Different Batches Of Cells Research Articles

Formation of factor 390 by cell extracts of Methanosarcina barkeri

Cell extracts of Methanosarcina barkeri converted coenzyme F420 in an ATP-dependent reaction to the adenylylated derivative factor 390. Although it was reported previously (L. M. Gloss and R. P. Hausinger, BioFactors 1:237-240, 1988) that whole cells were unable to perform this conversion, we observed the conversion in 7 of 11 extracts, all of which were prepared from different batches of cells.

Properties of 5‐hydroxytryptamine3 receptor‐gated currents in adult rat dorsal root ganglion neurones

1. Responses to 5-hydroxytryptamine (5-HT) were examined on rat dorsal root ganglion (DRG) neurones maintained in tissue cultures, by use of whole cell recording techniques. 2. 5-HT (usually 10 microM) evoked a depolarization associated with an increase in membrane conductance in 40% of DRG neurones. There was a considerable variation in the size and persistence of this response between different batches of cells. 3. The 5-HT response was mimicked by applying the agonists 2-methyl-5-HT (10 microM) and phenylbiguanide (10 microM). Responses were blocked by ICS 205-930 (100 nM), but not by methysergide (0.1-1.0 microM). 4. 5-HT currents could be carried by sodium and caesium ions, but not by choline ions. The amplitude and duration of the 5-HT responses were dependent on the concentration of divalent cations in the extracellular solution: both became greater when calcium and magnesium concentrations were decreased. 5. Staurosporine, a putative antagonist of protein kinases, inhibited responses to 5-HT.

Sertoli Cells in Culture Are Heterogeneous with Respect to Transferrin Release: Analysis by Reverse Hemolytic Plaque Assay*

It has been reported that not all Sertoli cells store the same product or respond morphologically to secretagogue stimulation. The following studies were performed to determine whether functional differences among these cells are also present with respect to the secretion of a product. Sertoli cells obtained from 18- to 20-day-old rats were cultured for 3 days and then subjected to reverse hemolytic plaque assays for transferrin (TF). Release of TF could be detected from only 62.7 +/- 0.47% (mean +/- SE; n = 4 experiments) of all cells in culture. Results obtained from immunocytochemical staining of different batches of cells from the same dispersions agreed quite closely with these plaque assay values, indicating that not all Sertoli cells in culture contain or secrete TF. Differences in the basal rate of TF release were observed among these secretors, as evidenced by a gradual appearance of plaques over an 8-h period. Addition of FSH, cAMP, or isoproterenol to the assay incubation mixture resulted in an acceleration in the rate of plaque formation. Although approximately twice as many secretors could be identified after 0.5 and 1 h of incubation in the presence of these agents than in their absence, it still required at least 4 h for the remainder of the TF cells to form plaques. This would indicate that only a portion of all TF secretors respond acutely to these modulators. Thus, our observations that not all Sertoli cells secrete TF, and those that do release this substance respond differently to at least three stimulatory agents demonstrate clearly that Sertoli cells are heterogeneous with respect to TF release. Moreover, these findings raise the possibility that differences in the functional capacity of individual cells may be an important factor contributing to the modulation of Sertoli cell secretion.

The polyol pathway and glucose 6-phosphate in human endothelial cells cultured in high glucose concentrations

In an attempt to identify the mechanisms underlying the ill effects of high glucose previously described in cultured human endothelial cells, we have investigated in these cells the activity of the polyol pathway and accumulation of glucose 6-phosphate, a powerful agent of non-enzymatic glycosylation. Sorbitol accumulation varied among different batches of cells (primary cultures). In presence of 5 mmol/l glucose the cellular sorbitol content ranged from 0.04 to 0.12 nmol/10(6) cells. When cells were exposed to 20 mmol/l glucose the sorbitol content increased by 2- to 3-fold to concentrations of 0.08-0.38 nmol/10(6) cells (p less than 0.01). Addition to the culture medium of 100 mumol/l Sorbinil, an inhibitor of aldose reductase, resulted in a substantial inhibition of sorbitol accumulation throughout the 14 days in culture, but the degree of inhibition varied inversely with the duration of cell exposure to high glucose (70% inhibition in cells exposed to high glucose and Sorbinil for 1-3 days versus 14% inhibition in cells exposed for 14 days, p less than 0.01). Sorbinil treatment failed to improve even slightly the abnormalities in cellular replication induced by high glucose. The cellular content of glucose 6-phosphate was augmented 3-fold by exposure to 20 mmol/l glucose (p less than 0.001). In conjunction with other studies these results indicate that in this model the polyol pathway is not an osmotically or metabolically important mechanism of glucotoxicity, and that the inhibitory activity of Sorbinil on the polyol pathway of human tissues may be a function of their length of exposure to hyperglycaemia. The consequences of intracellular accumulation of glucose 6-phosphate await investigations.

Analysis by plaque assays of GH and prolactin release from individual cells in cultures of male pituitaries. Evidence for functional heterogeneity within rat mammotrope and somatotrope populations.

In this study we evaluated the quantitative influence of GRF and TRH on the rate of hormone secretion from single cells in cultures of male pituitaries. To accomplish this, we dispersed pituitaries from male rats with trypsin and cultured them for 24 or 48 h. Reverse hemolytic plaque assays for GH and prolactin were then performed on retrypsinized cultures to identify individual cells that secreted these hormones. Mammotropes and somatotropes were found to comprise 31.4 +/- 1.8 and 32.2 +/- 0.9% (mean +/- SE, n = 3 experiments), respectively, of all cells in 24-hour cultures. Immunocytochemical staining of different batches of cells from the same dispersions corroborated the proportions of these two cell types. Differences in the rate of basal hormone secretion were observed within each of these cell populations as evidenced by the gradual appearance of prolactin and GH plaques over a 4-hour period when incubations were conducted in the absence of stimulatory secretagogues. Addition of increasing concentrations of GRF (1 X 10(-10) -1 X 10(-7) M) or TRH (1 X 10(-9) -1 X 10(-6) M) to these incubations resulted in dose-related increases in the rate of GH and prolactin plaque formation, respectively. Maximal plaque development by somatotropes could be induced within 30 min of administering large doses of GRF, indicating that most, if not all somatotropes are responsive to this secretagogue. In contrast, approximately one third of all mammotropes could not be stimulated to form plaques acutely when subjected to similar treatment with TRH. This observation suggests that mammotropes are heterogeneous with respect to TRH responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the mechanism of fluoropyrimidine cytotoxicity in l1210 cells: correlation with inhibition of thymidylate synthetase but not with incorporation into RNA.

The effects of 5-fluorouracil (FUra), 5-fluorouridine (FUrd), and 5-fluoro-2'-deoxyuridine (FdUrd) on L1210 cells were examined in an effort to determine whether the cytotoxicity of these fluoropyrimidines is more closely associated with incorporation of FUra residues into RNA or inhibition of thymidylate (dTMP) synthetase (5,10-methylenetetrahydrofolate: deoxyuridylate C-methyl-transferase, EC 2.1.2.45) by 5-fluoro-2'-deoxyuridylate (FdUMP). In different batches of cells exposed to equitoxic (LD50) doses of these drugs for 48 hr, the levels of free FdUMP, dUMP, and free dTMP synthetase were found to be very similar. However, the number of FUra residues incorporated into total cellular RNA were in the approximate ratio of 1:10:100 in cells treated with FdUrd, FUrd, and FUra, respectively. Although these results are consistent with a common DNA-directed mechanism of toxicity, thymidine (dThd), which should circumvent dTMP synthetase inhibition, did not rescue the cells from the effects of FUra. However, uridine (Urd), which should compete with FUra for incorporation into RNA, had no effect on the toxicity of FUra either. Urd at 10(-5) M did not decrease the amount of incorporation of 10(-7)M [(3)H]FUra into total RNA, but a limited fractionation of polysomal RNA showed about a 4-fold decrease of incorporation of FUra into mRNA in the presence of Urd. Urd and dThd did effectively decrease the cytotoxicity of FUrd and FdUrd, respectively. These observations suggest that cell rescue experiments may not be reliable indicators of the mechanism of cytotoxicity of antimetabolites with complex mechanisms of action.

Hexose transport in novikoff rat hepatoma cells a simple carrier with directional symmetry, but variable relative mobilities of loaded and empty carrier

The kinetics of transport of the non-metabolizable hexose, 3-O- methyl- d- glucose , have been measured in Novikoff rat hepatoma cells by both zero- trans entry and equilibrium exchange procedures. Transport conformed to a simple carrier model which operates symmetrically with respect to direction, but with greater mobility of the loaded than of the empty carrier. Although a complete kinetic description of the transporter can, in theory, be obtained by application of integrated equations describing the time course of substrate equilibrium across the membrane beginning from the zero- trans situation, statistical analysis of hypothetical data indicated that directional asymmetry or differential mobilities of loaded and empty carrier cannot be discerned reliably from such data alone. The difference in mobility of loaded and empty carrier, apparent in a comparison of zero- trans entry and exchange data, ranged from 1.5–7-fold in different batches of cells. It is concluded that the magnitude of the difference is not an inherent property of the transporter, but is determined physiologically, and may be involved in regulation of hexose transport.

Isolation of photosynthetic catalysts from cyanobacteria

Methods are described for the isolation of ferredoxins I and II, cytochrome c-553, cytochrome f, cytochrome c-550 and plastocyanin from large quantities of various cyanobacteria. The amino acid composition of cytochrome c-550 is reported. There is a variation in the relative amounts of these proteins in different batches of cells which may relate to the nutritional status of the organisms.

Ethanolamine Deaminase, a Cobamide Coenzyme-dependent Enzyme: I. PURIFICATION, ASSAY, AND PROPERTIES OF THE ENZYME

Abstract Ethanolamine deaminase, a clostridial enzyme that catalyzes the cobamide coenzyme-dependent conversion of ethanolamine to ammonia and acetaldehyde, has been obtained as a homogeneous protein. Isolation of the relatively stable enzyme was facilitated by its unique lack of solubility in solutions with an ionic strength greater than 0.11. The specific activities of apparently homogeneous preparations isolated from different batches of cells are slightly different. A spectrophotometric assay for the detection of less than 1 µg of purified enzyme was developed by coupling the formation of acetaldehyde in the deaminase reaction with DPNH oxidation in the presence of added alcohol dehydrogenase. The deaminase reaction has an absolute requirement for cobamide coenzyme. The apparent Km values for α-(5,6-dimethylbenzimidazolyl)cobamide coenzyme, α-benzimidazolyl)cobamide coenzyme, and α-(adenylyl)cobamide coenzyme are 1.5 x 10-6, 1.9 x 10-7, and 7.7 x 10-6 m, respectively. Of several compounds tested, only ethanolamine is a substrate for the enzyme. Potassium ion is required for catalytic activity, whereas Li+ and Na+ are competitive inhibitors. The pH optimum for the reaction is 6.8 to 8.2. p-Chloromercuriphenylsulfonate inhibited the deaminase 46% at 3 x 10-4 m. Like the dioldehydrase described by Lee and Abeles (15), ethanolamine deaminase is inactivated by incubation with cobamide coenzyme in the absence of substrate, and both the deaminase and dioldehydrase reactions have a detectable lag period at low levels of cobamide coenzyme.

Growth Studies of Nocardia Species. I. Respiration of Carbohydrates by Nocardia Rubra

SUMMARYRespiration studies of N. rubra confirmed earlier qualitative studies on carbohydrate utilization. The inherent high endogenous respiration of the cells was reduced to less than 10% by shaking overnight in phosphate buffer (pH 7). The pattern of utilization varied with the age of the cultures used, e.g., 4-day old being metabolically more active than 7-day old on a number of the sugars.When one of the less readily utilized sugars was replaced in part by glucose, the oxygen uptake was considerably higher than with either sugar alone, showing that glucose stimulated the utilization of the other sugar. This effect was observed in mixtures containing 15–50% glucose.Although the oxygen uptake/mg dry weight varied considerably with different batches of cells of the same age, the same general trends were observed within an age group. This variation may be due to the random mixtures of cells of different ages in the seemingly uniform inoculum used initially. This was overcome by using as inocula frozen stock suspensions of 24-hour shake cultures of N. rubra grown in a 0.07 M glucose medium.Such inocula were used in growth studies, in which the quantitative growth curves for several carbohydrates paralleled earlier growth studies. The patterns in mixtures of sugars confirmed the results from respiration studies.