Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2

An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ion-exchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40 degrees C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (K(m)) and the maximal rate of hydrolysis (V(max)) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate l-histidyl-beta-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract.     

Limited chloroplast gene transfer via recombination overcomes plastomegenome incompatibility between Nicotiana tabacum and Solanum tuberosum

Green cybrids with a new nucleus-chloroplast combination cannot be selected after protoplast fusion in the intersubfamilial Nicotiana-Solanum combination. As an approach to overcome the supposed plastomegenome incompatibility, a partial plastome transfer by genetic recombination has been considered. After fusions of protoplasts of a light-sensitive Nicotiana tabacum (tobacco) plastome mutant and lethally irradiated protoplasts of wild-type Solanum tuberosum (potato), a single green colony was recovered among 2.5×10⁴ colonies. The regenerated plants had tobacco-like (although abnormal) morphology, but were normally green, and sensitive to tentoxin, demonstrating chloroplast markers of the potato parent. Restriction enzyme analysis of the chloroplast DNA (cpDNA) revealed recombinant, nonparental patterns. A comparison with physical maps of the parental cpDNA demonstrated the presence of a considerable part of the potato plastome flanked by tobacco-specific regions. This potacco plastome proved to be stable in backcross and backfusion experiments, and normally functional in the presence solely of N. tabacum nucleus.     

Fluorescence-based viability assay for studies of reactive drug intermediates

Studies of drug toxicity, toxicologic structure-function relationships, screening of idiosyncratic drug reactions, and a variety of cytotoxic events and cellular functions in immunology and cell biology require the sensitive and rapid processing of often large numbers of cell samples. This report describes the development of a high-sensitivity, high-throughput viability assay based on (a) the carboxyfluorescein derivative 2'-7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) as a vital dye, (b) instrumentation capable of processing multiple small (less than 100 cells) samples, and (c) a 96-well unidirectional vacuum filtration plate. Double staining of cultured peripheral blood mononuclear cells with BCECF and propidium iodide (PI) showed no overlap between PI+ (nonviable) and BCECF+ (viable) cells by flow cytometric analysis. Optimal conditions were developed for dye loading and minimizing physical cell damage and fluorescence quench during the assay procedure. The ratio of BCECF fluorescence to internal standard fluorescent particles was linear from 40 to greater than 20,000 cells with a signal:noise ratio of approximately 3 at 40 cells/well. Sulfamethoxazole hydroxylamine (SMX-HA) was used as a model toxic drug metabolite to explore the validity of the BCECF procedure. SMX-HA, but not its parent compound sulfamethoxazole, resulted in a dose dependent loss of cellular fluorescence and the parallel accumulation of PI+ nonviable cells. When compared to the currently used tetrazolium dye reduction viability assay, the BCECF method was 3-fold more sensitive, greater than 10-fold faster, and required 1/10-1/100 the cell numbers.     

Lipid peroxidation in Ehrlich ascites cell mitochondria is not determined by the polyunsaturated fatty acid content of the membrane

Lipid peroxidation intensity is compared in Ehrlich Ascites Cell and in liver mitochondria, prepared from tumor bearing mice. Malondialdehyde formation is negligible in intact ascites tumour mitochondria, but it is significantly increased in permeabilised mitochondria and in isolated mitochondrial membranes. We suggest that the resistance against oxidative stress is a consequence of efficient protective mechanisms operating in the intact tumour mitochondria and the low level of polyunsaturated fatty acids under these circumstances cannot be the rate limiting factor in lipid peroxidation. Succinate, an effective inhibitor of mitochondrial lipid peroxidation in liver, cannot determine malondialdehyde formation in ascites tumour mitochondria.     

Assignment of human platelet GP2B (GPIIb) gene to chromosome 17, region q21.1-q21.3

Summary The platelet GPIIb-IIIa complex functions as a receptor for fibrinogen, fibronectin, and von Willebrand factor on activated platelets. This glycoprotein is a member of a broadly distributed family of structurally and immunologically related membrane receptors involved in cell-cell contact and cell-matrices interactions. GPIIb-IIIa is a heterodimer complex composed of GPIIb (the subunit), which consists of two disulfide-linked heavy and light chains, and GPIIIa (the subunit), which is a single polypeptide chain. Congenital absence of platelet GPIIb-IIIa in Glanzmann's thrombasthenia results in a severe bleeding disorder characterized by defective platelet aggregation and failure of fibrinogen to bind to platelets. The gene coding for GPIIb was located on 17q21.1-17q21.3 as determined by in situ hybridization with a 2650-pb GP2B (GPIIb) cDNA probe prepared from human megakaryocytes.     

Hepatocellular Carcinoma in Hepatitis B Surface Antigen Carriers in Eight Institutions

We reviewed records of all persons dying between 1979 and 1986 in eight California institutions for the mentally retarded. Autopsies had been done in 71% of the 1,181 deaths. Nine deaths were due to hepatocellular carcinoma, which invariably developed in carriers of hepatitis B surface antigen (HBsAg) and was fatal within four months of diagnosis. The mean age at death was 32.7 years. The incidence of hepatocellular carcinoma in HBsAg carriers was 140 times greater than in the US population. Persistent hepatitis B infection was probably etiologically related to hepatocellular carcinoma in this population, which is relatively free of exposure to other hepatocarcinogens.     

Metal Ion Interactions with Phosphoenolpyruvate Carboxylase from Crassula argentea and Zea mays

Metal ion interactions with phosphoenolpyruvate carboxylase from the CAM plant Crassula argentea and the C₄ plant Zea mays were kinetically analyzed. Fe²⁺ and Cd²⁺ were found to be active metal cofactors along with the previously known active metals Mg²⁺, Mn²⁺, and Co²⁺. In studies with the Crassula enzyme, Mg²⁺ yielded the highest V_max value but also generated the highest values of K_m(metal) and K_m(pep). For these five active metals lower K_m(metal) values tended to be associated with lower K_m(pep) values. PEP saturation curves showed more kinetic cooperativity than the corresponding metal saturation curves. The activating metal ions all have ionic radii in the range of 0.86 to 1.09 Å. Ca²⁺, Sr²⁺, Ba²⁺, and Ni²⁺ inhibited competitively with respect to Mg²⁺, whereas Be²⁺, Cu²⁺, Zn²⁺, and Pd²⁺ showed mixed-type inhibition. V_max trends with the five active metals were similar for the C. argentea and Z. mays enzymes except that Cd²⁺ was less effective with the maize enzyme. K_m(metal) values were 10- to 60-fold higher in the enzyme from Z. mays.     

Inhibition of macrophage activation by isoquinolinesulfonamides, phenothiazines, and a napthalenesulfonamide

The influence of isoquinolinesulfonamides (H-7 and H-8), phenothiazines(trifluoperazine and fluphenazine), and a naphthalenesulfonamide (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on stimulated superoxide anion production and phosphatidyl inositol (PI) cycle activity was investigated in the guinea pig alveolar macrophage. All five drugs were able to inhibit superoxide anion production stimulated by n-formyl-nel-leu-phe (FNLP), leukotriene B4 (LTB4), and phorbol-12,13-dibutyrate (PDB). The order of potency was trifluoperazine greater than or equal to fluphenazine greater than H-7 = W-7 greater than H-8. The dose response curves could be shifted to less efficacy by increasing extracellular calcium. By itself, W-7 markedly stimulated 45Ca+2 efflux, fluphenazine and trifluoperazine slightly stimulated 45Ca+2 efflux, while H-7 and H-8 had no effect on 45Ca+2 efflux from macrophages preloaded with 45Ca+2. Consistent with these results, W-7 markedly stimulated PI cycle activity, fluphenazine and trifluoperazine slightly stimulated PI cycle activity, while H-7 and H-8 had no significant effects on PI cycle activity. In addition, W-7 by itself was able to stimulate a weak and short-lived "burst" of superoxide anion production. In order to evaluate whether a site of action of the inhibitors was at protein kinase C and whether protein kinase C was involved in terminating the normally short-lived FNLP- and LTB4-stimulated macrophage activation, fluphenazine and H-7 were used to evaluate the duration of FNLP- and LTB4-stimulated PI cycle activity, at concentrations of the inhibitors that significantly blocked stimulated superoxide anion production. In all cases, FNLP and LTB4 still stimulated PI cycle activity, which still terminated even though protein kinase C was inhibited. These results suggest that all five drugs block protein kinase C, but H-7 was the most specific in its action at protein kinase C, while the phenothiazines and W-7 have multiple sites of action. In addition, these results suggest that protein kinase C may not function to cause the termination of FNLP- and LTB4-stimulated PI cycle activity and subsequent superoxide anion production.