Microcarrier culture of bowes melanoma cells in serum-free medium with Human plasma fraction IV-4+ V

Bowes melanoma cells were cultivated successfully in a serum-free medium which was constructed by the concept of maximum retention of proteins from fractionated human plasma having growth stimulatory activities. The cells could be cultivated in the serum-free medium without any adaptation period. The major serum-free component of the medium was the fraction IV-4 + V of the Cohn fractionation process of human plasma. Approximately six times increase of tissue-type plasminogen activator (t-PA) activity as compared with that in serum-free medium even though the cell growth was much slower. In addition, the growth stimulatory activities of thrombin and fibronectin were investigated during the cultivation of Bowes melanoma cells in this serum-free medium. These proteins contributed significantly to the enhanced growth of cells by reducing doubling time to 25 and 35 h as compared with 55 h in the serum-free medium without them. Especially, fibronectin supported cells to propagate near to the maximum cell density achieved in the medium with 10% FBS.     

Cyclic AMP and intracellular ionic activities innecturus gallbladder

Summary Open-tip and liquid ion-exchanger microelectrodes were used to study the effects of cAMP (6mm, added to the serosal medium) on apical membrane potential (E _m ) and intracellular sodium, potassium, and chloride activities (a _Na ⁱ ,a _K ⁱ ,a _Cl ⁱ ) inNecturus gallbladder under open-circuit conditions. Transepithelial potential difference (E _Tr ) was also measured. In the presence of cAMP,a _Cl ⁱ fell from about 1.5 times its equilibrium value to a level that corresponded to electrochemical equilibrium across the apical and basolateral cell membranes. Under these conditionsa _Na ⁱ decreased anda _K ⁱ increased,E _m was unchanged andE _Tr increased from virtually zero to a small but significant serosal positive value. The cAMP-induced increase ina _K ⁱ was abolished when Cl^–-free incubation media were used. Addition of the Ca⁺⁺-ionophore A23187 (0.5 g/ml) to the serosal medium had no effect onE _m ,E _Tr , ora _Cl ⁱ . When A23187 was added to the mucosal medium,E _m and the basolateral membrane potential hyperpolarized by about 20 mV and an increase in the outwardly directed electrochemical driving force for Cl^– was observed. These results indicate that cAMP inhibits coupled transapical Na–Cl entry into epithelial cells ofNecturus gallbladder and suggest that this inhibition may not be mediated by an increase in intracellular Ca⁺⁺ concentration.     

Intracellular ionic activities in frog skin

Summary Asymmetrical displacement currents are measured in the absence and in the presence of the lipophilic anion dipicrylamine (DPA) in the extracellular solution of nerve fibres of the frogRana esculenta. DPA (30nM-3 M) enhances the current by a component that has the properties expected for a translocation current of DPA ion across the lipid membrane. Analysis in terms of a single-barrier model yields the translocation rate constant (k), the total surface density of DPA absorbed to the membrane (N _t ), and the equidistribution voltage (). The value ofk of about 10⁴ s^–1 is similar to that for a solvent-free artificial bilayer formed by the Montal-Mueller method. The surface densityN _t varies with the DPA concentration as it does in the artificial bilayer, but is about tenfold smaller at all concentrations. The DPA ions sense an intrinsic electric field that is offset by a transmembrane voltage between 0 and 30 mV (inside positive). The part of the axolemma probed by the DPA ion appears as a thin (<2.5 nm), fluid bilayer of lipids. DPA ions seem, however, to be excluded from the major part of the axolemma as if this area is occupied by integral proteins or negative charges.     

Annuithrin,a new biologically active germacranolide from Helianthus annuus

Investigations on growth inhibition in Helianthus annuus led to the isolation of a new sesquiterpene lactone, a germacranolide with an α-methylene-γ-lactone moiety. The structure of this new germacranolide, annuithrin, was elucidated by spectroscopic methods. Its biological activity has been proven by growth inhibition in straight growth tests, antibacterial tests and inhibition of DNA-/RNA-synthesis in cells of the ascitic form of Ehrlich carcinoma.     

Influence of growth environment on the phosphoenolpyruvate: Glucose phosphotransferase activities of Escherichia coli and Klebsiella aerogenes: A comparative study

A consistent difference was found between glucose-limited cultures of Escherichia coli and Klebsiella aerogenes strains in the manner which their apparent cellular content of glucose: phosphoenolpyruvate phosphotransferase (glucose-PTS) varied with growth rate. With the former strains, activity increased as a function of growth rate; in the latter it decreased. However, under glucose-sufficient conditions (potassium-or ammonia-limitation) both species behaved similarly; the glucose-PTS activity was lower and bore no obvious relationship to the rate of glucose consumption expressed by the growing culture. These results are discussed in relation to the role of glucose as a regulator of glucose-PTS synthesis, and to the likely contribution which the glucose-PTS makes to the overall rate of glucose uptake, particularly by cells growing in glucose-sufficient environments.Abbreviation Glucose-PTS phosphoenolpyruvate phosphotransferase From May to November 1978 on study leave in the University of Amsterdam     

Effects of ion-pairing on calculations of ionic activities of major ions in freshwater

Ion-pairing has little effect on ionic activity calculations for weakly mineralized natural water (ionic strength < 2 mM). However, for more strongly mineralized freshwaters, corrections for ion-pairing are necessary if highly accurate ionic activities are required.     

Intimate coupling of creatine phosphokinase and myofibrillar adenosinetriphosphatase

ATPase and creatine phosphokinase (CPK) activities of isolated cardiac myofibrils were determined with ³²P γ-labeled ATP alone and with the addition of phosphorylcreatine (PC). With ATP and PC as substrates the label in the inorganic phosphate formed is greatly diluted indicating that the ATP formed by PC through CPK can reach the ATPase active site more readily than labeled ATP from the medium. The tight coupling of the ATPase and CPK activities further strengthens our view that PC serves an important role as high energy carrier between the energy producing sites (mitochondria) and the energy utilizing sites (myofibrils).     

Chemical characterization of a new Japanese variant of carbonic anhydrase I,CA INagasaki 1 (76 Arg→Gln)

A new inherited variant of carbonic anhydrase I (CA I), designated CA I_{Nagasaki 1} (CA I_{NGS 1}), was discovered during a survey of hemolysates from 5852 individuals from the cities of Hiroshima and Nagasaki in Japan. Analysis of the amino acid composition of a tryptic peptide from the CA I_{NGS 1} variant indicated that a glutaminyl residue was substituted for an arginyl residue at position 76. Heat degradation studies showed that the CA I_{NGS 1} variant was less stable than normal CA I. The CO₂ hydrase and esterase activities of the normal and variant carbonic anhydrases I, as well as the relative amounts of the two enzymes in heterozygotes, were similar.This work was supported in part by Contract E(11-1)-1552 with the Energy Research and Development Administration, Washington, D.C. (to J. V. Neel), and by U.S. Public Health Service Grant GM-24681.     

Local cold exposure of Andean Indians during normal and simulated activities.

Thermal resonses of Andean Indians were measured during several customary tasks associated with cold exposure in the highlands of southern Peru. These included surface temperature measurements of women while they washed clothing in the river and similar measurements of men while they constructed a diversion channel in the same river. A third test measured the effects of alcohol consumption on body temperatures during light activity. Women maintained slightly warmer hand than foot temperatures. Men maintained nearly equal hand and foot temperatures during the exposure period. Among male subjects the foot rewarmed at a faster rate than the hand. The results from the field studies compared favorably with results from earlier laboratory exposure tests. Comparisons between the river water exposure tests for males and females showed a consistent pattern where females maintained warmer hand and foot temperatures than males. These findings were in accord with previous laboratory studies among Quechua Indians and with the findings reported for other ethnic groups who experience natural cold stress. Alcohol ingestion appeared to have minimal effect in mitigating cold stress response during light activity. This finding was counter to earlier laboratory tests of resting subjects.