Characterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes in the lipopolysaccharide of the deep rough mutant I-69 Rd−/b+ of Haemophilus influenzae

Monoclonal antibodies against the lipopolysaccharide (LPS) of the deep rough mutant I-69 Rd⁻/b⁺ of Haemophilus influenzae were obtained after immunization of mice with sheep erythrocytes which had been coated with de- O -acylated LPS. Characterization of antibodies was performed by enzyme immuno assay (EIA) using LPS or neoglycoconjugates containing partial structures of LPS as solid-phase antigens and by haemagglutination with sheep erythrocytes coated with de- O -acylated LPS. Binding data were confirmed by EIA inhibition experiments using deacylated LPS or synthetic partial structures thereof. Three antibodies were specific for 3-deoxy- d - manno -octulopyranosonic acid- (Kdo) 5-phosphate, one for Kdo-4-phosphate, and one required, in addition to a Kdo-phosphate, parts of the phosphorylated glucosamine backbone of lipid A. All antibodies also bound in (i) Western blots to bacterial whole-cell lysates or isolated LPS separated by SDS–PAGE, (ii) bacterial colony blots, and (iii) immunofluorescence with live bacteria. The latter result indicated that Kdo-4- and Kdo-5-phosphate are synthesized by the bacteria and are not the result of phosphate migration.     

High-performance liquid chromatography of apolipoproteins in serum high-density lipoproteins

A simple and rapid method for apolipoprotein analysis in serum high-density lipoproteins (HDL) has been developed using high-performance liquid chromatography (HPLC) with sodium phosphate buffer (pH 7.0) containing 0.1% sodium dodecyl sulphate (SDS) as eluent. In contrast to the use of urea solution as an eluent, apolipoproteins can be analysed by applying an incubation mixure of HDL and the eluent buffer. A TSK-GEL column of G3000SW was found to be more profitable than G2000SW or G4000SW for analysis of HDL apolipoproteins. Elution patterns monitored by absorbance at 280 nm using a G3000SW column can give precise quantitative as well as qualitative information about apolipoproteins of molecular weight between 10⁴ and 10⁵. HPLC patterns of HDL apolipoproteins were compared between individual human subjects with various diseases. Elution profiles for lipid components in an incubation mixture were also examined.     

Unstable resistance of G mouse fibroblasts to ecotropic murine leukemia virus infection.

G mouse cells were resistant to N- and NB-tropic Friend leukemia viruses and to B-tropic WN 1802B. Though the cells were resistant to focus formation by the Moloney isolate of murine sarcoma virus, they were relatively sensitive to helper component murine leukemia virus. To amphotropic murine leukemia virus and to focus formation by amphotropic murine sarcoma virus, G mouse cells were fully permissive. When the cell lines were established starting from the individual embryos, most cell lines were not resistant to the murine leukemia viruses. Only one resistant line was established. Cloning of this cell line indicated that the resistant cells constantly segregated sensitive cells during the culture; i.e., the G mouse cell cultures were probably always mixtures of sensitive and resistant cells. Among the sensitive cell clones, some were devoid of Fv-1 restriction. Such dually permissive cells, and also feral mouse-derived SC-1 cells, retained glucose-6-phosphate dehydrogenase-1 and apparently normal number 4 chromosomes. The loss of Fv-1 restriction in these mouse cells was not brought about by any gross structural changes in the vicinity of Fv-1 on number 4 chromosomes.     

A tryptic peptide from beta-casein depresses protein synthesis and degradation and enhances ureogenesis in primary cultures of rat hepatocytes

1. A peptide which enhances ureogenesis in primary cultured hepatocytes of rats was isolated from a tryptic digest of bovine beta-casein. 2. The structure of the peptide was Ala-Val-Pro-Tyr-Pro-Gln-Arg which is located from 177th to 183rd residues from N-terminal of beta-casein. 3. The peptide also showed the activity to inhibit protein synthesis and protein degradation. 4. It also inhibited DNA synthesis of hepatocytes induced by insulin and/or epidermal growth factor.     

Cloning and characterization of the CYS3 (CYI1) gene of Saccharomyces cerevisiae.

A DNA fragment containing the Saccharomyces cerevisiae CYS3 (CYI1) gene was cloned. The clone had a single open reading frame of 1,182 bp (394 amino acid residues). By comparison of the deduced amino acid sequence with the N-terminal amino acid sequence of cystathionine gamma-lyase, CYS3 (CYI1) was concluded to be the structural gene for this enzyme. In addition, the deduced sequence showed homology with the following enzymes: rat cystathionine gamma-lyase (41%), Escherichia coli cystathionine gamma-synthase (36%), and cystathionine beta-lyase (25%). The N-terminal half of it was homologous (39%) with the N-terminal half of S. cerevisiae O-acetylserine and O-acetylhomoserine sulfhydrylase. The cloned CYS3 (CYI1) gene marginally complemented the E. coli metB mutation (cystathionine gamma-synthase deficiency) and conferred cystathionine gamma-synthase activity as well as cystathionine gamma-lyase activity to E. coli; cystathionine gamma-synthase activity was detected when O-succinylhomoserine but not O-acetylhomoserine was used as substrate. We therefore conclude that S. cerevisiae cystathionine gamma-lyase and E. coli cystathionine gamma-synthase are homologous in both structure and in vitro function and propose that their different in vivo functions are due to the unavailability of O-succinylhomoserine in S. cerevisiae and the scarceness of cystathionine in E. coli.     

Functional involvement of P-glycoprotein in blood-brain barrier.

P-glycoprotein, an active efflux pump of antitumor agents in multidrug-resistant tumor cells, exists in various normal tissues, including brain capillaries. To study the physiological function of P-glycoprotein expressed in brain capillary endothelium, we established nine mouse brain capillary endothelial cell (MBEC) lines and examined the transport of antitumor agents across the monolayer of MBEC epithelia. In the MBECs, the activities of alkaline phosphatase and gamma-glutamyl transpeptidase, specific markers for brain capillary endothelial cells, were about three times higher than those in other cells including human umbilical vein endothelial cells. By immunoblot analysis, P-glycoprotein was detected in all of the nine MBEC clones. The P-glycoprotein expressed in MBECs specifically bound [125I]iodoaryl azidoprazosin as that in multidrug-resistant cells, and efflux of vincristine was observed in the MBECs. When MBECs were grown on a porous filter membrane, they formed a monolayer of epithelium. By immunoelectron microscopic analysis, P-glycoprotein in MBEC epithelia was shown to be localized to the apical surface of the cells. Moreover, the unidirectional transepithelial transport of vincristine from basal side to apical side was demonstrated in vitro. These observations indicate that P-glycoprotein in brain capillary endothelium prevents vincristine from entering the central nervous system and thus may be one of the functional components of the blood-brain barrier.     

Identification of Elastase as a Secretory Protease from Cultured Rat Microglia

In the course of studying the secretory products of microglia, we detected protease activity in the conditioned medium. Various proteins (casein, histone, myelin basic protein, and extracellular matrix) were digested. The protease activity was characterized by using purified myelin basic protein as a substrate. Maximal activity was observed at neutral pH levels (7-8), which was different from the optimum pH level of proteolytic activity observed in the cell homogenate. The activity was inhibited approximately 60 and 50% by 1 mM phenylmethylsulfonyl fluoride and 40 microM elastatinal, respectively. In gel filtration, the major activity, which was inhibited in the presence of N-methoxysuccinyl-Ala-Ala-Pro-Val-methyl chloride, eluted at a position corresponding to a molecular mass of approximately 25 kDa. These results suggest that the major protease present in microglial conditioned medium is elastase or an elastase-like protease. This suggestion was confirmed by the finding that the 25-kDa protein band was stained with anti-elastase antiserum by western blotting. De novo synthesis of elastase in microglia was supported by [35S]methionine incorporation. In the presence of lipopolysaccharide, the secretory elastase decreased. These results demonstrate that microglia secrete proteases, one of which was identified as elastase. The significance of this enzyme production in physiological and pathological conditions is discussed.     

A high-resolution solid-state 13C-NMR study on crystalline bovine heart cytochrome-c oxidase and lysozyme. Dynamic behavior of protein and detergent in the complex.

We have recorded 100.6-MHz high-resolution solid-state 13C-NMR spectra of crystalline cytochrome-c oxidase from bovine heart muscle and hen egg-white lysozyme, to compare conformation and dynamics of a typical membrane-protein complex with those of lysozyme. The absence of severe interference with the solid-state 13C-NMR spectra, from both the line broadenings from paramagnetic centers and overlapping of intense detergent signals, provided spectral resolution of 13C-NMR feature of cytochrome-c oxidase crystals comparable to that of lysozyme crystal and better than that of dissolved or lyophilized samples. In fact, the observed peak intensities of the polar heads of the detergents BL8SY and Brij 35 were only about 10% and 3% of the anticipated values, respectively. The dynamic behavior of the backbone and side chains of cytochrome-c oxidase was compared with that of lysozyme on the basis of the 13C spin-lattice relaxation times (T1): the backbone of the cytochrome-c oxidase turned out to be more flexible than that of lysozyme. Molecular motions of the detergent molecules attached to the proteins are found to be highly heterogeneous. Detergent molecules undergo rapid tumbling motions in the crystals in about 10 ns as detected by T1. In addition to rapid motions, slow motions were detected by 1H spin-lattice relaxation time in the rotating frame (TH1 rho) and cross-polarization time (TCH), together with data from static spectra, indicating that the aliphatic portion of the detergent interacts more strongly with hydrophobic protein surfaces than do the polar heads.