Light microscopy of glial cells in turtles and birds

Summary Four classes of glial cells can be recognized in the central nervous system of turtles and birds on the basis of nuclear characteristics (methylene blue) and external morphology (Golgi technique). It seems likely that astrocytes and ependymal cells have a similar origin and function, but no evidence has been seen to indicate that transitional forms exist between astrocytes and oligodendrocytes or microgliacytes. Ependymal cells in the tectum and forebrain are covered by lamellate excrescences which are absent on cells in the spinal cord. Protoplasmic astrocytes are restricted to the gray matter. In the turtle they have an elongate shape characteristic of primitive elements, but stellate forms typical of mammals predominate in the bird. Fibrous astrocytes are abundant in the white matter. Endfeet are lacking in the turtle except on cells located near the pia; they are common for all elements in the bird and can sometimes be observed to outline the course of capillaries. Oligodendrocytes are identical to mammalian and amphibian forms. Small, round somata and long, thin processes are typical of types I and II while a tubular reticulum or membranous sheath characterizes type IV. The lack of a well defined somata and absence of transitional forms (type III) are compatible with the possibility that type IV is not a true cell type but corresponds to the inner cytoplasmic tongue of myelin. Microgliacytes are present in gray and white matter; they have a smaller overall size in the turtle and young chicken than in adult birds.Supported by a postdoctoral fellowship from the United States National Institutes of Health, NB 28,013-01Al.     

Nesting behavior ofHalictus scabiosæ in Switzerland (Hymenoptera,Halictidæ)

Summary The structure and populations of seven nests ofHalictus scabiosæ were studied in Geneva during July and August, 1964. Cells, containing brood of various ages, were radially arranged along the branching burrows. The one to six females in each nest did not belong to distinct worker and queen castes. This is in contrast to reports of distinct castes in what appears to be the same species from France. Most pollen collectors in the Geneva population were inseminated and some probably laid eggs.

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Zusammenfassung Die Struktur und die Bewohner von sieben Nestern vonHalictus scabiosæ, die im Juli und August 1964 in der Gegend um Genf gesammelt wurden, werden beschrieben. Zellen mit Brut verschiedenen Alters sind entlang verzweigter Gänge radiär angeordnet. Bis zu sechs Weibchen befanden sich in einem einzelnen Nest. Sie waren nicht in Königinnen und Arbeiterinnen differenziert, wie dies von Bienen aus Frankreich berichtet ist, die wahrscheinlich zur selben Art gehören. Die meisten Pollen-Sammlerinnen der Genfer Population waren befruchtet und einige von ihnen legten auch wahrscheinlich Eier.

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Résumé La structure et la population de sept nids deHalictus scabiosæ ont été étudiées à Genève durant les mois de juillet et d'août 1964. Des cellules contenant du couvain d'âge différent sont disposées en rayons le long de terriers qui se ramifient. Chaque nid était occupé par une à six femelles qui n'appartenaient pas à une caste distincte d'ouvrières ou de reine. La plupart des abeilles qui collectent le pollen furent inséminées et quelques-unes probablement pondèrent des ufs.

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Contribution No. 1303 from the Department of Entomology, The University of Kansas, Lawrence.     

A practical method for rescuing desired hybridomas during monoclonal antibody production

Summary We present a practical method for the rescue of previosly stable hybridoma clones which increases the proportion of desired cells in the population before cloning by limiting dilution. When the antibody activity of a culture supernatant was lower than that previously obtained, a precloning distribution at a density of 10 cells per microtiter well greatly improved the chances of obtaining a single active clone by subsequent limiting dilution. The Poisson distribution model was used to evaluate the method. Probabilities calculated clearly demonstrate the advantage of this precloning distribution step when attempting to isolate a hydridoma cell line that is relatively rare in a population. This work was supported in part by grants EY 06225 and EY 06226 from the National Eye Institute of the National Institutes of Health, Bethesda, MD and by an unrestricted departmental award from Research to Prevent Blindness, Inc.     

Characterization of Opioid Receptors in Cultured Neurons

The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors.     

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions

Summary A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor, bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins. Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, β-casein mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels. Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve as an excellent model in which the regulation of mammary development and gene expression can be investigated. This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.     

An abscisic acid analog inhibits abscisic acid-induced freezing tolerance and protein accumulation, but not abscisic acid-induced sucrose uptake in a bromegrass (Bromus inermis Leyss) cell culture

The application of abscisic acid (ABA), either as a racemic mixture or as optically resolved isomers, increases freezing tolerance in a bromegrass (Bromus inermis Leyss) cell culture and induces the accumulation of several heat-stable proteins. Two stereoisomers of an ABA analog, 23 dihydroacetylenic abscisyl alcohol (DHA), were used to study the role of ABA-induced processes in the acquisition of freezing tolerance in these cells. Freezing tolerance was unchanged in the presence of (–) DHA (LT₅₀ -9°C), and no increase in heat-stable protein accumulation was detected; however, the (+) enantiomer increased the freezing tolerance (LT₅₀ -13°C) and induced the accumulation of these polypeptides. All three forms of ABA increased freezing tolerance in the bromegrass cells, although (–) ABA was less effective than either (+) or (±) ABA when added at equal concentrations. Cells pretreated with 20 or 50 M (–) DHA displayed lower levels of freezing tolerance following the addition of 2.5, 7.5 or 25 M (±) ABA. Full freezing tolerance could be restored by increasing the concentration of (±) ABA to > 25 M. Pretreatment of cells with (–) DHA (20 or 50 M) had no effect on freezing tolerance when 25 M (+) ABA was added. The induction of freezing tolerance by 25 M (–) ABA was completely inhibited by the presence of 20 M (–) DHA. The accumulation of ABA-responsive heat-stable proteins was inhibited by pretreatment with 20 M (–) DHA in cells treated with 2.5 or 7.5M (+) ABA, and in cells treated with 25 M (–) ABA. The accumulation of these polypeptides was restored when (±) or (+) ABA was added at a concentration of 25 M. The analysis of proteins which cross-reacted with a dehydrin antibody revealed a similar inhibitory pattern as seen with the other ABA-responsive proteins. The effects of the various isomers of ABA and DHA on cell osmolarity and sucrose uptake was also investigated. In both cases, (±) and (+) ABA had pronounced effects on the parameters measured, whereas (–) ABA treated cells gave substantially different results. In both sucrose uptake and cell osmolarity, DHA had no significant effect on the results obtained following (±) or (+) ABA treatment. Maximum freezing tolerance was only observed in cells when both heat-stable protein accumulation and sucrose uptake were observed.Abbreviations ABA abscisic acid - DHA 2,3 dihydroacetylenicabscisyl alcohols - DMSO dimethyl sulfoxide - LT₅₀ temperature at which 50% of cells are killed The authors would like to acknowledge the technical assistance of Angela Bollman, Bruce Ewan and Angela Shaw. This work was supported by grants from the Natural Science and Engineering Research Council of Canada to L.V.G. and N.H.L., and a grant from the University of Saskatchewan to R.W.W.