Cloned fragment of diphtheria toxin linked to T cell-specific antibody identifies regions of B chain active in cell entry

The role of discrete domains of diphtheria toxin (DT) B chain in cytosol entry and cytotoxicity was investigated by linking a monoclonal antibody recognizing the human T cell-specific antigen T3 (UCHT1) to diphtheria toxin (UCHT1-DT), DT A subunit (UCHT1-DTA), or to a genetically engineered form of DT (UCHT1-MspSA) lacking the C-terminal 17-kDa portion of the B subunit. The N-terminal 21-kDa region of DT B chain increased toxicity of UCHT1-DTA 100-fold (UCHT1-MspSA) while addition of the C-terminal 17-kDa region (UCHT1-DT) increased toxicity 100-fold more. The cytotoxicity was dependent upon antibody binding as demonstrated by blocking toxicity with excess UCHT1. The differences in toxicity between these reagents were not due to differences in ADP-ribosylation activity of DT A chain, binding activity of the antibody moiety, extent of DT nicking, or the cross-linking method, so we conclude that the large differences in toxicity were due to the presence of different B chain domains. The large increase in toxicity by the C-terminal region of DT B did not appear to be caused by DT receptor binding. The lysosomotropic agent NH4Cl blocked the cytotoxic effect of DT, UCHT1-DT, and UCHT1-MspSA but not UCHT1-DTA.     

Protein Composition of PNS Myelin: Developmental Comparison of Control and Quaking Mice

Protein compositions were determined for sciatic nerve myelin isolated from young and adult control and quaking (Qk) mice. Age-related changes in the relative amounts of large (Pl) and small (Pr) basic proteins were found. In control animals, the ratio Pr/Pl increased with age, a change similar to that observed for the large (Bl) and small (Bs) CNS myelin basic proteins of adult mice. Pr/Pl also increased with age in the Qk mouse sciatic nerve, but only to the point that the value in the adult Qk mouse was similar to that observed for young control animals, a situation reminiscent of the effect of the Qk mutation on CNS basic proteins. Thus, our data suggest that the Qk mutation has a similar effect on peripheral nervous system (PNS) and CNS basic proteins. Our findings are consistent with recent electrophoretic and immunochemical data showing that PNS and CNS myelin basic proteins in rodents are analogous, and they suggest that the genetic program controlling basic protein expression is common to oligodendroglia and Schwann cells.     

Feeding and gut physiology in Acanthopleura spinigera (Mollusca)

The morphology and histology of the alimentary canal of the rock chiton Acanthopleura spinigera are described and the ability of regions of the gut to digest specific substrates investigated. The oesophagus is produced into a pair of thin-walled lateral pouches, the salivary glands or "sugar glands" which empty into the stomach. Folds of the capacious stomach are almost obscured by the large digestive gland over which is coiled the intestine. Histologically the gut consists of an outer layer of connective tissue, an inner muscular layer and a ciliated epithelium which varies in thickness from one region to the next. Proteases are most active in the stomach, digestive gland and anterior intestine at pH 6·5 and in the posterior intestine at pH 7·5-8·5. The digestion of lipoidal substance was greatest in the stomach and digestive gland and least in anterior intestine. There was little increase in the amount of digestion product obtained after 20 hours incubation. All regions of the alimentary canal and salivary gland were capable of digesting carbohydrates except that many low molecular weight carbohydrates were digested by salivary gland extracts only. The amylases were most active at pH 6–6·5. It is concluded that digestive enzymes are distributed throughout the intestinal tract but the amount of enzyme present varies from region to region, and is greatest just after feeding.     

PROTEIN COMPOSITION OF MYELIN OF THE PERIPHERAL NERVOUS SYSTEM

Abstract— Myelin was purified from the peripheral nervous system (PNS) of several species. The protein composition of these preparations was examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Proteins characteristic of all samples include, in order of increasing mobility: a series of high molecular weight proteins, the major peripheral nerve protein (P₀), two uncharacterized proteins, and two basic proteins (P₁ and P₂). Quantitative results, obtained by densitometry of gels stained with Fast Green showed differences in protein distribution, both between species, and from different types of nerves obtained from the same animal. The relative amounts of P₁ and P₂ proteins were the most variable; e.g. myelin from guinea-pig sciatic nerve had little or no P₂ protein, whereas 15 per cent of the myelin protein of beef posterior intradural root was Pz protein. P₀, P₁ and P₂ proteins from rabbit sciatic nerve and P₀ and P₂ proteins from beef dorsal and ventral intradural roots were purified and their amino acid compositions were determined. Our results indicated that the P₁ protein is very similar in size and amino acid composition to the basic protein of central nervous system myelin, whereas the P₀ and P₂ proteins are unique to the PNS.     

QUAKING MOUSE: ISOLATION AND CHARACTERIZATION OF MYELIN PROTEIN

A new technique, involving final purification on a continuous CsCl gradient, was utilized for the isolation of cerebral myelin from adult (4- to 6-month-old) quaking mice, littermate controls and young (10-day-old) normal mice. The yield of myelin from either adult quaking or normal young mice was 5-10 per cent of that from adult controls. After deli-pidation, myelin proteins were separated by polyacrylamide gel electrophoresis in buffers containing sodium dodecylsulphate. Two gel systems were utilized: (1) a high-resolution discontinuous electrophoresis system; and (2) a continuous system utilizing gels cross linked with ethylenediacrylate (EDA). The gels from the discontinuous system were stained with Fast Green and quantified by densitometry. The base lability of the EDA-linked gels permitted direct chemical determination of protein in specific bands. Myelin from brains of normal adult mice contained, as major components, one proteo-lipid and two basic proteins. There were also a number of high-molecular-weight proteins which represented a significant portion of the total. Myelin from quaking mice had qualitatively a similar distribution of proteins but the high-molecular-weight fraction comprised a much greater percentage of the total protein. The ratio of basic to proteolipid protein in preparations from quaking mice was considerably higher than that in the myelin from control mice. The distribution pattern of the myelin proteins from 10-day-old mice was quantitatively similar to that of quaking mice. Altogether the evidence supports the hypothesis that the quaking mutant provides a model of an immature nervous system with respect to myelination.     

Mechanical agitation of hybridoma suspension cultures: Metabolic effects of serum, pluronic F68, and albumin supplements

Metabolic effects of the medium supplements, fetal bovine serum (FBS), Pluronic F68, and bovine serum albumin (BSA) were compared for agitated bioreactor cultures of hybridoma cells. Agitation speeds up to 600 rpm, without entrainment of gas bubbles by sparging or vortex formation, allowed examination of cell interactions with turbulent fluid forces. For cultures in FBS-supplemented RPMI media, there was no significant effect of intense turbulent fluid shear on cell growth, metabolism, or antibody, production. Serum-free cultures (Pluronic F68 or BSA supplements) at 600 rpm demonstrated greatly increased glycolysis rates during exponential growth relative to controls. Nutrient limitations caused increased rates of decline of the viable cell concentrations and a reduction in final antibody titers by around 70%. The Pluronic F68 and BSA supplements did not lead to cell protection by modifying metabolism under conditions of intense turbulent fluid shear. Supplementing the protein-free medium with FBS reduced glycolysis rates in exponential growth phase, but this did not prevent a high rate of viable cell decline and low antibody titers. We concluded that FBS does not have a metabolic effect on cells subjected to intense turbulent fluid shear. Although the agitation conditions employed in this study were more intense than generally required for agitated bioreactor culture of hybridomas, we have demonstrated the importance of considering metabolic effects of turbulent fluid forces on cultures using nutrient-rich basal media, in addition to the considerations of gas bubble effects described by other workers. (c) 1992 John Wiley & Sons, Inc.     

New hydrazone derivatives of adriamycin and their immunoconjugates--a correlation between acid stability and cytotoxicity

New N-substituted hydrazine linkers were synthesized and their hydrazone derivatives of adriamycin were prepared. These functionalized adriamycin derivatives were conjugated with a monoclonal antibody, 5E9. The release rate of adriamycin from the hydrazones and from some of the conjugates was studied, and their relationship to the IC50's of the conjugate against 5E9-positive Daudi cells was investigated.     

Ethanol production from sucrose by immobilizedZymomonas mobilis cells in polyurethane foam

Summary The possibility of using polyurethane foam as a support for the immobilization ofZymomonas mobilis cells to carry out sucrose conversion to ethanol was investigated. Sucrose hydrolysis efficiencies of 90% and higher, volumetric reactor productivity of 20 gL^–1h^–1 and final ethanol concentration of 6.3% (v/v) at a dilution rate of 0.4 h^–1 show the good performance of polyurethane foams for whole cell immobilization.     

Partitioning the Components of Sexual Selection: Attractiveness and Agonistic Behaviour in Male Wax Moths,Achroia grisella (Lepidoptera: Pyralidae)

Two components of sexual selection in the lesser wax moth, Achroia grisella, are clearly defined: females choose males based on characters of their ultrasonic advertisement signals, and males display agonistic behaviour patterns in contests over signalling stations. We conducted a series of laboratory trials to determine whether successes in these two sexual selection arenas were related. Our trials showed that winners in contests over signalling stations were characterized by earlier initiation of both signalling and aggression. These characteristics may indicate higher levels of ‘motivation,’ but it is unclear whether this level is a fixed trait. There was no relationship, however, between any measure of a male's attractiveness to females and his success in winning competition trials. Thus, inter- and intra-sexual selection may be acting on different, uncorrelated traits in A. grisella.