pH dependence of the progression in NMR T-2 relaxation times in post-mortem muscle

Continuous NMR T-2 relaxation measurements were carried out on seven rabbit longissimus muscle samples in the period from 25 min to 28 h post-mortem at 200 MHz for H-1. To display differences in post-mortern pH progress and extent of changes in water characteristics during conversion of muscle to meat, three of the seven animals were pre-slaughter injected with adrenaline (0.5 mg/kg live weight 4 h before sacrifice) to differentiate muscle glycogen stores at the time of slaughter. Distributed analysis of T-2 data displayed clear differences in the characteristics of the various transverse relaxation components dependent on progress in pH, as did the water-holding capacity of samples 24 h postmortem. This reveals a pronounced effect of the progressive change in pH on the subsequent development in physical/chemical states of water during the conversion of muscle to meat. Finally, the relaxation characteristics are discussed in relation to supposed post-mortem processes of protein denaturation.

The Arabidopsis mutant iop1 exhibits induced over-expression of the plant defensin gene PDF1.2 and enhanced pathogen resistance

Jasmonate and ethylene are concomitantly involved in the induction of the Arabidopsis plant defensin gene PDF1.2. To define genes in the signal transduction pathway leading to the induction of PDF1.2, we screened for-mutants with induced over-expression of a beta-glucuronidase reporter, under the control of the PDF1.2 promoter. One mutant, iop1 (induced over-expressor of PDF1.2) produced small plants that showed induced over-expression of the pathogenesis-related genes PR-3, PR-4 and PR-1,2 (PDF1.2), combined with a down-regulated induction of PR-1 upon pathogen inoculation. The iop1 mutant showed enhanced resistance to a number of necrotrophic pathogens.

Refolding and purification of the human secreted group IID phospholipase A(2) expressed as inclusion bodies in Escherichia coli

The secreted phospholipases A(2) (sPLA(2)s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A(2) (hsPLA(2)-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA(2)-IID in Escherichia coli, the DNA-coding sequence for hsPLA(2)-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA(2)-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A(2). The refolded recombinant hsPLA(2)-IID demonstrated Ca(2+)-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA(2)-IID which will advance our understanding of the structure-function relationship and biological effects of the protein. (C) 2009 Elsevier Inc. All rights reserved.

The interaction of bothropstoxin-I (Lys49-PLA(2)) with liposome membranes

Bothropstoxin-I (BthTx-I) is a Lys49-PLA(2) from the venom of the snake Bothrops jararacussu, which permeabilizes biological and artificial membranes by a mechanism independent of lipid hydrolysis. This mechanism has been investigated by studying the interaction of nine single tryptophan BthTx-I mutants with negatively charged phospholipid membranes. Changes in the solvent exposure of the tryptophan in each mutant were evaluated comparing the rate of chemical modification (k(mod)) by bromosuccinamide with the maximum intrinsic tryptophan fluorescence emission wavelength (lambda(max)) in buffer and in the presence of 10% DMPA/90% DPPC liposomes. No changes in lambda(max). were observed, whereas k(mod) values for tryptophans at positions 7, 10, 31 and 125 were significantly reduced in the presence of lipids, suggesting that bound phospholipid decreases solvent accessibility at these positions. Since the half-lives of the fluorescence and chemical modification effects differ by at least six orders of magnitude, these results suggest that the bound phospholipid may interact with multiple locations on the protein surface over micro- to millisecond timescales. (C) 2009 Elsevier Ltd. All rights reserved.

Mapping of suramin binding sites on the group IIA human secreted phospholipase A(2)

Suramin is a polysulphonated napthylurea used as an antiprotozoal/anthelminitic drug, which also inhibits a broad range of enzymes. Suramin binding to recombinant human secreted group IIA phospholipase A(2) (hsPLA(2)GIIA) was investigated by molecular dynamics simulations (MD) and isothermal titration calorimetry (ITC). MD indicated two possible bound suramin conformations mediated by hydrophobic and electrostatic interactions with amino-acids in three regions of the protein. namely the active-site and residues located in the N- and C-termini, respectively. All three binding sites are located on the phospholipid membrane recognition surface, suggesting that suramin may inhibit the enzyme, and indeed a 90% reduction in hydrolytic activity was observed in the presence of 100 nM suramin. These results correlated with ITC data, which demonstrated 2.7 suramin binding sites on the hsPLA(2)GIIA, and indicates that suramin represents a novel class of phosphohpase A(2) inhibitor. (C) 2009 Elsevier Inc. All rights reserved.

Association Between Increased Platelet P-Selectin Expression and Obesity in Patients With Type 2 Diabetes A BARI 2D (Bypass Angioplasty Revascularization Investigation 2 Diabetes) substudy

OBJECTIVE- To determine whether obesity increases platelet reactivity and thrombin activity in patients with type 2 diabetes plus stable coronary artery disease. RESEARCH DESIGN AND METHODS- We assessed platelet reactivity and markers of thrombin generation and activity in 193 patients from nine clinical sites of the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D). Blood taken at the time of enrollment was used for assay of the concentration of prothrombin fragment 1.2 (PT1.2, released when prothrombin is activated) and fibrinopeptide A (FPA, released when fibrinogen is cleaved). Platelet activation was identified with the use of flow cytometry in response to 0, 0.2, and 1 mu mol/l adenosine diphosphate (ADP). RESULTS- Concentrations of FPA, PT1.2, and platelet activation in the absence of agonist were low. Greater BMI was associated with higher platelet reactivity in response to 1 mu m ADP as assessed by surface expression of P-selectin (r = 0.29, P < 0.0001) but not reflected by the binding of fibrinogen to activated glycoprotein IIb-IIIa. BMI was not associated with concentrations of FPA or PT1.2. Platelet reactivity correlated negatively with A1C (P < 0.04), was not related to the concentration Of triglycerides in blood, and did not correlate with the concentration of C-reactive peptide. CONCLUSIONS- Among patients enrolled in this substudy of BARI 2D, a greater BMI was associated with higher platelet reactivity at the time of enrollment. Our results suggest that obesity and insulin resistance that accompanies obesity may influence platelet reactivity in patients with type 2 diabetes.

The 2.0 angstrom crystal structure of a pocilloporin at pH 3.5: The structural basis for the linkage between color transition and halide binding

The pocilloporin Rtms5 and an engineered variant Rtms5(H146S) undergo distinct color transitions (from blue to red to yellow to colorless) in a pH-dependent manner. pK(a) values of 4.1 and 3.2 were determined for the blue (absorption lambda(max), 590 nm) to yellow (absorption lambda(max), similar to 453 nm) transitions of Rtms5 and Rtms5H(146). The pK(a) for the blue-yellow transition of Rtms5H(146S) increased by 1.4 U in the presence of 0.1 M KI, whereas the pK(a) for the same transition of Rtms5 was relatively insensitive to added halides. To understand the structural basis for these observations, we have determined to 2.0 A resolution the crystal structure of a yellow form of Rtms5(H146S) at pH 3.5 in the presence of iodide. Iodide was found occupying a pocket in the structure with a pH of 3.5, forming van der Waals contacts with the tyrosyl moiety of the chromophore. Elsewhere, it was determined that this pocket is occupied by a water molecule in the Rtms5(H141S) structure (pH 8.0) and by the side chain of histidine 146 in the wild-type Rtms5 structure. Collectively, our data provide an explanation for the observed linkage between color transitions for Rtms5(H146S) and binding to halides.

Carbamazepine can induce kidney water absorption by increasing aquaporin 2 expression

Background. Carbamazepine (Carba) is an anticonvulsant and psychotropic drug used widely for the treatment of intellectual disability and severe pains, but the incidence of hyponatremia is a common related occurrence. This hyponatremia is frequently attributed to a SIADH induced by this drug. It is also known that Carba is used to decrease the urinary volume in Diabetes Insipidus (DI) because it has an antidiuretic effect. Lithium (Li) is one of the most important drugs used to treat bipolar mood disorders. However Li has the undesirable capacity to induce DI. Nowadays, the association of these drugs is used in the treatment of patients with psychiatric and neurological problems. Methods. In vivo and in vitro (microperfusion) experiments were developed to investigate the effect of Carba in the rat Inner Medullary Collecting Duct (IMCD). Results. The results revealed that Carba was able to stimulate the V2 vasopressin receptor-Protein G complex increasing the water permeability (Pf) and water absorption. In vivo studies showed that in rats with lithium-induced DI, Carba decreased the urinary volume and increased the urinary osmolality. AQP2 expression was increased both in normal IMCD incubated with Carba and in IMCD from lithium-induced DI after Carba addition to the diet, when compared with the control. Conclusion. These results showed that the hyponatremia observed in patients using this anticonvulsant drug, at least in part, is due to the Carba capacity to increase IMCD`s Pf and that the Lithium-Carbamazepine association is beneficial to the patient.

Macromolecular assembly of polycystin-2 intracytosolic C-terminal domain

Mutations in PKD2 are responsible for approximately 15% of the autosomal dominant polycystic kidney disease cases. This gene encodes polycystin-2, a calcium-permeable cation channel whose C-terminal intracytosolic tail (PC2t) plays an important role in its interaction with a number of different proteins. In the present study, we have comprehensively evaluated the macromolecular assembly of PC2t homooligomer using a series of biophysical and biochemical analyses. Our studies, based on a new delimitation of PC2t, have revealed that it is capable of assembling as a homotetramer independently of any other portion of the molecule. Our data support this tetrameric arrangement in the presence and absence of calcium. Molecular dynamics simulations performed with a modified all-atoms structure-based model supported the PC2t tetrameric assembly, as well as how different populations are disposed in solution. The simulations demonstrated, indeed, that the best-scored structures are the ones compatible with a fourfold oligomeric state. These findings clarify the structural properties of PC2t domain and strongly support a homotetramer assembly of PC2.

Creatine in Type 2 Diabetes: A Randomized, Double-Blind, Placebo-Controlled Trial

GUALANO, B., V. DE. SALLES PAINNELI, H. ROSCHEL, G. G. ARTIOLI, M. NEVES JR, A. L. DE SA PINTO, M. E. DA SILVA, M. R. CUNHA, M. C. G. OTADUY, C. DA COSTA LEITE, J. C. FERREIRA, R. M. PEREIRA, P. C. BRUM, E. BONFA, and A. H. LANCHA JR. Creatine in Type 2 Diabetes: A Randomized, Double-Blind, Placebo-Controlled Trial. Med. Sci. Sports Exerc., Vol. 43, No. 5, pp. 770-778, 2011. Creatine supplementation improves glucose tolerance in healthy subjects. Purposes: The aim was to investigate whether creatine supplementation has a beneficial effect on glycemic control of type 2 diabetic patients undergoing exercise training. Methods: A 12-wk randomized, double-blind, placebo-controlled trial was performed. The patients were allocated to receive either creatine (CR) (5 g.d(-1)) or placebo (PL) and were enrolled in an exercise training program. The primary outcome was glycosylated hemoglobin (Hb(A1c)). Secondary outcomes included the area under the curve of glucose, insulin, and C-peptide and insulin sensitivity indexes. Physical capacity, lipid profile, and GLUT-4 protein expression and translocation were also assessed. Results: Twenty-five subjects were analyzed (CR: n = 13; PL: n = 12). Hb(A1c) was significantly reduced in the creatine group when compared with the placebo group (CR: PRE = 7.4 +/- 0.7, POST = 6.4 +/- 0.4; PL: PRE = 7.5 +/- 0.6, POST = 7.6 +/- 0.7; P = 0.004; difference = -1.1%, 95% confidence interval = -1.9% to -0.4%). The delta area under the curve of glucose concentration was significantly lower in the CR group than in the PL group (CR = -7790 +/- 4600, PL = 2008 +/- 7614; P = 0.05). The CR group also presented decreased glycemia at times 0, 30, and 60 min during a meal tolerance test and increased GLUT-4 translocation. Insulin and C-peptide concentrations, surrogates of insulin sensitivity, physical capacity, lipid profile, and adverse effects were comparable between the groups. Conclusions: Creatine supplementation combined with an exercise program improves glycemic control in type 2 diabetic patients. The underlying mechanism seems to be related to an increase in GLUT-4 recruitment to the sarcolemma.

Aerobic Exercise Training-Induced Left Ventricular Hypertrophy Involves Regulatory MicroRNAs, Decreased Angiotensin-Converting Enzyme-Angiotensin II, and Synergistic Regulation of Angiotensin-Converting Enzyme 2-Angiotensin (1-7)

Aerobic exercise training leads to a physiological, nonpathological left ventricular hypertrophy; however, the underlying biochemical and molecular mechanisms of physiological left ventricular hypertrophy are unknown. The role of microRNAs regulating the classic and the novel cardiac renin-angiotensin (Ang) system was studied in trained rats assigned to 3 groups: (1) sedentary; (2) swimming trained with protocol 1 (T1, moderate-volume training); and (3) protocol 2 (T2, high-volume training). Cardiac Ang I levels, Ang-converting enzyme (ACE) activity, and protein expression, as well as Ang II levels, were lower in T1 and T2; however, Ang II type 1 receptor mRNA levels (69% in T1 and 99% in T2) and protein expression (240% in T1 and 300% in T2) increased after training. Ang II type 2 receptor mRNA levels (220%) and protein expression (332%) were shown to be increased in T2. In addition, T1 and T2 were shown to increase ACE2 activity and protein expression and Ang (1-7) levels in the heart. Exercise increased microRNA-27a and 27b, targeting ACE and decreasing microRNA-143 targeting ACE2 in the heart. Left ventricular hypertrophy induced by aerobic training involves microRNA regulation and an increase in cardiac Ang II type 1 receptor without the participation of Ang II. Parallel to this, an increase in ACE2, Ang (1-7), and Ang II type 2 receptor in the heart by exercise suggests that this nonclassic cardiac renin-angiotensin system counteracts the classic cardiac renin-angiotensin system. These findings are consistent with a model in which exercise may induce left ventricular hypertrophy, at least in part, altering the expression of specific microRNAs targeting renin-angiotensin system genes. Together these effects might provide the additional aerobic capacity required by the exercised heart. (Hypertension. 2011;58:182-189.).

Guanosine promotes B16F10 melanoma cell differentiation through PKC-ERK 1/2 pathway

Malignant melanoma is one of the most lethal cancers. Nowadays, several anti-melanoma therapies have been employed. However, the poor prognosis and/or the increased toxicity of those treatments clearly demonstrate the requirement of searching for new drugs or novel combined chemotherapeutic protocols, contemplating both effectiveness and low toxicity. Guanosine (Guo) has been used in combination with acriflavina to potentiate the latter`s antitumor activity, through still unknown mechanisms. Here, we show that Guo induces B16F10 melanoma cell differentiation, attested by growth arrest, dendrite-like outgrowth and increased melanogenesis, and also reduced motility. A sustained ERK 1/2 phosphorylation was observed after Guo treatment and ERK inhibition led to blockage of dendritogenesis. Intracellular cyclic AMP was not involved in ERK activation, since its levels remained unchanged. Protein kinase C (PKC), in contrast to phospholipase C (PLC), inhibition completely prevented ERK activation. While the classical melanoma differentiation agent forskolin activates cAMP-PKA-Raf-MEK-ERK pathway in B16F10 cells, here we suggest that a cAMP-independent, PKC-ERK axis is involved in Guo-induced B16F10 differentiation. Altogether, our results show that Guo acts as a differentiating agent, with cytostatic rather than cytotoxic properties, leading to a decreased melanoma malignancy. Thus, we propose that Guo may be envisaged in combination with lower doses of conventional anti-melanoma drugs, in an attempt to prevent or diminish their adverse effects. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

Tube feeding with a diabetes-specific feed for 12 weeks improves glycaemic control in type 2 diabetes patients

Background and aims: Assess longer-term (12 weeks) effects of a diabetes-specific feed on postprandial glucose response, glycaemic control (HbA1c), lipid profile, (pre)-albumin, clinical course and tolerance in diabetic patients. Methods: In this randomized, controlled, double-blind, parallel group study 25 type 2 diabetic patients on tube feeding were included. Patients received a soy-protein based, multi-fibre diabetes-specific feed or isocaloric, fibre-containing standard feed for 12 weeks, while continuing on their anti-diabetic medication. At the beginning, after 6 and 12 weeks, several (glycaemic) parameters were assessed. Results: The postprandial glucose response (iAUC) to the diabetes-specific feed was lower at the 1st assessment compared with the standard feed (p = 0.008) and this difference did not change over time. HbA1c decreased over time in the diabetes-specific and not in the standard feed group (treatment*time:p = 0.034): 6.9 +/- 0.3% (mean +/- SEM) at baseline vs. 6.2 +/- 0.4% at 12 weeks in the diabetes-specific group compared to 7.9 +/- 0.3% to 8.7 +/- 0.4% in the standard feed group. No significant treatment*time effect was found for fasting glucose, insulin, (pre-) albumin or lipid profile, except for increase of HDL in the diabetes-specific group. Conclusions: The diabetes-specific feed studied significantly improved longer-term glycaemic control in diabetic patients. This was achieved in addition to on-going anti-diabetic medication and may affect clinical outcome. (C) 2009 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism.

Involvement of Astroglial Fibroblast Growth Factor-2 and Microglia in the Nigral 6-OHDA Parkinsonism and a Possible Role of Glucocorticoid Hormone on the Glial Mediated Local Trophism and Wound Repair

We have observed in previous studies that 6-hydroxydopamine (6-OHDA)-induced lesions in the nigrostriatal dopamine (DA) system promote increases of the astroglial basic fibroblast growth factor (FGF-2, bFGF) synthesis in the ascending DA pathways, event that could be modified by adrenosteroid hormones. Here, we first evaluated the changes of microglial reactivity in relation to the FGF-2-mediated trophic responses in the lesioned nigrostriatal DA system. 6-OHDA was injected into the left side of the rat substantia nigra. The OX42 immunohistochemistry combined with stereology showed the time course of the microglial activation. The OX42 immunoreactivity (IR) was already increased in the pars compacta of the substantia nigra (SNc) and ventral tegmental area (VTA) 2 h after the 6-OHDA injection, peaked on day 7, and remained increased on the 14th day time-interval. In the neostriatum, OX42 immunoreactive (ir) microglial profiles increased at 24 h, peaked at 72 h, was still increased at 7 days but not 14 days after the 6-OHDA injection. Two-colour immunofluorescence analysis of the tyrosine hydroxylase (TH) and OX42 IRs revealed the presence of small patches of TH IR within the activated microglia. A decreased FGF-2 IR was seen in the cytoplasm of DA neurons of the SNc and VTA as soon as 2 h after 6-OHDA injection. The majority of the DA FGF-2 ir cells of these regions had disappeared 72 h after neurotoxin. The astroglial FGF-2 IR increased in the SNc and VTA, which peaked on day 7. Two-colour immunofluorescence and immunoperoxidase analyses of the FGF-2 and OX42 IRs revealed no FGF-2 IR within the reactive or resting microglia. Second, we have evaluated in a series of biochemical experiments whether adrenocortical manipulation can interfere with the nigral lesion and the state of local astroglial reaction, looking at the TH and GFAP levels respectively. Rats were adrenalectomized (ADX) and received a nigral 6-OHDA stereotaxical injection 2 days later and sacrificed up to 3 weeks after the DA lesion. Western blot analysis showed time-dependent decrease and elevation of TH and GFAP levels, respectively, in the lesioned versus contralateral midbrain sides, events potentiated by ADX and worsened by corticosterone replacement. ADX decreased the levels of FGF-2 protein (23 kDa isoform) in the lesioned side of the ventral midbrain compared contralaterally. The results indicate that reactive astroglia, but not reactive microglia, showed an increased FGF-2 IR in the process of DA cell degeneration induced by 6-OHDA. However, interactions between these glial cells may be relevant to the mechanisms which trigger the increased astroglial FGF-2 synthesis and thus may be related to the trophic state of DA neurons and the repair processes following DA lesion. The findings also gave further evidence that adrenocortical hormones may regulate astroglial-mediated trophic mechanisms and wound repair events in the lesioned DA system that may be relevant to the progression of Parkinson`s disease.

Inhibition of phospholipase A(2) increases Tau phosphorylation at Ser214 in embryonic rat hippocampal neurons

Background: Arachidonic acid is released from cellular membranes by the action of phospholipase A(2) (PLA(2)) and is implicated in microtubule-associated protein Tau phosphorylation. Tau hyperphosphorylation affects its ability to stabilize microtubules. Objective: To determine the effect of PLA(2) inhibition on the phosphorylation state of Tau phosphoepitopes in primary cultures of hippocampal neurons. Methods: 4 DIC neurons were incubated at different concentrations of methyl-arachidonylfluorophosphonate (MAFP), an irreversible inhibitor of cPLA(2) and iPLA(2). Changes on Tau phosphorylation were determined by Western blotting with a panel of anti-Tau antibodies (C-terminal, Ser199/202, Ser202/205, Ser396 and Ser214). Results: The Ser214 site was hyperphosphorylated upon MAFP treatment. Significant differences were observed with 10 mu M (p = 0.01), 50 mu M (p = 0.01) and 100 mu M (p = 0.05) of MAFP. Less-intense changes were found in other phosphoepitopes. Conclusion: The present findings indicate that the phosphorylation of Ser214 is regulated by c- and/or iPLA(2), whereas other phosphoepitopes primarily regulated by GKS3b were not affected. (C) 2009 Elsevier Ltd. All rights reserved.