Absence of ganglioside GM1 in astroglial cells from newborn rat brain

An immunohistochemical method utilizing anti-ganglioside GM1 antiserum combined with the peroxidase-antiperoxidase technique was applied to a mixed cell population in primary cultures of newborn rat brain. Ganglioside GM1 was demonstrated to be present in neurons and oligodendroglia, but was absent in astroglia. This demonstration was confirmed using a newly developed biotinylated choleragen-avidin-peroxidase procedure. Primary cultures from newborn rat brain cells that had been subjected to a single treatment with trypsin (first passage) and then cultured for 14 days were predominately (95%) composed of astrocytes that stained positively for glial fibrillary acidic protein but were negative for GM1 ganglioside. This preparation contained only 0.34 nmol ganglioside NeuNAc per mg protein compared to 23.9 nmol gangliosidic NeuNAc/mg protein for a five day culture of newborn rat brain mixed cell culture that had not been subjected to passage. Prolongation of culture time from 5 to 21 days in the latter preparation reduced the ganglioside NeuNAc content to 4.9 nmol gangliosidic NeuNAc/mg protein as the proportion of astrocytes in the culture increased. Ganglioside GM1 could not be detected by TLC analysis of the lipid extract obtained from the “pure” astrocyte culture, although small amounts of GM3 and some polysialogangliosides were detected. About half of the label incorporated upon 24 h incubation of astrocytes in the presence of $\text{N-[}{}^3\text{H]acetylmannosammine}$ appeared in ganglioside GM3. It is concluded that astrocytes in mixed cell primary cultures from newborn rat brain, as well as astrocytes in astroglial preparations derived from such cultures, do not contain ganglioside GM1.     

Measurement of the relative electron-transport capacity of Photosystem I and Photosystem II in spinach chloroplasts

The ratio of Photosystem (PS) II to PS I electron-transport capacity in spinach chloroplasts was compared from reaction-center and steady-state rate measurements. The reaction-center electron-transport capacity was based upon both the relative concentrations of the PS II_α, PS II_β and PS I centers, and the number of chlorophyll molecules associated with each type of center. The reaction-center ratio of total PS II to PS I electron-transport capacity was about 1.8:1. Steady-state electron-transport capacity data were obtained from the rate of light-induced absorbance-change measurements in the presence of ferredoxin-NADP⁺, potassium ferricyanide and 2,5-dimethylbenzoquinone (DMQ). A new method was developed for determining the partition of reduced DMQ between the thylakoid membrane and the surrounding aqueous phase. The ratio of membrane-bound to aqueous DMQH₂ was experimentally determined to be 1.3:1. When used at low concentrations (200 μM), potassium ferricyanide is shown to be strictly a PS I electron acceptor. At concentrations higher than 200 μM, ferricyanide intercepted electrons from the reducing side of PS II as well. The experimental rates of electron flow through PS II and PS I defined a PS II/PS I electron-transport capacity ratio of 1.6:1.     

A 250-kilodalton cellular protein is induced by progestins in two human breast cancer cell lines MCF7 and T47D

We have studied the effect of R5020, a synthetic progestin, on the biosynthesis of cellular proteins extracted from the MCF7 and T47D human breast cancer cells, using gel electrophoresis. R5020 stimulates the synthesis, as measured after [35S]-methionine labelling, and the accumulation, as shown by silver staining, of a protein of molecular weight approximately equal to 250,000. The increase of the labelled 250-kilodalton protein was rapid (3 hours) and after 3 days this protein represented approximately equal to 6% of the total cellular proteins (approximately equal to 1 microgram/150,000 cells). The induction of the 250-kilodalton protein was obtained by physiologically active concentrations of several progestins and high concentrations of 5 alpha-dihydrotestosterone but not by estradiol or dexamethasone. It was inhibited by R486 , a progestin antagonist, but not by flutamide, an androgen antagonist. These results indicate a mediation by the progesterone receptor. The 250-kilodalton protein appears to be an excellent probe to study in cell culture the mechanism of action of progestin on human cells.     

Three-step preparation and purification of phosphorus-33-labeled creatine phosphate of high specific activity

Rabbit heart mitochondria were used as a source of enzymes for the synthesis of phosphoruslabeled creatine phosphate. This method is based on the coupled reaction between mitochondrial oxidative phosphorylation and mitochondrial-bound creatine kinase. It is possible to convert more than 90% of the inorganic phosphate (P_i) to creatine phosphate. The method used only small amounts of adenine nucleotides which led to a product with only slight nucleotide contamination. This could be removed by activated charcoal extraction. For further purification, a method for the removal of residual P_i is described.     

Changes in key regulatory enzymes of hepatic carbohydrate metabolism after glucose loading of starved rats

When glucose was given to starved rats there was an increase in both 6-phosphofructo 2-kinase and pyruvate kinase activity and a decrease in fructose 2,6-bisphosphatase activity 30 min and 60 min later. These changes were accompanied by an increase in glycogen deposition and by modest, but significant increases in fructose 2,6-bisphosphate levels at the same time. Metabolite measurements indicated that flux through 6-phosphofructo 1-kinase and pyruvate kinase were increased. These results suggest that although glycogen deposition may occur via the gluconeogenic pathway, glycolysis is activated at the same time by changes in the phosphorylation state of key regulatory enzymes as well as by the small rise in fructose 2,6-bisphosphate.     

Carrier mediated GABA translocation into rat brain mitochondria

GABA added to rat brain mitochondria causes oxidation of intramitochondrial NAD(P)H as well as inducing glutamate efflux from the mitochondrial matrix. The rate of NAD(P)H oxidation shows saturation characteristics, depends on GABA transport across the mitochondrial membrane and is inhibited by non-penetrant compounds and by the metal-complexing agent bathophenanthroline. These results show the existence of a specific GABA carrier. Inhibition studies strongly suggest the existence of two separate binding sites, namely the GABA binding site and the dicarboxylates binding site, as well as suggest the presence of a metal ion (ions) at GABA binding site. The occurrence of a GABA/GLUTAMATE antiport is proposed which allows a cyclical route to account for GABA synthesis and degradation in brain.     

Fructose 2,6-bisphosphate, sugar phosphates and adenine nucleotides in the regulation of glucose metabolism in the lactating rat mammary gland

Fructose 2,6-bisphosphate is present in the rat mammary gland, rising from a value of 1.4 nmol/g in pregnancy to 4.3 nmol/g tissue at 14 days lactation; the equivalent values calculated/ml intracellular water are 5.2 and 11.6 nmol, respectively. The tissue content of fructose 6-phosphate, fructose 1,6-bisphosphate, ATP and phosphoenolpyruvate remain relatively constant in the transition from pregnancy to the height of lactation. The changes in AMP, cyclic AMP, and citrate content of the mammary gland during lactation are such as to promote an increase in fructose 2,6-bisphosphate formation and flux through phosphofructokinase.     

Inositol trisphosphate activates pyruvate dehydrogenase in isolated fat cells

Inositol trisphosphate, when added to permeabilized rat fat cells, led to a several-fold increase of pyruvate dehydrogenase activity due to conversion of the inactive phospho form (PDHb) to the active, dephospho form (PDHa). It is suggested that inositol trisphosphate, probably through intracellular Ca2+-mobilisation, acts as a physiological mediator of insulin for activation of the mitochondrial PDH-complex.     

Lectin-like binding of Bacillus thuringiensis var. kurstaki lepidopteran-specific toxin is an initial step in insecticidal action

The two delta-endotoxins comprising the Bacillus thuringiensis var. kurstaki HD1 insecticidal protein crystal were separated. The lepidopteran-specific protoxin was activated in vitro and its mechanism of action investigated. Toxicity towards Choristoneura fumiferana CF1 cells was specifically inhibited by preincubation of the toxin with N-acetylgalactosamine and N-acetylneuraminic acid. The lectins soybean agglutinin and wheat germ agglutinin, which bind N-acetylgalactosamine, also inhibited toxicity. Since N-acetylneuraminic acid is not known to occur in insects, these results suggest that the toxin may recognise a specific plasma membrane glycoconjugate receptor with a terminal N-acetylgalactosamine residue.     

Microbial mineralization of organic phosphate in soil

Summary Phosphate-dissolving microorganisms were isolated from non-rhizosphere and rhizosphere of plants. These isolates included bacteria, fungi and actinomycetes. In broth cultures, Gram-negative short rod,Bacillus andStreptomyces species were found to be more active in solubilizing phosphate thanAspergillus, Penicillium, Proteus, Serratia, Pseudomonas andMicrococcus spp. The sterile soils mixed with isolated pure culture showed slower mineralization of organic phosphate than that of non-sterile soil samples at all incubation periods. Maximum amount of phosphate mineralization by isolated microorganisms were obtained at the 60th and the 75th day of incubation in sterile and non-sterile soils respectively. The mixed cultures were most effective in mineralizing organic phosphate and individuallyBacillus sp. could be ranked next to mixed cultures. Species ofPseudomonas andMicrococcus were almost the same as that of the control under both sterile and non-sterile conditions.