The influence of homologous plasma and fetal calf serum on human lymphocytic cortisol metabolism

Summary The influence of a) tissue culture medium (RPMI), b) homologous plasma (HP), and c) fetal calf serum (FCS) on lymphocytic cortisol metabolism was compared to that of phosphate buffered saline alone. RPMI was found to enhance the conversion rate 1.71 times, whereas HP and FCS enhanced it about 3.2 times. Raising the temperature of the HP and FCS to 100°C before incubation reduced the enhancing effect to the level of that obtained with RPMI.This work was supported by The Israel Cancer Research Fund N.Y. (George and Rose Blumenthal Research Fellowship for Hodgkins Disease). The authors are grateful to Dr Malchi and his staff from the Beilinson Hospital Blood Bank, for supplying us with buffy coats and to Mr Z. Klein for drawing the figure.     

In vitro development ofXenopus skin glands producing 5-hydroxytryptamine and caerulein

The granular glands of amphibian skin synthesize and store a large amount of bioactive amines and peptides which are structurally similar to mammalian brain-gut peptides. To investigate the development of peptide- and amine-producing cells in the granular glands, pieces of dorsal skin taken at various stages fromXenopus laevis tadpoles were cultured, and the contents of caerulein and 5-hydroxytryptamine (5-HT) were measured. When pieces of skin from tadpoles at stages 57 to 60 (Nieuwkoop and Faber stages) were cultured in a medium containing 10% fetal calf serum (FCS medium) or one containing FCS treated with charcoal (chFCS medium), the caerulein and 5-HT levels were increased for the six days of the incubation period. The caerulein content was lower in the chFCS medium than in the FCS medium. Addition of thyroxine to the chFCS medium had no significant effect on the caerulein content. These results show that the caerulein-and 5-HT-producing cells of the granular glands can develop in a culture system with FCS- or chFCS-containing media, and suggest that FCS contains substances which are absorbed by charcoal and stimulate development of the amine- and peptideproducing cells of the glands. In a preliminary search for correlation between caerulein and 5-HT synthesis, addition of 5-hydroxytryptophan (5-HTP), a precursor to 5-HT, to the FCS medium increased 5-HT content and, conversely, caused significant decrease in caerulein content, suggesting that accumulation of caerulein in the granular glands is influenced by the amount of 5-HT synthesis. These studies indicate that this culture system is a useful model for investigating the development of peptide- and amine-producing cells.     

Development of in vitro toxicity tests with cultures of freshly isolated rat hepatocytes

Freshly isolated and cultured hepatocytes were analyzed by two-parameter flow cytometry. The combined analysis of DNA and cellular protein content allowed the contribution of ploidy classes and of subpopulations within a ploidy class to be defined. Analysis of hepatocytes during exposure to dimethylsulfoxide (DMSO), phenobarbital (PB), low oxygen tension (4% O2) or fetal calf serum (FCS), provided insight into the dynamic response of individual ploidy classes as a function of culture time. By analogy with the age-dependent ploidy shifts in vivo, hepatocyte-cultures shift towards adult animals during exposure to DMSO and towards young animals when cultured at low pO2 (4% O2). FCS and phenobarbital disturb this constitutive ploidy balance. FCS increased the 2 N cell population, where stem cells probably respond to the proliferative stimuli provided by growth factors in the serum. Phenobarbital affects the liver-specific 4 N hepatocytes, which agrees with effects seen in liver after exposure in vivo. It is suggested that drug-induced pathological alterations in ploidy in hepatocyte cultures could serve as indicators of compounds, such as liver tumor promoters, which interfere with cell differentiation in liver. The heterotypic cell-cell interaction of freshly isolated hepatocytes with isolated, in vitro cultured, rat liver epithelial cells in co-cultures proved to be a valuable concept in toxicity testing: aldrin epoxidase, an enzyme system involved in xenobiotic metabolism, was stabilized for more than two weeks. After exposure to the three chemicals, 2-acetylaminofluoren, procarbazine and cyproterone-acetate, a preferential toxicity for each compound and cell population was established. Thus heterotypic cell cultures can considerably increase the amount of information available from in vitro studies. The final concept, combining monitoring of cellular DNA (ploidy) and protein content in hepatocyte cultures during and after exposure to a given test compound at tissue oxygen tension with the heterotypic cell-cell interaction, would create a more in vivo-like culture system. This would enhance the predictability of hepatocyte cultures and contribute to a more widespread use of the test system and as a result help to reduce the number of whole-animal tests.     

Development of in vitro toxicity tests with cultures of freshly isolated rat hepatocytes

Summary Freshly isolated and cultured hepatocytes were analyzed by two-parameter flow cytometry. The combined analysis of DNA and cellular protein content allowed the contribution of ploidy classes and of subpopulations within a ploidy class to be defined. Analysis of hepatocytes during exposure to dimethylsulfoxide (DMSO), phenobarbital (PB), low oxygen tension (5% O₂) or fetal calf serum (FCS), provided insight into the dynamic response of individual ploidy classes as a function of culture time. By analogy with the age-dependent ploidy shifts in vivo, hepatocyte-cultures shift towards adult animals during exposure to DMSO and towards young animals when cultured at low pO₂ (4% O₂). FCS and phenobarbital disturb this constitutive ploidy balance. FCS increased the 2 N cell population, where stem cells probably respond to the proliferative stimuli provided by growth factors in the serum. Phenobarbital affects the liver-specific 4 N hepatocytes, which agrees with effects seen in liver after exposure in vivo. It is suggested that drug-induced pathological alterations in ploidy in hepatocyte cultures could serve as indicators of compounds, such as liver tumor promoters, which interfere with cell differentiation in liver. The heterotypic cell-cell interaction of freshly isolated hepatocytes with isolated, in vitro cultured, rat liver epithelial cells in co-cultures proved to be a valuable concept in toxicity testing: aldrin epoxidase, an enzyme system involved in xenobiotic metabolism, was stabilized for more than two weeks. After exposure to the three chemicals, 2-acetylaminofluoren, procarbazine and cyproterone-acetate, a preferential toxicity for each compound and cell population was established. Thus heterotypic cell cultures can considerably increase the amount of information available from in vitro studies.The final concept, combining monitoring of cellular DNA (ploidy) and protein content in hepatocyte cultures during and after exposure to a given test compound at tissue oxygen tension with the heterotypic cell-cell interaction, would create a more in vivo-like culture system. This would enhance the predictability of hepatocyte cultures and contribute to a more widespread use of the test system and as a result help to reduce the number of whole-animal tests.     

Study of molecular events in cells by fluorescence correlation spectroscopy

To understand processes in a living cell, sophisticated and creative approaches are required that can be used for gathering quantitative information about large number of components interacting across temporal and spatial scales without major disruption of the integral network of processes. A physical method of analysis that can meet these requirements is fluorescence correlation spectroscopy (FCS), which is an ultrasensitive and non-invasive detection method capable of single-molecule and real-time resolution. Since its introduction about 3 decades ago, this until recently emerging technology has reached maturity. As commercially built equipment is now available, FCS is extensively applied for extracting biological information from living cells unattainable by other methods, and new biological concepts are formulated based on findings by FCS. In this review, we focus on examples in the field of molecular cellular biology. The versatility of the technique in this field is illustrated in studies of single-molecule dynamics and conformational flexibility of proteins, and the relevance of conformational flexibility for biological functions regarding the multispecificity of antibodies, modulation of activity of C5a receptors in clathrin-mediated endocytosis and multiplicity of functional responses mediated by the p53 tumor suppressor protein; quantitative characterization of physicochemical properties of the cellular interior; protein trafficking; and ligand-receptor interactions. FCS can also be used to study cell-to-cell communication, here exemplified by clustering of apoptotic cells via bystander killing by hydrogen peroxide.Received 15 July 2004; received after revision 13 October 2004; accepted 12 November 2004     

Ultrasonic comparison of two morphologically distinct melanosomes in malignant melanomas

Summary Ultrasonic measurement (0.333–200 MHz) of melanosomes isolated from B16 and Harding Passey (HP) mouse melanomas indicates that the partial wave resonance and principal relaxation of the 2 kinds of melanosomes are similar, but that their stochastic resonance is markedly different. The structure of the melanosomes appears basically amorphous, but linearly ordered and copolymeric in the molecular dimension of a segment composed of 5–6 zigzag units, which are packed closely in B16 and more openly in HP.Acknowledgment. This study was supported by Grants-in-Aid for Cancer Research, No. 580010030, No. 5839016 and No. 58480265, from the Ministry of Education, Science and Culture, by the Japan Ol'early Foundation and by the Alfred-Marchionini Foundation.     

Serotonin levels in fish brain: Effects of hydrostatic pressure and water temperature

Summary The contents of serotonin (5 HT) and its metabolite 5 hydroxy indoleacetic acid (5 HIAA) have been measured (HPLC technique) in the brains of eels exposed to different conditions of hydrostatic pressure and temperature (HP=1 or 101 ATA in winter, Tw=14°C, and in summer, Tw=19°C). It appears that an increase of Tw induces a significant increase of the 5 HT/5 HIAA ratio. In contrast, eels exposed at 101 ATA of HP for 1 h do not exhibit any modification in the 5 HT/5 HIAA brain ratio at a given temperature. The involvement of 5 HT under the conditions studied is discussed.     

Effect of diet on osmotic water flow across rat colon mucosa

Summary Osmotic water flow across colon mucosa was increased in rats adapted to a high protein diet (HP) compared to rats fed a high carbohydrate diet (HC). The diet-induced change of the osmotic permeability of the colon is probably a manifestation of a regulatory mechanism controlling intestinal water absorption.Supported by the Deutsche Forschungsgemeinschaft.The technical assistance of Miss Margitta Hosser is gratefully acknowledged.     

The use of 5-fluorocytosine and ketoconazole in the culture of the erythrocytic stages of Plasmodium falciparum and some tumor cell lines

In vitro culture systems are often contaminated by bacteria and fungi. It is therefore often necessary to supplement culture media with agents such as penicillin/streptomycin, gentamycin or amphotericin B. The latter cannot be used in the in vitro culture of erythrocytic stages of P. falciparum, and thus anti-fungal agents have not been regularly used in this system. We describe the prophylactic use of 5-fluorocytosine (5-FC) and ketoconazole (KTZ) in tissue cultures at concentrations up to 300 and 10 micrograms/ml respectively which have no effect on the growth of P. falciparum (FCR-3 strain). A melanoma cell line (C32) and a line of uterine carcinoma (C41) were also unaffected by similar concentrations of 5-FC and KTZ. When dissolved in complete culture medium (RPMI 1640) with 10% human plasma, the minimum inhibitory concentration of 5-FC for a susceptible strain of Candida remained below 2 micrograms/ml. These experiments suggest that 5-FC (at 50 micrograms/ml) alone or in combination with KTZ (at 1 microgram/ml) is a useful addition to the armamentarium of antimicrobials available to the tissue culture biologist for a variety of cell culture systems.     

The effect of hydrogen peroxide on the cyclin D expression in fibroblasts

Activation of mitogen-activated protein (MAP) kinase is essential for cyclin D1 expression and provides a link between mitogenic signalling and cell cycle progression. Hydrogen peroxide (H₂ O₂ ) activates MAP kinase; however, it is not known whether this leads to cyclin D expression. Sustained expression of cyclin D1 and D2 was observed when Her14 fibroblasts were incu-bated with 3 mM or higher H₂ O₂ concentrations. Similar results were obtained when cells were incubated in the presence of serum (FCS). However, the sustained expres-complex sion of cyclin D1 and D2 upon H₂ O₂ treatment was not due to the MAP kinase pathway, because MAP kinase kinase inhibitors did not inhibit cyclin D expression. Furthermore, cyclin D1 and D2 levels remained constant even after addition of a protein synthesis inhibitor, indicating that the effect of H₂ O₂ was not due to induction of protein synthesis. These results indicate that H₂ O₂ reversibly inhibits the ubiquitin-proteasome dependent degra-dation of cyclin D1 and D2, probably by transiently in-hibiting ubiquitination and/or the proteasome. Received 12 March 2001; received after revision 5 April 2001; accepted 9 April 2001     

Effect of pulsing electromagnetic fields on DNA synthesis in mammalian cells in culture

Summary DNA synthesis in Chinese hamster V79 cells was significantly enhanced when they were exposed to weak, pulsing electromagnetic fields generated by specific combinations of the pulse width (25s), frequency (10, 100 Hz) and magnetic intensity (2×10^–5 8×10^–5T). Conversely the DNA synthesis of cells in the fields at 4×10^–4 T was repressed to 80% of that in controls not exposed to the fields.This work was supported by Kaken Pharmaceutical Co., Ltd, Tokyo, Japan, and in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan and from the Foundation for Life Science Promotion Tokyo, Japan to I.K.     

北京温榆河浮游藻类与水质分析

2006年的监测结果显示,温榆河CODM。BOD5、NH4-N、TP和TN含量大于我国地表水环境质量V类标准的1．3～11．7倍,主要污染物为总氮、氨氯和总磷;五项指标（SS、SD、CODMn,TP、TN）的TSIM值为75．11～119．45,属于富营养型水体。温榆河4个监测断面共检出浮游藻类6门148种,绿藻种类最多（48％）,其次是硅藻（20．9％）、蓝藻（18．2％）,优势种群均为富营养型水体指示种。温榆河浮游藻类密度大,平均为3179．01×10^4 cells／L,绿藻占优势（75．6％）,蓝藻次之（14．6％）。     

Catecholamines in bovine semen

Summary In seminal plasma 5×10^–6 M noradrenaline was found to induced head-to-head association in bull spermatozoa. The sum of noradrenaline and adrenaline in freshly collected semen was 10 ng/ml (5.2×10^–8M), i.e., about 100 times lower than previously reported.     

Catalase and dehydroascorbate reductase in human polymorphonuclear leukocytes (PMN): Possible functional relationship

Summary In leukocytes (PMN) of individuals with Swiss type acatalasemia, the rate of dehydroascorbate reduction is 4 times normal. This observation suggests that the protective function served by catalase in human PMN is supported by dehydroascorbate reductase.This work was aided by grants to L.S. and R.B. from the American Cancer Society (CH-30B), the Grand Bethel of Oregon, International Order of Job's Daughters, and to S.W. and H.A. by grant number 3.384.74 from the Swiss National Science Foundation.     

Die Wirkung von Spirolactonen und von Herzglykosiden auf den Natrium- und Kaliumtransport an Erythrocyten

Summary Experiments made on human red cells showed that spirolactones (SC 5233 and SC 9420) added to the sample as dry powder (10^–3 g/ml) exert an inhibitory action on the active transport of sodium and potassium across the cell membrane. Spirolactone at its saturation concentration in serum (1.5×10^–5 g/ml), however, was ineffective.The inhibitory action was not affected by aldosterone or hydrocortisone.Experiments involving exposure of red cells to spirolactone and a cardiac glycoside simultaneously led to the conclusion that these substances do not have the same mechanism of action. The inhibition caused by spirolactone added to the inhibition due to a supramaximal concentration of ouabain (5 × 10^–4).

---

---

Diese Arbeit wurde unterstützt durch ein Stipendium der CIBA für naturwissenschaftliche, medizinische und technische Forschung.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptor-negative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [3H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10(-6) M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10(-5) M linoleic acid or 10(-5) M arachidonic acid but not by 10(-6) M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (Kd 0.54 nM +/- 0.11 nM; n = 6) than growth in medium supplemented with untreated serum (complete medium) (Kd = 1.68 nM +/- 0.48 nM; n = 6) (p less than 0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10(-5) M linoleic acid or 10(-5) M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10(-5) M stearic acid or 10(-5) M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10(-5) M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [3H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [3H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

Interferon-beta can induce the production of plasminogen activator by cultured human cancer cells

Three cultured human cell lines, renal cancer cells (ACHN), bladder cancer cells (EJ), and fibroblasts transformed in culture by Co-60 gamma rays (KMST-6), when treated with interferon-beta, produced 1.5 to 4 times as much plasminogen activator as the untreated control cultures. This enhanced production of PA was inhibited by cycloheximide or actinomycin D.     

Polarographic activity of the antitumor drug cis-dichlorodiammineplatinum (II). The effect of hydrolysis and trans-isomerization of the drug

Summary Electrochemical activities of cis-dichlorodiammineplatinum(II) (cis-DDP) and its trans isomer were studied by classical and differential pulse polarography (d.p.p.). It was shown that both isomers yielded a polarographic step or peak at about –1.6V (vs. Ag/AgCl), which corresponded to electroreduction of the complex and to catalytic hydrogen evolution. This signal was easily measurable with the aid of d.p.p. and was suitable for investigation of the extent of hydrolysis and trans-isomerization of cis-DDP leading to the formation of toxic products. The detection limit for determination of cis-DDP and its trans isomer by d.p.p. was 1×10^–6 mol/l.Acknowledgment. This work was supported in part by Lachema n.e., Brno (Czechoslovakia).     

Verlängerte Überlebenszeit von Hauthomotransplantaten bei Mäusen durch ein Methylhydrazinderivat

Summary Extended survival of skin homografts in mice was obtained by treatment with a methylhydrazine derivative (1-methyl-2-p-(isopropylcarbamoyl)-benzylhydrazine hydrochloride), representative of a new class of cytotoxic agents. Administration of 100 mg/kg prior to and continuously after the transplantation led to similar survival times as when 300 mg/kg was given daily only after the transplantation. In mice, the compound seems to be more effective than most of the drugs so far known to suppress transplantation immunity.