Motor representation of rhythmic jaw movements in the amygdala of guinea pigs.

OBJECTIVE: The areas of the amygdala contributing to rhythmic jaw movements and the movement patterns induced remain unknown. Therefore, the present study investigated the areas of the amygdala contributing to rhythmic jaw movements using repetitive electrical microstimulation techniques. DESIGN: Experiments were performed on head-restrained guinea pigs under ketamine-xylazine anesthesia. EMG activities in the masseter and digastric muscles and jaw movements were recorded. Short- and long-train electrical microstimulations of the amygdala were performed and the patterns of jaw movements induced were analyzed quantitatively. RESULT: The short-train stimulation induced short-latency EMG responses in the masseter and/or digastric muscles. The stimulation sites inducing short-latency EMG responses were distributed within the ventral part of the amygdala, which covered the medial, basal, and cortical nuclei. The long-train stimulation induced tonic jaw opening and two types of rhythmic jaw movements: those with or without lateral jaw shifts, which were characterized by a larger jaw gape and ipsilateral jaw excursion, respectively. Rhythmic jaw movements with lateral jaw shifts were characterized by overlapping masseter and digastric EMG activities. However, rhythmic patterns did not differ between the two types of rhythmic jaw movements. The stimulation sites that induced rhythmic jaw movements were more localized to the cortical nucleus. CONCLUSIONS: The present results suggest that the ventral part of the amygdala is involved in the induction of rhythmic jaw movements in guinea pigs. The putative roles of the limbic system in the genesis of functional (e.g., chewing) and non-functional (e.g., bruxism) rhythmic oromotor movements warrant further study.

After-effects of acute footshock stress on sleep states and rhythmic masticatory muscle activity during sleep in guinea pigs.

This study investigated the effects of acute footshock stress (FS) on the occurrence of rhythmic masticatory muscle activity (RMMA) during sleep in guinea pigs. Animals were prepared for chronic recordings from electroencephalogram, electrooculogram and electromyograms of neck and masseter muscles. The signals were recorded for six hours on the two successive days: the first day with stress-free condition (non-FS condition) and the second day with acute FS (FS condition). Sleep/wake states and RMMA were scored visually. Sleep variables and the frequency of RMMA occurring during non-rapid eye movement (NREM) sleep were compared during 6-h periods between the two conditions. Compared to non-FS condition, the amount of total sleep and NREM sleep significantly reduced during 2 h following the acute FS in the FS condition. Similarly, the frequency of RMMA significantly increased during 2 h following the acute FS for the FS condition compared to non-FS condition. During 2-6 h after FS in the FS condition, sleep variables and the frequency of RMMA did not differ from those without FS in the non-FS condition. These results suggest that acute experimental stress can induce transient changes in sleep-wake states and the occurrence of RMMA in experimental animals.

Changes in cortical, cardiac, and respiratory activities in relation to spontaneous rhythmic jaw movements in ketamine-anesthetized guinea pigs.

It has been reported that rhythmic jaw movements (RJMs) spontaneously occur in ketamine-anesthetized animals. The present study investigated the physiological processes that occur during the cortical, cardiac, and respiratory events which contribute to the genesis of RJMs in animals after supplemental ketamine injections. Fourteen guinea pigs were prepared to allow electroencephalographic, electrocardiographic, and electromyographic activities to be recorded from the digastric muscle, measurement of jaw movements, and nasal expiratory airflow under ketamine-xylazine anesthesia. Rhythmic jaw movements spontaneously occurred with rhythmic digastric muscle contractions, 23-29 minutes after injection of supplemental ketamine (12.5 and 25.0 mg kg-1 , intravenously). The cycle length of RJMs did not differ significantly between the two doses of ketamine (mean±SD: 12.5 mg kg-1 , 326.5 ± 60.0 ms; 25 mg kg-1 , 278.5 ± 45.1 ms). Following injection of ketamine, digastric muscle activity, heart and respiratory rates, and cortical beta power significantly decreased, while cortical delta and theta power significantly increased. These changes were significantly larger in animals given 25.0 mg kg-1 of ketamine than in those given 12.5 mg kg-1 . With the onset of RJMs, the levels of these variables returned to pre-injection levels, regardless of the dose of ketamine administered. These results suggest that, following supplemental ketamine injections, spontaneous RJMs occur during a specific period when the pharmacological effects of ketamine wear off, and that these RJMs are characterized by stereotypical changes in cardiac, respiratory, and cortical activities.

Comparison of rhythmic masticatory muscle activity during non-rapid eye movement sleep in guinea pigs and humans.

Rhythmic masticatory muscle activity can be a normal variant of oromotor activity, which can be exaggerated in patients with sleep bruxism. However, few studies have tested the possibility in naturally sleeping animals to study the neurophysiological mechanisms of rhythmic masticatory muscle activity. This study aimed to investigate the similarity of cortical, cardiac and electromyographic manifestations of rhythmic masticatory muscle activity occurring during non-rapid eye movement sleep between guinea pigs and human subjects. Polysomnographic recordings were made in 30 freely moving guinea pigs and in eight healthy human subjects. Burst cycle length, duration and activity of rhythmic masticatory muscle activity were compared with those for chewing. The time between R-waves in the electrocardiogram (RR interval) and electroencephalogram power spectrum were calculated to assess time-course changes in cardiac and cortical activities in relation to rhythmic masticatory muscle activity. In animals, in comparison with chewing, rhythmic masticatory muscle activity had a lower burst activity, longer burst duration and longer cycle length (P < 0.05), and greater variabilities were observed (P < 0.05). Rhythmic masticatory muscle activity occurring during non-rapid eye movement sleep [median (interquartile range): 5.2 (2.6-8.9) times per h] was preceded by a transient decrease in RR intervals, and was accompanied by a transient decrease in delta elelctroencephalogram power. In humans, masseter bursts of rhythmic masticatory muscle activity were characterized by a lower activity, longer duration and longer cycle length than those of chewing (P < 0.05). Rhythmic masticatory muscle activity during non-rapid eye movement sleep [1.4 (1.18-2.11) times per h] was preceded by a transient decrease in RR intervals and an increase in cortical activity. Rhythmic masticatory muscle activity in animals had common physiological components representing transient arousal-related rhythmic jaw motor activation in comparison to human subjects.

Biomonitoring of microcystin and aflatoxin co-occurrence in aquaculture using immunohistochemistry and genotoxicity assays

This work investigated the effects of co-occurring aflatoxin B1 (AFB1) and microcystin (MC) in aquaculture, using immunohistochemistry and genotoxicity methods. Tilapia (Oreochromis niloticus) were exposed to AFB1 by intraperitoneal and MC (cell extract of Microcystis aeruginosa) by intraperitoneal and immersion routes. The interaction of MC-AFB1 was evaluated co-exposing the intraperitoneal doses. Blood samples were collected after 8, 24, and 48h to analyze the micronucleus frequency and comet score. The interaction of MC-AFB1 showed a synergic mutagenic response by higher micronucleus frequency of co-exposed group. A slight genotoxic synergism was also observed in the comet score. Immunohistochemistry detected MC in al lthe fish liver tissues exposed to MC by intraperitoneal route, and only the immersed group with the highest dose of MC showed a positive response. Although MC was non-detectable in the edible muscle, the combination of immunohistochemistry with genotoxicity assay was an attractive biomonitoring tool in aquaculture, where the animals were frequently exposed to co-occurring synergic hazards.

Imunoistoquímica: detecção de microcistina em tilápia exposta ao extrato de Microcystis aeruginosa (Cyanobacteria) / Imunohistochemistry: detection of microcystin in tilápia exposed to Microcystis aeruginosa (Cyanobacteria) extract

A deterioração da qualidade de água pela piscicultura associa-se à eutrofização, com florescimento de cianobactérias. Microcystis aeruginosa destaca-se como principal produtora de microcistinas (MCs), grupo de hepatotoxinas com potencial promotor de tumor. No presente trabalho desenvolveu-se método imunoistoquímico para a detecção de MC em tilápias (Oreochromis niloticus) submetidas à injeção intraperitoneal (i.p.) ou imersão em extrato de M. aeruginosa BCCBUSP 262, empregando anticorpo monoclonal anti-MC (M8H5) e sistema polímero-peroxidase. As tilápias (N=42) foram submetidas a sete tratamentos, sendo três grupos inoculados i.p. com 2,0x105, 4,0x105 e 1,0x106 cels.Kg-1 de M. aeruginosa BCCBUSP 262 e quatro submetidos à imersão em diferentes concentrações do extrato da cianobactéria (variando de 1,0x104 a 1,0x105cel.mL-1). Analisando fígado e tecido muscular pelo ensaio imunoistoquímico, não se detectou marcação em tecido muscular. Todos os animais inoculados i.p. apresentaram marcação positiva para MC no fígado, mas em teste de imersão, apenas os expostos a maior dose (1,0x105 cels.mL- 1) apresentaram marcação positiva. Embora MC não seja detectada em tecido muscular, assim como no fígado de animais imersos em extrato de M. aeruginosa CCBUSP 262 em concentrações menores que 1,0x105 cels.mL-1, os resultados constituíram-se base para o desenvolvimento metodológico objetivando a aplicação da imunoistoquímica no diagnóstico rápido no controle de qualidade de pescados.

The deterioration of the water quality due to aquaculture is associated with eutrophication, with bloom of cyanobacteria. Microcystis aeruginosa is distinguished as main producer of microcystins (MCs), group of hepatotoxins with tumor promoter potential. In the present work immunohistochemical method for detection of MC in tilápia (Oreochromis niloticus), fish submitted to intraperitoneal injection (i.p.) or immersion in extract of M. aeruginosa BCCBUSP 262 was developed, using monoclonal antibody anti- MC (M8H5) and polymer peroxidase system. The tilápias (N=42) had been submitted to the seven treatments, three groups inoculated i.p. with 2.0x105, 4.0x105 and 1.0x106 cells. Kg-1 of M. aeruginosa BCCBUSP 262 and four groups exposed to the immersion in different extract concentrations of cyanobacterium. Analyzing liver and muscular tissue for immunohistochemical assay, muscular tissue was not stained. All the animals inoculated i.p. presented positive marking for MC in the liver, but in immersion test, only the ones exposed in the highest dose (1,0x105 cels.mL-1) presented positive marking. Although MC was not detected in muscular tissue, as well as in the liver of animals immersed in extract of M. aeruginosa BCCBUSP 262 in concentrations less than 1.0x105 cels.mL-1, the results would constitute in the base for the methodological development aiming the application of the immunohistochemistry in the rapid diagnosis in quality control of fish.

A comparison between enzyme immunoassay and HPLC for ochratoxin A detection in green, roasted and instant coffee

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for ochratoxin A (OTA) detection in green, roasted and instant coffees was developed using anti-OTA monoclonal antibody. Immunological reagents prepared were OTA-BSA (4.76 mg/mL), anti-OTA.7 MAb (2x10³-fold dilution) and HRP-anti IgG (10³-fold dilution). The detection limit was 3.73 ng OTA/g and correlation coefficients (r) between this immunoassay and high performance liquid chromatography were 0.98 for green coffee, 0.98 for roasted and 0.86 for instant. OTA levels detected by ic-ELISA were higher than by HPLC, with ELISA/HPLC ratio of 0.66 - 1.46 (green coffee), 0.96 - 1.11 (roasted) and 0.93 - 1.82 (instant). ELISA recoveries for OTA added to coffee (5 - 70 ng/g) were 81.53 percent for green coffee, 46.73 percent for roasted and 64.35 percent for instant, while recoveries by HPLC were 80.54 percent, 45.91 percent and 55.15 percent, respectively. Matrices interferences were minimized by samples dilution before carrying out the ELISA assay. The results indicate that MAb-based ic-ELISA could be a simple, sensitive and specific screening tool for OTA detection, contributing to quality and safety of coffee products.

ELISA competitivo indireto (ic-ELISA) baseado em anticorpos monoclonais foi desenvolvido para a detecção de ocratoxina A (OTA) em café verde, torrado e instantâneo. Os reagentes imunológicos necessários à reação consistiram de OTA-BSA (4,76 mg/mL), anti-OTA.7 MAb (diluído 2x10³) e anti IgG-HRP (diluído 10³), apresentando limite de detecção de 3,73 ng OTA/g. Os coeficientes de correlação (r) entre o imunoensaio e cromatografia líquida de alta eficiência (CLAE) foram de 0,98 (café verde), 0,98 (torrado) e 0,86 (instantâneo). Ic-ELISA detectou valores superestimados de OTA em relação a CLAE, com valor ELISA/CLAE variando de 0,66 - 1,46 (café verde), 0,96 - 1,11 (torrado) e 0,93 - 1,82 (instantâneo). As recuperações médias de OTA adicionada em café (5 - 70 ng/g) foram de 81,53 por cento (café verde), 46,73 por cento (torrado) e 64,35 por cento (instantâneo) por ELISA, em relação a 80,54 por cento, 45,91 por cento e 55,15 por cento por CLAE, respectivamente. A interferência de matriz no imunoensaio foi minimizada pela diluição das amostras previamente à análise por ELISA. O ic-ELISA desenvolvido pode ser considerado uma técnica alternativa simples, sensível e específica para análise de OTA, contribuindo para a qualidade e segurança de produtos de café.

Reliable indirect competitive ELISA used for a survey of ochratoxin A in green coffee from the North of Paraná State, Brazil.

The performance of an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a monoclonal antibody (mAb) for ochratoxin A (OTA) detection was evaluated in a comparative study with high-performance liquid chromatography (HPLC) analysis using 68 freshly harvested coffee samples from the North of Paraná State, Brazil. The anti-OTA mAb showed high specificity and low cross-reactivity with OTA analogues (OTB and OTalpha), but cross-reacted with OTC. This ic-ELISA showed a detection limit of 3.75 ngg-1 sample, when compared to 0.80 ngg-1 by HPLC, with an ic-ELISA/HPLC correlation coefficient of 0.90. As regards OTA analysis of these coffee samples, natural contamination was detected in 10 samples (14.7%) by both methods, where the ic-ELISA values (range 3.9-7.3 ngg-1) were 1.1 to 1.6-fold higher than HPLC data (2.7-4.7 ngg-1). Five samples (7.4%) were OTA positive (range 0.84-1.30 ngg-1) only by HPLC assay, probably due to the higher detection limit reached by ic-ELISA. OTA was undetectable in 53 samples (77.9%) by both methods, while all positive samples (range 0.84-7.30 ngg-1) showed OTA levels lower than 8 ngg-1 (maximum limit recommended by the European Union). The matrix interference of green coffee was minimized by dilution of sample extracts before carrying out the ELISA assay. This mAb-based ic-ELISA can be effectively applied for OTA screening in coffee, because it is simple, sensitive and sample preparation is easy.

Fumonisins in corn: correlation with Fusarium sp. count, damaged kernels, protein and lipid content

A contaminação natural por fungos e fumonisinas foi avaliada em 109 amostras de milho recém-colhido do Estado do Paraná e correlacionada com grãos ardidos (%). Além disso, grãos sadios e ardidos de 24 amostras de milho foram selecionados a fim de comparar o perfil da microbiota fúngica e níveis de fumonisinas. A correlação entre os teores de proteínas/lipídios e os níveis de fumonisinas também foi analisada nos 15 híbridos de milho mais freqüentemente cultivados no Estado do Paraná. A contagem total de fungos em 109 amostras de milho recém-colhido variou de 1,9x104 a 3,5x106 UFC/g, Fusarium sp. de 1,0x103 a 2,2x106 UFC/g e, níveis de fumonisinas de 0,13 a 20,38 µg/g. A contagem total de fungos/Fusarium spp. e níveis de fumonisinas apresentaram correlação positiva (p<0,05). Adicionalmente, houve uma correlação positiva entre grãos ardidos (%) e a contagem total de fungos/ Fusarium spp. (p < 0,05). Os níveis de fumonisinas nos grãos sadios variaram de 0,57 a 20,38 µg/g, enquanto que nos grãos ardidos variaram de 68,96 a 336,38 µg/g. Não foi observada correlação significativa entre os níveis de fumonisinas e os teores de proteínas/lipídios. Esses resultados ratificam a importância do monitoramento constante da contaminação por fungos toxigênicos e fumonisinas em milho e derivados a fim de garantir a qualidade e segurança dos produtos e minimizar o risco potencial à saúde humana e animal.

New Amylase Inhibitor Present in Corn Seeds Active In Vitro Against Amylase from Fusarium verticillioides.

A screening for specific amylase inhibitor levels against amylase from Fusarium verticillioides (Fusarium moniliforme), the most relevant mycotoxigenic fungus in corn, was conducted on 37 corn hybrids. The amylase inhibitor levels in these hybrids ranged from 5.5 to 16.0 amylase inhibitor units per gram of corn (AIU/g) in the MASTER and AG5011 hybrids, respectively. The hybrid with the maximum content of inhibitor was used as the source of this new protein. The inhibitor was partially purified using fractional precipitation, gel filtration on Sephadex G-75 column, high performance liquid chromatography (HPLC) Superose HR 10/30 column, and HPLC anion exchange chromatography, obtaining a 20.7-fold purification. Electrophoresis after denaturing and heating under reductive conditions showed an apparent 23.8 kDa molecular mass and an acidic isoelectric point of 5.4, which differs from previous molecular masses reported for other inhibitors present in corn seeds (14 and 22 kDa). This inhibitor showed activity against amylases from human saliva and pancreas, from the fungi F. verticillioides and Aspergillus flavus, and from the insects Acanthoscelides obtectus, Zabrotes subfasciatus, Tribolium castaneum, and Sitotroga cerealella. The mycoflora found in the corn grain indicated Fusarium sp. as the most prevalent fungi (81.1% of the samples), with a count ranging from 1.5 × 102 to 2.4 × 106 CFU/g of corn. The presence of fumonisins was detected in 21 out of the 37 hybrids studied, ranging from 0.05 to 2.67 µg of FB per gram of corn. No correlation could be established between this amylase inhibitor level in the corn seeds and the presence of Fusarium sp. or with the fumonisin content under the experimental conditions of the test.

Hepatitis B and C viruses infection, lifestyle and genetic polymorphisms as risk factors for hepatocellular carcinoma in Haimen, China.

A case-control study was carried out to investigate the impact of factors including virus infection, aflatoxin B1, microcystins, smoking/drinking and dietary habits as well as genetic polymorphisms of aldehyde dehydrogenase 2 (ALDH2) and cytochrome P4502E1 (CYP2E1), on susceptibility to hepatocellular carcinoma (HCC) in Haimen, China. A total of 248 patients with HCC and 248 sex-, age- and residence-matched population-based controls were recruited into the study. Virus infection, and ALDH2 and CYP2E1 gene polymorphisms were assessed in 134 paired cases and controls. By univariate analysis, hepatitis B virus (HBV) infection (odds ratio [OR]=9.75; 95% confidence interval [CI]=4.71-20.2), history of intravenous injection (OR=1.50; 95%CI=1.02-2.22), average income (OR=0.63; 95%CI=0.43-0.92), frequent intake of foods rich in protein, e.g., egg (OR=0.6; 95%CI=0.42-0.87), chicken (OR=0.53; 95%CI=0.35-0.79), pork (OR=0.67; 95%CI=0.46-0.98) and fresh fish (OR=0.58; 95%CI=0.39-0.87) significantly differed between cases and controls. However, peanut intake (OR=0.66; 95%CI=0.43-1.01), source of drinking water, including tap (OR=1.33; 95%CI=0.81-2.20), deep well (OR=0.94; 95%CI=0.56-1.55), shallow well (OR=0.85; 95%CI=0.55=1.30), river (OR=0.95; 95%CI=0.65-1.38), ditch (OR=1.09; 95%CI=0.76-1.55) and pond water (OR=1.0; 95%CI=0.14-7.10) were not significantly associated with risk. Univariate analysis also indicated that the 1-1 genotype of ALDH2 (OR=1.38; 95%CI=0.86-2.23) as well as the Pst1- and Rsa1-digested c1/c1 genotype of CYP2E1 (OR=1.36; 95%CI=0.81-2.28), was slightly more frequent in the case group. On multivariate analysis, HBV infection (OR=13.9; 95%CI=5.78-33.6) and history of intravenous injection (OR=2.72; 95%CI=1.24-6.00) were still associated with significantly increased risk of HCC, while frequent intake of fresh fish (OR=0.32; 95%CI=0.12-0.86) decreased this risk. These findings suggest that whereas peanut intake, water sources as well as genetic polymorphisms in ALDH2 and CYP2E1 do not significantly correlate with the risk of HCC, HBV infection is a main risk factor, and dietary items rich in protein, especially fresh fish, might protect against the risk of HCC in Haimen, China.

Determination of microcystins in lake water using reusable immunoaffinity column.

A reusable immunoaffinity column for purification of microcystins in lake water was prepared by coupling anti-microcystin-LR monoclonal antibodies to immunoaffinity support. Thanks to spherical shape of the immunoaffinity support Formyl-Cellulofine used in this study, applied solutions passed the column smoothly even when used repeatedly. Reusability of the column was examined by determining the recoveries of spiked microcystins-RR, -YR and -LR (100ng each) from lake water. After extraction with a Sep-Pak PS2 cartridge containing styrene-divinylbenzene copolymer, the extract was purified with the immunoaffinity column. The immunoaffinity column was regenerated by washing with Tris-HCl buffer containing bovine serum albumin for repeated uses. Recoveries of spiked microcystins from the first use of the column were 87-88%, and 83-88% from the second and third uses, and the recoveries gradually dropped to 63-77% from the 4-5th uses, the results of which indicated that the column could be used repeatedly for three times. The present method was applied to determine microcystins in water collected from three different lakes in Japan in 1999. In a sample from Lake Suwa, microcystins-RR and -LR were determined by high performance liquid chromatography with photodiode array detection and electrospray ionization-liquid chromatography/mass spectrometry.

Anti-Fusarium moniliforme activity and Fumonisin biodegradation by Corn and Silage microflora

A aplicaçäo de microroganismos visando controle de fungos micotoxigêncos ou detoxificaçäo em armazenagem consiste em uma área promissora, já que reduz contaminaçäo de ecossistema por resíduos agrotóxicos. Visando controle biológico, microorganismos isolados de milho e silagem foram analisados perante efeito anti-F, moniliforme (linhagem 113F) em associaçäo com a detoxificaçäo de fumonisinas. Após análise de 150 isolados, selecionou-se quatro bacilos Gram-positivos e uma levedura com melhor atividade inibitória. O halo de inibiçäo variou de 50 a 72.5 mm usando culturas íntegras e 25 a 52.5 mm, para extrato bruto de cultivo. Os isolados S9, S10, S69 (bacilos esporulados) e SE3071 (levedura) degradaram 43 por cento, 48 por cento, 83 por cento r 57 por cento de FB1 respectivamente, em relaçäo à concentraçäo inicial. O pH aumentou gradativamente com o tempo de incubaçäo

Screening and isolation of anti-Fusarium moniliforme compounds producing microorganisms from soil and corn

Fusarium moniliforme, fitopatógeno cosmopolita de milho, é responsável pela produçäo de micotoxina recentemente descoberta, denominada fumonisima. Com a finalidade de avaliar a inibiçäo de F. moniliforme, 34 antagonistas isolados de 60 amostras de solo e de 20 amostras de milho foram testados contra F. moniliforme 113F, produtor de fumonisina. O extrato bruto foi preparado com a cultura de microrganismos selecionados em caldo infuso de cérebro e coraçäo (BHI) e concentrados, adicionando-se etanol na proporçäo 1:1. A presença de organismos inibidores de F.moniliforme ocorreu em 29 amostras de solo, obtendo-se 36 microrganismos antagonistas. Referente ao milho, 15 amostras apresentaram microrganismos inibidores, permitindo o isolamento de 15 antagonistas. A caracterizaçäo destes 51 isolados demonstrou que 5 consistiram de leveduras, 3 cocos Gram-positivos , 3 cocos Gram-negativos e 40 bacilos Gram positivos, destacando-se a predominância do último grupo. Todos os 51 isolados