Mechanism of exocrine pancreatic insufficiency in streptozotocin-induced diabetes mellitus in rat: Effect of cholecystokinin-octapeptide

This study investigated the effects of cholecystokinin-octapeptide (CCK-8) on pancreatic juice flow and its contents, and on cytosolic calcium (Ca²⁺) and magnesium (Mg²⁺) levels in streptozotocin (STZ)-induced diabetic rats compared to healthy age-matched controls. Animals were rendered diabetic by a single injection of STZ (60 mg kg^–1, I.P.). Age-matched control rats obtained an equivalent volume of citrate buffer. Seven weeks later, animals were either anaesthetised (1 g kg^–1 urethane; IP) for the measurement of pancreatic juice flow or humanely killed and the pancreas isolated for the measurements of cytosolic Ca²⁺ and Mg²⁺ levels. Non-fasting blood glucose levels in control and diabetic rats were 92.40 ± 2.42 mg dl^–1 (n= 44) and >500 mg dl^–1 (n= 27), respectively. Resting (basal) pancreatic juice flow in control and diabetic anaesthetised rats was 0.56 ± 0.05 ul min^–1 (n= 10) and 1.28 ± 0.16 ul min^–1 (n= 8). CCK-8 infusion resulted in a significant (p < 0.05) increase in pancreatic juice flow in control animals compared to a much larger increase in diabetic rats. In contrast, CCK-8 evoked significant (p < 0.05) increases in protein output and amylase secretion in control rats compared to much reduced responses in diabetic animals. Basal [Ca²⁺]_i in control and diabetic fura-2-loaded acinar cells was 109.40 ± 15.41 nM (n= 15) and 130.62 ± 17.66 nM (n= 8), respectively. CCK-8 (10^–8M) induced a peak response of 436.55 ± 36.54 nM (n= 15) and 409.31 ± 34.64 nM (n= 8) in control and diabetic cells, respectively. Basal [Mg²⁺]_i in control and diabetic magfura-2-loaded acinar cells was 0.96 ± 0.06 nM (n= 18) and 0.86 ± 0.04 nM (n= 10). In the presence of CCK-8 (10^–8) [Mg²⁺]_i in control and diabetic cells was 0.80 ± 0.05 nM (n= 18) and 0.60 ± 0.02 nM (n= 10), respectively. The results indicate that diabetes-induced pancreatic insufficiency may be associated with derangements in cellular Ca²⁺ and Mg²⁺ homeostasis. (Mol Cell Biochem 261: 83–89, 2004)     

Effect of insulin on exocrine pancreatic secretion in healthy and diabetic anaesthetised rats

This investigation characterised the effects of exogenous insulin on exocrine pancreatic secretion in anaesthetised healthy and diabetic rats. Animals were rendered diabetic by a single injection of streptozotocin (STZ, 60 mg kg(-1) I.P.). Age-matched controls were injected citrate buffer. Rats were tested for hyperglycaemia 4 days after STZ injection and 7-8 weeks later when they were used for the experiments. Following anaesthesia (1 g kg(-1) urethane I.P.), laparotomy was performed and the pancreatic duct cannulated for collection of pure pancreatic juice. Basal pancreatic juice flow rate in diabetic rats was significantly (p < 0.001) increased whereas protein and amylase outputs were significantly (p < 0.001) decreased compared to control rats. Insulin (1 IU, I.P.) produced in healthy rats significant increases in pancreatic flow rate, amylase secretion and protein output compared to basal (p < 0.05). Insulin action also included a reduction in blood glucose (152.7 +/- 16.9 mg dl(-1), n = 6, prior to insulin and 42.0 +/- 8.4 mg dl(-1), n = 4, 100 min later). In fact, flow rate and glycaemia showed a strong negative correlation (p < 0.01, Pearson). Pretreatment with atropine (0.2 mg kg(-1), I.V.) abolished the effects of insulin on secretory parameters despite a similar reduction in glycaemia; in this series of experiments the correlation between flow rate and blood glucose was lost. In diabetic rats, insulin (4 IU, I.P.) did not modify exocrine pancreatic secretion. There was a fall in blood glucose (467.6 +/- 14.0 mg dl(-1), n = 10, prior to insulin and 386.6 +/- 43.6 mg dl(-1), n = 7, 120 min later). Rats, however, did not become hypoglycaemic. Similar results were observed in diabetic atropinized rats. The results of this study indicate that the effects of insulin on exocrine pancreatic secretion in anaesthetised healthy rats are mediated by hypoglycaemia-evoked vagal cholinergic activation.     

Cyclic AMP production and insulin releasing activity of synthetic fragment peptides of glucose-dependent insulinotropic polypeptide

Synthetic fragment peptides of glucose-dependent insulinotropic polypeptide (GIP) were evaluated for their ability to elevate cellular cAMP production and stimulate insulin secretion. In GIP receptor transfected CHL cells, GIP(4–42) and GIP(17–30) dose-dependently inhibited GIP-stimulated cAMP production (40±8%; p<0.01 and 15±6%; p<0.05, respectively), while GIP(1–16) exerted very weak agonist effects on cAMP production. In the clonal pancreatic -cell line, BRIN-BD11, GIP(1–16) demonstrated weak insulin releasing activity compared with native GIP. In contrast, GIP(4–42) and GIP (17–30) weakly antagonized the insulin releasing activity of the native peptide (23±6%; p<0.05 and 11±3%, respectively). These data demonstrate the critical role of the N-terminus and the involvement of regions of the C-terminal domain in generating full biological potency of GIP.     

Identification of a small molecule activator of novel PKCs for promoting glucose-dependent insulin secretion

Using an image-based screen for small molecules that can affect Golgi morphology, we identify a small molecule, Sioc145, which can enlarge the Golgi compartments and promote protein secretion. More importantly, Sioc145 potentiates insulin secretion in a glucose-dependent manner. We show that Sioc145 selectively activates novel protein kinase Cs (nPKCs; δ and ɛ) but not conventional PKCs (cPKCs; α, βI and βII) in INS-1E insulinoma cells. In contrast, PMA, a non-selective activator of cPKCs and nPKCs, promotes insulin secretion independent of glucose concentrations. Furthermore, we demonstrate that Sioc145 and PMA show differential abilities in depolarizing the cell membrane, and suggest that Sioc145 promotes insulin secretion in the amplifying pathway downstream of K_ATP channels. In pancreatic islets, the treatment with Sioc145 enhances the second phase of insulin secretion. Increased insulin granules close to the plasma membrane are observed after Sioc145 treatment. Finally, the administration of Sioc145 to diabetic GK rats increases their serum insulin levels and improves glucose tolerance. Collectively, our studies identify Sioc145 as a novel glucose-dependent insulinotropic compound via selectively activating nPKCs.     

Calcium-supported calpain degradation rates for cardiac myofibrils in diabetes

Objective The purpose was to investigate the calcium required for calpain-mediated degradation of selected cardiac myofibril proteins modified by diabetes, sulfhydryl (SH) and hydrophobic reagents.Methods: After 20 weeks of streptozotocin-induced (55 mg·kg^–1) diabetes, calcium sensitive calpain (1.5 U·ml^–1) degradation rates of purified cardiac myofibrillar proteins (1 mg·ml^–1) were measured,in vitro, and compared to degradation rates for N-ethylmaleimide (NEM) and 2-ptoluidinylnapthalene-6-sulfonate (TNS) treated samples.Results: Diabetes (blood glucose of 550±32 mg·dl^–1) reduced the yield of purified myofibrillar protein with minimal change in fibril protein composition. Total SH group reactivities (nmol·mg^–130min) were 220±21, 163±17 and 156±24 for control, diabetic and NEM-treated (0.5mM) myofibrils (p0.05). Calpain degradation rates were faster for all diabetic and SH modified myofibrillar proteins (p0.05), with a 45 and 35% reduction in the pCa₅₀ for a 37 kDa protein of diabetic and NEM-treated fibril complexes. For control myofibrils, both 100 and 200 uM TNS, reduced calpain degradation rates to a similar extent for all substrate proteins. In contrast, diabetic and NEM-treated samples showed a further reduction in calpain degradation rates with increasing TNS from 100 to 200 divi.Conclusion Our results support the hypothesis that in diabetes the calcium requirements for calpain degradation rates are reduced and dependent upon sulfhydryl group status and Ca²⁺-induced hydrophobic interactions, implicating a 37 kDa myofbillar-complexed protein.     

Preliminary compositional nutrient diagnosis norms for cowpea (Vigna unguiculata (L.) Walp.) grown on desert calcareous soil

This study calculated the compositional nutrient diagnosis (CND) norms of cowpea (Vigna unguiculata (L.) Walp.), as well as identified significant nutrient interactions of this crop growing in an irrigated calcareous desert soil. Three genotypes were distributed in rows in a 2-ha field. The soil showed high heterogeneity in its chemical properties. For statistical analysis, 86 foliar composite samples from healthy plants were used. Preliminary CND norms were developed using a cumulative variance ratio function and the ² distribution function. Means and standard deviations of row-centered log ratios V_X of five nutrients (N, P, K, Ca, and Mg) and a filling value R, which included all nutrients not chemically analyzed. Preliminary CND norms are: V_N^*=0.174±0.095, V_P^*=–2.172±0.234, V_K^*=–0.007±0.267, V_Ca^*=–0.022±0.146, V_Mg^*=–1.710±0.132, and V_R5^*=3.728±0.084. These CND norms are associated with dry bean yields higher than 1.88 t ha^–1, and are associated with the following foliar concentrations: 26.2 g N kg^–1, 2.5 g P kg^–1, 22.9 g K kg^–1, 21.6 g Ca kg^–1, and 4 g Mg kg^–1. Cowpea plants growing in desert calcareous soils took up lower amounts of N, P, and K than those considered as optimum in a previous report. Six interactions were strongly indicated for cowpea through principal component analyses: positive for Ca–Mg, and negative for N–Ca, N–Mg, Ca–P, Mg–P, and K–P. Furthermore, two interactions were identified using simple correlations, negative N–P and positive K–Ca.     

Adaptation of renal function to hypotonic medium in the winter flounder (Pseudopleuronectes americanus)

Summary The kidneys of winter flounders transferred to hypotonic medium were investigated for glomerular and tubular handling of fluid and electrolytes and for the urinary excretion of proteins. Media were sea water (925 mosm·kg^–1) and brackish water (70 mosm·kg^–1).In sea water, the urine was hypertonic to the plasma in 7 fish of this study. Urine flow rate was correlated with the GFR. After adaptation to brackish water a delay of 1 to 3 days was observed until the kidneys switched from fluid retention to the excretion of large amounts of dilute urine. GFR and urine flow rate were increased from 0.61±0.08 to 1.58±0.29 ml·h^–1·kg^–1 and from 0.14±0.02 to 0.68±0.08 ml·h^–1·kg^–1, respectively . With increased filtered load the tubular reabsorption of fluid decreased from 74±2.4% to 45±11.2%. The excretion rates of sodium and potassium were increased due to decreased fractional sodium and potassium reabsorption. The urinary excretion of divalent cations, however, was reduced because the net tubular reabsorption of calcium was increased and the net secretion of magnesium was diminished.Both the urinary total protein concentration and the protein pattern showed no significant change, but the rate of protein excretion was increased from 0.21±0.04 to 0.60±0.05 mg·h^–1·kg^–1. The comparison of protein patterns obtained from urine and serum samples revealed that high molecular weight (HMW) proteins prevail in the serum whereas low molecular weight (LMW) proteins dominate in the urine. The diminished quantity of the HMW-protein fraction in the urine thus may reflect size selectivity of the glomerular filtration barrier for serum proteins also in the winter flounder.Abbreviations BW brackish water - SW sea water - GFR glomerular filtration rate - HMW heigh molecular weight - LMW low molecular weight     

Diversity of K+ channels in the basolateral membrane of restingnecturus oxyntic cells

Summary Patch-clamp techniques have been applied to characterize the channels in the basolateral membrane of resting (cimetidine-treated, nonacid secreting) oxyntic cells isolated from the gastric mucosa ofNecturus maculosa. In cell-attached patches with pipette solution containing 100mm KCl, four major classes of K⁺ channels can be distinguished on the basis of their kinetic behavior and conductance: (1) 40% of the patches contained either voltage-independent (a) or hyperpolarization-activated (b), inward-rectifying channels with short mean open times (16 msec fora, and 8 msec forb). Some channels showed subconductance levels. The maximal inward conductanceg _max was 31±5 pS (n=13) and the reversal potentialE _rev was atV _p=–34±6 mV (n=9). (2) 10% of the patches contained depolarization-activated and inward-rectifying channels withg _max=40 ±18 pS (n=3) andE _rev was atV _p=–31±5 mV (n=3). With hyperpolarization, the channels open in bursts with rapid flickerings within bursts. Addition of carbachol (1mm) to the bath solution in cell-attached patches increased the open probabilityP _o of these channels. (3) 10% of the patches contained voltage-independent inward-rectifying channels withg _max=21±3 pS (n=4) andE _rev was atV _p=–24±9 mV (n=4). These channels exhibited very high open probability (P _o=0.9) and long mean open time (1.6 sec) at the resting potential. (4) 20% of the patches contained voltage-independent channels with limiting inward conductance of 26±2 pS (n=3) andE _rev atV _p=–33±3 mV (n=3). The channels opened in bursts consisting of sequential activation of multiple channels with very brief mean open times (10 msec). In addition, channels with conductances less than 6 pS were observed in 20% of the patches. In all nine experiments with K⁺ in the pipette solution replaced by Na⁺, unitary currents were outward, and inward currents were observed only for large hyperpolarizing potentials. This indicates that the channels are more selective for K⁺ over Na⁺ and Cl^–. A variety of K⁺ channels contributes to the basolateral K⁺ conductance of resting oxyntic cells.     

Momordica charantia fruit juice stimulates glucose and amino acid uptakes in L6 myotubes

The fruit of Momordica charantia (family: Cucurbitacea) is used widely as a hypoglycaemic agent to treat diabetes mellitus (DM). The mechanism of the hypoglycaemic action of M. charantia in vitro is not fully understood. This study investigated the effect of M. charantia juice on either ³H-2-deoxyglucose or N-methyl-amino-a-isobutyric acid (¹⁴C-Me-AIB) uptake in L6 rat muscle cells cultured to the myotube stage. The fresh juice was centrifuged at 5000 rpm and the supernatant lyophilised. L6 myotubes were incubated with either insulin (100 nM), different concentrations (1–10 g ml^–1) of the juice or its chloroform extract or wortmannin (100 nM) over a period of 1–6 h. The results were expressed as pmol min^–1 (mg cell protein)^–1, n= 6–8 for each value. Basal ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes by L6 myotubes after 1 h of incubation were (means ± S.E.M.) 32.14 ± 1.34 and 13.48 ± 1.86 pmol min^–1 (mg cell protein)^–1, respectively. Incubation of L6 myotubes with 100 nM insulin for 1 h resulted in significant (ANOVA, p < 0.05) increases in ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes. Typically, ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes in the presence of insulin were 58.57 ± 4.49 and 29.52 ± 3.41 pmol min^–1 (mg cell protein^–1), respectively. Incubation of L6 myotubes with three different concentrations (1, 5 and 10 g ml^–1) of either the lyophilised juice or its chloroform extract resulted in time-dependent increases in ³H-deoxy-D-glucose and ¹⁴C-Me-AIB uptakes, with maximal uptakes occurring at a concentration of 5 g ml^–1. Incubation of either insulin or the juice in the presence of wortmannin (a phosphatidylinositol 3-kinase inhibitor) resulted in a marked inhibition of ³H-deoxyglucose by L6 myotubes compared to the uptake obtained with either insulin or the juice alone. The results indicate that M. charantia fruit juice acts like insulin to exert its hypoglycaemic effect and moreover, it can stimulate amino acid uptake into skeletal muscle cells just like insulin. (Mol Cell Biochem 261: 99–104, 2004)     

Effect of Concomitant Administration of L-Glutamine and Cycloart-23-ene-3β, 25-diol (B2) with Sitagliptin in GLP-1 (7–36) Amide Secretion,Biochemical and Oxidative Stress in Streptozotocin - Nicotinamide Induced Diabetic Sprague Dawley Rats

Previously we have reported that, cycloart-23-ene-3β, 25-diol (called as B2) and L-glutamine stimulated glucagon like peptide-1 (GLP-1) (7–36) amide secretion diabetic rats. The objective of present investigation was to investigate the concomitant administration of cycloart-23-ene-3β, 25-diol+sitagliptin and L-glutamine+sitagliptin in streptozotocin - nicotinamide induced diabetic Sprague Dawley. Type 2 diabetes was induced in overnight fasted male Sprague Dawley rats pre-treated with nicotinamide (100 mg/kg, i.p.) followed by administration of streptozotocin (55 mg/kg, i.p.) 20 min after. The rats were divided into; I- non-diabetic, II- diabetic control, III- Sitagliptin (5 mg/kg, p.o.)+cycloart-23-ene-3β, 25-diol (1 mg/kg, p.o.), IV- Sitagliptin (5 mg/kg, p.o.)+L-glutamine (1000 mg/kg, p.o.). The concomitant treatment of cycloart-23-ene-3β, 25-diol and L-glutamine with sitagliptin was 8 weeks. Plasma glucose, body weight, food and water intake were determined every week. Glycosylated haemoglobin, lipid profile, plasma and colonic active (GLP-1) (7–36) amide, plasma and pancreatic insulin, histology of pancreata and biomarkers of oxidative stress were measured after 8^th week treatment. Concomitant administration of cycloart-23-ene-3β, 25-diol and L-glutamine with sitagliptin significantly (p<0.001) reduced plasma glucose, glyoxylated haemoglobin, lipid profile and oxidative stress parameters compared to diabetic control groups. Both concomitant treatment increased plasma and pancreatic insulin as well as plasma and colonic active (GLP-1) (7–36) amide secretion. Histological analysis by Gomori staining observed less destruction of pancreatic β cells. The result obtained from this study; it is concluded that concomitant administration of cycloart-23-ene-3β, 25-diol+sitagliptin and L-glutamine+sitagliptin showed additive antihyperglycaemic effect in diabetic rats.     

Freshwater to seawater acclimation of juvenile bull sharks (<Emphasis Type="Italic">Carcharhinus leucas</Emphasis>): plasma osmolytes and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase activity in gill,rectal gland,kidney and intestine

This study examined the osmoregulatory status of the euryhaline elasmobranch Carcharhinus leucas acclimated to freshwater (FW) and seawater (SW). Juvenile C. leucas captured in FW (3 mOsm l^–1 kg^–1) were acclimated to SW (980–1,000 mOsm l^–1 kg^–1) over 16 days. A FW group was maintained in captivity over a similar time period. In FW, bull sharks were hyper-osmotic regulators, having a plasma osmolarity of 595 mOsm l^–1 kg^–1. In SW, bull sharks had significantly higher plasma osmolarities (940 mOsm l^–1 kg^–1) than FW-acclimated animals and were slightly hypo-osmotic to the environment. Plasma Na⁺, Cl^–, K⁺, Mg²⁺, Ca²⁺, urea and trimethylamine oxide (TMAO) concentrations were all significantly higher in bull sharks acclimated to SW, with urea and TMAO showing the greatest increase. Gill, rectal gland, kidney and intestinal tissue were taken from animals acclimated to FW and SW and analysed for maximal Na⁺/K⁺-ATPase activity. Na⁺/K⁺-ATPase activity in the gills and intestine was less than 1 mmol Pi mg^–1 protein h^–1 and there was no difference in activity between FW- and SW-acclimated animals. In contrast Na⁺/K⁺-ATPase activity in the rectal gland and kidney were significantly higher than gill and intestine and showed significant differences between the FW- and SW-acclimated groups. In FW and SW, rectal gland Na⁺/K⁺-ATPase activity was 5.6±0.8 and 9.2±0.6 mmol Pi mg^–1 protein h^–1, respectively. Na⁺/K⁺-ATPase activity in the kidney of FW and SW acclimated animals was 8.4±1.1 and 3.3±1.1 Pi mg^–1 protein h^–1, respectively. Thus juvenile bull sharks have the osmoregulatory plasticity to acclimate to SW; their preference for the upper reaches of rivers where salinity is low is therefore likely to be for predator avoidance and/or increased food abundance rather than because of a physiological constraint.     

Hormonal effects on fatty acid binding and physical properties of rat liver plasma membranes

Summary The fluorescent fatty acids,trans-parimaric andcis-parinaric acid, were used as analogs of saturated and unsaturated fatty acids in order to evaluate binding of fatty acids to liver plasma membranes isolated from normal fed rats. Insulin (10^–8 to 10^–6 m) decreasedtrans-parinaric acid binding 7 to 26% whilecis-parinaric acid binding was unaffected. Glucagon (10^–6 m) increasedtrans-parinaric acid binding 44%. The fluorescence polarization oftrans-parinarate,cis-parinarate and 1,6-diphenyl-1,3,5-hexatriene was used to investigate effects of triiodothyronine, insulin and glucagon on the structure of liver plasma membranes from normal fed rats or from rats treated with triiodothyronine or propylthiouracil. The fluorescence polarization oftrans-parinarate,cis-parinarate, and 1,6-diphenyl-1,3,5-hexatriene was 0.300±0.004, 0.251±0.003, and 0.302±0.003, respectively, in liver plasma membranes from control rats and 0.316±0.003, 0.276±0.003 and 0.316±0.003, respectively, in liver plasma membranes from hyperthyroid rats (p<0.025,n=5). Propylthiouracil treatment did not significantly alter the fluorescence polarization of these probe molecules in the liver plasma membranes. Thus, liver plasma membranes from hyperthyroid animals appear to be more rigid than those of control animals. The effects of triiodothyronine, insulin and glucagon addedin vitro to isolated liver plasma membrane preparations were also evaluated as follows: insulin (10^–10 m) and triiodothyronine (10^–10 m) increased fluorescence polarization oftrans-parinaric acid,cis-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene in liver plasma membranes while glucagon (10^–10 m) had no effects. These hormonal effects on probe fluorescence polarization in liver plasma membranes were abolished by pretreatment of the rats for 7 days with triiodothyronine. Administration of triiodothyronine (10^–10 m)in vitro increased the fluorescence polarization of trans-parinaric acid in liver plasma membranes from propylthiouracil-treated rats. Thus, hyperthyroidism appeared to abolish thein vitro increase in polarization of probe molecules in the liver plasma membranes. Temperature dependencies in Arrhenius plots of absorption-corrected fluorescence and fluorescence polarization oftrans-parinaric acid,cis-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene were noted near 25°C in liver plasma membranes from triiodothyronine-treated rats and near 18°C in liver plasma membranes from propylthiouracil-treated rats. In summary, hormones such as triiodothyronine, insulin and glucagon may at least in part exert their biological effects on metabolism by altering the structure of the liver plasma membranes.     

Presence of galanin in human pancreatic nerves and inhibition of insulin secretion from isolated human islets

Summary In several animal species, galanin occurs in pancreatic nerves and inhibits insulin secretion. However, the presence and action of galanin in the human pancreas have not been established. Therefore, we examined the presence and nature of human pancreatic galanin-like immunoreactive material (GLIR) and the effects of galanin on glucose-stimulated insulin secretion from isolated human islets. Immunofluorescent staining of human pancreas revealed GLIR in fine varicose fibers in both islets and exocrine parenchyma. Furthermore, acid extracts of pancreas (n=3) and isolated islets (n=3) contained 0.17±0.06 and 0.23±0.11 pmol GLIR/mg protein. Human pancreatic GLIR coeluted with synthetic porcine galanin from Sephadex G-50. Moreover, synthetic porcine galanin inhibited glucose-stimulated insulin secretion from collagenase-isolated human islets at dose rates >10^-8 M. Thus, (1) human pancreas is innervated by galanin-containing nerves, (2) human pancreatic GLIR is of similar size as synthetic porcine galanin, and (3) porcine galanin inhibits glucose-stimulated insulin secretion from human islets. Therefore, galanin could be an important local regulator of insulin secretion in man.     

Energy requirements of the red kangaroo (<Emphasis Type="Italic">Macropus rufus</Emphasis>): impacts of age,growth and body size in a large desert-dwelling herbivore.

Generally, young growing mammals have resting metabolic rates (RMRs) that are proportionally greater than those of adult animals. This is seen in the red kangaroo (Macropus rufus), a large (>20 kg) herbivorous marsupial common to arid and semi-arid inland Australia. Juvenile red kangaroos have RMRs 1.5–1.6 times those expected for adult marsupials of an equivalent body mass. When fed high-quality chopped lucerne hay, young-at-foot (YAF) kangaroos, which have permanently left the mother's pouch but are still sucking, and recently weaned red kangaroos had digestible energy intakes of 641±27 kJ kg^–0.75 day^–1 and 677±26 kJ kg^–0.75 day^–1, respectively, significantly higher than the 385±37 kJ kg^–0.75 day^–1 ingested by mature, non-lactating females. However, YAF and weaned red kangaroos had maintenance energy requirements (MERs) that were not significantly higher than those of mature, non-lactating females, the values ranging between 384 kJ kg^–0.75 day^–1 and 390 kJ kg^–0.75 day^–1 digestible energy. Importantly, the MER of mature female red kangaroos was 84% of that previously reported for similarly sized, but still growing, male red kangaroos. Growth was the main factor affecting the proportionally higher energy requirements of the juvenile red kangaroos relative to non-reproductive mature females. On a good quality diet, juvenile red kangaroos from permanent pouch exit until shortly after weaning (ca. 220–400 days) had average growth rates of 55 g body mass day^–1. At this level of growth, juveniles had total daily digestible energy requirements (i.e. MER plus growth energy requirements) that were 1.7–1.8 times the MER of mature, non-reproductive females. Our data suggest that the proportionally higher RMR of juvenile red kangaroos is largely explained by the additional energy needed for growth. Energy contents of the tissue gained by the YAF and weaned red kangaroos during growth were estimated to be 5.3 kJ g^–1, within the range found for most young growing mammals.Abbreviations BMR basal metabolic rate - DEI digestible energy intake - MER maintenance energy requirement - MER_g maintenance plus growth energy requirement - PPE permanent pouch exit - RMR resting metabolic rate - YAF young-at-foot Communicated by I.D. Hume     

Chloride transport in apical membrane vesicles from bovine tracheal epithelium: Characterization using a fluorescent indicator

Summary Cl transport in apical membrane vesicles derived from bovine tracheal epithelial cells was studied using the Cl-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl) quinolinium. With an inwardly directed 50 mM Cl gradient at 23°C, the initial rate of Cl entry (J _Cl) was increased significantly from 0.32±0.12 nmol · sec^–1 · mg protein^–1 (mean±sem) to 0.50±0.07 nmol · sec^–1 · mg protein^–1 when membrane potential was changed from 0 to +60 mV with K/valinomycin. At 37°C, with membrane potential clamped at 0 mV, there was a 34±7% (n=5) decrease inJ _Cl from a control value of 0.37±0.03 nmol · sec^–1 · mg protein^–1 upon addition of 0.2mm diphenylamine-2-carboxylate. The following did not alterJ _Cl significantly (J _Cl values gives as percent change from control): 50mm cis Na (–1±5%), 0.1mm furosemide (–3±4%), 0.1mm furosemide in the presence of 50mm cis Na (–5±2%), 0.1mm H₂DIDS (–18±9%), a 1.5 pH unit inwardly directed H gradient (–7±7%), and 0.1mm H₂DIDS in the presence of a 1.5 unit pH gradient (4±18%). With inward 50mm anion gradients, the initial rates of Br and I entry (J _Br andJ ₁, respectively) were not significantly different fromJ _Cl.J _Cl was a saturable function of Cl concentration with apparentK _d of 24mm and apparentV _max of 0.54 nmol · sec^–1 · mg protein^–1. Measurement of the temperature dependence ofJ _Cl yielded an activation energy of 5.0 kcal/mol (16–37°C). These results demonstrate that Cl transport in tracheal apical membrane vesicles is voltage-dependent and inhibited by diphenylamine-2-carboxylate. There is no significant contribution from the Na/K/2Cl, Na/Cl, or Cl/OH(H) transporters. The conductive pathway does not discriminate between Cl, Br, and I and is saturable. The low activation energy supports a pore-type mechanism for the conductance.     

Comparison of soil nitrogen mineralization and nitrification in a mixed grassland and forested ecosystem in central Taiwan

We used the buried bag incubation method to study temporal patterns of net N mineralization and net nitrification in soils at Ta-Ta-Chia forest in central Taiwan. The site included a grassland zone, (dominant vegetation consists of Yushania niitakayamensis and Miscanthus transmorrisonensis Hayata) and a forest zone (Tsuga chinensis var. formosana and Yushania niitakamensis). In the grassland, soil concentration NH₄ ⁺ in the organic horizon (0.1–0.2 m) ranged from 1.0 to 12.4 mg N kg^–1 soil and that of NO₃ ^– varied from 0.2 to 2.1 mg N kg^–1 soil. In the forest zone, NH₄ ⁺ concentration was between 2.8 and 25.0 mg N kg^–1 soil and NO₃ ^–varied from 0.2 to 1.3 mg N kg^–1 soil. There were lower soil NH₄ ⁺ concentrations during the summer than other seasons. Net N mineralization was higher during the summer while net nitrification rates did not show a distinct seasonal pattern. In the grassland, net N mineralization and net nitrification rates were between –0.1 and 0.24 and from –0.04 to 0.04 mg N kg^–1 soil day^–1, respectively. In the forest zone, net N mineralization rates were between –0.03 and 0.45 mg N kg^–1 soil day^–1 and net nitrification rates were between –0.01 and 0.03 mg N kg^–1 soil day^–1. These differences likely result from differing vegetation communities (C₃ versus C₄ plant type) and soil characteristics.     

Organochlorine and organophosphorus pesticide concentrations in water,sediment, and selected organisms in Lake Naivasha (Kenya)

Water, sediment, red swamp crayfish (Procambarus clarkii) and black bass (Micropterus salmoides) from Lake Naivasha were analyzed for selected organochlorine and organophosphorus pesticide residues. The mean p,p'-DDT, o,p'-DDT and p,p'-DDE residue levels recorded in black bass (28.3 (± 30.0), 34.2 (±54.0) and 16.1 (±16.1) g kg^–1, respectively) and crayfish (4.6 (±5.1), 3.2 (±2.8), and 1.4 (±1.1) g kg^–1, respectively), were higher than previously recorded. This indicated recent usage of technical DDT in the lake's catchment. Levels of p,p'-DDT, higher than those of p,p'-DDE further emphasized this. Mean lindane, dieldrin, -endosulfan and aldrin concentrations in black bass were 100.5, 34.6, 21.6 and 16.7 g kg^–1, respectively. The same residues were detected at lower concentrations in crayfish at 2.0, 2.0, 2.0 and 1.9 g kg^–1, respectively. The higher fat content (3.7 ± 2.7% SD) in black bass (compared to 0.6 ± 0.3% in crayfish) accounted for the significantly higher residue concentrations in black bass. Organophosphate pesticides were the most commonly used pesticides in the lake's catchment, but none was detected in any of the samples. The results indicate that there is need for further work to identify sources and fate of pesticide contaminants, as well as to improve monitoring of pesticide use throughout the catchment.     

Mercury in the marine food chain in the Southern Ocean at Macquarie Island: an analysis of a top predator,Patagonian toothfish (<Emphasis Type="Italic">Dissostichus eleginoides</Emphasis>) and a mid-trophic species,the warty squid (<Emphasis Type="Italic">Moroteuthis ingens</Emphasis>)

The characteristics and habitat of the Patagonian toothfish (Dissostichus eleginoides) are typical of fish that accumulate high concentrations of mercury. In this study, mercury determinations were made on samples of muscle tissue from Macquarie Island toothfish and the Southern Ocean deepwater warty squid (Moroteuthis ingens). The analysis of mercury in the biological tissues was made by cold vapour-atomic absorption spectrometry following acid digestion. Performance of the analytical procedure was assessed by analysis of certified reference material (DORM-2, dogfish muscle). Mercury concentrations of 16 Macquarie Island toothfish ranged from 0.12 mg kg^–1 (550 g, 381 mm TL) to 0.59 mg kg^–1 (6,100 g, 823 mm TL), with a mean concentration of 0.33±0.12 mg kg^–1. A significant correlation was found between mercury and either toothfish weight or total length. The fish analysed were juveniles, which suggests that larger individuals would have higher mercury concentrations well exceeding food standard code limits for mercury in fish (typically 0.5 mg kg^–1). Warty squid, also from around Macquarie Island, had a low mean mercury concentration of 0.086 mg kg^–1 in mantle tissue; no significant correlation existed between mercury concentration and either squid mantle length or total weight. It is postulated that the squid have a mechanism, possibly involving the digestive gland, that prevents bioaccumulation of mercury in the mantle, and presumably other body tissues.     

Vegetative approach for improving the quality of water produced from soils in the westside of central California

Water reuse is a proposed strategy for utilizing or disposing of poor quality drainage water produced in the westside of central California. This 2-year field study evaluated the ability of two potential forage species to tolerate irrigation with water high in salinity, boron (B), and selenium (Se). The species used were: Sporobulus airoides var. salado (alkali sacaton) and Medicago sativa var. salado (alfalfa). After first year establishment with good quality water (<1 dS m^–1), the two species were furrow-irrigated with drainage effluent that had an average composition of sulfate-dominated salinity ((electrical conductivity (EC) of 6.2 dS m^–1)) B (5 mg l^–1), and Se (0.245 mg l^–1). Both crops were clipped monthly from June to October of each year. Total dry matter yields averaged between 11 and 12 mg ha^–1 for both crops irrigated with effluent for two growing seasons. Plant concentrations of Se ranged from a low of 1.3 mg kg^–1 in alkali sacaton to a high of 2.5 mg kg^–1 in alfalfa, while B concentrations ranged from a low of 60 mg kg^–1 in alkali sacaton to a high of 170 mg kg^–1 in alfalfa. Chemical composition of the soil changed as follows from preplant to post-irrigation after two seasons with drainage effluent: EC from 2.78 to 6.5 dS m^–1, extractable B from 1.9 to 5.6 mg l^–1, and no change in extractable Se at 0.012 mg l^–1 between 0 and 45 cm. Between 45 and 90 cm, EC values increased from 4.95 to 6.79 dS m^–1, extractable B from 2.5 to 4.8 mg l^–1, and no change in extractable Se at 0.016 mg l^–1. Increased salinity and extractable B levels in the soil indicate that management of soil salinity and B will be necessary over time to sustain long term reuse with poor quality water.     

Culture and Function of Electrofusion-Derived Clonal Insulin-Secreting Cells Immobilized on Solid and Macroporous Microcarrier Beads

In view of the advantages of the bulk production of clonal pancreaticbeta cells, an investigation was made of the growth and insulin secretoryfunctions of an electrofusion-derived cell line (BRIN-BD11) immobilizedon a solid microcarrier, cytodex-1 or a macroporous microcarrier,cultispher-G. For comparison, similar tests were performed usingBRIN-BD11 cells present in single cell suspensions or allowed toform pseudoislets. Similar growth profiles were recorded for eachmicrocarrier with densities of 4.4×10⁵±0.3 cells/ml and4.2×10⁵±0.2 cells/ml achieved using cytodex-1 andcultispher-G, respectively. Cell viability began to decline on day 5 ofculture. Insulin concentration in the culture medium reached a peak of26±2.0 ng/ml and 24±2.2 ng/ml for cells grown oncytodex-1 and cultispher-G, respectively. Cells grown on both types ofmicrocarrier showed a significant 1.5–1.8-fold acuteinsulin-secretory response to 16.7 mmol/l glucose. L-alanine (10 mmol/l) andL-arginine (10 mmol/l) also induced significant 3–4 fold increasesof insulin release. BRIN-BD11 cells immobilized on cytodex-1 or cultispher-Gout-performed single cell suspensions and pseudoislets in terms ofinsulin-secretory responses to glucose and amino acids. A 1.3-fold,2.2-fold and 1.7-fold stimulation of insulin secretion was observed forglucose, L-alanine and L-arginine respectively in single cellsuspensions. Corresponding increases for pseudoislets were1.6–1.8-fold for L-alanine and L-arginine, with no significantresponse to glucose alone. These data indicate the utility ofmicro-carriers for the production of functioning clonal beta cells.