Action of the C3b-inactivator on the cell-bound C3b.

The action of C3bINA and beta 1H on cell-bound C3b is described in this paper. The alpha-polypeptide of C3b that binds covalently to cell surfaces is cleaved by the C3bINA and beta 1H into two fragments: one of 60,000 (C3b alpha-60) and another of 40,000 (C3b alpha-40) daltons. The beta-chain of C3b is unaffected by the C3bINA and beta 1H. The three polypeptides, C3b alpha-60, C3b alpha-40, and C3 beta, are held together as a single unit by disulfide bonds. This unit, referred to as C3b' is covalently bound to cell surfaces via the C3b alpha-60 polypeptide. The conversion of C3b to C3b' by C3bINA and beta 1H abolishes the ability of the C3b-bearing cells to adhere to human erythrocytes as well as the ability to form, on the cell surface, the B, D, and properdin-dependent amplification C3-convertase. However, the agglutinability of the cells with either anti-C3c or anti-C3d is not affected. Treatment of the C3b'-bearing cells with trypsin releases fragments of C3b' into solution, leaving a polypeptide of 32,000 daltons covalently linked to the membrane. Since the trypsinized cells are agglutinable by anti-C3d but not by anti-C3c, the 32,000 dalton polypeptide appears to correspond antigenically to C3d.     

Purification and characterization of monkey salivary mucin.

Highly purified mucin was prepared from monkey (Macaca arctoides) extraparotid saliva by sequential chromatography on Sephadex G-200 (followed by reduction and alkylation of void volume materials), Sepharose CL-2B with 6 M urea, and CM52 cellulose with 6 M urea. Purity was critically ascertained by anion exchange chromatography, ultracentrifugal analysis, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide electrophoresis, and crossed immunoelectrophoresis. Use of crossed immunoelectrophoresis to examine mucin preparations has not been previously reported. This technique was useful for assessing purity and displaying charge and size microheterogeneity in the purified S-carboxymethylated mucin. Threonine and serine comprised 37.8% of the total amino acids while the oligosaccharide moiety contained N-acetyl-glucosamine, N-acetylgalactosamine, fucose, galactose, N-acetylneuraminic acid, and sulfate. Following alkaline borohydride treatment, the carbohydrate chains were found to be linked O-glycosidically between N-acetylgalactosamine and threonine (serine).     

Partitioning and utilization of energy during the larval development of the xanthid crab, Rhithropanopeus harrisii (Gould)

An energy budget is constructed for the larval development of the crab Rhithropanopeus harrisii (Gould) fed nauplii of the brine shrimp Artemia salina (L.). Between the first zoeal instar and the megalopa, there is a 5.4-fold increase in caloric consumption and a 13.2-fold increase in dry weight. Weight specific energy content increases through the zoeal stages and drops at the megalopa. Rate of oxygen consumption increases steadily throughout development. Assimilation, gross growth, and net growth efficiencies increase steadily through zoeal development and drop at the megalopa. Calories put into tissue production exceed those expended via respiration in zoeal stages II–IV; the reverse is true in zoeal stage I and the megalopa.

A total energy budget has been calculated and the partitioning of energy is discussed in relation to other physiological studies on this species.     

Inactivation of prophage lambda repressor in vivo.

Jacob &; Monod (1961) postulated that prophage A induction results from the inactivation of the λ repressor by a cellular inducer. Although it has been shown that the phage A repressor is inactivated by the recA gene product in vitro (Roberts et al., 1978), we wanted to determine the action of the “cellular inducer” in vivo. Our results have led to a new model, which defines the relationship between the “cellular inducer” and the recA gene product.In order to quantitate the action of the cellular inducer on the λ repressor, we made use of bacteria with elevated cellular levels of the λ repressor (hyperimmune lysogens). We determined the kinetics of repressor inactivation promoted by three representative inducing treatments: ultraviolet light irradiation, thymine deprivation and temperature shift-up of tif-1 mutants.The kinetics of repressor decay in wild-type monolysogens indicate that repressor inactivation is a relatively slow cellular process that takes a generation time to reach completion. Incomplete inactivation of the repressor without subsequent prophage development may occur in a cell. We call this phenomenon detected at the biochemical level “subinduction”. In hyperimmune lysogens. subinduction is always the case.A high cellular level of A repressor that prevents prophage λ induction does not prevent induction of a heteroimmune prophage such as 434 or 80. Although the cellular inducer does not seem specific for any inducible prophage, it does not inactivate two prophage repressors present in a cell in a random manner. We have called this finding “preferential repressor inactivation”. Preferential repressor inactivation may be accounted for by considering that the intracellular concentration of a repressor determines its susceptibility to the action of the inducer.In bacteria with varying repressor levels, a fixed amount of repressor molecules is inactivated per unit of time irrespective of the initial repressor concentration. The rate of repressor inactivation depends on the catalytic capacity of the cellular inducer that behaves as a saturated enzyme. In wild-type bacteria the cellular inducer seems to be produced in a limited amount, to have a weak catalytic capacity and a relatively short half-life. The amount of the inducer formed after tif-1 expression is increased in STS bacteria overproducing a tif-1-modified RecA protein. This result is an indication that a modified form of the RecA protein causes repressor inactivation in vivo.From the results obtained we propose a model concerning the formation of the cellular inducer. We postulate that the cellular inducer is formed in a two-step reaction. The is model visualises how the RecA protein can be induced to high cellular concentrations, even though the RecAp protease molecules remain at a low concentration. The latter accounts for the limited proteolytic activity found in vivo.     

High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion.

The protoplast fusion technique of Schaffner (W. Schaffner, Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980) has been adapted to introduce cloned herpes simplex virus genes into cultured mammalian cells. The technique involves digesting bacterial cell walls with lysozyme to produce protoplasts and then fusing the protoplasts to mammalian cells by treatment with polyethylene glycol. For monitoring transfer, protoplasts were labeled with the fluorescent dye fluorescein isothiocyanate before fusion. After fusion, greater than 50% of the mammalian cells were fluorescent, demonstrating that bacterial material was transferred with high frequency. Transfer of plasmid pBR325 occurred at frequencies of 1 to 2%, as measured by in situ hybridization. Fusion transfer of a chimeric plasmid consisting of the herpes simplex virus type 1 (strain KOS) EcoRI fragment F in pBR325 resulted in expression of some viral genomic sequences in about 5% of the mammalian cells, as detected by indirect immunofluorescence. One Ltk- cell in 300 to 500 was transformed to the TK+ phenotype after fusion with protoplasts carrying the chimeric plasmid pX1, which consists of pBR322 and the BamHI fragment coding for the herpes simplex virus type 1 thymidine kinase gene.     

Quantification of creative kinase isoenzymes by a radioimmunoassay

Summary It is not known whether loss of enzyme activity from the circulation is due to denaturation, inactivation or removal of intact enzyme molecules. This is in part due to the lack of an assay to measure enzyme protein concentration since available assays measure only enzyme activity. Radioimmunoassays for plasma enzymes and isoenzymes have not been possible because of oxidation in radioactive labelling by conventional methods and the problem of subunit dissociation. In the present study, antibodies specific to the B and M subunits of creatine kinase isoenzymes were obtained by immunization of rabbits with canine BB and MM creatine kinase. Anitgens (MM and BB) were radioactively labelled with ¹²⁵I by acylation, avoiding the problem of oxidation and subunit stabilized by mercaptoethanol (0.020 m) and Trisbuffer (1.6 m). A radioimmunoassay capable of detecting picogram amounts of CK isoenzymes was developed which measures the concentration of enzyme protein rather than activity. The method was shown to provide a sensitive quantitative method for analysis of plasma CK isoenzymes in dogs after myocardial infarction produced by coronary occlusion. This technique may provide a prototype for the development of radioimmunoassays for other plasma isoenzymes and should help to elucidate the nature of the disappearance of isoenzymes from the circulation.Work from the authors' laboratory was supported in part by the National Institutes of Health Grant HL 17646, SCOR in Ischemic Heart Disease     

Isolation and characterization of catalase deficient mutants of Salmonella typhimurium.

Summary Catalase deficient mutants (kat) ofSalmonella typhimurium have been isolated. The mutationskatA1, katC6 andkatD9 appear to map at about minute 10 on theSalmonella chromosome. ThekatC6 andkatD9 lesions are complemented by theE. coli F128 (lac+ pro+) episome but thekatA1 lesion is not.KatB2 maps at about minute 100. None of the mutants are oxygen sensitive; they grow as well as wild type bacteria, even when aerated.     

Identification of a transformation-specific protein induced by a Rous sarcoma virus.

Using antisera obtained from rats bearing Schmidt-Ruppin strain Rous sarcoma virus-induced tumors, we have idnetified a protein with an apparent molecular weight of 56,000 daltons and an isoelectric point of 6.3 in extracts of chick embryo fibroblasts transformed by a wild-type nondefective Rous sarcoma virus (Schmidt-Ruppin strain). This protein was not found in cells infected by trnasformation-defective mutants with either a partial or complete deletion of the src gene, nor in cells infected by a nontransforming avian leukosis virus. The 56,000 dalton molecular weight protein was found to be synthesized at both the permissive and nonpermissive temperatures in cells infected by either of two conditionallethal mutants that are temperature-sensitive in cell transformation. The amount of this protein, however, accumulated in cells infected by these temperature-sensitive mutants, relative to the structural polypeptides, differed significnatly from that seen with the nondefective virus. Pulsechase experiments indicate that the protein is extremely unstable, with a half-life of about 20 min, and does not serve as a precursor to any of the detectable virion polypeptides. Furthermore, incubation of the rat antiserum with purified, disrupted virus did not affect its immunoreactivity to this particular protein. We conclude that this 56,000 dalton molecular weight protein is a nonstructural protein specific to cells transformed by Rous sarcoma virus.     

Cellular immunity to Coccidioides immitis: In vitro lymphocyte response to spherules,arthrospores, and endospores

In vitro stimulation of incorporation of tritiated thymidine by human peripheral lymphocytes in response to two soluble antigens and three different intact but nonviable fungal forms of Coccidioides immitis was studied. Lymphocytes were obtained from three groups of subjects: healthy skin test positive, healthy skin test negative, and disseminated disease. Dose-response relationships to the intact forms (endospores, arthrospores, and spherules) were determined. Responses of lymphocytes from healthy skin test-positive subjects and subjects with disseminated disease were similar. Ranking of antigens by “potency” gave the following results: endospores = spherulin > mycelial filtrate > arthrospores = spherules. Endospores were the most potent of the intact forms in 10 of 11 subjects. The clear superiority of endospores over spherules is not due to differences in the total particle surface area available for presentation to the leukocytes. All antigens tested except spherules could discriminate between skin test-positive and skin test-negative subjects in this in vitro system. A T-cell-enriched, B-cell- and mono-cyte-depleted cell population demonstrated an active response to spherulin and to endospores. The variance of these finding with animal studies demonstrating spherules to be immunogenically superior when compared to endospores is discussed. This may have importance in future studies in humans of vaccines to C. immitis.     

Inhibitory effect of insulin on prostacyclin production by isolated rat adipocytes

Physiologic concentrations of insulin completely inhibited the norepinephrine-induced increment in the production of 6-keto-prostaglandin (PG) F_1α, the stable derivative of prostacyclin (PGI₂), by isolated rat adipocytes. The inhibition of PGI₂ production by insulin in isolated rat adipocytes supports the view that the elevated plasma level of 6-keto-PGF_1α in rats with non-ketotic diabetes mellitus and diabetic ketoacidosis is derived at least in part from production of PGI₂ by the adipocyte cell mass.