Glucose-6-phosphate dehydrogenase Boston

Summary A hitherto undescribed variant of erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) activity, G-6-PD Boston, is described in a 24-year-old Caucasian male of Polish-Jewish ancestry. A marked decrease in red cell G-6-PD activity was associated, in this individual, with a compensated hemolytic process. The electrophoretic mobility of the partially purified enzyme on cellulose acetate at pH 9.1 and on starch gel was indistinguishable from normal but the apparent K_m for both G-6-PD (18–21 M) and NADP (1.7–2.2) was significantly decreased. Preliminary evidence supports the concept that G-6-PD Boston may not be extremely rare among this particular population group.     

Polish variant of glucose-6-phosphate dehydrogenase (G-6-PD lublin)

Summary A new, favism-inducing variant of glucose-6-phosphate dehydrogenase in erythrocytes is described in a Polish family. The enzyme activity has been 0–4% of normal. The enzyme displayed normal heat stability, electrophoretic mobility 105–110% of normal, K_m for NADP: 16–22 M, K_m for G-6-P: 26 M, and the utilization of 2-deoxy-G-6-P: 2–3%.     

Glucose-6-phosphate dehydrogenase in the population of northern Thailand: Evidence for two common electrophoretic variants with deficient enzyme activity

Summary Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, identified by a dye decolorization test, was found in 101 (12.5 percent) of 811 male subjects from northern Tailand. Blood samples from 169 subjects with normal G-6-PD activity and from all 101 subjects with G-6-PD deficiency were examined by electrophoresis on cellulose acetate gel with the following results: In all samples with normal G-6-PD activity the enzyme had the electrophoretic mobility of type B G-6-PD. 73 of the 101 G-6-PD deficient samples had the same mobility and are therefore probably identical with the common Mediterranean variant B-. 16 of the 101 deficient samples contained an electrophoretically fast G-6-PD, and 1 sample a slow variant. In 11 deficient samples the enzyme could not be made visible. Kinetic studies on crude hemolysates suggest that the fast variant has a higher mean activity and heat stability in comparison to the B- variant.Established and supported by Stiftung Volkswagenwerk, Hannover.     

Inheritance of quantitative expression of erythrocyte glucose-6-phosphate dehydrogenase activity in the Negro—a twin study

Studies have been conducted on eight sets of monozygous and nine sets of dizygous female Negro twins, both members of whom were heterozygous for G-6-PD deficiency. Twins were studied both by assay of erythrocytic G-6-PD activity and by the methemoglobin elution test (MET). The MET is a procedure which identifies histochemically cells with appreciable G-6-PD activity and permits accurate determination of the percentage of such cells in heterozygotes. Monozygous twins showed significantly less within-pair variation than dizygous twins with both the MET and G-6-PD assay.Concerning the significantly greater agreement in MET results in monozygous twins than dizygous twins, our present working hypothesis is that X-chromosomal inactivation in the Negro female is genetically controlled, rather than random. However, certain alternate hypotheses allowing for random X-inactivation have not been excluded; these include somatic cell selection after random X-inactivation, and cell exchange between identical twins in utero/it. Studies in nontwin related heterozygotes now underway should help differentiate among these various possibilities.In addition to the studies on 17 pairs of female twins heterozygous for G-6-PD deficiency, 26 pairs of nondeficient female Negro twins have been studied by G-6-PD assay. Within-pair variation in monozygous twins was significantly less than within-pair variation in dizygous twins in all cases. The genetic influences detected with the G-6-PD assay in the female twins could theoretically be due to nonrandom X-inactivation, to genetically determined quantitative differences in enzyme activity (e.g., isoalleles), or to both. By appropriate calculations, based on the MET results, we have factored out the effects of X-inactivation on overall enzyme activity in the heterozygous deficient twins. After removal of the effect of X-inactivation, monozygous twins heterozygous for enzyme deficiency continue to show significantly less within-pair variation than dizygous twins. This finding indicates significant genetic influences on quantitative G-6-PD activity other than X-inactivation and other than the deficiency allele. This conclusion has been strengthened by studies on male twins where X-inactivation is not present.Supported by USPHS research grants AM-09381, HE-17544, AM-09919, and HE-03341, by USPHS Career Development Award 1-K3-AM-7959 (Dr. Brewer) and by U.S.A.E.C. Contract (11-1)-1552.     

GD (-) Aachen,a new variant of deficient glucose-6-phosphate dehydrogenase

Summary A deficient G-6PD variant was discovered in 4 males of one family from north-western Germany. Five generations of this family could be studied.The deficient G-6PD was a new variant, called Gd (-) Aachen. Its main characteristics are the following: severe enzyme deficiency in erythrocytes (3% of normal), contrasting with an almost normal activity in leukocytes; normal molecular specific activity (i.e., normal ratio enzyme activity/cross-reacting material); slow mobility in starch gel electrophoresis (92–94% of normal); increased Michaelis constant for glucose-6-phosphate (60–70 M) and NADP⁺ (20–25 M); decreased inhibition constant by NADPH with respect to NADP⁺ (7 M); increased inhibition by ATP; normal utilization of the substrate analogues; slightly biphasic pH curve; thermal instability, and normal activation energy of the enzymatic reaction.The relationships between the hematologic disorders (severe and frequent hemolytic crises) and the unfavorable kinetic modifications are discussed.with the technical assistance of Joelle Marie and Dominique CottreauDedicated to Prof. Dr. H. Schonenberg, Aachen, on his 60th birthday. The first results of this work were presented in part at the Kongress der Deutschen Kinderärzte, München.     

The occurrence of G-6-PD union in Thailand

Summary Partially purified G-6-PD from 4 deficient Thai males presenting with acute hemolytic anemia showed low Km G-6-P consumption, and very high consumption of 2d G-6-P, Gal-6-P, and dTPN. DPN consumption was zero. The enzyme had very biphasic pH optimum curve and very low heat stability. Electrophoretic mobility was 103–106% of normal. These properties are very similar to those of G-6-PD Union. The enzyme of these 4 subjects is thus named G-6-PD Union (Thai).

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Zusammenfassung Teilweise gereinigtes G-6-PD von 4 Thai-Männern, die wegen einer hämolytischen Anämie zur Behandlung kamen, zeigten eine niedrige km G-6-P, dagegen hohe 2d G-6-P, Gal-6-P und dTPN-Utilisation. DPN-Utilisation was O. Das Enzym zeigte eine starke biphasische pH-Optimum-Kurve und sehr geringe Hitzestabilität. Die elektrophoretische Wanderungsgeschwindigkeit war 103–106% des Normalen. Diese Eigenschaften sind sehr ähnlich denen des G-6-PD-Union. Deshalb wird das Enzym dieser drei Personen als G-6-PD-Union (Thai) bezeichnet.

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Supported by U.S. Public Health research grant AM 09805 from the National Institute of Arthritis and Metabolic Diseases and research grant no G3/181/74 from the World Health Organization.     

Osmotic measurements on stomatal cells ofCommelina communis L.

Summary Observations of aperture changes as sucrose is added to the solution bathing epidermal strips ofCommelina communis L. allow calculation of the osmotic changes required to open or close the stomatal pore, for comparison with changes in potassium content. With isolated guard cells, in strips in which all cells other than guard cells have been killed, the internal osmotic changes required are 83 mosmol kg^–1 m^–1 below 10m aperture, 129 mosmol kg^–1 m^–1 in the range 10–15 m, and 180 mosmol kg^–1 m^–1 above 15 m. For opening against subsidiary cell turgor in addition to guard cell turgor, in intact strips with live subsidiary and epidermal cells, these figures should each be increased by about 33 mosmol kg^–1 m^–1. A change in subsidiary cell turgor is magnified in its effects on the water relations of the guard cell by a factor greater than 3.7 for equal changes in the water potential of the two cells, or greater than 4.7 at constant volume of the guard cell.     

A case of 9p- syndrome

Summary In two studied variants of G-6-PD without chronic hemolysis in probands, sensitivity of enzymes to inhibition by NADPH was decreased. K_i for NADPH was 28 M in Gd Lublin and 19 M in Gd Pozna. Susceptibility to the oxidant-induced hemolysis was described in probands, as well as in patients hemizygous for two other variants of G-6-PD with increased K_i for NADPH. It is suggested that in these cases, the oxidant-induced hemolysis is aggravated by their inability to counteract the drop in NADPH concentration with an increase in G-6-PD activity.     

Characterization of glucose-6-phosphate dehydrogenase in Thailand

Summary Characterization of partially purified eryrhrocyte G-6-PD from 50 enzymedeficient males in 45 unrelated Thai families revealed 6 enzyme variants. Thirty-five subjects in 31 families had G-6-PD variant with normal electrophoretic mobility, slightly low Km G-6-P, normal substrate-analog utilization, normal pH-optimum curve, and slightly increased heat stability. This enzyme variant is called G-6-PD Mahidol.Six subjects had enzyme with fast electrophoretic mobility (106–108% of normal), low Km G-6-P, slightly increased substrate-analog utilization, biphasic pH-optimum curve, and slightly low to normal heat stability. This variant was identical to G-6-PD Canton.Five subjects had G-6-PD with fast electrophoretic mobility (103–106% of normal), low Km G-6-P, very high substrate-analog utilization except for DPN which it did not use as cofactor, markedly biphasic pH-optimum curve and very low heat stability. This variant is called G-6-PD Union (Thai).Two brothers had G-6-PD with normal electrophoretic mobility, low Km G-6-P, slightly increased substrate-analog utilization, biphasic pH-optimum curve and low heat stability. This variant is designated G-6-PD Siriraj.G-6-PD from one patient had slightly fast electrophoretic mobility, increased substrateanalog utilization, especially of DPN, and very low thermal stability. It is called G-6-PD Kan.One subject had G-6-PD with normal electrophoretic mobility, Km G-6-P, pH-optimum curve and heat stability, and increased substrate-analog utilization. This G-6-PD variant is named G-6-PD Anant.G-6-PD Mahidol is far more common than any other known variants in Thailand.

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Zusammenfassung Eine Charakterisierung von teilweise gereinigtem Erythrocyten-G-6-PD von 50 Männern mit Enzym-Defekt aus 45 nicht miteinander verwandten Thai-Familien ergab 6 Enzym-Varianten. 35 Personen in 31 Familien hatten eine G-6-PD-Variante mit normaler elektrophoretischer Wanderungsgeschwindigkeit, einen leicht verminderten G-6-P-Km-Wert, einer normalen Substratanalog-Verwertung, einer normalen pH-Optimum-Kurve und einer leicht erhöhten Hitze-Stabilität. Diese Enzym-Variante wurde G-6-PD Mahidol genannt.Sechs Personen hatten ein Enzym mit rascher elektrophoretischer Wanderung (106–108% der Norm), niedrigem Km für G-6-P, leicht erhöhter Substrat-Verwertung, einer biphasischen pH-Optimum-Kurve und normaler bis leicht erniedrigter Hitzestabilität. Diese Variante ist identisch mit G-6-PD Canton.Fünt Personen hatten G-6-PD mit rascher elektrophoretischer Wanderung (103–106%), niedrigem Km G-6-P, sehr hoher Substratanalog-Verwertung—mit Ausnahme von DPN, das nicht als Cofactor wirkte—, einer stark biphasischen pH-Optimum-Kurve und sehr geringer Hitze-Stabilität. Diese Variante wurde als G-6-PD Union (Thai) bezeichnet.Zwei Brüder hatten ein G-6-PD mit normaler elektrophoretischer Wanderung, niedrigem Km G-6-P, leicht erhöhter Substratanalog-Verwertung, einer biphasischen pH-Optimum-Kurve und geringer Hitze-Stabilität. Diese Variante erhielt den Namen G-6-PD Siriraj.G-6-PD eines Patienten hatte eine leicht erhöhte elektrophoretische Wanderungsgeschwindigkeit, eine erhöhte Substratanalog-Verwertung, besonders für DPN, und eine sehr geringe Hitze-Stabilität (G-6-PD Kan).Eine Person zeigte ein G-6-PD mit normaler elektrophoretischer Wanderungsgeschwindigkeit, Km G-6-P pH-Optimum-Kurve und Hitze-Stabilität. Nur die Substratanalog-Verwertung war erhöht. Diese Variante wurde G-6-PD Anant gennant.G-6-PD Mahidol ist die bei weitem häufigste Variante in Thailand.

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This investigation received financial support from the World Health Organization.     

Population study of erythrocyte glutathione reductase activity

Summary The activity of erythrocyte glutathione reductase (GR) was determined in a group of 87 prisoners from northern Thailand (65 with normal, 22 with deficient erythrocyte G-6-PD) without and with added FAD. The amount of stimulation by FAD was inversely related to the original activity suggesting that FAD stimulation in vivo is one of the main determinants of GR activity. 4 subjects showed insufficient stimulation by FAD. The binding of FAD to GR seemed to be closer to saturation in G-6-PD deficient subjects but the maximal stimulated activity of these subjects was higher than in the group with normal G-6-PD. This suggests that the marked increase of GR activity in G-6-PD deficient erythrocytes is due to increased binding of FAD and to a larger amount of stimulable enzyme. Original GR activity was positively correlated with the socio-economic status, and subjects who had taken riboflavin in the period prior to examination had a higher mean GR activity than those without vitamin intake. GR activity was markedly raised by administration of riboflavin and in G-6-PD deficient subjects glutathione stability was improved. In comparison to central European subjects more than 50 percent of the examined population are GR deficient. This seems to be due to a low dietary supply of riboflavin in most cases. The findings in 4 subjects with insufficient stimulation by added FAD raise the question whether hereditary forms of GR deficiency exist in this population.

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Zusammenfassung In einer Gruppe von 87 Strafgefangenen in Nordthailand (65 mit normaler, 22 mit defizienter G-6-PD) wurde die Aktivität der Glutathion-Reductase (GR) mit und ohne Zusatz von FAD bestimmt. Das Ausmaß der Stimulierung des Enzyms durch FAD verhielt sich reziprok zur ausgangsaktivität. Bei 4 Versuchspersonen war die Stimulierung durch FAD insuffizient. Die Bindung von FAD an GR war bei Versuchspersonen mit G-6-PD-Mangel näher am Sättigungspunkt. Aber auch die gesamte stimulierbare Aktivität der GR war bei Versuchspersonen mit G-6-PD-Mangel größer als bei Gesunden. Die deutliche Erhöhung der GR-Aktivität bei G-6-PD-Mangel scheint zwei Ursachen zu haben: vermehrte Bindung von FAD an das Enzym und Vermehrung der Gesamtmenge an stimulierbarem Enzym. In einer weiteren Gruppe war die Ausgangsaktivität der GR vom sozio-ökonomischen Status der Versuchsperson abhängig. Personen, die vor der Untersuchung Riboflavin-haltige Medikamente eingenommen hatten, hatten höhere Aktivitäten. Die GR-Aktivität wurde durch Einnahme von Riboflavin unter Kontrolle stark erhöht, und bei Versuchspersonen mit G-6-PD-Mangel wurde die Glutathion-Stabilität der Erythrocyten verbessert. Im Vergleich zu Mitteleuropäern sind mehr als die Hälfte der thailändischen Versuchspersonen als GR defizient einzustufen. Dies ist wahrscheinlich auf eine geringere Zufuhr an Riboflavin in der Nahrung zurückzuführen. Die Befunde bei 4 Versuchspersonen mit insuffizienter Stimulierung der GR durch FAD könnte durch einen erblichen Mangel an GR verursacht sein.

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Established and supported by Stiftung Volkswagenwerk.     

Ultrastructural localization of corticotropin, β-lipotropin,and αand β-endorphin in the porcine anterior pituitary

Summary The peroxidase-antiperoxidase immunocytochemical technique was used to identify the ACTH/endorphin cells in the porcine pituitary at the ultrastructural level and to determine the precise subcellular localization of the pro-ACTH/endorphin fragments. The cells display different aspects: 1) large, regular shapes with numerous and large secretory granules; 2) small, irregular and angular shapes with small granules aligned along the periphery of the cell; and 3) intermediate forms. The presence of and -endorphin not only in the same cells but also in the same secretory granules that contain ACTH and -LPH clearly indicates that both the precursor or its fragments and the abovementioned peptides are stored in the same granules and released simultaneously by the corticotropic cells. The presence of FSH in some corticotropic cells is also discussed.Abbreviations used in this Article ACTH corticotropin - -MSH -melanotropin (ACTH I–I3) - CLIP corticotropin-like intermediate lobe peptide (ACTH 18–39) - -LPH -lipotropin - -MSH -melanotropin (-LPH 41–58); -endorphin (-LPH 61–91); -endorphin (-LPH 61–76)     

Characterization of 2,3-butanediol-forming and valine-sensitive α-acetolactate synthase of Enterobacter cloacae

-Acetolactate synthase (-ALS) of Enterobacter cloacae ATCC 27613 was purified to homogeneity by ammonium sulphate precipitation, Sephadex G-200 gel filtration and hydroxyapatite affinity chromatography. The molecular weight of the enzyme was found to be 60 kDa by SDS–polyacrylamide gel electrophoresis and 200 kDa by gel filtration through Sephadex G-200, showing that the enzyme is a homotrimer. The K _m and V _max of the enzyme were 20 mM and 200 mol min^–1 mg (protein)^–1 respectively. The enzyme was optimally active at pH 6.0–8.0, 37 °C and showed concentration-dependent sensitivity to cofactors viz. FAD, NADP and NADPH and branched chain amino acids: leucine, isoleucine and valine. Substances like sodium formate, sodium acetate and sodium propionate, sugars and the selected intermediates of glycolytic pathway inhibited the enzyme. Glycerol, BSA and pyruvate-TPP stabilized the -ALS. The enzyme showed the properties of both a catabolic as well as an anabolic -ALS.     

Alzheimer's Aβ1–40 Peptide Modulates Lipid Synthesis in Neuronal Cultures and Intact Rat Fetal Brain Under Normoxic and Oxidative Stress Conditions

The effect of amyloid (A), the major constituent of the Alzheimer's (AD) brain on lipid metabolism was investigated in cultured nerve cells and in a fetal rat brain model. Differentiated (NGF) and undifferentiated PC12 cells or primary cerebral cell cultures were incubated with [¹⁴C]acetate in the absence or presence of A_1–40. Incorporation of label into lipid species was determined after lipid extraction and TLC separation. Phosphatidylcholine (PC) and phosphatidylserine (PS) synthesis was increased by A_1–40, in a dose dependent manner, an effect which was more pronounced in differentiated PC12 cells. A significant proportion of radioactivity (5–6%) was released into the medium with a radioactivity distribution similar to that of the cellular lipids. Cholesterol and PC were the highest labeled medium lipids. Increasing A_1–40 concentration up to 0.1 g/ml in cerebral cells but not in PC12 cells, caused a relative increase (1.5 fold) in release of PS, while that of PE decreased. Stimulation of PS release may possibly be associated with apoptotic cell death. A_1–40 peptide (5 g) was administered intraperitonealy into rat fetuses (18 days gestation) along with [¹⁴C]acetate (2Ci/fetus). After 24 h, the maternal-fetal blood supply was occluded for 20 min (ischemia) followed by 15 min reperfusion. Fetuses were killed and liver and brain tissue subjected to lipid extraction and radioactivity determination after TLC. A_1–40 peptide increased synthesis of different classes of lipids up to 20–40% in brain tissue compared to controls. Labeling of liver lipids was decreased by A_1–40 by 20–30%. A general decrease in synthesis of lipids was observed after ischemia/reperfusion. Our data suggest that A_1–40 peptide regulates normal lipid biosynthesis but under ischemia it compromises it. The latter finding may confirm the oxidative stress etiology in AD and suggests that A_1–40 modulation of lipid metabolism may have Alzheimer's pathological relevance, particularly at high peptide concentrations.     

Effect of β-alanyl-L-histidinato zinc on differentiation of osteoblastic MC3T3-El cells: Increases in alkaline phosphatase activity and protein concentration

The effect of -alany-L-histidinato zinc (AHZ) on bone cell function was investigated in osteoblastic MC3T3-E1 cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus AHZ (10^–7–10^–5 M) or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5 M) produced a remarkable increase of alkaline phosphatase activity and protein concentration in osteoblastic cells. Thus increases were seen with the prolonged cultivation (12–21 days). With the culture of 1, 3 and 12 days, the effect of AHZ (10^–6 M) to increase alkaline phosphatase activity and protein concentration was more intensive than the effect of zinc sulfate, (10^–6 M). The AHZ effects were completely abolished by the presence of cycloheximide (10^–6 M), indicating that AHZ stimulates protein synthesis in the cells. The present study suggests that AHZ has a stimulatory effect on cell differentiation, and that this effect is partly involved on protein synthesis in osteoblastic cells.     

Cell-wall-bound lytic activity in Chlorella fusca: function and characterization of an endo-mannanase

A cell-wall-degrading activity was solubilized from young cells and from mother cell walls of Chlorella fusca by treatment with LiCl. The cytoplasmic enzyme hexokinase was not detectable in these extracts. The LiCl-solubilized activity increased in the cell cycle parallel to the release of autospores. The enzyme was purified on a chromatofocusing column followed by gel filtration. Sodium dodecyl sulfate/polyacryl amide gel electrophoresis of the purified enzyme revealed a molecular weight of 44 kDa, whereas gel filtration indicated a molecular weight of 25 kDa. Cell-wall-lytic activity and -1,4-mannanase activity coeluted in gel filtration and were separated from -d-fucosidase activity. The enzyme degraded isolated cell walls and ivory nut mannan primarily to oligosaccharides with an estimated degree of polymerization 6. The soluble degradation products of the cell wall consisted of 92–96% mannose and 4–8% glucose. It is concluded that the cell-wall-lytic activity is caused by an endo-mannanase. In vivo, this enzyme probably degrades the mother cell wall and, after autospore release, remains bound to it as well as to the surface of the daughter cells by ionic forces. The identity of this bound enzyme with a soluble wall-degrading enzyme previously obtained from mother cells is discussed.     

Voltage-gated chloride currents in cultured canine tracheal epithelial cells

Summary Chloride ions (Cl^–) are concentrated in airway epithelial cells and subsequently secreted into the tracheal lumen by downhill flux through apical Cl^– channels. We have studied Cl^– currents in cultured canine tracheal cells using the whole-cell voltage-clamp technique. Ultrastructural techniques demonstrated that the cells used in the electrophysiological experiments possessed apical membrane specializations known to be present in the intact, transporting cell type. Cultured cells 2–6 days old were characterized by an input resistance of 3.4±0.8 G (n=11) and a capacitance of 63.8±10.8 pF (n=26). A comparison of 3 and 4 day-old cells with 5 and 6 day-old cells showed that the input resistance decreased almost 50%, and the cell capacitance and the inward and outward currents increased concomitantly approximately 200%. Cultured cells 3–4 days old held at –40 mV produced currents of 196±22 pA at 50 mV and –246±27 pA at –90 mV (n=212) with pipette and bath solutions containing primarily 140 KCl and 140 NaCl, respectively. The chloride channel blocker diphenylamine-2-carboxylate (DPC, 100 m) suppressed whole-cell currents by 76.8% at 60 mV; however, currents were unaffected by the stilbenes SITS (1mm) and DNDS (1–30 m). Replacement of K⁺ with Cs⁺ in the pipette solution did not affect the outward current, the current reversal potential, or the input resistance of the cells, indicating that the current was not significantly K⁺ dependent when the intrapipette solution was buffered to a Ca²⁺ concentration of 20nm. The Cl^–/Na⁺ permeability ratio was estimated to be greater than 11 as calculated from reversal potential measurements in the presence of an internal to external NaCl concentration ratio of 12. Current equilibrium permeabilities, relative to Cl^– were: I^– (2.9)NO ₃ ^– (1.1)Br^– (1.1)Cl^– (1.0)F^– (0.93)MeSO ₄ ^– (0.19)gluconate (0.18)aspartate (0.14). Depolarizations to potentials greater than 20 mV elicited a time-dependent component in the outward current in 71% of the cells studied. Currents inactivated with a double exponential time course at the most depolarized voltages. Recovery from inactivation was fast, holding potential-dependent, and followed a double exponential time course. Current amplitude was increased via a cAMP-dependent pathway as has been demonstrated for single Cl-selective channels in cell-attached patches from cultured canine and human tracheal epithelial cells. Forskolin, an activator of adenylate cyclase, produced a 260% increase in the outward current at +50 mV. In summary, cultured canine tracheal cells have a single resting conductance that is Cl^– selective, voltage-dependent, and modulated by a cAMP-dependent mechanism. This preparation appears to be appropriate for analysis of cellular modulation of airway Cl^– channels and Cl^– secretion.     

Effect of β-alanyl-L-histidinato zinc on protein components in osteoblastic MC3T3-El cells: Increase in osteocalcin,insulin-like growth factor-I and transforming growth factor-β

The effect of -alanyl-L-histidinato zinc (AHZ) on protein components in osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 3 days at 37°C in CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further 3 or 6 days. The homgenate of cells was analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The presence of AHZ (10^–7 to 10^–5 M) caused an appreciable increase of many protein components in cells. Especially, the 67 killo-dalton (kDa) and 44 kDa proteins which are the major components from control cells were clearly increased by the presence of AHZ. Furthermore, the concentrations of osteocalcin, insulin-like growth factor-I and transforming growth factor- in the culture medium secreted from osteoblastic cells were markedly increased by the presence of AHZ (10^–6 and 10^–5 M). The effect of AHZ was a greater than that of zinc sulfate (10^–6 and 10^–5 M). The present findings suggest that AHZ can increase many proteins which are involved in the stimulation of bone formation and cell proliferation in osteoblastic cells.     

Diverse effects of essential (n−6 andn−3) fatty acids on cultured cells

Fatty acids (FAs) have long been recognized for their nutritional value in the absence of glucose, and as necessary components of cell membranes. However, FAs have other effects on cells that may be less familiar. Polyunsaturated FAs of dietary origin (n–6 andn–3) cannot be synthesized by mammals, and are termed essential because they are required for the optimal biologic function of specialized cells and tissues. However, they do not appear to be necessary for normal growth and metabolism of a variety of cells in culture. The essential fatty acids (EFAs) have received increased attention in recent years due to their presumed involvement in cardiovascular disorders and in cancers of the breast, pancreas, colon and prostate. Manyin vitro systems have emerged which either examine the role of EFAs in human disease directly, or utilize EFAs to mimic thein vivo cellular environment. The effects of EFAs on cells are both direct and indirect. As components of membrane phospholipids, and due to their varying structural and physical properties, EFAs can alter membrane fluidity, at least in the local environment, and affect any process that is mediated via the membrane. EFAs containing 20 carbons and at least three double bonds can be enzymatically converted to eicosanoid hormones, which play important roles in a variety of physiological and pathological processes. Alternatively, EFAs released into cells from phospholipids can act as second messengers that activate protein kinase C. Furthermore, susceptibility to oxidative damage increases with the degree of unsaturation, a complication that merits consideration because lipid peroxidation can lead to a variety of substances with toxic and mutagenic properties. The effects of EFAs on cultured cells are illustrated using the responses of normal and tumor human mammary epithelial cells. A thorough evaluation of EFA effects on commercially important cells could be used to advantage in the biotechnology industry by identifying EFA supplements that lead to improved cell growth and/or productivity.Abbreviations AA arachidonic acid (20 carbons: 4 double bonds,n–6) - BHA butylated hydroxyanisole - BHT butylated hydroxytoluene - cAMP cyclic adenosine monophosphate - CHO Chinese hamster ovary - DAG diacylglycerol - DGLNA dihomo--linolenic acid (203,n–6) - DHA docosahexaenoic acid (226,n–3) - EFA essential fatty acid - EGF epidermal growth factor - EGFR epidermal growth factor receptor - EPA eicosapentaenoic acid (205,n–3) - FA fatty acid - FBS fetal bovine serum - GLNA -linolenic acid (183,n–6) - LA linoleic acid (182,n–6) - LNA -linolenic acid (183,n–3) - LT leukotriene - MDA malondialdehyde - NAD nicotinamide adenine dinucleotide - NDGA nordihydroguaiaretic acid - OA oleic acid (181,n–9) - PG prostaglandin - PKC protein kinase C - PUFA polyunsaturated fatty acid - SFM serum-free medium - TX thromboxane     

Genetic regulation of glucose 6-phosphate dehydrogenase activity in Drosophila melanogaster

Studies on two variants of X-linked enzyme, G6PD, in several inbred and outbred strains of Drosophila melanogaster suggest that (1) there is dosage compensation at this locus; (2) males have 20–33% more activity than females, due to enzyme-deficient eggs in the latter; (3) outcrossing Drosophila strains results in a significant rise in G6PD specific activity in such a way as to suggest the presence of two or more nonlinked loci specific in their effect on G6PD activity (the effect is twice as great in males as it is in females); (4) there is less A enzyme than B enzyme activity/mg protein in males, but they are equal in females; (5) the presence or absence of X-linked regulators for G6PD could not be ascertained.Aided by National Institutes of Health grants HD 00004, HD00486, and GM 14155.     

Control of Herpes simplex virus thymidine kinase gene expression in Saccharomyces cerevisiae by a yeast promoter sequence

Summary This study presents the first evidence that the 5 promoter region of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene (G-3-PD) promoter will permit expression of an adjacent foreign gene. The S. cerevisiae G-3-PD promoter was linked to the herpes simplex virus — thymidine kinase (HSV-TK) gene in a shuttle plasmid capable of autonomous replication in both yeast and Escherichia coli. Since the HSV-TK gene promoter is not functional in yeast, yeast cells containing these plasmids will express the HSV-TK gene and synthesize thymidine kinase only if the yeast promoter fragment is fused to the HSV-TK gene in the proper orientation. The 5 flanking sequences necessary for the expression of heterologous eukaryotic genes in S. cerevisiae are discussed.