RESUMO
Immune characteristics in 65 calves were evaluated in response to a Bacillus-based direct-fed microbial (DFM) provided in electrolyte scour treatment. Blood samples were analyzed for cell surface markers and α(1)-acid glycoprotein (AGP) concentration. AGP increased in scouring calves given electrolyte containing Bacillus at day 7 post-placement compared to scouring calves administered electrolyte alone and non-scouring calves, enhancing the inflammatory response for pathogen clearance. The Bacillus promotes T cell subsets including greater proportions of activated, mature cells (CD8(-)CD25(+), CD8(-)CD45RO(+), CD8(-)TCR1(+)) in calves given electrolyte containing Bacillus than scouring calves administered electrolyte alone and non-scouring calves. Also, the Bacillus may be alleviating inflammation at day 3 post-placement as the proportion of monocytes and granulocytes lacking L-selectin (CD172a(+)CD62L(-)) was greater in scouring calves given electrolyte compared to the other groups. Electrolyte containing Bacillus administered at the onset of scours influences components of innate and adaptive immune development during and following the scouring event.
Assuntos
Ração Animal/análise , Bacillus subtilis/fisiologia , Bovinos , Dieta/veterinária , Eletrólitos/uso terapêutico , Animais , Bovinos/imunologia , Doenças dos Bovinos/tratamento farmacológico , Indústria de Laticínios , Diarreia/tratamento farmacológico , Diarreia/veterinária , Suplementos Nutricionais , Glicoproteínas/metabolismo , Inflamação , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/fisiologia , Masculino , Probióticos/uso terapêuticoRESUMO
Platelets play a central role in maintaining biological hemostasis. Inappropriate platelet activation is responsible for thrombotic diseases such as myocardial infarction and stroke. Therefore, novel agents that can inhibit platelet activation are necessary. However, assays that monitor platelet aggregation are generally time-consuming and require high volumes of blood and specialized equipment. Therefore, a medium- to high-throughput assay that can monitor platelet aggregation would be considered useful. Such an assay should be sensitive, comparable to the "gold standard" assay of platelet aggregometry, and able to monitor multiple samples simultaneously but with low assay volumes. We have developed such a microtiter assay. It can assay an average of 60 independent treatments per 60 ml blood donation and demonstrates greater sensitivity than the current gold standard assay, namely platelet aggregation in stirring conditions in a platelet aggregometer. The microtiter plate (MTP) assay can detect known inhibitors of platelet function such as indomethacin, aspirin, and ReoPro. It is highly reproducible when using standard doses of agonists such as thrombin receptor-activating peptide (20 microM) and collagen (0.19 mg/ml). Finally, the MTP assay is rapid and sensitive and can detect unknown platelet-modulating agents from a library of compounds.
Assuntos
Avaliação Pré-Clínica de Medicamentos/instrumentação , Inibidores da Agregação Plaquetária/análise , Agregação Plaquetária/fisiologia , Sequência de Aminoácidos , Bioensaio , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Colágeno/farmacologia , Humanos , Dados de Sequência Molecular , Receptores de Trombina/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Trombina/farmacologiaRESUMO
Resistance to potato virus X (PVX) is determined by the product of a host resistance gene and a viral determinant specifying either virulence (resistance-breaking ability) or avirulence (resistance sensitivity). The viral coat protein is the determinant of resistance mediated by the host Nx gene while the 25 kDa movement protein is the determinant of Nb-mediated resistance. Group 1 and group 4 strains of PVX are avirulent or virulent respectively for both these determinants while group 2 and group 3 strains are virulent for one but avirulent for the other determinant. There are two alternative evolutionary mechanisms by which the various strain groups might have evolved: either by recombination between strains carrying virulence (or avirulence) determinants that evolved once only, or alternatively, by independent evolution of at least one virulence (or avirulence) determinant in distinct phylogenetic branches. These alternative hypotheses were investigated by (i) determining the complete genomic sequence of a group 1 and a group 4 strain and (ii) comparing the completely sequenced genomes of six isolates representative of the four strain groups. The analysis revealed the same phylogeny for all five PVX genes. Thus, there is no evidence that the PVX strain groups evolved by recombination.
Assuntos
Proteínas do Capsídeo , Evolução Molecular , Genoma Viral , Potexvirus/patogenicidade , Recombinação Genética , Regiões 5' não Traduzidas , Capsídeo/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , Potexvirus/classificação , Potexvirus/genética , Análise de Sequência de DNA , Solanum tuberosum/genética , Solanum tuberosum/virologia , VirulênciaRESUMO
Since calcium-activated neutral proteinase (calpain) activity and expression are significantly increased in activated glial/inflammatory cells in the central nervous system of animals with autoimmune demyelinating diseases, this enzyme may also play a role in peripheral organ systems in these diseases. In this study, the activity and expression of calpain and the endogenous inhibitor, calpastatin, were evaluated at transcriptional and translational levels in spleens of Lewis rats with acute experimental allergic encephalomyelitis (EAE) prior to the onset of clinical symptoms. Calpain activity and translational expression were increased by 475.5% and 44.3% respectively, on day 4 post-induction in adjuvant controls and animals with EAE. These levels remained elevated compared to normal controls on days 8 and 12. Calpastatin translational expression was similarly increased at these time points although transcriptional expression was not significantly altered at any time following induction of EAE. Likewise, transcriptional expression of mu-calpain was unchanged following induction, while small increases in m-calpain transcriptional expression were observed on days 2 and 8. Most calpain expression was observed in activated splenic macrophages at day 8 post-induction even though activated T cells were also calpain positive. In spinal cords of animals with EAE, calpain expression was significantly increased in rats with severe disease compared to those exhibiting only mild symptoms at day 12 post-induction. Thus, prior to symptomatic EAE, increased calpain activity and expression in peripheral lymphoid organs may play an important role in T cell migration and subsequent disease progression.
Assuntos
Proteínas de Ligação ao Cálcio/biossíntese , Calpaína/biossíntese , Encefalomielite Autoimune Experimental/enzimologia , Linfócitos/enzimologia , Macrófagos/enzimologia , Baço/patologia , Adjuvantes Imunológicos/farmacologia , Animais , Doenças Autoimunes/enzimologia , Doenças Autoimunes/genética , Doenças Autoimunes/patologia , Proteínas de Ligação ao Cálcio/genética , Calpaína/genética , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Indução Enzimática/efeitos dos fármacos , Ativação Linfocitária , Ativação de Macrófagos , Masculino , Biossíntese de Proteínas , Ratos , Ratos Endogâmicos Lew , Medula Espinal/enzimologia , Medula Espinal/patologia , Baço/imunologia , Fatores de Tempo , Transcrição GênicaRESUMO
Somatostatin (SRIF) is a 14-residue peptide hormone synthesized in the hypothalamus and pancreatic islets. SRIF-14 and an N-terminally extended form, SRIF-28, are generated by the proteolytic processing of an approx. 102-residue precursor prosomatostatin (proSRIF) at a single set of paired basic residues (Arg-Lys) and at a monobasic (Arg) site respectively. Previous work in our laboratory demonstrated that the propeptide of SRIF mediates intracellular sorting; we suggested that this information resides in the prohormone structure. To identify putative sorting domains we have investigated structural features of recombinant anglerfish proSRIF-II purified from Escherichia coli. Two species of proSRIF-II were obtained: a monomeric form and a disulphide-linked dimer. CD analyses revealed that monomeric proSRIF-II lacks appreciable periodic secondary structure; however, on slow heating (2 degrees C/min) and cooling, it assumed a predominantly alpha-helical conformation. When subjected to a second heating-and-cooling cycle, the alpha-helical conformation was maintained. In contrast, the dimeric form of proSRIF-II was predominantly alpha-helical and its helicity did not increase in response to heating and recooling. Our results suggest that proSRIF-II might exist in several different folding intermediate states.
Assuntos
Conformação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Somatostatina/química , Somatostatina/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Clonagem Molecular , Dimerização , Escherichia coli , Hipotálamo/metabolismo , Ilhotas Pancreáticas/metabolismo , Dados de Sequência Molecular , Mapeamento de Peptídeos , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Temperatura , Termodinâmica , TripsinaRESUMO
Phospholipid metabolism plays a central role in regulating vesicular traffic in the secretory pathway. In mammalian cells, activation of a Golgi-associated phospholipase D activity by ADP-ribosylation factor results in hydrolysis of phosphatidylcholine to phosphatidic acid (PA). This reaction has been proposed to stimulate nascent secretory vesicle budding from the trans-Golgi network. It is unclear whether PA itself or diacylglycerol (DAG), a metabolite implicated in yeast secretory vesicle formation, regulates budding. To distinguish between these possibilities we have used a permeabilized cell system supplemented with phospholipid-modifying enzymes that generate either DAG or PA. The data demonstrate that in mammalian cells accumulation of PA rather than DAG is a key step in regulating budding of secretory vesicles from the trans-Golgi network.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Complexo de Golgi/metabolismo , Ácidos Fosfatídicos/metabolismo , Animais , Células Cultivadas , Diacilglicerol Quinase/metabolismo , Diglicerídeos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol Diacilglicerol-Liase , Fosfolipase D/metabolismo , Adeno-Hipófise/metabolismo , Ratos , Fosfolipases Tipo C/metabolismoRESUMO
In demyelinating diseases such as multiple sclerosis (MS), myelin membrane structure is destabilized as myelin proteins are lost. Calcium-activated neutral proteinase (calpain) is believed to participate in myelin protein degradation because known calpain substrates [myelin basic protein (MBP); myelin-associated glycoprotein] are degraded in this disease. In exploring the role of calpain in demyelinating diseases, we examined calpain expression in Lewis rats with acute experimental allergic encephalomyelitis (EAE), an animal model for MS. Using double-immunofluorescence labeling to identify cells expressing calpain, we labeled rat spinal cord sections for calpain with a polyclonal millicalpain antibody and with mAbs for glial (GFAP, OX42, GalC) and inflammatory (CD2, ED2, interferon gamma) cell-specific markers. Calpain expression was increased in activated microglia (OX42) and infiltrating macrophages (ED2) compared with controls. Oligodendrocytes (galactocerebroside) and astrocytes (GFAP) had constitutive calpain expression in normal spinal cords whereas reactive astrocytes in spinal cords from animals with EAE exhibited markedly increased calpain levels compared with astrocytes in adjuvant controls. Oligodendrocytes in spinal cords from rats with EAE expressed increased calpain levels in some areas, but overall the increases in calpain expression were small. Most T cells in grade 4 EAE expressed low levels of calpain, but interferon gamma-positive cells demonstrated markedly increased calpain expression. These findings suggest that increased levels of calpain in activated glial and inflammatory cells in EAE may contribute to myelin destruction in demyelinating diseases such as MS.
Assuntos
Calpaína/biossíntese , Encefalomielite Autoimune Experimental/enzimologia , Inflamação/enzimologia , Neuroglia/enzimologia , Animais , Anticorpos Monoclonais/metabolismo , Galactosilceramidas/imunologia , Proteína Glial Fibrilar Ácida/biossíntese , Técnicas Imunoenzimáticas , Esclerose Múltipla/enzimologia , Ratos , Ratos Endogâmicos Lew , Medula Espinal/enzimologiaRESUMO
Homoeopathic remedies for migraine are widely available over the counter, statutorily offered by the national health service in the UK, and apparently popular with patients. Do they work? Sixty-three outpatients with migraine with or without aura by IHS criteria entered a 4-month randomized placebo-controlled, double-blind parallel-groups trial of individualized homoeopathic prophylaxis, the first month being baseline with all patients on placebo. Three patients (4.8%) dropped out, leaving 30 in each treatment group. There were chance differences in attack frequency and severity between the groups at baseline (attacks were more frequent but less severe in the placebo group). Both groups improved on therapy, but neither to a great extent on the primary outcome measure of attack frequency (verum: -19%; placebo: -16%). Reduction was mostly in mild attacks on placebo, more in moderate and severe attacks on homoeopathy. Few adverse events were reported. Overall, there was no significant benefit over placebo of homoeopathic treatment. The course of change differed between groups, and suggested that improvement reversed in the last month of treatment on placebo. On this evidence we cannot recommend homoeopathy for migraine prophylaxis, but cannot conclude that it is without effect.
Assuntos
Homeopatia , Transtornos de Enxaqueca/prevenção & controle , Adulto , Alcaloides/uso terapêutico , Animais , Atropa belladonna , Benzofenantridinas , Método Duplo-Cego , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Isoquinolinas , Masculino , Pessoa de Meia-Idade , Transtornos de Enxaqueca/tratamento farmacológico , Fitoterapia , Plantas Medicinais , Plantas Tóxicas , Estricnina , ViperidaeRESUMO
BACKGROUND: Plasma homocysteine levels are modulated by nutritional and genetic factors, among which is the enzyme methylenetetrahydrofolate reductase (MTHFR). A common defective (thermolabile) variant of this enzyme is causally associated with elevated plasma homocysteine, itself an independent risk factor for coronary heart disease. METHODS AND RESULTS: To examine the hypothesis that the allele (T) that codes for the thermolabile defect increases the risk of coronary heart disease, we studied 111 patients with clinical and objective investigational evidence of coronary heart disease and 105 control subjects. The frequencies of the thermolabile defect (T) in patients and control subjects were measured, and the prevalence of elevated plasma total homocysteine according to genotype was assessed. The frequency of the defective allele was higher in patients than in control subjects with an OR of 1.6 (95% CI, 1.1 to 2.4; P = .02). The OR in the coronary heart disease group for the homozygous TT genotype was 2.9 (95% CI, 1.2 to 7.2; P = .02); 17% of patients and 7% of control subjects had the TT genotype. Plasma total homocysteine levels were significantly associated with disease status, a relationship that matched the strength of the association between disease and homozygous inheritance of the defective enzyme. CONCLUSIONS: Homozygotes for the defective allele (T) are at increased risk of premature coronary heart disease. MTHFR, which modulates basal plasma homocysteine concentration, is folate dependent, and dietary supplementation or fortification with folic acid may reduce plasma homocysteine levels and consequent coronary risk in a significant proportion of the general population.
Assuntos
Doença das Coronárias/epidemiologia , Homocisteína/sangue , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Alanina/genética , Alelos , Doença das Coronárias/sangue , Doença das Coronárias/genética , Análise Mutacional de DNA , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2) , Pessoa de Meia-Idade , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Mutação Puntual/fisiologia , Fatores de Risco , Temperatura , Valina/genéticaRESUMO
It is now well-established that folic acid, taken peri-conceptionally, can reduce the risk of neural tube defects (NTDs). Recent work has demonstrated that an abnormality of homocysteine metabolism is a critical factor. The gene for 5,10 methylenetetrahydrofolate reductase, an enzyme important in homocysteine metabolism, was studied in relation to NTDs. To determine the frequency of the allele for the thermolabile form of the reductase, DNA samples were collected from people with NTDs, parents of people with NTDs, and normal controls. Of 82 people with NTDs, 15 (18.3%) were homozygous for the abnormal, thermolabile allele. This was significantly higher (p = 0.01) than the rate of 6.1% in the control population (odds ratio 3.47, 95% CI 1.28-9.41). This is the first specific genetic abnormality to be identified in NTDs. It explains the association between some NTDs and elevated homocysteine, given that the reductase is important in homocysteine metabolism. It also explains how folic acid supplementation prevents some NTDs, by overcoming a partial block in the conversion of 5,10 methylenetetrahydrofolate to 5 methyltetrahydrofolate. Genetic screening could identify women who will require folic acid supplements to reduce their risk of having a child with an NTD.
Assuntos
Defeitos do Tubo Neural/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Estudos de Casos e Controles , Genótipo , Humanos , Metilenotetra-Hidrofolato Redutase (NADPH2) , Razão de ChancesRESUMO
The bacteriologic and clinical effects of early antibiotic treatment of Campylobacter jejuni enteritis were studied. Erythromycin rapidly eliminated C. jejuni from stools, whereas trimethoprim-sulfamethoxazole did not. Despite its bacteriologic effectiveness, erythromycin did not reduce the duration or severity of diarrhea, abdominal pain, or other symptoms.
Assuntos
Infecções por Campylobacter/tratamento farmacológico , Enterite/tratamento farmacológico , Eritromicina/uso terapêutico , Sulfametoxazol/uso terapêutico , Trimetoprima/uso terapêutico , Adulto , Infecções por Campylobacter/microbiologia , Campylobacter fetus/efeitos dos fármacos , Criança , Combinação de Medicamentos/farmacologia , Combinação de Medicamentos/uso terapêutico , Enterite/microbiologia , Eritromicina/farmacologia , Fezes/microbiologia , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Sulfametoxazol/farmacologia , Fatores de Tempo , Trimetoprima/farmacologia , Combinação Trimetoprima e SulfametoxazolRESUMO
Milk specimens, 75 from cows immunized against cholera toxin and 35 from a human population in which enterotoxigenic Escherichia coli and rotaviral infections are endemic, were collected as paired filter paper and frozen whole milk samples. Each pair was tested for antibody activity against heat-labile E. coli and Vibrio cholerae enterotoxins. Additionally, 12 of the 35 paired human milk samples stored as frozen whole milk and dried on filter paper were tested for anti-rotavirus immunoglobulin A. Anti-enterotoxin and anti-rotavirus immunoglobulin A titers in milk dried on filter paper compared favorably with those of their frozen whole milk pairs. Filter paper samples offered considerable advantages for field collection, transportation, and storage over frozen liquid samples.