Phosphoinositide 3-kinase is involved in the glucagon-induced translocation of aquaporin-8 to hepatocyte plasma membrane

BACKGROUND INFORMATION: PI3K (phosphoinositide 3-kinase) mediates several signal transduction pathways in hepatocytes, including some involved in the regulation of vesicle trafficking. Hepatocytes express the water channel AQP8 (aquaporin-8) predominantly in an intracellular location, and it redistributes to the canalicular membrane, upon stimulation with the hormone glucagon, by a cAMP/protein kinase A-dependent mechanism. Since glucagon is capable of stimulating PI3K activity in hepatocytes and a cross talk between cAMP and PI3K has been suggested, in the present study, we examine whether PI3K activation is involved in the glucagon-induced translocation of AQP8. RESULTS: By quantitative immunoblotting of purified hepatocyte plasma membranes, we found that the preincubation of cells with two structurally different PI3K inhibitors, wortmannin or LY294002, prevented the glucagon-induced translocation of AQP8 to hepatocyte plasma membrane. Confocal immunofluorescence microscopy in cultured hepatocytes confirmed the dependence of the hormone-induced redistribution of AQP8 on PI3K activity. Functional studies showed that the PI3K inhibitors were also capable of preventing the glucagon-induced increase in hepatocyte osmotic membrane water permeability. CONCLUSIONS: Our results suggest that PI3K activation is involved in the glucagon-dependent signal transduction pathways leading to hepatocyte AQP8 translocation.     

Saccharopolyspora erythraea-catalyzed bioconversion of 6-deoxyerythronolide B analogs for production of novel erythromycins.

A method was developed for the large-scale bioconversion of novel 6-deoxyerythronolide B (6-dEB) analogs into erythromycin analogs. Erythromycin biosynthesis in Saccharopolyspora erythraea proceeds via the formation of a polyketide aglycone, 6-dEB, which is subsequently glycosylated, hydroxylated and methylated to yield the antibiotic erythromycin A. A modular polyketide synthase (PKS) directs 6-dEB synthesis using a dedicated set of active sites for the condensation of each of seven propionate units. Strategies based on genetic manipulation and precursor feeding are available for the efficient generation of novel 6-dEB analogs using a plasmid-based system in Streptomyces coelicolor. 6-dEB and 13-substituted 6-dEB analogs produced in this manner were fed to S. erythraea mutants which could not produce 6-dEB, yet retained their 6-dEB modification systems, and resulted in the generation of erythromycin A and 13-substituted erythromycin A analogs. Erythromycin B, C and D analogs were observed as intermediates of the process. Dissolved oxygen, temperature, the specific aglycone feed concentration, and pH were found to be important for obtaining a high yield of erythromycin A analogs. Cultivation conditions were identified which resulted in the efficient bioconversion of 6-dEB analogs into erythromycin A analogs, which this process demonstrated at the 100 l scale.     

Thymidylate synthase with a C-terminal deletion catalyzes partial reactions but is unable to catalyze thymidylate formation.

The V316Am mutant of Lactobacillus casei thymidylate synthase has a single amino acid deletion at the C-terminus which abolishes catalysis of dTMP formation. However, V316Am catalyzes two partial reactions which require covalent catalysis: a CH2H4folate-dependent exchange of the 5-hydrogen of dUMP for protons in water and a thiol-dependent dehalogenation of 5-bromo- and 5-iodo-dUMP. These reactions proceed with kcat and Km values similar to those of the wild-type TS-catalyzed reactions. dUMP, dTMP, and FdUMP are competitive inhibitors of the debromination reaction with Ki values similar to those obtained with wild-type enzyme. These results show that removal of the terminal valine does not alter the ability of the enzyme to bind to or form covalent bonds with nucleotide ligands. V316Am also forms a covalent ternary complex with FdUMP and CH2H4folate. However, the affinity of the TS-FdUMP complex for the cofactor is reduced, and the rate of covalent ternary complex formation and its stability are significantly lower than with wild-type TS. These results allow us to place the major defects of the mutation on steps that occur subsequent to initial CH2H4folate binding.     

Surface-exposed amino acids of eosinophil cationic protein play a critical role in the inhibition of mammalian cell proliferation

Eosinophil cationic protein (ECP) is a ribonuclease secreted from activated eosinophils that may cause tissue injure as a result of eosinophilic inflammation. ECP possesses bactericidal, antiviral and helminthotoxic activity and inhibits mammalian cell growth. The mechanism by which ECP exerts its toxicity is not known but it has been related to the ability of the protein to destabilise lipid bilayers. We have assessed the involvement of some cationic and aromatic surface exposed residues of ECP in the inhibition of proliferation of mammalian cell lines. We have constructed ECP mutants for the selected residues and assessed their ability to prevent cell growth. Trp10 and Trp35 together with the adjacent stacking residue are critical for the damaging effect of ECP on mammalian cell lines. These residues are also crucial for the membrane disruption activity of ECP. Other exposed aromatic residues packed against arginines (Arg75-Phe76 and Arg121-Tyr122) and specific cationic amino acids (Arg101and Arg104) of ECP play a secondary role in the cell growth inhibition. This may be related to the ability of the protein to bind carbohydrates such as those found on the surface of mammalian cells.     

Prolonged Ethanol Ingestion Increases Renal AQP2 and AQP3 Expression in Adult Ratsand in Their Offspring

This study evaluates the effect of prolonged ethanol ingestion on the renal ability to concentrate urine. Suckling Wistar rats born to mothers given ethanol before and during gestation and suckling periods (ethanol-exposed offspring) were used and the results were compared with those obtained from offspring of dams given diets containing no ethanol. Comparisons were also made between progenitors with or without prolonged ethanol ingestion. Body and kidney weights; arginine-vasopressin (AVP) and aldosterone plasma levels; plasma, urine and renal papillary osmolality; urine outflow; kidney AQP2, AQP3 and AQP4 expression and diencephalon AVP mRNA expression were determined. As compared with control offspring, the ethanol-exposed offspring present i) lower body and kidney weights; ii) lower urine outflow; iii) higher renal AQP2 and AQP3 mRNA; iv) higher renal AQP2 protein content and v) higher urine and renal papillary osmolality. These changes were also observed in the ethanol-treated progenitors, although they were of smaller magnitude. Plasma osmolality, renal AQP4 mRNA, AVP plasma levels and diencephalon AVP mRNA expression were not affected by the ethanol treatment. Plasma levels of aldosterone were only significantly increased in the ethanol-exposed suckling rats. It is concluded that maternal ethanol ingestion before and during gestation and suckling periods affects the renal function of the offspring, up-regulating renal AQP2 expression by an AVP-independent mechanism. Ethanol-treated progenitors manifest similar renal changes, although of lesser magnitude than the offspring.     

Associations between epicardial adipose tissue,subclinical atherosclerosis and high-density lipoprotein composition in type 1 diabetes

Background

The pathophysiology of cardiovascular complications in people with type 1 diabetes (T1DM) remains unclear. An increase in epicardial adipose tissue (EAT) and alterations in the composition of high-density lipoprotein (HDL) are associated with coronary artery disease, but information on its relationship in T1DM is very limited. Our aim was to determine the association between EAT volume, subclinical atherosclerosis, and HDL composition in type 1 diabetes.

Methods

Seventy-two long-term patients with T1DM without clinical atherosclerosis were analyzed. EAT volume and subclinical atherosclerosis were measured using cardiac computed tomography angiography. EAT was adjusted according to body surface to obtain an EAT index (iEAT). HDL composition was determined.

Results

The mean iEAT was 40.47?±?22.18 cc/m². The bivariate analysis showed positive associations of the iEAT with gender, age, hypertension, dyslipidemia, smoking, body mass index, waist circumference, insulin dose, and triglyceride (P?<?0.05). The iEAT correlated positively with small HDL, increased content of apolipoprotein (apo)A-II and apoC-III, and decreased content of apoE and free cholesterol. Multiple linear regression showed that age, apoA-II content in HDL, and waist circumference were independently associated with the iEAT. Fifty percent of the patients presented subclinical atherosclerotic lesions. These patients had a higher iEAT, and their HDL contained less cholesterol and more apoA-II and lipoprotein-associated phospholipase A2 than patients without subclinical atherosclerosis.

Conclusion

Alterations in the composition of HDL in TIDM are associated with increased iEAT and the presence of subclinical atherosclerosis. We propose that these abnormalities of HDL composition could be useful to identify T1DM patients at highest cardiovascular risk.

     

Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart

Isolated rat heart perfused with 1.5-7.5µM NO solutions or bradykinin, which activates endothelial NOsynthase, showed a dose-dependent decrease in myocardial O₂uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 µM NO, n = 18,P < 0.05) and to 1.2 ± 0.1 µM O₂ · min¹ · gtissue¹ (10 µM bradykinin, n = 10,P < 0.05). Perfused NO concentrations correlated with aninduced release of hydrogen peroxide (H₂O₂) inthe effluent (r = 0.99, P < 0.01). NO markedlydecreased the O₂ uptake of isolated rat heart mitochondria(50% inhibition at 0.4 µM NO, r = 0.99,P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b andcytochrome c, which accounts for the effects in O₂uptake and H₂O₂ release. Most NO was bound tomyoglobin; this fact is consistent with NO steady-state concentrationsof 0.1-0.3 µM, which affect mitochondria. In the intact heart,finely adjusted NO concentrations regulate mitochondrial O₂uptake and superoxide anion production (reflected byH₂O₂), which in turn contributes to thephysiological clearance of NO through peroxynitrite formation.

     

Changes in glucose 1,6-bisphosphate content in rat skeletal muscle during contraction.

Glucose 1,6-bisphosphate, fructose 2,6-bisphosphate, glycogen, lactate and other glycolytic metabolites were measured in rat gastrocnemius muscle, which was electrically stimulated in situ via the sciatic nerve. Both the frequency and the duration of stimulation were varied to obtain different rates of glycolysis. There was no apparent relationship between fructose 2,6-bisphosphate content and lactate accumulation in contracting muscle. In contrast, glucose 1,6-bisphosphate content increased with lactate concentration during contraction. It is suggested that the increase in glucose 1,6-bisphosphate could play a role in phosphofructokinase stimulation and in the activation of the glycolytic flux during muscle contraction.     

Immunological properties of rat phosphoglycerate mutase isozymes

In mammalian tissues three phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1) isozymes result from the homo-dimeric and hetero-dimeric combinations of two subunits (types M and B). Whereas rabbit antisera against type M subunit (purified from rat muscle) and against type BB isozyme (purified from rat brain) possessed a high degree of specificity, both antisera reacted with type BB and MM isozymes, as demonstrated by immunoneutralization and ELISA. Both the M subunit and B subunit were more immunoreactive than their respective dimeric isozymes. Subunits type M and B may possess common antigenic determinants, and some of these determinants may be sterically hindered in their dimeric structures.