Effect of CCK-8 on insulin-induced hyperphagia and hypothalamic orexigenic neuropeptide expression in the rat

The influence of cholecystokinin octapeptide (CCK-8) on normal and insulin-induced feeding and expression of orexigenic hypothalamic neuropeptides was investigated in male rats. CCK-8, administered during meals (4 microg/kg) or continuously (32 microg/kg over 60 min), blunted the stimulating effect of insulin (50 IU/kg) on feeding by reducing meal size (-60%; P<0.05 or -86%; P<0.0001, respectively). Rats without access to food and injected with IP insulin (50 IU/kg) showed increased hypothalamic mRNA levels of orexin (+30%; P<0.05) and melanin-concentrating hormone (+52%; P<0.05), as compared with ad libitum-fed and saline-injected control rats. Continuous IP infusion of CCK-8 (32 microg/kg) blunted these increases. Our results suggest that both orexin and melanin-concentrating hormone participate in the response to insulin hypoglycemia without food being present; these neurons may be involved in mechanisms related to insulin-induced hyperphagia. Signals triggered by peripheral CCK-8 act to decrease the expression of orexin and melanin-concentrating hormone. This may be associated with a reduction in hyperphagia.     

Modulation of gene expression by promoter mutants of the lambdacI857/pRM/pR system

Gene expression driven by the p(R) promoter of the lambdacI857/p(RM)/p(R) system results from inactivation of the temperature-sensitive CI857 repressor. The CI857 repressor, whose gene is transcribed by the divergently orientated p(RM) promoter, is destabilised at temperatures above 30 degrees C. In this study, the lambdacI857/p(RM)/p(R) system was modified by the introduction of a single (A-32G) and a double mutation (A-32G and T-41C). The mutated lambdap(R) expression modules, 32G and 32G/41C, tightly repressed the highly lethal phage PhiX174 lysis gene E at temperatures up to 37 and 39 degrees C, respectively. Expression of protein E and subsequent lysis of Escherichia coli was still induced by a temperature up-shift to 42 degrees C. The impact of the mutations on gene expression levels driven by the lambdap(R) and p(RM) promoters was evaluated at various temperatures using the lacZ reporter gene. Results indicate that the A-32G mutation confers a lambdap(R) promoter-down phenotype. The additional increase in the temperature stability of the 32G/41C expression system is due to the T-41C mutation leading to a higher p(RM) activity. The described lambdap(R) expression modules can be used to obtain a defined expression level at a given temperature and to tightly repress in particular highly lethal genes at different bacterial growth temperatures.     

Enterostatin decreases postprandial pancreatic UCP2 mRNA levels and increases plasma insulin and amylin

This study investigated the chronic effect of enterostatin on body weight and some of the associated changes in postprandial metabolism. Rats were adapted to 6 h of food access/day and a choice of low-fat and high-fat (HF) food and then given enterostatin or vehicle by an intraperitoneally implanted minipump delivering 160 nmol enterostatin/h continuously over a 5-day infusion period. Enterostatin resulted in a slight but significant reduction of HF intake and body weight. After the last 6-h food access period, enterostatin-treated animals had lower plasma triglyceride and free fatty acid but higher plasma glucose and lactate levels than control animals. Enterostatin infusion resulted in increased uncoupling protein-2 (UCP2) expression in various tissues, including epididymal fat and liver. UCP2 was reduced in the pancreas of enterostatin-treated animals, and this was associated with increased plasma levels of insulin and amylin. Whether these two hormones are involved in the observed decreased food intake due to enterostatin remains to be determined. As lipid metabolism appeared to be altered by enterostatin, we measured peroxisome proliferator-activated receptor (PPAR) expression in tissues and observed that PPARalpha, -beta, -gamma1, and -gamma2 expression were modified by enterostatin in epididymal fat, pancreas, and liver. This further links altered lipid metabolism with body weight loss. Our data suggest that alterations in UCP2 and PPARgamma2 play a role in the control of insulin and amylin release from the pancreas. This implies that enterostatin changes lipid and carbohydrate metabolic pathways in addition to its effects on food intake and energy expenditure.     

Obstructive sleep apnea: Plasma endothelin-1 precursor but not endothelin-1 levels are elevated and decline with nasal continuous positive airway pressure

Assessment of plasma endothelin-1 (ET-1) reveals conflicting results in cerebral and noncerebral conditions. Obstructive sleep apnea (OSA) syndrome has been used as a definite challenge for the investigation of endothelin measurements. Despite marked sleep-related breathing disturbances in untreated patients peripherally measurable ET-1 concentrations remained within the normal range and did not change after an appropriate therapy with continuous positive airway pressure (CPAP). In contrast, its precursor, big ET-1, was considerably elevated in untreated patients and dropped to normal values after long-term CPAP depending on compliance. Relatively stable big ET-1 elevations in untreated patients, during sleep and wakefulness, suggest that a general endothelial alteration beyond that explained by a direct impact of nocturnal breathing disturbances on the vascular system occurs. CPAP-therapy effectively lowered plasma big ET-1 in compliant patients and thus possibly their related risk for vascular diseases. Big ET-1 has been demonstrated to be a more appropriate marker of endothelial alteration than ET-1 because of its longer half-life. Simultaneous measurements are to be recommended.     

Construction of the first shuttle vectors for gene cloning and homologous recombination in Mycoplasma agalactiae

Mycoplasma agalactiae is a worldwide ruminant pathogen that causes significant economic losses by inflicting contagious agalactia in sheep and goats. The development of efficient control strategies requires a better understanding of the mycoplasma factors that promote successful infection. However, lack of genetic tools has been a major impediment in studying the pathogenic mechanisms of M. agalactiae. This study describes the identification and cloning of the M. agalactiae origin of replication (oriC) in order to construct the first shuttle vectors for targeted gene disruption, gene complementation and expression studies. Additionally, this report provides the first evidence of the occurrence of homologous recombination and the functionality of heterologous tetM determinant in this pathogen.     

Investigations on the expression of cytokines in the canine corpus luteum in relation to dioestrus

Control of luteal regression in the dog is still poorly understood. Unlike other domestic animal species, luteolysis is not prevented by hysterectomy. Indications that PGF(2alpha) may act as an endogenous luteolytic agent have been found only in pregnant animals during the prepartal decline of progesterone. Evidence from several species indicates that the immune system plays an important role in corpus luteum (Cl) function, possibly by the release of cytokines from immigrant immune cells. Hence, in the present experiment we attempted to examine the expression of cytokines in the canine Cl during the course of dioestrus (formation and regression of the Cl), using RT-PCR. Groups of 4-5 bitches were ovario-hysterectomised on days 5, 15, 25, 35, 45 and 60-80 after ovulation. Canine-specific primers for IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, TNFalpha, IFNgamma and TGFbeta1 were used. Positive and negative controls were included in all tests. Adequate expression was confirmed by sequencing selective samples of positive PCR products. The expression of mRNA for IL-8, IL-10, IL-12, TNF-alpha and TGF-beta1 was detected in all samples at each stage of dioestrus, without any obvious variations indicating a likely modulatory function of these cytokines in differentiation, maintenance or regression of the canine Cl. All tests for the expression of mRNA for IL-4, IL-1beta and IL-2 were negative. More negative than positive results were obtained when testing for the expression of mRNA for IL-6 and IFN-gamma, leading to the conclusion that expression of these two cytokines is at a low level, and no conclusion can be drawn as to their involvement in control of Cl function.     

Regulated expression of endothelial lipase by porcine brain capillary endothelial cells constituting the blood-brain barrier

Normal neurological function depends on a constant supply of polyunsaturated fatty acids to the brain. A considerable proportion of essential fatty acids originates from lipoprotein-associated lipids that undergo uptake and/or catabolism at the blood-brain barrier (BBB). This study aimed at identifying expression and regulation of endothelial lipase (EL) in brain capillary endothelial cells (BCEC), major constituents of the BBB. Our results revealed that BCEC are capable of EL synthesis and secretion. Overexpression of EL resulted in enhanced hydrolysis of extracellular high-density lipoprotein (HDL)-associated sn-2-labeled [(14)C]20 : 4 phosphatidylcholine. [(14)C]20 : 4 was recovered in cellular lipids, indicating re-uptake and intracellular re-esterification. To investigate local regulation of EL in the cerebrovasculature, BCEC were cultured in the presence of peroxisome-proliferator activated receptor (PPAR)- and liver X receptor (LXR)-agonists, known to regulate HDL levels. These experiments revealed that 24(S)OH-cholesterol (a LXR agonist), bezafibrate (a PPARalpha agonist), or pioglitazone (a PPARgamma agonist) resulted in down-regulation of EL mRNA and protein levels. Our findings implicate that EL could generate fatty acids at the BBB for transport to deeper regions of the brain as building blocks for membrane phospholipids. In addition PPAR and LXR agonists appear to contribute to HDL homeostasis at the BBB by regulating EL expression.     

Reconstructing the regulatory network controlling commitment and sporulation in Physarum polycephalum based on hierarchical Petri Net modelling and simulation

We reconstruct the regulatory network controlling commitment and sporulation of Physarum polycephalum from experimental results using a hierarchical Petri Net-based modelling and simulation framework. The stochastic Petri Net consistently describes the structure and simulates the dynamics of the molecular network as analysed by genetic, biochemical and physiological experiments within a single coherent model. The Petri Net then is extended to simulate time-resolved somatic complementation experiments performed by mixing the cytoplasms of mutants altered in the sporulation response, to systematically explore the network structure and to probe its dynamics. This reverse engineering approach presumably can be employed to explore other molecular or genetic signalling systems where the activity of genes or their products can be experimentally controlled in a time-resolved manner.     

Relationship between expression of the PM H<Superscript>+</Superscript>-ATPase,growth and ion partitioning in the leaves of salt-treated <Emphasis Type="Italic">Medicago</Emphasis> species

The role of the plasma membrane (PM) H⁺-ATPase (E.C. 3.6.1.3) in the plants response to salt stress was studied in the perennial leguminosae forage Medicago arborea L. and its close relative Medicago citrina (Font-Quer) Greuter, a species exposed to saline conditions in its original habitat. Plants were solution cultured for 8 days in 1 or 100 mM NaCl. Leaf growth and CO₂ assimilation were more inhibited by salt in M. arborea than in M. citrina. Both species were able to osmoregulate, and salt-treated plants maintained turgor potentials, with no differences between species. Contrasting ion distribution patterns showed that M. citrina was able to exclude Na⁺ from the leaves more selectively, while M. arborea had a greater buildup of leaf blade Na⁺. Isolation of purified PM and quantification of H⁺-ATPase protein by Western blot analysis against the 46E5B11D5 or AHA3 antibodies showed an increase in response to salt stress in the expanding (92%) and expanded leaves (87%) of M. citrina, while no differences were found in the corresponding leaves of M. arborea. The assay of H⁺-ATPase specific activity of the two leaf types in salinized M. citrina confirmed this increase, as activities increased with 55% and 104% for the expanded and expanding leaves, respectively, while no significant differences were found for either leaf type of salinized M. arborea. A possible role of the increased expression of the PM H⁺-ATPase for leaf expansion and ion exclusion in salt-stressed plants is discussed.