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1.
Curr Opin Struct Biol ; 71: 259-273, 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34592682

RESUMO

Accurate predictions from models based on physical principles are the ultimate metric of our biophysical understanding. Although there has been stunning progress toward structure prediction, quantitative prediction of enzyme function has remained challenging. Realizing this goal will require large numbers of quantitative measurements of rate and binding constants and the use of these ground-truth data sets to guide the development and testing of these quantitative models. Ground truth data more closely linked to the underlying physical forces are also desired. Here, we describe technological advances that enable both types of ground truth measurements. These advances allow classic models to be tested, provide novel mechanistic insights, and place us on the path toward a predictive understanding of enzyme structure and function.

2.
Science ; 373(6553)2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34437092

RESUMO

Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.


Assuntos
Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/química , Biocatálise , Domínio Catalítico , Flavobacterium/enzimologia , Hidrólise , Cinética , Microfluídica , Modelos Moleculares , Mutação , Oxigênio/metabolismo , Fosfatos/metabolismo , Conformação Proteica , Dobramento de Proteína , Termodinâmica
3.
Food Chem Toxicol ; 44(7): 964-73, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16426723

RESUMO

The oral toxicity of recombinant human lactoferrin (rhLF) produced in the milk of transgenic cows was investigated in Wistar rats by daily administration via oral gavage for 13 consecutive weeks, 7 days per week. The study used four groups of 20 rats/sex/dose. The control group received physiological saline and the three test groups received daily doses of 200, 600 and 2000 mg of rhLF per kg body weight. Clinical observations, growth, food consumption, food conversion efficiency, water consumption, neurobehavioural testing, ophthalmoscopy, haematology, clinical chemistry, renal concentration test, urinalysis, organ weights and gross examination at necropsy and microscopic examination of various organs and tissues were used as criteria for detecting the effects of treatment. Overall, no treatment-related, toxicologically significant changes were observed. The few findings that may be related to the treatment (lower cholesterol in high-dose females, lower urinary pH in high-dose males and females and very slightly higher kidney weight in high-dose females) were considered of no toxicological significance. Based on the absence of treatment-related, toxicologically relevant changes, the no-observed-adverse-effect level (NOAEL) was considered to be at least 2000 mg/kg body weight/day.


Assuntos
Animais Geneticamente Modificados/metabolismo , Lactoferrina/toxicidade , Leite/toxicidade , Animais , Comportamento Animal/efeitos dos fármacos , Contagem de Células Sanguíneas , Análise Química do Sangue , Bovinos , Ingestão de Líquidos/efeitos dos fármacos , Feminino , Humanos , Lactoferrina/química , Leite/química , Atividade Motora/efeitos dos fármacos , Nível de Efeito Adverso não Observado , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/toxicidade , Caracteres Sexuais
4.
J Comp Pathol ; 133(1): 1-13, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15904927

RESUMO

Experimental borrelia infection was induced in 62 specific--pathogen-free beagle dogs by exposure to Ixodes scapularis ticks harbouring the spirochaete Borrelia burgdorferi. Clinical signs of Lyme disease occurred in 39/62 dogs, the remaining 23 being subclinically infected. Clinical signs consisted of one to six episodes of transitory lameness with joint swelling and pain, most commonly affecting the elbow or shoulder joints. The polymerase chain reaction and culture demonstrated that the dogs remained infected for up to 581 days. At necropsy, gross findings consisted of lymphadenopathy in the area of tick attachment. Microscopical changes consisted of effusive fibrinosuppurative inflammation or nonsuppurative inflammation, or both, affecting synovial membranes, joint capsules and associated tendon sheaths. Plasma cells dominated areas of chronic inflammation, with CD3(+) T cells being present in lesser numbers. Microscopical signs of arthritis were polyarticular and more widespread than indicated by clinical signs, and most of the subclinically affected animals also had synovitis. In areas of tick attachment to the skin, hyperkeratosis and a mixture of suppurative and nonsuppurative dermatitis were encountered. Lymphadenopathy in superficial lymph nodes resulted from follicular and parafollicular hyperplasia. In 14/62 dogs, lymphoplasmacytic periarteritis and perineuritis were noted, resembling lesions found in human Lyme disease and syphilis, in which an underlying microangiopathy has been proposed.


Assuntos
Borrelia burgdorferi , Cápsula Articular/patologia , Doença de Lyme/patologia , Doença de Lyme/fisiopatologia , Animais , Artrite/microbiologia , Borrelia burgdorferi/isolamento & purificação , Modelos Animais de Doenças , Cães , Cápsula Articular/imunologia , Cápsula Articular/microbiologia , Doença de Lyme/complicações , Linfonodos/imunologia , Linfonodos/microbiologia , Linfonodos/patologia , Reação em Cadeia da Polimerase , Pele/imunologia , Pele/microbiologia , Pele/patologia
5.
Vet Pathol ; 38(3): 311-6, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11355661

RESUMO

Canine distemper virus (CDV) was previously considered to have a host range restricted to the canid family. In 1994, the virus was associated with sporadic outbreaks of distemper in captive felids. However, after severe mortality occurred in the Serengeti lions (Panthera leo), attention became focused on the pathogenesis of the virus and a concerted effort was made to identify the virus as CDV or a closely related feline morbillivirus. The present study was designed to explore the susceptibility of ferrets to challenge with two morbilliviruses isolated from lions and the protective effects of a modified-live mink distemper vaccine. Because mortality in ferrets infected with pathogenic CDV approaches 100%, the ferret was selected as a test animal. Two strains of lion morbillivirus were used as a challenge, A92-27/20 (California lion isolate) and A94-11/13 (Serengeti lion isolate). The two strains of lion morbillivirus were antigenically related to CDV (Rockborn strain), and ferrets were susceptible to both of the viruses when inoculated intraperitoneally. The inoculated ferrets were anorectic at 5-6 days postinoculation (PI), exhibited oculonasal discharge at 9-12 days PI, and became moribund at 12-22 days PI. Severe bilateral conjunctivitis was the typical clinical sign. Inclusion bodies characteristic of morbillivirus (eosinophilic, intranuclear, and intracytoplasmic) were distributed in many epithelial cells, including those of the skin, conjunctiva, gallbladder, liver, pancreas, stomach, trachea, lung, urinary bladder, and kidney. Virus was reisolated from selected lung tissues collected at necropsy and identified by CDV-specific immunofluorescence. Ferrets vaccinated with the mink distemper vaccine (Onderstepoort strain) were protected from challenge with the two lion strains, adding further support to the premise that the viruses are closely related to CDV.


Assuntos
Furões/virologia , Leões/virologia , Infecções por Morbillivirus/veterinária , Morbillivirus/patogenicidade , Vacinação/veterinária , Animais , Efeito Citopatogênico Viral , Vírus da Cinomose Canina/patogenicidade , Furões/imunologia , Imunofluorescência/veterinária , Histocitoquímica/veterinária , Masculino , Morbillivirus/classificação , Morbillivirus/imunologia , Infecções por Morbillivirus/imunologia , Infecções por Morbillivirus/patologia , Vacinas Virais/imunologia , Vacinas Virais/normas , Viremia/veterinária
6.
Food Chem Toxicol ; 39(3): 261-9, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11278058

RESUMO

A study was performed to provide data on the disposition, accumulation and toxicity of sodium iron EDTA in comparison with iron (II) sulfate in rats on administration via the diet for 31 and 61 days. Clinical signs, body weights, food consumption, food conversion efficiency, hematology, clinical chemistry and pathology of selected organs were used as criteria for disclosing possible harmful effects. Determination of iron and total iron binding capacity in blood plasma and non-heme iron analysis in liver, spleen and kidneys were used to assess the disposition and accumulation of iron originating from sodium iron EDTA or iron (II) sulfate. It was concluded that, under the conditions of the present study, iron is accumulated from the diet in liver, spleen and kidneys in a dose-dependent manner, and iron derived from FeEDTA is taken up and/or accumulated less efficiently in liver and spleen than iron from FeSO(4). Moreover, feeding iron up to 11.5 and 11.2 mg/kg body weight/day, derived from FeSO(4) and FeEDTA, respectively, did not result in tissue iron excess nor in any other toxicologically significant effects.


Assuntos
Ácido Edético/farmacocinética , Ácido Edético/toxicidade , Compostos Férricos/farmacocinética , Compostos Férricos/toxicidade , Ferro/farmacocinética , Animais , Peso Corporal/efeitos dos fármacos , Corantes , Dieta , Ingestão de Alimentos/efeitos dos fármacos , Análise de Alimentos , Ferro/sangue , Masculino , Ferroproteínas não Heme/sangue , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Distribuição Tecidual
7.
Food Chem Toxicol ; 37(9-10): 981-4, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10541454

RESUMO

Previously performed short-term (4-month) studies demonstrated that vitamins C and E, beta-carotene and selenium modulate growth of early putative preneoplastic acinar lesions induced in rat pancreas by azaserine. The present paper summarizes the results of long-term studies performed with azaserine-treated rats maintained on diets high in either beta-carotene, vitamins C and E or selenium. It appeared that rats given a diet high in beta-carotene, vitamin C or selenium, but not vitamin E, developed fewer pancreatic tumours than controls. The chemopreventive effects of these micronutrients were most pronounced when beta-carotene and/or selenium were given during the promotion phase of the carcinogenic process. Surprisingly, cell proliferation in azaserine-induced preneoplastic acinar lesions was higher in rats given beta-carotene and/or selenium via the diet in comparison to controls. It is considered unlikely that any antioxidant alone can be associated with protection against cancer. It is concluded that dietary supplementation of combinations of antioxidants may have practical application in chemoprevention of cancer.


Assuntos
Antioxidantes/farmacologia , Suplementos Nutricionais , Neoplasias Pancreáticas/prevenção & controle , Lesões Pré-Cancerosas/prevenção & controle , Animais , Ácido Ascórbico/farmacologia , Azasserina , Combinação de Medicamentos , Masculino , Neoplasias Pancreáticas/induzido quimicamente , Neoplasias Pancreáticas/patologia , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/patologia , Ratos , Ratos Wistar , Selênio/farmacologia , Fatores de Tempo , Vitamina E/farmacologia , beta Caroteno/farmacologia
8.
Mutat Res ; 443(1-2): 111-27, 1999 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-10415435

RESUMO

Epidemiologic investigations have suggested a relationship between dietary fat intake and various types of cancer incidences. Furthermore, epidemiologic studies as well as studies with animal models have demonstrated that not only the amount but also the type of fat consumed is important. At present, the mechanism by which dietary fat modulates carcinogenesis has not been elucidated. The effects of dietary fat on the development of tumours have been summarized in the present review with emphasis on colorectal, pancreas, breast and prostate cancer. It is concluded that influence on synthesis of prostaglandins and leukotrienes may be the universal mechanism by which dietary fats modulate carcinogenesis.


Assuntos
Neoplasias da Mama/etiologia , Neoplasias Colorretais/etiologia , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/metabolismo , Neoplasias/etiologia , Neoplasias Pancreáticas/etiologia , Neoplasias da Próstata/etiologia , Animais , Neoplasias da Mama/epidemiologia , Neoplasias Colorretais/epidemiologia , Feminino , Humanos , Leucotrienos/metabolismo , Masculino , Neoplasias Pancreáticas/epidemiologia , Prostaglandinas/metabolismo , Neoplasias da Próstata/epidemiologia , Ratos , Ratos Wistar
9.
Carcinogenesis ; 20(4): 651-6, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10223195

RESUMO

The objective of this experiment was to compare the effects of diets with either a non-fermentable fibre source (cellulose) or a fermentable fibre source [galacto-oligosaccharide (GOS)], combined with different levels of dietary fat, on the development of colorectal cancer. Male Wistar rats were fed AIN76-based diets with either a low or high level of cellulose, or a low or high level of GOS, for 9 months. The fat content of the diets was low, medium or high. All rats were treated with 1,2-dimethylhydrazine to induce colorectal tumours. Generally, the tumour incidence increased with increasing fat content in the diet. Despite marked faeces bulking, dietary cellulose either had no effect or an enhancing effect on the formation of colorectal tumours in general, although the development of carcinomas was decreased. GOS appeared to be highly protective against the development of colorectal tumours, as was demonstrated by an inhibitory effect on tumour incidence, multiplicity and size, regardless of the fat content of the diet. Neither fibre source influenced the bromodeoxyuridine labelling index determined in colon crypts or tumours. In animals fed high-GOS diets, the caecal content was significantly increased in weight and significantly decreased in pH. It was concluded that tumorigenesis was enhanced by increased fat content of the diet, and that the diets containing fermentable GOS conferred a greater protection against colorectal cancer than did the diets containing non-fermentable cellulose.


Assuntos
Celulose/farmacologia , Neoplasias Colorretais/prevenção & controle , Fibras na Dieta/farmacologia , Fermentação , Galactose/farmacologia , Oligossacarídeos/farmacologia , 1,2-Dimetilidrazina/toxicidade , Animais , Ácidos e Sais Biliares/metabolismo , Carcinógenos/toxicidade , Ceco , Celulose/metabolismo , Celulose/uso terapêutico , Celulose/toxicidade , Cocarcinogênese , Neoplasias Colorretais/etiologia , Gorduras na Dieta/toxicidade , Fibras na Dieta/metabolismo , Fibras na Dieta/uso terapêutico , Galactose/metabolismo , Galactose/uso terapêutico , Conteúdo Gastrointestinal , Concentração de Íons de Hidrogênio , Masculino , Oligossacarídeos/metabolismo , Oligossacarídeos/uso terapêutico , Óleos Vegetais/toxicidade , Ratos , Ratos Wistar , Organismos Livres de Patógenos Específicos , Óleo de Girassol
10.
Regul Toxicol Pharmacol ; 29(2 Pt 2): S11-28, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10341157

RESUMO

D-tagatose is an incompletely absorbed ketohexose (stereoisomer of D-fructose) which has potential as an energy-reduced alternative sweetener. In an earlier 90-day toxicity study, rats fed diets with 10, 15 and 20% D-tagatose exhibited increased liver weights, but no histopathological alterations. To determine whether there might be any toxicological relevance to this effect, three studies were conducted in male, adult Sprague-Dawley rats. In the first study, four groups received Purina diet (group A), Purina diet with 20% D-tagatose (group B), SDS diet (group C), or SDS diet with 20% D-tagatose (group D). For groups A and B, the 28-day treatment period was followed by a 14-day recovery period (Purina diet). Food remained available to all animals until the time of sacrifice. Groups of 10 rats were killed on days 14 (groups A and B), 28 (groups A-D), and 42 (groups A and B). Body weights, as well as weights of wet and lyophilized livers, were determined. The lyophilized livers collected on day 28 from groups A and B were analyzed for protein, total lipid, glycogen, DNA, and residual moisture. By day 14, relative wet liver weights had increased by 23% in group B. On day 28, the increase was 38% in group B and 44% in group D. At the end of the recovery period, the increase had diminished to 14% in group B. On day 28, liver glycogen content (in %) was significantly increased, and liver protein, lipid, and DNA contents were significantly decreased in group B compared to group A. Total amounts per liver of protein, total lipid, glycogen, and DNA were significantly increased. In the second study, four groups of 20 rats each received SDS diet with 0, 5, 10, and 20% D-tagatose for 29-31 days. The food was available until the time of sacrifice. At termination, plasma was obtained from 10 rats/group for clinicochemical analyses. Five rats/group were subjected to whole-body perfusion, followed by processing of livers for qualitative and quantitative electron microscopic examination. Livers of 6 rats/group were analyzed for acyl-CoA oxidase and laurate 12-hydroxylase (cytochrome P450 4A1) activity, DNA synthesis (Ki-67 index), and number of nuclei per unit area of tissue. Liver weights were significantly increased in linear relation to the D-tagatose intake. Plasma transaminases (but not glutamyl transferase and alkaline phosphatase) were increased in the high-dose group. Except for glycogen accumulation, no ultrastructural changes were seen on electron microscopic examination of livers of the control and high-dose groups. Morphometric analysis confirmed the increase of glycogen and the absence of alterations of endoplasmatic reticulum, mitochondria, and Golgi apparatus. The Ki-67 index did not differ between the groups. A dose-related decrease of the number of nuclei per unit area signified some hepatocellular hypertrophy. Acyl-CoA oxidase and CYP4A1 activity were significantly increased in the mid- and high-dose groups, but these increases were small and not accompanied by electron-microscopic evidence of peroxisome proliferation. In the third study, four groups received SDS diet (groups A and C) or SDS diet with 5% D-tagatose (groups B and D). All animals were killed on day 28. Groups A and B were fasted for 24 h before sacrifice; groups C and D had food available until sacrifice. Liver weights and liver composition were measured as in Study 1. Relative wet and dry liver weights were increased in response to the treatment in rats killed under the fed condition, but not in rats killed under the fasted condition. The livers of the treated rats (group D) had an increased glycogen content in comparison to the controls (group C). Taken together, these results demonstrate that D-tagatose at dietary levels of 5-20% increases liver glycogen deposition and relative liver weights in nonfasting rats. In fasted rats the 5% dose level is the no-effect level. (ABSTRACT TRUNCATED)


Assuntos
Hexoses/toxicidade , Glicogênio Hepático/metabolismo , Fígado/efeitos dos fármacos , Edulcorantes/toxicidade , Animais , Peso Corporal/efeitos dos fármacos , Ceco/anatomia & histologia , Ceco/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , DNA/biossíntese , Ingestão de Alimentos , Fígado/anatomia & histologia , Fígado/metabolismo , Fígado/ultraestrutura , Masculino , Microscopia Eletrônica , Tamanho do Órgão/efeitos dos fármacos , Proliferadores de Peroxissomos/toxicidade , Ratos , Ratos Sprague-Dawley
11.
Vet Immunol Immunopathol ; 67(3): 271-84, 1999 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-10195465

RESUMO

The up-regulation of the inducible nitric oxide synthase (iNOS) mRNA was determined by RT-PCR in 25 tissues each from 22 specific pathogen-free (SPF) dogs experimentally infected with Borrelia burgdorferi by tick exposure and from five uninfected control dogs. Using primers specific for a homologous region of the human and canine iNOS sequence, and canine macrophage mRNA, we isolated and partially sequenced canine iNOS. A sequence of 1775 bases was obtained and primers specific for canine iNOS mRNA constructed to investigate the expression of iNOS in dog tissues in response to infection with B. burgdorferi. In 12 out of 22 dogs infected with B. burgdorferi, acute lameness occurred within 55-82 days after infection whereas the other 10 dogs showed no or only mild clinical signs despite persistent infection up to Day 175. The numbers of iNOS mRNA-positive tissues in dogs with acute lameness were significantly higher than in dogs without lameness, while uninfected dogs showed only negligible iNOS expression. Dogs with acute lameness also had higher numbers of borrelia-positive tissues as well as higher scores in histopathological evaluations than infected dogs without lameness. Our results show that the expression of iNOS mRNA is related to the number of B. burgdorferi-positive tissues and the severity of inflammation as assessed by histopathology. These results implicate an up-regulation of the iNOS mRNA as part of the host's immune response to borrelia infection and a possible role for NO in the pathogenesis of canine Lyme arthritis.


Assuntos
Doenças do Cão/enzimologia , Doença de Lyme/veterinária , Óxido Nítrico Sintase/genética , RNA Mensageiro/biossíntese , Regulação para Cima , Animais , Artrite Infecciosa/enzimologia , Artrite Infecciosa/patologia , Artrite Infecciosa/veterinária , Grupo Borrelia Burgdorferi/isolamento & purificação , Doenças do Cão/patologia , Cães , Feminino , Humanos , Articulações/patologia , Coxeadura Animal/enzimologia , Coxeadura Animal/microbiologia , Coxeadura Animal/patologia , Doença de Lyme/enzimologia , Doença de Lyme/patologia , Macrófagos Alveolares/enzimologia , Masculino , Dados de Sequência Molecular , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Reação em Cadeia da Polimerase/veterinária , RNA Mensageiro/química
12.
Adv Vet Med ; 41: 309-24, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-9890024

RESUMO

During the last 40 years vaccines have been developed that have greatly reduced the incidence of infectious diseases of dogs. In general, modified live products have been superior to inactivated vaccines for dogs. It can be expected that recombinant and/or DNA vaccines may dominate the market in the future. Although most vaccines on the market are safe and efficacious, there have been exceptions where disease was induced by vaccination or dogs were not protected. The failure of protection may in part be due to variations in individual vaccine batches. Only potency tests but not efficacy tests are required, which may not be sufficient. For example, a virus titer in a vaccine may be meaningless if the minimum protective dose is not known. Overattenuated virus (e.g., CDV-Ond or parvovirus in cat cells) may have a high titer in tissue culture but is not immunogenic. The question of frequency of vaccination of dogs should be addressed. Annual revaccinations for CDV, CPV, and CAV are probably not needed. However, it would be desirable to collect more data to support less frequent vaccinations. Annual immunization for bacterial diseases such as kennel cough, Lyme disease, and leptospirosis should continue. It also would be desirable to develop more oro/nasal vaccines, perhaps combined with newly developed vectors that are less likely to induce undesirable side effects that may be seen after parenteral vaccination. Finally a word of warning against homeopathic "nosodes" to replace tested canine vaccines. They will appear highly effective as long as the majority of dogs remain vaccinated. As soon as a nonvaccinated dog population is large enough to allow virulent agents to spread, disease outbreaks will occur and we will be back where we began 40 years ago.


Assuntos
Infecções Bacterianas/veterinária , Vacinas Bacterianas , Doenças do Cão/imunologia , Imunização/veterinária , Vacinação/veterinária , Vacinas Virais , Viroses/veterinária , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/prevenção & controle , Gatos , Doenças do Cão/prevenção & controle , Cães , Imunização/métodos , Esquemas de Imunização , Vacinas de DNA , Viroses/imunologia , Viroses/prevenção & controle
13.
Crit Rev Toxicol ; 29(6): 515-42, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10628775

RESUMO

The safety of beta-carotene was reassessed by evaluating the relevant literature on the beneficial and adverse effects of beta-carotene on cancer and, in particular, by evaluating the results of toxicity studies. Beta-carotene appeared neither genotoxic nor reprotoxic or teratogenic, and no signs of organ toxicity have been found in subacute, subchronic, or chronic oral toxicity studies in experimental animals receiving doses of up to 1000 mg/day beta-carotene per kg body weight via the diet. Synthetic beta-carotene did not exert any carcinogenic effect in Sprague-Dawley rats or in CD1 mice. An enhanced risk of lung cancer was found in two human intervention studies. Although dose and (timing of) exposure, smoking status, and imbalance of antioxidant defense have been recognized as potential factors accounting for the outcome of these studies, a conclusive explanation has not yet been found. It is concluded that exposure to beta-carotene resulting in mean plasma concentrations of no more than 2.2 micromol/l (1.2 microg/ml) is safe to the general population. By contrast, in heavy smokers higher plasma concentrations may be associated with a higher lung cancer risk.


Assuntos
Antioxidantes/efeitos adversos , Neoplasias Pulmonares/induzido quimicamente , beta Caroteno/efeitos adversos , Absorção , Animais , Antioxidantes/farmacocinética , Antioxidantes/farmacologia , Transformação Celular Neoplásica , Quimioprevenção , Interações Medicamentosas , Humanos , Camundongos , Ratos , Ratos Sprague-Dawley , Neoplasias Cutâneas/prevenção & controle , Fumar/efeitos adversos , beta Caroteno/farmacocinética , beta Caroteno/farmacologia
14.
Vet Immunol Immunopathol ; 65(2-4): 259-66, 1998 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-9839878

RESUMO

The lion (Panthera leo) population in the Serengeti ecosystem was recently afflicted by a fatal epidemic involving neurological disease, encephalitis and pneumonia. The cause was identified as canine distemper virus (CDV). Several other species in the Serengeti were also affected. This report presents CDV H and P gene sequences isolated from Serengeti lions (Panthera leo), spotted hyenas (Crocuta crocuta), bat-eared fox (Otocyon megalotis) and domestic dog (Canis familiaris). Sequence analyses demonstrated that the four Serengeti species carry closely related CDV isolates which are genetically distinct from other CDV isolates from various species and locations. The results are consistent with the conclusions that: (1) a particularly virulent strain of CDV emerged among Serengeti carnivores within the last few years; (2) that strain has recognizable shared-derived (synapomorphic) genetic differences in both H and P genes when compared to CDV from other parts of the world; and (3) that the CDV strain has frequently crossed host species among Serengeti carnivores.


Assuntos
Carnívoros/virologia , DNA Viral/análise , Vírus da Cinomose Canina/genética , Cinomose/genética , África/epidemiologia , Animais , Sequência de Bases , Primers do DNA/química , Cinomose/epidemiologia , Cães , Genes Virais/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico
15.
Infect Immun ; 66(1): 247-58, 1998 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9423865

RESUMO

Canine synovial membrane explants were exposed to high- or low-passage Borrelia burgdorferi for 3, 6, 12, and 24 h. Spirochetes received no treatment, were UV light irradiated for 16 h, or were sonicated prior to addition to synovial explant cultures. In explant tissues, mRNA levels for the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), IL-1beta, and IL-8 were surveyed semiquantitatively by reverse transcription-PCR. Culture supernatants were examined for numbers of total and motile (i.e., viable) spirochetes, TNF-like and IL-1-like activities, polymorphonuclear neutrophil (PMN) chemotaxis-inducing activities, and IL-8. During exposure to synovial explant tissues, the total number of spirochetes in the supernatants decreased gradually by approximately 30%, and the viability also declined. mRNAs for TNF-alpha, IL-1alpha, IL-1beta, and IL-8 were up-regulated in synovial explant tissues within 3 h after infection with untreated or UV light-irradiated B. burgdorferi, and mRNA levels corresponded to the results obtained with bioassays. During 24 h of coincubation, cultures challenged with untreated or UV light-irradiated spirochetes produced similar levels of TNF-like and IL-1-like activities. In contrast, explant tissues exposed to untreated B. burgdorferi generated significantly higher levels of chemotactic factors after 24 h of incubation than did explant tissues exposed to UV light-treated spirochetes. In identical samples, a specific signal for IL-8 was identified by Western blot analysis. High- and low-passage borreliae did not differ in their abilities to induce proinflammatory cytokines. No difference in cytokine induction between untreated and sonicated high-passage spirochetes was observed, suggesting that fractions of the organism can trigger the production and release of inflammatory mediators. The titration of spirochetes revealed a dose-independent cytokine response, where 10(3) to 10(7) B. burgdorferi organisms induced similar TNF-like activities but only 10(7) spirochetes induced measurable IL-1-like activities. The release of chemotactic factors was dose dependent and was initiated when tissues were infected with at least 10(5) organisms. We conclude that intact B. burgdorferi or fractions of the bacterium can induce the local up-regulation of TNF-alpha, IL-1alpha, and IL-1beta in the synovium but that the interaction of viable spirochetes with synovial cells leads to the release of IL-8, which probably is a prime initiator of PMN migration during acute Lyme arthritis.


Assuntos
Grupo Borrelia Burgdorferi/imunologia , Interleucina-1/metabolismo , Interleucina-8/metabolismo , Doença de Lyme/imunologia , Membrana Sinovial/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anticorpos Bloqueadores , Grupo Borrelia Burgdorferi/efeitos da radiação , Fatores Quimiotáticos/análise , Quimiotaxia de Leucócito , Contagem de Colônia Microbiana , Meios de Cultivo Condicionados/análise , Testes Imunológicos de Citotoxicidade , Cães , Técnicas In Vitro , Interleucina-1/análise , Interleucina-1/genética , Interleucina-8/análise , Interleucina-8/genética , Neutrófilos/imunologia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/imunologia , Sonicação , Membrana Sinovial/metabolismo , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/genética , Raios Ultravioleta , Regulação para Cima
16.
J Wildl Dis ; 33(4): 823-32, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9391968

RESUMO

African wild dogs (Lycaon pictus) are endangered, with only 3,000-5,000 remaining in the wild. It is believed that wild dogs are unusually vulnerable to viral diseases, particularly rabies and canine distemper (CDV). However, canine distemper has been confirmed by laboratory diagnosis in only one free-living wild dog. The 43,000 km2 Selous Game Reserve (SGR; Tanzania) holds approximately 900 adult wild dogs. In a study area of 2,600 km2, the population maintained high density (> or = 1 dog/20.5 km2) from 1991 to 1996. The population was stable, varying 18% below and 9% above the mean density over the 6-yr period. Serum samples (n = 22) collected over 3 yr showed that most individuals were exposed to CDV (59%:95% confidence interval = 43-76% seropositive) and canine parvovirus (68%:95% CI = 54-81% seropositive), although none were seropositive for rabies (0%:95% CI = 0-17%). CDV titers were positively related to age, with no seropositive dogs younger than 1.9 yr. At least five of 13 dogs positive for CDV seroconverted during the study. Dogs with high CDV titers did not survive better in the years after sampling (mean survival +/- SE for those that died = 638 +/- 92 days,). Variation in mean litter size was inversely related to CPV exposure in the SGR and elsewhere. Annual mortality rates were low in comparison to other populations for all age classes (pups: 31 +/- 8%, n = 127, yearlings: 22 +/- 10%, n = 93, adults: 20 +/- 6%, n = 235). Annual mortality rates fluctuated little between 1992 and 1996. These data show that wild dog populations, like those of other canids, can remain stable and demographically healthy despite exposure to CDV and CPV.


Assuntos
Anticorpos Antivirais/sangue , Carnívoros , Viroses/veterinária , Animais , Animais Selvagens , Cinomose/epidemiologia , Vírus da Cinomose Canina/imunologia , Feminino , Masculino , Infecções por Parvoviridae/epidemiologia , Infecções por Parvoviridae/veterinária , Parvovirus Canino/imunologia , Prevalência , Raiva/epidemiologia , Raiva/veterinária , Vírus da Raiva/imunologia , Tanzânia/epidemiologia , Viroses/epidemiologia
17.
J Zoo Wildl Med ; 28(3): 312-8, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9365945

RESUMO

This report describes vaccine-induced canine distemper virus (CDV) infection in four European mink (Mustela lutreola) induced by the administration of a multivalent, avian-origin vaccine. Clinical signs consisting of seizures, ataxia, facial twitching, oculonasal discharge, hyperkeratosis of footpads, and anorexia developed 16-20 days postvaccination. Conjunctival smears from one animal were positive for CDV antigen by direct fluorescent antibody testing, confirming the clinical diagnosis. The four mink died 16-26 days postvaccination. Gross and microscopic lesions that were diagnostic for CDV infection included interstitial pneumonia, lymphoid depletion, nonsuppurative encephalitis, and dermatitis. Vaccine-strain virus was isolated from tissues of three animals. Cases of vaccine-induced distemper in mustelids using avian-origin vaccine have seldom been reported.


Assuntos
Vírus da Cinomose Canina/imunologia , Cinomose/induzido quimicamente , Vison , Vacinas Virais/efeitos adversos , Adenovirus Caninos/imunologia , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Antígenos Virais/análise , Antígenos Virais/imunologia , Dermatite/imunologia , Dermatite/patologia , Dermatite/veterinária , Cinomose/diagnóstico , Cinomose/epidemiologia , Feminino , Técnica Direta de Fluorescência para Anticorpo/veterinária , Incidência , Pulmão/patologia , Doenças Pulmonares Intersticiais/imunologia , Doenças Pulmonares Intersticiais/patologia , Doenças Pulmonares Intersticiais/veterinária , Linfonodos/patologia , Masculino , Paramyxoviridae/imunologia , Parvovirus Canino/imunologia , Vacinas Antirrábicas/efeitos adversos , Vacinas Antirrábicas/imunologia , Vírus da Raiva/imunologia , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologia
18.
Infect Immun ; 65(4): 1273-85, 1997 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9119462

RESUMO

Twenty 6-week-old specific-pathogen-free beagles were infected with Borrelia burgdorferi by tick challenge, and five uninfected dogs served as controls. During the study, all dogs were monitored for infection, clinical signs, and antibody response against B. burgdorferi. During episodes of lameness or postmortem, synovial fluids from each dog were examined for volume, cell number, polymorphonuclear leukocyte (PMN) content, cell viability, and chemotactic activity. Twenty-five tissues collected postmortem from each dog were tested for interleukin-8 (IL-8) mRNA, tumor necrosis factor alpha (TNF-alpha) mRNA, presence of live spirochetes, and histopathological changes. Thirteen infected dogs (group A), which seroconverted rapidly (maximum titers within 50 to 90 days), developed acute and severe mono- or oligoarthritis almost exclusively in the limb closest to the tick bite (median incubation period, 66 days). Synovial fluids of the arthritic joints collected during episodes of lameness had significantly elevated volume, cell count, PMN proportion, cell viability, and chemotactic activity for PMNs. The remaining joints of the same animals contained synovial fluids with elevated chemotactic activity and cell viability. Twelve dogs tested positive for IL-8 mRNA in multiple tissues (synovia, pericardium, and peritoneum), and 10 dogs expressed TNF-alpha mRNA, but only in the tributary lymph nodes of the inflamed joints. Histological examinations revealed severe poly- or oligoarthritis and moderate to severe cortical hyperplasia in draining lymph nodes of the inflamed joints in all 13 dogs. Seven infected dogs with mild or no clinical signs (group B) seroconverted slowly (peak titers after 90 days), and only some joint fluids showed chemotactic activity, which on average was lower than that in inflamed and noninflamed joints from dogs in group A. Four dogs expressed IL-8 mRNA (in the synovia and pericardium), and three dogs had TNF-alpha mRNA in tributary lymph nodes. Histologically, nonsuppurative arthritis was found in multiple joints, and mild to moderate cortical hyperplasia was found in draining lymph nodes. Five uninfected dogs without lameness (group C) had normal synovial fluids and tissues. In all infected dogs, live spirochetes were demonstrated more frequently in tissues of the somatic quadrant closest to the tick bite than in tissues further from the site of infection, suggesting that dissemination of B. burgdorferi occurs more by migration than by blood-borne spread. From these studies employing a canine model of B. burgdorferi infection, we conclude that IL-8 is involved in the pathogenesis of acute Lyme arthritis.


Assuntos
Grupo Borrelia Burgdorferi , Interleucina-8/metabolismo , Articulações/microbiologia , Doença de Lyme/microbiologia , Membrana Sinovial/microbiologia , Animais , Cães , Articulações/patologia , Doença de Lyme/metabolismo , Doença de Lyme/patologia , Membrana Sinovial/patologia , Regulação para Cima
19.
J Gen Virol ; 78 ( Pt 2): 367-72, 1997 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9018059

RESUMO

To characterize the variability of recent field isolates of canine distemper virus (CDV) from different hosts and geographical areas, we conducted nucleotide sequence analysis of the gene encoding the haemagglutinin (H), the attachment protein of this virus. Pronounced differences between field isolates were revealed in comparison to the Convac and Onderstepoort vaccine strains. The diversity of CDV appeared to exceed that determined for measles virus. Phylogenetic analysis also separated the field isolates of CDV from the vaccine strains and provided evidence for the existence of different contemporary genotypes of CDV. Isolates from a Greenlandic sledge dog and a Siberian seal formed a distinct lineage. The remaining isolates formed a group. This group contained two European isolates from mink and ferret, a single lineage comprising three European dog isolates, and another separate lineage of North American isolates from dog, javelina, raccoon and captive leopards.


Assuntos
Vírus da Cinomose Canina/genética , Variação Genética , Glicoproteínas/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Vírus da Cinomose Canina/classificação , Vírus da Cinomose Canina/isolamento & purificação , Cães , Dados de Sequência Molecular , Filogenia , Focas Verdadeiras , Vacinas Virais/genética
20.
J Clin Microbiol ; 35(1): 111-6, 1997 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8968890

RESUMO

In specific-pathogen-free dogs experimentally infected with Borrelia burgdorferi by tick exposure, treatment with high doses of amoxicillin or doxycycline for 30 days diminished but failed to eliminate persistent infection. Although joint disease was prevented or cured in five of five amoxicillin- and five of six doxycycline-treated dogs, skin punch biopsies and multiple tissues from necropsy samples remained PCR positive and B. burgdorferi was isolated from one amoxicillin- and two doxycycline-treated dogs following antibiotic treatment. In contrast, B. burgdorferi was isolated from six of six untreated infected control dogs and joint lesions were found in four of these six dogs. Serum antibody levels to B. burgdorferi in all dogs declined after antibiotic treatment. Negative antibody levels were reached in four of six doxycycline- and four of six amoxicillin-treated dogs. However, in dogs that were kept in isolation for 6 months after antibiotic treatment was discontinued, antibody levels began to rise again, presumably in response to proliferation of the surviving pool of spirochetes. Antibody levels in untreated infected control dogs remained high.


Assuntos
Grupo Borrelia Burgdorferi/isolamento & purificação , Doença de Lyme/microbiologia , Animais , Antibacterianos/uso terapêutico , Anticorpos Antibacterianos/sangue , Cães , Feminino , Doença de Lyme/sangue , Doença de Lyme/tratamento farmacológico , Doença de Lyme/fisiopatologia , Masculino , Reação em Cadeia da Polimerase , Fatores de Tempo
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