Alzheimer's disease-like APP processing in wild-type mice identifies synaptic defects as initial steps of disease progression.

BACKGROUND: Alzheimer's disease (AD) is the most frequent form of dementia in the elderly and no effective treatment is currently available. The mechanisms triggering AD onset and progression are still imperfectly dissected. We aimed at deciphering the modifications occurring in vivo during the very early stages of AD, before the development of amyloid deposits, neurofibrillary tangles, neuronal death and inflammation. Most current AD models based on Amyloid Precursor Protein (APP) overproduction beginning from in utero, to rapidly reproduce the histological and behavioral features of the disease within a few months, are not appropriate to study the early steps of AD development. As a means to mimic in vivo amyloid APP processing closer to the human situation in AD, we used an adeno-associated virus (AAV)-based transfer of human mutant APP and Presenilin 1 (PS1) genes to the hippocampi of two-month-old C57Bl/6 J mice to express human APP, without significant overexpression and to specifically induce its amyloid processing. RESULTS: The human APP, βCTF and Aβ42/40 ratio were similar to those in hippocampal tissues from AD patients. Three months after injection the murine Tau protein was hyperphosphorylated and rapid synaptic failure occurred characterized by decreased levels of both PSD-95 and metabolites related to neuromodulation, on proton magnetic resonance spectroscopy ((1)H-MRS). Astrocytic GLT-1 transporter levels were lower and the tonic glutamatergic current was stronger on electrophysiological recordings of CA1 hippocampal region, revealing the overstimulation of extrasynaptic N-methyl D-aspartate receptor (NMDAR) which precedes the loss of long-term potentiation (LTP). These modifications were associated with early behavioral impairments in the Open-field, Y-maze and Morris Mater Maze tasks. CONCLUSIONS: Altogether, this demonstrates that an AD-like APP processing, yielding to levels of APP, βCTF and Aβ42/Aβ40 ratio similar to those observed in AD patients, are sufficient to rapidly trigger early steps of the amyloidogenic and Tau pathways in vivo. With this strategy, we identified a sequence of early events likely to account for disease onset and described a model that may facilitate efforts to decipher the factors triggering AD and to evaluate early neuroprotective strategies.

Effect of diet composition and ration size on key enzyme activities of glycolysis-gluconeogenesis, pentose phosphate pathway and amino acid metabolism in liver of Sparus aurata

The effects of diet composition and ration size on the activities of key enzymes involved in intermediary metabolism were studied in the liver of gilthead sea bream (Sparus aurata). Highcarbohydrate, low-protein diets stimulated 6-phosphofructo 1-kinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) enzyme activities, while they decreased alanine aminotransferase (EC 2.6.1.2) activity. A high degree of correlation was found between food ration size and the activity of the enzymes 6-phosphofructo 1-kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase (positive correlations) and fructose-1,6-bisphosphatase (EC 3.1.3.11) (negative correlation). These correlations matched well with the high correlation also found between ration size and growth rate in starved fish refed for 22 d. Limited feeding (5 g/kg body weight) for 22 d decreased the activities of the key enzymes for glycolysis and lipogenesis, and alanine aminotransferase activity. The findings presented here indicate a high level of metabolic adaptation to both diet type and ration size. In particular, adaptation of enzyme activities to the consumption of a diet with a high carbohydrate level suggests that a carnivorous fish like Sparus aurata can tolerate partial replacement of protein by carbohydrate in the commercial diets supplied in culture. The relationship between enzyme activities, ration size and fish growth indicates that the enzymes quickly respond to dietary manipulations of cultured fish.

Effect of diet composition and ration size on key enzyme activities of glycolysis-gluconeogenesis, pentose phosphate pathway and amino acid metabolism in liver of Sparus aurata

The effects of diet composition and ration size on the activities of key enzymes involved in intermediary metabolism were studied in the liver of gilthead sea bream (Sparus aurata). Highcarbohydrate, low-protein diets stimulated 6-phosphofructo 1-kinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) enzyme activities, while they decreased alanine aminotransferase (EC 2.6.1.2) activity. A high degree of correlation was found between food ration size and the activity of the enzymes 6-phosphofructo 1-kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase (positive correlations) and fructose-1,6-bisphosphatase (EC 3.1.3.11) (negative correlation). These correlations matched well with the high correlation also found between ration size and growth rate in starved fish refed for 22 d. Limited feeding (5 g/kg body weight) for 22 d decreased the activities of the key enzymes for glycolysis and lipogenesis, and alanine aminotransferase activity. The findings presented here indicate a high level of metabolic adaptation to both diet type and ration size. In particular, adaptation of enzyme activities to the consumption of a diet with a high carbohydrate level suggests that a carnivorous fish like Sparus aurata can tolerate partial replacement of protein by carbohydrate in the commercial diets supplied in culture. The relationship between enzyme activities, ration size and fish growth indicates that the enzymes quickly respond to dietary manipulations of cultured fish.

The L-type voltage-gated calcium channel modulates microglial pro-inflammatory activity

Under pathological conditions, microglia, the resident CNS immune cells, become reactive and release pro-inflammatory cytokines and neurotoxic factors. We investigated whether this phenotypic switch includes changes in the expression of the L-type voltage-gated calcium channel (VGCC) in a rat model of N-methyl-d-aspartate-induced hippocampal neurodegeneration. Double immunohistochemistry and confocal microscopy evidenced that activated microglia express the L-type VGCC. We then analyzed whether BV2 microglia express functional L-type VGCC, and investigated the latter's role in microglial cytokine release and phagocytic capacity. Activated BV2 microglia express the CaV1.2 and CaV1.3 subunits of the L-type VGCC determined by reverse transcription-polymerase chain reaction, Western blot and immunocytochemistry. Depolarization with KCl induced a Ca2+ entry facilitated by Bay k8644 and partially blocked with nifedipine, which also reduced TNF-α and NO release by 40%. However, no nifedipine effect on BV2 microglia viability or phagocytic capacity was observed. Our results suggest that in CNS inflammatory processes, the L-type VGCC plays a specific role in the control of microglial secretory activity.

Expressional regulation of key hepatic enzymes of intermediary metabolism in European seabass (Dicentrarchus labrax) during food deprivation and refeeding

We hypothesized that the analysis of mRNA level and activity of key enzymes in amino acid and carbohydrate metabolism in a feeding/fasting/refeeding setting could improve our understanding of how a carnivorous fish, like the European seabass (Dicentrarchus labrax), responds to changes in dietary intake at the hepatic level. To this end cDNA fragments encoding genes for cytosolic and mitochondrial alanine aminotransferase (cALT; mALT), pyruvate kinase (PK), glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) were cloned and sequenced. Measurement of mRNA levels through quantitative real-time PCR performed in livers of fasted seabass revealed a significant increase in cALT (8.5-fold induction)while promoting a drastic 45-fold down-regulation of PK in relation to the levels found in fed seabass. These observations were corroborated by enzyme activity meaning that during food deprivation an increase in the capacity of pyruvate generation happened via alanine to offset the reduction in pyruvate derived via glycolysis. After a 3-day refeeding period cALT returned to control levels while PK was not able to rebound. No alterations were detected in the expression levels of G6PDH while 6PGDH was revealed to be more sensitive specially to fasting, as confirmed by a significant 5.7-fold decrease in mRNA levels with no recovery after refeeding. Our results indicate that in early stages of refeeding, the liver prioritized the restoration of systemic normoglycemia and replenishment of hepatic glycogen. In a later stage, once regular feeding is re-established, dietary fuel may then be channeled to glycolysis and de novo lipogenesis.

Expressional regulation of key hepatic enzymes of intermediary metabolism in European seabass (Dicentrarchus labrax) during food deprivation and refeeding

We hypothesized that the analysis of mRNA level and activity of key enzymes in amino acid and carbohydrate metabolism in a feeding/fasting/refeeding setting could improve our understanding of how a carnivorous fish, like the European seabass (Dicentrarchus labrax), responds to changes in dietary intake at the hepatic level. To this end cDNA fragments encoding genes for cytosolic and mitochondrial alanine aminotransferase (cALT; mALT), pyruvate kinase (PK), glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) were cloned and sequenced. Measurement of mRNA levels through quantitative real-time PCR performed in livers of fasted seabass revealed a significant increase in cALT (8.5-fold induction)while promoting a drastic 45-fold down-regulation of PK in relation to the levels found in fed seabass. These observations were corroborated by enzyme activity meaning that during food deprivation an increase in the capacity of pyruvate generation happened via alanine to offset the reduction in pyruvate derived via glycolysis. After a 3-day refeeding period cALT returned to control levels while PK was not able to rebound. No alterations were detected in the expression levels of G6PDH while 6PGDH was revealed to be more sensitive specially to fasting, as confirmed by a significant 5.7-fold decrease in mRNA levels with no recovery after refeeding. Our results indicate that in early stages of refeeding, the liver prioritized the restoration of systemic normoglycemia and replenishment of hepatic glycogen. In a later stage, once regular feeding is re-established, dietary fuel may then be channeled to glycolysis and de novo lipogenesis.

Berry polyphenol absorption and the effect of northern berries on metabolism, ectopic fat accumulation, and associated diseases

The prevalence of obesity and type 2 diabetes has increased at an alarming rate in developed countries. It seems in the light of current knowledge that metabolic syndrome may not develop at all without NAFLD, and NAFLD is estimated to be as common as metabolic syndrome in western population (23 % occurrence). Fat in the liver is called ectopic fat, which is triacylglycerols within the cells of non-adipose tissue. Serum alanine aminotransferase (ALT) values correlate positively with liver fat proportions, and increased activity of ALT predicts type 2 diabetes independently from obesity. Berries, high in natural bioactive compounds, have indicated the potential to reduce the risk of obesity-related diseases. Ectopic fat induces common endocrine excretion of adipose tissue resulting in the overproduction of inflammatory markers, which further induce insulin resistance by multiple mechanisms. Insulin resistance inducing hyperinsulinemia and lipolysis in adipocytes increases the concentration of free fatty acids and consequently causes further fat accumulation in hepatocytes. Polyphenolic fractions of berries have been shown to reverse inflammatory reaction cascades in in vitro and animal studies, and moreover to decrease ectopic fat accumulation. The aim of this thesis was to explore the role of northern berries in obesity-related diseases. The absorption and metabolism of selected berry polyphenols, flavonol glycosides and anthocyanins, was investigated in humans, and metabolites of the studied compounds were identified in plasma and urine samples (I, II). Further, the effects of berries on the risk factors of metabolic syndrome were studied in clinical intervention trials (III, IV), and the different fractions of sea buckthorn berry were tested for their ability to reduce postprandial glycemia and insulinemia after high-glucose meal in a postprandial study with humans (V). The marked impact of mixed berries on plasma ALT values (III), as well as indications of the positive effects of sea buckthorn, its fractions and bilberry on omental adiposity and adhesion molecules (IV) were observed. In study V, sea buckthorn and its polyphenol fractions had a promising effect on potprandial metabolism after high-glucose meal. In the literature review, the possible mechanisms behind the observed effects have been discussed with a special emphasis on ectopic fat accumulation. The literature review indicated that especially tannins and flavonoids have shown potential in suppressing diverse reaction cascades related to systemic inflammation, ectopic fat accumulation and insulin resistance development.

Structural studies on enzymes of biotechnological and biomedical interest

Structural studies of proteins aim at elucidating the atomic details of molecular interactions in biological processes of living organisms. These studies are particularly important in understanding structure, function and evolution of proteins and in defining their roles in complex biological settings. Furthermore, structural studies can be used for the development of novel properties in biomolecules of environmental, industrial and medical importance. X-ray crystallography is an invaluable tool to obtain accurate and precise information about the structure of proteins at the atomic level. Glutathione transferases (GSTs) are amongst the most versatile enzymes in nature. They are able to catalyze a wide variety of conjugation reactions between glutathione (GSH) and non-polar components containing an electrophilic carbon, nitrogen or sulphur atom. Plant GSTs from the Tau class (a poorly characterized class) play an important role in the detoxification of xenobiotics and stress tolerance. Structural studies were performed on a Tau class fluorodifen-inducible glutathione transferase from Glycine max (GmGSTU4-4) complexed with GSH (2.7 Å) and a product analogue Nb-GSH (1.7 Å). The three-dimensional structure of the GmGSTU4-4-GSH complex revealed that GSH binds in different conformations in the two subunits of the dimer: in an ionized form in one subunit and a non-ionized form in the second subunit. Only the ionized form of the substrate may lead to the formation of a catalytically competent complex. Structural comparison between the GSH and Nb-GSH bound complexes revealed significant differences with respect to the hydrogen-bonding, electrostatic interaction pattern, the upper part of -helix H4 and the C-terminus of the enzyme. These differences indicate an intrasubunit modulation between the G-and Hsites suggesting an induced-fit mechanism of xenobiotic substrate binding. A novel binding site on the surface of the enzyme was also revealed. Bacterial type-II L-asparaginases are used in the treatment of haematopoietic diseases such as acute lymphoblastic leukaemia (ALL) and lymphomas due to their ability to catalyze the conversion of L-asparagine to L-aspartate and ammonia. Escherichia coli and Erwinia chrysanthemi asparaginases are employed for the treatment of ALL for over 30 years. However, serious side-effects affecting the liver and pancreas have been observed due to the intrinsic glutaminase activity of the administered enzymes. Structural studies on Helicobacter pylori L-asparaginase (HpA) were carried out in an effort to discover novel L-asparaginases with potential chemotherapeutic utility in ALL treatment. Detailed analysis of the active site geometry revealed structurally significant differences between HpA and other Lasparaginases that may be important for the biological activities of the enzyme and could be further exploited in protein engineering efforts.

Metabolic syndrome – early cardio-metabolic, vascular and hepatic changes

Background: Metabolic syndrome (MetS) is a combination of several cardio-metabolic risk factors including obesity, hyperglycemia, hypertension and dyslipidemia. MetS has been associated with increased levels of apolipoprotein B (apoB) and low-density lipoprotein oxidation (OxLDL) and with an increased risk of cardiovascular disease and non-alcoholic fatty liver disease. Aims: To establish the relation of apoB and OxLDL with the MetS development and to determine the status of MetS as a risk factor for adverse liver changes and for subclinical atherosclerosis. Subjects and Methods: The present thesis is part of the two large scale population-based, prospective, observational studies. Cardiovascular Risk in Young Finns study was launched in 1980 including 3,596 subjects aged 3-18 years. Thereafter follow-up studies have been conducted regularly. In the latest follow-ups that were performed in 2001 (N=2,283) and 2007 (N=2,204), non-invasive ultrasound studies were introduced to the study protocol to measure subclinical atherosclerosis i.e. carotid intima-media thickness (IMT), carotid artery distensibility (Cdist) and brachial flow-mediated dilatation (FMD). Alanine-aminotransferase (ALT) and gammaglutamyltransferase (GGT) were measured in 2007 to assess liver function. The Bogalusa Heart Study is a long-term epidemiologic study of cardiovascular risk factors launched in 1972 in a biracial community of Bogalusa, Louisiana, USA. Total of 374 youths (aged 9-18 years at baseline in 1984-88) who underwent non-invasive ultrasound studies of the carotid artery as adults, were included in the analyses of the present thesis. Results: The odds ratios (95% confidence intervals) for MetS incidence during a 6-year follow-up by quartiles of apoB were 2.0(1.0-3.8) for the second quartile, 3.1(1.7-5.7) for the third quartile and 4.2(2.3-7.6) for the fourth quartile. OxLDL was not independently associated with incident MetS. Youth (aged 9-18 years) with MetS or with high body mass index were at 2-3 times the risk of having MetS, high IMT, and type 2 diabetes 24-years later as adults. IMT increased 79±7μm (mean±SEM) in subjects with MetS and 42±2μm in subjects without the MetS (P<0.0001) during 6- years. Subjects who lost the MetS diagnosis during 6-year follow-up had reduced IMT progression compared to persistent MetS group (0.036±0.005vs.0.079±0.010 mm, P=0.001) and reduced Cdist change compared to incident MetS group (-0.12±0.05vs.-0.38±0.10 %/mmHg, P=0.03) over 6-year follow-up. MetS predicted elevated ALT (β±SEM=0.380±0.052, P<0.0001 in men and 0.160±0.052, P=0.002 in women) and GGT (β±SEM=0.240±0.058, P<0.0001 in men and 0.262±0.053, P<0.0001 in women) levels after 6-years. Conclusions: These findings suggest that apoB may give additional information on early metabolic disturbances predisposing MetS. MetS may be used to identify individuals at increased risk of developing atherosclerosis and non-alcoholic liver disease. However, recovery from the MetS may have positive effects on liver and vascular properties.

Dissecting the molecular mechanisms of breast cancer bone metastasis for therapeutic targeting

Breast cancer that has metastasized to bone is currently an incurable disease, causing significant morbidity and mortality. The aim of this thesis work was to elucidate molecular mechanisms of bone metastasis and thereby gain insights into novel therapeutic approaches. First, we found that L‐serine biosynthesis genes, phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1) and phosphoserine phosphatase (PSPH), were up‐regulated in highly bone metastatic MDA‐MB‐231(SA) cells as compared with the parental breast cancer cell line. Knockdown of serine biosynthesis inhibited proliferation of MDA‐MB‐231(SA) cells, and L‐serine was essential for the formation of bone resorbing osteoclasts. Clinical data demonstrated that high expression of PHGDH and PSAT1 was associated with decreased relapse‐free and overall survival and with features typical of poor outcome in breast cancer. Second, RNA interference screening pointed out heparan sulfate 6‐O‐sulfotransferase 2 (HS6ST2) as a critical gene for transforming growth factor β (TGF‐β)‐induced interleukin 11 (IL‐11) production in MDA‐MB‐231(SA) cells. Exogenous heparan sulfate glycosaminoglycans heparin and K5‐NSOS also inhibited TGF‐β‐induced IL‐11 production in MDA‐MB‐231(SA) cells. Furthermore, K5‐NSOS decreased osteolytic lesion area and tumor burden in bone in mice. Third, we discovered that the microRNAs miR‐204, ‐211 and ‐379 inhibited IL‐11 expression in MDA‐MB‐231(SA) cells through direct targeting of the IL‐11 mRNA. MiR‐379 also inhibited Smad‐mediated signaling. Gene expression profiling of miR‐204 and ‐379 transfected cells indicated that these microRNAs down‐regulate several bone metastasis‐relevant genes, including prostaglandin‐endoperoxide synthase 2 (PTGS2). Taken together, this study identified three potential treatment strategies for bone metastatic breast cancer: inhibition of serine biosynthesis, heparan sulfate glycosaminoglycans and restoration of miR‐204/‐211/‐379.

Análise morfológica e fisiológica dos fígados e rins de ratas prenhes e seus fetos tratados pela associação zidovudina, lamivudina e ritonavir durante toda a prenhez

OBJETIVO: avaliar os efeitos da administração da associação zidovudina-lamivudina-ritonavir nos fígados e rins de ratas prenhes e seus conceptos do ponto de vista morfológico e fisiológico. MÉTODOS: 40 ratas albinas prenhes foram aleatoriamente divididas em 4 grupos: 1 controle (Ctrl: controle de veículo) e 3 experimentais (Exp1x, Exp3x e Exp9x). Estes últimos foram tratados por solução oral de zidovudina/lamivudina/ritonavir (Exp1x: 10/5/20 mg/kg; Exp3x: 30/15/60 mg/kg; Exp9x: 90/45/180 mg/kg). As drogas e o veículo foram administrados por gavagem, desde o 1º até o 20º dia de prenhez. No último dia do experimento, todos os animais foram anestesiados e sangue foi retirado da cavidade cardíaca para avaliação sérica das enzimas aspartato aminotransferase (AST) e alanina aminotransferase (ALT), por método calorimétrico, bem como da ureia, determinada por método cinético-enzimático, e creatinina, por método cinético-colorimétrico. Em seguida, fragmentos dos fígados e rins maternos e fetais foram coletados, fixados em formol a 10% e processados segundo os métodos histológicos para inclusão em parafina. Cortes com 5 µm de espessura foram corados pela hematoxilina-eosina (HE) e analisados por microscopia de luz. Na leitura das lâminas, considerou-se o padrão de normalidade para fígado e rins, tais como: hepatócitos, espaço porta íntegros e veias hepáticas bem definidas. Nos rins, a presença de corpúsculos renais, túbulos contorcidos e alças de Henle típicos. Nos fígados fetais considerou-se, ainda, a morfologia das células da linhagem eritrocitária nas diferentes fases do desenvolvimento, bem como os megacariócitos. Quando houve alteração da coloração padrão estabelecida para as estruturas hepáticas e renais, alteração na morfologia de núcleos, rompimento de limites de alguma organela citoplasmática, presença de congestão vascular, tudo isso foi entendido como provavelmente provocado pelas drogas em sua(s) dose(s) de aplicação. A avaliação estatística foi realizada por análise de variância (ANOVA), completada pelo teste de Tukey-Kramer (p<0,05). RESULTADOS: os fígados maternos dos grupos Ctrl, Exp1x e Exp3x mostraram hepatócitos típicos, espaço porta íntegros e veias hepáticas com aspecto normal. No fígado materno do grupo Exp9x, foram encontrados hepatócitos com sinais de atrofia e apoptose (eosinofilia citoplasmática e núcleos picnóticos). Além disso, identificou-se vasodilatação dos capilares sinusoides (congestão). Os rins maternos dos grupos Ctrl e Exp1x apresentaram-se normais, com corpúsculos renais, túbulos contorcidos e alças de Henle típicos. Já nos grupos Exp3x e Exp9x, foram encontrados congestão vascular, glomérulos pequenos ricos em células contendo núcleos hipercromáticos, sendo mais intensos no Exp9x. Com relação aos fígados e rins fetais, não foram observadas alterações morfológicas ou fisiológicas nos grupos estudados. Encontrou-se aumento significante nos níveis da AST (305,70±55,80; p<0,05) e da creatinina (0,50±0,09; p<0,05) no grupo Exp9x. CONCLUSÕES: nossos resultados evidenciam que a administração da associação zidovudina/lamivudina/ritonavir a ratas prenhes em altas doses causa alterações morfológicas e funcionais nos fígados e rins maternos. Não houve alterações nem morfológicas nem fisiológicas nos fígados e rins fetais.

Intoxicação experimental pelos frutos de Xanthium cavanillesii (Asteraceae) em bovinos

Os frutos moídos de Xanthium cavanillesii Schouw, foram administrados por via oral, em doses única ou repetidas, com intervalo semanal, a onze bovinos. Desses, quatro morreram. Doses únicas a partir de 5 g/kg foram letais para bovinos. Dose de 3 g/kg produziu sinais clínicos e recuperação em um bovino. Repetições de 4 doses de 3 g/kg para um bovino e 2 doses de 5 g/kg para outro bovino não foram tóxicas. Foram constatadas hipoglicemia e elevação dos níveis séricos de aspartato aminotransferase (AST) nos bovinos que apresentaram sinais clínicos da intoxicação. Os primeiros sinais clínicos nos animais que morreram foram observados entre 6 e 12 horas após a administração dos frutos. A evolução do quadro clínico variou entre 5h30min e 8 horas. O quadro clínico foi semelhante nestes animais sendo que os principais sinais clínicos foram anorexia, apatia, salivação profusa e tremores musculares. Ocorreram também hipomotilidade e atonia ruminal, cólicas abdominais, gemidos freqüentes, ranger de dentes, sudorese generalizada e endoftalmia. As alterações de locomoção observadas foram incoordenação motora, instabilidade do trem posterior, decúbito permanente com movimentos de pedalagem, espasmos musculares e opistótono. As alterações respiratórias foram aumento da freqüência respiratória, respiração laboriosa com ruídos e momentos de apnéia. Finalmente ocorria perda do reflexo palpebral, ausência de reflexo pupilar e morte. No bovino que se recuperou, os primeiros sinais clínicos foram observados 18 horas após a administração e evoluíram num período de aproximadamente 72 horas. Neste bovino, através de biópsias hepáticas, observou-se necrose hepática coagulativa centrolobular associada a congestão e hemorragias. Necrose hepática coagulativa massiva foi observado por biópsias hepáticas em um bovino que morreu, a partir de 12 horas após a administração dos frutos, associada com alterações nos níveis séricos de glicose e AST. As principais lesões encontradas na necropsia foram no fígado e consistiam de aumento do padrão lobular na superfície capsular e de corte, distensão da vesícula biliar e edema moderado da parede da vesícula biliar. A principal alteração microscópica era caracterizada por necrose coagulativa centrolobular ou massiva associada a congestão e hemorragia e alterações degenerativas nos hepatócitos circunjacentes.

Intoxicação experimental por monensina em eqüinos

Sete eqüinos foram tratados experimentalmente com monensina sódica. Dois desses animais receberem 3-4 kg/eqüino/dia de uma ração comercial sabidamente implicada em surtos naturais da intoxicação por monensina em eqüinos e que continha 180 ppm±20 da droga. Um eqüino recebeu uma única dose de 5 mg/kg e um outro recebeu 4 doses diárias de 1 mg/kg de monensina sódica originária de um premix. Esses quatro eqüinos morreram ou foram sacrificados in extremis, 3-8 dias após o início da administração da droga. Um quinto eqüino recebeu dose única de 5 mg/kg de monensina, ficou levemente doente e se recuperou. Dois eqüinos não desenvolveram sinais da intoxicação. Um desses eqüinos tinha recebido 40 doses diárias de 0,5 mg/kg de monensina e o outro recebeu 3 kg/dia de uma ração da mesma marca que a usada nas fazendas onde surtos de intoxicação por monensina foram detectados (mas de uma outra partida, mais tarde determinada como contendo menos de 5 ppm de monensina). O aparecimento dos sinais clínicos ocorreu de 2 a 5 dias após a administração da droga e a duração do quadro clínico variou de 24 a 76 horas. Os sinais clínicos incluíam taquicardia, arritmia, gemidos, incoordenação, sudorese, decúbito esternal, decúbito lateral, pedaleios e morte. Em cinco dos eqüinos intoxicados observaram-se marcadas elevações da atividade plasmática de creatina fosfoquinase e, em um eqüino, houve leve aumento da atividade plasmática de aspartato aminotransferase. Os principais achados de necropsia consistiram em áreas brancas ou amarelas, focais ou focalmente extensas e bilateralmente simétricas nos músculos esqueléticos. Essas lesões eram associadas a edema gelatinoso e translúcido das fáscias intermusculares. Quadríceps femoral, adutor, pectíneo, grácil, semi-membranáceo, supra-espinhal, subescapular, braquicefálico e quadríceps femoral estavam entre os músculos esqueléticos mais afetados. Os exames histológicos revelaram miopatia segmentar, multifocal a coalescente. Em dois eqüinos que receberam o premix de monensina [5 mg/kg (dose única) e 4 mg/kg (quatro doses diárias de 1 mg/kg)] não se observaram lesões macroscópicas ou microscópicas. Em nenhum dos dois eqüinos se observaram lesões macro ou microscópicas no miocárdio.

Avaliação clínico-laboratorial de bovinos Nelore infectados experimentalmente com Trypanosoma vivax

Avaliaram-se as alterações clínico-laboratoriais de seis bezerros Nelore, de ambos os sexos, inoculados experimentalmente com 10(7) organismos viáveis de Trypanosoma vivax, isolados de bovinos da região de Poconé, Estado de Mato Grosso. Os animais foram observados diariamente, durante 30 dias, quanto aos parâmetros de temperatura retal, volume globular (VG), parasitemia, produção de anticorpos, coloração de mucosas, comportamento e apetite. Determinaram-se os níveis séricos de aspartato aminotransferase (AST), fosfatase alcalina (FA), gama glutamiltransferase (GGT), creatina kinase (CK), colesterol, uréia, creatinina, cálcio, fósforo e o perfil eletroforético das proteínas séricas aos 4, 8, 12, 16, 23 e 30 dias pós-inoculação (DPI). Durante os 6 meses seguintes, os animais foram observados semanalmente, avaliando-se a temperatura retal, o VG e a parasitemia. T. vivax foi evidenciado a partir do terceiro e quarto DPI em todos os bezerros e persistiu até o 30° DPI em cinco dos seis animais em estudo. Ocorreu um decréscimo significativo (p<0,05) do valor médio do VG (25%) aos dez DPI. Os animais não apresentaram qualquer alteração no quadro clínico, bem como na avaliação da bioquímica sérica durante o período experimental. A soroconversão ocorreu aos 6 e 8 DPI, permanecendo todos os animais soropositivos nos 30 dias experimentais. Bovinos nelores jovens, infectados experimentalmente com T. vivax, foram capazes de estabelecer um equilíbrio na relação hospedeiro-parasita.

Fotossensibilização primária em eqüídeos e ruminantes no semi-árido causada por Froelichia humboldtiana (Amaranthaceae)

Fotossensibilização é freqüente em eqüinos no semi-árido da região Nordeste, mas jumentos, mulas, ovinos e bovinos são, também, afetados. A dermatite afeta, principalmente, áreas de pele despigmentadas e os animais se recuperam após serem retirados das pastagens. Para comprovar a etiologia da enfermidade Froelichia humboldtiana (Roem. et Schult.) Seub., coletada no campo foi administrada no mesmo dia da colheita ou após ser mantida em refrigerador por 1-4 dias, por períodos de 30 ou mais dias, ad libitum como único alimento, a 2 jumentos e um ovino branco e, como único alimento volumoso, a um eqüino branco. Esses animais não manifestaram sinais clínicos e as atividades séricas de gama-glutamiltransferase (GGT), aspartato-aminotransferase (AST) e alanino-aminotransferase (ALT) ficaram dentro dos valores normais. Em outro experimento, um ovino foi colocado a pastar diariamente, durante o dia, preso por uma corda em uma área que tinha exclusivamente F. humboldtiana, por um período de 26 dias. Lesões características de fotossensibilização foram observadas 4-5 dias após o início do experimento. Após cessar o consumo da planta, no 26º dia, o ovino recuperou-se totalmente em 30 dias. Em outro experimento, 4 ovinos foram também colocados, presos por cordas, na mesma área. Outros 4 permaneceram como controles em uma pastagem vizinha, mas sem F. humboldtiana. Lesões de pele, características de fotossensibilização foram observadas após 11-25 dias de consumo de F. humboldtiana. As atividades séricas de AST e GGT, e os níveis de bilirrubina sérica permaneceram dentro dos valores normais. No final do período de permanência em pastagens de F. humboldtiana, 2 ovinos foram abatidos e 2 foram retirados da pastagem. Os que foram abatidos não apresentaram lesões macroscópicas nem histológicas do fígado; os outros dois se recuperaram das lesões da pele 17 e 20 dias após o fim do pastejo. Uma égua e seu potro foram colocados na mesma pastagem com F. humboldtiana por um período de 44 dias. A égua, que não tinha áreas de pele despigmentadas não apresentou lesões, no entanto o potro desenvolveu dermatite nas áreas brancas de pele 25 dias após o início do pastejo. Após serem retirados da pastagem o potro recuperou-se totalmente em 15 dias. Estes experimentos indicam que F. humboldtiana causa fotossensibilização primária em animais domésticos. A ausência de lesões nos animais que ingeriram a planta após ser coletada e mantida em geladeira sugere que a planta perde sua toxicidade depois da coleta. A ausência de lesões oculares características da intoxicação por furocumarinas sugere que F. humboldtiana contém derivados da naftodiantrona, similares aos encontrados em Fagopyrum esculentum e Hypericum perforatum que não causam lesões oculares.