Free Amino Acid Content of Astroglial Cell Clones Derived from 8-Day Postnatal Mouse Cerebella

We have measured the free amino acid content of three distinct astroglial cell clones derived from permanent lines obtained after "spontaneous immortalization" of 8-day postnatal mouse cerebellar cultures; these clones show characteristics similar to the Golgi Bergmann glia cells, the fibrous astrocytes, and the velate protoplasmic astrocytes, i.e., the three main types of cerebellar astrocytes. The relative concentrations of amino acids that are thought to act as neurotransmitters were compared in confluent cultures of the different astroglial clones. The most striking result was a high concentration of glycine (20% of free amino acids), even in astroglial cells cultured in a glycine-free medium, a finding suggesting that glycine is synthesized by the astroglial clones. Furthermore, no gamma-aminobutyric acid (GABA) was detected. In contrast, a "neuron-like" clone derived from the same cerebellar culture contained GABA, whereas its glycine content was much lower than that of the astroglial clones. The present results, together with our previous finding of glycine synthesis in an astrocytic clone derived from 14-day postnatal mouse cerebella transformed by simian virus 40, indicate that a high glycine content may be characteristic of many cerebellar astroglial types.     

Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation

The influence of non-Saccharomyces yeast, Kluyveromyces lactis, on metabolite formation and the ethanol tolerance of Saccharomyces cerevisiae in mixed cultures was examined on synthetic minimal medium containing 20% glucose. In the late stage of fermentation after the complete death of K. lactis, S. cerevisiae in mixed cultures was more ethanol-tolerant than that in pure culture. The chronological life span of S. cerevisiae was shorter in pure culture than mixed cultures. The yeast cells of the late stationary phase both in pure and mixed cultures had a low buoyant density with no significant difference in the non-quiescence state between both cultures. In mixed cultures, the glycerol contents increased and the alanine contents decreased when compared with the pure culture of S. cerevisiae. The distinctive intracellular amino acid pool concerning its amino acid concentrations and its amino acid composition was observed in yeast cells with different ethanol tolerance in the death phase. Co-cultivation of K. lactis seems to prompt S. cerevisiae to be ethanol tolerant by forming opportune metabolites such as glycerol and alanine and/or changing the intracellular amino acid pool.     

Glutathione Content as an Indicator for the Presence of Metabolic Pathways of Amino Acids in Astroglial Cultures

Abstract: The intracellular content of glutathione in astroglia-rich primary cultures derived from the brains of newborn rats was measured to be 32.1 ± 5.4 nmol/mg of protein. During a 24-h incubation in a minimal medium lacking amino acids and glucose, the content of glutathione in these cultures was reduced to 52% of the original content. On refeeding of glucose, glutamate, glycine, and cysteine, glutathione was resynthesized. A maximal content of glutathione was found 4 h after refeeding, exceeding the amount of glutathione of untreated cultures by 72%. Maximal glutathione synthesis was observed only if glutamate, cysteine, and glycine were present. If successively each one of these amino acids was made limiting for the synthesis of glutathione, half-maximal contents of glutathione were found at 0.2 m M glutamate, 20 µ M cysteine, or 10 µ M glycine. Replacement of glutamate or glycine by other amino acids revealed the potential of astroglial cells to convert glutamine, aspartate, asparagine, proline, and ornithine into glutamate, and serine into glycine. These results demonstrate that the concentration of intracellular glutathione can serve as an indicator for the presence of metabolic pathways of amino acids in cultured cells.     

Utilization of Organic Nitrogen Sources by Two Phytoplankton Species and a Bacterial Isolate in Pure and Mixed Cultures

Algal production of dissolved organic carbon and the regeneration of nutrients from dissolved organic carbon by bacteria are important aspects of nutrient cycling in the sea, especially when inorganic nitrogen is limiting. Dissolved free amino acids are a major carbon source for bacteria and can be used by phytoplankton as a nitrogen source. We examined the interactions between the phytoplankton species Emiliania huxleyi and Thalassiosira pseudonana and a bacterial isolate from the North Sea. The organisms were cultured with eight different amino acids and a protein as the only nitrogen sources, in pure and mixed cultures. Of the two algae, only E. huxleyi was able to grow on amino acids. The bacterium MD1 used all substrates supplied, except serine. During growth of MD1 in pure culture, ammonium accumulated in the medium. Contrary to the expectation, the percentage of ammonium regenerated from the amino acids taken up showed no correlation with the substrate C/N ratio. In mixed culture, the algae grew well in those cultures in which the bacteria grew well. The bacterial yields (cell number) were also higher in mixed culture than in pure culture. In the cultures of MD1 and T. pseudonana, the increase in bacterial yield (number of cells) over that of the pure culture was comparable to the bacterial yield in mixed culture on a mineral medium. This result suggests that T. pseudonana excreted a more-or-less-constant amount of carbon. The bacterial yields in mixed cultures with E. huxleyi showed a smaller and less consistent difference than those of the pure cultures of MD1. It is possible that the ability of E. huxleyi to use amino acids influenced the bacterial yield. The results suggest that interactions between algae and bacteria influence the regeneration of nitrogen from organic carbon and that this influence differs from one species to another.     

Utilization of osmoprotective compounds by hybridoma cells exposed to hyperosmotic stress

A search was undertaken for osmoprotective compounds for mouse hybridoma cell line 6H11 grown in culture. When the osmolality of the growth medium was increased above the normal osmolality of 330 mOsmol/kg, growth rates were decreased in a dose-dependent fashion, reaching zero when the osmolality of the medium reached approx. 435 mOsmol/kg through the addition of KCl (60 mM), or 510 mOsmol/kg through the addition of NaCl (100 mM), or sucrose (175 mM). For NaCl or sucrose-stressed cultures, the inclusion of glycine betaine, sarcosine, proline, glycine, or asparagine in the growth medium gave a moderate to strong osmoprotective effect, measured as the ability of these compounds to enhance cell growth rates under hyperosmotic conditions. Inclusion of dimethylglycine may also give a strong osmoprotective effect under these stress conditions.In KCl-stressed cell cultures, addition of glycine betaine, sarcosine, or dimethylglycine gave strong osmoprotective effects. Of 38 compounds tested during NaCl stress, 7 gave weak osmoprotective effects and 25 gave no osmoprotective effect. The osmoprotective compounds accumulated inside the stressed cells. Accumulation was completed after 4 to 8 h, reaching intracellular concentrations of approx. 0.27 pmol/cell, or 0.15 M, in NaCl stressed cells (100 mM NaCl added).Glycine betaine, dimethylglycine, and sarcosine accumulation was observed only when these protectants were included in the medium. For all osmoprotectants, a growth medium concentration between 5 and 30 mM gave the maximal protective effect, with the exception of dimethylglycine, for which the optimum concentration was approx. 65 mM. Osmoprotective effects obtained with glycine, sarcosine, dimethylglycine, and glycine betaine, indicate that the more methylated compounds are the most effective protectants.The cellular content of glycine betaine and the glycine betaine uptake rate increased with medium osmolality in a linear fashion. Glycine betaine uptake was described by a model comprising a saturable component obeying Michaelis-Menten kinetics and a nonsaturable component. K(m) and V(max) for glycine betaine uptake were determined at 420 mOsmol/kg (50 mM NaCl added) and 510 mOsmol/kg (100 mM NaCl added). A K(m) value of approx. 2.5 mM was obtained at both medium osmolalities, while V(max) increased from 0.010 pmol/cell . h to 0.018 pmol/cell . h as the osmolality of the growth medium was increased, indicating an effect of medium osmolality on the maximal rate of transport rather than on the affinity of the transporters for glycine betaine. Hybridoma cells were not able to utilize the glycine betaine precursors choline or glycine betaine aldehyde for osmoprotection, suggesting that the cells lack part, or all, of the choline-glycine betaine pathway or the appropriate uptake mechanism.The uptake rate for glycine in NaCl-stressed hybridoma cells was approx. four times higher than the uptake rate for glycine betaine. Furthermore, if equimolar amounts of glycine betaine, glycine, sarcosine, and proline were simultaneously added to NaCl-stressed cell cultures, the intracellular concentrations of glycine, proline, and sarcosine were significantly higher than the concentration of glycine betaine.A 40% increase in hybridoma cell volume was observed when the growth medium osmolality was increased from 300 to 520 mOsmol/kg. (c) 1994 John Wiley & Sons, Inc.     

The effects of several factors on the growth of pure and mixed cultures of Azotobacter chroococcum and Bacillus subtilis

We studied the effect of a clay mineral, palygorskite, on the physiological activity of Azotobacter chroococcum and the phosphate-mobilizing bacterium Bacillus subtilis, as well as their mixed cultures, under various oxygen supply conditions during the utilization of phosphorus from readily and poorly soluble compounds (K2HPO4 x 3H2O) and (Ca3(PO4)2), respectively. During cultivation of the bacteria in a nutrient medium with Ca3(PO4)2, the number of microorganisms was higher than that observed in a medium with K2HPO4. An increase in oxygen mass transfer in the nutrient medium was followed by a rise in the number of Bacillus subtilis cells and an inhibition of Azotobacter chroococcum growth. An addition of palygorskite (5 g/l) into the nutrient medium stimulated the growth of both bacteria and stopped the decreasing growth of Azotobacter chroococcum at high values of oxygen mass transfer. The number of Bacillus and, particularly, Azotobacter cells was two to five times lower in a mixed culture than in a monoculture. These differences were less significant during the cultivation of mixed cultures in medium with palygorskite.     

Serine and glycine transport in fetal ovine hepatocytes

The role of hepatic serine and glycine transport in the regulation of the biosynthesis of serine by the fetal liver has not been studied. The goal of this study was to characterize serine and glycine transport and utilization at physiologic concentrations in primary cultures of fetal ovine hepatocytes. Primary culture of hepatocytes from mid gestation ( approximately 90 days) and term ( approximately 135 days) fetal sheep were studied after overnight serum free culture. At both gestational ages, the initial rate for sodium dependent 300 microM serine transport (1697+/-131 pmoles/min/mg protein at mid, 1765+/-544 at term) was fourfold greater than sodium dependent 300 microM glycine transport (309+/-54 at mid, 579+/-252 at term). At physiologic concentrations (300 microM), 69+/-7% of serine and 49+/-8% of glycine transport was sodium dependent. At term, sodium dependent serine transport has a V(max) of 1751+/-348 pmoles/min/mg protein and a K(m) of 159+/-111 microM. Sodium independent serine transport has a V(max) of 904+/-185 and a K(m) of 416+/-188 microM. Sodium dependent glycine transport has a V(max) of 410+/-69 and a K(m) of 2290+/-895 microM while sodium independent glycine transport exhibits non-saturable kinetics. Glycine (300 microM) sodium dependent transport was not inhibited by methyl-AIB while sodium dependent 300 microM serine transport was inhibited (31%). n-Ethylmaleimide inhibited sodium dependent serine and glycine transport by 36+/-9% and 37+/-2% respectively in term hepatocytes. Cysteine inhibited sodium dependent serine transport by 37%. Sodium independent glycine transport at 300 microM was higher in low glucose (1.1 mM) medium (881+/-76 pmoles/min/mg protein) compared to high glucose (5.5 mM) medium (510+/-60 P=0.004). There were no significant differences in serine or glycine incorporation into RNA, DNA, glycogen or lipid and protein. The predominance of serine transport over glycine at physiologic concentrations suggests that inward cellular amino acid transport of serine and glycine is not likely to be a regulatory mechanism that would favor serine biosynthesis in fetal ovine hepatocytes.     

Adult human retinal cells in culture. Identification of cell types and expression of differentiated properties

A method for culturing adult mammalian retinal neurons in serum-free N2 medium supplemented with nerve growth factor (NGF) is described. Identification of neurons in cultures of dispersed human retina was based upon morphology, immunocytochemical localization of bound tetanus toxin, and autoradiographic localization of 3H-neurotransmitter candidates (gamma-aminobutyric acid, glycine, dopamine) accumulated by high-affinity uptake mechanisms. Neurons would not attach to glass or plastic substrates, consequently the present studies were performed using neurons plated upon a feeder layer. Serum was required for the initial phase of attachment. The feeder layer was derived from retinal cells that had been plated on glass or plastic in the presence of serum and had later been passaged. Since these cells exhibited glial fibrillary acidic protein (GFAP) immunoreactivity, they were tentatively identified as being glial in origin. Under these conditions, neuron- and glia-specific properties were retained up to 28 days. The presence of interstitial retinol-binding protein (IRBP) in medium of cultures of neuronal cells on feeder layers was demonstrated by an immunoblot technique using rabbit antibovine IRBP antibodies. No IRBP was detected in medium in which the feeder layers alone had been cultured. IRBP biosynthesis was demonstrated by incubation of the cultures with [35S]methionine. Immunoprecipitable [35S]IRBP was detected only in medium from cultures containing neurons; cells of the feeder layer did not synthesize and secrete this glycoprotein. These findings are consistent with the hypothesis that IRBP, a 135K constituent of the interphotoreceptor matrix, is synthesized in vivo by a neuronal cell, specifically, the photoreceptors.     

The effects of osmotic upshock on the intracellular solute pools of Bacillus subtilis

The effects of hypersaline treatment (osmotic upshock) on solute accumulation have been studied in the Gram-positive bacterium Bacillus subtilis. Natural abundance 13C NMR spectroscopy studies revealed only proline as a major organic osmoticum in cells grown in defined medium (no exogenous organic solutes) and this finding was confirmed by amino acid analysis. Intracellular concentrations of both K+ and proline rose markedly after osmotic upshock. K+ influx from the medium was rapid (less than 1 h) but proline synthesis was a slower process (5-9 h). Proline synthesis appeared to be dependent on the prior accumulation of K+ and it is possible that K+ serves in some manner as the signal for increased proline synthesis. In cells upshocked in medium enriched in glycine betaine the endogenous synthesis of proline was repressed and glycine betaine served as the sole organic osmoticum. K+ was also accumulated under these conditions.     

Concentration of Free Amino Acids in Primary Cultures of Neurones and Astrocytes

The cellular distribution of free amino acids was estimated in primary cultures (14 days in vitro) composed principally of cerebellar interneurones or cerebellar and forebrain astrocytes. In cultured neural cells, the overall concentration of amino acids resembled that found in brain at the corresponding age in vivo. In the two neural cell types, there were marked differences in the distribution of amino acids, in particular, those associated with the metabolic compartmentation of glutamate. In neuronal cell cultures, the concentrations of glutamate, aspartate, and gamma-aminobutyric acid were, respectively, about three, four, and seven times greater than in astrocytes. By contrast, the amount of glutamine was approximately 65% greater in astroglial cell cultures than in interneurone cultures. An unexpected finding was a very high concentration of glycine in astrocytes derived from 8-day-old cerebellum, but the concentrations of both serine and glycine were greater in nerve cell cultures than in forebrain astrocytes. The essential amino acids threonine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine were all present in the growth medium, and small cellular changes in the contents of some of these amino acids may relate to differences in their influx and efflux during culturing and washing procedures. The present results, together with our previous findings, provide further support for the model assigning the "small" compartment of glutamate to glial cells and the "large" compartment to neurones, and also underline the metabolic interaction between these two cell types in the brain.     

Culture shock. Selective uptake and rapid release of a novel serum protein by endothelial cells in vitro

A novel protein has been purified from fetal calf serum and from serum-free bovine aortic endothelial cell conditioned culture medium. This protein consists of a single polypeptide chain of reduced Mr 70,000 (70K protein) and was separated from bovine serum albumin and other proteins by ion-exchange chromatography and immunoabsorption on Sepharose-coupled anti-70K protein antiserum. The 70K protein was shown to be structurally and immunologically distinct from bovine serum albumin, alpha-fetoprotein, and vitronectin by one- and two-dimensional peptide mapping, amino acid analysis, and enzyme-linked immunosorbent assay and/or immunoblotting. The 70K protein was located in endothelial cell cytoplasmic granules of irregular size and distribution. Metabolic radiolabeling studies showed that the 70K protein was not a biosynthetic product of these cells; its cytoplasmic location was due to a selective uptake from the fetal calf serum in which the cells were initially grown. After subconfluent cultures of endothelial cells were shifted to serum-free medium, nearly 80% of the total 70K protein that was measurable in the medium was released between 0 and 20 min. Moreover, sparse, rapidly proliferating cells released approximately 18-fold more 70K protein within 2 min as compared to dense, nonproliferating cultures. The concentration of 70K protein in fetal calf serum was estimated to be 400-600 micrograms/ml. Proliferating bovine aortic endothelial cells, 24 h after plating at an intermediate density, released approximately 250 pg of 70K protein/cell within the first 20 min after exposure to serum-free conditions. The data provide evidence for a novel protein in serum which is selectively internalized by endothelial cells in vitro and which in turn is released rapidly under conditions such as osmotic imbalance due to serum removal, or during periods of cellular proliferation, conditions which we term "culture shock."     

Ammonia production and amino acid metabolism by rat renal papillary epithelial cells in culture

A significant percentage of excreted ammonium is added to tubular fluid along the medullary collecting duct. However, it is not clear whether this ammonia is produced in the cortex and delivered into the medulla or is produced directly by medullary cells. To address this issue, rat epithelial cells derived from the renal papilla were grown in continuous culture and their ability to generate ammonia was examined. When grown in Dulbecco's modified Eagle's medium with 4 mM glutamine, these cells produced ammonia at a rate of approximately 27 nmol/10(6) cells/h. When these cells were grown in minimum essential medium without glutamine, ammonia production fell to 7 nmol/10(6) cells/ h. Increasing the glutamine concentrations of minimum essential medium to 4 mM increased ammonia production to slightly greater than 30 nmol/10(6) cells/ h. Increasing the media concentration of glutamate, glycine, or asparagine resulted in no significant increase in ammoniagenesis. Analysis of media amino acid concentration revealed that glutamine was the main amino acid consumed while alanine was the predominant amino acid produced. The glutaminase activity of these cells appears to be primarily phosphate-dependent, similar to that observed in vitro in papillary tubules. Alterations of K+ or H+ ion concentration did not alter ammoniagenesis, but addition of 2.5 mM ammonium chloride significantly reduced net ammonia production. It is concluded that rat papillary epithelial cells have the intrinsic ability to utilize glutamine to generate ammonia and alanine. In vivo ammonia produced locally in the medulla may contribute to final urinary ammonium excretion.     

Low external pH replaces 2,4-D in maintaining and multiplying 2,4-D-initiated embryogenic cells of carrot

A mixed culture comprised of both embryonic globules and nonembryogenic callus. was derived from seedling hypocotyls of Daucus carota cv. Scarlet Nantes on 2,4-D-containing medium using well-established methods. Then the mixed cultures were transferred to, and serially subcultured on, a hormone-free medium near pH 4. The medium contained 1 m M NH+ as the sole nitrogen source. When cultured in this way, embryonic globules were able to multiply without development into later embryo stages Nonembryogenic callus did not survive. Continuous culture of embryonic globules on this low pH hormone-free medium yielded cultures consisting entirely of preglobular stage proembryos (PGSPs). PGSP cultures have been maintained as such with continuous multiplication for nearly 2 years without loss of embryogenic potential. These hormone-free-maintained PGSPs continue their development to later embryo stages when cultured on the same hormone-free medium buffered at pH 5.8. We show that hormone-free medium near pH 4 can replace 2.4-D in its ability to sustain multiplication of 2,4-D-initiated embryogenic cells of carrot at an acceptable growth rate without their development into later embryo stages. This procedure provides selective conditions that do not permit the growth of nonembryogenic cells while providing an adequate environment for embryogenic cell proliferation and should prove invaluable in studying habituation.     

Utilization and formation of amino acids by chicken epiphyseal chondrocytes: comparative studies with cultured cells and native cartilage tissue

Utilization and production of amino acids by primary cultures of chicken growth plate epiphyseal chondrocytes grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum were investigated in both short-term (6-72 h) and long-term (3-24 day) cultures. Comparative studies were made on levels of free amino acids in chicken blood plasma and serum, and in extracellular fluids from different regions of growth plate cartilage and from two types of muscle. Chondrocytes rapidly consumed glutamine from the medium, and to lesser extents, various other amino acids. In contrast, free ammonia, alanine, glycine, glutamate, proline, and aspartate were released into the medium. The utilization of certain amino acids changed, depending on the stage of culture. Initially glutamate was released into the medium but after confluency was consumed. Conversely, histidine, lysine, and phenylalanine were initially utilized but later were released into the medium. Levels of total free amino acids in extracellular fluids of cartilage and muscle were higher than those in plasma and serum, while in cartilage the levels increased progressively from the resting to the hypertrophic zones. In these sequential regions certain amino acids increased proportionally, whereas others decreased. These interrelationships generally correlated closely with metabolism of amino acids by the cultured chondrocytes. They indicate that significant differences in amino acid metabolism exist between tissue areas and are reflected in the extracellular fluid composition. Accordingly, adjustment of specific amino acids may optimize culture conditions, enabling more normal phenotypic expression in vitro.     

外源甜菜碱提高恶臭假单胞菌(Pseudomonas putida)DLL-1耐盐性的研究

研究了外源甜菜碱对恶臭假单胞菌(Pseudomonas putida)DLL-1耐盐性的影响并对其渗透保护机制进行了初步的探讨;结果表明培养基中添加甜菜碱可以改善DLL-1细胞在高盐培养基中的生长情况,添加150mg/L的甜菜碱可以使DLL-1在1.2mol/L NaCl的基础盐培养基中生长,添加10mg/L的甜菜碱就足以显著缩短渗透胁迫条件下DLL-1细胞的延滞期和代时,增加生长量;和不添加对照相比,延滞期由24h缩短到6h,代时由60min缩短到35.7min,最大生长量OD610由1.29增长到1.57。在渗透胁迫条件下,细胞从外界快速吸收外源甜菜碱来代替自身相容性溶质的合成。     

The physiology and pathogenicity of Mycobacterium tuberculosis grown under controlled conditions in a defined medium

A chemically-defined culture medium was developed which supported batch growth of Mycobacterium tuberculosis, strain H37Rv, at a minimum doubling time of 14.7 h. This medium also facilitated chemostat culture of M. tuberculosis at a constant doubling time of 24 h. Chemostat growth was optimized at a dissolved oxygen tension of 20% (v/v) and 0.2% (v/v) Tween-80. Chemostat cultures were dispersed suspensions of single bacilli (1.5-3 microm long), or small aggregates, at a mean density of log10 8.3 cfu ml-1. A limited number of amino acids was utilized (alanine, asparagine, aspartate and serine were depleted by >50%; glycine, arginine, isoleucine, leucine and phenylalanine, by approximately 40%). Chemostat-grown cells were pathogenic in aerosol-infected guinea pigs, producing disseminated infection similar to that caused by plate-grown cells. Cells from chemostat culture were significantly more invasive for J774A.1 mouse macrophages than agar- or batch-grown cells. This study demonstrates the suitability of chemostat culture for the growth of pathogenic mycobacteria in a defined physiological state with potential applications for the controlled production of mycobacterial components for therapeutic and vaccine applications.     

Effect of growth substrates on morphology of Nocardia corallina.

Cells of Nocardia corallina ATCC 4273 form multiply branched coenocytic mycelia and subsequent fragment to spherical cells when grown on solidified complex media. In liquid shake cultures using complex media the organisms grow into pleomorphic but seldomly branched rods, divide as rods and then the rods fragment to spheres as the stationary phase is reached. In a defined liquid medium with glucose as carbon source, the organisms divide entively as spheres at a doubling time of 44 hrs. The addition of L-tyrosine, some fatty acids and tricarboxylic acid cycle intermediates or fructose to the glucose medium caused the cells to grow at considerably faster growth rates (2.8-8.5 hrs doubling times) and to undergo the shphre-rod-shpere growth cycle. Other amino acids, fatty acids or surgars added singly to the glucose medium did not produce the sphere to rod morphology change. Some amino acids when added to the medium in pairs effected sphere to rod morphopoiesis. None of these amino acids alone were effectors. Some of the culture grew as rods and the remainder as spheres when isoleucine and valine were added to the glucose medium. No other amino acid combination tested gave this result. The reason for the mixed growth response was traced to inhomogeneity of the parent culture. The life cycle of N. corallina is illustrated in a series of photomicrographs of two slide cultures.     

Nonneuronal cells mediate neurotrophic action of vasoactive intestinal peptide

The developmental regulation of neuronal survival by vasoactive intestinal peptide (VIP) was investigated in dissociated spinal cord-dorsal root ganglion (SC-DRG) cultures. Previous studies demonstrated that VIP increased neuronal survival in SC-DRG cultures when synaptic transmission was blocked with tetrodotoxin (TTX). This effect was further investigated to determine if VIP acted directly on neurons or via nonneuronal cells. For these studies, SC-DRG cells were cultured under conditions designed to provide preparations enriched for a particular cell type: astrocyte-enriched background cell (BG) cultures, meningeal fibroblast cultures, standard mixed neuron-nonneuron (STD) cultures, and neuron-enriched (N) cultures. Addition of 0.1 nM VIP to TTX-treated STD cultures for 5 d prevented the TTX-mediated death and the death that occurred naturally during development in culture, whereas the same treatment on N cultures did not prevent neuronal cell death. Conditioned medium from VIP-stimulated BG cultures prevented neuronal cell death when added to the medium (10% of total volume) of N cultures treated with TTX. The same amount of conditioned medium from BG cultures that were not treated with VIP had no protective action on N cultures. Conditioned medium from N or meningeal fibroblast cultures, either with or without VIP treatment, did not prevent TTX-mediated cell death in N test cultures. These data indicate that VIP increases the availability of neurotrophic survival-promoting substances derived from nonneuronal cultures, the most likely source being astroglial cells. This study suggests that VIP has a role in mediating a neuron-glia-neuron interaction that influences the trophic regulation of neuronal survival.     

Glycine release from Y79 retinoblastoma cells

Abstract: Glycine release, induced by a high concentration of potassium chloride (K⁺), was investigated in cultured human Y79 retinoblastoma cells. The cells were labeled by incubation with [2-³H]glycine prior to K⁺ depolarization. Depolarization with 55 m M K⁺ caused an immediate, Ca²⁺-dependent release of approximately 20% of the cellular radiolabeled glycine content. Chemical analysis of the intracellular free glycine content also showed that approximately 20%, 2.4 nmol/mg protein, was released after K⁺ depolarization. Glycine release from labeled Y79 cells was not stimulated by incubation with 55 mM choline chloride. Based on measurements with an amino acid analyzer, it is concluded that of the free amino acids contained in the Y79 cell, only glycine is specifically released into the extracellular fluid by K⁺ depolarization. Although the intracellular content of serine and glutamate decreased, these amino acids were not released from the cells. Further studies with [U-¹⁴C]serine suggest that serine is converted into glycine in Y79 cells. Veratridine also caused an immediate release of [2-³H]glycine from the cells, and this was blocked by tetrodotoxin. This suggests that the Y79 cells possess voltage-dependent Na⁺ channels. These results indicate that K ⁺ - and veratridine-stimulated glycine release occurs in Y79 retinoblastoma cells, providing additional evidence that this continuously cultured line may be a useful model for certain human retinal and central nervous system functions.     

Effect of vitamin K depletion and restoration on sphingolipid metabolism in Bacteroides melaninogenicus

Bacteroides melaninogenicus requires vitamin K for normal growth. Cells incubated in a vitamin K-free medium form defective cell envelopes. Studies with vitamin K-grown "K(+)" and vitamin K-depleted "K(-)" cells showed that [(14)C]choline and [(14)C]glycerol were not taken up, but several amino acids and acetate were incorporated to the same degree by both types of cultures. However, K(-) cells incorporated succinate to a greater degree than did K(+) cultures. The relative incorporation of succinate into ceramide phosphorylethanolamine and ceramide phosphorylglycerol was depressed compared with incorporation into phosphatidylethanolamine in K(-) cultures. B. melaninogenicus can be grown in serial subculture in the absence of vitamin K in the presence of 2.5 mg/ml of succinate. Under these conditions the relative incorporation of [2,3-(14)C]succinate and (32)P into ceramide phosphorylethanolamine and ceramide phosphorylglycerol is markedly depressed. Stimulation of phosphosphingolipid synthesis by vitamin K was shown by comparing the uptake of (32)P and lipid phosphorus levels of a succinate-grown, vitamin K-depleted culture supplemented with (32)P plus 0.1 micro g/ml vitamin K(1) with a similar culture supplemented with (32)P only. The phosphosphingolipids from the vitamin K-supplemented cells incorporated greater amounts of (32)P and had higher levels of phosphorus than did the ceramide phosphorylethanolamine and ceramide phosphorylglycerol of the culture without added vitamin K. It was further shown that vitamin K added to a vitamin K-depleted culture stimulated synthesis of ceramide phosphorylethanolamine and ceramide phosphorylglycerol 38 min and 60 min, respectively, following the addition of the vitamin; incorporation of (32)P into other phospholipids was unaffected.