Earthworm coelomocytes in vitro

Summary Contamination and low viability of earthworm coelomocytes in tissue culture have delayed in vitro studies. Using penicillin, streptomycin, tetracycline and Amphotericin B,Lumbricus terrestis coelomocytes were maintained viable and uncontaminated for 10 days at 15°C in medium L-15 supplemented with 5 to 10% fetal bovine serum. The coelomocytes survived for at least 10 days with 85% viability as assessed by trypan blue exclusion assays and phagocytosis of heat-killed yeast. Studies on the thymidine uptake, however, were negative. With the involvement of coelomocytes in tissue graft rejection, in vitro techniques can now be applied to study their capacity in the immune response. Supported in part by USPHS Research Grant 1 RO 1 HD09333-01 to E. L. Cooper.     

Amylolytic enzymes from Aspergillus hennebergi (A. niger Group): Purification and characterization of amylases from solid and liquid cultures

Two glucoamylases (I and II) were produced during solid-state culture of Aspergillus hennebergi (A. niger group) on cassava meal, whereas one glucoamylase and one alpha-amylase were synthesized by the mould in liquid culture. These glucoamylases were acidic proteins with thermotolerant activities. Glucoamylase I was not a glycoprotein, but glucoamylase II and the glucoamylase from liquid cultures contained 15% of sugars. The alpha-amylase was significantly less thermotolerant and of smaller molecular weight. The influence of culture conditions on the production of different amylases by the same Aspergillus strain on the same substrate is discussed.     

Synergistic Regulation of Cytosolic Ca2+ Concentration in Mouse Astrocytes by NK1 Tachykinin and Adenosine Agonists

The effects on cytosolic Ca2+ concentration of 2-chloroadenosine and [L-Pro9]-substance P, a selective agonist of NK1 receptors, were investigated on astrocytes from embryonic mice in primary culture. Cells responded to [L-Pro9]-substance P with a transitory increase in cytosolic Ca2+ which was of shorter duration when external Ca2+ was removed. A transient response to 2-chloroadenosine alone occurred. When simultaneously applied, [L-Pro9]-substance P and 2-chloroadenosine evoked a prolonged elevation of cytosolic Ca2+ (up to 30 min). This phenomenon was dependent on the presence of extracellular Ca2+, but insensitive to dihydropyridines, La3+, and Co2+, excluding the implication of voltage-operated Ca2+ channels. Arachidonic acid also induced a sustained elevation of cytosolic Ca2+, but did not increase further the response evoked by [L-Pro9]-substance P and 2-chloroadenosine. The activation of protein kinase C by a diacylglycerol analogue mimicked the effect of [L-Pro9]-substance P in potentiating the 2-chloroadenosine-evoked response. Like 2-chloroadenosine, pinacidil, which hyperpolarizes the cells by opening K+ channels, prolonged the elevation of cytosolic Ca2+ concentration induced by [L-Pro9]-substance P. Conversely, depolarization with 50 mM KCl canceled the effects of either pinacidil or 2-chloroadenosine applied with [L-Pro9]-substance P. Pertussis toxin pretreatment suppressed all the effects induced by 2-chloroadenosine.     

The use of photosynthesis inhibitor (DCMU) for in situ metabolic and primary production studies on soft bottom benthos

A selective chemical photosynthesis inhibitor, DCMU (Dichorophenyl-dimethylurea), dissolved in DMSO (Dimethyl sulfoxide) was substituted for the dark incubation method commonly used to measure the oxygen consumption in metabolic and primary production studies. We compared oxygen fluxes during light incubations with DCMU and dark incubations procedure, on soft bottom benthos. For this purpose, we studied the effects of different DCMU concentrations. A concentration of 5 · 10^–5 mol l^–1 inside a clear incubation enclosure completely inhibits photosynthesis without affecting the metabolism of soft bottom benthos.     

Isolation and phenotypic analysis of conditional-lethal, linker-insertion mutations in the gene encoding the largest subunit of RNA polymerase II in Saccharomyces cerevisme

Summary Linker-insertion mutagenesis was used to isolate mutations in the Saccharomyces cerevisiae gene encoding the largest subunit of RNA polymerase II (RP021, also called RPBI). The mutant rpo21 alleles carried on a plamid were introduced into a haploid yeast strain that conditionally expresses RP021 from the inducible promoter pGAL10. Growth of this strain on medium containing glucose is sustained only if the plasmid-borne rpo21 allele encodes a functional protein. Of nineteen linker-insertion alleles tested, five (rpo21-4 to –8) were found that impose a temperature-sensitive (ts) lethal phenotype on yeast cells. Four of these five is alleles encode mutant proteins in which the site of insertion lies near one of the regions of the largest subunit that have been conserved during evolution. Two of the is mutants (rpo21-4 and rpo21-7) display pleiotropic phenotypes, including an auxotrophy for inositol and a decreased proliferation rate at the permissive temperature. The functional relationship between RP021 and RP026, the gene encoding the 17.9 kDa subunit shared by RNA polymerases 1, 11, and III was investigated by determining the ability of increased dosage of RP026 to suppress the is phenotype imposed by rpo21-4 to –8. Suppression of the is defect was specific for the rpo21-4 allele and was accompanied by co-suppression of the inositol auxotrophy. These results suggest that mutations in the largest subunit of RNA polymerase II can have profound effects on the expression of specific subsets of genes, such as those involved in the metabolism of inositol. In the rpo21-4 mutant, these pleiotropic phenotypes can be attributed to a defective interaction between the largest subunit and the RP026 subunit of RNA polymerase II.     

Glucocorticoid hormones increase the activity of γ-glutamyltransferase in a highly differentiated hepatoma cell line

γ-Glutamyltransferase activity was detected in the plasma membrane of the highly differentiated hepatoma cell line Fao, (0.93 mU/mg cell protein). Dexamethasone (1 μM) provoked a 2–3-fold increase in the activity of the enzyme in the presence of fetal calf serum. Maximal induction occurred 48–72 h after addition of the glucocorticoid to the cell culture medium. The hormonal specificity was demonstrated by the relative potencies of several glucocorticoids and sex steroids: hydrocortisone and corticosterone increased γ-glutamyltransferase activity while tetrahydrocorticosterone and all sex steroids tested were ineffective. The effect of dexamethasone on γ-glutamyltransferase activity was specific since the activities of several other plasma membrane enzymes were not modified. The mechanism of the dexamethasone-induced increase in γ-glutamyltransferase activity was neither by modification of the affinity of the enzyme for its substrates nor by alteration of the subcellular distribution of the enzyme. This increase was prevented by cycloheximide and actinomycin D. The data presented are consistent with a specific glucocorticoid receptor-mediated induction of γ-glutamyltransferase activity in Fao cells. The kinetic parameters of the induction process by glucocorticoids are very similar to those found in adult rat liver. These results suggest that the Fao cell line is a very convenient system for the study of the molecular mechanisms of glucocorticoid effects on differentiated cells.     

Modifications of ganglioside composition in peripheral nerve of myelin deficient trembler mutant mouse

Ganglioside distribution was studied in peripheral nerves of normal controls and those of Trembler mutant mouse with defect in Schwann cell differentiation and myelination. Neuraminic acid content was considerably decreased in the mutant. Ganglioside distribution as evaluated by densitometry of resorcinol positive spots on thin-layer chromatography revealed a major peak for GDl_a in normal controls. In the mutant, the relative proportion was modified with qualitative modifications in the GDl_a area and a tremendous increase in GM₃ content. The relation with the intense Schwann cell proliferation observed in the mutant is discussed.