The biotinylation of the rabbit serotonin antibody and its application to immunohistochemical studies using the two-step ABC method

Histochemistry and Cell Biology - A newly modified and improved immunohistochemical method was devised for the demonstration of the serotonin neuron system in the central nervous system of the...     

Isoelectric focusing with reduced cathodic drift and migration into the anode chamber

An apparatus has been developed to reduce cathodic drift and migration into the anode chamber in vertical gel rod isoelectric focusing (IEF). In contrast to commercially available apparatuses, this apparatus can easily handle many more gels at one time, and the length, diameter and shape of its gel can be arbitrarily changed. In addition, high concentrations of detergent can be used to dissolve the protein samples, and removal of the gel cylinders from the glass tubes is easy.     

Absence of direct action of insulin on metabolism of the isolated perfused rat brain

The ability of insulin to influence directly the metabolism of the mammalian brain has been evaluated with an isolated, perfused rat brain preparation. Insulin was added to the perfusion fluid or was injected into the rat from which the isolated brain preparation was subsequently made. The spontaneous electrical activity of the brain, the rate of cerebral glucose consumption and the rate of efflux of K⁺ from the brain were not affected by insulin. We conclude that insulin either does not act directly on the brain or that its action is very small and/or very slow in comparison with its action on other tissues. We suggest that the effects on brain metabolism reported to occur after administering insulin and glucose to the intact animal may be secondary to the large stimulation of the metabolism of the liver and/or other organs.     

A genetic and biochemical study of streptomycin- and spectinomycin-resistance in Salmonella typhimurium

Summary In Salmonella typhimurium, streptomycin resistance can occur by mutation at the strA or the strB mutants have altered ribosomes which are refractory to the drug in cell-free amino acid incorporation systems, and in ³H-dihydrostreptomycin binding studies. StrB mutants, unlike the strA mutants, are resistant to several aminoglycoside antibiotics and resistance is not due to a mutational change in the cell's protein synthetic machinery. Spectinomycin resistant mutants of S. typhimurium also fall into two classes, only one of which is ribosomal in mechanism. The spcA and spcB loci are closely linked to strA, aroC, and argG on the S. typhimurium linkage map.     

Cellular Studies of X-Ray Induced Inhibition of Lens Regeneration

Whole-body X-irradiation of adult newts 0 to 3 days after lentectomy inhibits transformation of the dorsal iris epithelium into a lens in all cases. The first question raised was whether irradiation affects infiltration of the iris area by macrophages, and the phagocytic activities of these cell types in the iris epithelium (prominent phenomena in this system). The number of macrophages infiltrating into the iris epithelium, and their phagocytic activities (indicated by uptake of melanosomes) were not affected by irradiation under those conditions. The second group of experiments concerns the possible effects of irradiation on DNA replication of iris epithelial cells, which become transformed into lens cells in the non-irradiated system. Autoradiographic studies of iris epithelial cells in vivo revealed a significant suppressive effect of irradiation on the frequencies of cells incorporating ³H-thymidine 7 and 14 days after lentectomy. When autoradiography was applied to the primary pure culture of iris epithelial cells at different time intervals after the start of culture and irradiation in vitro , significant and persistent reduction of cell labelling due to irradiation, was demonstrated. Multiplication of spread cells in the iris epithelial culture was strongly and persistently inhibited throughout a period of 2 months. Inhibition of cell labelling and of cell multiplication was always accompanied by reduction in the extent of de-pigmentation of iris epithelial cells. De-pigmentation is one of the requirements for the cells become transformed into lens cells. The possible mechanism of radiation-induced inhibition of lens regeneration is discussed.     

Cytotoxic T lymphocyte triggering via CD16 is regulated by CD3 and CD8 antigens. Studies with T cell receptor (TCR)-alpha beta+/CD3+16+ and TCR-gamma delta+/CD3+16+ granular lymphocytes

The role of CD3 and CD8 Ag in CD16-mediated CTL triggering was studied in TCR-alpha beta+ and TCR-gamma delta+ granular lymphocytes (GL). In TCR-alpha beta+/CD3+4-8+16+ GL obtained from patients with GL-proliferative disorders, antibody-dependent cellular cytotoxicity was inhibited by anti-CD3 and anti-CD8 mAb. Anti-CD3 mAb also inhibited antibody-dependent cellular cytotoxicity activity of TCR-gamma delta+/CD3+4-8-16+ GL from a patient and that of TCR-gamma delta+/CD3+4-8+/-16+ T cell clones established from patients with proliferating TCR-gamma delta+ GL. In TCR-gamma delta+ T cell clones, cytotoxicity against Fc gamma R+ targets was induced by stimulation of CD16 Ag with anti-CD16 mAb, and such cytotoxicity was also inhibited by anti-CD3 mAb. These results indicate that CD3 and CD8 molecules play a regulatory role in CD16-mediated CTL triggering.     

Production and characterization of recombinant human neutrophil chemotactic factor

A putative mature human neutrophil chemotactic factor (NCF) corresponding to the C-terminal 72 amino acids of its precursor was directly produced in Escherichia coli by recombinant DNA technology. Human NCF was present in both the soluble and insoluble protein fractions of the homogenate of host cells, and it was partially purified as a water-soluble polypeptide from both fractions, separately. The partially purified NCF preparation was highly purified to an endotoxin-free homogeneous polypeptide by means of CM-Sepharose CL-6B column chromatography and gel filtration on Toyopearl HW-55. No difference between the human NCF preparations purified from both starting materials could be found concerning purity, primary structure, solubility, molecular weight, and chemotactic activity for human neutrophils. The amino acid sequence of recombinant human NCF was identical to the sequence deduced from the cDNA sequence. A methionine residue due to the translation initiation codon was removed. Recombinant human NCF was found to be biologically active and to exhibit chemotactic activity for human neutrophils in vitro and cause a neutrophil infiltration in vivo in mice.     

Pathway for the synthesis of triacylglycerols from monogalactosyldiacylglycerols in ozone-fumigated spinach leaves

When the upper leaf surface of spinach (Spinacia oleracea L.) plants was treated with [1-(14)C]acetate and grown for 2 days, (14)C was effectively incorporated into acyl moieties of leaf lipids in ratios approximately their composition by mass. Fumigation of the plants with ozone (0.5 microliter per liter) caused a redistribution of (14)C among lipid classes, i.e. a marked increase of (14)C content in triacylglycerol (TG) and 1,2-diacylglycerol (1,2-DG) and a decrease of label in monogalactosyldiacylglycerol (MGDG) without affecting (14)C distribution in leaf fatty acids. Label in both TG and 1,2-DG was found predominantly in their polyene molecular species. Since MGDG consists of similar polyene molecular species, the results indicate the synthesis of TG from MGDG via 1,2-DG. Label was also accumulated in tri- and tetragalactosyldiacylglycerol, products of galactolipid:galactolipid galactosyltransferase (GGGT). Moreover, there was a close relation between increases in the amounts of TG and the oligogalactolipids in ozonetreated leaves. These results indicate that MGDG was converted to 1,2-DG by GGGT and then to TG. In intact chloroplasts isolated from ozone-treated leaves, there was an enhanced production of free fatty acid (FFA), which was diminished by the addition of coenzyme A (CoA) and ATP, indicating that ozone stimulated the hydrolysis of MGDG to liberate FFA, which was in turn converted to acyl-CoA. The final step of TG synthesis, acylation of 1,2-DG with acyl-CoA, was confirmed by feeding with [1-(14)C]linolenic acid in leaf discs excised from ozone-fumigated leaves; (14)C was effectively incorporated into TG but not into 1,2-DG. These results demonstrate the synthesis of TG from 1,2-DG and FFA which were liberated from MGDG in ozone-fumigated spinach leaves.