甘草中光甘草定的提取和抗氧化活性研究

从光果甘草(Glycyrrhiza glabra L.)丙酮提取物中分离得到黄酮类单体成分-光甘草定(Glabridin),并对其抗氧化生物活性进行观察研究。以肝脏微粒体中的细胞色素P450/NADPH氧化系统作为体外抗氧化实验模型。微粒体中自由基诱发程度由探针物DCFH-DA的氧化产物DCF的浓度进行检测。以银杏叶提取物EGB761用作阳性对照物。结果显示,光甘草定以0.10,0.25和0.5 mg/mL浓度,分别抑制自由基浓度67%、73%和83%。在较低浓度时,其抗氧化活性强度与EGB761类似。以上结果说明,光甘草定在细胞色素P450/NADPH氧化系统中具有强抗自由基氧化作用。     

Synergistic Antifungal Effect of Glabridin and Fluconazole

The incidence of invasive fungal infections is increasing in recent years. The present study mainly investigated glabridin (Gla) alone and especially in combination with fluconazole (FLC) against Cryptococcus neoformans and Candida species (Candida albicans, Candida tropicalis, Candida krusei, Candida parapsilosis and Candida Glabratas) by different methods. The minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) indicated that Gla possessed a broad-spectrum antifungal activity at relatively high concentrations. After combining with FLC, Gla exerted a potent synergistic effect against drug-resistant C. albicans and C. tropicalis at lower concentrations when interpreted by fractional inhibitory concentration index (FICI). Disk diffusion test and time-killing test confirming the synergistic fungicidal effect. Cell growth tests suggested that the synergistic effect of the two drugs depended more on the concentration of Gla. The cell envelop damage including a significant decrease of cell size and membrane permeability increasing were found after Gla treatment. Together, our results suggested that Gla possessed a synergistic effect with FLC and the cell envelope damage maybe contributed to the synergistic effect, which providing new information for developing novel antifungal agents.     

Estrogenicity and Genotoxicity Detection in Different Contaminated Waters

Owing that Oreochromis niloticus is resistant to different aquatic environments, four contaminated sites were selected: decentralized engineered ecosystem (DEE) and conventional wastewater treatment plants (WWTP); urban lagoon; and gasoline-contaminated marshland. Endocrine disruption was assessed by alkali-labile phosphate (ALP) quantification, genotoxicity by micronuclei frequency, and morphological changes by hepatosomatic and gonadosomatic indexes. The ALP baseline of non-exposed O. niloticus males was low, compared with other fish species in the literature. Endocrine disruption was observed in fish exposed to raw wastewater of WWTP and DEE, discharge point of channeled river in the urban lagoon, and water from gasoline-contaminated marshland. After treatment in the DEE, the endocrine disruption effect was removed. The micronuclei frequency corroborated with the ALP results in most cases and proved to be a useful tool to assess genotoxicity in fish. The exposure time of 15 days was not enough to provoke morphological effects in most samples. However, in all gasoline-contaminated samples, the fishes showed an increase in the hepatosomatic index followed by a decrease in the gonadosomatic index. The tested biomarkers showed feasibility, sensibility, reproducibility, and were complementary in the assessment of chronic ecotoxicity; therefore, we recommend them to compose a suitable protocol for aquatic monitoring programs.     

In Vivo Estrogenicity of Nonylphenol and Its Ethoxylates in the Canadian Environment

The relative estrogenicity of nonylphenol and its ethoxylates has not been clearly demonstrated in the literature, despite the importance of this information for interpreting the environmental risk of these chemicals. There appears to be a discrepancy between the relative acute/chronic toxicity and estrogenicity reported in previous studies. These studies have suggested that the relatively higher concentrations of nonylphenol polyethoxylate metabolites (NP_nEO, NP_nEC) in municipal effluents may represent a risk to the environment. However, there is considerable uncertainty associated with the estimates of relative estrogenicity of these metabolites.

Plasma vitellogenin (Vg) was measured in rainbow trout (Oncorhynchus mykiss) after a 21-d flow-through exposure to concentrations of 1–250 μ g · L^{? 1} nonylphenol (NP), 1–280 μ g · L^{? 1} nonylphenol 1-ethoxylate (NP1EO), or 24–1450 μ g · L^{? 1} nonylphenol 1-ethoxycarboxylate (NP1EC). All three chemicals induced plasma Vg to varying degrees, their relative estrogenicity being NP > NP1EO > > NP1EC. Measurements of the relative potency of NP1EO and NP1EC compared to NP, yielded ratios of 0.22 and 0.03, respectively. These values are in general agreement with relative acute and chronic toxicity data in the literature. A re-evaluation of the estrogenicity of the biodegradation products of nonylphenol polyethoxylates in Canadian sewage treatment plant effluents was performed, using the relative estrogenicity determined in this study, and revealed that the contribution of alkylphenol polyethoxylates to effluent estrogenicity is significant but less than previously estimated. The ability of these chemicals, however, to act in concert with other estrogenic compounds such as 17β -estradiol, estrone, and 17α -ethinylestradiol, to provide a cumulative estrogenic exposure for the biota needs to be investigated.     

Estradiol metabolism in Ishikawa endometrial cancer cells

Estrogen-responsive human cells derived from a specimen of well differentiated endometrial adenocarcinoma (Ishikawa line) were incubated with [3H]estradiol (E2) at various concentrations and the medium was sampled at 3, 6 and 24 h to evaluate the kinetics of removal of the hormone and the formation of unconjugated or sulfated metabolites. The detectable products of metabolism were estrone and the conjugate estradiol-3-sulfate. The latter was identified by high pressure chromatography, before and after acetylation, oxidation, and hydrolysis. The disappearance of [3H]E2 from the medium was found to follow first order kinetics between 3 and 24 h, with half-lives increasing from 4.7 to 53 h as the initial concentrations of the hormone were raised from 10(-8) to 10(-6)M. At the lowest concentration, practically all of the [3H]E2 added to the cultures was converted to estradiol-3-sulfate in 24 h, whereas at 10(-6)M oxidation to estrone was quantitatively more important than sulfation. These results indicate the presence in Ishikawa cells of an estrogen sulfotransferase of low Michaelis constant for E2, and 17 beta-oxidoreductase activity that significantly contributes to the metabolism of E2 only at higher concentrations of substrate.     

Estrogenicity of the isoflavone metabolite equol on reproductive and non-reproductive organs in mice

Equol, a metabolite of the phytoestrogen daidzein, is present at significant levels in some humans who consume soy and in rodents fed soy-based diets. Equol is estrogenic in vitro, but there have been limited studies of its activity in vivo. We evaluated equol effects on reproductive and non-reproductive endpoints in mice. Ovariectomized age-matched (30-day-old) female C57BL/6 mice were fed phytoestrogen-free diets and given a racemic mixture of equol by daily injections (0, 4, 8, 12, or 20 mg [kg body weight](-1) day(-1)) or in the diet (0, 500, or 1,000 ppm) for 12 days. Mice were killed, and serum concentrations of total and aglycone equol were measured. Total serum equol concentrations ranged from 1.4 to 7.5 microM with increasing doses of injected equol, but uterine weight increased significantly only at 12 and 20 mg (kg body weight)(-1) day(-1). Dietary equol at 500 or 1,000 ppm produced total serum equol concentrations of 5.9 and 8.1 microM, respectively, comparable with those in rodents consuming certain high-soy chows; the proportion of equol present as the free aglycone was much lower with dietary administration than injections, which may be a factor in the greater biological effects induced by injections. Dietary equol did not significantly increase uterine weight. Increasing dietary and injected equol doses caused a dose-dependent increase in vaginal epithelial thickness. Uterine epithelial proliferation was increased by equol injections at 8-20 mg (kg body weight)(-1) day(-1) and 1,000 ppm dietary equol. Neither dietary nor injected equol decreased thymic or adipose weights. In conclusion, equol is a weak estrogen with modest effects on endpoints regulated by estrogen receptor alpha when present at serum levels seen in rodents fed soy-based diets, but quantities present in humans may not be sufficient to induce estrogenic effects, although additive effects of equol with other phytoestrogens may occur.     

Structural Aspects of The Inhibitory Effect of Glabridin on LDL Oxidation

The inhibitory effects of glabridin, an isoflavan isolated from licorice (Glycyrrhiza glabra) root, and its derivatives on the oxidation of LDL induced by copper ions or mediated by macrophages were studied, in order to evaluate the contribution of the different parts of the isoflavan molecule to its antioxidant activity. The peak potential (E_1/2) of the isoflavan derivatives, their radical scavenging capacity toward 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical and their ability to chelate heavy metals were also analyzed and compared to their inhibitory activity on LDL oxidation. In copper ion-induced LDL oxidation, glabridin (1), 4′-O-methylglabridin (2), hispaglabridin A (3), and hispaglabridin B (4), which have two hydroxyl groups at positions 2′ and 4′ or one hydroxyl at position 2′ on ring B, successfully inhibited the formation of conjugated dienes, thiobarbituric acid reactive substances (TBARS) and lipid peroxides, and inhibited the electrophoretic mobility of LDL under oxidation. Compounds 1–3 exhibited similar activities, whereas compound 4 was less active. In macrophage-mediated LDL oxidation, the TBARS formation was also inhibited by these isoflavans (1–4) at a similar order of activity to that obtained in copper ion-induced LDL oxidation. On the other hand, 2′-O-methylglabridin (5), a synthesized compound, whose hydroxyl at 2′-position is protected and the hydroxyl at 4′-position is free, showed only minor inhibitory activity in both LDL oxidation systems. 2′,4′-O-Dimethylglabridin (6), whose hydroxyls at 2′- and 4′-positions are both protected, was inactive. Resorcinol (7), which is identical to the phenolic B ring in glabridin, presented low activity in these oxidation systems. The isoflavene glabrene (8), which contains an additional double bond in the heterocyclic C ring, was the most active compound of the flavonoid derivatives tested in both oxidation systems. The peak potential of compounds 1–5 (300 μM), tested at pH 7.4, was similar (425–530 mV), and that for compound 6 and 8 was 1078 and 80 mV, respectively. Within 30 min of incubation, compounds 1, 2, 3, 4, 8 scavenged 31%, 16%, 74%, 51%, 86%, respectively, of DPPH radical, whereas compounds 5 and 6, which almost did not inhibit LDL oxidation, also failed to scavenge DPPH. None of the isoflavan derivatives nor the isoflavene compound were able to chelate iron, or copper ions. These results suggest that the antioxidant effect of glabridin on LDL oxidation appears to reside mainly in the 2′ hydroxyl, and that the hydrophobic moiety of the isoflavan is essential to obtain this effect. It was also shown that the position of the hydroxyl group at B ring significantly affected the inhibitory efficiency of the isoflavan derivatives on LDL oxidation, but did not influence their ability to donate an electron to DPPH or their peak potential values.     

同时提取光果甘草中光甘草定和甘草酸的工艺研究

本文主要对光果甘草中光甘草定、甘草酸两种重要活性成分的同时提取工艺进行了研究。通过单因素试验确定了影响提取的主要因素及适宜水平范围,通过正交试验确定了其最佳提取条件为:用含0.6%氨水的60%乙醇溶液作为提取剂,料液比为1∶20(w:v),在75℃条件下提取2 h。最佳提取条件下光甘草定的得率为0.238%±0.002%,甘草酸的得率为5.08%±0.03%。该工艺操作简单,投入一批原料,即可同时提取其中的光甘草定和甘草酸,为实现甘草资源高附加值的综合加工利用奠定了基础。     

The Inhibitory Effect of Glabridin from Licorice Extracts on Melanogenesis and Inflammation

Glabridin is the main ingredient in hydrophobia fraction of licorice extract affecting on skins. In this study, we investigated inhibitory effects of glabridin on melanogenesis and inflammation using cultured B16 murine melanoma cells and guinea pig skins. The results indicated that glabridin inhibits tyrosinase activity of these cells at concentrations of 0.1 to 1.0 μg/ml and had no detectable effect on their DNA synthesis. Combined analysis of SDS-polyacrylamide gel electrophoresis and DOPA staining on the large granule fraction of these cells disclosed that glabridin decreased specifically the activities of Tl and T3 tyrosinase isozymes. It was also shown that UVB-induced pigmentation and erythema in the skins of guinea pigs were inhibited by topical applications of 0.5% glabridin. Anti-inflammatory effects of glabridin in vitro were also shown by its inhibition of superoxide anion productions and cyclooxygenase activities. These data indicated that glabridin is a unique compound possessing more than one function; not only the inhibition of melanogenesis but also the inhibition of inflammation in the skins. By replacing each of hydroxyl groups of glabridin with others, it was revealed that the inhibitory effect of 2′-O-ethyl glabridin was significantly stronger than that of 4′-O-ethyl-glabridin on melanin synthesis in cultured B16 cells at the concentration of 1.0 mg/inl. With replacement of both of two hydroxyl groups, the inhibitory effect was totally lost. Based on these data, we concluded that two hydroxyl groups of glabridin are important for the inhibition of melanin synthesis and that the hydroxyl group at the 4’ position of this compound is more closely related to melanin synthesis.     

Hormonal control of proliferation in the Ishikawa endometrial adenocarcinoma cell line

Steroid hormone regulation of proliferation of the Ishikawa human endometrial adenocarcinoma cell line was investigated in defined tissue culture medium. Oestradiol increased cell number following treatment for greater than 8 days; 4-OH tamoxifen, used alone, induced growth in a similar manner to oestradiol and was not antagonistic when used in combination with oestradiol. Progesterone decreased cell number 4 days after treatment but thereafter the effect was lost; the effect of progesterone was abolished in the presence of Phenol Red, consistent with the oestrogenic properties of this indicator. Oestradiol together with progesterone for greater than 8 days resulted in maximal growth and was preceded by an apparent increase in synthesis of a protein of molecular weight 36 kDa pI 8.     

Glabridin induces oxidative stress mediated apoptosis like cell death of malaria parasite Plasmodium falciparum

Plants are known as the source of novel agents for developing new antimalarial drugs. Glabridin is a polyphenolic flavonoid, a main constituent in the roots of Glycyrrhiza glabra possesses various biological activities. However, its anti-plasmodial activity is unexplored. In the present work, it is for the first time demonstrated that glabridin inhibits Plasmodium falciparum growth in vitro with an IC₅₀ 23.9 ± 0.43 μM. Glabridin showed poor cytotoxicity in vitro with an IC₅₀ 246.6 ± 0.88 μM against Vero cell line and good selectivity index (9.6). In erythrocytic cycle, trophozoite stage was found to be most sensitive to glabridin. In silico study showed that glabridin inhibits Pf LDH enzyme activity by acting on NADH binding site. Glabridin induced oxidative stress by the generation of reactive oxygen and nitrogen species. Glabridin could induce apoptosis in parasite as evidenced by the depolarization of mitochondrial membrane potential (Δψ_m), activation of caspase like proteases and DNA fragmentation. These results indicate that glabridin exhibits antiplasmodial activity and is suitable for developing antimalarial agent from a cheap and sustainable source.     

The Ishikawa cells from birth to the present

More than 20 years have passed since the Ishikawa cell line, a well-differentiated human endometrial adenocarcinoma cell line, was established. Because this cell line bears estrogen and progesterone receptors, the cells have been used in numerous basic research areas such as reproductive biology and molecular science, and has been distributed to more than a hundred institutes. However, even the Ishikawa cells, after long-term culture, tend to transform into undifferentiated cells. In addition, it has been reported that estrogen and progesterone receptors disappeared from the cells that I distributed. I therefore attempted to establish well-differentiated cells from the parent Ishikawa cells and to produce a new and good quality supply of this cell line. I believe that it is very important for the investigator who established a cell line to be responsible for maintaining the quality of the cells. That is why I have not deposited this cell line in any cell bank. I would like to take this opportunity to report the history of Ishikawa cells from establishment to the present.     

Glabridin protects osteoblastic MC3T3-E1 cells against antimycin A induced cytotoxicity

Mitochondrial dysfunction, particularly respiratory chain disruption, is often responsible for aging-related bone diseases. In this study, the protective effects of glabridin, an isoflavan isolated from licorice root, against pharmacological inhibition of the respiratory chain were studied using osteoblastic MC3T3-E1 cells treated with antimycin A, which inhibits complex III of the electron transport system. Glabridin restored mitochondrial membrane potential dissipation, ATP loss, inactivation of complex IV, intracellular calcium elevation, and cytochrome c release that was induced by antimycin A treatment. This compound also prevented cell death. These results imply that glabridin protects osteoblasts from antimycin A-induced cell death via improved mitochondrial function. Glabridin scavenged ROS and mitochondrial superoxide anions generated by antimycin A. In addition, glabridin prevented antimycin A-induced nitrotyrosine increase and thioredoxin reductase inactivation, suggesting that glabridin may be useful for protecting mitochondria against a burst of oxidative stress. Since phosphoinositide 3-kinase (PI3K) and cAMP-response element-binding protein (CREB) signaling is known to be pro-survival, we determined whether PI3K and CREB activation is associated with the cytoprotective effects of glabridin in the MC3T3-E1 cells. Glabridin restored antimycin A-induced inactivation of PI3K and CREB, suggesting that PI3K and CREB-dependent pathways may be involved in glabridin-induced cytoprotective responses. Our study demonstrates that glabridin reduces mitochondrial dysfunction induced during aging, and could significantly prevent osteoblast damage in osteoporotic patients.     

Estrogen regulates DNA methyltransferase 3B expression in Ishikawa endometrial adenocarcinoma cells

It is well-known that exposure to unopposed estrogen is considered as an important risk factor for endometrial cancer. Recent studies have shown that over-expression of DNA methyltransferases (DNMTs) are involved in the development of endometrial cancer. Therefore, the present study was undertaken to elucidate the impact of estrogen on the expression of DNMTs in endometrial cancer. Ishikawa cell line was used. Flow cytometry analysis demonstrated that 17 β-estradiol (E₂) enhanced the cell proliferation with a peak at 10⁻⁸ M. Over-expression of DNMT3B treated with E₂ was confirmed by real-time PCR and western blotting analysis. Furthermore, the up-regulation of DNMT3B expression induced by E₂ was suppressed by the addition of ICI182780. However, we did not observe changes in the expression of DNMT1. Our study suggests that estrogen up-regulating the expression of DNMT3B in an ER-dependent pathway may be a possible mechanism for estrogen facilitates the malignant transformation of endometrial cancer cells.     

Regulation of SULT1E1 expression in Ishikawa adenocarcinoma cells by tibolone

Tibolone is used therapeutically as a hormone replacement agent and has beneficial effects on osteoporosis and hot flushes as well as libido in post-menopausal women without stimulatory effects in the breast and endometrium. The lack of effect in the endometrium is due in part to the tissue specific sulfation of tibolone and its active metabolites in endometrial tissues. Tibolone is metabolized into 3alpha-OH and 3beta-OH tibolone as well as the Delta4-isomer. Tibolone and the Delta4-isomer bind and activate progesterone and androgen receptors whereas 3alpha-OH and 3beta-OH tibolone activate the estrogen receptors. Human endometrium and Ishikawa endometrial adenocarcinoma cells express SULT1E1 that efficiently sulfates both 3-OH tibolone metabolites and has trace activity with tibolone but no activity with the Delta4-isomer. Treatment of Ishikawa cells with all four tibolone compounds resulted in the induction of SULT1E1 activity similar to the induction by progesterone. The induction of SULT1E1 was inhibited by RU486 indicating a role for the progesterone receptor. Sulfation of the tibolone compounds by Ishikawa cells and Ishikawa cells expressing physiological levels of SULT1E1 activity resulted in the sulfation of tibolone and the 3-OH metabolites but not Delta4-tibolone. These results indicate that the lack of endometrial stimulation involves induction of SULT1E1 and the selective sulfation and inactivation of the estrogenic 3-OH tibolones and interconversion of the tibolone metabolites to generate the progestagenic non-sulfated Delta4-isomer.     

Regulation of estrogen activity by sulfation in human Ishikawa endometrial adenocarcinoma cells.

Sulfation is an important conjugation reaction in the metabolism of steroids. Steroids sulfates do not interact with the appropriate hormone receptors; additionally, the presence of the charged sulfate moiety increases the aqueous solubility and excretion of most steroids. Estrogen sulfotransferase (EST) is the major form of human cytosolic ST involved in the conjugation of estrogens. EST is important in the inactivation of beta-estradiol (E2) during the luteal phase of the menstrual cycle. EST has a significantly higher affinity for the sulfation of E2 and 17alpha-ethinylestradiol (EE2) than for other potent estrogens such as diethylstilbestrol (DES) and equine estrogens. The ability of EST to sulfate these estrogenic compounds at physiologic concentrations is important in regulating their activation of the ER in estrogen responsive cells. Human Ishikawa endometrial adenocarcinoma (ISH) cells possess an estrogen receptor (ER)-regulated alkaline phosphatase (AlkPhos) which is used to assay ER activation. To study the effects of EST activity on the ER activation of different estrogenic compounds, ISH cells were stably transformed with an EST expression vector. Dose-response curves for the induction of AlkPhos activity by the different estrogenic compounds were generated with EST/ISH and control pcDNA/ISH cells. EST/ISH cells were 200-fold less sensitive to E2 and EE2 than were control cells. No differences were observed in the dose response curves for DES between EST/ISH and pcDNA/ISH cells. EST/ISH cells were approximately 3-10-fold less sensitive to the equine estrogens equilin and 17-equilin as compared to control cells. The ability of EST to decrease the ER activation of an estrogen correlates with the sulfation of these compounds at nanomolar concentrations by EST/ISH and pcDNA/ISH ISH cells. These results indicate that EST is capable of efficiently inactivating E2 and EE2 but is significantly less effective in inhibiting the ER binding of other potent estrogenic compounds.     

Biochemical composition of Streptocephalus dichotomus Baird Branchinella kugenumaensis (Ishikawa)

Summary Estimations of total nitrogen, non-protein nitrogen protein, amino acids, lipid, carbohydrate, ash and water contents are carried out for the two fairy shrimps Streptocephalus dichotomus and Branchinella kugenumaensis.The mean values for S. dichotomus are: T.N. - 12.13%; N.P.N. - 3.49%; Protein - 54.71%; Lipid - 11.01%; Carbohydrate - 7.43% and ash - 10.44% and for B. kugenumaensis are: T.N. - 12.63%; N.P.N. - 4.92%; Protein - 48.21%; Lipid - 17.14%; Carbohydrate - 8.93% and ash - 9.17% expressed as percentage dry weight.The protein ash contents are higher in S. dichotomus while non-protein nitrogen, lipid and carbohydrate are higher in B. kugenumaensis.These biochemical fractions show no significant difference between males and females in both S. dichotomus and B. kugenumaensis.The work on Streptocephalus dichotomus formed a part of a thesis submitted for the Degree of Doctor of Philosophy to the University of Madras in 1970.     

Moina weismanni Ishikawa, 1896 (Cladocera,Moinidae) in Central Europe

Moina weismanni Ishikawa is reported from Czechoslovakia, Hungary, and Yugoslavia. The species was formerly synonymized with Moina micrura Kurz, based on parthenogenotic females. In both species, parthenogenetic females have a characteristic postabdomen, antennules (characters formerly regarded as typical for a variety of M. micrura), and ephippium. Wales are quite different. Examined populations of both species are similar in length-to-width ratio. M. weismanni is typical for small, eutrophic but permanent, and for large temporary water bodies. M. micrura is restricted to the plankton.     

Effects of serine and glycine on proliferation of an Ishikawa cell variant

The proliferation of cells on an Ishikawa human endometrial adenocarcinoma line variant (Ishikawa-Var I) is markedly influenced by the medium used to culture them, viz. MEM vs BM (basal medium; DMEM/Ham's F12, 1/1, with additional glutamine and HETES), under serum-free conditions. Components of BM which are not present in MEM were systematically tested in order to identify those that might account for these differences. Cells were cultured for various periods of time, up to 8 days, in serum-free MEM to which the components to be tested were added. Cell population densities were evaluated using a fluorometric DNA assay when the cells were grown in multiwell plates, or by cell counting when the cells were cultured in plastic dishes. It was found that addition to MEM of a mixture of the amino acids that this medium lacks, significantly increased cell density. By testing individual amino acids at the concentrations present in BM, it could be demonstrated that addition of serine alone was sufficient to obtain the densities achieved with BM. Glycine, a metabolic precursor of serine, had a similar but smaller effect. None of the other missing compounds of BM was effective. Effects of serine on DNA synthesis were also estimated by measuring incorporation of [3H]thymidine for 1 h after a 24 h culture period in MEM. The effect of serine was similar and additive to that of 1% charcoal-treated fetal bovine serum. A serine concentration dependence studied either with this method or measuring DNA/well after 8 days in culture showed detectable effects at 0.005 mM concentration and maximal responses at about 0.025 mM. These findings are of potential importance in studies on regulatory mechanisms of cell proliferation. A possibility to be explored, for instance, is that serine added to the medium increases intracellular phosphatidylserine concentrations leading to increases in the activity of protein kinase C, a stimulator of cell proliferation in some systems.