Monoclonal antibody R24 distinguishes between different N-acetyl- and N-glycolylneuraminic acid derivatives of ganglioside GD3

Monoclonal antibody (MAb) R24 was previously shown to be directed toward ganglioside GD3 [Pukel, C. S., Lloyd, K. O., Travassos, L. R., Dippold, W. G., Oettgen, H. F., and Old, L. J. (1982) J. Exp. Med. 155, 1133-1147]. The structural specificity of the MAb has now been further characterized based on binding to structurally related glycolipids, including four GD3 derivatives with different N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc) substituents. Three assay systems (enzyme immunostaining on thin-layer chromatography, enzyme-linked immunosorbent assay, and immune adherence inhibition assay) were used. MAb R24 was found to react with (NeuAc-NeuAc-)GD3 and (NeuAc-NeuGc-)GD3 but not with (NeuGc-NeuAc-)GD3 or (NeuGc-NeuGc-)GD3. These results clearly indicate that the outer sialic acid (Sia) moiety of GD3 is crucial and must be a NeuAc residue, while the inner sialic acid is less involved in binding to the MAb and can be either NeuAc or NeuGc. The MAb was also found to cross-react weakly with two gangliosides, GT1a and GQ1b, but none of other gangliosides nor neutral glycolipids tested reacted. These findings suggest that the epitope detected by MAb R24 is the trisaccharide structure NeuAc alpha 2----8Sia alpha 2----3Gal-, which must be in a terminal position.     

Evaluation of tri-combinant vaccine for feline herpesvirus, calicivirus and panleukopenia virus infections in Japanese native cats

Tri-combinant vaccine consisting of attenuated feline herpesvirus (FHV) and feline calicivirus (FCV) and inactivated feline panleukopenia virus (FPLV), were evaluated for safety and efficacy, using Japanese native cats and the viral strains isolated in Japan. Thirty-eight 9- to 12-week-old kittens were inoculated intramuscularly and subcutaneously with the vaccine. Consequently, no adverse reaction was found, and protective efficacy was confirmed by challenge tests with the virulent strains of each virus. Serum-neutralizing antibodies against FCV and FPLV were maintained for at least one year after vaccination, whereas antibody against FHV disappeared in two cases at 24 weeks after vaccination. Application of this vaccine seemed effective for control of feline viral disease in cats for experimental use.     

Synthesis and secretion of an epidermal growth factor (EGF) by human fibroblast cells in culture

Human fibroblast (WS-1) cells in culture synthesized and secreted an epidermal growth factor which cross-reacted with human epidermal growth factor (hEGF) purified from human urine. hEGF secreted by the cells (designated as WS-1 EGF or fibroblast EGF) and hEGF isolated from urine (designated as urine EGF) were immunologically indistinguishable. The molecular weight of fibroblast EGF estimated by gel filtration was identical with that of hEGF from urine. On chromatofocusing chromatography, fibroblast EGF was eluted mainly at pH 4.26 as a sharp symmetric peak with a minor peak at pH 4.62, like urine EGF. These results suggested that EGF synthesized and secreted by human fibroblast cells is an identical molecule to that of hEGF in human urine.     

A highly sensitive enzyme immunoassay (EIA) system for mouse epidermal growth factor (mEGF)

A highly sensitive two-site enzyme immunoassay system for mouse epidermal growth factor (mEGF) was developed, based on the sandwiching of an antigen between anti-mouse EGF IgG antibody-coated on a polystyrene bead and anti-mouse EGF Fab' antibody-linked peroxidase (horseradish peroxidase, EC. 1.11.1.7). The procedure is simple and rapid compared to a bioassay. Also, the Fab' antibody-peroxidase complex is more stable than the 125I-labeled antibody. Purified mEGF is detectable at a concentration as low as 3 pg/ml. The detection range was 0.3 to 680 pg/sample with 0.1 ml samples. Levels of immunoreactive mEGF in extracts from adult male mice well agreed with those determined by a radioimmunoassay and a radioreceptor assay. The submaxillary gland contained an extremely high concentration of EGF, while other tissues had low levels of EGF.     

Characterization of the (Ca2+-Mg2+)ATPase purified by calmodulin-affinity chromatography from bovine aortic smooth muscle

A calmodulin inhibitor, trifluoperazine, suppresses ATP-dependent Ca2+ uptake into microsomes prepared from bovine aortic smooth muscle. From this microsomal preparation which we expected to contain calmodulin-dependent Ca2+-transport ATPase [EC 3.6.1.3], we purified (Ca2+-Mg2+)ATPase by calmodulin affinity chromatography. The protein peak eluted by EDTA had calmodulin-dependent (Ca2+-Mg2+)ATPase activity. The major band (135,000 daltons) obtained after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) accounted for about 80% of the total protein eluted. This major band was phosphorylated by [gamma-32P]ATP in a Ca2+-dependent manner. All the 32P incorporated into the major band was released by hydroxylaminolysis. The ATPase reconstituted in soybean phospholipid liposomes showed ATP, calmodulin-dependent Ca2+ uptake. The affinity of the ATPase for Ca2+, Km, was 7 microM and the maximum ATPase activity was 1.4 mumol/mg/min. These values were changed to 0.17 microM and 3.5 mumol/mg/min, respectively by the addition of calmodulin. The activity of the purified (Ca2+-Mg2+)ATPase was inhibited by orthovanadate, and the concentration required for half-maximal inhibition was about 1.8 microM which is close to that of plasma membrane ATPases. Judging from the effect of orthovanadate and the molecular weight, the purified (Ca2+-Mg2+)ATPase was considered to have originated from the plasma membrane not from the sarcoplasmic reticulum.     

Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains

Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.     

Influence of oxygen on the growth of Saccharomyces cerevisiae in continuous culture

Baker's yeast, Saccharomyces cerevisiae, was investigated for the combined influence of dissolved oxygen and glucose concentration in continuous culture. A reactor was operated at a range of dilution rates (0.1, 0.2, 0.25, 0.27, and 3.0 h(-1)), above and below the critical value that separates the oxidative and fermentation regions. For each dilution rate (D), steady states were established at each of five to ten different dissolved oxygen concentrations (DO) in the range of 0.01-5 mg/L. The use of on-line mass spectrometry facilitated the measurement of gaseous and dissolved O(2), CO(2), and ethanol. Intracellular carbohydrate, protein, RNA, DNA, lipid, and cytochrome concentrations were measured. Cell size measurements were reduced to specific surface areas. Cytochrome content showed up to 100% variation during a 20-day period of adaptation at D = 0.2 h(-1) to low DO. Eventually, the culture behaved the same at DO = 0.05 mg/L as it did initially at 3 mg/L. At D = 0.2, 0.25, and 0.27 h(-1), the transition between oxidation and fermentation was characterized by a critical DO which decreased with decreasing D. The X-D curves were shifted such that the critical D value was reduced with decreasing DO. Specific oxygen update rates varied with DO according to the saturation kinetics. Specific cell surface areas increased with decreasing DO. Cytochrome content generally decreased with decreasing DO, and Q(O(2) ) could be linearly related to the total cytochrome content, which exhibited a maximum at D = 0.27 h(-1).     

A Highly Sensitive Enzyme Immunoassay for Mouse β Nerve Growth Factor

Abstract: A sensitive two-site enzyme immunoassay system for mouse β nerve growth factor (NGF) was developed, based on the sandwiching of the antigen between anti-mouse β NGF antibody IgG coated to a polystyrene tube and anti-mouse β NGF antibody Fab'-linked β- d -galactosidase (β- d -galactoside hydrolase, EC 3.2.1.23). This method has the following advantages: (a) the procedures are simple and rapid compared to bioassay or two-site radioimmunoassay; (b) antibody Fab'-β- d -galactosidase complex is more stable than ¹²⁵I-labeled antibody; (c) purified β NGF is detectable at a concentration as low as 10 pg/ml. Our enzyme immunoassay was used to examine the levels of NGF in some tissues of mice. The submaxillary gland contained a high concentration of NGF. However, other tissues, such as the heart, brain, and skeletal muscle, and serum did not contain detectable NGF. These results support recent findings by other investigators that NGF was not found in the organs/tissues other than the submaxillary gland of mice.     

Flavonoids: potent inhibitors of arachidonate 5-lipoxygenase

Various flavonoids were found to be relatively selective inhibitors of arachidonate 5-lipoxygenase which initiates the biosynthesis of leukotrienes with the activity of slow reacting substance of anaphylaxis. Cirsiliol (3',4',5-trihydroxy-6,7-dimethoxyflavone) was most potent, and the enzyme partially purified from rat basophilic leukemia cells was inhibited by 97% at a concentration of 10 microM (IC50, about 0.1 microM). 12-Lipoxygenases from bovine platelets and porcine leukocytes were also inhibited but at higher concentrations (IC50, about 1 microM), and fatty acid cyclooxygenase purified from bovine vesicular gland was scarcely affected. The compound at 10 microM suppressed by 99% the immunological release of slow reacting substance of anaphylaxis from passively sensitized guinea pig lung (IC50, about 0.4 microM).     

Property and function of cell surface glycosyltransferases

Since a galactosyltransferase was first reported to be on the mammalian cell surface in the early 1970s, different classes of glycosyltransferase have been detected on the cell surface of various types of cell by enzymatic or immunological analysis. Although these surface enzymes are identical in catalytic property to those found in Golgi apparatus or endoplasmic reticulum where they are involved in biosynthesis of glycoconjugates, there obtained a monoclonal antibody that distinguishes these enzymes localized differently, and observed some differences in their protein chemistry and in their amounts expressed during cellular differentiation. Due to no availability of sugar donor intercellulary, the surface glycosyltransferases are participating in cellular interactions by recognizing and binding to the appropriate substrates on opposing cell surfaces and extracellular matrices, without showing any enzyme catalysis.