Effect of polyanions on the refolding of human acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) is unstable at physiological temperatures in the absence of polyanions such as heparin. Therefore, the effect of temperature on the kinetics of refolding of aFGF has been examined in the presence and absence of several polyanions. The protein folds into its native state at temperatures up to 30 degrees C without polyanions with an activation energy of approximately 14 kcal/mol, but does not acquire native structure above this temperature. When heparin, inositol hexasulfate, or sulfate ion are present, aFGF refolds below 30 degrees C with a slightly reduced activation energy (10-11 kcal/mol). In addition, the protein now also renatures between 30 and 50 degrees C with activation energies of 1-2 (heparin), 16 (inositol hexasulfate), and 7 (sulfate) kcal/mol. Trace heavy metals appear to inhibit the refolding process, but a molecular chaperone (bovine 70-kDa heat shock cognate protein) and a peptidylprolyl isomerase (the FK506-binding protein) have no effect. It is concluded that the rate of refolding of aFGF at physiological temperatures is probably controlled by the interaction of a native-like state of the protein with an unknown polyanionic species.     

Evidence for the presence of a glycosphingolipid-transfer protein in rat brain cytosol

Proteins in the postmicrosomal supernatant fraction of rat brain catalyzed the transfer of bovine brain galactocerebroside, sulfatide, and ganglioside GM1 from unilamellar liposomes to the rat erythrocytes or ghosts. The vesicles were made with egg yolk lecithin, cholesterol, 3H-labelled glycolipid, and a trace of [14C]triolein as a nonexchangeable marker. The routine assay of the glycosphingolipid transfer consisted of incubation of the donor liposomes with erythrocytes in the presence or absence of supernatant protein in physiological buffer at 37 degrees C for various time intervals. After the incubation, the erythrocytes were separated from the vesicles by centrifugation and the extent of protein-catalyzed transfer of labelled glycolipid in the membrane-bound total lipid fraction was determined by scintillation spectrometry. The fraction of [3H]glycosphingolipid transferred is represented by a change in the 3H/14C ratios at initial and subsequent time intervals. The glycosphingolipid transfer catalyzed by the supernatant protein was found to be logarithmic, whereas the protein-independent transfer was linear over a period of 3-4 h. The rate constant (K) and half time (t1/2) of the protein-catalyzed transfer reaction of cerebrosides and sulfatides were almost the same, while the transfer of ganglioside GM1 occurred at a slightly faster rate, probably owing to the greater aqueous solubility of this lipid. The transfer activity was also increased in a manner dependent on the amount of supernatant protein added up to 10 mg. The catalytic activity of the protein was lost when heated at 70 degrees C for 5 min. The pH optimum of the activity was around 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)     

traT gene sequences, serum resistance and pathogenicity-related factors in clinical isolates of Escherichia coli and other gram-negative bacteria

The R6-5 plasmid-specified outer membrane protein, TraT protein, has previously been shown to mediate resistance to bacterial killing by serum. Colony hybridization with a 700 bp DNA fragment carrying most of the traT gene was used to examine the prevalence of traT in Gram-negative bacteria, particularly strains of Escherichia coli, isolated from clinical specimens. traT was found in isolates of E. coli, Salmonella, Shigella and Klebsiella, but not in Pseudomonas, Aeromonas or Plesiomonas, nor in the few isolates of Enterobacter, Proteus, Acinetobacter, Citrobacter, Serratia or Yersinia that were examined. It was detected in a significantly higher proportion of the E. coli strains isolated from the blood of patients with bacteraemia/septicaemia or from faeces of patients with enteric infections (50-70%) than in that of strains isolated from normal faeces (20-40%). The incidence of traT in strains isolated from cases of urinary tract infections was variable. traT was found to be frequently associated with production of the K1 capsule and with the carriage of ColV plasmids, but not with the carriage of R plasmids, nor with serum resistance or the production of haemolysin.     

Immunoblotting identifies and antigen recognized by anti gp63 in the immune complexes of Indian kala-azar patient sera

In SDS-PAGE the immune complexes (IC) of kala-azar patient sera showed intense bands at 55 kDa and 20 kDa corresponding to heavy and light chains of immunoglobulins. In immunoblot experiment, kala-azar and normal IC after treatment with patient sera showed multiple bands of which the band at 55 kDa was most prominent in kala-azar IC. It is known that in kala-azar sera antihuman IgG is present, so the heavy band at 55 kDa region may be due to higher amount of IgG and/or other antigen(s) present at that region. Immunoblot experiments of kala-azar IC with anti gp63 also developed a major band at 55 kDa. It suggests that the antigen (55 kDa) and gp63 have common antigenic epitope (s). Normal IC did not react with anti gp63 indicating absence of this antigen in normal IC. Antigenic similarity between the IC antigen (55 kDa) and gp63 indicated that the former antigen may have been processed from gp63. In summary, identification of a parasite antigen (55 kDa) in IC of kala-azar patients sera may be useful in developing a serodiagnostic assay for visceral leishmaniasis. (Mol Cell Biochem130: 11–17, 1994)Abbreviations IC Immune Complexes - PEG Polyethylene Glycol (Mol wt 8000) - PBS Phosphate Buffer Saline - VL Visceral Leishmaniasis - AVL American Visceral Leishmaniasis - IgG Immunoglobulin G - TBS Tris Buffer Saline - SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis - gp63 A leishmanial surface glycoprotein of molecular mass 63,000 - TEMED N,N,N,N-Tetramethylethylenediamine     

Ibuprofen, a unique anti-inflammatory compound with antifungal activity against dermatophytes

Ibuprofen showed significant antifungal activity in vitro against dermatophytes at pH 5 (MIC: 5–40 μg ml^-1). In this respect it is comparatively more efficient than two well known and medically used antifungal compounds, benzoic and salicylic acids. This compound with anti-inflammatory activity which is not found in any other conventional antifungal organic acids, may have clinical prospects.     

Structural implications of spectroscopic characterization of a putative zinc finger peptide from HIV-1 integrase.

The N-terminal domain of human immunodeficiency virus (HIV-1) integrase (IN) contains the sequence motif His-Xaa3-His-Xaa23-Cys-Xaa2-Cys, which is strongly conserved in all retroviral and retrotransposon IN proteins. This structural motif constitutes a putative zinc finger in which a metal ion may be coordinately bound by the His and Cys residues. A recombinant peptide, IN(1-55), composed of the N-terminal 55 amino acids of HIV-1 IN was expressed in Escherichia coli and purified. Utilizing a combination of techniques including UV-visible absorption, circular dichroism, Fourier transform infrared, and fluorescence spectroscopies, we have demonstrated that metal ions (Zn2+, Co2+, and Cd2+) are bound with equimolar stoichiometry by IN(1-55). The liganded peptide assumes a highly ordered structure with increased alpha-helical content and exhibits remarkable thermal stability. UV-visible difference spectra of the peptide-Co2+ complexes directly implicate thiols in metal coordination, and Co2+ d-d transitions in the visible range indicate that Co2+ is tetrahedrally coordinated. Mutant peptides containing conservative substitutions of one of the conserved His or either of the Cys residues displayed no significant Zn(2+)-induced conformational changes as monitored by CD and fluorescence spectra. We conclude that the N terminus of HIV-1 IN contains a metal-binding domain whose structure is stabilized by tetrahedral coordination of metal by histidines 12 and 16 and cysteines 40 and 43. A preliminary structural model for this zinc finger is presented.     

Sister-chromatid exchange analyses in rodent maternal, embryonic and extra-embryonic tissues: Transplacental and direct mutagen exposures

Sister-chromatid exchange (SCE) analyses were conducted in maternal, embryonic and extraembryonic tissues of pregnant rats and mice. The various tissues were substituted in vivo with 5-bromodeoxyuridine (BrdU) by implantation of a BrdU tablet in pregnant animals at mid-gestation. Following maternal exposure to 5–20 mg/kg cyclophosphamide, embryonic liver cells demonstrated dose-dependent SCE increases up to 10-fold that of control. Rat embryos revealed little intralitter variability for this transplacental effect. Maternal marrow and yolk sac cells examined in the rat also underwent significant increases in SCE, although to different extents. While marrow SCE frequencies were similar to those of embryo liver, yolk sac SCE frequencies were generally much lower.

SCE analyses were also conducted in rat yold sac cells substituted in vivo with BrdU and subsequently explanted to whole-embryo culture. In vitro exposure to cyclophosphamide at concentrations up to 100 μg/ml had no SCE-inducing effect. However, similar exposures to phosphoramide mustard, a presumed metabolite of cyclophosphamide, caused dose-dependent increases in SCE up to 8-fold higher than control at 2 μg/ml. Thus, cyclophosphamide appears to require maternal metabolic activation in order to cause an increased SCE frequency in yolk sac cells. The system described permits versatile SCE analyses which can help to define relative maternal and embryo tissue-specific sensitivities to chemical-induced genetic damage.