The effect of conditioned medium obtained from <Emphasis Type="Italic">Scenedesmus subspicatus</Emphasis> on suspension culture of <Emphasis Type="Italic">Silene vulgaris</Emphasis> (<Emphasis Type="Italic">Caryophyllaceae</Emphasis>)

The effect of culture filtrate (conditioned medium, CM) containing cell exudates obtained from green alga, Scenedesmus subspicatus, on cell suspension of dicotyledonous plant Silene vulgaris was examined. The addition of diluted CM to the modified MS medium, supplemented with dicamba and BAP, stimulates cell biomass production. The biomass was composed of association of single non-dividing cells, cells during mitosis stage and cellular aggregates. Silene cells began mitotic divisions earlier in the presence of CM in medium when compared to control treatments. Results of performed bioassay showed that some factor or factors released by green alga to the culture medium could be responsible for sustained proliferation of phylogenetically distant species cells. Although it is still unclear which culture constituent influenced most the mitotic response of Silene suspension, results point at versatile stimulatory character of green alga exudates in higher plant cell culture.     

Arabinogalactan proteins are involved in cell aggregation of cell suspension cultures of <Emphasis Type="Italic">Beta vulgaris</Emphasis> L.

Arabinogalactan proteins (AGPs) are glycoproteins present at cell surfaces. Although exact functions of AGPs remain elusive, they are implicated in plant growth and development. The aim of this study was to evaluate the role of AGPs in the process of cell aggregation of Beta vulgaris L. suspension cultures. It was observed that B. vulgaris suspension cultures accumulated AGPs in parallel form to its cell growth. The AGPs maximum content in the stationary phase was 0.330 mg g⁻¹ dry weight (DW) in the cell wall (CW) and 1.534 mg g⁻¹ DW in the culture medium (CM), generating cell aggregates >500 μm (93.21% DW). The addition of tunicamycin (TM) caused a reduction of AGPs content in CW and CM of 46 and 64%, respectively. These changes were associated with inhibition of growth and the reduction of the cell aggregates >500 μm (50.0% DW). When TM was removed from the CM, cell growth, aggregation, and AGPs content on CW and CM were recovered. Precipitation of AGPs with Yariv reagent generated a reduction of 61.14% of AGPs content in CW and a total inhibition of AGPs secretion in CM. This Yariv treatment generated a reduction in the cell aggregates >500 μm of 51.31% of DW. When the Yariv reagent was removed from the culture, cells did not recover their AGPs accumulation. In addition, cell cultures did not recover their ability to grow and aggregate. These results indicate that AGPs are molecules required in the cellular aggregation process of B. vulgaris L. suspension cultures.     

Conditioned medium factor produced and released by <Emphasis Type="Italic">Desmosdemus subspicatus</Emphasis> and its effect on the cell cycle of the producer

Conditioned medium (CM) containing algal exudates released by cells of Desmodesmus subspicatus (green microalga) enhanced proliferation of the producer in a dilution-dependent manner, and twofold diluted CM (CM/2) appeared to be the most effective. Asynchronous and synchronized by light/dark regime (14/10 h) cultures of D. subspicatus were used to explain the effects of CM/2 on photosynthesis as well as growth and reproductive processes of cell cycle of the producer. During the light period, all control cells attained three commitment points (CP) triggering the reproductive processes and 20% of them, additionally the fourth one. This resulted in the formation of mainly eight and partly 16 autospores released from the parent cell during the dark period of the cell cycle. CM/2 markedly increased the number of cells that attained the fourth CP. Chlorophyll a fluorescence parameters, measured by OJIP test (O, J, I, and P-different steps of fluorescence induction curve), were applied to explain stimulatory effect of CM/2 on the rate of oxygen evolution. The number of cells as well as the parameter of their “vitality” (P.I) determined by OJIP analysis were equally enhanced by CM/2. The maximum yield of electron trapping and transport, as well as the fraction of active reaction centers, was also significantly higher in CM/2. The cells quickly produced and released CM factor (CMF) to the culture medium; its highest activity was observed in the middle light phase of the cell cycle. Dialysis, ion exchange chromatography, and solid-phase extraction with C₁₈ allowed reproducible extraction of the active compound from CM. Subjecting the extract to reverse-phase high-performance liquid chromatography led to one active fraction eluted as a peak with the shortest retention time indicating its hydrophilicity.     

Investigation of the dark metabolism of acetate in photoheterotrophically grown cells ofRhodospirillum rubrum

The mechanism of the aerobic dark assimilation of acetate in the photoheterotrophically grown purple nonsulfur bacteriumRhodospirillum rubrum was studied. Both in the light and in the dark, acetate assimilation inRsp. rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium. The assimilation of propionate was accompanied by the excretion of pyruvate. Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C₄-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate. These data implied that the citramalate (CM) cycle inRsp. rubrum cells can function as an anaplerotic pathway under aerobic dark conditions. This supposition was confirmed by respiration measurements. The respiration of cells oxidizing acetate depended on the presence of CO₂ in the medium. The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicated that citramalate and mesaconate were intermediates of the acetate assimilation pathway. The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle. The addition of 5 mM itaconate to extracts ofRsp. rubrum cells inhibited the activity of this enzyme by 85%. The data obtained suggest that the CM cycle continues to function inRsp. rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically.     

Age-related alterations in cell division and cell cycle kinetics in control and trimethyltin-treated lymphocytes of human individuals

Trimethyltin chloride induced age-related suppression of cell division and cell cycle kinetics in human peripheral blood lymphocytes cultured in RPMI 1640 culture medium supplemented with human AB serum, phytohemagglutinin and bromodeoxyuridine. A high frequency of M₁ (first metaphase) cells was seen in cultures treated with a high dose (C ₁ = 1.0 g per culture) and in lymphocytes from donors in the age range 40–70 years. The delay in cell division and cell cycle kinetics may indicate a longer duration in DNA synthesis induced by trimethyltin chloride in aged lymphocytes.     

GROWTH CONTROL OF DIFFERENTIATED FETAL RAT HEPATOCYTES IN PRIMARY MONOLAYER CULTURE : VI. Studies with Conditioned Medium and Its Functional Interactions with Serum Factors

Serum-deficient ≤0.00003% vol/vol) conditioned medium (CM) obtained from primary cultures of fetal rat hepatocytes initiates DNA synthesis and mitosis in homologous quiescent cultures. CM similarly prepared from 3T3 fibroblast cultures is inactive. At least two conditioning factors are involved in initiating DNA synthesis. The first of these, arginine, is obligatory, synthesized by the cells, and released into the culture medium. The second, a lipid or lipid-containing material, is stable to pH extremes (pH 2, pH 10) and chromatographs with an apparent R₁ ~0.5 on silica gel thin-layer plates using hexane-ether (4: 1) as the solvent system. It is suggested that these cultured hepatocytes enter or leave the G₀ or early G₁ phase of the cell cycle as determined in part by their capacity to use available conditioning factor and nutrient components of the medium, in particular, arginine. Serum factors including serum fraction I (4), insulin, and possibly, lipid-like conditioning material appear to initiate DNA synthesis by controlling cellular processes involved with the enhanced utilization and synthesis of growth-limiting nutrients.     

Manipulation of medium conditions and differentiation in the rat myogenic cell line L6

Myoblasts of the L6 rat cell line were grown in Ham's F12 nutrient medium containing 10% fetal calf serum (F12 + FCS). Although the cells were confluent by 6 days in culture, fusion was not observed even if cultures were maintained for 10–14 days. At least 80% of the cells in such confluent unfused cultures were in the G₁ phase of the cell cycle and less than 5% of the cells in confluent cultures synthesized DNA during a 4-day period. The synthesis of muscle-specific proteins (α-actin, β-tropomyosin, and myosin light chains LC1_emb and LC2_F) was negligible when compared to fused cultures of L6 cells grown for a similar time in Dulbecco's medium with 10% FCS (DME + FCS). When the unfused cultures were shifted from F12 + FCS to DME + FCS, DNA synthesis could be demonstrated in more than 95% of the cells and fusion occurred, indicating that neither proliferative nor myogenic capacity had been irreversibly lost. Raising the levels of calcium, varying the serum concentration from 0 to 20%, or the addition of medium components (present in DME but reduced or absent in F12) all failed to induce fusion in the L6 cells grown in F12. However, L6 cells will fuse in mixtures of F12 + FCS and DME + FCS. Fusion will also occur if L6 cells are grown at clonal density in F12 + FCS supplemented with calcium. While it has not been possible to determine why F12 + FCS is nonpermissive for L6 cells in confluent mass cultures, the results demonstrate that prolonged residence in the G₁ phase of the cell cycle is not a sufficient condition for L6 myoblast differentiation to occur.     

Characterization of human osteoblastic cells: Influence of the culture conditions

Summary Human osteoblastic cells were isolated enzymatically from adult human spongy bone and grown in MEM-Ham F12 1:1 medium supplemented with 2% Ultroser (USM). They were subcultured and examined for osteoblast features by morphological, histological, and biochemical approaches. The cells had a characteristic polyhedral morphology and produced a high level of alkaline phosphatase (ALKP). Confluent cultures were uniformly stained for ALKP and flow cytometry analysis with fluorescein diphosphate gave a single peak signal, reflecting a highly positive population, distinct from cultures of fibroblasts. The ALKP activity was stimulated by 1,25 (OH)₂ vitamin D₃. CD 44 was strongly expressed in these cultures, although osteoblasts are negative in vivo and osteocytes are positive. The main collagen synthesized was type I collagen and osteocalcin was produced after stimulation by vitamin D₃. 10 mM βGP induced mineralization and microprobe analysis of the crystals showed a composition close to hydroxyapatite. Changing the culture conditions to MEM-10% calf serum acted on cell behavior: it reduced the production of these biochemical markers of osteoblasts and the morphology became fibroblastlike with more rapid cell multiplication. The parameter most affected by the change in culture medium was ALKP, which was selected as the determinant criterion for defining an osteoblast culture. ALKP activity was then used to characterize a culture of cells seeded in a collagen gel.     

Cholinergic differentiation in neurogenic basal forebrain cultures

To study early events in the central nervous system (CNS) cholinergic development, cells from rat basal fore brain tissue were placed in culture at an age when neurogenesis in vivo is still active [embryonic day (E) 15]. The rapid mortality of these cells in defined medium, with 50% mortality after 5–10 h, was blocked completely by soluble proteins from the olfactory bulb (a basal forebrain target), extending earlier observations (Lambert, Megerian, Garden, and Klein, 1988). Treated cultures were capable of incorporating thymidine into DNA, and most cells incorporating ³H-thymidine (>90%) also stained positive for neurofilament, confirming neuronal proliferation in the supplemented cultures. A small percentage of ³H-thymidine labelled cells were glial fibrillary acidic protein (GFAP) positive, but growth factors that support astroglial proliferation [epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1)] were not sufficient for neuronal support. After 5 culture days with supplemented medium, almost 50% of the cells showed choline acetyltransferase (ChAT) immunofluorescence. The cholinergic neurons typically formed clusters separate from noncholinergic cells. These mature cultures did not develop if young cultures were treated with aphidicolin to block DNA synthesis. The data show that cultures of very young rat basal forebrain cells can be neurogenic, giving rise to abundant cholinergic neurons, and that early cell proliferation is essential for long-term culture survival.     

Ultraviolet radiation-induced neoplastic transformation of normal human cells,in vitro

Human foreskin cell cultures in scheduled DNA synthesis (S phase) of the cell cycle were exposed to UV irradiation at a dose of 10 J · m^?2 in the presence of insulin. These treated cell populations, when selectively passaged in a high amino acid supplemented complete growth medium (CM) after 20 Dulbecco's phosphate buffered saline (pH 6.8) (PDL), were able to be grown in soft agar. These treated cell populations were also grown in 1% serum supplemented growth medium and at 41°C in 10% serum supplemented growth medium. Cell populations 4–5 PDL after treatment exhibited altered colony morphology and altered lectin agglutination profiles but would not grow in soft agar. These events appeared to be associated with the early stages in the expression phase of the transformed phenotype. After 20 PDL, we observed that these cells would grow in soft agar at a frequency of 20 colonies/10⁵ cells seeded in soft agar. The cell populations derived from these colonies, when propagated and injected into the nude mice, formed myxofibromas at the injection sites rather than the type of tumor (fibrosarcoma) previously described for chemical carcinogen-induced neoplasms.     

An improved method of cell culture system from eye stalk,hepatopancreas, muscle,ovary, and hemocytes of <Emphasis Type="Italic">Penaeus vannamei</Emphasis>

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace’s insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO₂. Although the temperature preference of hemocyte cells was the same as ovarian cells, CO₂ supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies.     

Cytophotometric analysis of glycogen,protein and DNA of a glycogen-storing rat hepatoma (N13) cell line

This study examines the behavior of glycogenstoring rat hepatoma (N13) in vitro using cytophotometric techniques. A significant increase in glycogen is observed in these cells after 30 min incubation in a buffered solution containing 0.1 mM glucose, that is 80 times lower than the physiological glucose concentration in rat blood. N13 hepatoma cells grow exponentially in culture using RPMI 1640 tissue culture medium supplemented with 10% fetal bovine serum. During the first day in culture these cells store a large amount of glycogen and this increase is also observed in serum-free cultures. In more prolonged cultures the amount of glycogen per cell gradually becomes lower, although the culturing conditions are maintained. Similar variations of protein are also observed during the initial period of culture. DNA distribution does not show significant changes, although in serum-free cultures an increase in the proportion of cells in S and G₂/M phases is observed. The addition of glucagon, epinephrine and cyclic AMP derivatives to serum-free cultures does not impede the storage of glycogen. Nevertheless, addition of either 2 mM N⁶,O²-dibutyryl cyclic AMP or 0.1 mM 8-(4-chlorophenylthio)-cyclic AMP blocks the cell cycle at G₀/G₁ and glycogen content does not decrease after the first day in culture. We believe that this cell line offers an appropriated model to study glycogen metabolism and its involvement in the neoplastic process.     

Establishment and Growth of Embryogenic Suspension Cultures of Ocotea catharinensis Mez. (Lauraceae)

The focus of this study was to test the effects of 2,4-D, sucrose, culture media and initial inocula on the development of embryogenic suspension cultures of Ocotea catharinensis Mez. (Lauraceae). Suspension cultures were established in half-strength MS medium supplemented with 2% (w/v) sucrose either in the absence or in the presence of 2.2 μM 2,4-D, when higher cell viability was achieved. Under this culture condition the maximum fresh weight increase occurred in the fourth week. The cultures were yellow and consisted of a mixture of highly cytoplasmic single cells and small cell aggregates (<0.25 mm). The best proportion of inoculum per volume of medium for suspension culture development was 5% (w/w). Suspension cultures consisting of somatic embryos at the globular and cotyledonary stages (structures ranging from 1 to 3 mm) were successfully established on half-strength MS supplemented with 2% (w/w) sucrose through repetitive embryogenesis from the desiccated mature somatic embryos used as initial inoculum. The failure to initiate liquid cultures from non-desiccated mature somatic embryos was overcome by pre-treatment with air desiccation and reduction of the water content to 6.1 g H₂O g⁻¹ dry weight.     

Reversible accumulation of plant suspension cell cultures in g(1) phase and subsequent synchronous traverse of the cell cycle

The induction of DNA synthesis in Datura innoxia Mill. cell cultures was determined by flow cytometry. A large fraction of the total population of cells traversed the cell cycle in synchrony when exposed to fresh medium. One hour after transfer to fresh medium, 37% of the cells were found in the process of DNA synthesis. After 24 hours of culture, 66% of the cells had accumulated in G₂ phase, and underwent cell division simultaneously. Only 10% of the cells remained in G₀ or G₁. Transfer of cells into a medium, 80% (v/v) of which was conditioned by a sister culture for 2 days, was adequate to inhibit this simultaneous traverse of the cell cycle. A large proportion of dividing cells could be arrested at the G₀ + G₁/S boundary by exposure to 10 millimolar hydroxyurea (HU) for 12 to 24 hours. Inhibition of DNA synthesis by HU was reversible, and when resuspended into fresh culture medium synchronized cells resumed the cell cycle. Consequently, a large fraction of the cell population could be obtained in the G₂ phase. However, reversal of G₁ arrested cells was not complete and a fraction of cells did not initiate DNA synthesis. Seventy-four percent of the cells simultaneously reached 4C DNA content whereas the frequency of cells which remained in G₀ + G₁ phase was approximately 17%. Incorporation of radioactive precursors into DNA and proteins identified a population of nondividing cells which represents the fraction of cells in G₀. The frequency of cells entering G₀ was 11% at each generation. Our results indicate that almost 100% of the population of dividing cells synchronously traversed the cell cycle following suspension in fresh medium.     

TES and HEPES buffers in mammalian cell cultures and viral studies: Problem of carbon dioxide requirement

In order to evaluate TES and HEPES as a buffer system for cell culture, the proliferative capacities of cells of several mammalian cell lines in the medium buffered with either of these compounds were examined in cultures in stoppered and open flasks at high and low cell densities. When cultivated in stoppered flasks, cells grew equally well or even better in TES- and HEPES-buffered medium than in NaHCO₃-buffered medium irrespective of cell culture density. In open flasks or Petri dishes in TES- or HEPES-buffered medium, however, the proliferative capacity of cells in low density cultures was limited. The inhibition of cell growth in the latter condition was restored (1) as the cell density of the cultures increased; (2) by feeding continuously the cultures with the gas produced by high density cultures; (3) by introducing a small amount of CO₂ to the environment.These and other evidences presented suggest that, in agreement with the prevailing notions, CO₂ is required by cells as an essential nutrient for growth, and that the desired level of CO₂ in culture can be maintained efficiently by its production by even a small number of cells in culture as long as the culture flasks are stoppered. If flasks are not stoppered, however, the level of CO₂ tension is determined by an equilibrium between the rate of its production by the cells and that of escape from culture to air, resulting in the observed failure in growth of cells in TES- and HEPES-buffered medium at low cell densities unless cultures were further supplemented with added CO₂.     

Human endometrial carcinoma cells release factors which inhibit the growth of normal epithelial cells in culture

Autocrine and paracrine interactions between cells are important homeostatic mediators in normal tissues. Alterations to growth factor signalling pathways are likely to play a role in multistep carcinogenesis. In this study normal human endometrial epithelial cells (NHEC) after 3 days in culture were treated with serum-free medium conditioned for 24 h by log phase or confluent cultures of established RL95-2, HEC1A, or AN3CA endometrial carcinoma (EC) cell lines. By day 4, NHEC treated with either log phase or confluent conditioned medium (CM) showed a significant decrease (50–90% of control) in [³H]thymidine ([³H]TdR) incorporation. DNA synthesis was inhibited more by confluent than by log phase CM. By day 7, NHEC treated with CM exhibited fewer colonies per culture, fewer cells per colony, and an increased percentage of single cells. Several growth-regulatory gene products found in the nucleus or at the cell membrane have been shown to be expressed differently in normal and transformed cells. We selected the p53 and c-Ha-ras p21 proteins to further investigate the mechanism of alteration of proliferation in cells treated with carcinoma CM. Thus, by day 7, the percentage of NHEC with nuclear localization of wild type p53 (wt p53) was elevated by treatment with CM. In contrast, CM-treated EC cells continued to proliferate, and showed a decrease in the percentage of cells expressing nuclear wt p53 and an increase in the cytoplasmic expression of c-Ha-ras p21. Our studies show that EC cell lines release factors which inhibit the proliferation of NHEC, thus favoring the proliferation of EC cells.Abbreviations CM conditioned medium - EC endometrial adenocarcinoma - NHEC normal human endometrial epithelial cells     

Spirulina culture in sea-water

Summary Laboratory experiments using small raceway ponds have shown that Spirulina maxima can be adapted easily to grow in sea-water supplemented with nitrate, phosphate, bicarbonate, and Fe-EDTA. To prevent precipitate formation, phosphate was supplied by diffusion through a dialysis membrane; the amount of Na-bicarbonate added was low (100 ppm) and the pH was kept in the range 8.6–8.8 by bubbling CO₂ into the culture.No significant differences have been noticed in productivity or in the chemical composition of the biomass between cultures in sea-water and in the standard bicarbonate medium. Cultures subjected to light/dark cycles of 12/12 h showed a higher respiration rate in sea-water than in the bicarbonate medium.The higher weight loss in the sea-water medium in the dark was counterbalanced by an increased synthesis of carbohydrates during the light period.     

INORGANIC CARBON TRANSPORT IN RELATION TO CULTURE AGE AND INORGANIC CARBON CONCENTRATION IN A HIGH-CALCIFYING STRAIN OF EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE)1

The relationships among inorganic carbon transport, bicarbonate availability, intracellular pH, and culture age were investigated in high-calcifying cultures of Emiliania huxleyi (Lohmann) Hay & Mohler. Measurement of inorganic carbon transport by the silicone-oil centrifugation technique demonstrated that gadolinium, a potential Ca²⁺ channel inhibitor, blocked intracellular inorganic carbon uptake and photosynthetic ¹⁴CO₂₊ fixation in exponential-phase cells. In stationary-phase cells, the intracellular inorganic carbon concentration was unaffected by gadolinium. Gadolinium was also used to investigate the link between bicarbonate and Ca²⁺ transport in high-calcifying cells of E. huxleyi. Bicarbonate availability had significant and rapid effects on pH_i in exponential- but not in stationary-phase cells. 4′, 4′-Diisothiocyanostilbene-2,2′-disulfonic acid did not block bicarbonate uptake from the external medium by exponential-phase cells. Inorganic carbon utilization by exponential- and stationary-phase cells of Emiliania huxleyi was investigated using a pH drift technique in a closed system. Light-dependent alkalization of the medium by stationary-phase cells resulted in a final pH of 10.1 and was inhibited by dextran-bound sulphonamide, an inhibitor of external carbonic anhydrase. Exponential-phase cells did not generate a pH drift. Overall, the results suggest that for high-calcifying cultures of E. huxleyi the predominant pathway of inorganic carbon utilization differs in exponential and stationary phase cells of the same culture.     

GROWTH CHARACTERISTICS OF PHYTOPLANKTON DETERMINED BY CELL CYCLE PROTEINS: PCNA IMMUNOSTAINING OF DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

By immunohistochemistry and immunofluorescence methods, we observed that the analog of proliferating cell nuclear antigen (PCNA) in Dunaliella tertiolecta Butcher (Chlorophyceae) was exclusively located in the nucleus. Among positively stained cells, PCNA abundance varied, being highest in S-phase cells, lower in others, and undetectable in early G1- or late M-phase cells. In exponentially growing and partially synchronized cultures, the percentage of PCNA-stained cells (% PCNA-stained cells) oscillated in the photocycle (12:12 h LD). It increased during the light period and reached a peak (75%) before the onset of the dark period when the culture was mainly (71%) in the S phase of the cell cycle. The DNA synthesis inhibitor, hydroxyurea, depressed PCNA abundance, whereas no effect was detected for the mitosis inhibitor colchicine. We conclude that PCNA in D. tertiolecta is associated with the S phase of the cell cycle where it is accumulated and functioning. PCNA was used to characterize the growth pattern of cultures grown in different media, temperatures, and growth stages. The time lag between the PCNA-stained phase and the M phase was very short in a continuous culture grown in reduced f/2 medium at 22°C and was considerably longer in the cultures grown in f/2 at 15°C. When an exponentially growing culture grew older, % PCNA-stained cells decreased. In a late stationary culture where there was no net growth, a small number of cells were still cycling through the PCNA-stained phase and cell division. In the continuous culture grown at 22°C, the duration of the PCNA-stained phase (T_s) was 13 h. Calculations with this T_s and % PCNA-stained cells yielded a growth rate of 0.77 d^?1, which was close to that obtained by cell counts (0.69 d^?1). Taken together, the results suggest that PCNA is a useful indicator of growth status and a promising cell cycle marker for estimation of species-specific growth rate.     

Formation of N-Carboxymethyl Amino Acid from the Reaction of α-Amino Acid with Glyoxal

Enrichment cultures in a medium containing 0.1% methanol and 0.1% bicarbonate at pH 7.0 under anaerobic conditions in the light became mainly green in color. Forty-four enrichment cultures, which showed abundant growth, were obtained from 46 different sources and found to contain cells of methanol-utilizing bacteria and green algae as predominant members. From these enrichment cultures, two strains of bacteria and two strains of algae were isolated. The microorganisms isolated were designated as bacterium No. 7, bacterium No. 8, Chlorella sp. A-1 and Chlorella sp. B-1, respectively. Stable mixed cultures were easily formed by mixing the isolated cultures of bacteria and algae. Both methanol and bicarbonate were necessary for the growth of the mixed cultures under anaerobic-light conditions. Growth behavior of the mixed cultures was examined on a medium containing 0.1% methanol and 0.1 % bicarbonate at 30°C in the light (about 6000 lx). The maximum specific growth rate for the cultures, µ_max, was 0.092 hr^?1 (doubling time, 7.5 hr). The maximum cell yield was 0.87 g dry-cell weight per g of methanol used. The protein content of the biomass was 65%.