Prosthetic group content and ligand binding properties of a spinach catalase.

A recently discovered form of spinach catalase that contains both a novel heme and protoheme as prosthetic groups has been characterized using immunological and spectroscopic techniques. The enzyme appears to be a dimer of identical Mr 60,000 monomers. Extraction of the non-covalently bound prosthetic groups, followed by thin-layer chromatography of the extract, suggested that the novel heme contains four carboxylic acid side-chain groups. The resonance Raman spectrum of the resting enzyme indicates that the protoheme prosthetic group is five-coordinate and high-spin. The enzyme was shown to bind formate, azide and cyanide. Cyanide and azide binding to catalase are biphasic, suggesting the existence of two different binding sites for cyanide and azide in the enzyme. Results obtained from EPR and resonance Raman spectroscopies also support the hypothesis that two different ligand-binding sites are present in the enzyme. Western blots suggest that the Mr 60,000 peptide of the novel heme-containing catalase is similar or identical to that of a previously characterized, exclusively protoheme-containing, tetrameric catalase.

Ascites produced in recalcitrant hybridomas by backcrossing to a parental myeloma.

Hybridoma lines frequently lose their ability to produce ascites upon extended cultivation in vitro. We have reintroduced genes for tumor formation into three independent hybridoma lines by backcrossing them to the parental myeloma. The parental myeloma cells were eliminated by a brief in vitro selective period in HAT medium, after which the cells were inoculated into pristane-primed mice. In two out of three cases the resultant lines were able to produce ascites without further subcloning or other manipulations. The antibody retained its specificity as judged by immunoblotting. This method is a rapid and efficient approach for the reestablishment of ascites production in hybridoma lines.

Identification of a higher molecular weight protein that shows apparent cross-reactivity with anti-p21ras monoclonal antibodies on western blots.

We have characterized the immune cross-reactivity of several commercially available monoclonal antibodies prepared against the p21ras proteins and used in Western blotting experiments against human tissue homogenates. Under optimal conditions, only two bands were observed on Western blots. One of these comigrated with control p21ras protein. A second protein of apparent mobility corresponding to approximately 54 kDa was also observed with all four monoclonal antibodies tested. Protein sequencing by automated Edman degradation indicates that the 54 kDa species corresponds to human immunoglobulin heavy chain. Under suboptimal conditions, another high molecular weight species of apparent mobility 65 kDa was also observed to cross-react with some of the monoclonal antibodies tested. This 65 kDa species was identified by protein sequencing as human serum albumin. Coomassie blue staining of SDS-polyacrylamide gels indicates that serum albumin is a major contaminant of many surgically obtained human tissue samples, while p21ras and immunoglobulin heavy chain are present at much lower concentrations. These results may be of significance when using monoclonal antibodies to determine p21ras levels of whole tissue homogenates by dot-blot, slot-blot or microplate assays.

Monoclonal antibodies produced against antigenic determinants present in complex mixtures of proteins.

This overview provides information concerning the production of monoclonal antibodies (MAbs) against specific antigenic determinants present in complex mixtures of proteins. We review five specific techniques for the production of these antibodies (Abs): (a) So-called "shotgun," non-selective approach; (b) cascade procedure; (c) lymphocyte "panning"; (d) cyclophosphamide elimination of unwanted Ab producers; and finally (e) use of polyclonal antisera to extinguish unwanted antibody production. We discuss the relative advantages and disadvantages of these various procedures, and suggest alternative strategies by which specific MAbs might be generated.

Two screening methods show different antigen recognition patterns for four monoclonal antibodies to Candida albicans cell surface.

Four murine monoclonal antibodies (mAbs) showing similar reactivity with a cell-wall extract and mannan preparation obtained from Candida albicans were examined for epitope specificity. An enzyme-linked immunosorbent assay (ELISA) was used to determine total mAb binding to extracted cell-wall antigen when each mAb was reacted alone or in competition with a second mAb. This analysis suggested that three mAbs recognized the same determinant, which differed from that recognized by the fourth. The reactivity of these mAbs was also examined by indirect immunofluorescence assay with both yeast and germ tube forms of the dimorphic fungus. The three mAbs assigned the same epitope-specificity by ELISA showed two different patterns of reactivity with immunofluorescence. This discrepancy is discussed with respect to the postulated structure of mannan and the method of analysis.

Immunological, isoelectric, hydrophobic and molecular weight differences between soluble and ionically membrane-bound fractions of choline-o-acetyltransferase prepared from mouse and rat brain.

Choline-O-acetyltransferase (EC 2.3.1.6; ChAT) was prepared from synaptosomal fractions (P(2)) of mouse and rat brain in the presence of proteolytic inhibitors by the method of Gray and Whittaker (1962) as modified by (Salehmoghaddam and Collier, 1976). The P(2) fraction was hypo-osmotically shocked with glass distilled water and centrifuged to separate the cytoplasmic (S(3)) and vesicle-bound (P(3)) fractions. Fraction S(3) was saved for ChAT assay and compared with the ChAT fraction eluted from the P(3) by salt at a pH 7.4 or by detergent (Benishin and Carroll, 1983). These three fractions of ChAT were then compared by molecular weights, isoelectric points, immunoblotting with monoclonal or polyclonal antibodies and hydrophobicity. The results show that the S(3) fraction of ChAT has a molecular weight of 66 K(d), whereas the ionically-bound fraction of ChAT has a molecular weight of 73-78 K(d). SDS-PAGE of these two ChAT fractions followed by immunoblotting revealed the presence of two immunoreactive bands at 28-29 K(d) and 50-51 K(d) for the ionically bound ChAT fraction. Conversely, none of these antibodies immunostained any protein bands for the S(3) ChAT fraction even though one monoclonal antibody had been prepared against this ChAT fraction and the S(3) ChAT fraction had a similar specific activity prior to SDS-PAGE as did the salt solubilized ChAT fraction. However, anti-ChAT monoclonal antibody MB16 binds the native S(3) ChAT fraction in the co-precipitation assay. The S(3) fraction of ChAT had only one isoelectric point at pH 7.8, whereas the ionically bound and detergent soluble ChAT fractions had two isoelectric points at pH 8.1-8.15 and 7.45-7.5. The S(3) ChAT fraction also differed in hydrophobicity from the other two ChAT fractions. These differences between the S(3) and salt soluble ChAT fractions were not obviated by addition of Triton X-100 and thus could not be attributed to the association of lipids with either of the fractions. We conclude that the water soluble fraction of ChAT in central nerve terminals differs in its physical properties and its subcellular location from that which ionically binds to membranes.

Protein turnover and sensory traits of longissimus muscle from implanted and nonimplanted heifers.

Primary bovine muscle cell culture studies were conducted to determine whether implanting heifers had a direct effect on in vitro protein synthesis and degradation and to determine the effect of implanting heifers on longissimus muscle palatability. Feedlot heifers (n = 96) were administered one of six implant regimens to characterize their effect on in vitro amino acid uptake and protein degradation. Treatments consisted of: 1) a nonimplanted control (NI/NI); 2) no implant on d 1 and Revalor-H administered on d 84 of the experiment (NI/Rev); 3) Revalor-H on d 1, but no implant given at d 84 (Rev/NI); 4) Revalor-H administered on d 1 and d 84 (Rev/Rev); 5) Revalor-IH administered on d 1 and Revalor-H at d 84 (RIH/Rev); and 6) Synovex-H given at d 1 and Revalor-H administered at d 84 (Syn/Rev). Blood and longissimus lumborum muscle were collected 20 min postmortem, and serum and muscle extracts were incubated with primary bovine muscle cells. Implant treatments had minimal effects on shear force and sensory traits; however, steaks from Rev/Rev heifers were 0.31 kg more tender (P < 0.05) than steaks from NI/NI heifers. Serum protein synthesis and degradation were not affected (P > 0.10) by any implant treatment. When primary bovine muscle cells were treated with muscle extract, amino acid uptake was greater for heifers implanted with Rev/ Rev than for the average of all other treatments (P < 0.01). The Rev/Rev implant regimen also increased (P < 0.05) amino acid uptake compared with heifers treated with RIH/Rev, Syn/Rev, NI/NI, NI/Rev, or Rev/NI. Cellular protein degradation of the muscle cell culture treated with muscle extract tended (P < 0.10) to be higher in NI/NI-treated cells compared with the average of all implant treatments. In addition, cells treated with muscle extract from heifers implanted with Rev/Rev had lower (P < 0.05) protein degradation than the NI/NI control heifers. These results indicate that anabolic implant strategies can directly affect both muscle protein synthesis and degradation via effects that seem to be more autocrine than paracrine in nature.

Effects of biological type of beef steers on vitamin D, calcium, and phosphorus status.

Feedlot steers (n = 36) from three biological types (Bos indicus, Bos taurus-Continental, and Bos taurus-English) were used to determine the Ca, P, and vitamin D3 status of feedlot cattle. The USDA yield and quality grade traits were measured at slaughter, and the concentrations of vitamin D3 (VITD) and the metabolites 25-hydroxyvitamin D3 (25-OH D) and 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D) were determined in LM, liver, kidney, and plasma. Plasma and muscle Ca and P concentrations also were determined. Biological type of cattle affected a number of carcass traits. Carcasses from Bos taurus-English cattle had more marbling, resulting in higher quality grades (P < 0.05). Carcasses from Bos taurus-Continental cattle had lower calculated yield grades (P < 0.05) than did carcasses from cattle in the other biological types. In general, differences in carcass traits resulting from biological type were consistent with other reports. Plasma and LM Ca and P concentrations were not affected (P = 0.06) by biological type of cattle, indicating that Ca and P homeostasis is a conserved trait across the different types of cattle. Plasma VITD and 25-OH D concentrations were not affected (P = 0.41) by biological type, whereas plasma 1,25-(OH)2 D concentration was lower (P < 0.05) in Bos taurus-English cattle than in Bos taurus-Continental and Bos indicus cattle. Liver VITD and 25-OH D were not affected by biological type (P = 0.76), but liver 1,25-(OH)2 D concentration was greater (P < 0.05) in Bos indicus cattle than in Bos taurus-Continental cattle. Kidney vitamin D metabolite concentrations were not affected by biological type of cattle (P = 0.21). Muscle VITD concentration was greater (P < 0.05) in Bos taurus-English cattle than in the other two biological types, and muscle 25-OH D concentrations were greater (P < 0.05) in Bos taurus-English cattle than in Bos indicus cattle. Muscle 1,25-(OH)2 D concentration was less (P < 0.05) in the Bos taurus-Continental cattle than in the other two biological types. Cooking eliminated vitamin D metabolite differences among the biological types. Our results suggest that Bos indicus cattle had greater 1,25-(OH)2 D (the biologically active form) in tissues, and greater 1,25-(OH)2 D plasma concentrations than Bos taurus cattle. Thus, the need for VITD supplementation and optimal levels of Ca and P in feedlot diets might differ between Bos indicus and Bos taurus cattle.

Supplemental vitamin D3 concentration and biological type of beef steers. I. Feedlot performance and carcass traits.

Because of the Ca dependency of the calpains, oral supplementation of vitamin D3 (VITD) can increase the Ca content of muscle to activate the calpains and improve tenderness. Feedlot steers (n = 142) were arranged in a 4 x 3 factorial arrangement consisting of four levels of VITD (0, 0.5, 1, and 5 million IU/[steer x d]) for eight consecutive days antemortem using three biological types (Bos indicus, Bos taurus-Continental, and Bos taurus-English). Feedlot performance factors of ADG, DMI, and G:F were measured, and carcass quality, yield, and color data were collected. Plasma Ca and P concentrations were measured during d 4 to 6 of supplementation and at exsanguination, and carcass pH and temperature were measured in the LM at 3 and 24 h postmortem. Vitamin D3 treatment at 5 million IU/(steer x d) decreased ADG (P < 0.05) over the supplementation and feed intake for the last 2 d of feeding compared with untreated control steers. Likewise, G:F was decreased (P = 0.03) in steers supplemented with 5 million IU/d compared with controls. Overall, there was a linear decrease (P < 0.01) in ADG and G:F as a result of VITD supplementation. Plasma concentrations of Ca and P were increased (P < 0.05) by VITD concentrations of 1 and 5 million IU/(steer x d). All VITD treatments increased (P < 0.05) LM temperature at 3 h postmortem and pH at 24 h postmortem. Vitamin D3 treatments did not affect (P = 0.07) any other carcass measurements, including USDA yield and quality grade; thus, any improvements in meat tenderness as a result of VITD supplementation can be made without adversely affecting economically important carcass factors. Biological type of cattle did not interact with VITD treatment for any carcass or feedlot performance trait. Although feeding 5 million IU/(steer x d) of VITD for eight consecutive days had negative effects on performance, supplementing VITD at 0.5 million IU/ (steer x d) did not significantly alter feedlot performance.

Effect of supplemental vitamin D3 concentration on concentrations of calcium, phosphorus, and magnesium relative to protein in subcellular components of the longissimus and the distribution of calcium within longissimus muscle of beef steers.

The effect of supplementing diets with various levels of vitamin D3 to provide 0, 0.5, 1, and 5 million IU/(steer x d) for 8 d before slaughter on the mineral content and localization of Ca in LM and muscle fragments was studied during the postmortem aging process. Twelve feedlot steers of three biological types were given access to the four levels of vitamin D for 8 d before slaughter. Differential centrifugation techniques were used to determine the concentrations of minerals relative to protein in different muscle fragments on d 3 and 21 postmortem. Electron microscopy visualization of bound Ca indicated that vitamin D3 mobilized Ca from the sarcoplasmic reticulum and transverse tubule system into the myofibrils. Bound Ca was concentrated near the Z-line at the A-band/I-band juncture within the sarcomere. Supplementing steers with 1 and 5 million IU/(steer x d) of vitamin D3 increased (P < 0.05) Ca, P, and Mg concentrations per unit of protein in the cytosol. Soluble cytosolic Ca concentrations were greater (P < 0.05) on d 21 than on d 3 postmortem only when steers were supplemented with 5 million IU/d. Concentrations of Ca, P, and Mg in isolated tissues were increased (P < 0.05) in nuclei and myofibrilar proteins by supplementing steers with 1 and 5 million IU/ (steer x d) of vitamin D3. All supplemental vitamin D3 treatments also increased (P < 0.001) Mg concentrations in the cytosol, regardless of aging treatment, and increased Mg concentrations (P < 0.04) within the mitochondria at d 3 postmortem. Thus, supplementation of feedlot steers with vitamin D3 at levels of 0.5 to 5 million IU/(steer x d) increased Ca concentrations within respiring muscle, resulting in increased bound tissue Ca concentrations. When the respiring muscle was converted to meat, the increased bound tissue Ca resulting from vitamin D3 treatment released Ca concentrations into the cytosol during aging (P < 0.05). Results of this study indicate that vitamin D3 supplementation increased total cytosolic Ca, P, and Mg concentrations in meat.

Supplemental vitamin D3 concentration and biological type of steers. II. Tenderness, quality, and residues of beef.

Vitamin D3 was orally supplemented to determine the supplemental dose that improved beef tenderness in different cattle breed types. Feedlot steers (n = 142) were arranged in a 4 x 3 factorial arrangement consisting of four levels of supplemental vitamin D3 (0, 0.5, 1, and 5 million IU/steer daily) administered for eight consecutive days antemortem using three biological types (Bos indicus, Bos Taurus-Continental, and Bos Taurus-English). Warner-Bratzler shear force (WBSF) was measured at 3, 7, 10, 14, and 21 d postmortem, and trained sensory analysis was conducted at 7 d postmortem on LM, semimembranosus, gluteus medius, and supraspinatus steaks. Concentrations of vitamin D3 and the metabolites 25-hydroxyvitamin D3, and 1,25-dihydroxyvitamin D3 were determined in the LM, liver, kidney, and plasma. Biological type of cattle did not interact (P > 0.10) with vitamin D3 supplementation for sensory or tenderness traits, suggesting that feeding vitamin D3 for 8 d before slaughter affected the different biological types of cattle similarly. Supplementing steers with 0.5, 1, or 5 million IU/(steer(d) decreased (P < 0.05) LM WBSF at 7, 10, 14, and 21 d postmortem compared with controls, and vitamin D3 treatments of 0.5, 1, and 5 million IU decreased (P < 0.05) semimembranosus WBSF at 3, 7, and 14 d postmortem. In general, vitamin D3-induced improvements in WBSF were most consistent and intense in LM steaks. Sensory panel tenderness was improved (P < 0.05) by all vitamin D3 treatments in LM steaks. Sensory traits ofjuiciness, flavor, connective tissue, and off-flavor were not (P > 0.05) affected by vitamin D3 treatments. All vitamin D3 treatments decreased micro-calpain activity and increased muscle Ca concentrations (P < 0.05). Vitamin D3 concentrations were increased (P < 0.05) by supplementation in all tissues tested (liver, kidney, LM, and plasma); however, cooking steaks to 71 degrees C decreased (P < 0.05) treatment residue effects. The vitamin D metabolite 1,25-dihydroxyvitamin D3 was increased (P < 0.05) only in plasma samples as a result of the vitamin D3 treatments. These results indicate that supplementation with vitamin D3 at 0.5 million IU/steer daily for eight consecutive days before slaughter improved tenderness in steaks from different subprimal cuts by affecting muscle Ca concentrations, micro-calpain activities, and muscle proteolysis, with only a small effect on tissue residues of vitamin D3.

Serum resistance in Haemophilus ducreyi requires outer membrane protein DsrA.

Haemophilus ducreyi is resistant to killing by normal serum antibody and complement. We discovered an H. ducreyi outer membrane protein required for expression of serum resistance and termed it DsrA (for "ducreyi serum resistance A"). The dsrA locus was cloned, sequenced, and mutagenized. An isogenic mutant (FX517) of parent strain 35000 was constructed and characterized, and it was found to no longer express dsrA. FX517 was at least 10-fold more serum susceptible than 35000. DsrA was expressed by all strains of H. ducreyi tested, except three naturally occurring, avirulent, serum-sensitive strains. FX517 and the three naturally occurring dsrA-nonexpressing strains were complemented in trans with a plasmid expressing dsrA. All four strains were converted to a serum-resistant phenotype, including two that contained truncated lipooligosaccharide (LOS). Therefore, serum resistance in H. ducreyi does not require expression of full-length LOS but does require expression of dsrA. The dsrA locus from eight additional H. ducreyi strains was sequenced, and the deduced amino acid sequences were more than 85% identical. The major difference between the DsrA proteins was due to the presence of one, two, or three copies of the heptameric amino acid repeat NTHNINK. These repeats account for the variability in apparent molecular mass of the monomeric form of DsrA (28 to 35 kDa) observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since DsrA is present in virulent strains, is highly conserved, and is required for serum resistance, we speculate that it may be a virulence factor and a potential vaccine candidate.

Variable expression of a surface determinant during proliferation of Candida albicans.

The surface expression of an antigenic determinant that is present in the cell wall of Candida albicans was investigated with monoclonal antibody 24 (MAb24), an immunoglobulin M MAb. The proportion of the cell population that expressed the epitope under different growing conditions was determined by indirect immunofluorescence microscopy. More than 90% of stationary-phase yeast cells of strain B311 grown at 28 degrees C expressed the antigen. Less than 50% of yeast cells grown exponentially at 28 degrees C or either growing or stationary-phase yeast cells cultivated at 37 degrees C expressed the epitope. Germ tubes, which were induced at 37 degrees C from stationary-phase yeast cells grown at 28 degrees C, expressed the determinant on the parent yeast but not the hyphal portion of the germ tube. The change in antigen expression by stationary-phase cells grown at 28 degrees C, when they resumed growth by bud formation, suggested that antigen expression was lost by cells in the inoculum prior to the first cell division. By using the same assay, strong positive reactions were observed in stationary-phase cultures of other isolates of C. albicans, C. guilliermondii, C. stellatoidea, and C. tropicalis, but not with isolates of C. krusei, C. parapsilosis, or Torulopsis glabrata. The identification of the antigenic determinant as a carbohydrate was based on three observations: (i) interaction with a mannan preparation from the same organism, (ii) sensitivity of the antigen to periodate but not proteases, and (iii) coincidence of the migration of antigen during electrophoresis with material which stained intensely with carbohydrate but not with protein reagents. These observations suggest that the expression of the antigenic determinant of MAb24 is dependent on the growth conditions, growth state, and morphology of the cell and that the topography of the cell surface is dynamic.

Dynamic optimization of hybridoma growth in a fed-batch bioreactor.

This study addressed the problem of maximizing cell mass and monoclonal antibody production from a fed-batch hybridoma cell culture. We hypothesized that inaccuracies in the process model limited the mathematical optimization. On the basis of shaker flask data, we established a simple phenomenological model with cell mass and lactate production as the controlled variables. We then formulated an optimal control algorithm, which calculated the process-model mismatch at each sampling time, updated the model parameters, and re-optimized the substrate concentrations dynamically throughout the time course of the batch. Manipulated variables were feed rates of glucose and glutamine. Dynamic parameter adjustment was done using a fuzzy logic technique, while a heuristic random optimizer (HRO) optimized the feed rates. The parameters selected for updating were specific growth rate and the yield coefficient of lactate from glucose. These were chosen by a sensitivity analysis. The cell mass produced using dynamic optimization was compared to the cell mass produced for an unoptimized case, and for a one-time optimization at the beginning of the batch. Substantial improvements in reactor productivity resulted from dynamic re-optimization and parameter adjustment. We demonstrated first that a single offline optimization of substrate concentration at the start of the batch significantly increased the yield of cell mass by 27% over an unoptimized fermentation. Periodic optimization online increased yield of cell mass per batch by 44% over the single offline optimization. Concomitantly, the yield of monoclonal antibody increased by 31% over the off-line optimization case. For batch and fed-batch processes, this appears to be a suitable arrangement to account for inaccuracies in process models. This suggests that implementation of advanced yet inexpensive techniques can improve performance of fed-batch reactors employed in hybridoma cell culture.

Monoclonal antibody studies of ferredoxin:NADP+ oxidoreductase.

Eleven independent monoclonal antibodies, all IgG's, have been raised against the ferredoxin:NADP+ oxidoreductase of spinach leaves. All 11 monoclonal antibodies were able to produce substantial inhibition of the NADPH to 2,6-dichlorophenol indophenol (DCPIP) diaphorase activity of the enzyme, but none of the antibodies produced any significant inhibition of electron flow from NADPH to ferredoxin catalyzed by the enzyme. Spectral perturbation assays were used to demonstrate that antibody interaction with NADP+ reductase did not interfere significantly with the binding of either ferredoxin or NADP+ to the enzyme. Ultrafiltration binding assays were used to confirm that the monoclonal antibodies did not interfere with complex formation between ferredoxin and the enzyme. These results have been interpreted in terms of the likely presence of one or more highly antigenic epitopes at the site where the nonphysiological electron acceptor, DCPIP, binds to the enzyme. Furthermore, the results suggest that the site where DCPIP is reduced differs from both of the two separate sites at which the two physiological substrates, ferredoxin and NADP+/NADPH, are bound.

Characterization of mutant strains of Candida albicans deficient in expression of a surface determinant.

Monoclonal antibody (MAb) 17E4 reacts with a surface carbohydrate determinant and agglutinates cells of Candida albicans. Using this MAb, we have isolated N-methyl-N'-nitro-N-nitrosoguanidine-induced nonagglutinating mutants. Eleven of these were characterized for the presence and expression of the surface antigen recognized by MAb 17E4 by immunoblot analysis of whole cells and by fluorescence flow cytometry. Soluble cell wall extracts from five mutant strains were negative by immunoblot analysis. The reactivities of the strains with several other MAbs and commercial antisera (Candida Check; Iatron Laboratories, Tokyo, Japan) which also recognize carbohydrate determinants were examined by immunoblot analysis of whole cells. Mutant strains showed no or reduced expression of the MAb 17E4 antigen and could be placed into at least two distinct phenotypic classes. Recognition by the other MAbs tested showed a similar pattern, while recognition by the commercial antisera was unchanged in the mutant strains. 1H and 13C nuclear magnetic resonance spectral analysis of mannan prepared from the wild type and nonexpressing mutant-strain 4A showed that the spectra from the mutant strain were simpler than those of the wild type. Most of the beta-1,2 linkages and all of the C-1 phosphate linkages were absent in the 4A mannan spectra, which suggested that the mutant mannan lacked the phosphate-bound beta-1,2-linked mannooligosaccharides. The effect of the surface defect on the ability of mutant strain 4A to adhere and to invade host tissue was examined in two murine models. In ex vivo binding assays, strain 4A showed reduced binding to the marginal zone and increased binding to the white pulp of splenic tissue, decreased binding to kidney tissue, and no change in binding to liver tissue compared with the wild type. In vivo, no difference was observed in translocation of the wild type or strain 4A to liver following immuno-compromising treatment of infant mice which had been challenged with either strain by the oral-intragastric route.

Complex interaction between different proteinaceous components within the cell-wall structure of Candida albicans.

We have previously described a monoclonal antibody, MAb DC3:H10, which recognized an epitope preferentially expressed on the surface of Candida albicans germ tubes. In the present study we examined the MAb-reactive material further. Immunoblot analysis of the material purified partially by Sephadex G-200 and DEAE-Sephacel chromatography reacted with antibodies to the C. albicans C3d receptor (CR2). In an ELISA, MAb DC3:H10 captured antigen that was recognized by both anti-CR2 and anti-mp58 fibrinogen binding mannoprotein polyclonal antibodies. The MAb DC3:H10 failed to compete with either of these antisera in an ELISA. Indirect immunofluorescence (IIF) analysis showed differences in surface distribution for the MAb DC3:H10, the CR2, and the mp 58 epitopes. Dual labeling IIF experiments showed MAb DC3:H10 binding to be unaffected by binding of fibrinogen or anti-mp58 antibody. However, the binding patterns of MAb DC3:H10 were modified in the presence of anti-CR2 antibody, suggesting a complex interaction between these cell wall components.

Measurement of protein synthesis and degradation in C2C2) myoblasts using extracts of muscle from hormone treated bovine.

A detailed methodology is described for determination of treatment effects on muscle cell protein synthesis and muscle cell protein degradation in a cell culture system. C(2)C(22) mouse myoblasts were treated with growth media containing muscle extracts from bovine treated with different pharmaceutical agents. Radiolabeled amino acids were added to the growth media to determine treatment effects on protein synthesis and protein degradation. Percent protein synthesis was calculated by measuring amino acid uptake as a percentage over internal control. Percent protein degradation was measured using a pulse chase technique. These procedures will allow researchers to determine treatment effects on overall protein synthesis and degradation in vitro in a relatively short amount of time without excessive costs. A second benefit is that animals do not have to be fed radiolabeled feedstuffs. These procedures are not intended to elucidate the mechanisms behind pharmaceutical enhancement of muscle cell protein synthesis or protein degradation.