Electron probe microanalysis of potassium and phosphorus in mouse oocytes and zygotes

Intracellular concentrations of potassium and phosphorus were determined by Electron Probe Microanalysis in mouse mature oocytes and zygotes. The oocytes were characterized by insignificant variations in the concentrations of these elements in the cytoplasm: 60 +/- 4 and 103 +/- 6 mM, respectively. In zygotes, on the contrary, significant variations were observed: 64 +/- 16 and 84 +/- 14 mM, respectively. Changes in the potassium homeostasis during the first cell cycle have been discussed.     

Kinetic characterization of unspecific alkaline phosphatase at different villus sites of rat jejunum

Summary A quantitative histochemical method to determine the apparent Km and V _max values of rat intestinal unspecific alkaline phosphatase at different sites of the villi is described. Naphthol-As-Bi-phosphate (0.025–1.5 mM) is employed as substrate and Fast Blue B as coupling reagent, and the resulting azo-dye in the brush border membrane has an absorbance maximum at 550 nm. The ratio between the absorbance at 550 and 500 nm is constant as calculated from automatically recorded spectra at different intense dye deposits. Its absorbance is a linear function of incubation time up to 3 min and thickness of the slices up to 10 m both with medium (0.5 mM) and high (1.5 mM) substrate concentrations. Using the histochemical assay under comparable conditions in test tube experiments with homogenates of intestinal mucosa an app. Km of 0.26±0.081 mM (weighted regression analysis) and 0.28–0.084 mM (direct linear plotting) is determined, demonstrating an affinity to the histochemical substrate, which is about 10 times higher than for p-Nitro-phenyl-phosphate with the purified enzyme.The results obtained by scanning the total dye deposits along jejunal villi show considerable differences in enzymatic activity between single villi and an increase from the villus base up to the transition between medium and apical villus third. As well in the apical region as at the villus base saturation curves are obtained by determining the relationship between the absorbance and the substrate concentration under standard conditions (slice thickness 10 m, incubation time 3 min, 37°C, pH 8.3). Calculated by weighted regression analysis and direct linear plotting from the absorbance data of six female rats the medium app. kinetic data ±SD from the jejunal villi read as follows. Apical: Km=0.81±0.438 mM, V _max=3.99±1.217 absorbance units (A) and Km=0.87±0.428 mM, V _max=4.02±1.191 A, respectively. Basal: Km=0.82±0.261 mM, V _max=3.26±0.719 A and Km=0.77±0.184 mM, V _max=3.04±0.518 AU, respectively. As demonstrated by factorial analysis of variance only V _max is influenced by the villus position.Supported by the Deutsche Forschungsgemeinschaft GU 184/1     

The uptake of calcium by isolated chromaffin granules of the adrenal medulla

Bovine chromaffin secretory granules were purified by isopycnic Metrizamide gradient centrifugation and their Ca²⁺ sequestration pathways were characterized. The rate of Ca²⁺ sequestration at 37°C was first order, with a maximal uptake of 26.9 ±0.46 (mean ± S.D., n = 3) nmol Ca²⁺/mg protein and a first order rate constant (k) of 0.046 ± 0.002 min^–1. At 4°C the rate of uptake was substantially attenuated, with only 2.47 ± 0.2 (mean ± S.D, n = 3) nmol Ca²⁺/mg protein sequestered in 60 min. Ca²⁺ sequestration was 93% inhibited by 180 mM NaCl [I_50% of 78.7 ± 9.3 mM NaCl (mean ± S.D., n = 11)] but only slightly inhibited by KCl or MgCl₂. Ca ²⁺ sequestration was not stimulated by incubation with MgATP but was inhibited by 57% after incubation with 30 M monensin. Ca ²⁺ sequestration was dependent on extravesicular Ca ²⁺ with half-maximal sequestration at pCa²⁺ 6.81 ± 0.028 (mean ± S.D., n = 3). Sequestered Ca²⁺ could be exchanged with external ⁴⁵Ca²⁺, the exchange rate was first order (k of 0.042 ± 0.004: mean ± S.D., n = 3) and saturated at 27.7 ± 1.1 nmol Ca²⁺/mg (mean ± S.D., n = 3). The Ca²⁺/Ca²⁺ exchange system was totally inhibited by NaCl or KCl but only slightly by MgCl₂. About 75% of sequestered ⁴⁵Ca²⁺ could be released by incubation with NaCl, but only 8% was released by incubation with KCI. Half-maximal release of sequestered ⁴⁵Ca²⁺ required 69.3 ± 12.2 mM NaCl (mean ± S.D., n = 3). The Na⁺-induced release of sequestered ⁴⁵Ca²⁺ was rapid, t_0.5 of 2.80 ± 0.63 min (mean ± S.D., n = 3) and inhibited at 4°C. The concurrent incubation of chromaffin granules with ⁴⁵Ca²⁺ and either annexin proteins V or VI resulted in attenuated uptake of ⁴⁵Ca²⁺. These results suggest that Ca²⁺ uptake in adrenal chromaffin granules is regulated by Na⁺ and Ca²⁺ gradients and also possibly by annexins V and VI.Abbreviations EGTA ethylene glycol bis (-aminoethyl ether)-N,-N,N,N-tetraacetic acid - SDS Sodium dodecyl sulphate - PAGE Polyacrylamide gel electrophoresis - BSA bovine serum albumin - AI Annexin I - AIIt Annexin II tetramer - AIII Annexin III - AIV Annexin IV - AV Annexin V - AVI Annexin VI - k first order rate constant - AT total extent of Ca²⁺ uptake (nmol) - BufferA 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 5 mM EGTA - Buffer B 300 mM sucrose, 10 mM potassium phosphate (pH 7.0) and 1 mM EGTA - Buffer C 300 mM sucrose, 10 mM potassium phosphate (pH 7.0) - Buffer D 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.5 mM EGTA and 0.65 MM CaCl₂ - Buffer E 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.25 mM EGTA and 0.325 mM CaCl₂     

Physiological concentrations of purines and pyrimidines

The concentrations of bases, nucleosides, and nucleosides mono-, di- and tri-phosphate are compared for about 600 published values. The data are predominantly from mammalian cells and fluids. For the most important ribonucleotides average concentrations ±SD (M) are: ATP, 3,152±1,698; GTP, 468±224; UTP, 567±460 and CTP, 278±242. For deoxynucleosidestriphosphate (dNTP), the concentrations in dividing cells are: dATP, 24±22; dGTP, 5.2±4.5; dCTP, 29±19 and dTTP 37±30. By comparison, dUTP is usually about 0.2 M. For, the 4 dNTPs, tumor cells have concentrations of 6–11 fold over normal cells, and for the 4 NTPs, tumor cells also have concentrations 1.2–5 fold over the normal cells. By comparison, the concentrations of NTPs are significantly lower in various types of blood cells. The average concentration of bases and nucleosides in plasma and other extracellular fluids is generally in the range of 0.4–6 M; these values are usually lower than corresponding intracellular concentrations. For phosphate compounds, average cellular concentrations are: P_i, 4400; ribose-1-P, 55; ribose-5-P, 70 and P-ribose-PP, 9.0. The metal ion magnesium, important for coordinating phosphates in nucleotides, has values (mM) of: free Mg²⁺, 1.1; complexed-Mg, 8.0. Consideration of experiments on the intracellular compartmentation of nucleotides shows support for this process between the cytoplasm and mitochondria, but not between the cytoplasm and the nucleus.     

Use of a Hemoglobin-Trapping Approach in the Determination of Nitric Oxide in In Vitro and In Vivo Systems

We describe methods for measuring the release of nitric oxide (NO) derived from organic nitrates in vitro, using triple wavelength and difference spectrophotometry in the presence and absence of concentric microdialysis probes. These methods are based on the ability of NO to oxidize oxyhemoglobin (OxyHb) to methemoglobin (MetHb) quantitatively in aqueous solution. Isosorbide dinitrate (ISDN), a thiol-dependent organic nitrate, increased MetHb concentration in 45 min from 2.47 ± 0.47 to 4.15 ± 0.12 M (p < 0.05) and decreased OxyHb concentration from 2.13 ± 0.35 to 0.33 ± 0.26 M (p < 0.05) at 37°C. At 27°C, the OxyHb concentration was not significantly altered (2.04 ± 0.23 to 1.60 ± 0.04 M) by ISDN, nor was the MetHb concentration (from 2.68 ± 0.50 to 2.59 ± 0.25 M). Sodium nitroprusside (SNP), a thiol-independent organic nitrate, increased MetHb concentrations in 30 min from 4.21 ± 0.26 to 6.00 ± 0.56 M (p < 0.05) at 37°C, and from 4.23 ± 0.39 to 5.90 ± 0.43 M (p < 0.01) at 27°C. SNP also decreased OxyHb concentrations in 30 min from 1.99 ± 0.32 to 0.13 ± 0.12 M (p < 0.01) at 37°C, and from 2.25 ± 0.31 to 0.13 ± 0.09 M (p < 0.01) at 27°C. Difference spectrophometry indicated that 0.25-5 mM SNP significantly increased NO production in a dose-dependent fashion. This hemoglobin-trapping technique was also useful in quantifying the concentrations of NO released from SNP in aqueous solution in vitro, using concentric microdialysis probes. The NO concentration following exposure to SNP was 530 ± 50 nM, as determined using the difference spectrophotometric technique. To demonstrate the applicability of this technique to in vivo microdialysis, we implanted concentric microdialysis probes into hippocampus and cerebellum of conscious and anesthetized rats. Baseline NO concentrations in hippocampus of conscious and anesthetized rats were 11 ± 2 nM and 23 ± 9 nM, respectively, while in the cerebellum NO concentrations were 28 ± 9 nM and 41 ± 20 nM, respectively. These results demonstrate that microdialysis using a novel hemoglobin-trapping technique possesses adequate sensitivity to measure the NO levels produced from organic nitrates in aqueous solutions, and further document the applicability of this approach to in vivo systems.     

Effects of bovine Herpesvirus Type 5 on development of in vitro-produced bovine embryos

Bovine (Bos indicus) herpesviruses have been associated with reproductive disease. Type 1, the most studied species, is best known for its reproductive and respiratory effects. Type 5 (BoHV-5) has been detected in bull semen and aborted fetuses but not in oocytes and embryos. This study consisted of three experiments that evaluated (1) BoHV-5-infected oocytes matured in medium with fetal bovine serum (BoHV-FBS) or polyvinyl alcohol (BoHV-PVA) and fertilized by noninfected sperm; (2) noninfected oocytes fertilized by BoHV-5-infected sperm; and (3) infection of presumptive zygotes by BoHV-5. Each treatment involved nine drops of 15 to 20 oocytes. Infection with BoHV-5 was detected by polymerase chain reaction and in situ hybridization assay, and fertilization capacity and embryonic development were assessed using in vitro culture. Experimentally induced infection was obtained in all experiments, and vertical transmission of BoHV-5 by gametes was confirmed. The cleavage rate was reduced (P = 0.0201) in BoHV-FBS (80.4 ± 8.9%; mean ± SD) compared with that of noninfected oocytes (89.9 ± 6.5%); neither differed from BoHV-PVA (87.3 ± 7.1%), and the resulting embryo production rate was not significantly different among groups. Rates of cleavage (87.5 ± 7.5% vs. 92.2 ± 5.5%, control vs. infected) and development of embryos (41.7 ± 9.9% vs. 44.3 ± 7.7% to morula/blastocyst/expanded blastocyst [M/B/EB] and 39.6 ± 10.3% vs. 40.8 ± 9.2% to blastocyst/expanded blastocyst/hatching blastocyst [B/EB/HB] stages) were not compromised by infected sperm (P = 0.1462, P = 0.5402, and P = 0.8074, respectively). However, presumptive zygotes directly infected 1 d after fertilization produced a lower number (P = 0.0140 to M/B/EB and P = 0.002 to B/EB/HB stages) of in vitro-produced embryos (31.6 ± 4.6 vs. 25.0 ± 5.5 and 31.6 ± 4.6 vs. 20.2 ± 5.4; control vs. infected). In conclusion, BoHV-5 infected gametes and was transmissible to the embryo during in vitro development. As zygotes infected 1 d after fertilization had compromised development, BoHV-5 has the potential to be a pathogen with economic consequences.     

Growth and physiology of potassium-limited chemostat cultures of Paracoccus denitrificans

In potassium-limited chemostat cultures of Paracoccus denitrificans the maximum specific growth rate (µ_max) was found to depend on the input potassium concentration: At 0.21mM µ_max was 0.10–0.11 h^-1; at 0.44 mM 0.15–0.16 h^-1 and at 0.66 mM 0.20–0.21 h^-1. The plots of the specific rates of oxygen-, succinate-and potassium consumption against gave straight lines. The intracellular potassium concentration was a linear function of and varied from 1% (0.13 M) at a value of 0.034 h^-1 to 2.2% (0.29 M) at =0.26 h^-1; the potassium concentration gradient and the potassium concentration in the culture fluid in the steady state were dependent on the input potassium concentration. The potassium concentration gradient varied from 8,900-1,200. At all values 20–25% of the total energy production was used for potassium transport. 350,100 and 30 ATP molecules were calculated to be required to maintain one potassium ion intracellular during 1 h at values of 0.034, 0.197 and 0.257 h^-1 respectively. It is concluded that the amount of circulation of potassium is dependent on the potassium concentration gradient or on the potassium concentration in the culture in the steady state. The dependency of µ_max on the input potassium concentration was explained by the assumption that at low input potassium concentrations the net uptake of potassium (influx-efflux) is not rapidly enough to maintain the high potassium gradient in the existing cells and to establish it in the newly formed cells. At high values and at high input potassium concentrations µ_max is limited by the specific rate of oxygen consumption, which was found to be 11–12 mmol O₂ g dry weight^-1 h^-1 at µ_max for potassium-, succinate-and sulphate-limited chemostat cultures.     

Effects of copper sulphate concentrations during in vitro maturation of bovine oocytes

The objectives were to evaluate

1) copper (Cu) concentrations in plasma and follicular fluid (FF) from cattle ovaries; 2) the effects of supplemental Cu during in vitro maturation (IVM) on DNA damage of cumulus cells and glutathione (GSH) content in oocytes and cumulus cells; and 3) supplementary Cu during IVM on subsequent embryo development. Copper concentrations in heifer plasma (116 ± 27.1 μg/dL Cu) were similar (P > 0.05) to concentrations in FF from large (90 ± 20.4 μg/dL Cu) and small (82 ± 22.1 μg/dL Cu) ovarian follicles in these heifers. The DNA damage in cumulus cells decreased with supplemental Cu concentrations of 4 and 6 μg/mL (P < 0.01) in the IVM medium (mean ± SEM index of DNA damage was: 200.0 ± 27.6, 127.6 ± 6.0, 46.4 ± 4.8, and 51.1 ± 6.0 for supplementation with 0, 2, 4, and 6 μg/mL Cu respectively). Total GSH concentrations increased following supplementation with 4 μg/mL Cu (4.7 ± 0.4 pmol in oocytes and 0.4 ± 0.04 nmol/10⁶ cumulus cells) and 6 μg/mL Cu (5.0 ± 0.5 pmol in oocytes and 0.5 ± 0.05 nmol/10⁶ cumulus cells, P < 0.01) compared with the other classes. Cleavage rates were similar (P ≥ 0.05) when Cu was added to the IVM medium at any concentration (65.1 ± 2.0, 66.6 ± 1.6, 72.0 ± 2.1, and 70.7 ± 2.1 for Cu concentrations of 0, 2, 4, and 6 μg/mL). Percentages of matured oocytes that developed to the blastocyst stage were 18.7 ± 0.6, 26.4 ± 0.03, and 29.0 ± 1.7% for 0, 2, and 4 μg/mL Cu, and was highest (33.2 ± 1.6 %) in oocytes matured with 6 μg/mL Cu (P > 0.01). There was an increase (P > 0.05) in mean cell number per blastocyst obtained from oocytes matured with 4 and 6 μg/mL Cu relative to 0 Cu (IVM alone) and 2 μg/mL Cu. In conclusion, Cu concentrations in the FF and plasma of heifers were similar. Adding copper during oocyte maturation significantly increased both intracellular GSH content and DNA integrity of cumulus cells. Since embryo development was responsive to copper supplementation, we inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Cu concentrations during IVM.     

Effect of Ammonia and Methionine Sulfoximine on myo-Inositol Transport in Cultured Astrocytes

Ammonia causes astrocyte swelling which is abrogated by methionine sulfoximine (MSO). Since myo-inositol is an important osmolyte, we investigated the effects of ammonia and MSO on myo-inositol flux in cultured astrocytes for periods up to 72 hours. Uptake of myo-inositol was significantly decreased by 26.7 (P < 0.05) and 39.3 (P lt; 0.006) percent after 48 hours of exposure to 5 or 10 mM ammonia, respectively. The maximum rate of uptake was 14.0 ± 0.5 nmol/hour/mg protein which was reduced to 7.45 ± 0.27 and 7.02 ± 0.57 nmoles/hour/mg protein by 5 or 10 mM ammonia, respectively. The Kms by Michaelis-Menten equation for the control, and in the presence of 5, or 10 mM ammonia were 32.5 ± 4.52, 44.4 ± 5.82, and 39.3 ± 7.0 M, respectively. Kms by Hanes-Woolf plot for the control, 5, or 10 mM ammonia were 25, 45, and 40 M, respectively. Treatment of astrocytes with either 5 or 10 mM NH₄Cl for 6 hours caused a decrease in myo-inositol content by 66% and 58%, respectively. MSO (3 mM) partially diminished the ammonia-induced inhibition of myo-inositol uptake and decreased myo-inositol content by 31% after 24 hours. Additionally, ammonia increased myo-inositol efflux briefly through the fast efflux component but had little effect on myo-inositol efflux through the slow efflux component of astrocytes exposed to ammonia for up to 72 hours. Predominantly decreased myo-inositol influx coupled with brief efflux through the fast component may represent an adaptive response to diminish the extent of ammonia-induced astrocyte swelling.     

Two types of calcium-dependent channels of potassium outward current in the somatic membrane of Helix pomatia neurons

Characteristics of delayed potassium outward current were investigated during voltage clamp experiments on nonidentified intracellularly perfused neurons isolated from the snailHelix pomatia. A calcium-dependent potassium curent displaying special properties was shown to exist, apart from the voltage-operated potassium currents dependent on intracellular calcium ions . This type of current increases with a rise in the extracellular concentration of calcium ions , is not blocked by intracellular application of 10 mM EGTA and 77 mM fluoride, and may be suppressed by adding 1.5 mM cobalt ions to the extracellular fluid. This current, unlike , only takes a few milliseconds to peak, after which it fades to a steady level, comparable with that of .A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 2, pp. 185–191, March–April, 1987.     

Morphological development of normal and phyllody expressing Rosa hybrida cv. Motrea flowers

A large number of soybean (Glycine max L. Merr.)flowers and young pods abscise rather than develop into mature pods. Flower andpod drop or abortion accounts for the majority of total reproductive abscissionand influences potential soybean yield. The objectives of this study were todetermine the patterns of flower, pod and seed development under treatmentswiththe growth regulators, 2-(2,4-dichlorophenoxy) propanoic acid (2,4-DP) and6-benzylaminopurine (BAP), applied at the early reproductive stages, and toexamine the association of reproductive abscission with growth characteristicsand agronomic traits, including seed yield and seed weight. Small seeded [cvPungsan (11.1±0.4 g100-seed^–1)] and large seeded [cv Manlee(21.0±0.5 g 100-seed^–1)]genotypes were separately planted in the greenhouse and field, and treated witheither 2,4-DP or BAP. 2,4-DP (a synthetic auxin) and BAP (a syntheticcytokinin)were each applied at three concentrations (i.e. high, intermediate or low):0.12mM, 0.08 mM, 0.04 mM, and 1.5mM, 1 mM, 0.5 mM respectively. High andlow concentrations were employed for greenhouse experiments to examine thenumber of flowers per plant in pots. With the exception of low BAP (0.5mM) treatment in Pungsan, all treatments increased total podnumberwith various numbers of seeds per pod. Low 2,4-DP (0.04 mM) inbothgenotypes or BAP (0.5 mM) in Manlee significantly reduced flowerabortion and delayed abscission of pods in both genotypes, resulting inincreased pod setting rates. Under field conditions using intermediateconcentrations, 1 mM BAP significantly increased 100-seed weightto22.3 g at R1 in Manlee and 11.9 g at R3 in Pungsan.BAP (1 mM) at R3 in Pungsan significantly improved seed yield(40.1g plant^–1). Maturity was not significantlyaffected by either application in Manlee, but was significantly affected by BAPin Pungsan. In Pungsan, 2,4-DP increased pod number, plant height and nodenumber, but decreased 100-seed weight in Pungsan treated at R1, causing nosignificant change of seed yield. This study suggested that exogenousregulatorssignificantly influenced reproductive and growth characteristics, andconsequently seed yield, but increase of pod number was not always beneficialfor seed yield.     

Calmodulin selectively modulates the guanylate cyclase activity by repressing the lipid phase separation temperature in the inner half of the bilayer of rat brain synaptosomal plasma membranes

The association of [¹²⁵I-]calmodulin with rat brain synaptosomal plasma membranes, when incubated for 1 h at 25° in the presence or in absence of 20 M Ca²⁺, follows a sigmoid path with a Hill coefficient h=1.79±0.12 and h=1.72±0.11, respectively. The total association of calmodulin with the membrane increased approx. 60%–80% at all the range of calmodulin concentrations used in the presence of 20 M Ca²⁺. A three fold increase of guanylate cyclase activity was shown in the presence of low concentrations of calmodulin (up to 10 mM); higher concentrations (up to 40 mM) however, led to a progressive inhibition of the enzyme activity with respect to maximal stimulation. Calmodulin increased the lipid fluidity of synaptosomal plasma membranes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(r_o/r)-1]^–1. Arrhenius-type plots of [(r_o/r)-1]^–1 indicated that the lipid separation of the membrane at 22.7±1.2° was perturbed by calmodulin such that the temperature was reduced to 16.3±0.9° and 15.5±0.8° in the absence or in the presence of 20 M Ca²⁺. Arrhenius plots of guanylate cyclase and acetylcholinesterase activities exhibited brak points at 25.7±1.4° and 22.3±1.0° in control synaptosomal plasma membranes, respectively. The break point for the guanylate cyclase was reduced to 16.3±0.9° in calmodulin treated synaptosomal plasma membranes whereas that of acetylcholinesterase remained unaffected (21.1±0.9°). The allosteric properties of guanylate cyclase by Mn-GTP (as reflected by changes in the Hill coefficient) were modulated by calmodulin while those of acetylcholinesterase by fluoride (F^–) were not altered. We propose that calmodulin achieves these effects through asymmetric perturbations of the membrane lipid structure and that increase in membrane fluidity of the inner leaflet of the membrane induced by calmodulin may be an early key event to the process of neurotransmitter release.     

Biosorption and solubilization of copper oxychloride fungicide by Aspergillus niger and the influence of calcium

The biosorption of copper oxychloride fungicide particulates(1 m diameter), at concentrations ranging from 25 to 500 ppm active ingredient (ai), by pelleted mycelium of Aspergillus niger grown on Czapek Dox medium was evaluated. The concentration of the fungicide adsorbed to the mycelium, remaining suspended or solubilized in the medium, was determined by analysis of its copper content (CuF)using atomic absorption spectrophotometry (AAS). 2-day-old pellets exhibited highbiosorption efficiency ranging from 97 ± 1.0 to 88 ± 1.2% of the initially added fungicide concentrations, respectively, within 10 min. However, underthe same conditions, amounts of the removed fungicide by 6-day-old mycelial pellets were significantly lower and ranged from 0.5 ± 0.03 to 0.15 ± 0.01%. Scanning electron microscopy studies of 2-day-old pellets supplemented with thefungicide revealed predominant aggregations of clumps and dense particulates on the hyphal tips. The adsorbed CuF of 125 ppm ai fungicide subsequently decreased from 7.5 ± 0.5 to 2.1 ± 0.1 mol Cu (mg dry wt)^-1 after 12 h incubation. Simultaneously, the soluble portion of CuF remaining in the medium increased from 0.9 ± 0.6 to4.9 ± 0.2 mol Cu ml^-1. The presence of 50 mM CaCl₂ resulted in a decrease of the adsorbed CuF to 3.5 ± 0.5 mol Cu (mg dry wt)^-1 and solubilizedcopper in the medium increased to 5.9 ± 0.8 mol Cu ml^-1. Additionally, the cellular copper contents attained after 2 h were 0.08 ± 0.01 and 0.16 ± 0.007 mol Cu (mg dry wt)^-1 in absence and presence of calcium, respectively. The addition of calcium to glucose-starved pellets greatly increased the medium [H⁺] which was conclusively discussed in relation to Ca²⁺/H⁺ exchangecapacity of the fungal cells. These results are of potential environmental,biotechnological and agricultural importance.     

Nonidentity of the cDNA sequence of human breast cancer cell malic enzyme to that from the normal human cell

A cDNA coding for human breast cancer cell cytosolic NADP⁺-dependent malic enzyme was obtained. This cDNA is composed of a length of 2084 base pairs, with 1698 base pairs coding for 565 amino acid residues and a length of 386 base pairs representing a 3-noncoding region. Comparing this nucleotide sequence with that from the normal human tissue [Loeber, G., Dworkin, M. B., Infante, A., and Ahorn, H. (1994),FEBS Lett. 344, 181–186] reveals that three nucleotides in the open reading frame and the length of 3-noncoding region of the cDNA are different. One of the changes results in a substitution of serine at position 438 for proline, which, however, may not cause significant changes in the predicted secondary structure. A partial cDNA lacking the first 84 nucleotides in the open reading frame was successfully cloned and expressed functionally inEscherichia coli cells. ItsK _m value forl-malate (1.21±0.11 mM) is four times higher than that for the natural human breast cancer cell malic enzyme (0.29±0.04 mM) but similar to that for the full-length recombinant enzyme (1.06±0.07 mM). TheK _m values for Mn²⁺ and NADP⁺ (0.26±0.03 and 0.97±0.4M, respectively) are similar to those for the natural enzyme (0.12±0.02 and 1.9±0.3M, respectively) or the recombinant wild-type enzyme (0.56±0.04 and 0.44±0.02M, respectively). A recombinant pigeon liver malic enzyme without the first 13 amino acid residues was used for comparison. TheK _m values forl-malate and Mn²⁺ of the truncated enzyme (11.2±0.9 mM and 61.2±4.6M, respectively) are over 40 times larger than those for the natural pigeon liver malic enzyme (0.21±0.02 mM and 1.06±0.08M, respectively) or the recombinant wild-type enzyme (0.25±0.01 mM and 1.48±0.05M, respectively). We suggest that the N-terminus of malic enzyme may be required for the substrate binding during the catalytic cycle.     

Characterization of the inhibitory effects of erythromycin and clarithromycin on the HERG potassium channel

Both erythromycin and clarithromycin have been reported to cause QT prolongation and the cardiac arrhythmia torsade de pointes in humans, however direct evidence documenting that these drugs produce this effect by blocking human cardiac ion channels is lacking. The goal of this study was to test the hypothesis that these macrolide antibiotics significantly block the delayed rectifier current (I_Kr) encoded by HERG (the human ether-a-go-go-related gene) at drug concentrations, temperature and ionic conditions mimicking those occurring in human subjects. Potassium currents in HEK 293 cells stably transfected with HERG were recorded using a whole cell voltage clamp method. Exposure of cells to erythromycin reduced the HERG encoded potassium current in a concentration dependent manner with an IC₅₀ of 38.9 ± 1.2 M and Hill Slope factor of 0.4 ± 0.1. Clarithromycin produced a similar concentration-dependent block with an IC₅₀ of 45.7 ± 1.1 M and Hill Slope factor of 1.0 ± 0.1. Erythromycin (25–250 M) and clarithromycin (5 or 25 M) also produced a significant decrease in the integral of the current evoked by an action potential shaped voltage clamp protocol. The results of this study document that both erythromycin and clarithromycin significantly inhibit the HERG potassium current at clinically relevant concentrations.     

Mechanisms whereby glucose deprivation triggers metabolic preconditioning in the isolated rat heart

Transient glucose deprivation of the heart [GLU (-)] confers a preconditioning-like protection against subsequent ischemic/reperfusion (I/R). The mechanisms involved remain unclear. We hypothesized that GLU (-) would induce the classic ischemic preconditioning activated signaling cascade. Potential metabolic consequences and putative cell signaling events induced by transient glucose deprivation were evaluated as candidate mediators of this cardioprotection.Isolated glucose-perfused rat hearts were subjected to 30 min global ischemia followed by 30 min reperfusion (index I/R). Cardiac contractile recovery following I/R was used as the functional end-point in these studies. Metabolic preconditioning was stimulated by 15 min GLU (-) followed by 10 min glucose repletion prior to the index I/R. The potential metabolic consequences of GLU (-) were evaluated by using excess octanoate (11 mM OCT Hi) or 11 mM 2-deoxy-D-glucose (2-DG) in place of GLU (-) and by combining GLU (-) with fuels known to inhibit glycolysis supply (20 mM pyruvate or 1 mM octanoate, OCT Lo). The roles of -adrenoceptors, -adrenoceptors, adenosine receptors, protein kinase C (PKC) and mitochondrial K_ATP channels were investigated using inhibitors prazosin (10 M), propranolol (10 M), 8-(p-sulfophenyl) theophylline, (SPT 100 M), chelerythrine (CHEL 10 M) and 5-hydroxydecanoate (5 HD 100 M) respectively.GLU (-) increased mechanical recovery (59.8 ± 4.0 vs. 32.3 ± 4.7%; p < 0.01). Protection was abolished by pyruvate 26.6 ± 3.1; SPT 36.6 ± 3.0; CHEL 35 ± 4.8 or 5 HD 23.8 ± 3.3%. In a separate set of experiments, the specificity of SPT in this model was tested by preconditioning with adenosine (100 M) (34.7 ± 4 vs. control 16.8 ± 1.3%, p = 0.01) and blocking this protection with the same dose of SPT (16.3 ± 1.5%) used in the GLU (-) studies. Protection was unaltered by prazosin (50.2 ± 3.3%), propranolol (55.5 ± 4.0%), or OCT Lo (50.2 ± 2.5%). Protection was not mimicked by OCT Hi (35.6 ± 3.8%) or 2-DG (34 ± 4.3%).Transient glucose deprivation does not seem to achieve preconditioning-like cardioprotection by decreased glycolysis. Rather, the signal system may involve enhanced adenosine release, PKC, and activation of the mitochondrial K_ATP channel.     

Caecal water and electrolyte absorption and the effects of acetate and glucose, in dehydrated, low-NaCl diet hens

Summary In vivo electrolyte transport and water absorption from the caeca of dehydrated, low-NaCl diet hens are reported. In the absence of luminal glucose or acetate, net electrolyte transport rates and water absorption are small. When physiological concentrations of acetate (40 mM) are included in the perfusate, Na⁺ transport and water absorption increase significantly (P<0.01): 38±7 eqNa⁺/caecum kg·h and 256±33 l H₂O/caecum · kg · h.A similar increase in water absorption occurs with the inclusion of 15 mM glucose in the perfusate (219±30 l H₂O/caecum · kg · h), however both net Na⁺ and Cl^– absorption increase: 28±6 eq Na⁺/caecum · kg · h and 21±5 eq Cl^–/caecum kg · h.These pronounced increases in electrolyte and water absorption are not accompanied by any significant increase in transmural potential difference.The data presented establish caeca as important sites in the recuperation of water and electrolytes in dehydrated, low-NaCl diet hens.Abbreviations ECPD electrochemical potential difference - PD (transmural) potential difference - PEG polyethylene glycol     

Concentrations of signal transduction proteins mediating exercise and insulin responses in rat extensor digitorum longus and soleus muscles

Differences in the concentrations of signal transduction proteins often alter cellular function and phenotype, as is evident from numerous, heterozygous knockout mouse models for signal transduction proteins. Here, we measured signal transduction proteins involved in the adaptation to exercise and insulin signalling in fast rat extensor digitorum longus (EDL; 3% type I fibres) and the slow soleus muscles (84% type I fibres). The EDL and soleus were excised from four rats, the proteins extracted and subjected to Western blots for various signal transduction proteins. Our results show major differences in signal transduction protein concentrations between EDL and soleus. The EDL to soleus concentration ratios were: Calcineurin: 1.43 ± 0.10; ERK1: 0.38 ± 0.18; ERK2: 0.61 ± 0.16; p38, : 1.36 ± 0.15; p38/ERK6: 0.95 ± 0.11; PKB/AKT: 1.44 ± 0.08; p70S6k: 6.86 ± 3.58; GSK3: 0.69 ± 0.03; myostatin: 1.95 ± 0.43; NF-B: 0.32 ± 0.10 (values >1 indicate higher expression in the EDL, and values <1 indicate higher expression in the soleus). With the exception of p38/ERK6, the concentration of each signal transduction protein was uniformly higher in one muscle than in the other in all four animals. These experiments show that signal transduction protein concentrations vary between fast and slow muscles, presumably reflecting a concentration difference on a fibre level. Proteins that promote particular functions such as growth or slow phenotype are not necessarily higher in muscles with that particular trait (e.g. higher in larger fibres or slow muscle). Interindividual differences in fibre composition might explain variable responses to training and insulin. (Mol Cell Biochem 261: 111–116, 2004)     

Effect of 2,6-Diisopropylphenol and Halogenated Anesthetics on Tetraphenylphosphonium Uptake by Rat Brain Synaptosomes: Determination of Membrane Potential

The effect of 2,6-diisopropylphenol (propofol) in comparison to that of the halogenated anesthetics enflurane, isoflurane, and halothane on tetrapenylphosphonium uptake by rat brain synaptosomes was studied. A direct method to separately measure the synaptosomal and the mitochondrial transmembrane potential by using the tetraphenylphosphonium cation (TPP⁺) was utilized. The latter is a lipophylic charged molecule which distributes between two compartments according to the transmembrane electrical potential in the presence or absence of 60 mM KCl as a synaptosomal membrane depolarizing agent. After previously reporting the damages induced by general anesthetics on isolated mitochondria, the aim of this paper was to study their possible action on the synaptosomal membrane potential and whether or not drugs concentrations damaging isolated mitochondria are also effective on synaptosomal mitochondria. The results indicated that, in the presence of glucose, mitochondria included in synaptosomes were able to maintain a transmembrane potential of 202 ± 8 mV (mean ± SD) while the synaptosomal membrane showed a potential of 78 ± 8 mV (mean ± SD). When anesthetic concentrations (0.6–1 mM propofol, 10–40 M enflurane, 30–50 M isoflurane, 8–15 M halothane) that impair mitochondrial energy metabolism were used, the synaptosomal transmembrane potential was maintained and, in addition, a slight increase of the TPP⁺ taken up was observed as the anesthetic concentration was increased.     

Supply and compartmentalization of potassium in mesophyll cells of the needles of spruce,Picea abies (L.) Karst.

Summary The water and potassium content and the relative vacuolar volume ( = V_vacuole/V_cell) of mesophyll cells of the needles of healthy 21-yearold spruce trees [Picea abies (L.) Karst.] were determined. In 5-year-old needles was 0.626 ± 0.178 (ovx ± SD). Potassium concentrations in the bulk tissue water ranged from about 65 to 105 mM. Simulations were made using this information and a simple two-compartmental model of the cell with the bulk cytoplasm and the vacuole and assuming that the minimum cytoplasmic and vacuolar K⁺ concentrations are 100–150 mM and 10–15 mM respectively. It is shown that a K⁺ content of needles below 50 mmol/1 tissue water would be precarious for maintenance of normal physiological and metabolic performance.