RESUMO
BACKGROUND: Traditionally, the Arctic diet has been derived entirely from locally harvested animal and plant species; however, in recent decades, imported foods purchased from grocery stores have become widely available. The present study aimed to examine Inuvialuit, traditional or nontraditional dietary patterns; nutrient density of the diet; dietary adequacy; and main food sources of energy and selected nutrient intakes. METHODS: This cross-sectional study used a culturally appropriate quantitative food frequency questionnaire to assess diet. Traditional and nontraditional eaters were classified as those consuming more or less than 300 g of traditional food daily. Nutrient densities per 4184 kJ (1000 kcal) were determined. Dietary adequacy was determined by comparing participants' nutrient intakes with the Dietary Reference Intakes. RESULTS: The diet of nontraditional eaters contained, on average, a lower density of protein, niacin, vitamin B12 , iron, selenium, zinc, omega-3 fatty acids (P ≤ 0.0001), vitamin B6 , potassium, thiamin, pantothenic acid (P ≤ 0.001), riboflavin and magnesium (P ≤ 0.05). Inadequate nutrient intake was more common among nontraditional eaters for calcium, folate, vitamin C, zinc, thiamin, pantothenic acid, vitamin K, magnesium, potassium and sodium. Non-nutrient-dense foods (i.e. high fat and high sugar foods) contributed to energy intake in both groups, more so among nontraditional eaters (45% versus 33%). Traditional foods accounted for 3.3% and 20.7% of total energy intake among nontraditional and traditional eaters, respectively. CONCLUSIONS: Diet quality and dietary adequacy were better among Inuvialuit who consumed more traditional foods. The promotion of traditional foods should be incorporated in dietary interventions for this population.
Assuntos
Cultura , Dieta , Alimentos , Inuíte , Adulto , Regiões Árticas , Estudos Transversais , Fibras na Dieta , Proteínas Alimentares/administração & dosagem , Ingestão de Energia , Feminino , Humanos , Masculino , Micronutrientes/administração & dosagem , Minerais/administração & dosagem , Territórios do Noroeste , Avaliação Nutricional , Valor Nutritivo , Vitaminas/administração & dosagemRESUMO
Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.
Assuntos
Quimera , Hepacivirus/fisiologia , Fígado/virologia , Replicação Viral , Animais , Transplante de Células , Hepacivirus/genética , Homozigoto , Humanos , Camundongos , Camundongos SCID , RNA Viral/isolamento & purificação , TransgenesRESUMO
Myristoylated alanine-rich C-kinase substrate (MARCKS), a prominent substrate for conventional and novel protein kinase C (PKC) isoforms, is involved in the regulation of membrane-cytoskeletal interactions. Addition of [gamma-32P]ATP to the membrane fraction of digitonin-permeabilized C6 glioma cells resulted in phosphorylation and release of MARCKS, indicating involvement of an active membrane-bound kinase. Pretreatment of cells with 2 microM 4 beta-12-O-tetradecanoyl-phorbol-13-acetate (beta-TPA) for 18 h downregulated conventional (PKC alpha) and novel (PKC delta) isoforms of PKC by > 90% in both membrane and soluble fractions, but did not inhibit the rate of ATP-dependent phosphorylation or release of MARCKS, or decrease levels of membrane-bound PKC zeta or PKC mu. MARCKS phosphorylation was inhibited by staurosporine, bis-indolylmaleimide (a PKC-specific inhibitor), Gö6983 (inhibits all isoforms except PKC mu), and a peptide from the calmodulin-binding domain of MARCKS, but was unaffected by EGTA or Gö6976 (inhibits cPKCs and PKC mu). Peptide mapping indicated similar in vivo and in vitro phosphorylation at serine residue(s) known to be phosphorylated by PKC. These findings support a novel mechanism by which MARCKS may be regulated by an atypical PKC isoform in phorbol ester-downregulated cells.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Proteína Quinase C/metabolismo , Proteínas/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Transporte Biológico Ativo , Cálcio/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular , Isoenzimas/metabolismo , Cinética , Substrato Quinase C Rico em Alanina Miristoilada , Fosforilação , Ratos , Transdução de Sinais , Solubilidade , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
MARCKS (myristoylated alanine-rich C-kinase substrate) is known to interact with calmodulin, actin filaments, and anionic phospholipids at a central basic domain which is also the site of phosphorylation by protein kinase C (PKC). In the present study, cytochalasin D (CD) and calmodulin antagonists were used to examine the influence of F-actin and calmodulin on membrane interaction of MARCKS in C6 glioma cells. CD treatment for 1 h disrupted F-actin filaments, increased membrane bound immunoreactive MARCKS (from 51% to 62% of total), yet markedly enhanced the amount of MARCKS translocated to the cytosolic fraction in response to the phorbol ester 4beta-12-O-tetradecanoylphorbol 13-acetate. In contrast, CD treatment had no effect on phorbol ester-stimulated phosphorylation of MARCKS or on translocation of PKC alpha to the membrane fraction. Staurosporine also increased membrane association of MARCKS in a PKC-independent manner, as no change in MARCKS phosphorylation was noted and bis-indolylmaleimide (a more specific PKC inhibitor) did not alter MARCKS distribution. Staurosporine inhibited the phorbol ester-induced translocation of MARCKS but not of PKC alpha in both CD pretreated and untreated cells. Calmodulin antagonists (trifluoperazine, calmidazolium) had little effect on the cellular distribution or phosphorylation of MARCKS, but were synergistic with phorbol ester in translocating MARCKS from the membrane without a further increase in its phosphorylation. We conclude that cytoskeletal integrity is not required for phosphorylation and translocation of MARCKS in response to activated PKC, but that interaction with both F-actin and calmodulin might serve to independently modulate PKC-regulated localization and function of MARCKS at cellular membranes.
Assuntos
Actinas/metabolismo , Calmodulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Proteínas/metabolismo , Animais , Membrana Celular/metabolismo , Citocalasina D/farmacologia , Citoesqueleto/metabolismo , Ativação Enzimática/efeitos dos fármacos , L-Lactato Desidrogenase/análise , Substrato Quinase C Rico em Alanina Miristoilada , Ésteres de Forbol , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Ratos , Estaurosporina/farmacologia , Trifluoperazina/farmacologia , Células Tumorais CultivadasRESUMO
Although alien to man, the ability to endure the freezing of extracellular body fluids during the winter has developed in several species of terrestrially hibernating frogs and turtles as well as in many species of insects and other invertebrates. Wood frogs, for example, can endure freezing for at least 2 weeks with no breathing, no heart beat or blood circulation, and with up to 65% of their total body water as ice. Our studies are providing a comprehensive view of the requirements for natural freezing survival and of the physical and metabolic protection that must be offered for effective cryopreservation of vertebrate organs. Molecular mechanisms of natural freeze tolerance in lower vertebrates include: 1) control over ice crystal growth in plasma by ice nucleating proteins, 2) the accumulation of low molecular weight cryoprotectants to minimize intracellular dehydration and stabilize macromolecular components, and 3) good ischemia tolerance by all organs that may include metabolic arrest mechanisms to reduce organ energy requirements while frozen. Cryomicroscopy of tissue slices and magnetic resonance imaging (MRI) of whole animals is revealing the natural mode of ice propagation through an organism. MRI has also revealed that thawing is non-uniform; core organs (with high cryoprotectant levels) melt first, facilitating the early resumption of heart beat and blood circulation. Studies of the production and actions of the natural cryoprotectant, glucose, in frogs have shown its importance in maintaining a critical minimum cell volume in frozen organs and new work on the metabolic effects of whole body dehydration in 3 species of frogs has indicated that adaptations supporting freeze tolerance grew out of mechanisms that deal with desiccation resistance in amphibians. Studies of the regulation of cryoprotectant glucose synthesis by wood frog liver have shown the role of protein kinases and of alpha and beta adrenergic receptors in regulating the glycemic response, and of changes in membrane glucose transporter proteins to facilitate cryoprotectant distribution.
Assuntos
Criopreservação , Espaço Extracelular/fisiologia , Congelamento , Fígado/ultraestrutura , Imageamento por Ressonância Magnética , Trifosfato de Adenosina/metabolismo , Animais , Temperatura Corporal/fisiologia , Fosforilases/metabolismo , Ranidae/metabolismo , Tartarugas/metabolismoRESUMO
The effects of anoxic submergence (16 h at 15 degrees C) on cellular mRNA contents were assessed in five organs of anoxia tolerant turtles Trachemys scripta elegans. Poly(A)+ RNA was extracted from liver, red and white skeletal muscle, kidney and heart of control and anoxic turtles, as well as from heart and kidney of turtles allowed 24 h aerobic recovery (at 15 degrees C) after anoxia exposure. Poly(A)+ RNA content increased by 30% in white muscle from anoxic turtles relative to control animals but was unchanged by metabolic state in other organs. Extracted mRNA was translated in vitro in a wheat germ lysate system and the 35S-labelled polypeptides that were produced were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Overall translational activity of the mRNA pool [cpm 35S-methionine incorporated per microgram poly(A)+ RNA] was altered by anoxia exposure in three organs, increasing by 38 and 18% in liver and kidney and decreasing by 42% in red muscle. Anoxia exposure also led to qualitative changes in the protein products that resulted from in vitro translation. Sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed the presence of a novel 19.5-kDa polypeptide in liver of anoxia-exposed animals as well as increased amounts of two other proteins at 28.6 and 79.9 kDa. In heart a new translation product of 26.8 kDa appeared in anoxia, and in kidney a 32.8-kDa polypeptide was produced during the aerobic recovery period after anoxia exposure. Anoxia stimulated the appearance of a 37.5-kDa protein in red skeletal muscle but anoxic red muscle also lost proteins of 40, 32, and 28.2 kDa that were present in aerobic controls. Anoxia exposure did not change the proteins produced by in vitro translation in white muscle. The results suggest that anoxia exposure triggers rapid cellular responses in T. s. elegans that modify translatable mRNA populations in organs, leading to new protein transcripts.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Adaptação Fisiológica , Hipóxia/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Tartarugas/genética , Anaerobiose , Animais , Autorradiografia , Eletroforese em Gel de Poliacrilamida , Imersão , Técnicas In Vitro , RNA Mensageiro/metabolismoRESUMO
Microsomal triacylglycerol hydrolase (TGH) hydrolyzes stored triacylglycerol in cultured hepatoma cells (Lehner, R., and Vance, D. E. (1999) Biochem. J. 343, 1-10). We studied expression of TGH in murine liver and found both protein and mRNA increased dramatically at 27 days after birth. Nuclear run-on assays demonstrated that this was due to increased transcription. We cloned 542 base pairs upstream of the transcriptional start site of the murine TGH gene. Electrophoretic mobility shift assays demonstrated enhanced binding of hepatic nuclear proteins from 27-day-old mice to the murine TGH promoter, yielding three differentially migrating complexes. DNase I footprint analysis localized these complexes to two distinct regions: site A contains a putative Sp binding site, and site B contains a degenerate E box. We transfected primary murine hepatocytes with a series of 5'-deletion constructs upstream of the reporter luciferase cDNA. Positive control elements were identified in a segment containing site A. Competitive electrophoretic mobility shift assays and supershift assays demonstrated that site A binds Sp1 and Sp3. Transcriptional activation assays in Schneider SL-2 insect cells demonstrated that Sp1 is a potent activator of the TGH promoter. These experiments directly link increased TGH expression at the time of weaning to transcriptional regulation by Sp1.
Assuntos
Lipase/genética , Fígado/enzimologia , Fator de Transcrição Sp1/fisiologia , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA Complementar/química , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Genes Reporter , Lipase/biossíntese , Lipoproteínas VLDL/metabolismo , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Fator de Transcrição Sp3 , Spodoptera , Fatores de Transcrição/metabolismoRESUMO
Although alien to man, the ability to endure the freezing of extracellular body fluids during the winter has developed in several species of terrestrially hibernating frogs and turtles as well as in many species of insects and other invertebrates. Wood frogs, for example, can endure freezing for at least 2 weeks with no breathing, no heart beat or blood circulation, and with up to 65 per cent of their total body water as ice. Our studies are providing a comprehensive view of the requirements for natural freezing survival and of the physical and metabolic protection that must be offered for effective cryopreservation of vertebrate organs. Molecular mechanisms of natural freeze tolerance in lower vertebrates include: 1) control over ice crystal growth in plasma by ice nucleating proteins, 2) the accumulation of low molecular weight cryoprotectants to minimize intracellular dehydration and stabilize macromolecular components, and 3) good ischemia tolerance by all organs that may include metabolic arrest mechanisms to reduce organ energy requirements while frozen. Cryomicroscopy of tissue slices and magnetic resonance imaging (MRI) of whole animals is revealing the natural mode of ice propagation through an organism. MRI has also revealed that thawing is non-uniform; core organs (with high cryoprotectant levels) melt first, facilitating the early resumption of heart beat and blood circulation. Studies of the production and actions of the natural cryoprotectant, glucose, in frogs have shown its importance in maintaining a critical minimum cell volume in frozen organs and new work on the metabolic effects of whole body dehydration in 3 species of frogs has indicated that adaptations supporting freeze tolerance grew out of mechanisms that deal with desiccation resistance in amphibians. Studies of the regulation of cryoprotectant glucose synthesis by wood frog liver have shown the role of protein kinases and of (alpha and beta adrenergic receptors in regulating the glycemic response, and of changes in membrane glucose transporter proteins to facilitate cryoprotectant distribution.