpha-helical coiled-coil is an ideal model for studying protein stability and subunit interactions.

We have designed de novo a two-stranded alpha-helical coiled-coil which consists of two identical 35-residue polypeptide chains arranged in a parallel and in-register alignment. Their structure is stabilized by interchain hydrophobic interactions from hydrophobes at positions "a" and "d" of a repeating heptad sequence. The formation and stability of the coiled-coil is dependent on peptide concentration due to the monomer-dimer equilibrium. In contrast, that coiled-coil containing an inter-helical disulfide bond does not show any concentration dependence in the guanidine hydrochloride denaturation experiments as expected. Replacement of one large hydrophobic Leu residue in each chain with Ala significantly decreases coiled-coil stability in both the reduced and oxidized coiled-coils [decreases in transition midpoint of 1.6M (2.3-0.7) and 2.4M (5.3-2.9), respectively]. A large pH dependence on coiled-coil stability is observed over the pH range 4 to 7 (transition midpoints at pH 4, 5, 5.5, 6 and 7 were 3.8, 3.2, 2.0, 1.2 and 0.7M, respectively). The increasing stability with decreasing pH correlates with the protonation of the Glu acid side-chains and reduction of intrachain repulsions between Glu-Glu side-chains in positions i, i + 3 or i, i + 4 along each alpha-helix of the coiled-coil. In addition, coiled-coil stability increases with increasing ionic strength.     

Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase.

Site-directed mutagenesis was used to examine the catalytic importance of 2 histidine and 4 arginine residues in Escherichia coli periplasmic acid phosphatase (EcAP). The residues that were selected as targets for mutagenesis were those that were also conserved in a number of high molecular weight acid phosphatases from eukaryotic organisms, including human prostatic and lysosomal acid phosphatases. Both wild type EcAP and mutant proteins were overproduced in E. coli using an expression system based on the T7 RNA polymerase promoter, and the proteins were purified to homogeneity. Examination of the purified mutant proteins by circular dichroism and proton NMR spectroscopy revealed no significant conformational changes. The replacement of Arg16 and His17 residues that were localized in a conserved N-terminal RHGXRXP motif resulted in the complete elimination of EcAP enzymatic activity. Critical roles for Arg20, Arg92, and His303 were also established because the corresponding mutant proteins exhibited residual activities that were not higher than 0.4% of that of wild type enzyme. In contrast, the replacement of Arg63 did not cause a significant alteration of the kinetic parameters. The results are in agreement with a previously postulated distant relationship between acid phosphatases, phosphoglycerate mutases, and fructose-2,6-bisphosphatase. These and earlier results are also consistent with the conclusion that 2 histidine residues participate in the catalytic mechanism of acid phosphatases, with His17 playing the role of a nucleophilic acceptor of the phospho group, whereas His303 may act as a proton donor to the alcohol or phenol.     

Kallistatin: a novel human tissue kallikrein inhibitor. Purification, characterization, and reactive center sequence.

A novel human tissue kallikrein inhibitor designated as kallistatin has been purified from plasma to apparent homogeneity by polyethylene glycol fractionation and successive chromatography on heparin-Agarose, DEAE-Sepharose, hydroxylapatite, and phenyl-Superose columns. A purification factor of 4350 was achieved with a yield of approximately 1.35 mg per liter of plasma. The purified inhibitor migrates as a single band with an apparent molecular mass of 58 kDa when analyzed on SDS-polyacrylamide gel electrophoresis under reducing conditions. It is an acidic protein with pI values ranging from 4.6 to 5.2. No immunological cross-reactivity was found by Western blot analyses between kallistatin and other serpins. Kallistatin inhibits human tissue kallikrein's activity toward kininogen and tripeptide substrates. The second-order reaction rate constant (ka) was determined to be 2.6 x 10(4) M-1 s-1 using Pro-Phe-Arg-MCA. The inhibition is accompanied by formation of an equimolar, heat- and SDS-stable complex between tissue kallikrein and kallistatin, and by generation of a small carboxyl-terminal fragment from the inhibitor due to cleavage at the reactive site by tissue kallikrein. Heparin blocks kallistatin's complex formation with tissue kallikrein and abolishes its inhibitory effect on tissue kallikrein's activity. The amino-terminal residue of kallistatin is blocked. Sequence analysis of the carboxyl-terminal fragment generated from kallistatin reveals the reactive center sequence from P1' to P15', which shares sequence similarity with, but is different from known serpins including protein C inhibitor, alpha 1-antitrypsin, and alpha 1-antichymotrypsin. The results show that kallistatin is a new member of the serpin superfamily that inhibits human tissue kallikrein.     

Human lysosomal protective protein. Glycosylation, intracellular transport, and association with beta-galactosidase in the endoplasmic reticulum.

In lysosomes beta-galactosidase and neuraminidase acquire a stable and active conformation through their association with the protective protein. The latter is homologous to serine carboxypeptidases and has cathepsin A-like activity which is distinct from its protective function towards the two glycosidases. To define signals in the human protective protein important for its intracellular transport, and to determine the site of its association with beta-galactosidase, we have generated a set of mutated protective protein cDNAs carrying targeted base substitutions. These mutants were either singly transfected into COS-1 cells or cotransfected together with wild type human beta-galactosidase. We show that all point mutations cause either a complete or partial retention of the protective protein precursor in the endoplasmic reticulum. This abnormal accumulation leads to degradation of the mutant proteins probably in this compartment. Only the oligosaccharide chain on the 32-kDa subunit acquires the mannose 6-phosphate recognition marker, the one on the 20-kDa subunit seems to be merely essential for the stability of the mature protein. In cotransfection experiments, wild type beta-galactosidase and protective protein appear to assemble already as precursors, soon after synthesis, in the endoplasmic reticulum. Mutated protective protein precursors that are retained in the endoplasmic reticulum or pre-Golgi complex interact with and withhold normal beta-galactosidase molecules in the same compartments, thereby preventing their normal routing.     

In vivo cytotoxic T lymphocyte induction with soluble proteins administered in liposomes.

The in vivo induction of a CTL response usually requires that Ag be endogenously synthesized so that appropriate processing can occur. In most of the few examples where successful CTL induction was reported with proteins and peptides, unacceptable adjuvants or means of Ag formulation were used. In the present report, liposomes were used to incorporate the soluble proteins OVA and beta-galactosidase. This simple and convenient to use approach, which requires minimal amounts of Ag, results in priming for a CD8+ CTL response and the establishment of immunologic memory. The liposome approach may not only prove a convenient means of inducing CTL responses in vivo but may also be useful to study the mechanisms of Ag processing.     

Genotoxicity of heated cooking oil vapors.

Epidemiological studies of lung cancer in Chinese women indicated that factors other than cigarette smoking are related to lung cancer risk. A case-control study suggested that indoor air pollution, particularly from cooking oil emissions, may be involved. Condensates of volatile emissions from rapeseed and soybean cooking oils were prepared and found to be genotoxic in short-term tests including the Salmonella mutation assay, SV50 forward-mutation assay, and sister-chromatid exchange assay, as well as the micronucleus assay in mouse bone marrow. In contrast, condensates from rapeseed oil with butylated hydroxyanisole or hydrogenated rapeseed oil were not mutagenic, implicating oxidation products as the cause for mutagenicity. Peanut oil and lard condensates were not mutagenic in any assay. The association of exposure to Chinese rapeseed cooking-oil emissions and lung-cancer risk may be related to the mutagenic component of these condensates.     

Oleanane glycosides from Glycyrrhiza yunnanensis roots.

From the roots of Glycyrrhiza yunnanensis, collected in Yunnan, China, six new oleanane-type triterpene glycosides named yunganosides A1, B1, C1, D1, E2 and F2 were isolated together with hypaphorine. The structures of these glycosides were established by spectroscopic and chemical means.     

UNC-5, a transmembrane protein with immunoglobulin and thrombospondin type 1 domains, guides cell and pioneer axon migrations in C. elegans.

The unc-5 gene is required for guiding pioneering axons and migrating cells along the body wall in C. elegans. In mutants, dorsal migrations are disrupted, but ventral and longitudinal movements are largely unaffected. The gene was tagged for molecular cloning by transposon insertions. Based on genomic and cDNA sequencing, the gene encodes UNC-5, a transmembrane protein of 919 aa. The predicted extracellular N-terminus comprises two immunoglobulin and two thrombospondin type 1 domains. Except for an SH3-like motif, the large intracellular C-terminus is novel. Mosaic analysis shows that unc-5 acts in migrating cells and pioneering neurons. We propose that UNC-5 is a transmembrane receptor expressed on the surface of motile cells and growth cones to guide dorsal movements.     

Influence of dissolved oxygen concentration on the biosynthesis of cephalosporin C.

Cephalosporin C was produced by a highly productive strain of Cephalosporium acremonium under industrial production conditions by fed-batch cultivation in a 40-l stirred-tank reactor using a complex medium containing 50 g l-1 peanut flour. The influence of dissolved oxygen concentration (pO2, DOC), which was maintained at different constant levels between 5 and 40% of its saturation value, during the production phase by means of a parameter-adaptive pO2-controller, on the cephalosporin C biosynthesis, was investigated. The concentrations of cephalosporin C (CPC) and its precursors penicillin N (PEN N), deacetoxycephalosporin C (DAOC), and deacetylcephalosporin C (DAC) were monitored by on-line HPLC. The concentrations of amino acids, valine (VAL), cysteine (CYS), alpha-amino-adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV) were determined by off-line HPLC. By reducing the pO2 in the production phase from 40 to 5% of its saturation value, the CPC concentration diminished from 7.2 to 1.1 g l-1 and the PEN N concentration increased from 2.57 to 7.65 g l-1. The DAC concentration also dropped from 3.13 to 0.42 g l-1; however, the DAOC concentration was less influenced. The concentrations of AC and ACV were also less affected. The small DOC did not lead to an accumulation of the intermediate AC and ACV during the production phase. With increasing DOC in the range of 5-20%, the maximal specific production rate, the cell mass concentration-based and the substrate-based yield coefficients for CPC increased almost linearly, and fell back for PEN N.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentration and detection of hepatitis A virus and rotavirus from shellfish by hybridization tests.

A modified polyethylene glycol precipitation method for concentration of virus followed by a new method to recover nucleic acid was used to detect hepatitis A virus (HAV) and rotavirus (SA11) in shellfish (oysters and hard-shell clams) by hybridization tests. Infectious virus, seeded into relatively large quantities of shellfish, was recovered consistently, with greater than 90% efficiency as measured by either in situ hybridization (HAV) or plaque assay (rotavirus SA11). Viral nucleic acid for dot blot hybridization assays was extracted and purified from virus-containing polyethylene glycol concentrates. Separation of shellfish polysaccharides from nucleic acid was necessary before viral RNA could be detected by dot blot hybridization. Removal of shellfish polysaccharides was accomplished by using the cationic detergent cetyltrimethylammonium bromide (CTAB). Use of CTAB reduced background interference with hybridization signals, which resulted in increased hybridization test sensitivity. After polysaccharide removal, dot blot hybridization assays could detect approximately 10(6) physical particles (corresponding to approximately 10(3) infectious particles) of HAV and 10(4) PFU of SA11 rotavirus present in 20-g samples of oyster and clam meats. These studies show continuing promise for the development of uniform methods to directly detect human viral pathogens in different types of shellfish. However, practical applications of such methods to detect noncultivatable human viral pathogens of public health interest will require additional improvements in test sensitivity.