Poly- and Monoclonal Antibodies Against Recombinant Rat Brain Calbindin D-28K Were Produced to Map Its Selective Distribution in the Central Nervous System

Many processes in the CNS depend on calcium. The calcium signal is transduced into an intracellular response via Ca2(+)-binding proteins, including calbindin D-28K. In many laboratories, polyclonal antibodies against chicken intestinal calbindin D-28K have been used to study its localization in the brain (normal and degenerated) of various species, including humans, but some of these antisera cross-reacted with other proteins, including calretinin. We purified recombinant rat brain calbindin D-28K to raise antisera in rabbits and purified a recombinant rat-chicken calbindin D-28K hybrid protein to immunize mice for the generation of monoclonal antibodies. These antisera were highly specific for calbindin D-28K, as demonstrated by two-dimensional Western blotting analysis. Immunohistochemical analyses combined with in situ hybridization studies demonstrated that calbindin D-28K in the Purkinje cells of the cerebellum is independent of vitamin D. The antibodies described here will be important tools for studying the regulation of expression of calbindin D-28K and its biological function in the brain and in the PNS.     

The brain organization of butterflyfishes

Synopsis The encephalization indices of angelfishes (Pomacanthidae) and butterflyfishes (Chaetodontidae) are typical of advanced perciform fishes: both families lie in the upper part of the polygon of teleost indices. The chaetodontids seem to be a little more encephalized than pomacanthids. The general morphology of the brains in both families is very similar: small olfactory bulbs, large optic tectum and a cerebellum which covers the brain structures in front of it like a cap. This morphology is shared by another family of the coral reef biotope, the Acanthuridae. The histological architecture is also typical of advanced teleosts, with a cortex-like pallium, a laminated nucleus geniculatus (= pretectalis superficialis), a complex valvula cerebelli and a corpus glomerulosum with a clear neuropile centre. The quantitative analysis of the main subdivisions of the brain, either from relative volumes or from indices, shows small olfactory bulbs (microsmy) but important telencephalic and diencephalic centres, large tectal centres (vision) and large cerebellum (precise locomotion). Many of these peculiarities are shared by other fishes inhabiting coral reefs. The differences between the two families seem to be primarily correlated with food habits: the angelfishes, which are sponge-feeders and may have an overweight due to the ballast of the sponge-skeleton in their digestive tract, and which do not need either such good vision or such precise locomotion to pick up their prey, could be a little less encephalized than the butterflyfishes.     

Characterization and co-culture of novel nontransformed cell lines derived from rat endometrial epithelium and stroma

Summary Normal and neoplastic growth of epithelial cells depends on mutual interactions between epithelial and stromal cells. As a tool for the study of the underlying molecular mechanisms, we have developed temperature-sensitive, nontransformed cell lines derived from rat uterine epithelium and stroma by transfecting primary cultures with a temperature-sensitive mutant of the SV40 large T antigen. The epithelial and stromal cell lines obtained shared relevant morphological characteristics with the primary cells from which they were derived. Immunocytochemical analysis showed that the epithelial cell lines expressed the intermediate filament cytokeratin, whereas the stromal lines expressed the intermediate filament vimentin. Alkaline phosphatase activity was present in all cell lines examined. All cell lines were anchorage dependent and did not form foci. One epithelial cell line expressed oxytocin mRNA, a gene product recently shown to be highly expressed in vivo in the uterine epithelium at term. If grown on Matrigel, this cell line formed domelike structures, a further characteristic of its differentiated phenotype. In an attempt to reconstitute an endometrium in vitro, epithelial cells were seeded on top of a layer of stromal cells. Paraffin cross sections showed that this in vitro system consisted of a bilayer structure. Four to five cuboidal epithelial cells were typically anchored atop one stromal cell, forming an endometriumlike tissue. The present in vitro system should provide a useful model for further studies on endometrial functions and epithelial/stromal cell interactions at a molecular level.     

Drosophila melanogaster acetylcholinesterase: Identification and expression of two mutations responsible for cold- and heat-sensitive phenotypes

Ace ^IJ29 and Ac ^IJ40 are cold- and heat-sensitive variants of the gene coding for acetylcholinesterase in Drosophila melanogaster. In the homozygous condition, these mutations are lethal when animals are raised at restrictive temperatures, i.e., below 23° C for Ace ^IJ29 or above 25° C for Ace ^IJ40. The coding regions of the gene in these mutants were sequenced and mutations changing Ser³⁷⁴ to Phe in Ace ^IJ29 and Pro⁷⁵ to Leu in Ace ^IJ40 were found. Acetylcholinesterases bearing these mutations were expressed in Xenopus oocytes and we found that these mutations decrease the secretion rate of the protein most probably by affecting its folding. This phenomenon is exacerbated at restrictive temperatures decreasing the amount of secreted acetylcholinesterase below the lethality threshold. In parallel, the substitution of the conserved Asp²⁴⁸ by an Asn residue completely inhibits the activity of the enzyme and its secretion, preventing the correct folding of the protein in a non-conditional manner.     

Alzheimer's disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells

Background: Paired helical filaments (PHFs) are a characteristic pathological feature of Alzheimer's disease; their principal component is the microtubule-associated protein tau. The tau in PHFs (PHF-tau) is hyperphosphorylated, but the cellular mechanisms responsible for this hyperphosphorylation have yet to be elucidated. A number of kinases, including mitogen-activated protein (MAP) kinase, glycogen synthase kinase (GSK)-3α, GSK-3β and cyclin-dependent kinase-5, phosphorylate recombinant tau in vitro so that it resembles PHF-tau as judged by its reactivity with a panel of antibodies capable of discriminating between normal tau and PHF-tau, and by a reduced electrophoretic mobility that is characteristic of PHF-tau. To determine whether MAP kinase, GSK-3α and GSK-3β can also induce Alzheimer's disease-like phosphorylation of tau in mammalian cells, we studied the phosphorylation status of tau in primary neuronal cultures and transfected COS cells following changes in the activities of MAP kinase and GSK-3.Results Activating MAP kinase in cultures of primary neurons or transfected COS cells expressing tau isoforms did not increase the level of phosphorylation for any PHF-tau epitope investigated. But elevating GSK-3 activity in the COS cells by co-transfection with GSK-3α or GSK-3β decreased the electrophoretic mobility of tau so that it resembled that of PHF-tau, and induced reactivity with eight PHF-tau-selective monoclonal antibodies.Conclusion Our data indicate that GSK-3α and/or GSK-3β, but not MAP kinase, are good candidates for generating PHF-type phosphorylation of tau in Alzheimer's disease. The involvement of other kinases in the generation of PHFs cannot, however, be eliminated. Our results suggest that aberrant regulation of GSK-3 may be a pathogenic mechanism in Alzheimer's disease.     

Production of a membrane-bound proteins,the human gamma-glutamyl transferase,by CHO cells cultivated on microcarriers,in aggregates and in suspension

Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h^–1, the highest cell density (near 1.3×10⁶ cells ml^–1), and the highest enzyme activity around 300 mU ml^–1, which corresponded to a specific cellular level of 20 mU 10^–5 cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems.     

Microbial transformation of ethylpyridines

Pyridine and its derivatives have been found as pollutants in the environment. Although alkylpyridines constitute the largest class of pyridines contaminating the environment, little information is available concerning the fate and transformation of these compounds. In this investigation ethylpyridines have been used as model compounds for investigating the biodegradability of alkylpyridines. A mixed culture of ethylpyridine-degrading microorganisms was obtained from a soil that had been exposed to a variety of pyridine derivatives for several decades. The enrichment culture was able to degrade 2-, 3-, and 4-ethylpyridine (100 mg/L) at 28° C and pH 7 within two weeks under aerobic conditions. The degradation rate was greatest for 2-ethylpyridine and least for 3-ethylpyridine. Transformation of ethylpyridines was dependent on substrate concentration, pH, and incubation temperature. Studies on the metabolic pathway of 4-ethylpyridine revealed two products; these chemicals were identified by MS and NMR analyses as 4-ethyl-2(1H)-pyridone and 4-ethyl-2-piperidone. 6-Ethyl-2(1H)-pyridone was determined to be a product of 2-ethylpyridine degradation. These results indicate that the transformation mechanism of ethylpyridines involves hydroxylation and reduction of the aromatic ring before ring cleavage.