Immunoaffinity purification of type I protein kinase C

We designed a simple procedure for the purification of type I protein kinase C, using immunoaffinity chromatography with a monoclonal antibody, MC-1b, obtained by rescreening hybridoma cells available for an affinity ligand. Western blotting demonstrated that MC-1b specifically reacted with type I protein kinase C, and the enzyme molecule dissociated from MC-1b-coupled Sepharose 4B with mild eluants such as thiocyanate retained the kinase activity. A 1148-fold purification was achieved and 210 micrograms of type I protein kinase C was obtained from three rabbit brains, by means of a two-step procedure, using DEAE-cellulose and immunoaffinity chromatography. The resultant preparation was homogeneous, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis hydroxylapatite chromatography, and immunological analysis using MC-1a, MC-2a, and MC-3a.     

Retinal Gangliosides in RCS Mutant Rats

Abstract: The distribution of retinal gangliosides was studied in normal and mutant rats with retinal dystrophy at 30 and 180 days of age. The loss of photoreceptor cells in the retinal dystrophic RCS rats was not associated with a significant reduction in the relative distribution of any of the major retinal gangliosides. The loss of photoreceptors, however, caused a marked increase in total retinal ganglioside concentration. These findings suggest that photoreceptor cells contain a low concentration of gangliosides and that no major retinal ganglioside is localized or concentrated in these cells. The cellular localization and function of the most abundant retinal ganglioside, G_D3, is discussed.     

Differential Cellular Enrichment of Gangliosides in the Mouse Cerebellum: Analysis Using Neurological Mutants

Abstract: The cellular distribution of gangliosides in the cerebellum was studied in a series of adult mouse mutants that lose specific populations of neurons. The weaver ( wv ) mutation destroys the vast majority of granule cells, whereas the Purkinje cell degeneration mutation ( pcd ) destroys the vast majority of Purkinje cells. The staggerer ( sg ) and lurcher ( Lc ) mutations, on the other hand, destroy the vast majority of both granule and Purkinje cells. A proliferation of reactive glial cells, which occurs as a consequence of neuronal loss, has been reported in the sg/sg and pcd/pcd mutants, but not in the wv/wv mutant. Compared with the normal (+/+) mice, the concentration (μg/100 mg dry weight) of G_D1a was significantly reduced in those mutants that lost granule cells, but was not reduced in the pcd/pcd mutant. The concentration of G_TIa, on the other hand, was significantly reduced in those mutants that lost Purkinje cells, but was not reduced in the wv/wv mutant. A significant elevation in the concentration of G_D3, which may be related to the proliferation of reactive glial cells, was observed in the pcd/pcd, sglsg , and Lc /+ mutants, but was not observed in the wv/wv mutant. Because these ganglioside abnormalities were confined to the cerebellum, they cannot result from genetic defects in ganglioside metabolism. Instead, these abnormalities result from a differential enrichment of gangliosides in neural membranes. Our findings suggest that G_DT1a is more heavily concentrated in granule cells than Purkinje cells, whereas the opposite appears true for G_Tla. It also appears that GD3 is enriched in reactive glial cells and may play an important role during the morphological transformation of neural membranes.     

A granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by carrageenin-induced inflammatory cells of mice

Seven days after subcutaneous injection of 2% carrageenin solution in mice, the induced inflammatory tissue was isolated and treated with 0.1% trypsin. When the dispersed cells were incubated in a nutrient medium containing 5--20% calf serum, the cells grew adhering to the culture-dish surface and colony-stimulating factor (CSF) was accumulated in the medium gradually. Addition of lipopolysaccharide (Escherichia coli endotoxin) in the cell culture did not enhance the CSF production. It was shown by isoelectrofocusing that the majority of the produced CSF was an acidic molecule (pI = 3.8), while the treatment of this CSF with neuraminidase yielded a less acidic CSF species (pI = 5.1). Upon gel-filtration chromatography in the presence of 6 M guanidine HCl, the CSF exhibited an apparent molecular weight of 42,000. On the other hand, polyacrylamide gel-electrophoresis in the presence of 0.1% sodium dodecyl sulfate gave a molecular weight estimate of 65,000. Microscopic examination of the bone marrow cell culture showed that about one-third of the colonies were granulocytic. Addition of prostaglandin E₁(PGE₁) in the bone marrow cell culture significantly inhibited the action of the CSF, while prostaglandin D₂ was less inhibitory than PGE₁. The result suggests that the cells isolated from the inflammatory tissue are capable of producing CSF, which may have a role for proliferation and maturation of the mononuclear phagocytes at the site of inflammation.     

Plasma levels of 16 alpha-hydroxypregnenolone, 16 alpha-hydroxyprogesterone and 16 alpha-hydroxydehydroepiandrosterone in the fetus and neonates.

Simultaneous determination of unconjugated 16 alpha-hydroxypregnenolone (16 alphaOH-Preg), 16 alpha-hydroxyprogesterone (16 alphaOH-Prog) and 16 alpha-hydroxydehydroepiandrosterone (16 alphaOH-DHEA) in fetal and neonatal plasma was performed utilizing a newly developed radioimmunoassay. In all neonates, the three 16 alpha-hydroxysteroid levels were consistently higher in umbilical cord plasma than in the maternal peripheral circulation. 16 alpha-OH-Preg in the umbilical arterial plasma increased from 11.2 +/- 3.1 at 24 weeks to 29.7 +/- 12.0 ng/ml at term, 16 alphaOH-Prog from 15.5 +/- 3.2 to 34.3 +/- 11.0 ng/ml and 16 alphaOH-DHEA from 5.1 +/- 1.2 to 5.9 +/- 1.0 ng/ml. In the anencephalic neonates, only 16 alphaOH-Preg showed an increase pattern under ACTH priming. 16 alpha-OH-Preg levels for normal full term neonates remain relatively constant at the first 24 hr and show a slight decrease at 3 days post partum. In small full term neonates, 16 alphaOH-Preg levels in umbilical arterial plasma are considerably higher than in normal neonates and remain at roughly equivalent levels for the first 5 days post partum. 16 alphaOH-Prog and 16 alphaOH-DHEA levels in umbilical arterial plasma in normal and small full term neonates are almost equal and both groups show a rapid decrease during the first 24 hr. Comparison with findings of the three 16 alpha-hydroxysteroids in fetal and neonatal plasma is discussed.     

An improved method for the syntheses of p-aminophenyl 1-thio-β-d-glycosides

The condensation of the appropriate acetylglycosyl bromides with p-amino-benzenethiol in the presence of sodium methoxide afforded p-aminophenyl 1-thio-β-d-glucopyranoside, 1-thio-β-d-galactopyranoside, 1-thio-β-d-xylopyranoside, and 2-acetamido-2-deoxy-1-thio-β-d-glucopyranoside. p-Aminophenyl 1-thio-β-d-glucopyranosiduronic acid was synthesized by condensation of methyl (2,3,4-tri-o-acetyl-β-d-glucopyranosyl bromide)uronate with p-aminobenzenethiol, followed by saponification with sodium hydroxide.     

Oxygen concentration-dependent induction of a 140-kDa protein in magnetic bacterium Magnetospirillum magnetotacticum MS-1

Abstract The magnetic bacterium Magnetospirillum magnetotacticum prefers a microaerobic habitat and should be able to sense oxygen. Therefore, the bacterium was cultured under atmospheres containing 0–5% O₂ and analyzed for oxygen-dependent changes in the levels of its protein components by sodium dodecyl sulfate-polyccrylamide gel electrophoresis (SDS-PAGE). The analysis revealed a marked anaerobic induction of a 140-kDa protein, which was suppressed when M. magnetotacticum was switched from microaerobic (<1% O₂) to aerobic (>1% O₂) growth conditions. Although its function remains to be determined, the 140-kDa protein may serve as a useful tool to gain insight into the physiology of the organism.     

A chitin sulfate-like polysaccharide from the test of the tunicate Halocynthia roretzi

Considerable amounts of water-soluble polysaccharides were found in the test of the tunicate Halocynthia roretzi. After fractionation with ethanol, a chitin sulfate-like polysaccharide was isolated. From the results of chemical analysis, optical rotation, infrared spectrum and alkaline treatment, it is suggested that the predominant structure for this polysaccharide is (1→4)-2-acetamido-2-deoxy-6-O-sulfo-β-D-glucopyranan.