Sphingomyelinase activities in neuronal cell cultures.

Human and rat brains have been previously demonstrated to contain three sphinomyelinases, one lysosomal with a pH optimum of 5.0, one with a pH optimum of 7.4 and a dependence on magnesium and another with a pH optimum of 7.0 and no divalent cation requirement. Using brain cell cultures and clonal cell lines of both neuronal and glial origin the activities of the pH 5.0 and pH 7.4 (magnesium-dependent) sphingomyelinase were examined. Sphingomyelinase activity measured at pH 5.0 was found in all the cell lines tested including G26, C6, N18 (differentiated and undifferentiated), mouse “L” cells, human skin fibroblasts, fetal mouse brain surface cultures and fetal mouse brain reaggregate cultures. However, pH 7.4 (magnesium-dependent) sphingomyelinase activity was found only in the N18 cell lines and the reaggregate cultures suggesting the probable localization of this activity in neurons. Although the pH 7.4 sphingomyelinase activity was found in the fetal mouse brain used for the surface cultures this activity was rapidly lost. This enzyme may play an important role in neuronal development and maturation.     

Antiproliferative action of valorphin in cell cultures.

The antiproliferative effects of the haemoglobin beta-chain fragment (33-39) (valorphin or VV-haemorphin-5) were studied in a panel of tumour cell lines and normal cells of different origin, using various methods of activity determination (trypan blue inclusion test, sulphorhodamine B staining, MTT staining, flow cytometry and clonogenic test). Valorphin suppressed the proliferation of tumour cells by 25%-95%, depending on the cell line. The maximal valorphin activity was detected in transformed cells of fibroblastic (L929) and epithelial (MCF-7) origin, transformed haematopoietic cells (K562, HL-60) being less sensitive. In normal cells, valorphin activity was several fold lower (10%-15%). A study of the dynamics of cell proliferation in L929 cells using a visual cell count and flow cytometry showed that valorphin induced reversible and relatively short (24 h) S-phase arrest of cell proliferation, accompanied by a reversible increase of cell size. The proliferation delay was followed by a comparatively long period of reversible resistance of the cells to the peptide (96 h) when the cells are dividing at normal rate. The same dynamics were demonstrated for A549, MCF-7 and primary murine breast carcinoma cells. On the basis of the data obtained, a pattern of regulation of cell growth by valorphin is suggested.     

Chemical potential of Aphelandra sp. cell cultures

Six different callus lines and three different suspension culture lines were established from plants of two Aphelandra species (Acanthaceae). All established lines were analyzed for secondary metabolite accumulation. A discrepancy between secondary metabolites accumulated in the plants and in the cell cultures could be observed. All established Aphelandrasp. cell cultures produced verbascoside (acteoside) as the major extractable metabolite. Time course experiments were carried out to investigate the relationship between cell growth and verbascoside production. In the present study it was shown that verbascoside accumulation was growth dependent and positively related to the presence of 2,4-D in the medium. The conditions in which verbascoside represents ca. 18% of cell culture weight have been defined. Free polyamines were detected in the cell culture lines cultivated in MS liquid medium (cysteine 10 mg l^-1, thiamine 1 mg l^-1, 2,4-D 1 mg l^-1, kinetin 0.2 mg l^-1 and sucrose 30 g l^-1). Putrescine and spermidine accumulated within 8 days to a maximum of 8.4 μmol g^-1 of dry wt and 2.6 μmol g^-1 of dry wt respectively and thereafter their concentration decreased rapidly. There was no evidence for the presence of spermine or any other type of free or conjugated polyamines in the tested cell culture lines. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chromosomal mosaicism in amniotic fluid cell cultures.

Over the past 6 years, using in situ processing methods, we have identified 32 cases of mosaicism in amniotic fluid cell cultures prepared from 1,100 samples. Two of these (45,X/46,XX and 46,XX/47,XX, + 21) were called true mosaics because multiple colonies demonstrated the same abnormal chromosome complement, and on subsequent evaluation of the newborn blood or fetal tissues, mosaicism was confirmed. Of the remaining cases, 29 were designated as pseudomosaics because only single or partial colonies exhibited an aberrant chromosome complement, 12 having a trisomy 2 line. In the final case, a double trisomy was demonstrated in only one of eight colonies in the first culture, but in the culture from a repeat sample an additional two colonies showed the same double trisomy. Since no abnormal cells were observed in infant blood, it was postulated that the mosaicism may only have been present in the extraembryonic tissues. It is our conviction that the use of these cloning methods should diminish the danger of misdiagnosis in genetic amniocentesis.     

Ethanol-responsive gene expression in neural cell cultures.

We have studied the molecular mechanisms underlying neuronal adaptation to chronic ethanol exposure. NG108-15 neuroblastoma cells were used to perform a detailed analysis of ethanol-induced changes in neuronal gene expression. High resolution, quantitative two-dimensional (2-D) gel electrophoresis of in vitro translation products showed both dose-dependent increases and decreases in specific mRNA abundance following treatment with ethanol at concentrations seen in actively drinking alcoholics (50-200 mM). Dose response curves for representative members of the increasing or decreasing response groups had very similar profiles, suggesting that similar mechanisms may regulate members of a response group. Some mRNAs that increased with ethanol treatment appeared identical to species induced by heat shock while other mRNAs were only induced by ethanol. We conclude that chronic ethanol exposure can produce specific coordinate changes in expression of neuronal mRNAs, including some members of the stress protein response. However, the overall pattern of ethanol-responsive gene expression is distinct from the classical heat shock subgroup of stress proteins response. Changes in gene expression and specifically, mechanisms regulating a subset of stress protein expression, could be an important aspect of neuronal adaptation to chronic ethanol seen in alcoholics.     

Progesterone biotransformation by plant cell suspension cultures.

Progesterone was converted to 5alpha-pregnane-3alpha-ol-20-one, delta4-pregnene-20alpha-ol-3-one, delta4-pregnene-14alpha-ol-3,20-dione, delta4-pregnene-7beta,14alpha-diol-3,20-dione, and delta4-pregnene-6beta,11alpha-diol-3,20-dione by cell cultures of Lycopersicon esculentum. Cell cultures of Capsicum frutescens (green) metabolized progesterone to delta4-pregnene-20alpha-ol-3-one in very high yield, and Vinca rosea yielded delta4-pregnene-20beta-ol-3-one and delta4-pregnene-14alpha-ol-3,20-dione. A stereospecific reduction of the keto groups and a double bond and stereospecific introduction of hydroxyl groups at the 6, 11, and 14 positions have been observed. The mono- and dihydroxylated progesterones have not previously been reported as metabolic products of progesterone by plant cell systems and represent de novo hydroxylation of a nonglycosylated steroid.     

A heartwood pigment in Dalbergia cell cultures.

In an extensive survey of the genera Baphia, Caesalpinia, Dalbergia, Haematoxylon, and Pterocarpus, we have identified a number of species whose cell cultures accumulated pigments similar to those in heartwood. Thirteen rosewood (Dalbergia) species produced a purple quinonemethide pigment in the callus that was apparently identical between the species. The pigment was first purified from D. retusa cell culture and its structure was elucidated by mass, infrared, and detailed 1H and 13C NMR and NOE spectroscopic studies including 2D experiments (COSY, NOESY, HMQC, and HMBC). Retusapurpurin A (1a) is a C(30) isoflavan of novel skeleton whose formation can be rationalized to occur via regioselective oxidative coupling of an isoflavan to 4,4'-dihydroxy-2'-methoxychalcone. Retusapurpurin A was also isolated from D. parviflora heartwood and cell culture indicating that stress metabolism pathways that are shared with heartwood-type secondary metabolism subpathways are initiated in Dalbergia cell cultures. Therefore, Dalbergia cell cultures afford a good model system for studying heartwood-type metabolic differentiation.     

Growth kinetics of vitis vinifera cell suspension cultures: I. Shake flask cultures

Vitis vinifera cell suspension cultures carried out in shake flasks were closely examined for biomass growth and cell division in relation to carbohydrate, NH(4), NO(3)PO(4), and dissolved oxygen (DO)consumption. After inoculation, the oxygen uptake rate of the cultures measured on-tine was observed to increase continuously to a maximum value of 3.8 mmol O(2)L(-1)h(-1) at day 7 when cell division ceased and dissolved oxygen reached its lowest level of 17% air saturation. During this first phase of growth, the specific oxygen uptake rate remained constant at approximately 0.6 mmol 02 O(2) g(-1) dw h(-1)or approximately 2.2 mumol O(2), (10(6) cells)(-1) h(-1) whereas dry biomass concentration increased exponentially from 1.5 to 6.0 g dw L(-1). Thereafter, dry biomass concentration increased linearly to approximately 14 g dw L(-1) at day 14 following nitrate and carbohydrate uptake. During this second phase of growth, the biomass wet-to-dry weight ratio was found to increase in an inverse relationship with the estimated osmotic pressure of the culture medium. This corresponded to inflection points in the dry and wet biomass concentration and packed cell volume curves. Furthermore, growth and nutrient uptake results suggest that extracellular ammonium or phosphate ion availability may limit cell division. These findings indicate that cell division and biomass production of plant cell cultures may not always be completely associated, which suggests important new avenues to improve their productivity. (c) 1995 John Wiley & Sons, Inc.     

Oxygenation of cell cultures

Submersed cultures are increasingly being used for fermentation with animal cells. Reactor design is particularly important in these operations, because of the sensitivity of the cells to shear. In addition to the usual aeration methods, open-pore membranes or pure diffusion membranes are used for oxygenation in order to avoid gas bubbles. The various oxygenation methods are described in the present article [1]. Design principles for surface aeration, bubble columns, loop reactors, and stirred tanks, as well as oxygenation with Accurel or silicone membranes, are presented and discussed specifically for the low oxygen inputs desired in cell cultures. The scale laws are formulated, and special reference is made to problems of scale up. The various oxygenation methods are finally compared on the basis of the design principles presented, with particular attention to mechanical stress on the cells and to the laws of scale translation.List of Symbols A Interfacial area - a =A/V, Specific interfacial area - c ^* Saturation concentration - c Gas concentration in the liquid phase - d Impeller diameter - d ₂ Outside diameter of tubular membrane - d ₁ Inside diameter of tubular membrane - d Diameter under stretched conditions - d _p Particle diameter - d _L Diameter of sparger holes - D Reactor diameter - D _L Draught tube diameter - Gas/liquid diffusion coefficient - e Eccentricity - Fr Froude number - G Mass flow - g Acceleration due to gravity - h Height of impeller blade - H Filling height - Hy Henry constant for the liquid phase - Hy _s Henry constant of the membrane material - k Overall mass transfer coefficient - k _L Gas-liquid interface mass transfer coefficient - L Length of the tubular membrane - L Length of the streched turbulare membrane - n Impeller speed - Ne P/ n³d⁵, Newton number - O₂-partial pressure in the membrane - O₂-partial pressure in the reactor - P Impeller power - q Gas throughput - r Cell specific respiration rate - Re Reynolds number - Sc , Schmidt number - Sh , Sherwood number - u Liquid velocity - Root mean square velocity of turbulent fluctuations Superficial gas velocity - V Filled reactor volume - V _s Sparged volume - X Cell concentration - Energy dissipation - Dynamic viscosity - Temperature - Kinematic viscosity - Density of the liquid - Surface tension - Shear stress     

Characterization of proton extrusion in sunflower cell cultures.

Cell suspensions derived from callus root tips of sunflower (Helianthus annuus L., cv. enano) were obtained in order to assess the effects of different chemical and physical agents on cell H+ extrusion. Cell H+ efflux was sensitive to temperature, pH, inhibitors of plasmalemma H(+)-ATPase and Ca2+ and K+ concentrations in the assay medium, as well as to the light intensity at which cells were cultivated. Thus, in the darkness and at 60 mumol/m2/s of illumination, a strong inhibition of H+ extrusion was detected as compared to cells grown at 30 mumol/m2/s. H+ extrusion by cells grown at 30 mumol/m2/s was unaffected by the presence of calcium in the assay medium, while at 60 mumol/m2/s such an activity increased when calcium was removed. These results provide the basis for the use of cell suspensions as an appropriate model to investigate the involvement of membrane-associated processes in plant tolerance mechanisms to different environmental stresses.     

Nicotinamide deamidase released by neuroblastoma cell cultures.

Abstract— An enzymic activity which catalyses the deamidation of nicotinamide to nicotinic acid has been found in the growth media of neuroblasts and glioblasts cultivated in vitro. Some properties of the crude nicotinamide deamidase from the growth medium of neuroblastoma M1 cells have been studied.