Co-occurrence of swine cysticercosis due to Taenia solium and Taenia hydatigena in ethnic minority villages at the Thai-Myanmar border.

As part of the international joint projects working towards the control of taeniosis/cysticercosis in Asia Pacific, epidemiological studies on Taenia solium cysticercosis have been carried out in high-incidence populations, such as minority groups in Thailand. To assess the epidemiology of cysticercotic infections in pigs in the hill-tribe minority villages (Karen) in Tak province, Thailand, we conducted serological screening and necropsies. The patterns of antibody response to T. solium antigens were then investigated using immunoblot assays. Of the 188 pig serum samples tested for antibody responses to partially purified low-molecular-weight antigens of T. solium cyst fluid, positive responses were detected in 37 samples (19.7%). Based on these results, 16 pigs (10 seropositive and 6 seronegative) were necropsied for investigation of cysticerci and intestinal parasites. All seropositive pigs were coinfected with both T. solium and Taenia hydatigena cysticerci, except one, which was infected with T. hydatigena alone. Three of the six seronegative pigs were confirmed to be infected with T. hydatigena. Pigs infected with T. solium showed much stronger antibody responses than those infected with T. hydatigena. Our results demonstrate the co-occurrence of two swine cysticercoses due to T. solium and T. hydatigena in the studied areas. This study also reveals the importance of direct confirmation of the presence of cysticerci by necropsy after serological screening. In addition to the prevalence of swine cysticercosis in these endemic areas, our findings also reveal potential implications for the development of serological diagnostic assays for swine cysticercosis.

Artificial sweeteners and mixture of food additives cause to break oral tolerance and induce food allergy in murine oral tolerance model for food allergy.

BACKGROUND: Processed foods are part of daily life. Almost all processed foods contain food additives such as sweeteners, preservatives and colourants. From childhood, it is difficult to avoid consuming food additives. It is thought that oral tolerance for food antigens is acquired during early life. If tolerance fails, adverse immune responses to food proteins may occur. OBJECTIVE: We hypothesized that food additives prevent acquisition of oral tolerance and aimed to verify the safety of food additives. METHODS: We induced experimental oral tolerance in mice for ovalbumin (OVA), a food antigen, by previous oral treatment with OVA before sensitization with OVA injections. Food additives were administered at the induction of oral tolerance, and food allergy was induced by repeated administration of OVA. Symptoms of food allergy were defined as a change in body temperature and allergic diarrhoea. RESULTS: Saccharin sodium and a mixture of food additives inhibited acquisition of oral tolerance. Hypothermia and allergic diarrhoea with elevation of OVA-specific IgE were induced in the murine model of oral tolerance. Analyses of antigen-presenting cells in mesenteric lymph nodes showed that food additives affected their manner of migration. Additionally, food additives decreased the proportion of CD25hi regulatory T cells among CD4+ T cells in the mesenteric lymph nodes. CONCLUSIONS AND CLINICAL RELEVANCE: A large amount of food additives may prevent acquisition of oral tolerance. Intake of food additives in early life may increase the risk of food allergies.

Development of a real-time PCR assay for the quantification of Ma-LMM01-type Microcystis cyanophages in a natural pond.

UNLABELLED: Microcystis aeruginosa forms toxic cyanobacterial blooms throughout the world where its infectious phages are thought to influence host population dynamics. To assess the cyanophage impact on the host dynamics, we previously monitored Ma-LMM01-type phage abundance using a real-time PCR with a primer set designed based on the sequence of Microcystis phage Ma-LMM01; and we estimated the phage-infected host cell abundance. However, a recent study shows the Ma-LMM01 g91 gene sequence belongs to the smallest group, group III, of the three genotype groups, suggesting Ma-LMM01-type phage abundance was underestimated. Therefore, to re-evaluate the effect of Ma-LMM01-type phages on their hosts, we monitored the abundance of Ma-LMM01-type phages using real-time PCR with a new primer set designed based on the sequences of genotype groups I-III. We found phage abundance between 10(3) and 10(4) ml(-1) using the new primer set in samples where previously these phages were not detected using the old primer set. The frequency of Ma-LMM01-type phage-infected cells to Ma-LMM01-type phage-susceptible host cells may be as high as 30%, suggesting the phages may occasionally affect not only shifts in the genetic composition but also the dynamics of Ma-LMM01-type phage-susceptible host populations. SIGNIFICANCE AND IMPACT OF THE STUDY: Phages are one of the factors that may control the ecology of their host blooms. Therefore, it is essential to estimate phage abundance to understand phage impact on host populations. A real-time PCR assay was improved to detect a larger range of Microcystis cyanophages in natural surroundings where no phages were detected using a previous method by re-designing a new primer set based on sequences from three Ma-LMM01-type phage genetic groups. The new method allows us to determine the distribution, dynamics and infection cycle of the phage to help understand the interaction between the phages and the hosts.

Recombinant AgB8/1 ELISA test vs. commercially available IgG ELISA test in the diagnosis of cystic echinococcosis.

The diagnosis and clinical management of cystic echinococcosis (CE) rely on imaging and serology, the latter still having a complementary role as its accuracy in assessing cyst viability is unsatisfactory. We used an experimental IgG ELISA test based on the recombinant antigen rEgAgB8/1 cloned from Echinococcus granulosus to differentiate active from inactive/cured CE infection, comparing its performance to that of a commercially available ELISA test used routinely in our hospital laboratory. Both tests were performed on sera from 88 patients with hepatic echinococcal cysts, grouped according to cyst stage based on ultrasonographical morphology, and on 17 patients surgically treated for echinococcosis and 18 patients with nonparasitic hepatic cysts included as controls. Tests' performances did not differ significantly, but the overall concordance between tests drastically dropped when groups were analysed separately. Further longitudinal studies should evaluate whether these discrepancies reflect the different ability of either test to predict the evolution of cysts over time. Although the recombinant-AgB8/1-based ELISA test seems to have no clinical advantage over the commercially available ELISA test in the assessment of hepatic CE cyst viability, the easiness of production and reproducibility of high-quality recombinant antigens makes rEgAgB8/1 a valid candidate for use in CE ELISA diagnostic tests.

The first report on cystic echinococcosis in a cat caused by Echinococcus granulosus sensu stricto (G1).

A case of cystic echinococcosis (CE) in a domestic cat is described from Saint Petersburg, Russia. Ultrasonography showed numerous cysts with hyperechoic walls and anechoic contents within the cat's abdominal cavity. Molecular identification based on mitochondrial DNA genes indicated that the causative agent was Echinococcus granulosus sensu stricto (G1 strain). This is the first report of CE in a cat caused by E. granulosus sensu stricto with molecular confirmation.

Cell surface organization by the membrane skeleton.

Single-particle tracking and laser tweezers have facilitated the observation of the mechanics of molecular interactions in the plasma membrane of living cells at the level of single (or a few) molecules at nanometer/piconewton precision. These techniques have recently revealed that the membrane skeleton provides both confining and binding effects on the movement of membrane proteins, and that it can play a pivotal role in the molecular organization of the plasma membrane, especially in the formation of special membrane domains.

Single-molecule imaging of EGFR signalling on the surface of living cells.

The early events in signal transduction from the epidermal growth factor (EGF) receptor (EGFR) are dimerization and autophosphorylation of the receptor, induced by binding of EGF. Here we observe these events in living cells by visualizing single molecules of fluorescent-dye-labelled EGF in the plasma membrane of A431 carcinoma cells. Single-molecule tracking reveals that the predominant mechanism of dimerization involves the formation of a cell-surface complex of one EGF molecule and an EGFR dimer, followed by the direct arrest of a second EGF molecule, indicating that the EGFR dimers were probably preformed before the binding of the second EGF molecule. Single-molecule fluorescence-resonance energy transfer shows that EGF-EGFR complexes indeed form dimers at the molecular level. Use of a monoclonal antibody specific to the phosphorylated (activated) EGFR reveals that the EGFR becomes phosphorylated after dimerization.

Compartmentalized structure of the plasma membrane for receptor movements as revealed by a nanometer-level motion analysis.

Movements of transferrin and alpha 2-macroglobulin receptor molecules in the plasma membrane of cultured normal rat kidney (NRK) fibroblastic cells were investigated by video-enhanced contrast optical microscopy with 1.8 nm spatial precision and 33 ms temporal resolution by labeling the receptors with the ligand-coated nanometer-sized colloidal gold particles. For both receptor species, most of the movement trajectories are of the confined diffusion type, within domains of approximately 0.25 microns2 (500-700 nm in diagonal length). Movement within the domains is random with a diffusion coefficient approximately 10(-9) cm2/s, which is consistent with that expected for free Brownian diffusion of proteins in the plasma membrane. The receptor molecules move from one domain to one of the adjacent domains at an average frequency of 0.034 s-1 (the residence time within a domain approximately 29 s), indicating that the plasma membrane is compartmentalized for diffusion of membrane receptors and that long-range diffusion is the result of successive intercompartmental jumps. The macroscopic diffusion coefficients for these two receptor molecules calculated on the basis of the compartment size and the intercompartmental jump rate are approximately 2.4 x 10(-11) cm2/s, which is consistent with those determined by averaging the long-term movements of many particles. Partial destruction of the cytoskeleton decreased the confined diffusion mode, increased the simple diffusion mode, and induced the directed diffusion (transport) mode. These results suggest that the boundaries between compartments are made of dynamically fluctuating membrane skeletons (membrane-skeleton fence model).

Barriers for lateral diffusion of transferrin receptor in the plasma membrane as characterized by receptor dragging by laser tweezers: fence versus tether.

Our previous results indicated that the plasma membrane of cultured normal rat kidney fibroblastic cell is compartmentalized for diffusion of receptor molecules, and that long-range diffusion is the result of successive intercompartmental jumps (Sako, Y. and Kusumi, A. 1994. J. Cell Biol. 125:1251-1264). In the present study, we characterized the properties of intercompartmental boundaries by tagging transferrin receptor (TR) with either 210-nm-phi latex or 40-nm-phi colloidal gold particles, and by dragging the particle-TR complexes laterally along the plasma membrane using laser tweezers. Approximately 90% of the TR-particle complexes showed confined-type diffusion with a microscopic diffusion coefficient (Dmicro) of approximately 10(-9) cm2/s and could be dragged past the intercompartmental boundaries in their path by laser tweezers at a trapping force of 0.25 pN for gold-tagged TR and 0.8 pN for latex-tagged TR. At lower dragging forces between 0.05 and 0.1 pN, particle-TR complexes tended to escape from the laser trap at the boundaries, and such escape occurred in both the forward and backward directions of dragging. The average distance dragged was half of the confined distance of TR, which further indicates that particle-TR complexes escape at the compartment boundaries. Since variation in the particle size (40 and 210 nm, the particles are on the extracellular surface of the plasma membrane) hardly affects the diffusion rate and behavior of the particle-TR complexes at the compartment boundaries, and since treatment with cytochalasin D or vinblastin affects the movements of TR (Sako and Kusumi as cited above), argument has been advanced that the boundaries are present in the cytoplasmic domain. Rebound of the particle-TR complexes when they escape from the laser tweezers at the compartment boundaries suggests that the boundaries are elastic structures. These results are consistent with the proposal that the compartment boundaries consist of membrane skeleton or a membrane-associated part of the cytoskeleton (membrane skeleton fence model). Approximately 10% of TR exhibited slower diffusion (Dmicro approximately 10(-10)-10(-11) cm2/s) and binding to elastic structures.

Regulation mechanism of the lateral diffusion of band 3 in erythrocyte membranes by the membrane skeleton.

Mechanisms that regulate the movement of a membrane spanning protein band 3 in erythrocyte ghosts were investigated at the level of a single or small groups of molecules using single particle tracking with an enhanced time resolution (0.22 ms). Two-thirds of band 3 undergo macroscopic diffusion: a band 3 molecule is temporarily corralled in a mesh of 110 nm in diameter, and hops to an adjacent mesh an average of every 350 ms. The rest (one-third) of band 3 exhibited oscillatory motion similar to that of spectrin, suggesting that these band 3 molecules are bound to spectrin. When the membrane skeletal network was dragged and deformed/translated using optical tweezers, band 3 molecules that were undergoing hop diffusion were displaced toward the same direction as the skeleton. Mild trypsin treatment of ghosts, which cleaves off the cytoplasmic portion of band 3 without affecting spectrin, actin, and protein 4.1, increased the intercompartmental hop rate of band 3 by a factor of 6, whereas it did not change the corral size and the microscopic diffusion rate within a corral. These results indicate that the cytoplasmic portion of band 3 collides with the membrane skeleton, which causes temporal confinement of band 3 inside a mesh of the membrane skeleton.

Cytoplasmic regulation of the movement of E-cadherin on the free cell surface as studied by optical tweezers and single particle tracking: corralling and tethering by the membrane skeleton.

The translational movement of E-cadherin, a calcium-dependent cell-cell adhesion molecule in the plasma membrane in epithelial cells, and the mechanism of its regulation were studied using single particle tracking (SPT) and optical tweezers (OT). The wild type (Wild) and three types of artificial cytoplasmic mutants of E-cadherin were expressed in L-cells, and their movements were compared. Two mutants were E-cadherins that had deletions in the COOH terminus and lost the catenin-binding site(s) in the COOH terminus, with remaining 116 and 21 amino acids in the cytoplasmic domain (versus 152 amino acids for Wild); these are called Catenin-minus and Short-tailed in this paper, respectively. The third mutant, called Fusion, is a fusion protein between E-cadherin without the catenin-binding site and alpha-catenin without its NH2-terminal half. These cadherins were labeled with 40-nm phi colloidal gold or 210-nm phi latex particles via a monoclonal antibody to the extracellular domain of E-cadherin for SPT or OT experiments, respectively. E-cadherin on the dorsal cell surface (outside the cell-cell contact region) was investigated. Catenin-minus and Short-tailed could be dragged an average of 1.1 and 1.8 micron by OT (trapping force of 0.8 pN), and exhibited average microscopic diffusion coefficients (Dmicro) of 1.2 x 10(-10) and 2.1 x 10(-10) cm2/s, respectively. Approximately 40% of Wild, Catenin-minus, and Short-tailed exhibited confined-type diffusion. The confinement area was 0.13 micron2 for Wild and Catenin-minus, while that for Short-tailed was greater by a factor of four. In contrast, Fusion could be dragged an average of only 140 nm by OT. Average Dmicro for Fusion measured by SPT was small (0.2 x 10(-10) cm2/s). These results suggest that Fusion was bound to the cytoskeleton. Wild consists of two populations; about half behaves like Catenin- minus, and the other half behaves like Fusion. It is concluded that the movements of the wild-type E-cadherin in the plasma membrane are regulated via the cytoplasmic domain by (a) tethering to actin filaments through catenin(s) (like Fusion) and (b) a corralling effect of the network of the membrane skeleton (like Catenin-minus). The effective spring constants of the membrane skeleton that contribute to the tethering and corralling effects as measured by the dragging experiments were 30 and 5 pN/micron, respectively, indicating a difference in the skeletal structures that produce these two effects.

Single-molecule analysis of chemotactic signaling in Dictyostelium cells.

Single-molecule imaging techniques were used to reveal the binding of individual cyclic adenosine 3',5'-monophosphate molecules to heterotrimeric guanine nucleotide-binding protein coupled receptors on the surface of living Dictyostelium discoideum cells. The binding sites were uniformly distributed and diffused rapidly in the plane of the membrane. The probabilities of individual association and dissociation events were greater for receptors at the anterior end of the cell. Agonist-induced receptor phosphorylation had little effect on any of the monitored properties, whereas G protein coupling influenced the binding kinetics. These observations illustrate the dynamic properties of receptors involved in gradient sensing and suggest that these may be polarized in chemotactic cells.

Reconstitution of brefeldin A-induced golgi tubulation and fusion with the endoplasmic reticulum in semi-intact chinese hamster ovary cells.

The fungal metabolite brefeldin A (BFA) induces the disassembly of the Golgi complex in mammalian cells. The drug seems to accentuate tubule formation and causes the subsequent fusion with the endoplasmic reticulum (ER). To investigate the biochemical requirements and kinetics of BFA-induced Golgi disassembly, we have reconstituted the process of green fluorescent protein-tagged Golgi complex disassembly in streptolysin O-permeabilized semi-intact Chinese hamster ovary cells. For quantitative analysis of the morphological changes to the Golgi complex in semi-intact cells, we developed a novel morphometric analysis. Based on this analysis, we have dissected the BFA-induced Golgi disassembly process biochemically into two processes, Golgi tubule formation and fusion with the ER, and found that the formation is induced by only ATP and the residual factors in the cells and that the subsequent fusion is mediated in an N-ethylmaleimide-sensitive factor-dependent manner via Golgi tubules. Tubulation occurs by two pathways that depend on either microtubule integrity or exogenously added cytosol. In the presence of GTPgammaS, coat protein I inhibited the Golgi tubule fusion with the ER but showed no apparent effect on tubulation. Additionally, we analyzed the kinetics of tubulation and fusion independently in nocodazole-treated and -untreated semi-intact cells and found that tubulation is a rate-limiting step of the Golgi disassembly.

Gastrointestinal helminths and Taenia spp. in parenteral tissues of free-roaming pigs (Sus scrofa indicus) from hilltribe village at the western border of Thailand.

A serological survey of pig cysticercosis was conducted in a hill-tribe village at Thai-Myanmar border, Tak province of Thailand in 2012. Sixteen backyard pigs were examined for pig cysticercosis and gastrointestinal helminth infection. In addition to cysticerci of Taenia solium and Taenia hydatigena found outside the gut, nine other helminth species were found in guts: Echinostoma malayanum, Pseudanoplocephala crawfordi, Ascarops dentata, Physocephalus sexalatus, Gnathostoma doloresi, Ascaris suum, Globocephalus sp., Oesophagostomum dentatum and Bourgelatia diducta. The study presents a report for the first time of adult tapeworm, P. crawfordi infection in pigs from Thailand. For medical importance, E. malayanum, P. crawfordi, G. doloresi and A. suum have been confirmed as potentially zoonotic helminths and pigs may act as one of the reservoir hosts for human helminthiases. Pigs of both gender and all ages appeared to be exposed to the parasites equally and did not show any significant difference to these helminth species in richness and total intensity.

A novel phosphoglycolipid archaetidyl(glucosyl)inositol with two sesterterpanyl chains from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1.

The structures of two novel polar lipids (AGI and AI) of an aerobic hyperthermophilic archaeon, Aeropyrum pernix, were elucidated. AGI and AI were the only two major lipids and accounted for 91 mol% and 9 mol%, respectively, of total polar lipids of this organism. The core lipid of A. pernix total lipids consisted solely of 2,3-di-O-sesterterpanyl-sn-glycerol (C25,25-archaeol). The molecular weights of the free acid forms of AGI and AI were shown by FAB-mass spectrometry to be 1196 and 1034, respectively. AI was completely hydrolyzed by phosphatidylinositol-specific phospholipase C, while AGI was not hydrolyzed at all under the same condition for the hydrolysis of AI. The molar ratio of phosphate, myo-inositol, and glucose in AGI was 1.0:0.97:0.95. The positions of linkages between myo-inositol and glucose, and between myo-inositol and phosphate in AGI were determined by NMR analyses of intact AGI and glucosylinositol prepared from AGI. Finally, it was concluded that the structures of AGI and AI were 2,3-di-O-sesterterpanyl-sn-glycerol-1-phospho-1'-(2'-O-alpha-D-glu cosyl)- myo-inositol (C25,25-archaetidyl(glucosyl)inositol) and 2,3-di-O-sesterterpanyl-sn-glycerol-1-phospho-myo-inositol (C25,25-archaetidylinositol), respectively. This is the first example that a core lipid of whole polar lipids is composed of only one species C25,25-archaeol in one organism and that glucosylinositol is found in a polar lipid as a polar head group.

Coupling of beta-cell desensitization by hyperglycemia to excessive stimulation and circulating insulin in glucose-infused rats.

Nondiabetic rats were infused with glucose for 48 h to maintain moderate or marked hyperglycemia (mean blood glucose 13.2 +/- 0.7 or 22.8 +/- 0.3 mM, respectively). The two levels of hyperglycemia increased plasma insulin levels severalfold but decreased the insulin response to 27 mM glucose by 19 and 95%, respectively, versus saline infusion. Diazoxide (5 mg.kg-1.h-1), when continuously infused during the hyperglycemia protocols, completely inhibited the glucose-induced rise in plasma insulin levels. Diazoxide transformed beta-cell insensitivity to stimulation: glucose-induced insulin release was thus increased 318% after moderate hyperglycemia and 707% after marked hyperglycemia. These stimulatory effects of diazoxide were reversed by exogenous insulin infusion (8 or 2 U/24 h) in a dose-dependent manner. It is concluded that excessive beta-cell stimulation rather than glucotoxicity underlies hyperglycemia-induced beta-cell insensitivity. Effects of hyperinsulinemia can form part of the mechanisms whereby excessive stimulation affects beta-cell secretion.

Evaluation of clinical and serological data from Taenia solium cysticercosis patients in eastern Inner Mongolia Autonomous Region, China.

Neurocysticercosis (NCC) is one of the major causes of neurological disease in China. ELISA and immunoblotting using glycoproteins purified by preparative isoelectric focusing were used to detect human cysticercosis in Tongliao area, Inner Mongolia, China in 1998. Approximately 89% (39 of 44 inpatients and outpatients with suspected NCC at Tongliao City Hospital) were residents of Inner Mongolia. About 53% were male and 77% were of working age (18-59 years), and 32% were farmers. Immunoblotting and ELISA showed a high correlation. Of the 44 patients, 31 positive by cerebral computed tomography (CT) scan were confirmed serologically to have cysticercosis. In the ELISA, patients with no lesions by CT scan had lower OD values, similar to those of normal serum. These findings confirm that both ELISA and immunoblotting assays are sufficiently sensitive to detect asymptomatic or symptomatic cysticercosis patients.

A 48-hour lipid infusion in the rat time-dependently inhibits glucose-induced insulin secretion and B cell oxidation through a process likely coupled to fatty acid oxidation.

Short- and long-term effects of hyperlipidemia with elevated FFA on insulin secretion were investigated. Male Sprague-Dawley rats were fed ad libitum and additionally infused with Intralipid 10%, 1.0 ml/h. After 3 h of Intralipid the response to 27 mM glucose in isolated perfused pancreas was enhanced by 86%, P less than 0.02. After 6 h of Intralipid enhancement had subsided. After 48 h of Intralipid glucose-induced insulin release was inhibited by 49%, from 1950 +/- 177 microU/min after saline to 1003 +/- 232 microU/min after Intralipid, P less than 0.02. Inhibition was glucose-selective since responses to other secretagogues (1 mM 3-isobutyl-1 methylxanthine, 10 mM octanoate, or 5 mM alpha-ketoisocaproic acid) were unaffected as were pancreatic contents of insulin (2284 +/- 111 mU/pancreas after saline, 2566 +/- 131 mU/pancreas after Intralipid). In isolated islets from 48 h lipid infused rats production of [14-C]CO2 from D[U-14-C]glucose was decreased (P less than 0.02) in parallel with the insulin response to 27 mM glucose. Glucose-induced secretion was partially normalized by in vitro exposure to a carnitine palmitoyl-transferase I inhibitor (Etomoxir). Effects of a 48 h lipid infusion were also tested during hyperglycemia. Rats were infused with glucose, and hyperglycemia was enhanced by dexamethasone (25 micrograms/24 h). Hyperglycemia depressed glucose-induced secretion from perfused pancreas from 2072 +/- 22 microU/min after saline + dexamethasone to 1185 +/- 155 microU/min after glucose + dexamethasone, P less than 0.01). Intralipid, added to the latter protocol, further inhibited glucose-induced secretion to 437 +/- 87 microU/min, P less than 0.005. Hyperlipidemia is concluded to be associated with short term stimulation but long term inhibition of glucose-induced insulin secretion. Evidence indicates that inhibition depends on fatty acid oxidation, is coupled to decreased glucose oxidation and operates both during normo- and hyperglycemia.

Multiple abnormalities in insulin responses to nonglucose nutrients in neonatally streptozotocin diabetic rats.

Insulin responses to nutrient secretagogues were investigated in neonatally streptozotocin-injected (n-STZ) rats, i.e. an animal model of noninsulin-dependent diabetes. In the perfused pancreas 16 mM L-glutamine induced and 10 mM octanoate tended to induce (P less than 0.2) higher responses in n-STZ than in nondiabetic rats. Addition of 3.9 mM glucose potentiated responses to glutamine and octanoate more in n-STZ (3.3- and 3.4-fold) than in nondiabetic rats (1.5- and 1.9-fold). Conversely, the succinate derivative succinate monomethylester (Succ ME) induced lesser response in n-STZ rats (57% of that in nondiabetic rats) and coperfusion with 3.9 mM glucose increased the response less in n-STZ (1.4-fold) than in nondiabetic rats (3.8-fold). Pyruvate (20 mM) mimicked the potency of 3.9 mM glucose, i.e. pyruvate potentiated the response to Succ ME only nonsignificantly (1.2-fold) in n-STZ but markedly (4.9-fold) in nondiabetic rats. Dichloroacetate (20 mM) failed to affect the response to Succ ME together with pyruvate in n-STZ rats. To investigate the role of hyperglycemia for octanoate-induced secretion, nondiabetic rats were made hyperglycemic by 48-h glucose infusions. Octanoate-induced secretion from perfused pancreas was enhanced 3.8-fold after moderate hyperglycemia (13.2 +/- 0.6 mM) and 17-fold after marked hyperglycemia (22.7 +/- 0.6 mM). This positive association between response and degree of hyperglycemia was not found with a nonnutrient secretagogue, 3-isobutyl-1-methylxanthine. Results with glutamine and octanoate indicate that oxidation of nonglucose nutrients which normally do not regulate secretion is enhanced secondary to chronic hyperglycemia. Results with Succ ME and pyruvate suggest that early steps of oxidation of glucose are impaired in n-STZ rats.

Purification and characterization of phosphoenolpyruvate carboxylase from the hyperthermophilic archaeon Methanothermus sociabilis.

Phosphoenolpyruvate carboxylase (PEPC) was purified for the first time from hyperthermophilic archaeon Methanothermus sociabilis, growing autotrophically with an optimum at 88 degrees C. The optimum temperature for enzyme activity was similar to that for growth and was 85 degrees C. The native enzyme was a homotetramer of 240 kDa molecular mass and the subunit displayed an apparent molecular mass of 60 kDa. The archaeal PEPC was insensitive to various metabolites which are known as allosteric effectors for most bacterial and eucaryal counterparts. The enzyme showed extreme thermostability such that there remained 80% of the enzyme activity after incubation for 2 h at 80 degrees C. These results implied that archaeal PEPC was significantly different from bacterial and eucaryal entities.