Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H2O2 in HL-1 mouse cardiac muscle cells

Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H2O2), but not angiotensin II, stimulated MIF expression in HL-1 cells. H2O2-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H2O2-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.

Myocardial ischemic preconditioning upregulated protein 1(Mipu1):zinc finger protein 667 - a multifunctional KRAB/C2H2 zinc finger protein

Myocardial ischemic preconditioning upregulated protein 1 (Mipu1) is a newly discovered upregulated gene produced in rats during the myocardial ischemic preconditioning process. Mipu1 cDNA contains a 1824-base pair open reading frame and encodes a 608 amino acid protein with an N-terminal Krüppel-associated box (KRAB) domain and classical zinc finger C2H2 motifs in the C-terminus. Mipu1 protein is located in the cell nucleus. Recent studies found that Mipu1 has a protective effect on the ischemia-reperfusion injury of heart, brain, and other organs. As a nuclear factor, Mipu1 may perform its protective function through directly transcribing and repressing the expression of proapoptotic genes to repress cell apoptosis. In addition, Mipu1 also plays an important role in regulating the gene expression of downstream inflammatory mediators by inhibiting the activation of activator protein-1 and serum response element.

PPARγ induces growth inhibition and apoptosis through upregulation of insulin-like growth factor-binding protein-3 in gastric cancer cells

Peroxisome proliferator activator receptor-gamma (PPARγ) is a ligand-activated transcriptional factor involved in the carcinogenesis of various cancers. Insulin-like growth factor-binding protein-3 (IGFBP-3) is a tumor suppressor gene that has anti-apoptotic activity. The purpose of this study was to investigate the anticancer mechanism of PPARγ with respect to IGFBP-3. PPARγ was overexpressed in SNU-668 gastric cancer cells using an adenovirus gene transfer system. The cells in which PPARγ was overexpressed exhibited growth inhibition, induction of apoptosis, and a significant increase in IGFBP-3 expression. We investigated the underlying molecular mechanisms of PPARγ in SNU-668 cells using an IGFBP-3 promoter/luciferase reporter system. Luciferase activity was increased up to 15-fold in PPARγ transfected cells, suggesting that PPARγ may directly interact with IGFBP-3 promoter to induce its expression. Deletion analysis of the IGFBP-3 promoter showed that luciferase activity was markedly reduced in cells without putative p53-binding sites (-Δ1755, -Δ1795). This suggests that the critical PPARγ-response region is located within the p53-binding region of the IGFBP-3 promoter. We further demonstrated an increase in PPARγ-induced luciferase activity even in cells treated with siRNA to silence p53 expression. Taken together, these data suggest that PPARγ exhibits its anticancer effect by increasing IGFBP-3 expression, and that IGFBP-3 is a significant tumor suppressor.

Dexmedetomidine decreases the inflammatory response to myocardial surgery under mini-cardiopulmonary bypass

Cardiopulmonary bypass (CPB) with extracorporeal circulation produces changes in the immune system accompanied by an increase in proinflammatory cytokines and a decrease in anti-inflammatory cytokines. We hypothesize that dexmedetomidine (DEX) as an anesthetic adjuvant modulates the inflammatory response after coronary artery bypass graft surgery with mini-CPB. In a prospective, randomized, blind study, 12 patients (4 females and 8 males, age range 42-72) were assigned to DEX group and compared with a conventional total intravenous anesthesia (TIVA) group of 11 patients (4 females and 7 males). The endpoints used to assess inflammatory and biochemical responses to mini-CPB were plasma interleukin (IL)-1, IL-6, IL-10, interferon (INF)-γ, tumor necrosis factor (TNF)-α, C-reactive protein, creatine phosphokinase, creatine phosphokinase-MB, cardiac troponin I, cortisol, and glucose levels. These variables were determined before anesthesia, 90 min after beginning CPB, 5 h after beginning CPB, and 24 h after the end of surgery. Endpoints of oxidative stress, including thiobarbituric acid reactive species and delta-aminolevulinate dehydratase activity in erythrocytes were also determined. DEX+TIVA use was associated with a significant reduction in IL-1, IL-6, TNF-α, and INF-γ (P<0.0001) levels compared with TIVA (two-way ANOVA). In contrast, the surgery-induced increase in thiobarbituric acid reactive species was higher in the DEX+TIVA group than in the TIVA group (P<0.01; two-way ANOVA). Delta-aminolevulinate dehydratase activity was decreased after CPB (P<0.001), but there was no difference between the two groups. DEX as an adjuvant in anesthesia reduced circulating IL-1, IL-6, TNF-α, and INF-γ levels after mini-CPB. These findings indicate an interesting anti-inflammatory effect of DEX, which should be studied in different types of surgical interventions.

Carbohydrate composition of ripe pineapple (cv. perola) and the glycemic response in humans

Brazil is the third largest producer of pineapple (Ananas comosus) and the market for fresh pineapple is sustained by the Hawaii and Perola cultivars. In this work the Perola cultivar was divided into three main parts, shell, core and pulp, for characterization. Moisture in the pulp was higher (between 10 and 15%) than in the shell and core. The amount of protein was higher in the core (35%) than in the pulp and shell. Perola contained relatively low concentrations of total ascorbic acid in the edible parts, although higher levels of ascorbic acid in the shell. Citric acid corresponded to almost 60% of the total organic acids. The total soluble sugars [~7-12% (FW)] were predominantly sucrose, fructose and glucose. The core had almost twice as much total sugar (12%) than the pulp (6.8%). The amount of insoluble dietary fiber was around 1%, and the soluble fiber was less than 0.1%. The pulp showed the highest concentration of polyphenols (0.49%) and antioxidant activity (33 µmol.g-1) out of the parts. The consumption of the pineapple pulp or core produced a high glycemic index (~93%), but considering the glycemic load, this fruit can be considered as low dietary.

Instrumental color and sensory acceptance of soy-based emulsions: a response surface approach

Response Surface Methodology (RSM) was applied to evaluate the chromatic features and sensory acceptance of emulsions that combine Soy Protein (SP) and red Guava Juice (GJ). The parameters analyzed were: instrumental color based on the coordinates a* (redness), b* (yellowness), L* (lightness), C* (chromaticity), h* (hue angle), visual color, acceptance, and appearance. The analyses of the results showed that GJ was responsible for the high measured values of red color, hue angle, chromaticity, acceptance, and visual color, whereas SP was the variable that increased the yellowness intensity of the assays. The redness (R²adj = 74.86%, p < 0.01) and hue angle (R²adj = 80.96%, p < 0.01) were related to the independent variables by linear models, while the sensory data (color and acceptance) could not be modeled due to a high variability. The models of yellowness, lightness, and chromaticity did not present lack of fit but presented adjusted determination coefficients bellow 70%. Notwithstanding, the linear correlations between sensory and instrumental data were not significant (p > 0.05) and low Pearson coefficients were obtained. The results showed that RSM is a useful tool to develop soy-based emulsions and model some chromatic features of guava-based emulsions through RSM.

Lactose intolerance and cow's milk protein allergy

Abstract Adverse reactions to food intake have very diverse etiology and symptomatology. Regarding milk, its food allergy is presented as lactose intolerance, the sugar in milk, or allergy to milk protein. Despite having different symptomatology, confusions among allergic conditions to dairy and its mediators are common. Milk protein allergy originates from protein components present in milk, causing reactions to either the protein fractions in emulsion (caseins) or in whey (milk albumin). The allergic reaction is type IV mediated by T lymphocytes. The allergic reaction produces severe cellular damage and it triggers physical, mental and emotional symptomatology that may vary in time, intensity and severity. Lactose intolerance is originated by total or partial absence of the enzyme that digests this disaccharide. Lactose intolerance can be primary or congenital and secondary; the former being more rare and severe, the latter being more common. Lactase deficiency can be diagnosed by symptoms associated with cramping and diarrhea. Thus, the objective of this study was to conduct a review of available literature on cow’s milk protein allergy and lactose intolerance.

Physiological response of cowpea seeds to salinity stress

This work aimed to evoluate physiological response of cowpea (Vigna unguiculata L. Walp) seeds submitted to salt stress. Seeds of cultivars 'Epace-10'; 'Canapu' and 'Pitiúba' of cowpea, were submitted to germination test in germinator at 25(0)C, in "germitest" papers imbibed in distilled water or in 0, 10, 50, 100 and 200mol m-3 NaCl solutions. At the first and second counting of the germination test, normal seedlings were accounted, weighted and dried, obtaining data for vigor, total germination, fresh matter weight and dry matter weight. The seedlings hypocotyls, root and total length were measured total proteins content in cotyledons were obtained from germinating seeds. The presence of salt at concentrations higher than 50mol m-3 NaCl affect the germination, seedlings growth and cotyledons total protein synthesis of all cowpea cultivars. The seeds of cultivar pitiúba were is more tolerant to salinity, than the cultivars Canapu and Epace-10.

Molecular patterns behind immunological and metabolic alterations in lysinuric protein intolerance

Lysinuric protein intolerance (LPI) is a recessively inherited disorder characterised by reduced plasma and increased urinary levels of cationic amino acids (CAAs), protein malnutrition, growth failure and hyperlipidemia. Some patients develop severe immunological, renal and pulmonary complications. All Finnish patients share the same LPIFin mutation in the SLC7A7 gene that encodes CAA transporter y+LAT1. The aim of this study was to examine molecular factors contributing to the various symptoms, systemic metabolic and lipid profiles, and innate immune responses in LPI. The transcriptomes, metabolomes and lipidomes were analysed in whole-blood cells and plasma using RNA microarrays and gas or liquid chromatography-mass spectrometry techniques, respectively. Toll-like receptor (TLR) signalling in monocyte-derived macrophages exposed to pathogens was scrutinised using qRT-PCR and the Luminex technology. Altered levels of transcripts participating in amino acid transport, immune responses, apoptosis and pathways of hepatic and renal metabolism were identified in the LPI whole-blood cells. The patients had increased non-essential amino acid, triacylglycerol and fatty acid levels, and decreased plasma levels of phosphatidylcholines and practically all essential amino acids. In addition, elevated plasma levels of eight metabolites, long-chain triacylglycerols, two chemoattractant chemokines and nitric oxide correlated with the reduced glomerular function in the patients with kidney disease. Accordingly, it can be hypothesised that the patients have increased autophagy, inflammation, oxidative stress and apoptosis, leading to hepatic steatosis, uremic toxicity and altered intestinal microbe metabolism. Furthermore, the LPI macrophages showed disruption in the TLR2/1, TLR4 and TLR9 pathways, suggesting innate immune dysfunctions with an excessive response to bacterial infections but a deficient viral DNA response.

Adaptations of skeletal muscle pyruvate dehydrogenase kinase in response to food-restriction in mitochondrial subpopulations

University, 2006 Dr. Sandra J. Peters Pyruvate dehydrogenase (PDH) catalyses the decarboxylation of pyruvate, to form acetyl-CoA. PDH activity is down-regulated by intrinsic PDH kinases (predominantly PDK2 and PDK4 isoforms), but the understanding of the PDK isoform distribution and adaptation to nutritional stresses has been restricted to mixed mitochondrial populations, and not delineated between subsarcolemmal (SS) and intermyofibrillar (IMF) subpopulations. SS and IMF mitochondria exhibit distinct morphological and biochemical properties; however the functional differences are not well understood. This study investigated the effect of fed (FED) versus 48 h total foodrestriction (FR) on rat red gastrocnemius muscle PDK2 and 4 isoform content in SS and IMF mitochondria. PDK4 content was ~3-5 fold higher in SS mitochondria compared to IMF (p=0.001), and increased with FR -3-4- fold in both subpopulations (p<0.001). PDK2 was -2.5-4 fold higher in SS mitochondria compared to IMF (p=0.001), but PDK2 was unaltered with FR. Citrate synthase activity (|imol/min/mg mitochondrial protein) was not different between either subpopulation. As well there were no significant differences between mitochondrial subpopulations in PDH complex components in both fed and FR states. These results demonstrate that there is a markedly higher content of both PDK isofonns in SS compared to IMF mitochondria. Although PDK2 does not increase in either subpopulation in response to FR, PDK4 increases to a similar extent in both SS and IMF after 48 h food-restriction.

Amputation and heat induced protein synthesis in the regenerating forelimb of Notophthalmus viridescens

This thesis compares the responses of regenerating forelimb tissues of the newt Notophthalmu..f vlridescens to the stresses of hyperthermia and ID.echanical injury of amputation. In particular, both quantitative and qualitative changes in the synthesis of soluble proteins in stump tissues, including those of the heat shock protein family (HSP70-1ike) were examined. Results from SDS-PAGEfluorography indicate that the trauma of amputation mimics the heat shock response both quantitatively and temporally in its transient repression of the synthesis of most normal cellular proteins, and qualitatively. in the locaJized expression of two unique proteins (hsp30 and hsp70). Fluorography of proteins separated by twodimensional gets revealed that thelCl4:alizedt amputation induced 70kDa protein (amp70) was distinct from the more basic newt hsp/hsc70 isoforms. Although limb amputation resulted in an increase in the synthesis of HSP70 mRNA analogous to that induced by heat 3.b.OCKf amp70 did not cross-react with murine monoclonal antibodies directed against both the inducible and cognate HSP70 proteins of the human. Thus, the possible relationship of amp70 to other members of the HSP70-1ike protein family remains unclear. Western analyses indicated that the levels of the constitutive form of HSP70 (hsc70) were found to be regulated in a stage-dependent manner in the distal stump tissues of the regen,erating forelimb of the newt. The highest levels were found in the mid-late bud stage, a period during which rapidly dividing blastema cells begin to redifferentiate in a proximodistal direction. Immediately after amputation) hsc70 synthesis and accumulation was depressed below steady-state levels measured in the unamputated limb~ The results are discussed in light of a possible role for HSPs and amputatio~ induced proteins in the epimorphic regeneration of the amphibian limb.

An investigation into differences between indoleacetic acid and fusicoccin in their influence on RNA synthesis, protein synthesis and growth in Avena coleoptile tissue

Growth stimulation of Avena coleoptile tissue by indoleacetic acid (IAA) and fusicoccin (FC) was compared by measuring both their influence on RNA and protein synthesis during IAA or FC stimulated growth. FC stimulated growth more than IAA during the initial four hour exposure, after which the growth rate gradually declined to the control rate. FC, but not IAA, increased the uptake of 3H-Ieucine into tissue and the specific radioactivity of extracted protein. Cycloheximide inhibited the incorporation of 3H-Ieucine into protein by approximately 60% to 70% in all cases. In the presence of cycloheximide 3H-radioactivity accumulated in FC-treated tissue, whereas IAA did not seem to influence 3H-accumulation. These results suggest that FC stimulated leucine uptake into the tissue and that increased specific activity of coleoptile protein is due to increased leucine uptake, not an increased rate of protein synthesis. There was no measurable influence of IAA and/or FC on RNA and protein synthesis during the initial hours of a growth stimulation. Inhibitors of RNA and protein synthesis, actinomycin D and cycloheximide, respectively, severely inhibited IAA enhanced growth but only partially inhibited FC stimulated growth. The data are consistent with suggestions that a rapidly turning over protein participates in IAA stimulated growth, and that a continual synthesis of RNA and proteins is an absolute requirement for a long term growth response to IAA. On the contrary, FC-stimulated growth exhibited less dependency on the transcription and translation processes. The data are consistent with proposals suggesting different sites of action for FC and IAA stimulated growth. l?hen compared to CO2-free air, CO2 at 300 ppm had no significant influence on coleoptile growth and protein synthesis in the presence or absence of lAA or FC. Also, I mM malate, pH 6.0 did not influence growth of coleoptiles in the presence or absence of lAA. This result was obtained despite reports indicating that 300 ppm CO2 or I mM malate stimulates growth and protein synthesis. This lack of difference between CO2-treated and untreated tissue could indicate either that the interstitial space CO2 concentration is not actually different in the two treatments due to significant endogenous respiratory CO2 or else the data would suggest a very loose coupling between dark CO2 fixation and growth. IAA stimulated the in vivo fixation of 14c-bicarbonate (NaHI4c03) by about 25% and the addition of cycloheximide caused an inhibition of bicarbonate fixation within 30 min. Cycloheximide has also been reported to inhibit IAA-stimulated H+ excretion. These data are consistent with the acid growth theory and suggest that lAA stimulated growth involves dark CO2 fixation. The roles of dark CO2 fixation in lAA-stimulated growth are discussed.

The role of learning and growth hormone-releasing factor in the control of protein intake in rats /

Both learning and basic biological mechanisms have been shown to play a role in the control of protein int^e. It has previously been shown that rats can adapt their dietary selection patterns successfully in the face of changing macronutrient requirements and availability. In particular, it has been demonstrated that when access to dietary protein is restricted for a period of time, rats selectively increase their consumption of a proteincontaining diet when it becomes available. Furthermore, it has been shown that animals are able to associate various orosensory cues with a food's nutrient content. In addition to the role that learning plays in food intake, there are also various biological mechanisms that have been shown to be involved in the control of feeding behaviour. Numerous studies have documented that various hormones and neurotransmitter substances mediate food intake. One such hormone is growth hormone-releasing factor (GRF), a peptide that induces the release of growth hormone (GH) from the anterior pituitary gland. Recent research by Vaccarino and Dickson ( 1 994) suggests that GRF may stimulate food intake by acting as a neurotransmitter in the suprachiasmatic nucleus (SCN) and the adjacent medial preoptic area (MPOA). In particular, when GRF is injected directly into the SCN/MPOA, it has been shown to selectively enhance the intake of protein in both fooddeprived and sated rats. Thus, GRF may play a role in activating protein consumption generally, and when animals have a need for protein, GRF may serve to trigger proteinseeking behaviour. Although researchers have separately examined the role of learning and the central mechanisms involved in the control of protein selection, no one has yet attempted to bring together these two lines of study. Thus, the purpose of this study is to join these two parallel lines of research in order to further our understanding of mechanisms controlling protein selection. In order to ascertain the combined effects that GRF and learning have on protein intake several hypothesis were examined. One major hypothesis was that rats would successfully alter their dietary selection patterns in response to protein restriction. It was speculated that rats kept on a nutritionally complete maintenance diet (NCMD) would consume equal amount of the intermittently presented high protein conditioning diet (HPCD) and protein-free conditioning diet (PFCD). However, it was hypothesized that rats kept on a protein-free maintenance diet (PFMD) would selectively increase their intake of the HPCD. Another hypothesis was that rats would learn to associate a distinct marker flavour with the nutritional content of the diets. If an animal is able to make the association between a marker flavour and the nutrient content of the food, then it is hypothesized that they will consume more of a mixed diet (equal portion HPCD and PFCD) with the marker flavour that was previously paired with the HPCD (Mixednp-f) when kept on the PFMD. In addition, it was hypothesized that intracranial injection of GRF into the SCN/MPOA would result in a selective increase in HPCD as well as Mixednp-t consumption. Results demonstrated that rats did in fact selectively increase their consumption of the flavoured HPCD and Mixednp-f when kept on the NCMD. These findings indicate that the rats successfully learned about the nutrient content of the conditioning diets and were able to associate a distinct marker flavour with the nutrient content of the diets. However, the results failed to support previous findings that GRF increases protein intake. In contrast, the administration of GRF significantly reduced consumption of HPCD during the first hour of testing as compared to the no injection condition. In addition, no differences in the intake of the HPCD were found between the GRF and vehicle condition. Because GRF did not selectively increase HPCD consumption, it was not surprising that GRF also did not increase MixedHP-rintake. What was interesting was that administration of GRF and vehicle did not reduc^Mixednp-f consumption as it had decreased HPCD consumption.

Pyruvate dehydrogenase activity in response to skeletal muscle contraction at two stimulation frequencies in pyruvate dehydrogenase kinase 2 knockout mice

Pyruvate dehydrogenase (PDH) plays an important role in regulating carbohydrate oxidation in skeletal muscle. PD H is deactivated by a set of PD H kinases (PD K 1-4) with PDK2 and 4 being the predominant isoforms in skeletal muscle. PDK2 is highly sensitive to pyruvate inhibition, and is the most abundant isoform, while PDKI and 4 protein content are normally lower. This study examined the PDK isoform content and PDHa activation in muscle at rest and 10 and 40 Hz stimulation from PDK2 knockout (PDK2KO) mice to delineate the role of PDK2 in activating the PDH complex during low and moderate intensity muscle contraction. PDHa activity was lower in PDK2KO mice during contraction while total PDK actitvity was -4 fold lower. PDK4 protein was not different, however PDKI partially compensated for the lack of PDK2 and was -56% higher than WT. PDKI is a very potent inhibitor of the PDH complex due to its phosphorylation site specificity and allosteric regulation. These results suggest that the site specificity and allosteric regulatory properties of the individual PDK isoforms are more important than total PDK activity in determining transformation of the complex and PDHa activity during acute muscle contraction.

Phosphorylation of Skeletal Muscle Pyruvate Dehydrogenase Phosphatase in Response to Insulin Stimulation

Pyruvate dehydrogenase phosphatase (PDP) regulates carbohydrate oxidation through the pyruvate dehydrogenase (PDH) complex. PDP activates PDH, enabling increased carbohydrate flux towards oxidative energy production. In culture myoblasts, both PDP1 and PDP2 undergo covalent activation in response to insulin–stimulation by protein kinase C delta (PKCδ). Our objective was to examine the effect of insulin on PDP phosphorylation and PDH activation in skeletal muscle. Intact rat extensor digitorum longus muscles were incubated (oxygenated at 25°C, 1g of tension) for 30min in basal or insulin–stimulated (10 mU/mL) media. PDH activity increased 58% following stimulation, (p=0.057, n=11). Serine phosphorylation of PDP1 (p=0.047) and PDP2 (p=0.006) increased by 29% and 48%, respectively (n=8), and mitochondrial PKCδ protein content was enriched by 45% in response to stimulation (p=0.0009, n=8). These data suggest that the insulin–stimulated increase in PDH activity in whole tissue is mediated through mitochondrial migration of PKCδ and subsequent PDP phosphorylation.