Postnatal D2-CAM/N-CAM Sialylation State Is Controlled by a Developmentally Regulated Golgi Sialyltransferase

Abstract: Golgi-enriched fractions have been isolated from rat brain of increasing postnatal age and defined by electron microscopy and distribution of marker enzymes. The expression of sialyltransferase activity associated with these fractions has been demonstrated to developmentally decrease and this appeared to be, in part, dependent on endogenous competitive inhibition. The developmental regulation of this activity paralleled the sialylation state of the neural cell adhesion molecule (D2-CAM/N-CAM) and could be demonstrated to be capable of endogenously sialylating this protein in the isolated Golgi fractions. In 12-day-old animals the majority of the transferred [¹⁴C]sialic acid was found to be associated with the high-molecular-weight [>200 kilodaltons (kd)] form of D2-CAM/N-CAM, indicative of the protein having been heavily sialylated. Sialylation of the individual D2-CAM/N-CAM polypeptides was also demonstrated in both 12-day and adult animals and transfer was evident only in the 180-kd and 115-kd components and not in the 140-kd component. In contrast, Golgi-enriched fractions prepared from adult animals showed little capability of heavily sialylating D2-CAM/N-CAM to any significant extent.     

Effect of carbon source on enzymes involved in glycerol metabolism inNeurospora crassa

Specific activities of eight enzymes involved in glycerol metabolism were determined in crude extracts of three strains ofNeurospora crassa after growth on six different carbon sources. One of the strains was wild type, which grew poorly on glycerol as sole carbon source; the other two were mutant strains which were efficient glycerol utilizers. A possible basis for this greater effeciency of glycerol utilization was catabolite repression of glyceraldehyde kinase by glycerol in wild type, and two-fold higher glycerate kinase activity in the mutant strains after growth on glycerol, thus apparently allowing two routes for glyceraldehyde to enter the glycolytic pathway in the mutant strains but only one in wild type. The preferential entry of glyceraldehyde to the glycolytic pathway through glycerate was suggested by the lack of glyceraldehyde kinase in all three strains after growth on one or more of the carbon sources and the generally higher levels of aldehyde dehydrogenase and of glycerate kinase than of glyceraldehyde kinase.     

Mitogenic response of rat lung and tracheal epithelial cells in monolayer primary cultures—Modulation of TNF-α expression

Epithelial cells isolated from rat lung and trachea were grown on monolayers and their response to a number of hormones and growth factors were studied. Maximum proliferative response in serum containing media was observed when insulin, cholera toxin and cortisol were present together. However, these additives when present independently showed a marginal response. The synergism, due to these factors in promoting growth was seen very early in culture (day 4) as shown by thymidine labelling studies, On examining the indices of early mitogenesis, such as the expression ofc-myc, our data suggests that these factors stimulate the expression ofc-myc within 4 h. With respect to expression of TNF-α mRNA, this study suggests a possible modulation of TNF-α expression in response to these mitogens that stimulate proliferation maximally. Whether this expression of TNF-α by these epithelial cells is due to a maximal proliferative stimulus and/or is an early step in the cascade of intracellular signalling events is to be investigated in detail.     

Potassium and Taurine Release Are Highly Correlated with Regulatory Volume Decrease in Neonatal Primary Rat Astrocyte Cultures

Abstract: Neonatal rat primary astrocyte cultures were swollen by exposure to hypotonic buffer. Using an electrical impedance method for determination of cell volume coupled with on-line measurements of efflux of radioactive ions or amino acids, we have investigated the role of K⁺ (using ⁸⁶Rb), taurine, and d -aspartate (an analogue of glutamate) in regulatory volume decrease (RVD). Addition of 1 m M quinine, 10 µ M nimodipine, 100 µ M BAPTA-AM, 10 µ M trifluoperazine, or a calcium-free buffer significantly ( p < 0.0001) inhibited RVD. This was accompanied by inhibition of ⁸⁶Rb release but an increase in d -[³H]-aspartate release, which was proportional to the degree to which RVD was inhibited. These results support a regulatory role for calcium in RVD and show that inhibition of calcium entry from the extracellular fluid, intracellular calcium sequestration, inhibition of calcium-activated K⁺ channels, and inhibition of calmodulin all inhibit RVD. Because d -[³H]aspartate efflux profiles increase as RVD is inhibited, it is unlikely that d -aspartate release is a main determinant of RVD. In contrast, [³H]taurine release was increased by 1 m M quinine and inhibited by 10 µ M trifluoperazine. The net release of K⁺ and taurine is highly correlated with the degree of RVD, implicating a regulatory role for both K⁺ and taurine release in RVD.     

Expression and function of a retinoic acid receptor in budding ascidians

 Retinoic acid is thought to induce transdifferentiation of multipotent epithelial stem cells in the developing buds of the ascidian Polyandrocarpa misakiensis. We isolated a cDNA clone from this species, named PmRAR, encoding a retinoic acid receptor (RAR) homologue. PmRAR clusters with other RARs on phylogenetic trees constructed by three different methods. Within the cluster, PmRAR is on a separate branch from all the subtypes of RARs, suggesting that RAR subtypes arose in the ancestral vertebrates after divergence of vertebrates and urochordates. The embryos of another ascidian species Ciona intestinalis were co-electroporated with a mixture of a PmRAR expression vector and a lacZ reporter plasmid containing vertebrate-type retinoic acid response elements. The expression of lacZ depended on the presence of both retinoic acid and PmRAR, suggesting that PmRAR is a functional receptor. PmRAR mRNA is expressed in the epidermis and mesenchyme cells of the Polyandrocarpa developing bud. The mRNA is not detectable in the mesenchyme cells in the adult body wall, but its expression can be induced by retinoic acid in vitro. These results suggest that the PmRAR is a mediator of retinoic acid signalling in transdifferentiation during asexual reproduction of protochordates. Received: 6 April 1998 / Accepted: 27 July 1998     

Immunoelectrophoretic approach to the metabolic regulation of glutamine synthetase in Rhodopseudomonas capsulata E1F1: role of glutamine

Anti-glutamine synthetase serum was raised in rabbits by injecting purified glutamine synthetase (GS) of the phototrophic bacterium Rhodopseudomonas capsulata E1F1. The antibodies were purified to monospecificity by immunoaffinity chromatography in GS-sepharose gel. These anti-GS antibodies were used to measure the antigen levels in crude extracts from bacteria, grown phototrophically with dinitrogen, nitrate, nitrite, ammonia, glutamate, glutamine or alanine as nitrogen sources. The amount of GS detected by rocket immunoelectrophoresis was proportional to Mn²⁺-dependent transferase activity measured in the crude extracts. Addition of GS inhibitor l-methionine-d,l-sulfoximine (MSX) to the actively growing cells promoted increased antigen levels, that were not found in the presence of glutamine or chloramphenicol. The ammonia-induced decrease in GS relative levels was reverted by MSX. GS levels remained constant when phototrophically growing cells were kept in the dark.Abbreviations GS glutamine synthetase - MOPS 2-(N-morpholine) propane sulfonate - MSX l-methionine-d,l-sulfoximine