Effect of substance P on nerve fiber regeneration in tissue culture

Explants of the ganglion trigeminale from chick embryos (PNS) and of the hippocampus from fetal rats (CNS) were cultivated in maximow chambers with growth medium or maintanance medium. Varied concentrations of substance P (SP . 3 CH3COOH . 4 H2O) were added. 1. The effect of substance P (SP) is related to concentration. In the presence of 10(-7)M SP in the growth medium and of 10(-4)M SP in the maintanance medium the cultivation of PNS cultures indicates positive results. These doses are suitable. 2. Within the first 24 hours in vitro SP stimulates the index of area in PNS cultures. The index of characterizes the relation of the outgrowth zone to the explant. In CNS cultures a significant difference of this effect was not observed. 3. The index of growth of nerve fibers may compare the test cultures with the control cultures. SP significantly increases the index of fiber growth in PNS cultures. A stimulation of CNS cultures was observed, significance was not found. 4. From the beginning of the cultivation with SP up to 48 hours in vitro the growth of nerve fibers significantly increases in the treated cultures in comparison with the control cultures. After this time the growth of nerve fibers decreased and a morphological conformity of test cultures and controls was observed. 5. The role of SP is discussed in specific activity on PNS tissue in vitro. The reactive neurons may be from the medio dorsal group of cells of the sensible ganglion.     

Comparative Physiology of Apple Rootstocks: II. The Effect of pH and Aeration on the Growth of Malling Apple Rootstocks in Water Cultures

Three Malling apple rootstocks, M.IX, M.II, and M.XVI, weregrown in aerated and non-aerated nutrient solutions at pH 3·6,4·0, and 5·5, and their growth and survival comparedwith rootstocks grown in sand and in soil. No plants survivedin the non-aerated cultures due to severe bacterial and fungalattack on the roots. In the aerated series a marked differencein susceptibility of rootstocks to root infection was found,M.XVI being considerably more resistant than M.II or M.IX. Morerootstocks survived at the lower pH's (4·0 and 3·6)but among surviving plants of M.XVI growth was much less thanat pH 5·5. It would appear therefore that factors makingfor survival do not of necessity make for optimum growth. Insand and soil cultures all plants survived.     

Lignin release and photomixotrophism in suspension cultures of Picea abies

The effect of different concentrations of sucrose (0-4%) and of two growth regulators (0–50 μ M 2,4-D and 0–25 μ M kinetin) was tested on growth and chlorophyll content of suspension cultures of Picea abies (L.) Karst. originating from chlorophyllous embryo callus in an elevated CO₂ (2%) atmosphere. A continuous chlorophyllous suspension culture was achieved on a medium containing 2% sucrose and a low level of organic nitrogen (0.25 m M arginine and 0.5 m M glutamine) supplemented with 2,4-D (0.5 μ M ) and kinetin (2.5 μ M ). The same medium with 4% sucrose gave the best growth response, but a negative correlation between chlorophyll level and growth was observed. The chlorophyllous cultures grew slowly in a medium with low (0.5%) sucrose or without any carbohydrate source, suggesting photomixotrophism. A high concentration of kinetin inhibited both growth and chlorophyll synthesis. Release of lignin into the nutrient medium was observed in several experiments, especially in slow-growing cultures supplemented with sucrose. Only a few successive passages of suspensions that produced lignin could be cultured before cell death. The cultures releasing lignin may be unique for studies on synthesis and biodegradation of this very complex compound.     

Regulation of T-cell activation and T-cell growth factor (TCGF) production by hydrogen peroxide

Activated macrophages are known to release a variety of immunoregulatory substances including the low-molecular-weight substances hydrogen peroxide and lactate. We report here that lactate but not hydrogen peroxide is capable of supporting a substantial production of T-cell growth factor (TCGF) in cultures of accessory cell-depleted splenic T-cell populations after stimulation with concanavalin A. Hydrogen peroxide and its biosynthetic precursor superoxide anion (O2-) mediate, however, a strong augmentation of the TCGF production by accessory cell-depleted T-cell populations in the presence of lactate. Lactate inhibits the incorporation of [3H]thymidine in short-term cultures (18-26 hr) of accessory cell-depleted T cells. This confirms the rule that (optimal) production of T-cell growth factor requires a growth inhibitory signal. Concentrations of hydrogen peroxide which augment TCGF production most effectively (i.e., 1 X 10(-5) M) do not inhibit the incorporation of [3H]thymidine; and higher concentrations (3 X 10(-5)-1 X 10(-4) M) of hydrogen peroxide inhibit both the production of TCGF and the incorporation of [3H]thymidine. In agreement with the augmenting effect of hydrogen peroxide on TCGF production, it was observed that the proliferative response in mixed lymphocyte cultures is suppressed by catalase and augmented by 1 X 10(-5) M H2O2. Proliferative and cytotoxic responses in mixed lymphocyte cultures with an external source of interleukin 2 (IL-2) in contrast, are not augmented by 1 X 10(-5) M H2O2. The relatively high concentration of 1 X 10(-4) M hydrogen peroxide was found to inhibit the proliferative responses in mixed lymphocyte cultures with or without external IL-2 but not the cytotoxic response in the presence of IL-2. This indicates that CTL precursor cells may be relatively resistant against H2O2.     

Changes in the synthesis of minor cartilage collagens after growth of chick chondrocytes in 5-bromo-2'-deoxyuridine or to senescence

Analyses were made of the minor collagens synthesized by cultures of chondrocytes derived from 14-day chick embryo sterna. Comparisons were made between control cultures, cultures grown for 9 days in 5-bromo-2'-deoxyuridine (BrdU) and clones of chondrocytes grown to senescence. Separation of minor collagens from interstitial collagens was achieved by differential salt precipitation in the presence of carrier collagens in acid conditions. The precipitate at 0.9 M NaCl 0.5 M acetic acid from control cultures was shown by CNBr peptide analysis to contain only the alpha 1(II) chain of type II collagen, whereas after BrdU treatment or growth to senescence synthesis of only alpha 1(I) and alpha 2(I) chains occurred. The synthesis of type III collagen was not detected. Analysis of the precipitate at 2.0 M NaCl, 0.5 M HAc from control cultures demonstrated the synthesis of 1 alpha, 2 alpha and 3 alpha chains together with the synthesis of short chain (SC) collagen of Mr 43000 after pepsin digestion. After BrdU treatment or growth to senescence alpha chains were isolated which possessed the migration positions on polyacrylamide gel electrophoresis (PAGE), or the elution positions on CM-cellulose chromatography, of the alpha 1(V) and alpha 2(V) chains of type V collagen. In addition, for BrdU-treated but not for control cultures, intracellular immunofluorescent staining was observed with a monoclonal antibody which specifically recognizes an epitope present in the triple helix of type V collagen. Synthesis of short chain (SC) collagen was not detected after BrdU treatment or growth to senescence. These results suggest that chick chondrocytes grown in conditions known to cause switching of collagen synthesis from type II to type I collagen also undergo a switch from the synthesis of 1 alpha, 2 alpha and 3 alpha chains to the synthesis of the alpha 1(V) and alpha 2(V) chains of type V collagen. It appears that there are several cartilage-specific collagens which together undergo a regulatory control to the synthesis of collagens typical of other connective tissues.     

Cell size and mutual cell adhesion. II. Evidence for a relation between cell size, long-range electrostatic repulsion and intercellular adhesiveness during density-regulated growth in suspension.

The strength of the long-range electrostatic repulsion forces on HeLa cells is measured by agglutinative titration using low molecular weight polylysine (M.W. 11,000). Repulsion forces, found to be present on the smaller HeLa cells from density-inhibited suspension cultures, are weakened by incubation of the cells in hypotonic NaCl solutions. Repulsion forces, found to be absent on the larger cells from fast growing cultures, can be induced on these cells by incubation in hypertonic NaCl solutions. Both effects of anisotonicity are reversible, and disappear on restoration of the medium to normal tonicity. Induction of repulsion forces on fast growing cells is prevented by previous treatment of the cells with neuraminidase. Neuraminidase also abolishes repulsion on density-inhibited cells. It is proposed that alterations of the cell size, produced by anisotonicity or occurring during growth in isotonic suspension medium, affect mutual cell adhesiveness by modifying the strength of the repulsion forces generated by cell surface sialic acids.     

Vanilla Flavour Production Through Biotransformation Using Capsicum frutescens Root Cultures

Normal roots of Capsicum frutescens were excised from tissue-cultured plants into half strength Murashige and Skoog's medium with 2.23 μM naphthalene acetic acid. Maximum growth of cultured roots was 6.5 g fresh weight 40 ml^-1, as recorded on day 20. Even though normal roots were unable to accumulate capsaicin, they contained other phenylpropanoid intermediates and vanillylamine, as detected by HPLC analysis. Normal roots of Capsicum frutescens were treated with ferulic acid and protocatechuic aldehyde in order to study their biotransformation ability. Ferulic acid, which is the nearest precursor to vanillin, when fed at concentrations of 1 and 2 mM led to the accumulation of vanilla flavour metabolites, vanillin being the major one. In cultures treated with 1 and 2 mM ferulic acid, maximum vanillin accumulation of 12.3 and 16.4 μM was observed, on day 6 after precursor addition, respectively. Feeding of ferulic acid and β-cyclodextrin complex (2 mM each) enhanced the accumulation of biotransformed products. Moreover, vanillin accumulation was recorded as 24.7 μM on day 6 after precursor addition, which was 1.5 times higher than in cultures fed with ferulic acid (2 mM) alone. When ferulic acid was fed along with β-cyclodextrin (1 mM each) to cultures growing in a three-litre bubble column bioreactor, the maximum vanillin production of 10.7 μM was obtained; other vanilla flavour metabolites were also formed after 9 days of precursor addition. Root cultures could also biotransform protocatechuic aldehyde wherein a maximum vanillin production of 7.9 μM was recorded on day 6 after precursor addition. The bioconversion efficiency was observed to be 5-7% in case of ferulic acid fed cultures and 3.2% in case of protocatechuic aldehyde fed cultures suggesting the possible channelling of precursors to alternate biosynthetic pathways such as lignin.     

Hepatopancreas cell cultures from mud crab, <Emphasis Type="Italic">Scylla paramamosain</Emphasis>

Hepatopancreas is an important digestive and endocrine organ in crustacean. However, there are few reports on cell cultures from crabs. Here, the cell cultures of hepatopancreas from Scylla paramamosain was studied in vitro. Both the primary cell culture and subculture were grown in Leibovitz’ L-15 medium, M199 medium, or a specially designed medium for S. paramamosain (MSP). The results showed that hepatopancreas cells in vitro grew in compact clusters in 2–3 d. Four types of cells could be identified. They were embryo cells, fibrillar cells, resorptive cells, and blister-like cells, respectively. Some of these cells could be subcultured for three generations. The MSP supported the best survival of these hepatopancreas cells, while M199 medium was the least effective of these three media. Fetal bovine serum and crab muscle extracts as supplements stimulated growth, but the crab hemolymph inhibited cell growth. Taken together, MSP is an appropriate medium for hepatopancreas cell cultures from S. paramamosain and can support cultures through several passages.     

A Spot Test for Catechol 2:3-Oxygenase in Bacteria

A simple and quickly performed colour test is described, in which organisms containing catechol 2:3-oxygenase (meta-pyrocatechase) produce from catechol a yellow coloured intermediate, α-hydroxymuconic semialdehyde. The yellow colour is produced most consistently, rapidly and intensely by 2–4 day old cultures from 2·7–27 mM solutions of catechol. The purple-brown colour produced by some organisms is discussed.
Using growth from nutrient agar plates, the test was applied to cultures of 239 Gram negative bacteria; all of the 64 cultures which gave a positive result had been in contact previously with aromatic substrates and many of them could oxidize phenol. The significance of a positive reaction is discussed briefly.     

The Effect of Various Aliphatic Aldehydes and Related Compounds on the Growth of Dipodascus aggregatus

The growth of Dipodascus aggregatus in cultures inoculated with cells from the acceleration phase of growth was stimulated by the saturated and unbranched aliphatic C(3) C(4) , and C(6) to C(11) aldehydes (80 μM]. Nonanal was most active in stimulating growth. The C(12) aldehyde inhibited growth. The C(5) , aldehyde generally inhibited growth. - Nonan did not affect growth. 2-Nonanone and 5-nonanone promoted growth insignificantly. - In cultures inoculated with cells from the exponential phase growth was unaffected or even inhibited by all the aldehydes tested. The C(4) , C(5) , C(10) , and C(11) aldehydes inhibited growth to a larger extent than nonanal.     

Abelson virus-infected cells can exhibit restricted in vitro growth and low oncogenic potential.

We have designed a method for growing bone marrow cells infected with Abelson murine leukemia virus which permits examination of target cell growth early after infection. This culture system increases the efficiency of target cell growth by favoring rapid growth of a mixed population of adherent cells in the primary culture. The nonadherent Abelson virus-infected cell populations expressed pre-B-cell differentiation markers characteristic of Abelson virus-transformed cells (mu-heavy chains of immunoglobulin M and terminal deoxynucleotidyltransferase). Early after infection, these cell populations exhibited restricted in vitro and in vivo growth properties which differed from those of an established Abelson virus-transformed cell line, 2M3. These included a marked dependency upon the adherent cell layer for growth and viability, a lower efficiency of agar colony formation, and a lower capacity for tumor production in syngeneic animals. Growth of the early populations could be maintained in the absence of the adherent cell layer by using conditioned medium from long-term adherent cell cultures established in the absence of viral infection. After passage of the populations for several weeks, the in vitro growth properties gradually shifted toward that of the 2M3 cell line. Twelve-week-old populations grew independently of the adherent cell layer and showed an increased efficiency of agar colony formation. These data indicate that many lymphoid target cells exhibit an intermediate transformed phenotype when infected with Abelson virus. Growth of these cells in culture is mediated via a synergistic interaction between intracellular expression of the viral transforming gene and an exogenous growth-promoting activity which can be provided by cultures of adherent bone marrow cells.     

Biochemical evidence for formate transfer in syntrophic propionate-oxidizing cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei

The hydrogenase and formate dehydrogenase levels in Syntrophobacter fumaroxidans and Methanospirillum hungatei were studied in syntrophic propionate-oxidizing cultures and compared to the levels in axenic cultures of both organisms. Cells grown syntrophically were separated from each other by Percoll gradient centrifugation. In S. fumaroxidans both formate dehydrogenase and hydrogenase levels were highest in cells which were grown syntrophically, while the formate-H(2) lyase activities were comparable under the conditions tested. In M. hungatei the formate dehydrogenase and formate-H(2) lyase levels were highest in cells grown syntrophically, while the hydrogenase levels in syntrophically grown cells were comparable to those in cells grown on formate. Reconstituted syntrophic cultures from axenic cultures immediately resumed syntrophic growth, and the calculated growth rates of these cultures were highest for cells which were inoculated from the axenic S. fumaroxidans cultures that exhibited the highest formate dehydrogenase activities. The results suggest that formate is the preferred electron carrier in syntrophic propionate-oxidizing cocultures of S. fumaroxidans and M. hungatei.     

Mycoplasmal infection of lymphocyte cell cultures: Infection withM. salivarium

Summary Many conclusions concerning cell culture mycoplasmas are based on data from studies in fibroblast cultures. Some conclusions may not be valid in other types of differentiated cell cultures.M. salivarium was isolated from 35 human lymphocyte cultures (HLC), 34 from the same laboratory. The organism grew to more than 10⁸ colony forming units (CFU) per ml of lymphocyte suspensions and was readily detectable by microbiological culture, uridine phosphorylase, and uridine/uracil assays. Direct mycoplasmal assays on HLC by DNA fluorochrome staining and scanning electron microscopy (SEM) yielded artifacts that interfered with diagnosis. For DNA and SEM of HLC, inoculation into indicator cell cultures is recommended.M. salivarium infection of HLC did not produce any immediate difference in growth rates; however, infected cultures eventually died 14 to 29 passages after infection in contrast to uninfected controls. The same organism in 3T6 fibroblasts effected a 60% decrease in growth rate. AlthoughM. salivarium is a frequent isolate from the oral cavity, it is a rare cell culture isolate.M. salivarium was able to initiate growth over a wide pH range, grew as well in cell cultures as in cell-free media, and was resistant to 50 μg per ml of gentamycin, tylocine, kanamycin, and erythromycin. By C₀t_1/2 analysis,M. salivarium had a genomic molecular weight of 4.2×10⁸ daltons.M. salivarium did not increase chromosome aberrations in one HLC. Some of these results have application to infection of HLC by other mycoplasmal species. These studies were supported by contracts NO1-AG-82117 from the National Institute on Aging, NO1-GM-9-2101 from the National Institute of General Medical Sciences, and Grant RO1-A1-15748 from the National Institute of Allergy and Infectious Diseases.     

Choline Derivatives Involved in Osmotolerance of Penicillium fellutanum

Penicillium fellutanum is osmotolerant and xerotolerant when cultured in a low-phosphate medium containing 3 M NaCl. Glycerol and erythritol accumulated in cultures with NaCl concentrations up to 2 M; glycerol was the only detectable polyol in cultures containing 3 M NaCl. In cultures with 3 M NaCl, the intracellular levels of glycine betaine and choline-O-sulfate were 22- and 2.6-fold greater (70 and 46 mM), respectively, than those of cultures without added NaCl. The levels of glycine betaine and glycerol decreased in mycelia transferred from a medium containing 3 M NaCl into a fresh medium without added NaCl. NaCl at 3 M inhibited mycelial mass accumulation; this inhibition was partially corrected by supplementation of cultures with glycine betaine (2 mM) or choline-O-sulfate (10 mM). The presence of exogenous choline chloride (2 mM) in plate cultures protected the cells from stress from 3 M NaCl. The data suggest that glycine betaine and choline-O-sulfate are secondary osmoprotectants which are effective at the point that the cell is incapable of synthesizing more glycerol.     

Responses to estradiol in a human endometrial adenocarcinoma cell line (Ishikawa)

A human endometrial adenocarcinoma cell line (Ishikawa) has been found to be estrogen responsive. The growth stimulatory effects of estradiol (10(-8) M) could be clearly demonstrated when cell cultures containing the hormone were compared with the maximal cell density achieved in control cultures. The approx. 3-fold increase in cell density observed 2-3 weeks after plating, with frequent medium changes, could by blocked by a 100-fold molar excess of the antiestrogen trans-4-monohydroxytamoxifen. When added to hormone-free cultures that had reached a plateau level of cell numbers on day 14 after plating, estradiol (10(-8) M) caused the resumption of proliferation: after 6 days in the presence of the hormone, the cultures contained nearly twice the cell numbers of controls. Effects of estradiol on Ishikawa cells were also evident from the several-fold increases in the levels of specific progesterone binders provoked by the hormone at 10(-9)-10(-6) M concentrations. Cells injected into nude mice formed tumors which contained estrogen and progesterone binders. The availability of a fast-growing (doubling time approx. 30 h) endometrial cancer cell line responsive to estradiol at near physiologic levels will facilitate biochemical studies of hormonal effects on the human endometrium.     

The effect of PGE2 on the activation of quiescent lung fibroblasts

The effect of prostaglandin E2 (PGE2) on fibroblast proliferation was examined. The presence of PGE2 for 24 h inhibited the growth of quiescent cells stimulated with serum, platelet-derived growth factor and macrophage-derived factors. Maximal inhibition of nuclear labeling with [3H]thymidine occurred at concentrations greater than 10(-7) M. The inhibitory effect of PGE2 was less potent in exponentially growing cells and was not the result of conversion of PGE2 to PGA2 during incubation in growth medium. The G1 phase was determined to be 12-14 h in untreated cultures. The extent of growth inhibition by PGE2 was similar with addition of PGE2 at 0, 3, 6, or 9 h following restimulation of quiescent cell cultures. Approximately 25% of the cells that enter S phase are refractory to PGE2-induced growth inhibition. Short-term exposure to PGE2 (5 min and 30 min) caused substantial growth inhibition. The serum-induced proliferation was also inhibited by the cAMP analogue, dibutyrl cAMP. Our results suggest that PGE2 affects a distinct subpopulation of cells. Restimulation of quiescent cells treated with PGE2 for 24 h, indicated that release from PGE2 exposure is associated with prolongation of the G1 phase of the cell cycle.     

Effect of Nitrogen Source on Growth and Trichloroethylene Degradation by Methane-Oxidizing Bacteria

The effect of nitrogen source on methane-oxidizing bacteria with respect to cellular growth and trichloroethylene (TCE) degradation ability were examined. One mixed chemostat culture and two pure type II methane-oxidizing strains, Methylosinus trichosporium OB3b and strain CAC-2, which was isolated from the chemostat culture, were used in this study. All cultures were able to grow with each of three different nitrogen sources: ammonia, nitrate, and molecular nitrogen. Both M. trichosporium OB3b and strain CAC-2 showed slightly lower net cellular growth rates and cell yields but exhibited higher methane uptake rates, levels of poly-β-hydroxybutyrate (PHB) production, and naphthalene oxidation rates when grown under nitrogen-fixing conditions. The TCE-degrading ability of each culture was measured in terms of initial TCE oxidation rates and TCE transformation capacities (mass of TCE degraded/biomass inactivated), measured both with and without external energy sources. Higher initial TCE oxidation rates and TCE transformation capacities were observed in nitrogen-fixing mixed, M. trichosporium OB3b, and CAC-2 cultures than in nitrate- or ammonia-supplied cells. TCE transformation capacities were found to correlate with cellular PHB content in all three cultures. The results of this study suggest that the nitrogen-fixing capabilities of methane-oxidizing bacteria can be used to select for high-activity TCE degraders for the enhancement of bioremediation in fixed-nitrogen-limited environments.     

The Effect of Nonanal on Dipodascus aggregatus II. Influence of the Prehistory of the Inoculum and of the Presence of Ethanol

Nonanal, added in ethannlic solution, in concentrations lower than 40 to 80 μM did not affect the growth of Dipodascus aggregatus, provided the inoculum had been harvested from the exponential phase of growth. Growth could even be inhibited by 80 μM. If the inoculum had been grown to the exponential phase and then for another period, to the acceleration phase, in fresh liquid medium, growth was strongly promoted by 80 μM nonanal. If cells from the exponential phase were grown for another period in the supernatant fluid of centrifuged cultures from the exponential phase, 80 μM affected growth in the following way: in five different experiments growth was not stimulated, in one experiment undoubtedly promoted, and weakly stimulated in another one. The growth of cultures inoculated with cells grown only on malt agar was not affected by 80 μM nonanal. Pretreatment of cells, harvested from the acceleration phase, with nonanal (80 μM) in the presence of ethanol did not diminish the growth-promoting action of nonanal on the cultures inoculated with these cells. Nonanal, in the absence of ethanol, in a concentration of 10 μM did not affect the growth of cells, harvested from the acceleration phase, whereas 100 μM nonanal strongly inhibited growth. An attempt is made to explain the results starting from the endogenous metabolism.     

Influence of Manganese on Growth of a Sheathless Strain of Leptothrix discophora

Mn²⁺ exerted various effects on the growth of Leptothrix discophora strain SS-1 in batch cultures depending on the concentration added to the medium. Concentrations of 0.55 to 5.5 μM Mn²⁺, comparable to those in the environment from which strain SS-1 was isolated, decreased cell yield and prolonged stationary-phase survival, but did not affect growth rate. Elevated concentrations of 55 to 910 μM Mn²⁺ also decreased cell yield and prolonged survival, but growth rate was decreased as well. The addition of 1,820 μM Mn²⁺ caused a decline in cell numbers followed by an exponential rise after 80 h of incubation, indicating the development of a population of cells resistant to Mn²⁺ toxicity. When 360 μM Mn²⁺ or less was added to growth flasks, Mn²⁺ was oxidized to manganese oxide (MnO_x, where x is ~2), which appeared as brown particles in the medium. Quantification of Mn oxidation during growth of cultures to which 55 μM Mn²⁺ was added showed that nearly all of the Mn²⁺ was oxidized by the beginning of the stationary phase of growth (15 to 25 h). This result suggested that the decrease in cell yield observed at low and moderate concentrations of Mn²⁺ was related to the formation of MnO_x, which may have bound cationic nutrients essential to the growth of SS-1. The addition of excess Fe³⁺ to cultures containing 55 μM Mn²⁺ increased cell yield to levels near those found in cultures with no added Mn²⁺, indicating that iron deprivation by MnO_x was at least partly responsible for the decreased cell yield.     

Cell cycle distributions, growth characteristics, and variation in prolactin and growth hormone production in cultured rat pituitary cells.

Clonal strains of rat pituitary tumour cells (GH3 cells) spontaneously produce and secrete prolactin and growth hormone. Chromosome analysis and DNA ploidy measurements revealed that the GH3 cells in the present study were triploid and had a decreased chromosome number compared to the parent strain. Monolayer cultures of these cells grow exponentially for 6-7 days with a mean doubling time of 54 h. Cell cycle distributions and phase durations were determined by micro-flow fluorometric measurements of cellular DNA content combined with computer calculations. During exponential growth the cell cycle distribution did not change (65.4% cells with a G1 phase DNA content, 24.9% with an S phase DNA content, and 9.7% with a (G2 + M) phase DNA content). Counting of mitoses gave 1.4% cells in M phase. The 3H-Tdr labeling indices were determined by autoradiography, and the results were in good agreement with the number of cells in S phase as calculated by micro-flow fluorometry. The phase durations were: Ts=15.9 h, TG2=6.2 h, TM=1.1 h, and TG1=30.9 h. TS and TM calculated from 3H-Tdr labeled and Colcemid treated cultures gave corresponding results. In plateau phase cultures the number of cells with a G1 DNA content increased to 80% and the number of cells with an S phase DNA content decreased to between 5% and 10%. The specific production of prolactin and growth hormone determined by radioimmunoassay showed two and four-fold increases respectively, during exponential growth. The hormone values decreased to initial or subinitial values (day 2 values) when approaching plateau phase. We conclude: that changes in the cell cycle distribution of the cell population cannot be responsible for the spontaneous alterations in hormone production during growth and that most of the hormone-producing cells must be in the G1 phase.