Transport of thymidine during the cell cycle in mitotically synchronized CHO cells

The kinetics of total uptake of thymidine into the cell were determined for cells which had been mitotically synchronized, plated into scintillation vials and pulsed with five concentrations of [³H]-thymidine at various times during the cell cycle. From Lineweaver-Burk plots of these rates, V_max and K_m values were determined for the transport of thymidine. The V_max values ranged from a low of 2.0 pmoles/ min/10⁶ cells in mid-G1 to a high of 99.7 in mid-S before a decline in late S and G2. K_m values displayed only a 5-fold range in values.     

A study of the substrate specificity of aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2

A systematic study was made of the ability of aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2 to hydrolyse different peptide substrates. The enzyme showed a marked preference for substrates containing arginine as the N-terminal residue but, to a lesser extent, was also capable of cleaving other residues such as lysine and leucine. There was a tendency for the activity to increase with the hydrophobicity index of the C-terminal residue of dipeptide substrates. It was also observed that the enzyme tended to have higher affinities but lower V _max values for tripeptides with hydrophobic C-terminal residues. The values determined for K _m and V _max increased with chain length for oligopeptides of the general formula Lys-Phe-(Gly) _n , the optimum, as determined from V _max/K _m, being when n = 4. Typical K _m values for the most effective substrates were in the range 0.2–0.6 mM.     

Purification and partial characterization of alanine dehydrogenase from Streptomyces aureofaciens

Alanine dehydrogenase was purified to near homogeneity from cell-free extract of Streptomyces aureofaciens, which produces tetracycline. The molecular weight of the enzyme determined by size-exclusion high-performance liquid chromatography was 395 000. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis was 48 000, indicating that the enzyme consists of eight subunits with similar molecular weight. The isoelectric point of alanine dehydrogenase is 6.7. The pH optimum is 10.0 for oxidative deamination of L-alanine and 8.5 for reductive amination of pyruvate. K _M values were 5.0 mM for L-alanine and 0.11 mM for NAD⁺. K _M values for reductive amination were 0.56 mM for pyruvate, 0.029 mM for NADH and 6.67 mM for NH₄Cl.Abbreviation AlaDH alanine dehydrogenase     

The isomerization of D-glucose into D-fructose catalyzed by whole-cell immobilized glucose isomerase. The dependence of the intrinsic rate of reaction on substrate concentration,pH, and temperature

The relation between the intrinsic rate of the glucose-fructose isomerization catalyzed by whole-cell immobilized glucose isomerase and the substrate concentration can be described with the kinetic model The numerical values of k₁, k_?1, k₂, and k_?2 have been determined from low-conversion experiments starting from pure glucose or fructose solutions, and are presented as a function of pH and temperature. The difference between the overall chemical reaction rate determined in high-conversion experiments and that calculated from the individual k₁, k_?1, k₂, and k_?2 values is less than 10%.     

Oxygen uptake kinetics of Pseudomonas chlororaphis grown in glucose- or glutamate-limited continuous cultures

Oxygen uptake and glucose and glutamate oxidation kinetics of the heterotrophic bacterium Pseudomonas chlororaphis grown in glucose- or glutamate-limited cultures under oxygen-saturating or oxygen-limiting conditions were determined. K _m values for oxygen were 1.4– 5.6 μM. Only in the case of glucose were significantly lower K _m values and enhanced specific oxygen affinity (V _max/K _m) per cell found under oxygen-limiting conditions. Both K _m and specific affinity values for glucose and glutamate oxidation were apparently affected by oxygen concentration, although a statistically significant enhancement of the oxidation kinetics was found only for glutamate. The kinetic data found for P. chlororaphis support the conclusion that the outcome of competition for oxygen with Nitrosomonas europaea in the rhizosphere of oxygen-releasing macrophytes will primarily be determined by oxidation kinetics of the electron donor instead of the oxygen uptake kinetics of the respective organisms. Received: 20 September 1996 / Accepted: 5 February 1997     

Kinetics Characterization of Taurocholic Transport in Lactobacillus reuteri

Taurocholic acid transport in Lactobacillus reuteri CRL 1098 was determined. The bile acid is incorporated inside the cells by an active and saturable transport showing a typical kinetics of Michaelis-Menten with values of K _m and V _max of 0.35 mm and 20 mm, respectively. Received: 30 May 2000/Accepted: 5 July 2000     

Diurnal changes in chlorophylla fluorescence and carotenoid composition inOpuntia ficus-indica,a CAM plant,and in three C3 species in Portugal during summer

Summary Diurnal changes in chlorophylla fluorescence were determined in four species, differing in life form, in Portugal during the summer of 1989. These includedOpuntia ficus-indica, a CAM plant, andHelianthus annuus, Ficus carica andArbutus unedo, three C₃ species. Steady state fluorescence yield,F _S, and maximum fluorescence yield,F _M′, were determined at different times of the day. Using the model of Genty et al. (1989), the photon use efficiency of photosystem II electron transport,φ _e, was calculated from (F _M′−F _S)/F _M′. Diurnal changes in relative rate of non-cyclic electron transport through photosystem II,J _e, were derived by multiplyingφ _e by the incident photon flux density (PFD). WhenJ _e, determined for each species for various points in time throughout the day, was plotted against corresponding values of PFD, the light response curves obtained showed thatJ _e was linearly dependent on PFD in low light and approached saturation in high light. The highest values ofJ _e were observed inHelianthus annuus, followed byOpuntia ficus-indica, Ficus carica andArbutus unedo. The proportion of the xanthophyll zeaxanthin to total carotenoids, determined around noon, was inversely related to maximum rates ofJ _e.     

Inhibition Profiles of Some Symmetric Sulfamides Derived from Phenethylamines on Human Carbonic Anhydrase I,and II Isoenzymes

In this work, the inhibitory effect of some symmetric sulfamides derived from phenethylamines were determined against human carbonic anhydrase (hCA) I, and II isoenzymes, and compared with standard compound acetazolamide. IC₅₀ values were obtained from the Enzyme activity (%)-[Symmetric sulfamides] graphs. Also, K_i values were calculated from the Lineweaver-Burk graphs. Some symmetric sulfamides compounds ( 11 – 18 ) demonstrated excellent inhibition effects against hCA I, and II isoenzymes. These compounds demonstrated effective inhibitory profiles with IC₅₀ values in ranging from 21.66–28.88 nM against hCA I, 14.44–30.13 nM against hCA II. Among these compounds, the best K_i value for hCA I (K_i: 8.34±1.60 nM) and hCA II (K_i: 16.40±1.00 nM) is compound number 11 . Besides, the IC₅₀ value of acetazolamide used as a standard was determined as hCA I, hCA II 57.75 nM, 49.50 nM, respectively. Moreover, in silico ADME-Tox study showed that all synthesized compounds ( 11 – 18 ) had good oral bioavailability in light of Jorgensen's rule of three, and of Lipinski's rule of five.     

KINETICS OF COMPETITIVE INHIBITION OF NEUTRAL AMINO ACID TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER

The transport of tryptophan across the blood-brain barrier is used as a specific example of a general approach by which rates of amino acid influx into brain may be predicted from existing concentrations of amino acids in plasma. The kinetics of inhibition of [¹⁴C]tryptophan transport by four natural neutral amino acids (phenylalanine, leucine, methionine, and valine) and one synthetic amino acid (α-methyl tyrosine) is studied with a tissue-sampling, single injection technique in the barbiturate-anesthetized rat. The equality of the K₁ (determined from cross-inhibition studies) and the K_m (determined from auto-inhibition data) for neutral amino acid transport indicate that these amino acids compete for a single transport site in accordance with the kinetics of competitive inhibition. Based on equations derived for competitive inhibition, apparent K_m values are computed for the essential neutral amino acids from known data on amino acid transport K_m and plasma concentrations. The apparent K_m values make possible predictions of the in vivo rates of amino acid influx into brain based on given plasma amino acid concentrations. Finally, a method is presented for determining transport constants from saturation data obtained with single injection techniques.     

Effects of sex and dietary phosphorus level on the apparent metabolizable energy and nutrient digestibility in broiler chickens

The influence of sex of broilers and dietary phosphorus (P) level on energy utilization and apparent ileal digestibility of N, P and phytate-P were investigated. The AME_N was determined using 3- and 6-week old broilers, while the apparent ileal digestibility were determined only with 6-week old birds. Sex of broilers had no effect on the AME_N values determined during week 3. During week 6, the AME_N values for male broilers were higher (P?0.01) than those for the females. An interaction (P?0.05) between sex and dietary P level was also observed. AME_N values determined with male broilers were lower in the adequate-P diet compared to those in the low-P diet, whereas no effect of P level was observed in females. Female broilers tended (P?0.10) to have a higher ileal N digestibility than the males, but a significant (P?0.01) sex ×?P level interaction was observed. Males receiving the adequate-P diet had a lower N digestibility than those receiving the low-P diet, whereas the opposite was true in the females. Ileal phytate P degradation in males was higher than in females (0.282 vs. 0.234), but the differences were not significant. Increasing dietary P level increased ileal P digestibility in both the males and females, but the improvements were greater in the females, as indicated by a significant sex × P level interaction.     

Effect of ionic strength on the pKa of ligands bound to DNA

The pK_a of 3,8-diamino-6-phenyl-phenanthridine (DAPP), a nonquaternary analog of ethidium bromide, has been determined spectrophotometrically as a function of sodium ion concentration both free in solution and complexed to DNA. Unwinding angle determinations with this compound were determined with Col El DNA using ethidium bromide as a standard. The unwinding angle for DAPP was 24 ± 2° relative to 26° for ethidium, and this suggests that DAPP binds in a manner quite similar to ethidium and with no significant outside bound DAPP under these experimental conditions. Isobestic behavior was obtained on spectrophotometric pH titration above pH 5 as long as the ratio of DNA-phosphate to ligand was between 100 and 300 and the DNA phosphate concentration was approximately 0.01M or greater. The loss of isosbestic behavior which occurred below pH 5 is probably due to titration of the 8 amino group of the ligand complexed to DNA. To circumvent this problem, pK_a values and the extinction coefficient of the acidic species were both determined by a computer program using experimental data obtained above pH 5. The pK_a of the free compound has only a minor dependence on ionic strength, while the pK_a of the ligand bound to DNA in an intercalated complex depends strongly on the sodium ion concentration. The pK_a of the DAPP-DNA complex is a linear function of –log[Na⁺] as predicted by the ion-condensation theory of polyelectrolytes. It was determined that DAPP is essentially completely bound to DNA under the conditions of these experiments by (1) determination of apparent pK_a values as a function of total DNA concentration, (2) calculation of binding constants for the neutral species of DAPP, and (3) spectral analysis of the protonated and neutral species of DAPP bound to DNA relative to DAPP free in solution. These results support the ion-condensation theory; provide an independent method for measuring ψ*, the average number of counterions associated per phosphate of DNA in the intercalated conformation; and illustrate that there are no specific pH effects or absolute pK_a values for ligands bound to DNA, but only ionic-strength-dependent results.     

Determination of oocyte membrane permeability coefficients and their application to cryopreservation in a rabbit model

Having an effective means to cryopreserve human oocytes would offer more flexibility in healthcare services for infertility patients, and obviate cryopreservation of preimplantation embryos. It is essential to establish good animal models for human oocyte cryopreservation and the rabbit is a good candidate. Attempts to improve oocyte cryopreservation are often empirical, with results often being irreproducible. Cryopreservation protocols may be optimized by modeling the changes in oocyte volume and the associated damages incurred during the addition and dilution of cryoprotective agents (CPA). The objectives of the current study were to determine cryobiological properties of rabbit oocytes, including the isotonic volume, osmotically inactive cell fraction (V_b), hydraulic conductivity (L_p), permeability (P_s) to dimethylsulfoxide (Me₂SO), ethylene glycol (EG), and glycerol (GLY) and to examine the correlation between cell volume excursions and viability. This has led to the development of the accumulative osmotic damage (AOD) model associated with the processes of CPA addition/dilution. Mature rabbit oocytes were perfused with 15% (V/V) CPA medium (dissolved in 1× PBS). The osmotic responses of the oocytes were videotaped. A two-parameter model was fit to the experimental data to determine the values of L_p and P_s. Oocyte volumes reached upon equilibration with 285, 600, 900, and 1200 mOsm (milliosmolal) solutions of non-permeating compounds were plotted in a Boyle van’t Hoff plot. The average radius of rabbit oocytes in an isotonic solution was determined to be 55.7 ± 1.2 μm (n = 16). The rabbit oocyte exhibited an “ideal” osmotic response in the range from iso-osmolity to 1200 mOsm. The V_b was determined to be 20% of the isotonic value with r² = 0.97. The values of L_p were determined to be 0.79 ± 0.26, 0.82 ± 0.22, and 0.64 ± 0.16 μm min⁻¹ atm⁻¹ and the P_s values were determined to be 2.9 ± 1.3, 2.7 ± 1.3, and 0.27 ± 0.18 × 10⁻³ cm min⁻¹ for Me₂SO, EG and GLY, respectively. There were no significant differences (p > 0.05) between values for L_p and P_S in the presence of the Me₂SO and EG. However, these values were significantly different from the values in presence of GLY. We calculated the AOD values of those oocytes that experienced the process of CPA additions/dilutions and found that these values were highly correlated to the development rates of these oocytes after parthenogenetic activation (r = −0.98).     

In vivo nuclear magnetic resonance studies on the lugworm Arenicola marina. I. Free inorganic phosphate and free adenylmonophosphate concentrations in the body wall and their dependence on hypoxia

The cytoplasmic concentrations of free inorganic phosphate and free AMP in the body wall of the lugworm Arenicola marina were estimated in order to verify their proposed regulatory role in glycogenolysis during anaerobiosis (Kamp 1993). Using in vivo ³¹P nuclear magnetic resonance spectroscopy the concentration of free inorganic phosphate was determined to be 4.7±0.7 mmol·1^-1 (±standard deviation, n=3) varying with season (Juretschke and Kamp 1995). These values were two to three times lower than those measured in perchloric acid extracts. In contrast, values for the phosphagen phosphotaurocyamine assessed biochemically in the extracts and by in vivo nuclear magnetic resonance were very similar. During the transition from normoxia to hypoxia the concentration of free inorganic phosphate increased to the same extent and at the same rate as the concentration of phosphotaurocyamine decreased. A discrepancy was also found for the biochemically determined AMP and ADP concentrations in the extract and those derived from the equilibrium constants of the taurocyamine kinase (ADP_free) and adenylate kinase (AMP_free) reactions. Calculated concentrations of ADP_free and AMP_free in normoxic specimens were about two or even four orders of magnitude lower than the values determined in extracts. During hypoxia the concentrations of AMP and ADP increase moderately when measured biochemically in extracts, while the values for ADP_free and AMP_free rise three- and nine-fold during the first 3 h of hypoxia. Thereafter, the levels stay constant due to a progressive acidosis. If during hypoxia pH_i is stabilized by addition of buffering substances to the incubation medium, both ADP_free and AMP_free rise continuously. The significant changes found for the concentrations of free inorganic phosphate and AMP_free support their assumed regulatory role in glycogenolysis during anaerobiosis, though these AMP_free values seem too low to actually activate glycogen phosphorylase. The strong effect of pH_i on the levels of ADP_free and AMP_free suggest a mechanism by which acidosis prevents a continuing increase of glycogenolysis (ATP-producing pathway) during prolonged anaerobiosis (protective effect of acidosis).Abbreviations ADP _free cytoplasmic adenosine diphosphate - AK adenylate kinase - AMP _free cytoplasmic adenosine monophosphate - CK creatine kinase - GPase glycogen phosphorylase - MW molecular weight - Int P _i integral of P_i-signal - NMR nuclear magnetic resonance - P _i-free cytoplasmic inorganic phosphate - PCA perchlori acid - PFK 6-phosphofructokinase - PME phosphomonoester - PPA phenylphosphonic acid - (P)Tc (phospho)taurocyamine - S _f saturation factor - Sw sea water - TcK taurocyamine kinase - TRA triethanolamine - TRIS tris(hydroxymethyl)aminomethane     

The role of temperature in enhancing the insecticidal activity of Bacillus thuringiensis against Spodoptera littoralis larvae

The present study scrutinised how far temperature would affect the velocity of the insecticidal activity of Bacillus thuringiensis, as the rapidity of pest control achievements is of a great concern. Third instar Spodoptera littoralis larvae were treated with Bt at three concentration levels under five different temperatures (15°C, 20°C, 25°C, 30°C and 35°C). LT₅₀s were evaluated in each case. The LT₅₀ values showed various levels of reductions as temperature and/or Bt concentration increased, indicating that the velocity of mortality (1/LT₅₀) and/or the rapidity of Bt activity was almost temperature dependant. However, relatively high and low reduction percentages in the LT₅₀ values on the elevation of 5°C were obtained at lower and higher temperature ranges, respectively. The temperature coefficient, Q ₁₀ values, determined within narrow ranges (5°C) showed great reductions when temperature increased from 15°C to 20°C at all Bt concentrations. Raising temperature by 5°C above 20°C or 25°C almost caused similar Q ₁₀ values indicating constant increase in the response of Bt activity within 20–30°C temperature range. Q ₁₀ values over 30°C were comparatively very low. This proved that decrease in Q ₁₀ values due to the rise of temperature was dependant on the starting temperature.     

Calcium-stimulated myofibrillar ATPase activity correlates with shortening velocity of muscle fibres in Xenopus laevis

Summary The iliofibularis muscle ofXenopus laevis is reported to contain five types of fibres which have different force—velocity relationships. Ten fibres of each type were selected on the basis of succinate dehydrogenase activity, cross-sectional area and location in the muscle, in order to assess the validity of the fibre type classification.Maximum calcium-stimulated myofibrillar ATPase activity (V _max) and apparent Michaelis constant (K _m) for ATP were determined for these 50 fibres from serial sections. The values obtained varied according to the type of fibre. Type 1 had the highest and type 5 the lowest values forK _m andV _max.In a separate experiment, single freeze-dried fibres were used to determine the relationship between their ATP content and apparentK _m for ATP. There was a tendency for high ATP concentrations in fibres with highK _m values.When myofibrillar ATPase activity was related to the maximum velocity of shortening of the five fibre types, a significant correlation was found. It is concluded that calcium-stimulated myofibrillar ATPase histochemistry allows an estimate of the maximum shortening velocity of muscle fibres fromXenopus laevis.     

Cellular drug efflux and reversal therapy of cancer

A prevalent form of multidrug resistance (MDR) in cancer cells is caused by an ATP-dependent drug efflux pump; this pump catalyzes the rapid exit of cytotoxic chemotherapy drugs from the cells. The Michaelis equation can be used to describe drug efflux through the MDR pump at a low drug substrate concentration [S]. The inhibition mechanism of an MDR reversal agent can be characterized when two different values of [S] are used to determine two values for the half-inhibition of efflux through the pump (I ₅₀). The reaction is noncompetitive when the two values ofI ₅₀ are identical; the reaction is competitive when an increase in [S] produces a significant increase in the value ofI ₅₀ TheI ₅₀ has been determined for several different reversal agents with the substrate rhodamine 123. The inhibition potency observed is: cyclosporin A >DMDP>amiodarone>verapamil>quinidine>quinine>propranolol. Chemotherapy drugs that are potent inhibitors of the MDR pump could be used for the treatment of MDR neoplasia.     

Water permeability of isolated cuticular membranes: The effect of cuticular waxes on diffusion of water

Summary The water permeability of astomatous cuticular membranes isolated from Citrus aurantium L. leaves, pear (Pyrus communis L.) leaves and onion (Allium cepa L.) bulb scales was determined before and after extraction of cuticular waxes with lipid solvents. In pear, the permeability coefficients for diffusion of tritiated water across cuticular membranes (CM) prior to extraction [P _d(CM)] decreased by a factor of four during leaf expansion. In all three species investigated P _d(CM) values of cuticular membranes from fully expanded leaves varied between 1 to 2×10^-7 cm^-3 s^-1·P _d(CM) values were not affected by pH. Extraction of cuticular waxes from the membranes increased their water permeability by a factor of 300 to 500. Permeability coefficients for diffusion of THO across the cutin matrix (MX) after extraction [P _d(MX)] increased with increasing pH. P _dvalues were not inversely proportional to the thickness of cuticular membranes. By treating the cutin matrix and cuticular waxes as two resistances acting in series it was shown that the water permeability of cuticles is completely determined by the waxes. The lack of the P _d(CM) values to respond to pH appeared to be due to structural effects of waxes in the cutin matrix. Cuticular membranes from the submerse leaves of the aquatic plant Potamogeton lucens L. were three orders of magnitude more permeable to water than the cuticular membranes of the terrestrial species investigated.Abbreviations CM cuticular membrane - MX cutin matrix - WAX waxes This study was supported by a grant from the Deutsche Forschungsgemeinschaft.     

Synthesis of sulfonamide-containing N-hydroxyindole-2-carboxylates as inhibitors of human lactate dehydrogenase-isoform 5

N-Hydroxyindole-2-carboxylates possessing sulfonamide-substituents at either position 5 or 6 were designed and synthesized. The inhibitory activities of these compounds against isoforms 1 and 5 of human lactate dehydrogenase were analysed, and K_i values of the most efficient inhibitors were determined by standard enzyme kinetic studies. Some of these compounds displayed state-of-the-art inhibitory potencies against isoform 5 (K_i values as low as 5.6 μM) and behaved as competitive inhibitors versus both the substrate and the cofactor.     

Effect of methylation on the side‐chain pKa value of arginine

Arginine methylation is important in biological systems. Recent studies link the deregulation of protein arginine methyltransferases with certain cancers. To assess the impact of methylation on interaction with other biomolecules, the pK_a values of methylated arginine variants were determined using NMR data. The pK_a values of monomethylated, symmetrically dimethylated, and asymmetrically dimethylated arginine are similar to the unmodified arginine (14.2 ± 0.4). Although the pK_a value has not been significantly affected by methylation, consequences of methylation include changes in charge distribution and steric effects, suggesting alternative mechanisms for recognition.     

DNA base composition of extremely halophilic cocci

Summary The percentage guanine+cytosine (GC) content of the DNA of 9 extremely halophilic cocci were determined from melting temperature (T _m) and the E ₂₆₀/E ₂₈₀ ratio at pH 3. The values found ranged from 60.5–65.8, with an average 62.6% GC.