Differentiation of lens and pigment cells in cultures of neural retinal cells of early chick embryos

Dissociated cells of neural retinas of 3.5-day-old chick embryos (stages 20–21) were cultured as a monolayer in order to examine their differentiation in vitro. These cells started to grow actively soon after inoculation and formed a confluent sheet within which neuroblast-like cells with long cytoplasmic processes were differentiated by 8 days. At about 16 days the differentiation of both lentoid bodies and foci of pigment cells was observed, while neuronal structure disappeared. The numbers of lentoid bodies and foci of pigmented cells continued to increase up to 30 days, when primary cultures were terminated. The increase in δ-crystallin content, as measured by quantitative immunoelectrophoresis assay using rabbit antiserum against δ-crystallin, was consistent with the increase in the number of lentoid bodies in cultures. The amount of α-crystallin per culture, estimated by the same technique as above, reached a maximum at 16 days and decreased slightly during further culture. The differentiation of both lentoid bodies and pigment cells was observed also in cultures of the second generation. The results demonstrate that cells of the undifferentiated neuroepithelium of 3.5-day-old embryonic retinas can achieve at least three differentiations, neuronal, lens, and pigment cells, in vitro. We discuss several differences between the present results and the previous ones from in vitro cultures of 8- to 9-day-old embryonic neural retinas.     

An improved rapid assay of glycogen synthase

As an alternative to rapid filtration washing the glycogen free of any unreacted UDP-[¹⁴C]-glucose by ascending chromatography (ethanol:water, 2:1) can be used. This technique also makes the filter paper assay of glycogen synthase much faster: The samples are ready for liquid scintillation counting in 30 min. Among the other advantages offered by this procedure, we should also mention that blanks are very low, large volumes of ethanol can be saved, and the unreacted UDP-[¹⁴C]glucose can be recovered by elution and recycled (it migrates with the front of the solvent).     

Effects of density-dependent inhibition of growth on phospholipid metabolism in monolayer cultures of animal cells.

The onset of density-dependent inhibition of growth is correlated with a change in the synthesis of the major phospholipids of either BHK-21 (hamster) and 3T3 (mouse) cells. The change consists of inhibition of phosphatidylcholine synthesis while the synthesis of phosphatidylethanolamine remains constant or increases. The incorporation of radioactive precursors reflects turnover rates, since no significant differences were observed in phospholipid composition. No major differences in fatty acyl chains of these same phospholipids were observed as a function of growth rate despite considerable individuality of relative patterns of polar lipids in both cell lines. No other density-dependent differences in phospholipid metabolism were observed which were common to both cell lines. These results suggest that major aspects of growth regulation may be metabolic rather than compositional and that regulation of the enzymes of lipid metabolism may be useful probes of effector mechanisms in growth control.     

Zinc accumulation and metabolism in primary cultures of adult rat liver cells. Regulation by glucocorticoids

Adult rat liver parenchymal cells were isolated by the collagenase perfusion technique and cultured as a monolayer for up to 20 h. The quantity of zinc accumulated from the extracellular environment was significantly increased by adding physiological concentrations of certain glucocorticoids to the medium. The degree of stimulation was directly related to glucocorticosteroids potency. Sex steroids, certain peptide hormones and prostaglandins E₂ and F_2α did not influence zinc accumulation.Control cells exhibited a decline of zinc accumulation after 4 h in culture although uptake processes were still operative. When dexamethasone, the most potent glucocorticoid used, was present in the medium the cells accumulated zinc at a linear rate greater than that seen in control cells, for at least 20 h. The dexamethasone-induced stimulation of zinc accumulation was relatively specific since ⁴⁵Ca, ¹⁴C-labelled amino acids and [³⁵S]cystine accumulation was not influenced by the hormone. A lag of 4 h was observed before an effect of dexamethasone on zinc accumulation could be detected. Moreover, the hormone-stimulated phase of accumulation was blocked when the cells were simultaneously incubated with either actinomycin D or cycloheximide. The additional complement of zinc accumulated by the dexamethasone-treated cells was localized in the cytosol fraction. Gel filtration and ion-exchange chromatorgraphy confirmed that this additional cytosol zinc was bound to metallothionein. [³⁵S]Cystine was incorporated into metallothionein in hormone-treated cells indicating that the protein was synthesized de novo during periods of enhanced zinc accumulation.     

The induction of suppressor T cells by lipopolysaccharide in human peripheral blood lymphocyte cultures in the presence of fetal calf serum

Alveolar macrophages (AM) were collected by repeated endobronchial lavage from mice, rats, guinea pigs, and rabbits, and titrated into cultures of mitogen-stimulated syngeneic or autochthonous lymphocytes. Significant species differences were detected in regard to AM activity in the cultures. AM from guinea pigs and mice stimulated PHA-induced lymphoproliferation, while those from rats and rabbits were inhibitory; blood or peritoneal macrophages were not inhibitory in any of the species examined.     

A rapid assay for prolyl 3-hydroxylase activity.

An assay is reported for prolyl 3-hydroxylase activity. The method is based on the release of tritiated water (THO) during 3-hydroxylation of a 2,3-T-l-proline-labeled (T = tritium) polypeptide substrate in which all prolyl residues recognized by prolyl 4-hydroxylase have been converted to 4-hydroxyprolyl residues. The formation of THO was essentially linear with enzyme concentration and time, and the K_m for the polypeptide substrate was about 3.4 × 10^?8m. A linear correlation was found between THO release and the synthesis of 3-hydroxyproline, the latter being analyzed by amino acid analyzer. The assay is simple, rapid, sensitive, and reproducible, and it is specific even in tissue samples containing a large excess of prolyl 4-hydroxylase activity.     

Colorimetric determination of catechol siderophores in microbial cultures

A highly sensitive spectrophotometric method for the selective detection of catechol compounds such as catechol siderophores (e.g., enterobactin) is described. The basis of the method involves the ability of the vicinal aromatic hydroxyl groups under acidic conditions to bring about a reduction of Fe3+ (from ferric ammonium citrate) to Fe2+. Detection of Fe2+ in the presence of Fe3+ is made with 1,10-phenanthroline under previously established conditions. The assay mixture is heated at 60 degrees C for 1 h to accelerate the development of color which is subsequently measured at 510 nm. The Beer-Lambert law is obeyed over the range of 0.16 to 60 microM 2,3-dihydroxybenzoic acid. Compared to the Arnow nitration method, the assay is more responsive, is approximately seven times more sensitive, and is effective with catechols substituted at positions 3 and 4. The method gives positive results with catechols such as DL-DOPA, L-dopamine, (+/-)-epinephrine, and DL-norepinephrine. Very rapid color development is obtained with ascorbic acid and p-diols, while m-diols are poorly detected. Low degrees of reactivity are shown by hydroxylamino and hydroxamate compounds. Phenolic, sulfydryl, indolyl, and quinonyl derivatives do not interfere with the reaction. The method has been adapted to determine catechol compounds in the culture medium of bacterial cells grown at different iron concentrations.     

A rapid,sensitive assay for starch phosphorylase and ADPglucose pyrophosphorylase

Starch phosphorylase (EC 2.4.1.1) and ADPglucose pyrophosphorylase (EC 2.7.7.27) activities can be measured very accurately and quickly using a recording pH meter. Activities less than 0.28 nmol/min are readily measured.     

Spectrophotometric assay of phenolpthalein in biological fluids.

An assay for phenolphthalein in biological fluids has been developed utilizing methods previously applied to the assay of bromosulphalein and to the deconjugation of steroidal compounds in urine. Intestinal perfusate, serum, and urine samples containing phenolphthalein are deproteinized with acidified acetone, the samples dried, and the phenolphthalein redissolved in ethanol. Color is developed with 0.5 m glycine buffer, pH 12, and the samples read at 550 nm after blanking the spectrophotometer with one of the replicates to which acidic glycine buffer is added. To measure conjugated phenolphthalein in urine, the sample is incubated overnight with β-glucuronidase/arylsulphatase prior to phenolphthalein determination as noted above. This method gives an accurate assay of phenolphthalein to 10^?5m concentrations with coefficients of variation between 2 and 8% and with no resulting interference from hemoglobin or bilirubin.     

The use of hormone-supplemented serum-free media in primary cultures.

Recent advances in tissue culture and endocrinology have made possible the growth of established cell lines in hormone-supplemented serum-free media. The hormone requirements differ for different cell types but are similar or identical for the same cell types. The hormone supplements derived for four different cell types, a melanoma, GH3 pituitary tumor, and testicular cell lines TM3 and TM4 are used in preparing primary cultures for organs to detect melanoma metastasis, and grow normal pituitary and normal Leydig and Sertoli cells, respectively. This hormone supplementation and the concomitant elimination or reduction of the serum requirement is shown to have several advantages in the preparation of primary cultures including prolonged viability and function, partial or total selection of the desired cell type and inhibition of fibroblast overgrowth.It is felt that such culture systems will significantly expand the range of problems which can be approached using primary culture systems.     

Histidine regulation in Salmonella typhimurium: XVI. A sensitive radiochemical assay for histidinol dehydrogenase

A sensitive radiochemical assay for measurement of histidinol dehydrogenase is presented. The method is based upon separation of the product of the reaction. [¹⁴C]histidine, from the substrate, [¹⁴C]histidinol, on small Dowex 50 columns. The assay can be performed on cell extracts or on toluenized cells and is approximately 100 times more sensitive than previously reported assays for this enzyme.[¹⁴C]histidinol is obtained in high yields through conversion of uniformly labeled ¹⁴C-glucose by a strain of Salmonella typhimurium derepressed for the histidine operon and blocked at the histidinol dehydrogenase step. Accumulated [¹⁴C]histidinol is purified from the culture supernatant by ion-exchange chromatography.This sensitive assay has facilitated measurement of reduced levels of histidine operon expression in promoter mutants, and has been adapted for study of histidine operon regulation in a cell free protein synthesizing system.     

An enzymatic microassay for lactose

An enzymatic microassay for lactose using a lactase enzyme derived from Saccharomyces fragilis is described. The assay uses 50-μl samples, provides 100% hydrolysis of lactose, and is sensitive within the range of 12.5–500 nmol per sample. The assay has been validated against an assay for ¹⁴C lactose which involves thin-layer chromatographic isolation of lactose. The assay is sufficiently sensitive for use in physiologic studies.     

Synthesis and turnover of lipids in monolayer cultures of BHK-21 cells.

An aryl azide, 2,4-dinitro-5-fluorophenylazide, has been prepared and characterized with respect to the rate of nucleophilic displacement of the active fluorine by amine groups. This reaction occurs readily under mildly alkaline conditions and at temperatures below 30 °C, which makes it suitable for modifying free amine groups, even those of active enzymes. The resulting 2,4-dinitro-5-azidophenylamine derivative is a photoaffinity label. Absorption of light of wavelengths shorter than 450 nm causes generation of the highly reactive aryl nitrene that covalently inserts into its immediate environment.     

Modulation of a neural antigen during metamorphosis in Drosophila melanogaster

Transplantation of the acousticolateral placode to the evacuated eye position in embryos of the frog Rana pipiens has been used to force axons of the VIIIth cranial nerve to penetrate the diencephalon. These ectopic axons establish a growth trajectory that is strikingly similar to their normal growth trajectory within the medulla oblongata despite the fact that no other axons within the diencephalon normally follow this route. The result is discussed in terms of the "blueprint" and substrates pathway hypotheses which have been advanced to explain the initial development of axon tracts within the central nervous system.     

Lactose biosynthesis: A sensitive radiochemical assay

A method is presented for the determination of lactose biosynthesis from labeled glucose, galactose, or other precursors based upon the addition of samples of the reaction mixture (after removal of the tissue or biosynthetic enzymes) to each of two strains of Escherichia coli. While both strains can metabolize glucose and galactose, only one is able to hydrolyze lactose. The sugars are converted by the bacteria largely to cell material and carbon dioxide. The difference between the residual, nonvolatile, soluble radioactivity in the medium from the two bacterial cultures represents the lactose unused by the strain unable to hydrolyze it.     

An ultrastructural study of the recovery of Chinese hamster ovary cells after freezing and thawing

The effect of freezing on the recovery of Chinese hamster tissue cells has been studied by freezing cells at a rate known to give high recovery and comparing these under the electron microscope with nonfrozen trypsinized cells for periods up to 14 hr after treatment. The main areas of damage were the cell surface and the cytoskeletal framework of the cell. The microfilament and microtubule systems underlying the cell membrane were shown to be disrupted in both the frozen and nonfrozen cells but repolymerization and reorganization was shown to be retarded for a longer period in the frozen cells. A greater degree of surface blebbing was observed in the frozen cells and heterochromatin was densely stained. The delay in return of the frozen cell to a normal morphology and physiology may be due to the need for the cell to repair sublethal cell damage before normal physiological processes can continue.     

Superoxide dismutase assay using alkaline dimethylsulfoxide as superoxide anion-generating system

Alkaline dimethylsulfoxide as a superoxide anion-generating system in association with cytochrome c as a superoxide anion-indicating scavenger has been used to develop a new assay for superoxide dismutase. The assay is sensitive (one unit of enzymatic activity is provided by 110 ng of purified copper-containing superoxide dismutase) and highly specific. The nature of this system prevents the usual interferences and its simplicity allows for multiple, rapid measurements of superoxide dismutase activity in biological preparations using either normal or automated procedures.     

Exposure to 4-aminopyridine prevents depressant effects of opiates on sensory-evoked dorsal-horn network responses in spinal cord cultures

The depressant effects of morphine (0.1-1 microM) on sensory-evoked dorsal-horn network responses in explants of mouse spinal cord with attached dorsal root ganglia (DRGs) were rapidly restored after addition of 4-aminopyridine (4-AP; 0.1 mM) and major components of these cord responses were stably maintained in the presence of the opiate. Moreover, prior exposure of cord-DRG explants to 0.1 mM 4-AP prevented the depressant effects of 0.1 microM morphine on DRG-evoked dorsal-horn responses, and the effects of 1-10 microM morphine were at least partly antagonized. Increased Ca++ levels (5 microM) attenuated the depression of dorsal horn responses by 1-10 micro M morphine and these effects of Ca++ were greatly enhanced in the presence of 4-AP--in some cultures, concentrations of morphine as high as 100 micro M were strongly antagonized during test periods up to 2 hours. Receptor assays showed that 0.1 mM 4-AP +/- 5 mM Ca++ had no effect on stereospecific opiate binding, indicating that the antagonist actions of these agents in our cultures do not occur at the level of the opiate receptor. The relevance of our in vitro studies of 4-AP antagonism of opiate-depressant effects on sensory-evoked dorsal-horn network responses for analyses of problems in opiate analgesia has been strengthened by a recent report demonstrating that 4-AP does, in fact, reverse morphine analgesia in rats, as determined by tail flick tests.     

Temperature sensitivity of muscle and neuron differentiation in embryonic cell cultures from the Drosophila mutant, shibire.

The sex-linked temperature-sensitive mutation, shibire^ts1, which causes, at the restrictive temperature, adult paralysis and pleiotropic morphological defects in embryonic, larval, and pupal development, has been shown to exhibit temperature-sensitive inhibition of differentiation in embryonic cultures in vitro. When shi cultures were incubated at 30°C for 24 hr, both muscle and neuron differentiation were inhibited more than 90% compared to control shi cultures incubated at 20°C. Heat shift experiments showed that the temperature-sensitive periods for neuron and muscle differentiation occurred at 11 to 18 and 14 to 16 hr, respectively, where zero time was the initiation of gastrulation in donor embryos. Short heat pulses (4 and 8 hr) which extended into the temperature-sensitive period resulted in moderate inhibition of differentiation; greater inhibition occurred as the duration of the pulses increased. In contrast, heating wild-type Oregon-R cultures at 30°C for 24 hr did not inhibit muscle cell differentiation and inhibited neuron differentiation relatively little. The temperature-sensitive period in shibire for muscle differentiation occurred well after myoblast division, during the period of myocyte elongation, aggregation, and fusion, whereas that for neuron differentiation took place during a period of enzyme synthesis (acetylcholinesterase and choline acetyltransferase) and axon elongation. Thus, the shi temperature-sensitive gene product affects at least two different cell types, in vitro, at different times during differentiation.     

Increase in DNA synthesis in Werner''s syndrome cells by hybridization with normal human diploid and HeLa cells

The dominance or recessiveness of the senescent phenotype in cells from patients with Werner's syndrome (WS cells) was investigated using cell fusion. The [³H]thymidine labeling index of normal human diploid fibroblast cell X WS cell heterodikaryons was considerably lower than that of normal homodikaryons, but was significantly higher than that of WS homodikaryons. The labeling index of WS cell X HeLa cell heterodikaryons was the same as that of HeLa homodikaryons. The labeling indices of heterodikaryons obtained by fusion between various strains of premature aging cells were as low as those of parental homodikaryons. These results indicate: (1) the senescent phenotype of WS cells appears to be partially recessive to the phenotype of normal cells and completely recessive to that of HeLa cells; (2) the marked inhibition of DNA synthesis in normal nuclei in heterodikaryons with WS cells could be due to ‘senescent factor(s)’ in WS cells; and (3) no complementation phenomenon was observed among genetically different premature aging cells, probably due to ‘senescent factor(s)’.