3′-Azido-3′-deoxy-5′-O-isonicotinoylthymidine: A Novel Antiretroviral Analog of Zidovudine. II. Stability in Aqueous Media and Experimental and Theoretical Ionization Constants

Abstract

Degradation of 3′-azido-3′-deoxy-5′-O-isonicotinoylthymidine (AZT-Iso), an antiretroviral derivative of zidovudine, was investigated in buffer pH 7.4, µ = 300 mOsm at 37, 50 and 60°C, and in water (pH 6.6, 37°C), giving zidovudine (AZT) and isonicotinic acid (INA) as products. The rate constants were determined by reversed-phase HPLC showing pseudo-first-order kinetics related to the residual amount of AZT-Iso. In this way, the studied compound was demonstrated to be 153 times more stable in water than in buffer solution at 37°C. The analytical method was conveniently validated demonstrating to be a rapid and accurate stability-indicating technique. In addition, experimental and theoretical values of pKa were determined.     

3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine: a novel antiretroviral analog of zidovudine. III. Solubility studies

The pH-solubility behavior and solubility of 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine (AZT-Iso), an antiretroviral derivative of zidovudine with important biological activity, was studied in water, ethanol, ethanol: water, and n-octanol. The N-pyridine pKa value was determined from its pH-solubility profile, which was in accordance with that of the experimental value of methyl isonicotinate. Also, the ethanol cosolvency in ethanol:water mixtures at 25 degrees C was studied, and log-linear and nonlinear solubilization models were applied to the experimental solubility AZT-Iso data, which allowed us to predict its solubility in those solvent mixtures at a determined content of cosolvent.     

Angiotensin converting enzyme: substrate inhibition

Phosphate, borate, and Tris inhibit angiotensin converting enzyme (ACE), but HEPES buffer is inert. Measurements of substrate inhibition were made in HEPES buffer at pH 7.0 and 25 degrees C and 37 degrees C. Substrate inhibition was marked and goes to completion. A new equation for substrate inhibitions enables one, under favorable circumstances, to determine whether there is cooperativity in the binding of substrate to the inhibitory and active sites. Cooperativity does occur with ACE using Hipp-His-Leu as substrate. The kinetic parameters were measured (Km = 0.21 mM, K* = 0.65 mM at 37 degrees C). The enzyme concentration (1.94 X 10(-8) M) was determined by titration with lisinopril so that kcat (5 X 10(3) at 37 degrees C) could be determined. Using this value and the molecular weight the specific activity of ACE was calculated for different common buffers. The specific activity in HEPES calculated from Vmax was 33.7 units/mg at 37 degrees C.     

Lability in storage of 3H-dopamine and 3H-norepinephrine in crude synaptosome (P2) and vesicle-associated fractions of rat brain

Crude synaptosome (P2) fractions prepared from rat striatum and hypothalamus, preloaded with 3H-dopamine (DA) or 3H-norepinephrine (NE), were incubated at 37 degrees C for 5 min. The addition of reserpine at a concentration of 0.1 microM to the striatal synaptosomes substantially depleted 3H-DA to about 45% of control values, but had no effect on 3H-NE. An analogous difference in sensitivity to reserpine, though less pronounced, was observed between 3H-DA and 3H-NE loaded into hypothalamic synaptosomes. Preloaded synaptosome fractions prepared from striatum and hypothalamus were also lysed under hypoosmotic conditions, filtered, and then washed with 130 mM KH2PO4 buffer, pH 7.4, maintained at 0 degrees or 37 degrees C. Washing with 0 degrees C buffer produced no appreciable change in the amount of 3H-DA or 3H-NE retained by the hypoosmotic-resistant subsynaptosomal fractions. Increasing the temperature of the wash buffer to 37 degrees C, however, elicited a volume-dependent depletion of 3H-DA about 2.5-fold higher than that obtained for 3H-NE. Consistent with this finding, the retention of 3H-DA by a crude vesicle fraction prepared from striatum was found to be significantly less than the retention of 3H-NE following 4.5 and 6 min of incubation at 20 degrees C. Thus, in intact synaptosomes, 3H-DA appears to be stored in a form that is more susceptible than 3H-NE to depletion by reserpine, and this effect may be related to differences between the intravesicular storage stability of DA and NE.     

Simultaneous saccharification and fermentation of Kanlow switchgrass pretreated by hydrothermolysis using Kluyveromyces marxianus IMB4

A thermotolerant yeast strain named Kluyveromyces marxianus IMB4 was used in a simultaneous saccharification and fermentation (SSF) process using Kanlow switchgrass as a feedstock. Switchgrass was pretreated using hydrothermolysis at 200 degrees C for 10 min. After pretreatment, insoluble solids were separated from the liquid prehydrolyzate by filtration and washed with deionized water to remove soluble sugars and inhibitors. Insoluble solids were then hydrolyzed using a commercial cellulase preparation and the released glucose was fermented to ethanol by K. marxianus IMB4 in an SSF process. SSF temperature was 37, 41, or 45 degrees C and pH was 4.8 or 5.5. SSF was conducted for 7 days. Results were compared with a control of Saccharomyces cerevisiae D(5)A at 37 degrees C and pH 4.8. Fermentation by IMB4 at 45 and 41 degrees C ceased after 3 and 4 days, respectively, when a pH 4.8 citrate buffer was used. Fermentation continued for all 7 days using IMB4 at 37 degrees C and the control. When pH 5.5 citrate buffer was used, fermentation ceased after 96 h using IMB4 at 45 degrees C, and ethanol yield was greater than when pH 4.8 citrate buffer was used (78% theoretical). Ethanol yield using IMB4 at 45 degrees C, pH 5.5 was greater than the control after 48, 72, and 96 h (P < 0.05).     

Synthesis and characterisation of a new pH-sensitive amphotericin B--poly(ethylene glycol)-b-poly(L-lysine) conjugate

This paper reports on the synthesis, characterisation, and efficiency of a new intravenous conjugate of amphotericin B (AMB). Twelve molecules of AMB were attached to block copolymer poly(ethylene glycol)-b-poly(L-lysine) via pH-sensitive imine linkages. In vitro drug release studies demonstrated the conjugate (M(w)=26,700) to be relatively stable in human plasma and in phosphate buffer (pH 7.4, 37 degrees C). Controlled release of AMB was observed in acidic phosphate buffer (pH 5.5, 37 degrees C) with the half-life of 2 min. The LD(50) value determined in vivo (mouse) is 45 mg/kg.     

Formation of a phospholipid-linked pyrrolecarbaldehyde from model reactions of D-glucose and 3-deoxyglucosone with phosphatidyl ethanolamine

Phospholipid-linked 'advanced glycation end products' (AGEs) are supposed to play an important role for lipid oxidation in vivo. The identification of the pyrrolecarbaldehyde 1-[2-formyl-5-(hydroxymethyl)-1 H-pyrrol-1-yl]-4,10-dioxo-7-(tetradecanoyloxy)-3,5,9-trioxa- 4lambda5-phosphatricosan-4-olate (7) from model reactions of D-glucose or 3-deoxyglucosone (4, 3-DG) with phosphatidyl ethanolamine (PE) is described. A preparation method is given for 1-(2-hydrox?ethyl)-5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde (8). Independent syntheses as well as unequivocal structural characterization are reported for the substitution products of 8 1-(2-hydroxyethyl)-5-(methoxymethyl)-1H-pyrrole-2-carbaldehyde (9a) and 5-(ethoxymethyl)-1-(2-hydroxyethyl)-1H-pyrrole-2-carbaldehyde (9b). For all these compounds, chromatographic and spectroscopic data were established by GLC-MS and HPLC with diode array detection (DAD). PE and D-glucose or 3-DG 4 were either incubated at pH 7.4, 100 degrees C for 3 h or at pH 7.4, 37 degrees C for 5 weeks in neat buffer or ethanol buffer mixtures. The phospholipid fraction was purified on a C18 solid-phase extraction column and cleaved with ethanolic potassium hydroxide. The carbaldehyde 8, released in this process, was identified bs GLC-MS and quantified by HPLC-DAD. Formation of 7 is favored in the ethanol buffer reactions relative to those in buffer solution only although the amounts determined from the 37 degrees C incubations generally are very low. It seems likely, therefore, that phospholipid-linked pyrrolecarbaldehydes, such as 7, are biomarkers rather than effectors of membrane damage in vivo.     

Absence of Na+,K(+)-ATPase regulation of endosomal acidification in K562 erythroleukemia cells. Analysis via inhibition of transferrin recycling by low temperatures.

Transferrin (Tf) acidification has been shown to be limited to pH 6 in murine Balb/c 3T3 fibroblasts, human A549 epidermoid carcinoma cells, and Chinese hamster ovary cells and is followed by alkalinization during recycling. In contrast, Tf acidification in the human erythroleukemic cell line K562 proceeds to below pH 5.5, and alkalinization of internal Tf during recycling is not observed. To explore the regulation of endosomal pH in K562 cells, we determined whether the existence of an early endosome of pH 6 could be demonstrated in K562 cells. The kinetics of Tf internalization, acidification, and recycling were determined at temperatures which block recycling of Tf in 3T3 cells. As in 3T3, Tf recycling in K562 was inhibited at 24 degrees C and below. At these temperatures, Tf internalization and acidification were delayed relative to 37 degrees C, yet the minimum pH achieved was below 5.5. Temperatures at or below 19 degrees C resulted in a complete block in recycling (at least over 40 min), which was rapidly reversible by incubation at 37 degrees C. Ouabain (a specific inhibitor of the Na+,K(+)-ATPase) had no effect on K562 Tf acidification, indicating that K562 endosomal pH is probably not regulated by the Na+,K(+)-ATPase. The results suggest that differentiation of the early endocytic pathway in erythroid cells involves changes such that the pH of Tf-containing compartments is not limited to 6 by the Na+,K(+)-ATPase.     

Kinetics of pH-dependent fusion between 3T3 fibroblasts expressing influenza hemagglutinin and red blood cells. Measurement by dequenching of fluorescence

Fusion between membranes of 3T3 fibroblasts expressing hemagglutinin (HA) from the Japan strain of influenza virus and human red blood cells (RBC) was measured using an assay for lipid mixing based on the relief of self-quenching (dequenching) of fluorescence of the lipid probe octadecylrhodamine (R18). The probe was incorporated into the membrane of intact RBC at self-quenching concentrations, and the RBCs were bound to the 3T3 cells. Fusion, which allowed movement of R18 into 3T3 cell membranes, was monitored by spectrofluorometry as an increase in fluorescence. Upon lowering the pH below 5.4, the fluorescence increased after a delay of about 30 s at 37 degrees C, and leveled off within 2 min. In control experiments where R18 RBCs bound to 3T3 cells expressing the uncleaved precursor hemagglutinin (HA0) were incubated at 37 degrees C and low pH, no fluorescence increase was observed. This indicated that the R18 dequenching occurred as a result of HA-induced fusion of plasma membranes. Fusion showed a very steep pH dependence with a threshold at pH 5.4 and a maximum at pH 5.0, similar to HA-induced fusion seen previously using cell biological techniques. The fusion rate increased and the delay for the onset of fusion decreased as the temperature was raised above 20 degrees C. Low pH activation of the fusion process at 37 degrees C could be partially arrested by raising the pH after 2-10 s, but not after 15 s, indicating that the irreversible pH-activated conformational change of HA necessary for fusion was complete within about 15 s. Analysis of the data indicates that the pH-induced membrane fusion activity of HA is a highly cooperative event.     

Some molecular properties of rat-liver lysosomal beta-glucuronidase isoenzymes.

The isoenzymes of rat-liver lysosomal beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase (EC 3.2.1.31)) were inactivated at different rates at 0 degrees C in 3M guanidinium chloride solutions adjusted to pH 5.0 In 4 M urea buffered by 0.01 M glycylglycine, pH 7.0 isoenzymes I, III, and V were reversibly inhibited 80%. Sodium dodecyl sulfate (SDS), 0.1% in 0.01 M phosphate buffer, pH 7.0 irreversibly inhibited at 37 degrees C all five isoenzymes. Sedimentation analysis showed that loss of catalytic activity in these denaturing media is accompanied by dissociation into slower sedimenting subunits. SDS gel electrophoresis revealed that the isoenzymes are apparently tetramers made up of different proportions of subunits alpha, beta, and gamma having apparent molecular weights of 62,900, 60,200, and 58,700, respectively. The three subunits appear to be glycoproteins.     

Analysis of Cell Wall Constituents of Biocide-Resistant Isolates from Dental-Unit Water Line Biofilms

In past years, the significance of microbial resistance to biocides has increased. Twenty biocide-resistant bacterial strains were isolated from dental-unit water line biofilm. All strains resisted high biocide concentrations (up to 100 mug ml(-)1): sodium dodecyl sulphate, hydrogen peroxide, sodium hypochlorite, phenol, Tween 20, ethylenediaminetetraacetic acid, chlorohexidine gluconate, and povidine iodine. Among bacteria, biocide sensitivity is based on permeability of biocides through the cell wall. Gram-positive bacteria are more permeable and susceptible to biocides, whereas Gram-negative bacteria have a more complex cell wall and are the least sensitive bacteria. The present study was designed to study the effect of biocides on the cell wall of biocide-resistant bacteria. Peptidoglycan (PG), diaminopimelic acid (DAP), and teichoic acid contents of the cell wall were determined in L-broth and L-broth supplemented with biocides at different temperatures (37 degrees C and 45 degrees C) and pH levels (7 and 9). In general and Gram staining-specific comparison, a significant increase (p < 0.05) in the DAP content of biocide-resistant bacteria was observed at pH 7 and at both temperatures. In tubing-specific comparison, a significant increase in the amount of teichoic acid in air water tubing (37 degrees C at pH 9) and DAP in patient tubing (pH 7 at both temperatures) was observed. In main water pipe, a significant decrease (p > 0.05) in PG content was noticed at 45 degrees C and pH 9. Overall, a significant increase in DAP content may be an important constituent in the manifestation of isolate resistance against various biocides.     

Reversible induction of actin rods in mouse C3H-2K cells by incubation in salt buffers and by treatment with non-ionic detergents

Incubating conditions which induced actin paracrystal-like intracellular structures (actin rods) were investigated by using several cell lines. We have found that an incubation of cells of a mouse fibroblastic cell line, C3H-2K, in an isotonic solution of NaCl containing 1 mM MgCl2, 1 mM CaCl2 and 10 mM MES, pH 6.5, induced disintegration of stress fibers and formation of actin rods in the cytoplasm. Actin rods were induced also by incubating in salt buffers in which Na+ of the above solution was substituted by most cations except K+ or Rb+. When the actin rod-forming cells were transferred back to DMEM containing 10% FBS, actin rods disappeared and stress fibers subsequently re-formed within 1 h at 37 degrees C. Although the induction was observed in NaCl buffer at a wide range of pH values (5.5-10), the optimal pH was 6.5. Formation of actin rods is dependent upon cellular metabolism, as it was inhibited at 4 degrees C, or by metabolic inhibitors. Incubation in NaCl buffer induced actin rods in HeLa, L, NRK, BALB/c 3T3 and Swiss 3T3 cells, but not in CEF or MEF cells. A decrease in cell volume was observed parallel with the induction of actin rods, except for CEF and MEF cells. Alterations in intracellular concentrations of Na, K or Ca were not correlated with the induction, however. Actin rods were also induced in C3H-2K cells by a brief treatment with non-ionic detergents. Tween 80 at concentrations as low as 0.003% was effective for the induction, but did not increase the passive membrane transport of p-nitrophenylphosphate. In contrast to the induction by NaCl buffer, treatment with Tween 80 induced numerous tiny actin rods at 4 degrees C, which became larger when further incubated at 37 degrees C. Double immunofluorescence staining with anti-actin antibody and anti-vinculin antibody showed that vinculin plaques remained at least in an early stage of the actin rod formation. We discuss the mechanism for the induction of actin rods based upon the present findings.     

Estimation of protease activity in soils at low temperatures by casein amendment and with substitution of buffer by demineralized water

The aim of this work was to modify the method of Ladd and Buttler (1972), by substituting Tris-HCl buffer (pH 8.52) with demineralized water (DEMI H(2)O), in order to assess its suitability for measurement of casein-protease activity at pH levels close to those of real soil in H(2)O. Measurements were undertaken over a range of incubation temperatures from 3 to 49 degrees C. Testing was performed on one organic soil and two different mineral soils. The substitution of Tris-HCl buffer by DEMI H(2)O at 49 degrees C decreased casein-protease activity to 67.25% in mineral soil and to 53.76% in organic soil. With decreasing temperature casein-protease activity decreased the most in organic soil, i.e., 0.07% of original its value at 3 degrees C. The incubation period was extended to maximally 336 h at 3 degrees C to totally obtain >10.0% of L-tyrosine equivalents released at optimum or close to optimum temperature and pH conditions. The Q(10) values of casein-protease activity measured after substituting Tris-HCl buffer with DEMI H(2)O were unexpectedly high. Between the temperatures of 3 and 49 degrees C Q(10) ranged from 3.46 to 4.25, whereas between 3 and 25 degrees C Q(10) ranged from 6.78 to 11.08. Therefore, the modified method of Ladd and Buttler (1972) presented can be used for measurement of soil casein-protease activity under pH conditions close to that of real soil pH and at an averaged soil temperatures measured in the field. This modification makes possible an expression of soil casein-protease activity potential - when being combined with measurements of casein-protease activity under optimum or close to optimum temperature and pH conditions, if high concentration of casein is present.     

Inhibition of phosphate transport in human erythrocytes by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl)

Treatment of intact human erythrocytes with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) leads to inhibition of anion transport as measured by [32P]phosphate exchange for intracellular chloride. Inhibition is rapid at 37 degrees C (80% inhibition, 1.7 mM NBD-Cl, 3 min, pH 6.9) and not reversed by washing the cells with 1% bovine serum albumin in isotonic sucrose citrate buffer. Pretreatment of cells with N-ethylmaleimide and p-chloromercuribenzenesulfonic acid enhanced transport inhibition by NBD-Cl. Transport inhibition caused by brief incubations of erythrocytes with NBD-Cl could be almost completely reversed with dithiothreitol or beta-mercaptoethanol. Prolonged incubation (60 min, 37 degrees C, pH 6.4, sucrose-citrate buffer) following NBD-Cl treatment leads to partial reversal of transport inhibition. The residual inhibition is then only partially reversed by dithiothreitol treatment. Reversal of transport inhibition of dithiothreitol or beta-mercaptoethanol may be prevented by incubation of the erythrocytes with sodium dithionite. Phosphate transport was readily inhibited by other tyrosine-directed reagents, tetranitromethane (55% inhibition, 1.6 mM, 3 min, 37 degrees C, pH 8.3 in sucrose-citrate medium) and p-nitrobenzene sulfonyl fluoride (31% inhibition, 1.8 mM, 3 min, 37 degrees C, pH 8.1 in sucrose-citrate medium) but not by N-acetylimidazole (10% inhibition, 37.5 mM, 30 min, 37 degrees C, pH 7.5). These results suggest that NBD-Cl inhibits anion exchange by two mechanisms; a rapid inhibition reversible by sulfhydryl reagents, possibly due to modification of a tyrosine residue(s), and a slower irreversible inhibition due to modification of an essential amino group in the transporter.     

Ca2+-calmodulin binding to caldesmon and the caldesmon-actin-tropomyosin complex. Its role in Ca2+ regulation of the activity of synthetic smooth-muscle thin filaments.

We measured the concentration of calmodulin required to reverse inhibition by caldesmon of actin-activated myosin MgATPase activity, in a model smooth-muscle thin-filament system, reconstituted in vitro from purified vascular smooth-muscle actin, tropomyosin and caldesmon. At 37 degrees C in buffer containing 120 mM-KCl, 4 microM-Ca2+-calmodulin produced a half-maximal reversal of caldesmon inhibition, but more than 300 microM-Ca2+-calmodulin was necessary at 25 degrees C in buffer containing 60 mM-KCl. The binding affinity (K) of caldesmon for Ca2+-calmodulin was measured by a fluorescence-polarization method: K = 2.7 x 10(6) M-1 at 25 degrees C (60 mM-KCl); K = 1.4 x 10(6) M-1 at 37 degrees C in 70 mM-KCl-containing buffer; K = 0.35 x 10(6) M-1 at 37 degrees C in 120 mM-KCl- containing buffer (pH 7.0). At 37 degrees C/120 mM-KCl, but not at 25 degrees C/60 mM-KCl, Ca2+-calmodulin bound to caldesmon bound to actin-tropomyosin (K = 2.9 x 10(6) M-1). Ca2+ regulation in this system does not depend on a simple competition between Ca2+-calmodulin and actin for binding to caldesmon. Under conditions (37 degrees C/120 mM-KCl) where physiologically realistic concentrations of calmodulin can Ca2+-regulate synthetic thin filaments, Ca2+-calmodulin reverses caldesmon inhibition of actomyosin ATPase by forming a non-inhibited complex of Ca2+-calmodulin-caldesmon-(actin-tropomyosin).     

An acidic protease from the grass carp intestine (Ctenopharyngodon idellus)

The acidic Protease was extracted from the intestine of the grass carp (Ctenopharyngodon idellus) by 0.1 M sodium phosphate buffer, pH 7.0 at 4 degrees C after neat intestine was defatted with acetone, and partially purified by ammonium sulfate precipitation, gel filtration chromatography and ionic exchange chromatography. SDS-PAGE electrophoresis showed that the enzyme was homogeneous with a relative molecular mass of 28,500. Substrate-PAGE at pH7.0 showed that the purified acidic protease has only an active component. Specificity and inhibiting assays showed that it should be a cathepsin D. The optimal pH and optimal temperature of the enzyme were pH2.5 and 37 degrees C, respectively. It retained only 20% of its initial activity after incubating at 50 degrees C for 30 min. The enzyme lost 81% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride (PMSF). Its V(max) and K(m) values were determined to be 3.57 mg/mL and 0.75 min(-1), respectively.     

Variability in gas production by Escherichia coli in enrichment media and its relationship to pH.

Variability in gas production in multiple subcultures of Escherichia coli was assessed in two selective enrichment media and in lactose peptone water. Considerable variability occurred with all media at 37 and 44 degrees C. Addition of buffer increased gas production and decreased variability. The relationships between pH, growth, and gas production were complex. In buffered media, viable counts increased by 269 x 10(6) to 382 x 10(6)/U of pH fall, whereas in unbuffered media, they increased by 9.45 x 10(6) to 30.37 x 10(6)/U of pH fall. In buffered and unbuffered media, pH fell as gas production rose. However, variability in gas production among individual subcultures was not associated with changes in pH.     

Influence of temperature on enzyme activity determination in serum : L-aspartate aminotransferase isoenzymes]

The influence of temperature on activity assays of the isoenzymes of L-aspartic aminotransferase in described. For this purpose, isolated human isoenzymes were added to inactivated serum. Half-saturation constants were determined at 17.8 degrees C, 25 degrees C, 30 degrees C, and 37 degrees C, and the substrate saturation and pH curves were recorded. The cytoplasmatic (c) and mitochondrial (m) GOT showed temperature-dependent differences in the half-saturation constants for the substrates L-aspartate and 2-oxoglutarate. For both isoenzymes pH 7.4 is considered the optimum regardless of the temperature of measurement, and Tris-HCl is the optimal buffer. In the Arrhenius plot there is a bent at 27 degrees C for both isoenzymes. Thermal denaturation as a possible reason for this deviation from the linearity in the Arrhenius plot could be ruled out.     

Influence of temperature and pH on the in vitro adherence of Candida albicans.

Yeast adherence to epithelial cells is a very important step in colonization and infection caused by these opportunistic pathogens. This phenomenon may be modified in vitro by many factors. The aim of this work was to find out how variations in pH and temperature modify the in vitro adherence of Candida albicans to epithelial cells. We worked with epithelial buccal cells and a yeast strain according to Gibbons and Van Houte technique with slight modifications. In the first assay, adherence at 28 degrees C and 37 degrees C, and three pH values, 6, 7.2 and 8.4 were simultaneously studied. We did not find significant variations in adherente capacity, but a slight increase was detected at pH 7.2 and 37 degrees C. In the second assay, temperature was fixed at 37 degrees C and four pH values were studied: 3, 4, 5, and 7.2. We find a highly significant difference (p < 0.001) between adherente at pH = 7.2 with respect to the other pH values. According to these results C. albicans adherence to epithelial buccal cells, in vitro, is produced at 37 degrees C and pH 7.2 in optimal conditions.     

Characterization of Adenosine deaminase (ADA) in hemolymph of Biomphalaria glabrata.

Adenosine is an important signaling molecule for many cellular events. Adenosine deaminase (ADA) is a key enzyme for the control of extra- and intra-cellular levels of adenosine. Activity of ADA was detected in hemolymph of B. glabrata and its optimum assay conditions were determined experimentally. The pH variation from 6.2 to 7.8 caused no significant change in ADA activity. Using adenosine as a substrate, the apparent Km at pH 6.8 was 734 micromols.L(-1). Highest activity was found at 37 degrees C. Standard assay conditions were established as being 15 minutes of incubation time, 0.4 microL of pure hemolymph per assay, pH 6.8, and 37 degrees C. This enzyme showed activities of 834 +/- 67 micromol.min(-1).L(-1) (25 degrees C) and 2029 +/- 74 micromol.min(-1).L(-1) (37 degrees C), exceeding those in healthy human serum by 40 and 100 times, respectively. Higher incubation temperature caused a decrease in activity of 20% at 43 degres C or 70% at 50 degrees C for 15 minutes. The ADA lost from 26% to 78% of its activity when hemolymph was pre-incubated at 50 degrees C for 2 or 15 minutes, respectively. Since the ADA from hemolymph presented high levels, it can be concluded that in healthy and fed animals, adenosine is maintained at low concentrations. In addition, the small variation in activity over the 6.2 to 7.8 range of pH suggests that adenosine is maintained at low levels in hemolymph even under adverse conditions, in which the pH is altered.