A simple RT-PCR-based strategy for screening connexin identity

Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx) proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s) can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain) that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins.

Involvement of the caudal raphe nuclei in the feeding behavior of rats

Involvement of the caudal raphe nuclei (raphe pallidus, RPa; raphe magnus, RMg, and raphe obscurus, ROb) in feeding behavior of adult rats was studied by measuring c-Fos protein expression, in animals submitted to the "meal-feeding" model of food restriction in which the rats were fed ad libitum only from 7:00 to 9:00 h, for 15 days. The experimental groups submitted to chronic fasting, named 'search for food' (SF), 'ingestion of food' (IF) and 'satiety of food' (SaF) were scheduled after a previous study in which the body weight and the general and feeding behaviors were evaluated by daily monitoring. Acute, 48-h fasting (AF) was used as control. In the chronic group, the animals presented a 16% reduction in body weight in the first week, followed by a continuous, slow rise in weight over the subsequent days. Entrainment of the sleep-wake cycle to the schedule of food presentation was also observed. The RPa was the most Fos immunopositive nucleus in the chronic fasting group, followed by the RMg. The ANOVA and Tukey test (P<0.05) confirmed these results. The IF group was significantly different from the other three groups, as also was the number of labeled cells in the RPa in SF and IF groups. Nevertheless, no significant difference was observed between RMg and RPa, or RMg and ROb in the SaF and AF. However, it is interesting to observe that the groups in which the animals were more active, searching for or ingesting food, presented a larger number of labeled cells. These results suggest a different involvement of the caudal raphe nuclei in the somatic and autonomic events of feeding behavior, corroborating the functions reported for them earlier.

Meal-feeding scheme: twenty years of research in Brazil

Naomi Shinomiya Hell was the first researcher to investigate the physiological adaptations to a meal-feeding scheme (MFS) in Brazil. Over a period of 20 years, from 1979 to 1999, Naomi's group determined the physiological and metabolic adaptations induced by this feeding scheme in rats. The group showed the persistence of such adaptations even when MFS is associated with moderate exercise training and the performance to a session of intense physical effort. The metabolic changes induced by the feeding training were discriminated from those caused by the effective fasting period. Naomi made an important contribution to the understanding of the MFS but a lot still has to be done. One crucial question still remains to be satisfactorily answered: what is the ideal control for the MFS?

Effect of dietary (n-3) highly unsaturated fatty acids on growth and survival of fat snook (Centropomus parallelus, Pisces: Centropomidae) larvae during first feeding

The effect of rotifers, Brachionus rotundiformis (S-type), fed three different diets: A (rotifer fed Nannochloropsis oculata), B (rotifer fed N. oculata and baker's yeast, 1:1), and C (rotifer fed N. oculata and baker's yeast, 1:1, and enriched with Selcoâ), was evaluated based on the survival, growth and swim bladder inflation rate of fat snook larvae. Rotifers of treatment A had higher levels (4.58 mg/g dry weight) of eicosapentaenoic acid (EPA) than B (1.81 mg/g dry weight), and similar levels (0.04 and 0.06 mg/g dry weight, respectively) of docosahexaenoic acid (DHA). Rotifers of treatment C had the highest levels of EPA (13.2 mg/g dry weight) and DHA (6.08 mg/g dry weight). Fat snook eggs were obtained by spawning induction with human chorionic gonadotropin. Thirty hours after hatching, 30 larvae/liter were stocked in black cylindric-conical tanks (36-liter capacity). After 14 days of culture, there were no significant differences among treatments. Mean standard length was 3.13 mm for treatment A, 3.17 mm for B, and 3.39 mm for C. Mean survival rates were very low (2.7% for treatment A, 2.3% for B, and 1.8% for C). Swim bladder inflation rates were 34.7% for treatment A, 27.1% for B, and 11.9% for C. The lack of differences in growth and survival among treatments showed that the improvement of the dietary value of rotifer may not have been sufficient to solve the problem of larval rearing. Some other factor, probably pertaining to the quality of the larvae, may have negatively influenced survival.

Limited-sampling strategy models for estimating the pharmacokinetic parameters of 4-methylaminoantipyrine, an active metabolite of dipyrone

Bioanalytical data from a bioequivalence study were used to develop limited-sampling strategy (LSS) models for estimating the area under the plasma concentration versus time curve (AUC) and the peak plasma concentration (Cmax) of 4-methylaminoantipyrine (MAA), an active metabolite of dipyrone. Twelve healthy adult male volunteers received single 600 mg oral doses of dipyrone in two formulations at a 7-day interval in a randomized, crossover protocol. Plasma concentrations of MAA (N = 336), measured by HPLC, were used to develop LSS models. Linear regression analysis and a "jack-knife" validation procedure revealed that the AUC0-¥ and the Cmax of MAA can be accurately predicted (R²>0.95, bias <1.5%, precision between 3.1 and 8.3%) by LSS models based on two sampling times. Validation tests indicate that the most informative 2-point LSS models developed for one formulation provide good estimates (R²>0.85) of the AUC0-¥ or Cmax for the other formulation. LSS models based on three sampling points (1.5, 4 and 24 h), but using different coefficients for AUC0-¥ and Cmax, predicted the individual values of both parameters for the enrolled volunteers (R²>0.88, bias = -0.65 and -0.37%, precision = 4.3 and 7.4%) as well as for plasma concentration data sets generated by simulation (R²>0.88, bias = -1.9 and 8.5%, precision = 5.2 and 8.7%). Bioequivalence assessment of the dipyrone formulations based on the 90% confidence interval of log-transformed AUC0-¥ and Cmax provided similar results when either the best-estimated or the LSS-derived metrics were used.

Malnutrition during lactation as a metabolic imprinting factor inducing the feeding pattern of offspring rats when adults: The role of insulin and leptin

The aim of the present study was to determine the impact of malnutrition during early postnatal life and the feeding pattern of rat offspring when adults (2 months and 1 year old). In comparison with rats normally fed during lactation, we observed that adult offspring displayed a faster process of feeding reduction when a protein-free diet was offered. In addition, we studied the concentration of insulin and leptin in the lactating pups (10 days) and when these offspring became adult after the onset of a new feeding pattern induced by the protein-free diet. When the diet was changed at 60 days, the offspring malnourished during lactation displayed, after 3 days, a food intake reduction around 41.4 vs 14.2% of the control group. At 10 days of life, plasma leptin and insulin were higher in the malnourished pups when compared with normally fed rats (leptin: 4.6 ± 0.8 vs 2.25 ng/ml; insulin: 0.73 ± 0.12 vs 0.22 ± 0.03 ng/ml) while at 60 days they showed reduction of both hormones when compared with the control group (leptin: 1.03 ± 0.25 vs 1.43 ± 0.5 ng/ml; insulin: 0.54 ± 0.3 vs 0.61 ± 0.4 ng/ml). Despite the different food intake reductions, the malnourished and control rats displayed a similar reduction of insulin and leptin after 3 days of protein-free diet (from 60 to 63 days). The data suggest that the high concentration of insulin and leptin found at 10 days in the malnourished pups may elicit a sustained long-term and unique feeding pattern.

The effect of feeding on the respiratory activity of the sloth

The aim of the present study was to confirm whether feeding influences the resting breathing rate and to observe possible alterations in blood gas and pH levels produced by feeding in unanesthetized sloths (Bradypus variegatus). Five adult male sloths (4.1 ± 0.6 kg) were placed daily in an experimental chair for a period of at least 4 h for sitting adaptation. Five measurements were made for each sloth. However, the sloths one, two and five were studied once and the sloths three and four were studied twice. Breathing rate was determined with an impedance meter and the output signal was digitized. Arterial blood samples were collected for blood gas analysis with a BGE electrolytes analyzer and adjusted for the animal's body temperature and hemoglobin content. The data are reported as mean ± SD and were collected during the resting period (8:00-10:00 h) and during the feeding period (16:00-18:00 h). The mean breathing rate increased during mastication of ymbahuba leaves (rest: 5.0 ± 1, feeding: 10 ± 1 bpm). No significant alterations were observed in arterial pH (rest: 7.42 ± 0.05, feeding: 7.45 ± 0.03), PCO2 (rest: 35.2 ± 5.3, feeding: 33.3 ± 4.4 mmHg) or PO2 (rest: 77.5 ± 8.2, feeding: 78.4 ± 5.2 mmHg) levels. These results indicate that in unanesthetized sloths 1) feeding evokes an increase in breathing rate without a significant change in arterial pH, PCO2 or PO2 levels, and 2) the increase in breathing rate produced by feeding probably is due to the act of mastication.

Tomato and garlic by gavage modulate 7,12-dimethylbenz[a]anthracene-induced genotoxicity and oxidative stress in mice

Chemoprotection by dietary agents is a promising strategy for cancer prevention. The aim of the present study was to evaluate the combined effect of tomato and garlic against 7,12-dimethylbenz- [a]anthracene (DMBA)-induced genetic damage and oxidative stress in 12-14-week-old male Swiss albino mice. The animals were randomized into experimental and control groups and divided into eight groups of five animals each. Group 1 animals were injected intraperitoneally with 35 mg/kg body weight DMBA suspended in peanut oil as a single dose. Groups 2-4 animals received tomato (500 mg/kg body weight), garlic (125 mg/kg body weight) and a combination of tomato and garlic for 5 days by gavage, respectively, followed by DMBA 1.5 h after the final feeding. The doses of tomato and garlic correspond to the average human daily consumption. Animals in groups 5, 6 and 7 received tomato alone, garlic alone and tomato + garlic combination, respectively, for 5 days. Group 8 animals received the same volume of water and served as control. The incidence of bone marrow micronuclei and the extent of lipid peroxidation and the concentrations of antioxidants glutathione, glutathione peroxidase and glutathione-S-transferase were measured in the liver, 48 h after DMBA exposure. Increased frequency of micronuclei and enhanced lipid peroxidation accompanied by compromised antioxidant defenses were observed in DMBA-treated animals. Although pretreatment with tomato or garlic significantly reduced the frequency of DMBA-induced bone marrow micronuclei, the combination of tomato and garlic exhibited more profound effect in inhibiting DMBA-induced genotoxicity and oxidative stress. We suggest that a broad spectrum of antimutagenic and anticlastogenic effects can be achieved through an effective combination of functional foods such as tomato and garlic.

A strategy for obtaining social benefits from the gene revolution

The strategy described in the present paper offers details about the possibility for Brazil to play a more substantial role in the gene revolution. If successfully applied, the powerful science-based technology currently available in Brazil can contribute to extend the benefits of the gene revolution to the poorest countries, very much like the Green Revolution did in the past, thereby reducing the hunger syndrome which claimed the lives of millions of people in some Asian countries, particularly Pakistan and India, decades ago. In his visit to Brazil in February 2004, Norman Borlaug had the opportunity to witness the success of Brazilian agriculture. At a Conference held at ESALQ - Superior School of Agriculture Luiz de Queiroz in Piracicaba, SP, Brazil, he stated that the 21st century revolution will come from Brazil in the area of agriculture. He also said that reducing hunger is essential for the world to achieve socioeconomic stability. A central question remains unanswered: who will fund this revolution? The FAO 2003-2004 Annual Report listed the barriers preventing the gene revolution from reaching the poorest countries: inadequate regulatory procedures - Intellectual Property Rights and Biosafety, poorly functioning seed delivering systems and weak domestic plant breeding capacity; all are discussed in this paper.

Cardiovascular responses to locomotor activity and feeding in unrestrained three-toed sloths, Bradypus variegatus

Heart rate (HR) and systolic (SBP), diastolic (DBP) and mean (MBP) blood pressure were recorded by biotelemetry in nine conscious unrestrained sloths for 1 min every 15 min over a 24-h period. The animals were allowed to freely move in an acoustically isolated and temperature-controlled (24 ± 1ºC) experimental room with light-dark cycle (12/12 h). Behavior was closely monitored through a unidirectional visor and classified as resting (sitting or suspended), feeding (chewing and swallowing embauba leaves, Cecropia adenops), or locomotor activity around the tree trunk or on the room floor. Locomotor activity caused statistically significant increases in SBP (+8%, from 121 ± 22 to 131 ± 18 mmHg), DBP (+7%, from 86 ± 17 to 92 ± 10 mmHg), MBP (+8%, from 97 ± 19 to 105 ± 12 mmHg), and HR (+14%, from 84 ± 15 to 96 ± 15 bpm) compared to resting values, indicating a possible major influence of the autonomic nervous system on the modulation of cardiac function during this behavior. During feeding, the increase in blood pressure was even higher (SBP +27%, from 119 ± 21 to 151 ± 21 mmHg; DBP +21%, from 85 ± 16 to 103 ± 15 mmHg; MBP +24%, from 96 ± 17 to 119 ± 17 mmHg), while HR remained at 14% (from 84 ± 15 to 96 ± 10 bpm) above resting values. The proportionally greater increase in blood pressure than in HR during feeding suggests an increase in peripheral vascular resistance as part of the overall response to this behavior.

The oral motor capacity and feeding performance of preterm newborns at the time of transition to oral feeding

The objective of the present study was to determine the oral motor capacity and the feeding performance of preterm newborn infants when they were permitted to start oral feeding. This was an observational and prospective study conducted on 43 preterm newborns admitted to the Neonatal Intensive Care Unit of UFSM, RS, Brazil. Exclusion criteria were the presence of head and neck malformations, genetic disease, neonatal asphyxia, intracranial hemorrhage, and kernicterus. When the infants were permitted to start oral feeding, non-nutritive sucking was evaluated by a speech therapist regarding force (strong vs weak), rhythm (rapid vs slow), presence of adaptive oral reflexes (searching, sucking and swallowing) and coordination between sucking, swallowing and respiration. Feeding performance was evaluated on the basis of competence (defined by rate of milk intake, mL/min) and overall transfer (percent ingested volume/total volume ordered). The speech therapist's evaluation showed that 33% of the newborns presented weak sucking, 23% slow rhythm, 30% absence of at least one adaptive oral reflex, and 14% with no coordination between sucking, swallowing and respiration. Mean feeding competence was greater in infants with strong sucking fast rhythm. The presence of sucking-swallowing-respiration coordination decreased the days for an overall transfer of 100%. Evaluation by a speech therapist proved to be a useful tool for the safe indication of the beginning of oral feeding for premature infants.

An improved strategy for evaluating the extent of chronic arterial baroreceptor denervation in conscious rats

There is no index or criterion of aortic barodenervation, nor can we differentiate among rats that have suffered chronic sham, aortic or sino-aortic denervation. The objective of this study was to develop a procedure to generate at least one quantitative, reproducible and validated index that precisely evaluates the extent of chronic arterial barodenervation performed in conscious rats. Data from 79 conscious male Wistar rats of about 65-70 days of age with diverse extents of chronic arterial barodenervation and used in previous experiments were reanalyzed. The mean arterial pressure (MAP) and the heart rate (HR) of all rats were measured systematically before (over 1 h) and after three consecutive iv bolus injections of phenylephrine (PHE) and sodium nitroprusside (SNP). Four expressions of the effectiveness of barodenervation (MAP lability, PHE ratio, SNP ratio, and SNP-PHE slope) were assessed with linear fixed models, three-level average variance, average separation among levels, outlier box plot analysis, and overlapping graphic analysis. The analysis indicated that a) neither MAP lability nor SNP-PHE slope was affected by the level of chronic sodium intake; b) even though the Box-Cox transformations of both MAP lability [transformed lability index (TLI)] and SNP-PHE slope [transformed general sensitivity index (TGSI), {((3-(ΔHRSNP-ΔHRPHE/ΔMAPSNP-ΔMAPPHE))-0.4-1)/-0.04597}] could be two promising indexes, TGSI proved to be the best index; c) TLI and TGSI were not freely interchangeable indexes for this purpose. TGSI ranges that permit differentiation between sham (10.09 to 11.46), aortic (8.40 to 9.94) and sino-aortic (7.68 to 8.24) barodenervated conscious rats were defined.

Effect of air-temperature and diet composition on the drying process of pellets for japanese abalone (Haliotis discus hannai) feeding

The aim of this research was to study the effect of air-temperature and diet composition on the mass transfer kinetics during the drying process of pellets used for Japanese Abalone (Haliotis discus hannai) feeding. In the experimental design, three temperatures were used for convective drying, as well as three different diet compositions (Diets A, B and C), in which the amount of fishmeal, spirulin, algae, fish oil and cornstarch varied. The water diffusion coefficient of the pellets was determined using the equation of Fick's second law, which resulted in values between 0.84-1.94×10-10 m²/s. The drying kinetics was modeled using Page, Modified Page, Root of time, Exponential, Logarithmic, Two-Terms, Modified Henderson-Pabis and Weibull models. In addition, two new models, referred to as 'Proposed' models 1 and 2, were used to simulate this process. According to the statistical tests applied, the models that best fitted the experimental data were Modified Henderson-Pabis, Weibull and Proposed model 2, respectively. Bifactorial analysis of variance ANOVA showed that Diet A (fishmeal 44%, spirulin 9%, fish oil 1% and cornstarch 36%) presented the highest diffusion coefficient values, which were favored by the temperature increase in the drying process.

Acetic acid as an intervention strategy to decontaminate beef carcasses in mexican commercial slaughterhouse

Beef can be contaminated during the slaughter process, thus other methods, besides the traditional water washing, must be adopted to preserve meat safety. The objective of this study was to evaluate the effect of 2% acetic acid interventions on the reduction of indicator bacteria on beef carcasses at a commercial slaughterhouse in Mexico. Reduction was measured by the count of mesophilic aerobic bacteria (TPC), total coliform (TC), and fecal coliform (FC) (log CFU/ cm²). Among the different interventions tested, treatments combining acetic acid solution sprayed following carcass water washing had greater microbial reduction level. Acetic acid solution sprayed at low pressure and longer time (10-30 psi/ 60 s) reached higher TPC, TC, and FC reductions than that obtained under high pressure/ shorter time (1,700 psi/ 15 s; P<0.05). Exposure time significantly affected microbial reduction on carcasses. Acetic acid solution sprayed after carcass washing can be successfully used to control sources of indicator bacteria on beef carcasses under commercial conditions.