ALBERT

Notes: Differentiated Malpighian tubules of Periplaneta americana nymphs consist of four distinct regions. The distal, middle, and proximal regions are similar to the same regions in adult tubules. However, the transparent portion of the middle region was found to have ultrastructural characteristics different from those of the longer opaque segment of the middle region and the two other tubule regions. This newly distinguished region is called the lower middle region. Transitional zones, areas where cells show characteristics of two adjacent regions, are apparent between the distal and middle regions and between the middle and lower middle regions.The middle region of primary tubules undergoes an increase in autophagic activity and a modification of its basal infoldings and microvilli shortly before each molt. An increase in autophagic activity is also observed in the lower middle region near the time of molting.

Notes: The vascular anatomy of five beavers (Castor canadensis) was studied by dissection and injection of arteries and veins with vinyl acetate. There is extensive countercurrent arrangement of arteries and veins distal to and including the common iliac artery and veins. Two types of countercurrent vessels occur (1) a venae comitantes type in which two or three veins surround a central artery, and (2) a modified rete type. The retia are located proximal to the large flat tail and the webbed hind feet. Two bypass veins are described for the feet and tail and the significance of these structures in temperature regulation is stressed.

Notes: The epithelial cell line, H4-II-E derived from Reuber hepatoma H35 has no significant activity of ornithine carbamoyltransferase (OCT, EC 2.1.3.3) and is not able to grow in arginine-deprived medium.A multi-step selection procedure is described which selects from H4-II-E populations, cells with OCT activity which can grow in arginine-deficient, ornithine-supplemented media.

Notes: In a myeloid leukemia cell line, the inducibilities of the Fc receptor, phagocytosis and cell motility were compared. Thymidine analogues such as BUdR, BCdR and IUdR blocked the induction of phagocytosis and motility but not induction of the Fc receptor. This BUdR susceptibility in the induction of phagocytosis and motility was lost in a BUdR resistant line which was isolated for its growth capability in a high concentration of BUdR. Actinomycin D and puromycin brought about a marked decrease in the inducibility of phagocytosis but not in that of the Fc receptor.This led us to the following conclusion: There is a genetic control in the inducibility of phagocytosis and motility in this cell line, and the incorporation of BUdR into cellular DNA results in the DNA becoming unresponsive to a differentiation-stimulating factor. In contrast, gene activation does not seem to be necessary for induction of the Fc receptor.The order of induction of several differentiation markers was also discussed.

Notes: The relative rates of the initiation and elongation phases of protein synthesis have been determined in heat- and cold-shocked CHO cells from measurements of the incorporation of 35S-methionine into N-terminal and internal positions of growing peptides by a modified Edman degradation. When the cells are shifted from 37°C to temperatures between 10°C and 34°C, the rate of initiation is at first reduced more extensively than that of elongation. After 20 to 30 minutes at the lower temperature, however, the cells undergo a metabolic adjustment which includes increasing the rate of initiation until it corresponds to the rate of elongation at that temperature. Calculated apparent energies of activation for initiation and elongation are in reasonable agreement with those determined in other mammalian cells. When the cooled cells are returned to 37°C, the rates of initiation and elongation recover immediately but do not exceed the control values. Exposure to elevated temperature (43°C) causes an immediate cessation of initiation and thus a delayed inhibition of elongation; upon return to 37°C, the rate of initiation is transiently elevated above the control rate, and the rate of elongation returns to the control rate after a 2- to 3-minute delay. Hence, a factor which leads to supranormal rates of initiation may accumulate at high but not at low temperatures.

Notes: Peptide production in senescent and presenescent human foreskin fibroblasts was measured using 2-dimensional polyacrylamide gel electrophoresis. This procedure permits the visualization of a cohort of the major peptides being produced. Among this cohort of over 500 peptides only two were found to differ in relative amount in that more was being produced in senescent cells. This difference was confirmed by measurements of the relative intensity of the peptide spot. This difference was senescent cell-specific and not due to the differences in rate of growth of senescent and non-senescent cells.

Notes: HeLa (substrain Ho) grown in serum free medium showed an increase in the specific activity of alkaline phosphatase when fetal calf serum (10%) was added to the medium (9.7 nmoles/sec/mg protein to 86.8). Under the same conditions, eight intracellular enzymes showed no increase in activity. Similar results were obtained using a different serum or medium, and with a second strain of HeLa (substrain ATC).For a given set of growth conditions, the effect of serum was dependent on its concentration and required one or more culture generations to develop. The type of isozyme expressed did not change. Neither zinc nor a total serum lipid extract would substitute for serum. The enzyme expressed by HeLaHo was not induced by prednisolone, while that in HeLaATC was. However, for cells grown in excess prednisolone without serum, the specific activity was 25% of that found for cells grown with prednisolone and serum. Cortexolone, an antagonist of prednisolone, was without effect for HeLaHo grown in A3 medium with or without serum.The serum factor had the following characteristics. It was not lost on dialysis, treatment with DNase and RNase, or removal of lipoproteins. It was reduced after heating by 65% and after treatment with Pronase by 82%.The data are interpreted to indicate the presence of a factor (s) in serum, probably a protein, which is involved in stimulating alkaline phosphatase specific activity.

Notes: Elevation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) activity by glucocorticoids was shown to be dependent on the concentration of hormone in the medium over a range of 5 × 10-10 to 1 × 10-8 M, although the presence of steroid in the assay at 10-5 M elicited no increase in activity. There was a demonstrated time dependence for the addition of dexamethasone i.e., from zero to six hours after serum removal, addition of hormone resulted in the same peak activity; addition at 12 hours gave slight elevation but resulted in an extended maintenance of the peak level of activity; addition at 24 hours showed no effect. When cycloheximide was added at the above times, subsequent kinetics showed identical decay of the enzyme activities from control and treated cultures at 6 and 24 hours, but at 12 hours the activity from dexamethasone treated cells exhibited an extended lag before the onset of decay, which then proceeded at the same rate as the control. The continuous presence of the hormone was not necessary for the induction to continue and the addition of Actinomycin D to cultures incubated in the presence of hormone resulted in an immediate decay of catalytic activity without evidence of “superinduction”. The addition of progesterone at the same time as dexamethasone resulted in a concentration-dependent inhibition of the augmentation, suggesting the involvement of the glucocorticoid receptor in the aug-mentation, suggesting the involvement of the glucocorticoid receptor in the elevation of HMG-CoA reductase activity. Flow microfluorometric (FMF) analysis of hormone treated cells indicated a delayed entrance into the DNA synthesis (S) phase of the cell cycle. The temporal relationships between this cell cycle perturbation and HMG-CoA reductase elevation are discussed.

Notes: Human α-L-fucosidase, purified from placenta, was taken up from the culture medium by skin fibroblasts from patients with fucosidosis (α-L-fucosidase deficiency). The rate of uptake was low (uptake coefficient = 6 × 10-4 ml.mg-1.h-1). Intracellular α-L-fucosidase activity was directly proportional to enzyme in the medium up to an activity of at least 40 nmoles/min/ml. No evidence for saturation of specific cell-surface receptors was seen. However, uptake was reduced by 75% by 1 mM mannose-6-phosphate and by 50% by 1 mM glucose-6-phosphate, suggesting that uptake may be mediated by a receptor recognising a phosphorylated sugar or an analagous compound. Enzyme taken up by the cells was most active in subcellular fractions enriched with lysosomes and had an isozyme pattern, by isoelectric focusing, identical to that of the original enzyme preparation.Fucosidosis fibroblasts were shown to accumulate low molecular-weight, fucose-containing compounds to a level several times greater than control cells. This stored material was eluted from Sephadex G-25 as an asymmetrical peak with an elution volume of approximately twice the void volume of the column. Addition of placental α-L-fucosidase to the culture medium of fucosidosis fibroblasts prevented excessive accumulation of fucose-containing material and accelerated the breakdown of material accumulated prior to enzyme uptake.

Notes: The stability of both rapidly and slowly degraded proteins in wild type CHO cells is similar to that in three ts aminoacyl-tRNA synthetase mutants at both permissive and non-permissive temperatures, although the degree of tRNA charging in the synthetase mutants differs considerably with temperature. These results indicate that the altered rate of protein breakdown seen under a variety of physiological conditions in eukaryotic systems is not mediated by uncharged tRNA.

Notes: The manganese content of the egg and embryo of the Medaka, Oryzias latipes was determined by activation analysis. A remarkable increase in the amount of manganese in the egg was observed within one hour after fertilization. The rate of increase was reduced by the gastrula stage and the concentration of manganese remained unchanged at a later stage. The accumulation of manganese by the Oryzias egg was discussed in relation to the effect of manganese on respiratory enzyme systems.

Notes: Growth of a human leukemic T-cell line (CEM C7) in 10-6 M dexamethasone results in inhibition of growth and rapid loss of cell viability after a delay of approximately 18 to 24 hours. Analysis of dexamethasonetreated cells by flow-microfluorometry showed that they were arrested in the G1 phase of the cell cycle. Loss of cell viability began at the same time as G1 accumulation was first detectable, and 20% of all cells were found to be blocked in G1 at this time suggesting that loss of viability and G1 arrest were coincident events. Half-maximal and maximal effects on both viability and G1 arrest after 48 hours in steroid were nearly identical with respect to steroid concentration and corresponded to half-maximal and full occupancy of glucocorticoid specific receptor by hormone, consistent with a glucocorticoid receptor mediated mechanism for both phenomena. Most non-viable cells were arrested in G1, and accumulation of cells in G1 was irreversible; removal of steroid in the presence of colcemid did not result in a decreased fraction of G1 cells. Furthermore, dexamethasone treatment did not protect cells against the effects of 33258 Hoechstamplified killing of bromodeoxyuridine substituted cells exposed to light. These results show that dexamethasone arrests these leukemic cells in G1 and strongly suggest that dexamethasone-treated cells are killed upon entry into G1.

Notes: During synchronous differentiation of embryonic chick muscle cells in culture, the Na-dependent uptake of an amino acid analog, α-amino isobutyric acid (AIB) undergoes an abrupt, transient increase. The increase in AIB uptake is concomitant with the rapid fusion of mononucleated myoblasts, and precedes the accumulation of muscle-specific proteins. Subsequently, Nadependent AIB transport diminishes markedly during postfusional differentiation of myotubes. The rate of AIB uptake is increased by insulin both before and after myoblast fusion. This stimulation by insulin is restricted to the Nadependent component of total AIB uptake but is apparently not the result of insulin-mediated increase in the trans-membrane Na gradient.

Notes: The characterization of a temperature-sensitive Chinese hamster cell mutant has been continued with the aim of localizing the apparent defect in glycoprotein synthesis (Tenner et al., 1977). Although the mutation is lethal, a demonstration of the ability of the mutant cells to support proliferation of Mengo virus at the nonpermissive temperature indicates that the general metabolic processes of the cells remain intact at a time when glycoprotein synthesis is severely depressed. A quantitative study of protein synthesis on membrane-associated polysomes suggests that the synthesis of the polypeptide portion of the glycoproteins at 40.8°C may be normal. The investigation of lipidsaccharide molecules which have been implicated in the formation and transfer of the oligosaccharide „core„ to polypeptide acceptors shows that mutaant cells at the nonpermissive temperature are capable of synthesizing these lipid saccharides normally, and that the pool of the dolichyl oligosaccharide is maintained at a constant level independent of the temperature. The rate of formation of the lipid-oligosaccharide, however, is reduced in intact mutant cells at the nonpermissive temperature. Further investigations show this decreased rate to be the result of an increased half life of the lipid-oligosaccharide at 40.8°C. These data indicate that the temperature-sensitive step in glycoprotein biosynthesis is the transfer of the oligosaccharide core from the lipid-oligosaccharide intermediates to the nascent polypeptide chain. The data presented also provide evidence that the lipid-saccharide intermediates, previously described mainly in in vitro systems, are in fact involved in the glycosylation of a majority, if not all, of the mannose-containing glycoproteins in intact, growing hamster cells.

Notes: Addition of insulin to nonproliferating serum-free cultures of secondary chicken embryo (CE) cells caused a 30% to 50% increase in cell number. Addition of any one of several glucocorticoids (dexamethasone, cortisol, or corticosterone) to the cultures two days before insulin addition increased the mitogenic effect of insulin by about twofold at each insulin concentration tested. This glucocorticoid stimulation of cell proliferation was “permissive” because in the absence of insulin glucocorticoids caused little increase in cell number (usually less than 15%). Glucocorticoids were maximally active at low concentrations (e.g., 10-10 M dexamethasone). Steroids without glucocorticoid activity were inactive over a wide range of concentrations. Glucocorticoids increased the mitogenic response to insulin largely by increasing the percentage of cells that insulin stimulated to synthesize DNA.The maximum mitogenic effect of insulin upon CE cells rapidly decreased after the cells were serially subcultured. After only nine population doublings (4 passages) in culture, the response to insulin was diminished by about 70%. The mitogenic effect of insulin plus dexamethasone declined similarly during serial subculture, and was always about twofold greater than the effect of insulin alone. The cells maintained their mitogenic responsiveness to serum as these responses decreased.In contrast to the growth promoting influence of glucocorticoids in the presence of insulin, glucocorticoids inhibited the mitogenic response of CE cells to serum. This result may resolve our above findings with reports that glucocorticoids inhibit the proliferation of CE cells.

Notes: Much controversy regarding the relationship between nutrients and serum in regulation of cell growth can be reconciled by recognizing that serum contains multiple factors which regulate different events in the cell cycle. Serum was fractioned into a platelet-derived growth factor (PDGF), which induces cells to become competent to synthesize DNA, and plasma which allows competent cells to traverse G0/G1 and enter the S phase. Nutrients are not required for the cellular response to PDGF; however amino acids are required for plasma to promote the entry of PDGF-treated, competent cells into S phase. The nutrient independent, PDGF-modulated, growth regulatory event (competence) is located 12 hours prior to the G1/S phase boundary in quiescent, density-arrested Balb/c-3T3 cells. The nutrient dependent, plasma-modulated event is located six hours prior to the G1/S phase boundary and corresponds in time to a plasma dependent growth arrest point. Moreover, plasma controls the concentration of amino acids required for DNA synthesis. Infection of density-arrested Balb/c-3T3 cells with SV40 overrides both the nutrient independent and the nutrient dependent growth regulatory events.

Notes: In the wild-type strains, 156 and 168, of Paramecium primaurelia, the alleles G156 and G168 expressed at medium temperature specify two immunologically distinguishable surface antigens 156G and 168G, whose phenotypic expression shows allelic exclusion, the majority of heterozygotes being phenotypically [156G] while a small minority is phenotypically [156G-168G]. At high temperature, the antigens coded by another locus, generally the D locus, are expressed. This system, displaying both intergenic and interallelic exclusion, provides favourable material to analyze the respective roles of the genome, of the antigens expressed and of the environmental conditions, in particular temperature, on the regulation of the expression of surface antigens.This analysis was carried out by studying the variations of the expression of surface antigens as a function of temperature, culture medium and previously expressed antigens in different genetic situations (a) in homozygotes: the wild-type strains 156 and 168, and the isogenized strains “G156 isogenic 168” carrying the G156 allele in a 168 genetic background; (b) in heterozygotes of the two phenotypic classes of heterozygotes, [156G] and [156G-168G]. The results show that (1) the thermal stability of the expression of a given surface antigen and its rate of re-appearance at the cell surface depend on its own specificity: (2) in heterozygotes [156G-168G], the stability of the expression of the antigen 156G is modified and “adjusted” to that of the less stable surface antigen 168G, and (3) the surface antigen itself exerts a positive control on the maintenance of its own expression.An interpretative model of “transmembranous control” is proposed to account for the regulation of the expression of surface antigens in Paramecium.

Notes: Murine erythroleukemic cells were induced to differentiate along the erythroid pathway by Me2SO and HMBA. These inducers caused an early decrease in the transport of glucose and amino acids, both in non-synchronized and in synchronized cultures. Careful analysis of the transport parameters in synchronized cultures showed a cyclic fluctuation of the Vmax but no significant change of the Km. In the presence of the inducers, however, a modification of the Km and Vmax of both carriers was observed which was not dependent on cell cycle. This modification is very early and precedes the transient arrest of the cells in G1 reported previously. In addition, a Me2SO-resistant cell line (DR10) does not show any changes in the transport of glucose and amino acids when incubated with Me2SO. However, there is an effect on the transport when incubated with HMBA which induces differentiation of 50% of the cells.These data support the hypothesis that an early effect of the inducers on the plasma membrane may be a necessary prerequisite for initiation of differentiation in murine erythroleukemic cells.

Notes: The biosynthesis of NAD has been examined in 3T3 cells. The net synthesis of pyridine nucleotides does not occur when cells are cultured in the absence of performed pyridine ring compounds; however, growth continues normally for up to four cell doublings resulting in cells with a total pyridine nucleotide content that is reduced by as much as 12-fold. The mechanism that adjust the relative amounts of NADP and NAD are also altered such that the amount of NADP relative to NAD increases 5-fold. Both nicotinate and nicotinamide can be used as a precursor for NAD biosynthesis, however nicotinate is utilized less efficiently than nicotinamide. The presence of functional pathways for the biosynthesis of NAD from nicotinate via nicotinate mononucleotide and nicotinate adenine dinucleotide and from nicotinamide via nicotinamide mononucleotide has been demonstrated by identification of biosynthetic intermediates following short term exposure of cells to radiolabelled precursors. When cells are grown in Dulbecco's modified Eagle's medium which contains 33 μM nicotinamide the biosynthesis of NAD proceeds by a single pathway with nicotinamide mononucleotide as the only intermediate. Nicotinamide ribonucleoside which previously has been postulated to be an intermediate in the conversion of nicotinamide to NAD is not an intermediate in NAD biosynthesis.

Notes: Activities related to Na-K transport were measured in cell cultures of ground squirrel kidney cortex in order to compare these cells with those of intact kidney and of continuous cell lines. A microsomal preparation containing plasma membrane Na,K-ATPase from fresh kidney showed twice the activity of a similar preparation from 72-hour cultured cells. Na,K-ATPase of homogenates of 72-hour cells showed one-third to one-fourth the specific activity of that from 6-hour cultured cells. The associated K-dependent phosphatase activity also declined as a function of time in culture. The ouabain-sensitive influx of K into 6-hour cultured cells was twice as great as the K influx into 72-hour cells. The number of sites binding 3H-ouabain in intact cultured cells declined 81% on a cell protein basis between 6 and 72 hours in culture. This decline in ouabain binding sites was relatively greater than that of K influx, so that the K turnover number increased over this same time period.The decline in ouabain-sensitive K influx during culture was complementary to an increase in furosemide-sensitive K influx. Measurements of unidirectional and net K fluxes showed that there were three components of K influx into 3-day cultured cells: ouabain-sensitive Na:K exchange, furosemide-sensitive K:K exchange, and K diffusion. In the 6-hour cultures, however, there was no furosemide-sensitive K:K exchange.Thus, after three days in culture ground squirrel kidney cells lose a feature characteristic of the original parent cells (high Na,K-ATPase activity), and gain a feature common to many undifferentiated cultured cells (furosemidesensitive K:K exchange).

Notes: We have studied the effects of theophylline treatment on pigmentation characteristics and growth of two B16 melanoma cell lines, HFH-18 and P/140. Cell counts of control and theophylline-treated cultures confirmed that the drug inhibits cell growth. Light and electron microscope cytochemistry with the L-dopa reaction indicated that the two cell lines differ in their ability to transfer Golgi-associated tyrosinase to developing premelanosomes. The results of these experiments, considered with results of electrophoretic analyses and activity measurements by the Pomerantz method, also provide evidence that increased tyrosinase synthesis occurs in response to theophylline treatment. In addition, results indicate that theophylline induces changes in the rate of synthetic or degradative posttranslational modification of tyrosinase. Measurements of intracellular cyclic AMP levels by radioimmunoassay in control cultures and in theophylline- and α-MSH-treated cultures were made. Although the hormone induced spectacular increases in cyclic AMP levels, theophylline produced no detectable change. These results indicate that theophylline differs from α-MSH because theophylline-induced changes in pigmentation may not require the participation of intracellular cyclic AMP.

Notes: Phorbol esters stimulate 2-deoxy-D-glucose (DG) uptake in rodent and human cell cultures. The potent tumor promoting agent, 12-0-tetradecanoyl phorbol-13 acetate (TPA), induces a 12-fold stimulation in confluent 3T3 cells and a 2.5-fold stimulation in HeLa cells. When a series of macrocyclic diterpenes are assayed, their relative potencies in stimulating DG uptake in 3T3 cells correlate with other known biologic effects of these compounds. On a molar basis, TPA is a much more potent stimulator of DG transport than insulin or epidermal growth factor. In HeLa cells, the ED50 value of the TPA effect is 0.2 nM. The increase in DG uptake occurs immediately after the addition of TPA, reaches a maximum at 90 minutes, persists for at least three hours after removal of TPA from the medium, and is temperature dependent. The stimulation is not inhibited by cycloheximide or actinomycin D. As in control cells, DG uptake in TPA treated cells is inhibited by p-hydroxymercuribenzoate, phloridzin, cytochalasin B, and dexamethasone. Although the precise mechanism is not known, evidence is presented that the TPA stimulation of DG uptake is due to enhanced transport of the sugar rather than to effects of intracellular metabolism. The enhanced transport may be secondary to a more generalized change in membrane structure.

Notes: Three cell lines of mouse erythroleukemia transformed by Friend virus (FLC), namely 745, F4-1, and 3BM-78, were grown for six days in the absence or in the presence of 1.5% (v/v) dimethylsulfoxide (DMSO) and compared cytochemically for naphtol-AS D-chloroacetate esterase (E), alkalinephosphatase (AP), myeloperoxidase (MP) and periodic acid Schiff (PAS) reaction activity. In the absence of inducer only 1-2% of slightly E positive cells could be found. E positivity greatly increased in 3BM-78 and F4-1 but poorly in 745 cells, after treatment with DMSO. Unlike E reaction, AP and MP reactions were positive in about 5% 3BM-78 and F4-1 cells without DMSO, but there were no positive cells after DMSO treatment. All three lines were always PAS negative. Hemoglobin synthesis (benzidine staining) was intensively induced by DMSO in all three lines. Morphologically after DMSO treatment, FLC matured displaying characteristics of basophilic megaloblastoid cells. The emergence of specific esterase activity, a marker of granulocytes, in FLC differentiating along the erythroid pathway, suggests that in these leukemia cells the genetic determinants for leukopoietic differentiation are retained and capable of being expressed phenotypically.

Notes: The potential of nanomelic chondrocytes to synthesize chondroitin sulfate was investigated by providing the mutant cells with p-nitrophenyl-β-D-xyloside, a compound which acts as an artificial acceptor for glycosaminoglycan synthesis. Under these conditions the synthesis of chondroitin sulfate in nanomelic and normal chondrocytes is comparable. The chondroitin sulfate synthesized by the mutant is indistinguishable in molecular size and composition from that synthesized by similarly treated normal chondrocytes.

Notes: The equilibrium distribution of 5,5-dimethyloxazoladine 2,4-dione (DMO) between intra- and extracellular volume was used to estimate intracellular pH (pHi) in Tetrahymena pyriformis. In control experiments, DMO was found to equilibrate rapidly in response to a pH gradient. Under normal growth conditions, pHi was constant over a finite range of external pH, being maintained near pH 7.1 over the external pH range 5.25 to 7.3. This same range of external pH was also optimal for growth. pHi was monitored during the cell cycle of a synchronous population of T. pyriformis GL. The cells were synchronized either by starvation/refeeding or heat shock. Under both conditions, there were two alkaline shifts of approximately 0.4 pH units per cell cycle. These shifts in pH retained a constant remporal relationship to S phase and were not affected by changes in the time, duration, or magnitude of cytokinesis.

Notes: Methods for the induction of an exudate of polymorphonuclear neutrophilic leukocytes (PMN) in the peritoneal cavity of C57BL, BALB/c, SJL and CBA mice were analysed. Peritoneal exudates in male mice were highly enriched for PMN (80-90%) three hours after a single injection of calcium caseinate whereas eosionophils comprised less than 1% of the exudate population. Female mice were a less satisfactory source of PMN because the proportion of eosinophils in the exudate was variable. Purification of PMN from peritoneal exudate cells was performed on the basis of light scattering using a Becton-Dickinson cell sorter or by density gradient centrifugation with graded polyvinylpyrroliodone-coated silica particles (Percoll). Both techniques yielded approximately 97% pure PMN preparations. Electrophoretic analysis of the PMN proteins revealed an abundance of lactoferrin and actin, but several other proteins were also present in high concentrations. Proteolytic degradation of several high molecular weight proteins (〉90,000) was prevented by the addition of phenylmethylsulphonyl fluoride (PMSF) and ethylene diamine tetracetic acid (EDTA). Surface iodination, using diphenyl, tetrachloroglycouril (IODO-DEN), indicated that there were six tyrosine-containing proteins present on the external cell membrane. The apparent molecular weights of these surface proteins ranged from 185,000 to 90,000 and the major 125I-labeled protein had an apparent molecular weight of 90,000. Neither actin nor lactoferrin was labeled with 125I unless cell viability was lost during the iodination procedure. Standard conditions for labeling the cell surface only, required low iodide and IODO-GEN concentrations. Biosynthetic labeling of PMN using 35S-methionine increased the sensitivity of detection for most of the proteins, but some of the granule storage proteins (such as lactoferrin) were not effectively labeled within three hours.

Notes: Clones of Chinese hamster ovary (CHO) cells were isolated by single-step selection for resistance to killing by Concanavalin A (ConA) and certain cellular and membrane properties were examined. The ConA-resistant isolates were only about 2-fold more resistant than wild type cells to the selecting lectin, but exhibited pleiotropic temperature-sensitivity for growth, markedly altered morphology and adherence, and significant differences in susceptibility to other agents such as colchicine. Two revertants to full temperature-resistance were isolated from different ConA-resistant mutants. One revertant clone had reacquired wild type sensitivity to ConA while the other revertant remained ConA-resistant. The two series of wild type, ConA-resistant, and temperature revertant clones were analyzed for altered mobility of cell surface glycoproteins using lactoperoxidase/125I and galactose oxidase/[3H]borohydride labelling procedures. The ConA-resistant clones showed increased mobility on polyacrylamide gels of three classes of labelled proteins, in the molecular weight ranges 225,000, 200,000, and 130,000 daltons. These changes persisted in the temperature-revertant that remained ConA-resistant, while two of the altered protein classes were restored to wild type mobility in the revertant that regained ConA-sensitivity. Cell hybridization experiments indicated that the temperature-sensitive phenotypes of different ConA-resistant isolates are recessive and noncomplementing, implying that the same gene is affected in each case. The reversions to temperature resistance appear to be recessive suppressor mutations in different genes.

Notes: Bovine corneal and aortal endothelial cell cultures were established from primary explants and subcultured for at least 40 passages. With both cell, exogenous thymidine, folate or folinate markedly increased the proliferation of these cells and decreased their serum requirement in Medium 199.Medium 199 supplemented with thymidine was particularly useful for cell survival at low densities; clones were readily produced when single cells were plated as low as 0.07 cells. cm-2. In contrast to the results of others, neither fibroblast growth factor nor epidermal growth factor were necessary for cell proliferation or survival at low densities.

Notes: The binding of [3H]tuftsin to normal and in vivo stimulated mouse peritoneal macrophage populations was studied at 22°C. The [3H]tuftsin binding to thioglycollate-stimulated macrophages was shown to be rapid and saturable, with an equilibrium dissociation constant (KD) (calculated from a Scatchard plot) of 5.3 × 10-8 M. The calculated number of binding sites per macrophage amounts to approximately 72,000. Binding competition studies with unlabelled tuftsin yielded a KD of 5.0 × 10-8 M. [3H] [N-Acetyl-Thr1]tuftsin, an inactive analog of tuftsin, failed to bind specifically to thioglycollatestimulated macrophages. [N-Acetyl-Thr1]tuftsin and the tripeptide [Des-Arg4]tuftsin failed to compete for tuftsin binding sites, while [D-Arg4]tuftsin, an analog with small tuftsin-like activity, exhibited a low degree of inhibition of [3H]tuftsin binding. Thus a rather high degree of specificity is involved in the binding of the tetrapeptide.Normal as well as six different macrophage populations induced by stimulation with thioglycollate, concanavalin-A, starch, mineral oil, glucan and Bacillus Calmette Guerrin (BCG), exhibited a similar degree of binding of [3H]tuftsin. Corynebacterium parvum (CP)-stimulated macrophages, on the other hand, showed a 6- to 10-fold-lower capacity for tuftsin binding.See Note added in proof on p. 62. Under similar experimental conditions, mouse fibroblast and lymphocyte preparations revealed no detectable specific binding.Tuftsin augmented the phagocytic response of normal and stimulated macrophages assessed both for phagocytosis mediated via the Fc-receptor and via non-specific receptors. CP-stimulated macrophages did not exhibit an increased phagocytic response upon treatment with tuftsin.

Notes: Infection of BALB/c mice with Rauscher leukemia virus (RLV) gives rise to pronounced erythrocytopoiesis manifesting in splenomegaly and is associated with progressive development of anemia. In the spleen erythroid colony forming units (CFU-E) increase exponentially up to 800-fold that of normal levels by the third week of infection. In vitro these CFU-E are dependent on erythropoietin for colony formation, their erythropoietin requirements being higher than that of CFU-E from normal mice. Numbers of CFU-E in spleen and degree of splenomegaly in anemic RLV infected mice were also shown to be modified by red blood cell transfusion, but progression of the disease was not stopped. Erythroid burst forming units (BFU-E) were also responsive to erythropoietin. However, a small proportion of cells also formed BFU-E colonies at concentrations which did not support growth of normal marrow BFU-E.When compared to normal, CFU-E found in RLV-infected spleen have similar velocity sedimentation rates. However, buoyant density separation of leukemic spleen cells indicated that CFU-E were more homogeneous (modal density 1.0695 g/cm3) than CFU-E from normal spleen. Analysis of physical properties of CFU-E and the nonhemoglobinized erythroblast-like cells, which accumulate in the spleen showed that they differed mainly in their distribution of cell diameter.Our findings show that erythroid progenitor cells in RLV infected mice are responsive to erythropoietin in vitro. Also in vivo erythropoiesis appears to be under control of erythropoietin but other factors which lead to progression of RLV disease apparently exist. Most proerythroblast-like cells, which are characteristic of this disease, apparently lack the potential to form colonies and may be more mature than CFU-E.

Notes: We are examining the relationship of RNA metabolism and de novo pyrimidine synthesis as parameters of malignant transformation. These initial experiments on normal hamster embryo fibroblasts have shown that excreted nucleosides are markers for intracellular RNA metabolism. We employed affinity chromatography to concentrate the nucleosides in the medium and sensitive column chromatographic procedures to quantitatively measure them. The excretion of pyrimidine nucleoside from hamster embryo fibroblasts in culture was found to be dependent on the growth stage of the cells, with the greatest accumulation occurring during cell quiescence. The major nucleoside excretion products, uridine and cytidine, were both normal end products of RNA metabolism and the major nucleoside excretion products from cultured cells. The modified nucleosides N-1-methylguanosine, N-2-methylguanosine, N-2-dimethylguanosine, N-4-acetylcytidine, N-1-methylinosine, pseudouridine, N-1-methyladenosine, N-3-methylcytidine, and 5-methylcytidine were found, as were several unidentified nucleosides.

Notes: A transplantable methylcholanthrene-induced fibrosarcoma of female BALB/c mice (the MC-2 fibrosarcoma) was dissociated by combined mechanical and enzymatic means, then fractionated by isopycnic centrifugation in linear albumin gradients.In some experiments recovered cells were both cultured in soft nutrient agar and inoculated subcutaneously into syngeneic recipients. In these experiments a highly significant correlation was observed between subsequent colony number and rapid growth phase tumor size suggesting identity of clonigenic and tumorigenic cells.It was consistently found that clonigenic cells were markedly depleted from the low density extremes of the cell density distribution profiles suggesting that the low density neoplastic cells had irreversibly left the growth fraction.With increasing tumor age, sequential studies showed that both total and clonigenic cell density distribution profiles were variable, showing no obvious trend, suggesting that in the age (13-35 days) and size (2-8 g) range studied growth fraction changes had little selective effect on cells of any specific density. These results imply that a marked selective depletion of low density clonigenic cells (or selective accumulation of low density non-proliferative cells) must mainly occur during an earlier phase of tumor growth. Studies on several other murine solid tumors also showed maximal depletion of clonigenic cells from the least dense fractions, suggesting that this situation may be common.

Notes: A variant of murine L5178Y lymphoma resistant to procaine hydrochloride (PH) was selected by exposing the cells to gradual increments of PH in the growth medium until the cell grew exponentially in the presence of 1.5 mM PH. Using cinephotomicrography, it was observed that the majority of cells that initially succumbed to PH failed to undergo successful mitosis. With respect to chromosomal, cell size distribution and flow microfluorometric analyses, the PH-resistant cells are very similar to a spontaneous tetraploid cell line (R1T) previously cloned. The isolated cells, designated R1/P, were also found to be cross-resistant to analogues of PH, namely, lidocaine, tetracaine and dibucaine. The naturally-occurring tetraploid cell line (R1T) was also found to be more resistant to local anesthetics, although not to the same extent as R1/P cells. Since the enzyme that hydrolyzes procaine appears to be absent in all these lymphoid cell lines, the difference in resistance does not appear to depend on differences in the ability of these cells to remove the agent. It is suggested that an alteration in the structure and/or function of the plasma membrane in R1/P cells have rendered them either less sensitive to the membrane-perturbing effects of the local anesthetics or less permeable to local anesthetics molecules. The ability of local anesthetics to affect membranes and cytoskeleton structures may play a role in the genesis and/or selection of these cell variants.

Notes: Cultured Friend murine erythroleukemia cells (Friend cells) are induced to undergo erythroid differentiation when grown in the presence of dimethylsulfoxide (DMSO) and other compounds. The effects of unifilar substitution of bromouracil (BU) for thymidine in the DNA (BU-DNA) of Friend cells were examined. Cells were grown in the presence of 5-bromodeoxyuridine (BrdU) for one generation, then centrifuged and resuspended in medium containing DMSO without BrdU. These cells exhibited a delay in the appearance of heme-producing, benzidine-reactive (B+) cells and a decreased rate of cell proliferation in comparison to the control not containing BU-DNA. A transient inhibition of entry into S phase was observed when control cells or cells containing BU-DNA were grown in the presence of DMSO for 10 to 20 hours. This transient inhibition was increased in the BrdU culture. Thus, BU-substitution in Friend cells alters other cellular functions in addition to erythroid differentiation. The rate of increase in the percent of cells committed to differentiate (those forming B+ colonies in plasma clots) was similar in the BrdU and control cultures until 40 to 50 hours. After this time, a delay in the appearance of committed cells was observed in the BrdU culture. The effect of BrdU on the appearance of B+ cells was more pronounced and occurred earlier than its effect on the rate of commitment. Therefore, the delay in the appearance of B+ cells in the BrdU culture was due primarily to perturbation of post-commitment events such as the accumulation of hemoglobin.We also examined the effect on growth and differentiation after BrdU was incorporated during different intervals of S phase in cells synchronized by centrifugal elutriation or by double thymidine block and hydroxyurea treatment. The delay in the appearance of B+ cells and inhibition of cell proliferation were only observed when BrdU was incorporated in the first half of S phase. BrdU (10 μM) had no effect on growth or differentiation when present during late S or G1 and G2. These results, using two very different methods to achieve cell synchrony, indicate that the effects of BrdU on growth and differentiation described above are due to its incorporation into DNA sequences replicating during early S.

Notes: Somatic cell hybrids between either normal human fibroblasts, phenotypically normal mouse fibroblasts or mouse peritoneal macrophages and HT1080 human diploid fibrosarcoma cells were studied for their ability to form tumors in nude mice. The results of this study indicate that tumorigenic behavior is expressed as a dominant trait in both human-human and mouse-human hybrid cells.

Notes: The interaction of mitogenic factors on a single cell type and the comparative activity of a given factor in diverse cell types have been studied byapplying the principles of Michaelis-Menten kinetics to clonal growth data. Such comparisons are facilitated by derivation of two parameters; Kmmitogen, the mitogen concentration that gives half-maximal clonal growth and a theoretical maximal growth rate, RMAXT. Both parameters are analogous to the Km and VMAX as applied to enzymatic reactions. Use of these parameters permits meaningful comparisons between cells with different growth rates.Using kinetic analysis of dose-response data, we found that normal human epithelial cells require 200 times more fetal bovine serum protein (FBSP) than a malignant line to multiply at their respective half-maximal rates. Further, the KmFBSP of normal cells was reduced to that of the malignant line by the inclusion of growth factors (EGF or FGF, and hydrocortisone) in the medium. On the other hand, even though greater levels of serum were required when growth factors and hydrocortisone were not present, their inclusion did not alter RMAXT. Interactions between mitogenic factors were shown to be unidirectional. Although EGF reduced the KmFBSP, FBSP did not change the KmEGF. The same type of analysis revealed that hydrocortisone, which potentiated the mitogenic activity of EGF did not change the KmEGF. Kinetic analysis of cell growth should prove useful in studies on the relation between growth and tumor promotion as well as in the evaluation of growth-inhibiting chemotherapeutic agents.

Notes: Binding of Concanavalin A to mouse L cells which were grown in serum free, chemically defined medium and depleted of their membrane sterol by blocking their de novo sterol synthesis was investigated. Kinetic analysis of binding data implied positive cooperativity, with two different affinities for Con A, in both experimental and control cultures. The amount of Con A bound to the cell surface at saturation was approximately 0.5 picomoles per mg cellular protein in controls and approximately 1.0 picomoles per mg cellular protein in 25-hydroxycholesterol treated cultures (which had a reduced sterol concentration of up to 50% in their plasma membranes). This phenomenon was reversed when cholesterol or mevalonate was added to the inhibited cultures to compensate for their inability to synthesize sterol. Our findings indicate that lectin binding to specific glycoprotein receptors is influenced by membrane lipid composition.

Notes: A novel variant of S49 mouse lymphoma cells is described which is resistant to growth arrest and cytolysis by dibutyryl cyclic AMP but, in contrast to previously described variants, has normal cyclic AMP-dependent protein kinase. The variant is also resistant to N6-monobutyryl cAMP but is sensitive to killing by 8-bromo cAMP and cholera toxin. Extracts of the variant appear to contain wild type levels of both O2′-butyrylesterase and cyclic AMP phosphodiesterase activities. Accumulation of exogenous [3H]dibutyryl cyclic AMP is reduced in the variant suggesting a defect in either uptake or secretion of the analog or its metabolic products. Accumulation of cyclic AMP in variant cells after stimulation of adenylate cyclase with either isoproterenol or cholera toxin is also reduced compared with wild type cells, although cyclase activity of membranes prepared from the variant cells is normal. Extracellular accumulation of cyclic AMP after stimulation of variant cells with isoproterenol is greater than that found with wild type cells. It is concluded that the variant has an alteration in its cyclic AMP secretion mechanism resulting in more efficient extrusion of cyclic AMP than in wild type cells.

Notes: We have produced somatic cell hybrids between totipotent mouse teratocarcinoma cells and rat hepatoma cells. These hybrids were tested for the expression of liver specific functions expressed in the hepatoma cell parent and for their ability to differentiate when injected into nude mice. The results of this study indicate that hybrid cell clones do not resemble either of the parental cells, since they do not produce albumin and tyrosine aminotransferase that are expressed in the rat hepatoma parent, and are incapable of forming either teratocarcinoma or hepatomas when injected in experimental animals.

Notes: Adhesion between cells of different tissues from chick embryos was studied by the modified collecting particle (Pessac et al., 1977a) and stationary monolayer assays. These assays measure the number of 3H labeled cells that adhere to the surface of cell aggregates and tissue fragments or on top of cell monolayers. The numbers of cells from a given suspension that adhere to identical or different cells can be compared. Intercellular adhesion may be considered as tissue specific if, in identical conditions, more cells adhere to identical cells than to cells from different tissues. Neural retina, cerebrum, optic tectum, liver and kidney cells from 9-day-old chick embryos showed no tissue specificity of adhesion, while heart cells adhered preferentially to collecting heart fragments.

Notes: Hepatocytes were isolated by established procedures from freshly-excised livers of ovariectomized rats. Integrity of the cells was verified by DNA, protein, and calcium contents, and by dye exclusion. The cells also showed progressive increments in oxidation to 14CO2 of [26-14C]cholesterol during one to five hours' incubation. Analysis was undertaken of cellular reactivities toward estrogen and the hepatocarcinogen dibutylnitrosamine (DBN). Binding and retention of [3H]estradiol-17β (E2β) by isolated liver cells was specific for E2β, saturable, temperature-dependent, and maximal after 30-minute incubation. The apparent dissociation constant for the binding process at 22°C is 2 × 10-9 M, and the total number of binding-sites at saturation corresponds to approximately 3,400 E2β molecules per liver cell. To probe for steroid binding-sites at their external surfaces, cells were incubated 30 minutes with mounted 17β-estradiol-17-hemisuccinyl:albumin:nylon fibers. The covalentlyimmobilized estrogen (1 ng/mg albumin) was accessible for interaction with antiserum directed against 17β-estradiol-17-hemisuccinyl:albumin. Significant numbers of isolated liver cells were retained by estrogen-derivatized fibers at 22°C after extensive washes. Binding was markedly reduced by incubation at 4°C and by prior exposure to free E2β (× 10-8 M), but not to the relatively inert estradiol-17α (E2α). Fiber-bound cells could be dislodged by brief incubation in 150 mOsM saline with 2 × 10-7 M E2β or diethylstilbestrol, but not E2α, cortisol, progesterone, or testosterone, and recovered intact. Cells that had been retained by the fibers and those that were not adherent were collected and washed under identical conditions, then plated in serum-free, chemically-defined medium at 37°C. After 72 hours, specific binding of E2β by the fiber-binding cells during 30 minutes' incubation was 2.5-fold that of cells which had not bound the immobilized steroid. Similarly, stimulation of the oxidation to 14CO2 of [26-14C]cholesterol by E2β was greater in fiber-binding than in non-binding liver cells after three hours' incubation. In the absence of added mitogen, thymidine incorporation into macromolecular form (20 hours), and cell proliferation (48 hours) were significantly greater in fiber-binding cells as compared to non-binding hepatocytes. Moreover, in parallel experiments, when cells were exposed to 1 × 10-9 M estrogens or to 1 × 10-4 M nitrosamines to assess the capacities of these substances to increase basal thymidine incorporation, total DNA, and cell numbers, only those cells with estrogen-binding sites at their surfaces showed significant E2β- and DBN-induced increments in these parameters as compared with paired controls that had been treated with E2α or the noncarcinogen diphenylnitrosamine. These data indicate that the accessibility of hormone-binding components at the plasma membrane may contribute to the capacity of a given liver cell to respond to E2β, as well as to other known hepatocarcinogens.

Notes: The genetic approach to the problem of cellular growth control is limited by the availability of recessive mutations in cell lines which are capable of growth control in vitro. The CHO cell line has yielded many recessive mutations including, for example, tsH1, a temperature sensitive leucyl-tRNA synthetase mutant, which under non-permissive conditions rapidly shuts down protein synthesis and generates uncharged tRNA. Both CHO and tsH1 are transformed, however, and do not respond to environmental stimuli with the coordinated regulation of macromolecular processes observed in normal diploid fibroblasts. We describe here the isolation and characterization of growth control revertants obtained from both CHOwt and tsH1. The best of these GRC+L-73, isolated from tsH1, had 20 chromosomes, one less than tsH1, had normal fibroblastic morphology, would not grow in suspension, required high serum concentrations for growth, grew to relatively low cell densities at saturation in monolayer culture and showed a stationary phase characterized by arrest in a G1-like state with maintenace of high viability for several weeks. It is expected that this line as well as a ts revertant GRC+LR-73 will greatly facilitate the genetic investigation of growth control and, in particular, will help to elucidate the role of uncharged tRNA in the regulation of macromolecular synthesis in mammalian cells.

Notes: Treatment of adult rats with dexamethasone resulted in an increase in cardiac muscle weight but a decrease in skeletal muscle weight. The different response of skeletal and cardiac muscles to the glucocorticoid was also reflected by a dexamethasone-induced enhancement of myofibrillar protease activity in the gastrocnemius muscle and an inhibition of a similar proteolytic activity in the heart. Newborn rats also exhibit the same, tissue-specific response to the glucocorticoid hormone. Consequently, the difference between cardiac and skeletal muscle responsiveness to conditions of wasting was investigated in culture. Average rates of degradation of intracellular proteins were determined in cultured cells derived from rat skeletal and cardiac muscle by following the release of radioactivity from cells prelabelled with 14C-phenylalanine. The release of label into the TCA soluble medium as measured during 12 hours of incubation, conformed to a first-order reaction and both cell types were found to degrade intracellular proteins at a similar rate. After 12 hours of incubation in a complete Ham F-10 medium supplemented with serum approximately 18% of total cellular protein was degraded. Incubation in a minimal medium or serum-deprivation enhanced the average rate of proteolysis to a value of 29% degradation at 12 hours indicating that intracellular proteolysis in these cells is responding to nutritional deprivation by increased activity. However, addition of glucose (22.2 nM) or dexamethasone (10-6M) to the incubation medium failed to affect the rate of net protein degradation. Under no experimental condition could a difference be found between the proteolytic response of skeletal muscle cells to that of cardiac muscle cells and both cell types displayed similar changes in rates of protein degradation under various nutritional and hormonal conditions in culture. Thus, protein sparing in the heart of intact animals under catabolic conditions which enhance protein loss in skeletal muscle can probably not be ascribed to intrinsic differences in the direct response of cellular proteases to the tested hormones and nutrients. Rather, an extracellular factor(s) is apparently required for induction of the differential response of these tissues in the intact animal to protein wasting conditions. Alternatively, cells in culture might have lost the property of differential degradative response which operates in vivo.

Notes: In the past, it has been difficult to grow human diploid fibroblast cells at clonal densities. Newly devised cell culture media and rigorously controlled environmental conditions have greatly increased the ease with which such cells can be cloned. The present work was undertaken to determine whether, under appropriate conditions, diploid fibroblast cells from human embryonic lung, grow as well at clonal densities as in mass culture. The parameters studied were: (1) population doubling time, (2) in vitro proliferative capacity, (3) attachment, (4) percentage of non-dividing cells. In all cases essentially the same results were obtained for cultures at clonal densities and mass cultures. These results indicate that the behavior of these types of cells in clonal culture can be used to infer the behavior of individual cells and clones within a mass culture.

Notes: RNA synthesis has been investigated in resting and growing cells of a culture of Scarlet rose. The rates of messenger RNA (mRNA) and ribosomal RNA (rRNA) synthesis are five- and ten-fold higher, respectively, in the growing culture. In stationary phase cultures, newly synthesized 26S and 18S rRNA do not appear in the cytoplasm in equimolar amounts. Rather, the 26S/18S ratio of [3H]-uridine labeled rRNA of stationary cells ranged from 0.9 to 1.3 while the ratio of the corresponding fraction from growing cells was 1.6 to 2.0. A similar result was obtained when cells were labeled with [3H-CH3] methionine. Pulse chase experiments demonstrated that the nascent pre-rRNA in resting cells could be chased into polysomes. These data are interpreted to indicate that a major part of the regulation of rRNA synthesis in stationary cells is at the level of the processing of the rRNA transcript.

Notes: The growth of cervical carcinoma cell (HeLa) cultures in a hyperosmolar environment stimulates increased production of the onco-developmental peptides human choriogonadotropin (hCG) and follitropin (FSH). This effect was observed in two sublines examined in this study, HeLa65 and HeLa71. hCG and FSH were measured by radioimmunoassay using antiserum against the β-subunit of the hormone dimer, thus insuring immunochemical specificity. The amounts of hCG and FSH produced by HeLa65 and HeLa71 cells cultured in hyperosmolar medium were 2- to 50-fold higher than corresponding hormone levels in basal cultures. Synthesis of gonadotropins depended on concentration and duration of exposure to hyperosmolar medium. Levels of culture medium osmolality effective in inducing hormone production also inhibit the incorporation of 14C-thymidine into acid-insoluble macromolecules. Hyperosmolality thus stimulates the ectopic production of gonadotropic hormones while retarding cellular growth and nucleic acid synthesis.

Notes: Wild-type Cloudman S91 melanoma cells have a retarded rate of division when agents which raise cyclic AMP levels such as melanotropin, protaglandin E1, or cholera toxin are supplemented to the culture medium. A mutant cell line was isolated which had the opposite response, i.e., the mutant grew very slowly unless agents which raised cyclic AMP levels were present (Pawelek et al., '75a). In this report evidence is presented indicating that the molecular basis for the mutant phenotype resides in the major cyclic AMP-dependent protein kinase found in the cells. The mutant kinase had increased thermolability and an elevated activation constant for cyclic AMP over the corresponding wild-type kinase. It is proposed that the elevated requirement for cyclic AMP for the proliferation of cAdep cells is related to the elevated activation constant of this kinase, suggesting that the kinase is a positive regulator of proliferation in Cloudman S91 cells.

Notes: Cytoplasts prepared from L929 or Hepa-2 cells were separated from whole cells using density gradients of renografin. Using this technique, cytoplasts can be isolated from cell lines which cannot be routinely enucleated with an efficiency of 100%. The purified cytoplasts excluded the vital dye trypan blue and were utilized in nuclear transplantation experiments to reconstruct whole viable cells capable of division. In addition, the renografin gradient technique was used to separate the newly reconstructed cells from any contaminating “non-renucleated” cytoplasts. This will permit immediate biochemical characterization of cytoplasmic-nuclear hybrid cells without interference from contaminating cytoplasts.

Notes: The toxicity and extent of growth inhibition produced by chloramphenicol (CAP) in CAPs Chinese hamster cells (line V79-5) was found to be dependent on the type and concentration of hexose in the medium. In high levels of glucose (6.5 mM), cultures of CAPs cells underwent 7-8 population doublings in the presence of 100 μg/ml CAP and viability then dropped rapidly. In contrast, lower concentrations of glucose (1.0 mM) permitted only limited growth (2-3 doublings) in the presence of 100 μg/ml CAP, but the cells remained viable and apparently quiescent for prolonged periods of time. The growth potential of V79-5 cells in CAP appeared specifically dependent on glucose, as mannose and galactose could not substitute for glucose. The toxicity of CAP to these cells seemed to be determined primarily by the number of cell doublings in the presence of the drug.A CAPR derivative of V79-5, designated 5-3, was analyzed in order to determine whether the requirement for glucose for cell growth in the presence of CAP also occurred in cells that were isolated as resistant to the drug. In order to rigorously control the hexose in the medium, some experiments were performed with medium containing dialysed, instead of whole, fetal calf serum. It was seen that the growth of the CAPR line in the presence (but not the absence) of 100 μg/ml CAP was dependent on glucose in the medium. Thus, resistance to CAP in these cells appears to be a conditional state, dependent on glucose for expression. Furthermore, the glucose auxotrophy of these cells in the presence of CAP suggests that CAP is still affecting some activities in cells isolated as CAPR.

Notes: Heat-synchronized Tetrahymena pyriformis have been subjected to 5-, 15- and 30-minute pulses of hydrostatic pressure in the range 100-300 atm, without being simultaneously subjected to significant heats of compression. The pressure-induced division delays depend on (1) the level of pressure used, (2) the length of pressure pulse and (3) the time after the synchronizing treatment at which the pressure is applied. A pressure-dependent inhibition of 3H-leucine incorporation into protein was also measured. Comparison of the effects of pressure with those of pulse treatments of cycloheximide and emetine on cell division and protein synthesis revealed that the physical agent produced characteristically different responses from those of the chemical agents. Of particular interest was the fact that the division delays induced by pressures of 200 atm and above were greater than those observed after treatments with cycloheximide and emetine which produced comparable levels of protein synthesis inhibition. Pressure also delayed cells if it was applied at a time when addition of chemical inhibitors had little effect on the first synchronous division. The results show that inhibition of protein synthesis by pressure cannot entirely account for pressure-induced effects on cell division. The possibility that pressure may also directly affect other processes, such as the assembly of proteins into structures required for division, is discussed.

Notes: A study was made of the transport of a variety of amino acids by uninfected and Rous sarcoma virus-infected chicken embryo fibroblasts. Following a period of amino acid starvation, transformed, but not normal cells, showed increased levels of transport for α-aminoisobutyric acid, proline and alanine, three amino acids which are transported primarily by the A transport system. There was no starvation-induced increase in the transport of leucine, phenylalanine, lysine, or cycloleucine. In the absence of starvation, normal and transformed cells exhibited comparable rates of amino acid transport. Cycloheximide was able to block the increase in uptake. The enhanced uptake was characterized by an increase in Vmax for transport and little change in Km.The data demonstrate that an alteration in the regulation of the A amino acid transport system is an early event in malignant transformation by Rous sarcoma virus. However, since this alteration is made manifest only following a period of starvation, our findings suggest that increased amino acid uptake does not play a role in generating the other manifestations of the transformed state seen in cell culture.

Notes: 2-Deoxyglucose transport was characterized in human polymorphonuclear leukocytes (PMN) and rabbit alveolar macrophages (AM). The Km was 1 mM for human PMN and 1.6 mM for rabbit AM, and the Vmax was 0.66 × 10-3 μmoles/45 sec/106 PMN and 5.09 × 10-4 μmoles/45 sec/106 AM. The rate of 2-deoxyglucose transport was the same before and after phagocytosis in PMN from normal individuals and three patients with chronic granulomatous disease, as well as rabbit AM. Studies of the kinetics of 2-deoxyglucose transport and intracellular fate of 2-deoxyglucose in human PMN indicate that the nature of the membrane transport system is not altered by phagocytosis. The results support the concept that the plasma membrane is mosaic in character with geographically separate transport and phagocytic sites.

Notes: The phenotype of a ouabain-resistant Aedes albopictus cell line has been partially characterized. Treatment of ouabain-sensitive cells with 0.005-1.0 mM ouabain resulted in an 80% reduction in the uptake of 86rubidium (86Rb+), an ion with an affinity for the K+ pump binding site; ouabain-resistant cells showed only a 40% reduction with 1.0 mM ouabain. When ouabain-sensitive cells were incubated in the presence of ouabain (0.1 mM) for one and one-half to three hours, the molar ratio of intracellular Na+/K+ rose from 0.2 to 4.2. In ouabain-resistant cells, a similar treatment had very little effect. Based on [3H] ouabain-binding studies, ouabain-resistant cells were estimated to have 60% fewer binding sites per cell than ouabain-sensitive cells. The spontaneous mutation rate from ouabain sensitivity to ouabain resistance was calculated to be 1-6 X 10-8 mutations/cell/generation, a value similar to that reported for mammalian cells at the analogous locus.

Notes: Tunicamycin, an antibiotic that inhibits protein glycosylation, elicited a rapid depletion of insulin binding activity at the surface of 3T3-L1 adipocytes. Disappearance of insulin receptors occurred more rapidly in the presence of tunicamycin than when protein synthesis was inhibited by cycloheximide and was accompanied by a diminution in sensitivity of the adipocytes to the acute effects of insulin and anit-insulin receptor antibody on hexose uptake and metabolism.

Notes: T98 and T98G are two related cell lines that were derived from a human glioblastoma multiforma tumor. T98G has almost twice as many chromosomes as T98, suggesting that it is a polyploid variant of T98. Three aspects of control of cellular proliferation were studied in T98 and T98G cells in comparison to WI-38 normal human diploid cells. WI-38 cells have the following properties: (1) they can undergo only a limited number of population doublings in vitro; (2) they cannot proliferate without anchorage; and (3) they become arrested in G1 phase under stationary phase conditions. T98 cells differ from normal cells in all three of these properties, as do many other transformed cell lines. However, the derivative of T98, namely T98G, expresses an unique combination of normal and transformed aspects of the control of cellular proliferation. T98G cells are like normal cells in that they become arrested in G1 phase under stationary phase conditions, yet they also exhibit the transformed characteristics of anchorage independence and immortality. Thus, T98G cells demonstrate that transformation to immortality and anchorage independence can exist without concomitant loss of the normal mechanism for G1 arrest in response to stationary phase conditions. This result supports the hypothesis that each of these three aspects of control of cellular proliferation can be altered independently. Partially transformed cell lines, such as T98G, should be useful for sorting out the biochemical changes associated with transformation in each of these aspects.

Notes: The relationship of cell surface changes to proliferative decline of human diploid fibroblasts was investigated using the concanavalin A-mediated red blood cell adsorption assay. The amount of the red blood cells adsorbed to human diploid fibroblasts via concanavalin A increased continously from the early phases of cell passage up through cell senescence, while the amount of 3H-concanavalin A binding did not change to a significant extent. The red blood cell adsorption is not a function of cell cycle phase and time spent in culture. Cocultivation of young cells with old cells also did not affect the adsorption capacity of respective cells. Thus, the concanavalin A-mediated red blood cell adsorption can be expected to serve as a new cell surface marker for aging in vitro. Using this marker, it was revealed that transient cell size or 3H-thymidine incorporating capacity do not have a direct relationship with the division age of a cell. Small rapidly dividing cells in old populations resemble large slowly dividing or nondividing cells of the same populations and differ from small rapidly dividing cells in young populations, in terms of cell surface properties.

Notes: Normal rat hepatocytes have been fused with highly differentiated rat hepatoma cells. Some of the hybrids express a physiologically significant level of activity of the urea cycle enzyme ornithine carbamoyltransferase (OCT), a liver-specific function not found in the hepatoma cells. These hybrids have 10% of the adult rat liver OCT specific activity, incorporate 3H-ornithine into protein arginine, and can be selectively grown in arginine-free medium supplemented with ornithine. Somatic cell hybridization of normal differentiated cells with highly differentiated neoplastic cells of the same tissue type may be useful as a general method for obtaining permanent cell lines with new tissue-specific phenotypes.

Notes: This report strongly suggests that two compartments in Tetrahymena thermophila contain peptidase activity: the cytoplasm and the outer cell surface.Determinations of amino acid concentrations in the extracellular medium upon incubation of cells with peptides suggest that the surface-bound peptidase activity hydrolyses di- and tri-phenylalanine equally fast on a molar basis.Growth experiments designed to characterize the in vivo peptidase specificities showed that both T. thermophila and T. pyriformis can use L-leucyl-L-leucine, but not L-leucyl-D-leucine as a leucine donor. These results are independent of whether the cells form food vacuoles or not.

Notes: Stimulation of Balb/c-3T3 cell growth by TPA requires factors found in serum. We examined the interaction between TPA and serum growth factors in the stimulation of cell growth. The number of cells synthesizing DNA (incorporating 3H-thymidine) within 24 to 30 hours after the addition of TPA and the growth factors to density-inhibited Balb/c-3T3 cultures in serum-free medium was determined by autoradiography. With no additions or with TPA (30-300 ng/ml) alone, only 3-7% of cells synthesized DNA. However, TPA synergistically promoted DNA synthesis in combination with each of the defined serum growth fractions, platelet derived growth factor and platelet poor plasma. TPA also synergistically promoted DNA synthesis in combination with purified growth factors including fibroblast growth factor, insulin (10-6-10-5 M), and epidermal growth factor. In all conditions, TPA enhancement of DNA synthesis also resulted in an increase in cell number. Because TPA synergistically enhanced the activity of each growth factor tested, it did not act identically to any of the growth factors.

Notes: Prostaglandin generation by human peripheral blood mononuclear cells is enhanced during co-culture with human thyroid cells. The objective of the present study was to determine the influence of various sera on this process.Human thyroid adenoma cell monolayers were cultured with normal human peripheral blood mononuclear cells for three days in the presence of a variety of sera, or serum fractions. Prostaglandin E (PGE) in the medium was measured by bioassay or by radioimmunoassay. Significantly more PGE was generated in cultures containing fetal calf serum than in those containing human serum. This difference was not abolished by dialysis of the human serum. When the 50% (NH4) 2SO4 precipitate of the serum was used, PGE generation was similar to that in fetal calf serum, indicating the presence of an inhibitory factor in human serum. The degree of this inhibitory activity was similar in autologous and heterologous human serum, as well as in normal subjects and patients with Graves' disease. Gel filtration and ion-exchange chomatography of human serum showed the inhibitor to co-migrate with albumin. Evidence presented suggests that the inhibitor is not albumin itself but is, instead, a factor tightly bound to albumin. Inhibitory activity was also found in rabbit, goat, rat and cow serum.Prostaglandins are potent modulators of immune-cell function. These data indicate that this process may be modulated by a factor in mammalian serum. The relative absence of this factor in fetal serum may have important implications in regard to the profound changes which occur in the immune system after birth.

Notes: We have recently found that aphidicolin, a tetracyclic diterpene-tetraol produced by several fungi, blocks DNA synthesis of sea urchin embryos by interfering with the activity of DNA polymerase α. These cells fail to proliferate in the presence of aphidicolin.In continuation of these studies, we determined the drug-sensitive stage in the first cell cycle of the sea urchin Clypeaster japonicus embryo.In continuous exposure to aphidicolin (2 μg/ml) from five minutes after fertilization, mitotic division of the embryo was completely suppressed. Embryos were exposed to the drug at progressively later intervals and their capability for cytokinesis was examined. Evidence was thereby obtained that aphidicolin acts at the S-period to inhibit DNA synthesis resulting in developmental arrest of the embryo.

Notes: A pseudodiploid clone of Chinese hamster cells transformed in vitro with Simian virus 40 (SV40) was isolated from soft agar and injected into nude mice through three successive passages with a short in vitro cultivation between each animal inoculation. The original clone and the three subsequent tumor populations were characterized in regard to SV40 T antigen staining, modal chromosome number, and Giemsa-banded karyotype. All tumor cell lines maintained the pseudodiploid mode, as well as the positive SV40 T antigen staining. Nonrandom chromosomal changes included loss of one of the X chromosomes, additions of abnormal variants of chromosomes No. 1 and No. 2, the appearance of unidentified marker chromosomes, and the loss of autosomes No. 5, No. 6, and No. 11. The deletion of one of the X chromosomes occurred with about the same frequency in all cell lines. Additions of abnormal chromosomes No. 1 and No. 2 tended to recur more often in the tumor cell lines than in the original clone. The appearance of marker chromosomes, as well as the loss of autosomes No. 5, No. 6, and No. 11 demonstrated a correlation with tumorigenicity. Yet, the three successive passages of the cells through the animal did not select for a tumor population with a single, homogeneous karyotype.

Notes: The objective of this study was to determine the points in the cell cycle at which normal and transformed cells become arrested as a result of polyamine deprivation. Treatment of normal (human fibroblast line PA2 and mouse 3T3) and transformed (CHO, HeLa and SV3T3) cells with methylglyoxal bis-(guanyl-hydrazone) resulted in a significant decrease in the levels of spermidine and spermine which was associated with an inhibition of growth. Examination of the prematurely condensed chromosomes (PCC) of the polyaminedepleted cells, revealed that normal fibroblasts were preferentially arrested in early G1 phase while a majority of cells in the transformed lines were blocked in S phase. A close examination of the PCC of the transformed cells indicated a significant decrease in the number of DNA replication sites suggesting that polyamines have an important role in DNA chain initiation.

Notes: Cholera toxin was used in an attempt to inhibit epidermal growth factor stimulated 3T3 cell division. Instead, cholera toxin alone at low concentrations (10-10 M), was able to stimulate cell division and could augment EGF stimulated cell division. The mitogenic effect of cholera toxin can occur despite a dramatic increase in the intracellular levels of cAMP in 3T3 cells. Cholera toxin stimulated mitogenesis could not be mimicked by choleragenoid, the binding but inactive subunit of cholera toxin, or by other agents which elevate cAMP levels in 3T3 cells.

Notes: A mutant (A204) of Chinese hamster ovary cells (CHO-K1), which is deficient in dihydroorotate (DHO) dehydrogenase (E.C. 1.3.3.1) activity, has been isolated by a replica plating procedure. The mutant does not show a requirement for exogenously added pyrimidines. Examination of intact cells shows that the mutant accumulates a large amount of carbamyl aspartate and is markedly but not totally deficient in biosynthesis of orotate from earlier precursors of pyrimidine biosynthesis, including aspartate and dihydroorotic acid, when compared to wild-type cells. Analysis of cell-free extracts of mutant and wild-type cells shows that the mutant is deficient in DHO dehydrogenase activity, possessing ca. 5% of the wild-type activity. This evidence leads to the conclusion that this mutant, A204, is in fact partially deficient in DHO dehydrogenase, and that in these cells it is this enzyme which carries out the fourth step of de novo pyrimidine biosynthesis.

Notes: Plasma membranes isolated from normal and RSV transformed chick embryo fibroblasts were phosphorylated in vitro using endogenous protein kinase and ATP (γ32P) and the labeled phosphoproteins were analyzed by SDS-PAGE. A number of protein phosphorylation changes were observed following transformation, however in most cases they were relatively small quantitative differences. The four major changes were in proteins of 47,000, 58,000, 75,000 and 135,000 daltons. Decreased phosphorylation of the 47,000 dalton polypeptide was found in transformed cell membranes but this alteration was shown to be due to differences in cell growth rather than transformation. Increased phosphorylation of the 75,000 dalton protein was at least partially related to virus infection. However, increased phosphorylation of the 58,000 and 135,000 dalton polypeptides were entirely transformation specific.

Notes: Incorporation of (14C)choline and (3H)myo-inositol into the total lipid fraction, incorporation of (14C)acetate into the sterol fraction and incorporation of (3H)thymidine into DNA were studied in human lymphocyte cultures. Concanavalin A induced an increase in the incorporation of these labels with the following features: (a) Phospholipid synthesis was increased promptly. The lag time for the increase in sterol synthesis and DNA synthesis were 5 hours and 27 hours respectively; (b) The increase in phospholipid synthesis and sterol synthesis was proportional to ConA concentration initially. Cells treated with a high concentration of ConA showed very low levels of DNA synthesis; (c) The increase in phospholipid synthesis could be abolished immediately by α-Methyl-Mannoside. α-Methyl-Mannoside blunted but did not abolish the increase in sterol synthesis. α-Methyl-Mannoside enhanced DNA synthesis of those cells which had been treated by a high concentration of ConA; and (d) Selective inhibition of sterol synthesis with 25-hydroxycholesterol did not prevent the increase in phospholipid synthesis, but it blocked the increase in DNA synthesis. Supplement of LDL, HDL or total lipoproteins to lymphocyte cultures was effective in preventing the inhibition of DNA synthesis by 25-hydroxycholesterol. These results suggest that in lymphocyte activation by ConA phospholipid synthesis, sterol synthesis and DNA synthesis were sequentially increased. The rate of cellular commitment to mitogenesis was proportional to ConA concentrations. High concentrations of ConA arrested the cell growth at a postcommitment point in the G1 phase. Enhanced phospholipid synthesis was a precommitment event. Enhanced sterol synthesis was a postcommitment event and reflected the requirement of an increased cholesterol supply for the passage of cell growth through G1.

Notes: The homologous compounds of the 4-alkoxy- and 4-alkylamino-series inhibit the exchange transport of glucose in human erythrocytes; they show a competitive inhibition with one or two inhibitor molecules which become bound to a singular site of the transport system for glucose. The importance of length of hydrocarbon chain of the localanesthetics for the mode of their action is discussed.

Notes: Iodinated proteins were degraded after injection into HeLa cells at first-order rates with half-lives varying from three hours for the trout nonhistone chromosomal protein, HMG-T, to 60 hours for whale myoglobin. Fluoresceinated-bovine serum albumin (fl-BSA) was degraded almost twice as fast as unmodified BSA. The rate of degradation of 125I-BSA was very similar in eight cell lines of mouse, human, monkey and rat origin. Microinjected proteins were analyzed on SDS-acrylamide gels after injection, and for BSA and immunoglobin G, all remaining intracellular 125I migrated at the molecular weight of the injected proteins. By contrast, more than 80% of the extracellular 125I chromatographed as iodotyrosine. With the exception of fl-BSA, which exhibited perinuclear accumulation in approximately one-half of the injected cells, autoradiography showed that throughout the period of study the injected proteins remained dispersed in the cytoplasm.

Notes: Cell division in fertilized sea urchin eggs was reversibly inhibited when the ketoaldehyde phenyl glyoxal (PG) at a concentration of 0.1 mM was added to eggs for ten minutes prior to the formation of the mitotic spindle. We investigated whether inhibition of mitosis was due to PG binding to the cell surface (as previously suggested by Stein and Berestecky, '74) or to some intracellular effect. When 14C-PG was added to eggs, label was readily taken up into the egg cytoplasm; very little label was associated with the egg surface. In the cytoplasm PG combined with equimolar amounts of reduced glutathione (GSH), decreasing the levels of cellular GSH to less than 15% of normal and accounting for at least 50% of the PG taken up by eggs. The concentrations of oxidized and protein-bound glutathione were unaffected by PG treatment. We showed that glyoxalase enzymes were present in sea urchin eggs and were capable of metabolizing the PG-GSH complex, thereby restoring GSH to normal levels after PG was removed from the sea water. Though some other effect of PG cannot be ruled out, the major fate of PG in eggs was to combine with GSH, and the transient decrease in GSH which resulted could lead to inhibition of mitosis. While other reports (Nath and Rebhun, '76; Oliver et al., '76) have shown that reagents which oxidize GSH disrupt microtubule-related events, our results showed that such inhibition could be caused by decreased GSH levels alone.

Notes: PGE1 elicited a slow, dose-dependent membrane depolarization with an increase in membrane conductance in the somatic cell hybrid TCX11. The ED 50 was 1-2 × 10-8 M with maximal responses at 1-5 × 10-7 M. Dopamine (DA) reversed the effect of PGE1 and caused the membrane potential and resistance to return to control levels. Chronic exposure of cells (measured in minutes) to DA alone would not cause this hyperpolarization. 5-HT was also tested and failed to consistently reverse the PGE1 effects. Chlorpromazine antagonized the effects of DA on the PGE1 response. The electrophysiological results reported here using TCX11 cells are discussed in light of previously reported biochemical results describing interactions of PGE1 and DA, and the electrophysiological effects of DA alone.

Notes: Mutants temperature-sensitive for growth have been isolated from the established line of Chinese hamster fibroblasts Wg1A. These mutants, together with the ones previously isolated by Roscoe et al. ('73), have been characterized with regard to their cell cycle properties. Most of them become arrested in the G1 phase of the cell cycle when incubated under restrictive conditions. By performing temperature shift experiments with synchronous cultures, the execution steps of most of the mutated functions have been located within the second half of the G1 phase.

Notes: Total cellular calcium levels do not change when 3T3-4a cells stop proliferating due to serum depletion, or when serum-arrested quiescent cells are incubated for up to 44 hours in calcium-deficient medium (∼10 μM Ca++). Upon stimulation with dialyzed serum cells enter S and progress through at least one cycle even at extremely low calcium levels in the culture medium (≥10 μM). Cells divide until a final cell density is attained which is proportional to the calcium concentration in the medium and cells reversibly arrest in G1. Cells which arrested in G1 in medium containing ≤26 μM Ca++ in the presence of excess serum can be stimulated to enter S in response to added calcium after a prereplicative phase of 14 to 16 hours. Serum does not affect 45Ca-uptake in these cells. Benzo[a]pyrene transformed 3T3 (BP3T3) cells have a 100-200 times lower Ca++-requirement than 3T3 cells but arrest in G1 at low Ca++ levels. In contrast, SV40-virus transformed 3T3 (SV3T3) cells that grow without restriction in monolayer cultures have even lower Ca++-requirements for growth than BP3T3 cells and have no Ca++-sensitive restriction point. Therefore, 3T3 and BP3T3 cells have retained the capacity to sense intracellular Ca++-pool sizes and to arrest in G1 at subthreshold cellular Ca++-levels.

Notes: Elemental concentrations in the cytoplasm and nucleus of post-natal mouse myocytes were measured at 2, 4, 8, 16, 32 days and in adults using electron probe X-ray microanalysis. Using an analysis of variance test, significant age dependent changes were found in intracellular potassium, sulfur, phosphorus, and chlorine concentations (mg/kg dry weight), while sodium and magnesium concentrations did not show significant changes. The application of t tests following linear regression analyses (age versus concentration for each element) did, however, give a significant slope for cytoplasmic sodium, potassium, sulfur, and phosphorus values. The findings correspond closely with an emission spectroscopy-titrimetric study of whole heart ventricle of the same developmental period (Hazelwood and Nichols, '70).

Notes: The objective of the present study was to determine the effects of insulin on amphibian hepatocytes in primary culture. Hepatocytes were isolated from adult bullfrogs by collagenase perfusion and maintained as monolayers in serum-free medium. Cells cultured in the continuous presence of insulin exhibited a relatively constant rate of protein secretion over the first four to five days, whereas controls showed an almost three-fold decrease over the same time period. The decline in secreted proteins was equally represented in most exported proteins, except that serum albumin secretion showed twice as much of a decrease relative to the other proteins.The maintenance of protein secretion by insulin was the result of its effect on protein synthesis. The rate of protein synthesis was measured by the incorporation of (3H)-leucine into protein using culture medium containing 0.5 mM leucine, a condition where the specific radioactivity of leucyl-tRNA was shown to be equal to that of (3H)-leucine in the medium. Cultures maintained with insulin for 60 hours synthesized protein at two to three times the rate found in non-insulin treated controls whose rate of protein synthesis was first detectably decreased after nine hours of culture in the insulin-free medium.Sedimentation profiles of polyribosomes from hepatocytes maintained for 60 hours without insulin showed proportionately fewer ribosomes in large polysomes and more in monosomes and free ribosomal subunits than ribosomes from cells cultured with insulin. This result suggests that the decrease in protein synthesis found in the absence of insulin is due to a defect in initiation. Insulin does not exert its effect by regulating cellular levels of ATP; no change in ATP content was found in cells maintained with or without insulin.The results show that insulin maintains high levels of protein synthesis and secretion in amphibian hepatocytes. The hepatocytes in monolayer culture provide a system to study the molecular mechanisms involved in the translational control of protein synthesis by insulin.

Notes: The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA in Chinese hamster ovary (CHO) cells and in mitogen-stimulated human lymphocytes during their progression through the cell cycle. Green and red fluorescence of individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry.CHO cells were synchronized by selective detachment at mitosis. Their rate of progression through G1 and subsequently through S phase correlated with the content of stainable RNA. The mean duration of the G1 phase was 5.2 hours for cells with high RNA content (highest 25 percentile population) and 8.1 hours for cells with low RNA (lowest 25 percentile). The duration of S phase was 5.9 and 7.5 hours for high- and low-RNA, 25 percentile subpopulations, respectively.Lymphocytes synchronized at the G1/S boundary by hydroxyurea or 5-fluorodeoxyuridine showed extremely high intecellular variation with respect to content of stainable RNA. After release from the block they traversed S phase at rates linearly proportional to the content of stainable RNA. The duration of S phase was five hours for cells with high RNA-, six to nine hours for cells with moderate RNA- and up to 27 hours for cells with minimal RNA-content.The data suggest that the rate of progression the cell cycle of individual cells within a population may be correlated with the number of ribosomes per cell.

Notes: Stimulation of postconfluent Swiss 3T3 cells in serum-free medium with 4.3 mM Ca2+ results in marked increases in both released and cell-associated plasminogen activator (PA). Increased release of PA commenced approximately 10 to 12 hours post-stimulation and continued to increase steadily until 48 hours at which time the stimulated cells (4.3 mM Ca2+) released approximately 14 times more PA than control cells (1.8 mM Ca2+). Sr2+, like Ca2+, also stimulates PA synthesis/release either in the presence or in the absence of 1.8 mM Ca2+ whereas an excess of Mg2+ inhibits Ca2+ stimulation. Supranormal [Pi] in the medium stimulates PA synthesis/release in the presence of 1.8 mM Ca2+. Further, optimal stimulation by 4.3 mM Ca2+ requires a normal level of Pi (1.0 mM). Elevation of medium [Ca2+] or [Pi] results in an enhanced uptake of Ca2+. The facts that cycloheximide treatment completely abolishes the Ca2+ stimulatory effect and that an increase in cell associated PA precedes release indicate that PA release is coupled to synthesis of new PA. Ca2+ stimulation of PA synthesis/release also requires continuous energy production and RNA as well as protein synthesis. A hypothesis is proposed to explain the relationship between stimulation of PA production and its enhanced release from cells stimulated by elevated [Ca2+] or [Pi] in the media. The possibility that PA release may be an example of the phenomenon of membrane shedding as opposed to secretion is discussed.

Notes: Binding of either low density lipoprotein (LDL) or Concanavalin A (ConA) to actively growing vascular endothelial cells is associated with a redistribution of the appropriate cell surface receptor sites which form patches and caps. This receptor lateral mobility is greatly restricted when endothelial cells reach confluence and adopt the configuration of a cell monolayer composed of closely apposed and non-overlapping cells. In this case, although the cells still exhibit specific LDL binding to the appropriate cell surface receptor sites, neither the binding of LDL nor of ConA induces a receptor redistribution. The lack of LDL receptor redistribution correlates with a marked decrease in the rate of LDL internalization. In contrast, no such a density-dependent changes are observed in cell types which grow on top of each other and form multiple cell layers at confluence. Thus, neither LDL nor ConA induced cap formation in either sparse or confluent smooth muscle cell cultures and the same rate of LDL internalization is observed at both cell densities. Similarly, adsorptive endocytosis of cationized LDL (which enters the cell independently of the LDL receptor sites) was not correlated with a detectable receptor redistribution, nor was it significantly affected by changes in cell density and spatial organization.The formation of a confluent cell monolayer resting on an underlying basement membrane might therefore provide, via a change in membrane dynamics, a mechanism whereby the endothelium of large blood vessels can function as a protective barrier against the high circulating levels of LDL in plasma.

Notes: The three xanthine derivatives, caffeine, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) produced dose-dependent increases in cyclic AMP concentrations in HeLa cells after long term treatment. Only IBMX produced increases over the first 60 minutes, with a peak of approximately 5-fold control values five to 10 minutes after the addition of the drug. About four hours after the addition of either 0.67 or 1.0 mM IBMX there was a second peak in the concentration of cyclic AMP which was at least as large and usually larger than the peak observed at five to ten minutes. Neither caffeine nor theophylline increased cyclic AMP concentrations above control values until one hour after addition of the compounds, and there was no indication of a peak in the concentration at four hours. Between 24 and 72 hours, all three compounds produced elevations in cyclic AMP levels that were steadily maintained. At any given concentration, the order of potency was IBMX 〉 theophylline 〉 caffeine. If the xanthine derivatives were removed from the medium after 24 hours of treatment, the cyclic AMP concentrations fell to control levels within one hour. Treatment with 5-iodo-2′-deoxyuridine (IdUrd) or hydrocortisone alone did not change the levels of cyclic AMP, nor did the presence of these inducers of alkaline phosphatase activity alter the effects of the xanthine derivations on cyclic AMP concentrations. The data showed a significant correlation between the magnitude of the increase in cyclic AMP concentrations over the period from 24 to 72 hours and the degree of inhibition by the xanthine derivatives of the induction of alkaline phosphatase activity.

Notes: The ability to synthesize inosinetriphosphate was demonstrated in blood cells as well as in a variety of tissue extracts in spite of the presence of ITP pyrophosphohydrolase. At the expense of having sub-optimal conditions, an assay system was selected that completely repressed the hydrolyzing enzyme, thus permitting the accumulation of ITP.In an attempt to define the biosynthetic pathway of ITP, and since guanylate kinase has been implicated in the formation of ITP, the rate of synthesis of ITP and GTP in cell extracts was compared.The comparison of the specific activities of the [14C]-labeled hypoxanthine and guanine moieties of the inosine and guanosine phosphates formed during incubation with [8-14C]-inosine and [8-14C]-guanosine respectively, revealed striking differences in the relative rates of isotope incorporation. Tentative mechanisms are proposed to explain these differences.The data obtained thus far does not discard the possibility that ITP may be formed by stepwise phosphorylation and (or) by direct pyrophosphorylation of IMP.

Notes: The effect of insulin on hexose transport in cultured human skin fibroblasts.Studies were carried out on cultures of human skin fibroblasts to explore the effect of insulin on hexose transport in serum-starved monolayers. Insulin (100 mU/ml) stimulated 2-deoxy-D-glucose transport (30% above control values) after 30 minutes exposure time, the response being similar up to four hours exposure to insulin. In several experiments (n = 22) employing three cell strains, insulin (100 mU/ml) exposure led to variable stimulation of 2-deoxy-D-glucose transport (an average of 37% above control values, with a range of 0 = 120%). The insulin-induced stimulation of 2-deoxy-D-glucose transport showed a dose dependency with increasing amounts of insulin, the response being maximal at an insulin concentration of 100 mU/ml. Kinetic analysis of 2-deoxy-D-glucose transport showed that insulin addition resulted in a slight change in the transport Km (3.13 to 4.06 mM) and a 1.8-fold increase in the transport Vmax (17.6 nanomoles/mg protein/min to 32.1 nanomoles/mg protein/min). Insulin also stimulated the transport of 3-0-methyl-D-glucose while the hexokinase activity of the cells was not affected. Further, this insulin-induced stimulation of sugar transport was not blocked by cycloheximide. The results indicate that insulin stimulated the stereospecific carrier-mediated of hexose transport in cultured human skin fibroblasts.

Notes: Stable variants of the macrophage-like cell line J774.2, defective in adenylate cyclase and protein kinase activities, were selected by cloning cells resistant to the growth-inhibitory effect of cholera toxin and 8-bromoadenosine 3′:5′ cyclic monophosphoric acid (8 Br-cAMP), respectively. These variants were analyzed for their ability to respond to cyclic AMP-mediated enhancement of phagocytosis and cyclic AMP-mediated inhibition of plasminogen activator secretion and growth. The adenylate cyclase variants were unaffected by cholera toxin but were sensitive to 8 Br-cAMP-mediated inhibition of plasminogen activator secretion and growth. One of these variants exhibited a defect in phagocytosis that could be corrected by 8 Br-cAMP. The protein kinase variants exhibited normal basal phagocytosis that could not be stimulated by either 8 Br-cAMP or cholera toxin; they were also insensitive to cyclic AMP-mediated inhibition of plasminogen activator secretion and growth. The studies demonstrate that the three effects of cyclic AMP in J774.2-inhibition of growth and plasminogen activator secretion, and enhancement of basal Fc-mediated phagocytosis-are mediated by a cyclic AMP-dependent protein kinase. The results support the usefulness of variants in cyclic nucleotide metabolism in understanding the regulation of differentiated cell function by cyclic AMP.

Notes: The effect of phytohemagglutinin (PHA) on lymphocytes was examined with respect to free intracellular water volume and intracellular [K+]. At a cell concentration of 30 × 106 lymphocytes/ml in modified Hank's Buffered Salt Solution (HBSS) in the presence of 10% human AB serum, addition of PHA at 3 mg/ml resulted in a 24-27% decrease in free intracellular water space within 30 to 60 minutes and a return to control level after three hours. A larger change in intracellular water (44%) was observed under similar conditions in the absence of serum. The absolute intracellular K+ content did not change after PHA addition, but the cell water volume decrease arising from PHA addition resulted in a 29% increase in intracellular [K+] at 60 minutes. The decrease in lymphocyte water volume induced by PHA was also observed for concanavalin A which stimulates lymphocyte proliferation, but not for wheat germ lectin, an agglutinating agent which is not mitogenic. Thus, volume regulation may be closely associated with the mitogenicity of these compounds.

Notes: When serum is made rate-limiting for clonal multiplication of human diploid fibroblasts, the presence of a 2-oxocarboxylic acid in the medium becomes essential. The requirement is independent of the 20 amino acids and glucose. Glyoxylic, pyruvic, 2-oxoglutaric, and oxalacetic acids are most effective. The types of 2-oxocarboxylic acids that support multiplication are oxidized substrates for several, pyridine nucleotide-linked intracellular oxidoreductases. The requirement is not satisfied by carboxylic acids, oxidized substrates for oxidoreductases that are not linked to pyridine nucleotides, or by nonspecific electron acceptors. The quantitative requirement for 2-oxocarboxylic acids in cell multiplication is markedly affected by the concentration of serum proteins in the medium. Therefore, 2-oxocarboxylic acid metabolism may be related to the mechanism by which serum growth factors regulate cell multiplication.

Notes: HeLa cell mitochondria were allowed to incorporate 3H-thymidine in a cell free system and the effect of ethidium bromide, cytosine arabinoside and cytosine arabinoside triphosphate on the labeling of mitochondrial DNA was studied. The labeled products, isolated by sedimentation velocity in CsCl-ethidium bromide two-step gradients, showed similar sedimentation profiles as in vivo labeled mtDNA. Cytosine arabinoside triphosphate and ethidium bromide strongly inhibited the labeling of mitochondrial DNA, whereas cytosine arabinoside appeared to be much less effective. Tritiated deoxycytidine was found to be incorporated by isolated mitochondria, whereas cytosine arabinoside was shown to enter the mitochondrial acid-soluble pool but not to be incorporated in acid-insoluble form. These results are in agreement with the previously reported findings of in vivo experiments.

Notes: Normal C57 Black mouse embryo cells did not form colonies in agarose, but rare variant (ar+) cells able to grow in agarose were detected. Fluctuation analysis showed that ar+ variants arose by spontaneous mutation in the cultured cells. The frequency of ar+ variants was increased by treating cells with N-methyl-N'nitro-N-nitrosoguanidine or ethyl methane sulphonate, or by abortive infection by human adenovirus type 5. Induced ar+ cells were fibroblastic; most grew slowly and had slightly reduced saturation density and increased serum requirement, but formed colonies in agarose. Fourteen of twenty ar+ clones induced by Ad5 were T antigen negative and two of these were also negative when tested for viral DNA. Six clones contained a few cells that were T antigen positive when first tested, but were negative when retested later. The ar+ variants were tumorigenic in athymic and in normal syngeneic mice. The results suggest that the ar+ phenotype can arise by spontaneous or chemically-induced mutation, and can be induced by adenovirus by a process different from classical transformation.

Notes: When calcium is removed from culture medium, motility of cultured cells is decreased. The effect is rapid, reversible and pronounced. Decreased motility is observed with normal mouse Balb/c 3T3 cells, mouse L929 cells, rat kidney fibroblasts and chick embryo fibroblasts. The calcium dependence of movement can be observed both with individual cells and with the movement of the margin of a monolayer into a wound. Magnesium will not substitute for calcium to maintain motility. Strontium will substitute, but is not as effective as calcium for maintaining cell movement. Low concentrations of the divalent cation ionophore A23187 (0.5-1 μm) partially reverse the reduced migration observed at low calcium concentrations. These results are consistent with the hypothesis that movement of non-muscle cells occurs through mechanisms similar to those important in the contraction of muscle.

Notes: Mitotic HeLa cells (M cells) synthesize protein at about 25% of the rate of S phase cells. This decrease in protein synthesis is due to a reduction in the rate of initiation. However, extracts prepared from M cells are almost as active in protein synthesis as S cell extracts. Both cell extracts are quite active in in vitro initiation of protein synthesis. Moreover, two steps in initiation, binding of Met-tRNAf to 40S ribosomal subunits and binding of mRNA to ribosomes, show similar activity in both extracts. The difference in protein synthesizing activity observed in vivo is largely eliminated in the preparation of cell-free systems. The ribosomes of M cells contain small mot wt RNA, which inhibits protein synthesis in vitro. This RNA, which has possibly a nuclear origin, may be a cause of the reduction in the rate of protein synthesis in M cells.

Notes: Friend erythroleukemic cells can be induced by a variety of agents to synthesize hemoglobin and to exhibit other characteristics suggesting erythroid maturation. Upon induction of hemoglobin synthesis with dimethyl-sulfoxide (DMSO), the chloride flux in Friend cells gradually increases, until after five days of exposure to DMSO (when the hemoglobin content of the cells approaches that of the mature erythrocyte) the flux is three times the value in non-induced cells. A similar flux increase is observed in the presence of a different type of inducer, hypoxanthine, but no increase in flux is seen in the mutant cell line, TG-13, which does not synthesize hemoglobin after DMSO treatment. Thus, the flux increase seems to be associated with the induction process, rather than being a direct effect of the inducing agent. After DMSO treatment, the sulphate flux decreases and the chloride/sulphate selectivity increases, as would be expected if the cells were becoming more like red cells. On the other hand, the sensitivity of the chloride flux to the inhibitor, furosemide, and to temperature is the same in the induced as in the non-induced Friend cells, and different from that of the mature red cell. Thus, the anion transport properties of the induced Friend cell are different from those of both the non-induced Friend cell and the mature erythrocyte. Either the system in the induced cell represents an intermediate stage in the development of the mature red cell characteristics, or else the maturation of transport function in the Friend cell differs from that in normal erythrocyte precursors.