GIP-dependent expression of hypothalamic genes

GIP (glucose dependent insulinotrophic polypeptide), originally identified as an incretin peptide synthesized in the gut, has recently been identified, along with its receptors (GIPR), in the brain. Our objective was to investigate the role of GIP in hypothalamic gene expression of biomarkers linked to regulating energy balance and feeding behavior related neurocircuitry. Rats with lateral cerebroventricular cannulas were administered 10 μg GIP or 10 microl artificial cerebrospinal fluid (aCSF) daily for 4 days, after which whole hypothalami were collected. Real time Taqman? RT-PCR was used to quantitatively compare the mRNA expression levels of a set of genes in the hypothalamus. Administration of GIP resulted in up-regulation of hypothalamic mRNA levels of AVP (46.9±4.5 %), CART (25.9±2.7 %), CREB1 (38.5±4.5 %), GABRD (67.1±11 %), JAK2 (22.1±3.6 %), MAPK1 (33.8±7.8 %), NPY (25.3±5.3 %), OXT (49.1±5.1 %), STAT3 (21.6±3.8 %), and TH (33.9±8.5 %). In a second experiment the same set of genes was evaluated in GIPR(-/-) and GIPR(+/?) mice to determine the effect of lack of GIP stimulation on gene expression. In GIPR(-/-) mice expressions of the following genes were down-regulated: AVP (27.1±7.5 %), CART (28.3±3.7 %), OXT (25.2±5.8 %), PTGES (23.9±4.5 %), and STAT3 (8.8±2.3 %). These results suggest that AVP, CART, OXT and STAT3 may be involved in energy balance-related hypothalamic circuits affected by GIP.     

The aadA Gene of Plasmid R100 Confers Resistance to Spectinomycin and Streptomycin in Myxococcus xanthus

Plasmids with the aadA gene from plasmid R100, which confers resistance to the aminoglycosides spectinomycin and streptomycin in Escherchia coli, can be introduced into wild-type Myxococcus xanthus, strain DK1622, by electroporation. Recombinant M. xanthus strains with integrated plasmids carrying the aadA gene acquire resistance to high levels of these antibiotics. Selection for aadA in M. xanthus can be carried out independently of, or simultaneously with, selection for resistance to kanamycin. The kinds and frequencies of recombination events observed between integrative plasmids with aadA and the M. xanthus chromosome are similar to those observed after the transformation of yeast. Cleavage of integrative plasmid DNA at a site adjacent to a region of homology between the plasmid and the M. xanthus genome favors the targeted disruption of M. xanthus genes by allele replacement.     

Comparisons of the release of vasodilator substances from left and right cardiac chambers of the isolated perfused rabbit heart: Implications for intraventricular thrombus formation

Prostacyclin (PgI₂) and endothelium-derived nitric oxide (EDNO) are produced by the arterial and venous endothelium. In addition to their vasodilator action on vascular smooth muscle, both act together to inhibit platelet aggregation and promote platelet disaggregation. EDNO also inhibits platelet adhesion to the endothelium. EDNO and PgI₂ have been shown to be released from the cultured endocardial cells. In this study, we examined the release of vasoactive substances from the intact endocardium by using isolated rabbit hearts perfused with physiological salt solution (95% O₂/5% CO₂, T = 37 °C). The right and left cardiac chambers were perfused through separate constant-flow perfusion loops (physiological salt solution, 8 ml min⁻¹). Effluent from left and right cardiac, separately, was bioassayed on canine coronary artery smooth muscle, which had been contracted with prostaglandin F_{2α_}(2 × 10⁻⁶ M) and no change in tension was exhibit. However, addition of calcium ionophore A23187 (10⁻⁶ M) to the cardiac chambers’ perfusion line induced vasodilation of the bioassay coronary ring, 61.4 ± 7.4% versus 70.49 ± 6.1% of initial prostaglandin F_2α contraction for the left and right cardiac chambers perfusate, respectively (mean ± SEM, n = 10, p > 0.05). Production of vasodilator was blocked totally in the left heart but, only partially blocked in the right heart by adding indomethacin (10⁻⁵ M) to the perfusate, respectively, 95.2 ± 2.2% versus 41.5 ± 4.8% (mean ± SEM, n = 10, p < 0.05). 6-Keto prostaglandin F_1α, measured in the endocardial superfusion effluent was also higher for the left cardiac chambers than for the right at the time of stimulation with the A23187, respectively, 25385.88 ± 5495 pg/ml (n = 8) versus 13,132.45 ± 1839.82 pg/ml (n = 8), (p < 0.05). These results showed that cyclooxygenase pathway plays major role in generating vasoactive substances for the left cardiac chamber endocardium; while it is not the main pathway for the right ventricular endocardium at which EDNO and PgI₂ could act together and potentiate their antithrombogenic activities in isolated perfused rabbit heart. This may be an explanation for the intraventricular thrombus mostly seen in left ventricle rather than in right ventricle as a complication of myocardial infarction.     

Contributions of the hippocampus and medial prefrontal cortex to energy and body weight regulation

The effects of selective ibotenate lesions of the complete hippocampus (CHip), the hippocampal ventral pole (VP), or the medial prefrontal cortex (mPFC) in male rats were assessed on several measures related to energy regulation (i.e., body weight gain, food intake, body adiposity, metabolic activity, general behavioral activity, conditioned appetitive responding). The testing conditions were designed to minimize the nonspecific debilitating effects of these surgeries on intake and body weight. Rats with CHip and VP lesions exhibited significantly greater weight gain and food intake compared with controls. Furthermore, CHip-lesioned rats, but not rats with VP lesions, showed elevated metabolic activity, general activity in the dark phase of the light-dark cycle, and greater conditioned appetitive behavior, compared with control rats without these brain lesions. In contrast, rats with mPFC lesions were not different from controls on any of these measures. These results indicate that hippocampal damage interferes with energy and body weight regulation, perhaps by disrupting higher-order learning and memory processes that contribute to the control of appetitive and consummatory behavior.     

Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

Background  

Tight junctions are an intercellular adhesion complex of epithelial and endothelial cells, and form a paracellular barrier that restricts the diffusion of solutes on the basis of size and charge. Tight junctions are formed by multiprotein complexes containing cytosolic and transmembrane proteins. How these components work together to form functional tight junctions is still not well understood and will require a complete understanding of the molecular composition of the junction.     

2D NMR studies of aminoglycoside antibiotics. Use of relayed coherence transfer for 1H resonance assignment and in situ structure elucidation of amikacin derivatives in reaction mixtures

Phase-sensitive 2D 1H/1H COSY spectra can be used to identify the structures of individual pure specimens of the aminoglycoside antibiotic amikacin and its N-hemisuccinyl derivatives. However, even at 500 MHz the 2D chemical shift dispersion does not allow for unambiguous assignment of all cross-peaks. By use of 2D relayed coherence transfer experiments (RELAY) optimized to detect two-step 1H/1H scalar interactions in which one of the J-values is small, sufficient additional correlations can be obtained from the frequency-isolated resonances to allow facile tracing of all scalar connectivities. Complete assignments of the 1H NMR spectra of amikacin, its 6'-N-hemisuccinamide, and a novel bis(acylate) [gamma-N-(p-vinylbenzoyl)amikacin 6'-N-hemisuccinamide] were obtained for aqueous media. The NMR spectrum of amikacin free base was also assigned in dimethyl sulfoxide solution. The RELAY experiment can be extended to the analysis of reaction mixtures, which allows for the identification and resonance assignment of regioisomeric amikacin haptens in the mixture state. All of the N-monohemisuccinyl isomers of amikacin have been identified in reaction mixtures through the RELAY experiment. The relative reactivities of the amino functions of amikacin toward acylating agents were found to be 6'-N greater than 3-N equal to or greater than 3"-N equal to or greater than gamma-N. However, this reactivity order is altered after the initial acylation event.     

Triiodothyronine depresses the NAD-linked glycerol-3-phosphate dehydrogenase activity of cultured neonatal rat heart cells

The activity of NAD-linked alpha-glycerol-3-phosphate dehydrogenase (NAD-G3PDH; EC 1.1.1.8) was depressed by 35% when the thyroid hormone 3,3',5-triiodo-L-thyronine (20 micrograms/liter) was added to the serum-free, hormonally supplemented medium of cultured neonatal rat heart cells. The degree of depression was greater (65%) when the medium contained normal serum levels of hydrocortisone and insulin. There is a dramatic inverse dose-response relationship between triiodothyronine levels and NAD-G3PDH activity. The classic elevation by thyroid hormones of the FAD-linked alpha-glycerol-3-phosphate dehydrogenase (FAD-G3PD; EC 1.1.99.5) was observed concurrently. The medium-glucose depletion rate in triiodothyronine-free cells was depressed 32% through 11 days-in-culture, indicating reduced glycolytic activity. The activities of nine other metabolically important enzymes which were measured during this study, including hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphofructokinase, pyruvate kinase, malate dehydrogenase, NAD-isocitrate dehydrogenase, NADH cytochrome c reductase, and succinic cytochrome c reductase, did not respond to varying triiodothyronine concentrations.     

Halothane-Induced Alterations of Glucose and Pyruvate Metabolism in Rat Cerebra Synaptosomes

Synaptosomes isolated from rat cerebra were used to study the effects of the inhalational anesthetic, halothane, on cholinergic processes. To identify possible mechanisms responsible for the depression of acetylcholine synthesis, we examined the effects of halothane on precursor metabolite metabolism involved with supplying the cytosol with acetyl-CoA for acetylcholine synthesis. Three percent halothane/air (vol/vol) depressed 14CO2 evolution from labeled pyruvate and glucose. Steady-state 14CO2 evolution from [1-14C]glucose was depressed 84% by halothane, while 14CO2 evolution from [6-14C]glucose and [3,4-14C]glucose was decreased 67 and 52%, respectively, when compared with control conditions. Halothane inhibited the activities of both pyruvate dehydrogenase (14% depression) and ATP-citrate lyase (32% depression). Total synaptosomal acetyl-CoA concentrations were unaffected by halothane. Three percent halothane/air (vol/vol) caused a 77% increase in medium glucose depletion rate from 1.38 nmol (mg protein)-1 min-1 to 2.44 nmol (mg protein)-1 min-1. Production of lactate by the synaptosomes in the presence of halothane increased by 231% from a control rate of 1.44 nmol (mg protein)-1 min-1 to 4.77 nmol (mg protein)-1 min-1. Lactate production rate from pyruvate was also enhanced by 56% in the presence of halothane. These data lend support to the concept that the NAD+/NADH potential may be involved in the halothane-induced depression of acetylcholine synthesis.     

The formation of plasma membrane blebs in hepatocytes exposed to agents that increase cytosolic Ca2+ is mediated by the activation of a non-lysosomal proteolytic system

Exposure of isolated hepatocytes to extracellular ATP, cystamine or ionophore A23187 was associated with an increase in cytosolic Ca2+ concentration, a stimulation of intracellular proteolysis, and the appearance of plasma membrane blebs which preceded the loss of cell viability. Both bleb formation and cell killing were prevented when inhibitors of Ca2+-activated neutral proteases, such as antipain or leupeptin, were included in the incubation medium, whereas inhibitors of lysosomal proteases had no effect. Thus, the activation of a Ca2+-dependent, non-lysosomal proteolytic system appears to be responsible for the plasma membrane blebbing and, ultimately, the cytotoxicity associated with treatment of hepatocytes with agents that disrupt intracellular Ca2+ homeostasis.