Functional involvement of TMF/ARA160 in Rab6-dependent retrograde membrane traffic

The small GTPase Rab6 regulates retrograde membrane traffic from endosomes to the Golgi apparatus and from the Golgi to the endoplasmic reticulum (ER). We examined the role of a Rab6-binding protein, TMF/ARA160 (TATA element modulatory factor/androgen receptor-coactivator of 160 kDa), in this process. High-resolution immunofluorescence imaging revealed that TMF signal surrounded Rab6-positive Golgi structures and immunoelectron microscopy revealed that TMF is concentrated at the budding structures localized at the tips of cisternae. The knockdown of either TMF or Rab6 by RNA interference blocked retrograde transport of endocytosed Shiga toxin from early/recycling endosomes to the trans-Golgi network, causing missorting of the toxin to late endosomes/lysosomes. However, the TMF knockdown caused Rab6-dependent displacement of N-acetylgalactosaminyltransferase-2 (GalNAc-T2), but not beta1,4-galactosyltransferase (GalT), from the Golgi. Analyses using chimeric proteins, in which the cytoplasmic regions of GalNAc-T2 and GalT were exchanged, revealed that the cytoplasmic region of GalNAc-T2 plays a crucial role in its TMF-dependent Golgi retention. These observations suggest critical roles for TMF in two Rab6-dependent retrograde transport processes: one from endosomes to the Golgi and the other from the Golgi to the ER.     

Rab cascades and tethering factors in the endomembrane system

Rab GTPases are key proteins that determine organelle identity and operate at the center of fusion reactions. Like Ras, they act as switches that are connected to a diverse network of tethering factors, exchange factors and GTPase activating proteins. Recent studies suggest that Rabs are linked to each other via their effectors, thus coordinating protein transport in the endomembrane system. Within this review, we will focus on selected examples that highlight these issues.     

Leishmania donovani: dyskinetoplastid cells survive and proliferate in the presence of pyruvate and uridine but do not undergo apoptosis after treatment with camptothecin

We have shown that treatment with luteolin in leishmanial cells causes loss of mt-DNA and induces apoptosis through mitochondria dependent pathway [Sen, N., Das, B.B., Ganguly, A., Banerjee, B., Sen, T., Majumder, H.K., 2006. Leishmania donovani: intracellular ATP level regulates apoptosis-like death in luteolin induced dyskinetoplastid cells. Experimental Parasitology, in press]. Here, we report that mitochondrial DNA depleted leishmanial cells require exogenous sources of pyruvate and uridine to survive and proliferate. The presence of pyruvate and uridine in a growing media help them to produce sufficient amount of glycolytic ATP to maintain the mitochondrial membrane potential in the absence of their functional ETC. Treatment of wild type cells with CPT causes generation of ROS that leads to apoptosis. But unlike the normal cells ROS was not generated in these mt-DNA depleted cells after treatment with CPT. Taken together we have shown for the first time that dyskinetoplastid cells are auxotrophic for pyruvate and uridine and apoptosis cannot be induced in these cells in the presence of CPT. Therefore, the presence of mitochondrial DNA is absolutely necessary for the cytotoxicity of CPT in kinetoplastid parasites.     

Plasmodium yoelii: the effect of second blood meal and anti-sporozoite antibodies on development and gene expression in the mosquito vector, Anopheles stephensi

The sporogonic development of the malaria parasite takes place in the mosquito and a wide range of factors modulates it. Among those, the contents of the blood meal can influence the parasite development directly or indirectly through the mosquito response to the infection. We have studied the effect of a second blood meal in previously infected mosquitoes and the effect of anti-sporozoite immune serum on parasite development and mosquito response to the infection. The prevalence and intensity of infection and gene expression of both Plasmodium yoelii and Anopheles stephensi was analyzed. We verified that a second blood meal and its immune status interfere with parasite development and with Plasmodium and mosquito gene expression.     

Variable actin dynamics requirement for the exit of different cargo from the trans-Golgi network

Efficient post-Golgi trafficking depends on microtubules, but actin filaments and actin-associated proteins are also postulated. Here we examined, by inverse fluorescence recovery after photobleaching, the role of actin dynamics in the exit from the TGN of fluorescent-tagged apical or basolateral and raft or non-raft-associated cargoes. Either the actin-stabilizing jasplakinolide or the actin-depolymerising latrunculin B variably but significantly inhibited post-Golgi traffic of non-raft associated apical p75NTR and basolateral VSV-G cargoes. The TGN-exit of the apical-destined VSV-G mutant was impaired only by latrunculin B. Strikingly, the raft-associated GPI-anchor protein was not affected by either actin toxin. Results indicate that actin dynamics participates in the TGN egress of both apical- and basolateral-targeted proteins but is not needed for apical raft-associated cargo.     

Post-ruminal or intravenous infusions of carbohydrates or amino acids to dairy cows 2. Late lactation

The objectives of this study were to compare the effects of post-ruminal and intravenous infusions of wheat starch or glucose (CHO) or a mixture of amino acids (AA) on milk protein yield, nitrogen (N) utilisation, plasma metabolites and mammary extraction rate of dairy cows in late lactation. Eight cow, ruminally fistulated, was assigned to two 4 × 4 Latin squares during 14-day periods, where the last 7 days were for infusions. Infusions were: (1) starch in the abomasum (SP), (2) glucose in the blood (GB), (3) AA in the abomasum (AP), and (4) AA in the blood (AB). The experiment started 165 ± 4 days (mean ± s.e.) post partum (milk yield 22.5 ± 1.1 kg) Daily amounts of nutrients infused were 257, 283, 233, and 260 g for SP, GB, AP and AB, respectively. The cows were fed a basal diet consisting of a concentrate mixture and grass silage (55:45 on a dry-matter (DM) basis), where total dry-matter intake (DMI) was 13.3 kg/day. Milk production was affected by site of infusion within substrate, whereas infusion substrates within infusion site (CHO or AA) were of minor importance. Responses to intravenous infusions (GB or AB) were similar to those in early lactation, but more pronounced. Compared with SP infusion, GB infusion increased ( P < 0.05) milk yield, energy-corrected milk (ECM), protein and lactose yield by 1.4 and 0.9 kg, 38 and 59 g, respectively. The AB infusion had 1.4 and 1.3 kg, 51, 52 and 50 g higher ( P < 0.05) milk yield, ECM, protein, fat and lactose yields than the AP infusion, respectively. N balance data indicated higher losses of metabolic faecal nitrogen (MFN) by abomasal than by intravenous infusions, but the catabolism of AA was lower than in early lactation indicated by no difference ( P < 0.05) in urinary N excretion between treatments. Intravenous AA infusion increased plasma glucose and insulin above that of intravenous glucose infusion. The treatment effects on plasma insulin concentrations were higher in late than in early lactation, suggesting a higher sensitivity in late lactation even at similar negative energy balance. Compared with the SP infusion, GB infusion showed lower ( P < 0.05) concentrations of essential AA (EAA) and branched-chain AA (BCAA) resulting in a higher AA utilisation because of a higher milk protein production. AP infusion increased ( P < 0.05) plasma non-essential AA concentration compared with AB infusion, but infusion site of AA had no effect ( P>0.05) on plasma EAA or BCAA. It is concluded that it is the nutrient supply and not the lactation stage per se that is important for the response in milk production. Nevertheless, stage of lactation affects the N metabolism and the response in plasma hormone concentrations even when cows are in negative energy balance in both lactation stages.     

Metabolic syndrome in Yup'ik Eskimos: the Center for Alaska Native Health Research (CANHR) Study

Objective: This study investigated the prevalence of metabolic syndrome and its defining components among Yup'ik Eskimos. Research Methods and Procedures: A cross‐sectional study design that included 710 adult Yup'ik Eskimos ≥18 years of age residing in 8 communities in Southwest Alaska. The prevalence of metabolic syndrome was determined using the recently updated Adult Treatment Panel III criteria. Results: The prevalence of metabolic syndrome in this study cohort was 14.7%, and varied by sex with 8.6% of the men and 19.8% of the women having metabolic syndrome. This is lower than the prevalence of 23.9% in the general U.S. adult population. The most common metabolic syndrome components/risk factors were increased waist circumference and elevated blood glucose. High‐density lipoprotein (HDL) cholesterol levels in Yup'ik Eskimos were significantly higher, and triglycerides lower than levels reported in National Health and Nutritional Examination III. Discussion: Compared with other populations, metabolic syndrome is relatively uncommon in Yup'ik Eskimos. The higher prevalence among Yup'ik women is primarily explained by their large waist circumference, suggesting central body fat accumulation. Further increases in metabolic syndrome risk factors among Yup'ik Eskimos could lead to increases in the prevalence of type 2 diabetes and cardiovascular disease, once rare in this population.     

Glucose dysregulation in obese children: predictive, risk, and potential protective factors

Objective: The aim of our study was to determine the prevalence of impaired glucose tolerance (IGT) and type 2 diabetes (DM2) in obese children and adolescents of Greek origin and compare our data with pertinent literature findings in an attempt to uncover predictive, risk, and preventive factors. Research Methods and Procedures: A total of 117 obese children and adolescents 12.1 ± 2.7 years old underwent a 2‐hour oral glucose tolerance test (OGTT). Insulin resistance (IR) and β‐cell function were estimated using the homeostasis model assessment (HOMA)‐IR and the insulinogenic index, respectively. Results: A total of 17 patients (14.5%) had IGT, and none had DM2. The overall prevalence rates of both IGT and DM2 in our subjects were lower than those reported in a recent multiethnic U.S. study. Nevertheless, the difference between our IGT data and those of the U.S. study was due mostly to the prepubertal subjects (9% vs. 25.4%), whereas no difference was observed in the pubertal population (18% vs. 21%). Fasting glucose, insulin, and HOMA‐IR values were not predictive of IGT. The absolute value of insulin at 2 hours of the OGTT combined with the time‐integrated glycemia (AUCG) can strongly predict IGT, whereas higher area under the curve for insulin (AUCI) values were found to be protective. Discussion: In ethnic groups less prone to diabetes development, IGT or DM2 in obese subjects is more likely to develop at puberty than at the prepubertal stage. It is advisable that physicians caring for obese adolescents perform an OGTT for early detection of IGT because HOMA‐IR values, although higher in IGT subjects and indicative of IR, cannot predict IGT.     

Potential of intestinal electrical stimulation for obesity: a preliminary canine study

Objective: The aims of this study were to investigate the therapeutic potential of intestinal electrical stimulation (IES) for obesity. Experiments were performed to investigate the effects of IES on food intake, gastric tone, gastric accommodation, and its possible pathway. Research Methods and Procedures: Ten normal dogs and six dogs with truncal vagotomy were used in this study. Each dog was equipped with a gastric cannula for the measurement of gastric tone and accommodation by barostat and one pair of duodenal serosal electrodes for IES. The experiment on food intake was composed of both control session without IES and IES session after a 28‐hour fast. The experiment on gastric tone and accommodation was performed in the fasting and fed states and composed of three sessions: control, IES, and IES with N^G‐nitro‐l ‐arginine. Results: IES significantly reduced food intake in the normal dogs (459.0 vs. 312.6 grams, p < 0.001). The food intake was negatively correlated with the fasting gastric volume during IES. IES significantly decreased fasting gastric tone in the normal dogs reflected as a decrease in gastric volume (89.1 vs. 261.3 mL, p < 0.01), which was abolished by vagotomy and N^G‐nitro‐l ‐arginine. Discussion: IES reduces food intake and inhibits gastric tone in the fasting state. The inhibitory effect of IES on gastric tone is mediated by both vagal and nitrergic pathway.     

Fatness, fitness, and insulin sensitivity among 7- to 9-year-old children

Objective: The purpose of this study was to examine the relationships among fatness and aerobic fitness on indices of insulin resistance and sensitivity in children. Research Design and Methods: A total of 375 children (193 girls and 182 boys) 7 to 9 years of age were categorized by weight as normal‐weight, overweight, or obese and by aerobic fitness based on a submaximal physical working capacity test (PWC). Fasting blood glucose (GLU) and insulin (INS) were used to calculate various indices of insulin sensitivity (GLU/INS), the homeostasis model assessment (HOMA), and the quantitative insulin sensitivity check index (QUICKI). Surrogate measures of pancreatic β cell function included the insulinogenic index (INS/GLU) and the HOMA estimate of pancreatic β‐cell function (HOMA %B). Results: Insulin sensitivity and secretion variables were significantly different between the normal‐weight children and the overweight and obese subjects. Fasting insulin (FI), HOMA, QUICKI, and INS/GLU were significantly different between the overweight and obese subjects. Likewise, the high fitness group possessed a better insulin sensitivity profile. In general, the normal‐weight–high fit group possessed the best insulin sensitivity profile and the obese‐unfit group possessed the worst insulin sensitivity profile. Several significant differences existed among the six fat‐fit groups. Of particular note are the differences within BMI groups by fitness level and the comparison of values between the normal‐weight–unfit subjects and the overweight and obese subjects with high fitness. Conclusions: The results indicate that aerobic fitness attenuates the difference in insulin sensitivity within BMI categories, thus emphasizing the role of fitness even among overweight and obese children.