Effect of the three-dimensional organization of liver cells on the biogenesis of the γ-glutamyltransferase fraction pattern

Context Four gamma-glutamyltransferase (GGT) fractions with different molecular weights (big-, medium-, small- and free-GGT) are detectable in human plasma. Objective Verify if liver cells can release all four GGT fractions and if the spatial cell organization influences their release. Methods Hepatoma (HepG2) and melanoma (Me665/2/60) cells were cultured as monolayers or spheroids. GGT released in culture media was analysed by gel-filtration chromatography. Results HepG2 and Me665/2/60 monolayers released the b-GGT fraction, while significative levels of s-GGT and f-GGT were detectable only in media of HepG2-spheroids. Bile acids alone or in combination with papain promoted the conversion of b-GGT in s-GGT or f-GGT, respectively. Conclusions GGT is usually released as b-GGT, while s-GGT and f-GGT are likely to be produced in the liver extracellular environment by the combined action of bile acids and proteases.     

Developmental variations of plasma gamma-glutamyltransferase fractions in humans and in laboratory mammalians

Plasma samples from human cord blood, and fetuses, newborns, and adults of different mammalians species were analyzed by gel-filtration chromatography, to ascertain whether gamma-glutamyltransferase (GGT) fractions reflect liver maturation. Human cord blood plasma showed higher b-, m-, and s-GGT fraction as compared to adult women. In rat and mouse fetuses and in newborns, b-GGT was the most abundant fraction. As in adult humans, in adult rats, mice, rabbits, sheep, and mini pigs, f-GGT was the most abundant fraction. GGT fractions are a common feature of all mammalian species tested. Their pattern changes seem to reflect liver postnatal maturation, function.     

Cultured human cells release soluble γ-glutamyltransferase complexes corresponding to the plasma b-GGT

Serum γ-glutamyltransferase (GGT) is thought to derive from the liver, but its values predict morbidity and mortality for several diseases, such as cardiac infarction, stroke, diabetes, renal failure and cancer. We assessed total GGT and its fractions in the culture supernatants of human cell lines (melanoma, prostate cancer, bronchial epithelium) by gel filtration chromatography. We also compared the GGT elution profile in plasma and the corresponding very-low-density lipoprotein (VLDL) fraction. All the cell lines tested released soluble GGT whose activity increased in parallel with the cell growth. Released GGT presented a molecular weight of 2000?kDa, identical to the b-GGT fraction of human plasma and corresponding to that of VLDL. But ultracentrifugation studies showed that b-GGT had a higher density than VLDL. The b-GGT present in human plasma can be produced by tissues other than the liver, thus explaining the increase of serum GGT observed in diseases of other organs.     

Alcohol, smoking and body build: obesity as a result of the toxic effect of 'social' alcohol consumption

Previous work showed that obesity in the average human male is not due to increased caloric intake. To test the hypothesis that 'social' ethanol consumption causes obesity by a hepatotoxic mechanism, the relationships between alcohol intake, cigarette smoking, serum gamma-glutamyl transpeptidase (GGT) and body build were investigated in 816 adult patients, 491 males and 325 females. A large part of the Broca index variance could be explained by hepatic damage as reflected by the GGT level. The higher the GGT, the more overweight were the subjects. Hyperinsulinemia may be the pathogenetic link; insulin is the strongest known blocker of lipolysis. Almost the total variation of obesity with GGT, however, occurred in the range of GGT up to 25 U/l, which is usually, but nevertheless erroneously, considered to be the normal range. This effect was independent of sex and age. Normal GGT is below 10 U/l, which is found on average in females aged less than 20 years. Females tolerate less alcohol than males. Although GGT is as high in females as in males around age 30, males drink about three times as much ethanol. For the same GGT the Broca index is significantly higher in females than in males. GGT generally increases with age; maximum GGT is reached in females in the age group 21-40 years (due to the change in drinking habits around 1968), declining thereafter; in males at age 50. Obesity per se is not correlated with a high GGT. In the females there are hormonal factors influencing obesity. Although in the females GGT decreases on average after age 40, obesity increases (due to the decrease in estrogens). After age 50 ethanol tolerance in males decreases: they reduce their alcohol consumption, and yet the GGT remains high. -Cigarette smoking is a factor which independently influences obesity. Although people who smoke tend also to drink more alcohol, smokers are significantly leaner than nonsmokers. On average males smoke about twice as heavily as females; this contributes to the fact that on average males are leaner than females despite their higher alcohol consumption. Due to lower consumption the influence of ethanol and smoking on body build is smaller in females than in males.     

Broad substrate Cytochrome P450 monooxygenase activity in the cells of Aspergillus terreus MTCC 6324

Cytochrome P450 (CYP) monooxygenase activities with different category of substrates namely, alkanes, alkane derivatives, alcohols, aromatic compounds, organic solvents, and steroids were detected in the cells of Aspergillus terreus. High CYP specific activity was observed when methanol (5.6+/-0.017 U mg(-1)), acetone (7.76+/-0.02 U mg(-1)), dimethylsulphoxide (DMSO) (9.70+/-0.005 U mg(-1)), n-hexadecane (4.39+/-0.02 U mg(-1)), or n-octadecane (4.23+/-0.01 U mg(-1)) were used as substrates. Significant CYP specific activity was also detected when naphthalene (3.80+/-0.002 U mg(-1)) was used as substrate. The CYP catalysis of n-hexadecane had followed both terminal and sub terminal oxidations. The activity was localized in the cytosol of n-hexadecane grown cells, while, it was apparently distributed in light mitochondrial fraction and microsomal fraction of glucose grown cells. The substrate specificities of CYP present in all the locations were similar irrespective of the substrates used for the growth. Heme staining of the microsomal fraction containing CYP and other proteins in SDS-PAGE showed single heme protein band with corresponding molecular weight of 110 kDa.     

Preferential distribution of long chain polyunsaturated fatty acids in phospatidyl ethanolamine fraction of guinea pig alveolar apical membranes

We investigated the fatty acid distribution in guinea pig alveolar apical membranes at different developmental stages. Fatty acid composition of the purified membranes isolated from guinea pig fetuses (at 65 day, term=68 day), neonates (day 1) and adult males was determined. The levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) were higher in the adult guinea pig alveolar apical membrane phosphatidylethanolamine (PE) fraction (9. 3+/-2.2 and 2.9+/-1.0%, respectively) while in other phospholipids (PL) fractions their levels were low or absent (P<0.01). Furthermore, levels of AA and DHA in the PE fraction of apical membrane increased significantly from fetal (6.6+/-3.0 and 0.8+/-0.4%, respectively) to neonatal life (10.3+/-1.5 and 3.0+/-0.8%, respectively). Increase in the level of DHA (almost four-fold) was much more pronounced than that of AA (P<0.05). As for guinea pig alveolar membranes, EPA and AA were mostly present in the PE fraction in pulmonary adenocarcinoma derived cells (A549 cells), a parallel model of type II pneumocytes, with the levels of AA around three-fold greater than that of EPA, Binding of radiolabelled fatty acids to A549 cells showed no significant differences between the maximum uptake achieved for different fatty acids (AA, 1.7+/-0.2, EPA, 2.3+/-0.3, LA, 1.7+/-0.2, OA, 2.0+/-0.2nmol/mg protein, P>0.5). Once the fatty acids were taken up by these cells AA was mostly identifiable in the monoacylglycerol (MAG) fraction, whereas EPA was equally distributed between the MAG and PL fractions. Oleic acid was mainly present in the triglyceride (TAG) fraction whereas LA was evenly distributed between the TAG, MAG, and PL fractions. Our data demonstrate a preferential distribution of AA and DHA in PE fractions of alveolar apical membranes during development.     

Hydrolysis of cis and trans isomers of retinyl palmitate by retinyl ester hydrolase of pig liver

Relative retinyl ester hydrolase activities of pig liver homogenates (n = 4) toward 9,13-cis-, 13-cis-, 9-cis-, and all-trans-retinyl palmitate were 6.8 +/- 0.5 (SE), 5.7 +/- 0.5, 2.4 +/- 0.1, and 1, respectively. The range of apparent Km values for the four isomers was 142 to 268 microM, and the pH optima were 8-9 in all cases. Peak activities of retinyl ester hydrolase activities in pig liver cytosol toward 13-cis- and all-trans-retinyl palmitate were found in the 20 to 40% and in the 60 to 80% saturated ammonium sulfate (AS) fractions, respectively. By use of size-exclusion chromatography in 2 M KCl, hydrolase activity eluted at volumes corresponding to greater than 2000, 180, and 15 kDa from the 20-40% AS fraction, and at 180 kDa from the 60-80% AS fraction. On the basis of molecular size, different substrate specificities, detergent effects, and susceptibilities to inhibition by phenylmethylsulfonyl fluoride, we conclude that at least three distinct retinyl ester hydrolases are present in pig liver cytosol.     

The antioxidant power and level of lipid peroxidation products in the sera of apparently healthy adult males

The aim of this study was to determine the antioxidant potential of the serum and the level of lipid oxidation products in the sera of apparently healthy adult males. The "antioxidant power" of the serum, defined as the ability to reduce ferric ions by antioxidants from the serum (FRAP), was taken as the indicator of total antioxidation potential. The formation of lipid oxidation products was evaluated as thiobarbituric reactive species serum test (TBARS). The ferrous oxidation in xylenol orange version 2 (FOX2) assay coupled with triphenylphosphine was used for measurement of total sera hydroperoxides (ROOHs). The following biochemical variables were determined in the sera: aspartat aminotranspherase (AST), alanine aminotranspherase (ALT), gamma-glutamyl transpherase (GGT), bilirubin, glucose, creatinine, cholesterol, triglycerides and hemoglobin. Blood sera from apparently healthy subjects (166 adult males) were analyzed. Median age of study participants was 36 years (range 25-50 years). The X +/- SD sera FRAP level of the 166 apparently healthy adult males was 1047 +/- 131 micromol/L (779-1410 range). The X +/- SD level of sera TBARS was 1.2 +/- 0.3 micromol/L of the sera (0.5-2.2 range). Compared with the level of TBARS in the sera, the level of FOX2-ROOH was significantly higher The X +/- SD level of lipid hydroperoxides in the fresh sera, determined as FOX2, was 3.9 +/- 1.5 micromol/L of the sera (1.9-6.9 range). Observation of correlation between FRAP and determined biochemical variables in the sera have confirmed a statistically significant linear correlation between sera FRAP and bilirubin, hemoglobine, glucose, ALT and triglycerides (p < 0.05). The results of sera FRAP, TBARS and FOX2 levels can help in estimating the antioxidant status of humans. Significant correlation between the antioxidant power of blood serum and particular biochemical parameters indicates the complexity of defence mechanisms and various molecules involved in increasing the reduction power of the serum.     

Effects of aging on responses to furosemide

Furosemide causes both diuretic and non-diuretic changes in renal function. We compared responses to intravenous furosemide 0.5 mg.kg-1 in 38 subjects (30 males, 8 females) aged 18 to 30 with those in 14 subjects (9 males, 5 females) aged 50 and over. There were no consistent differences attributable to gender. Older persons showed greater natriuresis (47 percent in males and 26 percent in females) but their increment in plasma renin activity was markedly reduced. The urinary excretion of thromboxane B2 was elevated in older subjects (58 +/- 10 vs. 30 +/- 4 ng/4 h, p less than 0.05 for males; 48 +/- 7 vs. 29 +/- 4 ng/4 hr, p less than 0.05 for females) while that of 6-keto prostaglandin F1 alpha was not different. While differences in the diuretic response to furosemide may be due to pharmacokinetic differences, the non-diuretic response differences may reflect age related changes in renal prostaglandin synthesis.     

Serum gamma-glutamyltransferase within its normal range predicts a chronic elevation of alanine aminotransferase: a four year follow-up study

BACKGROUND: Previous epidemiological and experimental studies support the concept that serum gamma-glutamyltransferase (GGT) activity within its normal range is related to oxidative stress. Since oxidative stress plays a crucial role in the pathogenesis of various liver diseases, serum GGT may predict development of liver damage. METHODS: A total of 6,523 healthy male workers with normal alanine aminotransferase (ALT, <35 U/l) in a steel manufacturing company were followed for four years. Liver damage was defined as a chronic elevation of serum ALT (both 2001 and 2002). RESULTS: After adjusting for age, body mass index, alcohol consumption, cigarette smoking, exercise, and baseline value of ALT, in comparison with the group whose GGT level was <10 U/l, the adjusted relative risks for elevated ALT level among those with GGT levels 10-19, 20-29, 30-39, and over 40 U/l was 1.0, 2.5, 4.7, 7.4, and 12.0, respectively (P for trend <0.01). More importantly, this association was similarly observed even among non-drinkers; the corresponding relative risks were 1.0, 1.8, 3.8, 5.6, and 6.2 (P for trend <0.01). However baseline ALT did not predict abnormal GGT level four years later. CONCLUSION: Serum GGT levels within normal range predict incidence of chronic elevation of ALT. Oxidative stress might explain this relationship.     

Isolation and partial characterisation of immunoglobulin from southern bluefin tuna Thunnus maccoyii Castelnau

Specific and total serum immunoglobulins were extracted by immunoaffinity, mannan-binding protein and Protein A affinity chromatography from southern bluefin tuna (Thunnus maccoyii Castelnau) immunised with rabbit IgG, and from non-immunised southern bluefin tuna. SDS-PAGE in 10% reducing gels revealed two heavy chains with molecular weights of approximately 74.6 +/- 1.3 kDa and 71.2 +/- 0.9 kDa, and two light chains with molecular weights of approximately 29 +/- 1.2 kDa and 28 +/- 1.0 kDa. Under non-reducing, but denaturing, conditions in 4% and 5% SDS-PAGE gels, a high molecular weight and a low molecular weight fraction were demonstrated. By gel filtration using Sephacryl HR 300 a molecular weight of 845 kDa, consistent with a tetramer, was obtained for the high molecular weight fraction, and a molecular weight of 168 kDa, consistent with a monomer, was obtained for the low molecular weight fraction. The extinction coefficient at A280 for the purified immunoglobulin (Ig) was determined to be 1.24. Tuna a-rabbit IgG Ig was reactive with all non-reduced mammalian IgG antigens tested, suggesting that common conformational antigenic determinants were recognised.     

The source of urinary epidermal growth factor in humans

To clarify the source of human urine EGF, we studied EGF renal clearance in 20 healthy, young adult subjects. Immunoreactive EGF was measured hourly in EDTA plasma, heparin plasma, serum and urine of 12 males and 8 females during a 3 h study period. Plasma and urine creatinine and creatinine clearance were measured and calculated hourly. Mean (and SEM) creatinine clearance was similar in males and females (118 +/- 12 vs 105 +/- 6 ml/min). EGF was not detectable in plasma, whereas relatively high levels were measured in serum (2.5 +/- 0.25 vs 1.5 +/- 0.18 ng/ml in males and females respectively p less than 0.05). Urine EGF excretion averaged 1641 +/- 233 ng/h in males and 1507 +/- 191 ng/h in females (p greater than 0.05). A significant correlation was observed between urine creatinine and urine EGF concentrations in both male (r = 0.98, p less than 0.01) and female (r = 0.94, p less than 0.01) subjects. EGF immunoreactivity in urine and serum eluted from G-75 sephadex columns similarly to recombinant 6000 Mr hEGF. Urine excretion of EGF approximated 1.5 micrograms/h or 25 ng/mg creatine. The high concentrations of EGF found in urine in the face of non-detectable levels of EGF in plasma favor the hypothesis that EGF in urine is derived from kidney synthesis and secretion. The significant positive correlation between urine creatinine and urine EGF suggests a functional correlation between glomerular filtration and the process of tubular EGF excretion.     

The impact of maternal serum on development of enolase activity in fetal rat brain cell culture

The effect of gestational age on serum-mediated changes in enolase activity was tested in a fetal rat brain cell culture. After 6 days exposure to graded concentrations (10 and 20%) of nonpregnant female rat sera, enolase activity in brain cell cultures increased from 2.83 +/- 0.03 to 3.74 +/- 0.19 mumol/min/mg protein, P less than 0.01. By contrast, similar concentrations of 20-day maternal serum progressively decreased enzyme activity from 1.52 +/- 0.14 to 1.19 +/- 0.08 mumol/min/mg protein. The inhibitory effect was apparent at 14 days gestation and became progressively greater during late gestation to reach a maximum at 20 days. Combining equal concentrations of 20-day pregnant with either nonpregnant or adult male serum neutralized the inhibitory activity. When serum from 20-day pregnant rats was partitioned by a dialysis membrane with a 50,000 MW pore size, inhibitory activity could be similarly neutralized by male or nonpregnant female serum. When 20-day maternal serum was passed successively through filters with a greater than 300,000, 100,000, and 50,000 MW exclusion, the inhibitory activity was apparent in all fractions excluded by a molecular weight of 50,000. No inhibition was apparent in fractions that were not excluded by 50,000 MW pore size. Inhibition of enolase activity was greatest in the fraction with MW greater than 300,000. Binding of IGF II could also be demonstrated in this fraction. Binding of IGF II was evident in the fraction greater than 100,000 MW but could not be demonstrated in fractions with a lower molecular weight. The presence of mRNA for IGF II in 20-day fetal rat brain cell cultures was evident when total cellular RNA was analyzed by an RNAase protection assay. It is proposed that a high-molecular-weight component of maternal serum in late gestation can bind endogenously generated IGF II. Such binding, by depleting the necessary growth factors, could inhibit in vitro growth and development of enolase activity.     

Facilitated glucose transporter of human erythrocyte: proteolytic mapping of the [3H]cytochalasin B photoaffinity-labeled transporter polypeptide

The human erythrocyte D-glucose transporter is an integral membrane glycoprotein with an heterogeneous molecular mass spanning a range 45-70 kDa. The protein structure of the transporter was investigated by photoaffinity labeling with [3H]cytochalasin B and fractionating the labeled transporter according to molecular mass by preparative SDS-polyacrylamide gel electrophoresis. Each fraction was digested with either papain or S. aureus V8 proteinase, and the labeled proteolytically derived peptide fragments were compared by SDS polyacrylamide gel electrophoresis. Papain digestion yielded two major peptide fragments, of approx. molecular mass 39 +/- 2 and 22 +/- 2 kDa; treatment with V8 proteinase resulted in two fragments, with mass of 24 +/- 2 and 15 +/- 2. Proteolysis of each transporter fraction produced the same pattern of labeled peptide fragments, irrespective of the molecular mass of the original fractions. The binding characteristics of [3H]cytochalasin-B-labeled transporter to Ricinis communis agglutinin lectin was examined for each transporter molecular mass fraction. It was found that higher-molecular-mass fractions of intact transporter had a 2-fold greater affinity for the lectin than lower-molecular-mass fractions (i.e., 67 kDa greater than 45 kDa fraction). However, proteolytically derived labeled peptide fragments from each fraction had minimal affinity for the lectin. These results suggest that the labeled peptide fragments have been separated from the glycosylated regions of the parent transporter protein. The present findings indicate that, although transporter proteins have an apparently heterogeneous molecular mass, some regions of the protein share a common peptide. Furthermore, the glycosylated regions appear to be located some distance from the [3H]cytochalasin-B-labeled site(s).     

Characterization of (Na+, K+)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii

To better understand the adaptive strategies that led to freshwater invasion by hyper-regulating Crustacea, we prepared a microsomal (Na+, K+)-ATPase by differential centrifugation of a gill homogenate from the freshwater shrimp Macrobrachium olfersii. Sucrose gradient centrifugation revealed a light fraction containing most of the (Na+, K+)-ATPase activity, contaminated with other ATPases, and a heavy fraction containing negligible (Na+, K+)-ATPase activity. Western blotting showed that M. olfersii gill contains a single alpha-subunit isoform of about 110 kDa. The (Na+, K+)-ATPase hydrolyzed ATP with Michaelis Menten kinetics with K5, = 165+/-5 microM and Vmax = 686.1+/-24.7 U mg(-1). Stimulation by potassium (K0.5 = 2.4+/-0.1 mM) and magnesium ions (K0.5 = 0.76+/-0.03 mM) also obeyed Michaelis-Menten kinetics, while that by sodium ions (K0.5 = 6.0+/-0.2 mM) exhibited site site interactions (n = 1.6). Ouabain (K0.5 = 61.6+/-2.8 microM) and vanadate (K0.5 = 3.2+/-0.1 microM) inhibited up to 70% of the total ATPase activity, while thapsigargin and ethacrynic acid did not affect activity. The remaining 30% activity was inhibited by oligomycin, sodium azide and bafilomycin A. These data suggest that the (Na+, K+)-ATPase corresponds to about 70% of the total ATPase activity; the remaining 30%, i.e. the ouabain-insensitive ATPase activity, apparently correspond to F0F1- and V-ATPases, but not Ca-stimulated and Na- or K-stimulated ATPases. The data confirm the recent invasion of the freshwater biotope by M. olfersii and suggest that (Na+, K+)-ATPase activity may be regulated by the Na+ concentration of the external medium.     

Immunostimulatory polysaccharides from Chlorella pyrenoidosa. A new galactofuranan. measurement of molecular weight and molecular weight dispersion by DOSY NMR

Fractionation of the hot water extract of Chlorella pyrenoidosa was performed using a combination of ethanol precipitation, size exclusion chromatography, and anion exchange chromatography. One fraction contained a new polysaccharide, and this compound was shown to be a 1-->2-linked beta-d-galactofuranan from its 1D and 2D (1)H and (13)C NMR spectra, with a molecular weight of 15 kDa from DOSY NMR measurements. A number of other fractions were shown to have the same repeating unit as the previously identified arabinogalactan. However, arabinogalactans from different fractions were shown by DOSY NMR to have different molecular weights, which ranged from 27 to 1020 kDa. Agreement with molecular weights measured for some of these fractions by SEC-MALS was very good, further confirming the relationship established by Viel et al. between molecular weights of neutral polysaccharides and self-diffusion coefficients. The smaller molecular weight polysaccharides, the galactofuranan and the 27 and 50 kDa arabinogalactans, were shown to be close to monodisperse by analysis of the distributions of the self-diffusion coefficients for the polymers. The larger arabinogalactans had considerable variation in their molecular weights (188 +/- 109 kDa and 1020 +/- 370 kDa). Only the two larger arabinogalactans showed immunostimulatory activity.     

Chronic resistance training in women potentiates growth hormone in vivo bioactivity: characterization of molecular mass variants

This investigation determined the influence of acute and chronic resistance exercise on responses of growth hormone (GH) molecular variants in women. Seventy-four healthy young women (23 +/- 3 yr, 167 +/- 7 cm, 63.8 +/- 9.3 kg, 26.3 +/- 4.0% body fat) performed an acute bout of resistance exercise (6 sets of 10 repetition maximum squat). Blood samples were obtained pre- and postexercise. Resulting plasma was fractionated by molecular mass (fraction A, >60 kDa; fraction B, 30-60 kDa; and fraction C, <30 kDa) using chromatography. Fractionated and unfractionated (UF) plasma was then assayed for GH using three different detection systems (monoclonal immunoassay, polyclonal immunoassay, and rat tibial line in vivo bioassay). Subjects were then matched and randomly placed into one of four resistance exercise training groups or a control group for 24 wk. All experimental procedures were repeated on completion of the 24-wk resistance training programs. After acute exercise, immunoassays showed consistent increases in UF GH samples and fractions B and C; increases in fraction A using immunoassay were seen only in the monoclonal assay. No consistent changes in bioactive GH were found following acute exercise. Conversely, chronic exercise induced no consistent changes in immunoassayable GH of various molecular masses, whereas, in general, bioassayable GH increased. In summary, although acute exercise increased only immunoactive GH, chronic physical training increased the biological activity of circulating GH molecular variants. Increased bioactive GH was observed across all fractions and training regimens, suggesting that chronic resistance exercise increased a spectrum of GH molecules that may be necessary for the multitude of somatogenic and metabolic actions of GH.     

Effect of calcium on rat intestinal alkaline phosphatase activity and molecular aggregation

Two fractions of rat intestinal alkaline phosphatase (IAP) were detected by Western blot: 168 +/- 6 and 475 +/- 45 kDa. The low molecular weight fraction constitutes 43% of the isolated proteins exhibiting 82% of the enzymatic activity, and a heavier fraction constitutes 57% of the isolated proteins and has 18% of the enzymatic activity. Calcium produced an increase of the 475-kDa form to the detriment of the 168-kDa form. This work also describes the kinetic and structural changes of IAP as a function of calcium concentration. With [Ca2+] < 10 mmole/L, the Ca(2+)-IAP interaction fitted a binding model with 7.8 +/- 4.4 moles of Ca2+ /mole of protein, affinity constant = 19.1 +/- 8.4 L/mmole, and enzymatic activity increased as a linear function of [Ca2+] (r = 0.946 p < 0.01). On the other hand, with [Ca2+] > 10 mmole/L the data did not fit this model and, the enzymatic activity decreased as a function of [Ca2+] (r = - 0.703 p < 0.05).     

Toxicity of hexachlorobenzene in Meriones unguiculatus: effects on thyroid and liver

The effect of in vivo administered hexachlorobenzene (HCB) on liver and thyroid was studied on Meriones unguiculatus. HCB (1.6, 4, and 16 mg/kg of body weight) has been administered orally to meriones for 30 days. At the end of the experiment, the body weight of the animals did not show significant change. However, the higher dose of HCB treatment led to a pronounced hepatic hypertrophy comparatively to controls. Histological observations revealed many cytomorphological alterations. Cellular necrosis, periportal, and centrolobular vein congestion and cytoplasmic vacuolisation were noted and correlated with the administered doses of HCB. The higher dose of HCB induced modifications in the activities of hepatic transaminases and on thyroid hormones levels: ALAT activity level was more pronounced in males (170+/-24.7 U/l vs. 52.66+/-8.29 U/l in controls) than in females (120+/-12.47 U/l vs. 56+/-5 U/l in controls). However, ASAT activity increased significantly only in females (259+/-29 U/l vs. 244.66+/-18 U/l in controls). Plasma total triiodothyronine (TT3) and total thyroxine (TT4) levels seemed to be sex-dependent in intoxicated animals, since TT4 decreased significantly in males (21.95+/-7.46 nmol/l vs. 40.59+/-1.08 nmol/l in controls) and TT3 in females (1.42+/-0.11 nmol/l vs. 3.96+/-0.48 nmol/l in controls).     

Gill (Na+,K+)-ATPase in diadromous, freshwater palaemonid shrimps: species-specific kinetic characteristics and alpha-subunit expression

To better comprehend physiological adaptation to dilute media and the molecular mechanisms underlying ammonia excretion in palaemonid shrimps, we characterized the (Na+,K+)-ATPase from Macrobrachium amazonicum gills, disclosing high- (K(0.5) = 4.2+/-0.2 micromol L(-1); V = 33.9+/-1.9 U mg(-1)) and low-affinity (K(0.5) = 0.144+/-0.010 mmol L(-1); V = 232.9+/-15.3 U mg(-1)) ATP hydrolyzing sites. Stimulation by Na+ (K(0.5) = 5.5+/-0.3 mmol L(-1); V = 275.1+/-15.1 U mg(-1)), Mg2+ (K(0.5) = 0.79+/-0.06 mmol L(-1); V = 261.9+/-18.3 U mg(-1)), K+ (K(M) = 0.88+/-0.04 mmol L(-1); V = 271.8+/-10.9 U mg(-1)) and NH4(+) (K(M) = 5.0+/-0.2 mmol L(-1); V = 385.9+/-15.8 U mg(-1)) obeys single saturation curves, activity being stimulated synergistically by NH4(+) and K+. There is a single K+ binding site, NH4(+) binding to a second, exclusive site, stimulating activity by 33%, modulating K+ affinity. (Na+,K+)-ATPase activity constitutes approximately 80% of total ATPase activity (K(Iouabain) = 147.5+/-8.9 micromol L(-1)); Na+-, K+-, Ca2+-, V- and F(o)F(1)-ATPases are also present. M. amazonicum microsomal fractions possess approximately 2-fold less (Na+,K+)-ATPase alpha-subunit than M. olfersi, consistent with a 2.6-fold lower specific activity. These differences in (Na+, K+)-ATPase stimulation by ATP and ions, and specific activities of other ATPases, suggest the presence of distinct biochemical adaptations to life in fresh water in these related species.