Modeling the Ethanol Tolerance of the Probiotic Yeast Saccharomyces cerevisiae var. boulardii CNCM I-745 for its Possible Use in a Functional Beer.

Saccharomyces yeasts are able to ferment simple sugars to generate levels of ethanol that are toxic to other yeasts and bacteria. The tolerance to ethanol of different yeasts depends also on the incubation temperature. In this study, the ethanol stress responses of S. cerevisiae and the probiotic yeast S. boulardii CNCM I-745 were evaluated at two temperatures. The growth kinetics parameters were obtained by fitting the Baranyi and Roberts model to the experimental data. The four-parameter logistic Hill equation was used to describe the ethanol tolerance of the yeasts at the temperatures of 28 and 37 °C. Adequate determination coefficients were obtained (R2 > 0.91) in all cases. S. boulardii grown at 28 °C was selected as the yeast with the best ethanol tolerance (6-8%) for use in the elaboration of functional craft beers.

Risk factors, clinical features and outcomes of visceral leishmaniasis in solid-organ transplant recipients: a retrospective multicenter case-control study.

Visceral leishmaniasis (VL) is a rare disease in solid-organ transplant (SOT) recipients. Therefore, little is known about the risk factors and disease behavior in the transplant setting. This multicenter, matched case-control study (1:2 ratio) was designed to determine the risk factors, clinical features and outcomes of VL among this population. Control and case subjects were matched by center, transplant type and timing. Thirty-six VL cases were identified among 25 139 SOT recipients (0.1%). VL occurred 5.7-fold more frequently in Brazil than in Spain, presenting a median time of 11 months after transplantation. High-dose prednisone in the preceding 6 months was associated with VL. Patients were diagnosed over 1 month after symptom onset in 25% of cases. Thirty-one patients (86%) were febrile upon diagnosis, 81% exhibited visceromegaly and 47% showed pancytopenia. Concomitant infection was common. Parasites were identified in 89% of patients; the remaining patients were diagnosed by serology. The majority of the patients received amphotericin B. Relapses occurred in 25.7% of cases, and the crude mortality rate was 2.8%. VL after SOT is related to the VL prevalence in the general population. Delayed diagnosis frequently occurs. Liposomal amphotericin is the most commonly used therapy; mortality is low, although relapses are common.

Predictors of the short- and long-term survival of HIV-infected patients admitted to a Brazilian intensive care unit.

The outcomes of HIV-infected patients requiring critical care have improved. However, in developing countries, information about HIV-infected patients admitted to intensive care units (ICUs) is scarce. We describe the prognosis of HIV-infected patients admitted to a Brazilian ICU and the factors predictive of short- and long-term survival. A historical cohort study, including HIV-infected patients admitted to a Brazilian ICU at an HIV/AIDS reference hospital, was conducted. Survivors were followed up for 24 months after ICU discharge. Demographic, clinical and laboratory data, disease severity scores and mortality were evaluated. Data were analysed using survival and regression models. One hundred and twenty-five patients were studied. In-ICU and in-hospital mortality rates were 46.4% and 68.0%, respectively. Multivariate analysis showed that the in-ICU mortality was significantly associated with APACHE (Acute Physiology and Chronic Health Evaluation) II scores (odds ratio [OR], 1.11; 95% confidence interval [CI], 1.03-1.11), mechanical ventilation (OR, 6.39; 95% CI, 1.29-31.76), tuberculosis treatment (OR, 2.62; 95% CI, 1.03-6.71), use of antiretroviral therapy (OR, 0.19; 95% CI, 0.05-0.77) and septic shock (OR, 4.38; 95% CI, 1.78-10.76). Septic shock was also associated with long-term survival (hazard ratio, 3.0; 95% CI, 1.31-6.90). In-hospital and in-ICU mortality were higher than those reported for developed countries. ICU admission mostly due to AIDS-related diseases may explain these differences.

Efecto de la variedad y la fertilización en indicadores de calidad proteica in-vitro de dos variedades y una línea de garbanzo (cicer arietinum) / Variety and fertilization effect on indicators of protein quality in-vitro of two varieties and one line of chickpea (cicer arietinum)

Protein quality in relation to the degree of fertilization of two varieties (Costa and Blanco Sinaloa) and an advanced line (Hoga L10) of chickpeas obtained from the Campo Experimental Costa de Hermosillo, Sonora, Mexico from INFAP was evaluated. They were grown in similar conditions with 3 levels of nitrogen fertilization (60,120 and 200K/Ha.). Protein and non-protein nitrogen, digestibility and available lysine were analyzed. Significant differences between the varieties and the advanced line in protein and non-protein nitrogen and available lysine were found. The effect of the degree of fertilization on protein quality was significant, showing that a level ofl20KI Ha an increase is observed in protein and non-protein nitrogen and in the percent of digestibility. No significant differences were observed between 60 and 200KIHa. Available lysine was higher at the 200KIHa level. These indicators of protein quality can be used to complement commercial and agricultural aspects that are evaluated for improvement and selection of new varieties of chickpeas.

En este trabajo se evaluó la calidad proteica in-vitro en dos variedades y una línea avanzada de garbanzo, así como su relación con el grado de fertilización aplicado durante su cultivo. Se utilizaron dos variedades comerciales de garbanzo (Costa y Blanco Sinaloa) y una línea avanzada Hoga L10, sembradas en condiciones ambientales similares, con tres niveles de fertilización de nitrógeno (60, 120 y 200K/Ha.), las cuales fueron obtenidas del Campo Experimental Costa de Hermosillo, Sonora, México del INIFAP. Se llevó a cabo un análisis químico proximal, determinaciones de calidad proteica como: nitrógeno proteico y no proteico utilizando extracciones con ácido tricloroacético, digestibilidad in-vitro por el método multienzimático y lisina disponible. A excepción de digestibilidad in-vitro se observaron diferencias significativas entre las variedades y línea avanzada en: nitrógeno proteico, no proteico y lisina disponible. El efecto de la fertilización fue observado en todos las determinaciones de calidad proteica, los resultados indicaron que al aumentar el nivel de fertilización a 120K/Ha se obtiene un incremento significativo en el porcentaje de nitrógeno proteico y no proteico, así como el porcentaje de digestibilidad. Entre los niveles de 60 y 200K/Ha no se observaron diferencias significativas. La lisina disponible fue mayor en las variedades y líneas con nivel de fertilización de 200K/Ha. Estos indicadores de calidad proteica pueden complementar a los aspectos agronómicos y comerciales que son evaluados en los programas de mejoramiento, en la selección de nuevas variedades de garbanzo para su explotación comercial.

Raman spectroscopy for the identification of pigments and color measurement in Dugès watercolors.

Spectroscopic and colorimetric analysis of a representative set of Dugès watercolor paintings was performed. These paintings were the result of scientific studies carried out by the zoologist Alfredo Dugès, who recorded the fauna of the Mexican Republic between 1853 and 1910. Micro-Raman spectroscopy, with an excitation wavelength of 830 nm, and colorimetric techniques were employed in order to understand if different colors with the same hue were reproduced using the same pigments. The color coordinates of the measured areas were obtained in the CIEL*a*b* color space. Raman analysis showed that, in some cases, to reproduce colors with the same hue the pigment employed was not the same. Pigments identified in the watercolors were vermilion, carbon-based black, lead white, gamboge and chrome yellow, Prussian and ultramarine blue. Some of these pigments have been used since ancient times, others as Prussian blue, chrome yellow and synthetic ultramarine blue arrived to the market at the beginning of the 18th and 19th centuries, respectively. Furthermore, regarding the white color, instead of left the paper unpainted, lead white was detected in the eye of a bird. The green color was obtained by mixing Prussian blue with chrome yellow. The results of this work show the suitability of using Raman spectroscopy for watercolor pigment analysis and colorimetric techniques to measure the color of small areas (246 microm x 246 microm) that was the case for the lead white pigment.

Postnatal decrease of sodium current density in rat pituitary melanotropes following the onset of dopaminergic innervation.

Peptide secretion from rat melanotropes is tonically inhibited by a dopaminergic synaptic input that develops after birth and acts through D2 dopamine receptors. In this study, whole-cell Na(+) currents were recorded from melanotropes that were isolated from rat pituitary intermediate lobes at postnatal days 1-20 (P1-P20) and maintained in culture for 5-24 h. Coincident with the development of innervation, melanotropes exhibited a progressive decrease in peak Na(+) current density from P3 to P14. The decrease involved a 50% reduction in maximal Na(+) conductance with no detectable changes in channel gating. Subcutaneous injections of the D2 antagonist sulpiride, applied from P11 to P13, restored melanotrope Na(+) channel activity to pre-innervation levels. Thus, the activation of D2 receptors by the dopaminergic input reduces the functional expression of Na(+) channels in melanotropes.

Hepatosplenic schistosomiasis in field-based studies: a combined clinical and sonographic definition.

A combined clinical and sonographic classification of hepatosplenic schistosomiasis mansoni to be used in field-based studies is proposed herein. Seven hundred forty one individuals out of 892 (83%), living in an area endemic for schistosomiasis in Brazil, have been submitted to clinical and ultrasound examinations. Based on two stool examinations the overall prevalence for schistosomiasis in this area was 73%. Abdominal palpation was performed with patients in dorsal decubit, during deep breath, by two experienced physicians and a portable ultrasound was used for the evaluation of liver fibrosis, portal collaterals and spleen size. Four groups of individuals were identified using data obtained by abdominal palpation and ultrasound examination: (1) palpable spleen and intense periportal thickening in 9 individuals (1.2%); (2) spleen not palpable and intense periportal thickening in 15 (2%); (3) palpable spleen with light to moderate periportal thickening in 32 (4.3%), and (4) palpable spleen with a normal liver on ultrasound in 30 (4%). The definition of hepatosplenic schistosomiasis in field-based studies as the finding of Schistosoma mansoni eggs in the stools in an individual with splenomegaly is not acceptable anymore. Abdominal ultrasound should be combined with clinical examination to accurately identify hepatosplenics in endemic areas for schistosomiasis.

L-type calcium channel activity regulates sodium channel levels in rat pituitary GH3 cells.

1. The effects of chronic pharmacological modulation of L-type Ca2+ channel activity on the cell surface expression of Na+ channels were examined in GH3 cells. 2. Prolonged inhibition (4-5 days) of L-channels with nimodipine caused a 50-60 % decrease in the peak amplitude of whole-cell Na+ currents recorded with the patch-clamp technique. On the contrary, prolonged exposure to the L-channel agonist Bay K 8644 induced an approximately 2.5-fold increase in peak Na+ current. In both cases, there were only minor changes in cell capacitance and no significant changes in Na+ channel gating properties. 3. Measurements of the specific binding of radiolabelled saxitoxin to intact cells showed that nimodipine treatment reduced the number of cell surface Na+ channels, whereas treatment with Bay K 8664 produced the opposite effect. The dual regulation of Na+ channel abundance explained the mentioned changes in Na+ current amplitude. 4. Plasma membrane Na+ channels had a half-life of approximately 17 h both in control cells and in cells treated with Bay K 8644, as estimated from the rate of decay of peak Na+ current after inhibition of protein synthesis with cycloheximide. Actinomycin D, an inhibitor of gene transcription, and also cycloheximide, occluded the stimulatory effect of Bay K 8644 on Na+ current density when measured over a 24 h period. 5. These findings indicate that the entry of Ca2+ through L-type channels influences in a positive way the number of functional Na+ channels in GH3 cells, and suggest that Ca2+ influx stimulates either Na+ channel gene expression or the expression of a regulatory protein that promotes translocation of pre-assembled Na+ channels into the plasma membrane.

Calcium block of Na+ channels and its effect on closing rate.

Calcium ion transiently blocks Na+ channels, and it shortens the time course for closing of their activation gates. We examined the relation between block and closing kinetics by using the Na+ channels natively expressed in GH3 cells, a clonal line of rat pituitary cells. To simplify analysis, inactivation of the Na+ channels was destroyed by including papain in the internal medium. All divalent cations tested, and trivalent La3+, blocked a progressively larger fraction of the channels as their concentration increased, and they accelerated the closing of the Na+ channel activation gate. For calcium, the most extensively studied cation, there is an approximately linear relation between the fraction of the channels that are calcium-blocked and the closing rate. Extrapolation of the data to very low calcium suggests that closing rate is near zero when there is no block. Analysis shows that, almost with certainty, the channels can close when occupied by calcium. The analysis further suggests that the channels close preferentially or exclusively from the calcium-blocked state.

Ca2+ current expression in pituitary melanotrophs of neonatal rats and its regulation by D2 dopamine receptors.

1. We have examined the voltage-dependent Ca2+ channel activity of rat melanotrophs during the early postnatal period. The cells were dissociated from pituitary intermediate lobes, kept in culture for 5-24 h and then subjected to whole-cell patch-clamp experiments. 2. Like their adult counterparts, neonatal melanotrophs were able to generate Na+ currents, K+ currents and Ca2+ currents in response to membrane depolarization. Ca2+ currents were carried by both low- and high-threshold Ca2+ channels. 3. High-threshold Ca2+ current density decreased sharply between postnatal day 4 (P4) and P12. This period coincides with the onset of dopaminergic innervation within the intermediate lobe. Accordingly, the developmental decrease in Ca2+ current density was largely reversed by chronic in vivo treatment with sulpiride, a dopamine D2 receptor antagonist. 4. Prolonging the time in culture from 5 h to 8 days did not significantly alter the Ca2+ channel activity of P3 melanotrophs, whereas the high-threshold Ca2+ current in previously innervated (P14) melanotrophs stayed small for the first 24 h and then increased 3-fold during the subsequent 4-5 days. This increase required RNA and protein synthesis and was prevented by adding D2 agonists to the culture medium. 5. These results provide evidence for a postnatal suppression of high-threshold Ca2+ current expression in pituitary melanotrophs mediated by presynaptic dopamine neurons through D2 dopamine receptors.

Induction of classical lactotropes by epidermal growth factor in rat pituitary cell cultures.

Long term incubation of pituitary tumor GH3 cultures with epidermal growth factor (EGF) induces reciprocal changes in PRL and GH production. However, it is not known whether EGF alters the cellular composition of these cultures. Another unanswered question is whether chronic treatment with EGF stimulates PRL secretion from nonneoplastic pituitary cells. In this study, GH3 cells and pituitary cells from neonatal (10-day-old) rats were cultured for 6 and 2 days, respectively, in the absence or presence of 5 nM EGF. Cells containing PRL and/or GH were then enumerated using light microscopic immunocytochemistry. In addition, neonatal pituitary cells were subjected to reverse hemolytic plaque assays for PRL. EGF treatment drastically increased the proportion of classical lactotropes (cells that secrete only PRL) in the GH3 cultures, from about 0.5% to 8% of all cells, without modifying the percentage of GH-positive cells. A similar action of EGF was observed in the primary cultures. Moreover, EGF enhanced by 240% the amount of PRL secreted from the neonatal lactotrope population during 1-h incubations under basal conditions. This effect was mediated by a selective increase in the relative number of PRL secretors forming large plaques. The results suggest that EGF promotes the differentiation of classical lactotropes in both GH3 cultures and pituitary cultures from neonatal rats, and that these cells are characterized by a high basal rate of PRL secretion.

Long-term regulation of calcium channels in clonal pituitary cells by epidermal growth factor, insulin, and glucocorticoids.

In rat pituitary GH3 cells, epidermal growth factor (EGF) and insulin stimulate prolactin production, whereas glucocorticoids exert the opposite effect. In the present study, GH3 cells were subjected to whole-cell patch clamp to assess the chronic actions of such regulatory factors on voltage-dependent calcium currents. Before the electrical recording, cells were grown 5-6 d either under standard conditions or in the presence of 5 nM EGF, 100 nM insulin, 1 microM dexamethasone or 5 microM cortisol. EGF induced a twofold selective increase in high-threshold calcium current density. Insulin and glucocorticoids, on the other hand, specifically regulated low-threshold Ca channels. Current density through these channels increased by 70% in insulin-treated cells, and decreased by 50% in cells exposed to dexamethasone or cortisol. Other Ca channel properties investigated (conductance-voltage curves, deactivation rates, time course and voltage dependence of low-threshold current inactivation) were unaffected by the chemical messengers. The alterations in current density persisted for many hours after removing the regulatory factors from the culture medium. In fact, the stimulatory action of EGF on high-threshold current lasted > 3 d. The results suggest that the control of prolactin production by the factors tested involves regulation of the surface density of functional Ca channels in the plasma membrane.

Comparison of lactotrope subtypes of neonatal and adult male rats: plaque assays and patch-clamp studies.

We examined the differences in lactotrope number and function between pituitary cultures from neonatal (10-day-old) and adult male rats. Basal hormone release was measured with the reverse hemolytic plaque assay. Whole cell Ba2+ currents through Ca2+ channels were recorded from identified prolactin (PRL) secretors with the patch-clamp technique. Lactotropes were classified in two groups according to the relative amount of PRL released: small-plaque (SP) secretors accounted for 6% of all cells in both neonatal and adult pituitary cultures, whereas large-plaque (LP) secretors comprised 13% of the adult pituitary cells but were scarce in cultures from neonates. Simultaneous plaque assays for PRL and growth hormone (GH) showed that in adults as well as in neonates the number of SP and LP secretors was similar to the number of lactosomatotropes (PRL cells that also release GH) and classical lactotropes (PRL-only cells), respectively. Ba2+ current density at positive membrane potentials was markedly higher in adult LP secretors than in neonatal or adult SP lactotropes. We conclude that the appearance of LP secretors constitutes a major postnatal change within the rat lactotrope population. These cells present a large activity of high-threshold Ca2+ channels in the plasma membrane, release PRL at high basal rates, and may correspond to classical lactotropes. The results further suggest that neonatal lactotrope-like cells persist during development and give place to adult SP secretors.

Lactotrope subtypes are differentially responsive to calcium channel blockers.

Pituitary cultures from adult rats contain two subtypes of prolactin (PRL) cells, small-plaque (SP) and large-plaque (LP) lactotropes, which exhibit distinct rates of basal secretion and thereby form PRL plaques of different sizes in reverse hemolytic plaque assay experiments. In the present study, we have used plaque assays to examine the effects of omega-conotoxin (omega-CgTx) and nifedipine, which block Ca2+ entry through high voltage-activated (HVA) channels in the plasma membrane, on basal PRL secretion from single male rat lactotropes. We found that omega-CgTx, like nifedipine, is a potent inhibitor of PRL secretion. In addition, we observed that both drugs decrease the number of cells forming large PRL plaques, while promoting a comparable increase in the abundance of small plaque formers. The results indicate that blocking the HVA Ca channels preferentially suppresses PRL release from LP lactotropes, and suggest that the inhibited PRL secretors tend to behave functionally as SP lactotropes.

Differential expression of Na channels in functional subpopulations of rat lactotropes.

We have investigated the voltage-dependent Na channel activity of single lactotropes in pituitary cultures from adult male rats by recording whole cell Na+ currents under voltage clamp. Cells were identified by their ability to secrete prolactin in the basal state as measured with the reverse hemolytic plaque assay. We found that cells with elevated secretory rates [large-plaque (LP) lactotropes] present relatively large Na+ currents in response to depolarization, whereas Na+ currents in cells secreting prolactin at low rates [small-plaque (SP) lactotropes] are small or are not detectable. The maximum amplitude of the inward Na+ current, normalized by cell capacitance, is about sixfold larger, on the average, in LP lactotropes than in SP lactotropes. Complete block of the Na channels with external tetrodotoxin inhibits by approximately 72% the amount of prolactin secreted from the entire cell population over a period of 1 h, an effect that depends on a drastic reduction in the proportion of LP lactotropes. The results indicate that Na channel activity promotes basal prolactin secretion in male rat lactotropes and suggest that differences in Na channel expression contribute to explain the functional heterogeneity of these pituitary cells.

Calcium ion as a cofactor in Na channel gating.

Calcium ions in the external medium stabilize the resting state of voltage-dependent channels, including Na channels. This effect of calcium on channel gating is usually explained in terms of the surface charge hypothesis, which proposes that local adsorption of calcium ion to the outside of the membrane alters the intramembranous electric field, thus influencing channel behavior indirectly. Calcium ion has also been shown to block Na channels, most strongly at negative voltage. We have examined these two apparently separate effects of calcium, the gating effect and Ca block, and find the two are closely correlated. We propose that calcium (or a suitable substitute) is an essential cofactor in normal gating and that it produces gating and blocking effects by binding within the channel.

Calcium channels and basal prolactin secretion in single male rat lactotropes.

Ba2+ currents through voltage-dependent Ca channels and basal prolactin secretion were measured in single, cultured lactotropes by the combined use of whole cell patch-clamp recording and the reverse hemolytic plaque assay. Measurements of plaque area, a cumulative index of the relative amount of prolactin released by a cell per unit time, indicate that lactotropes can be grouped in two main subpopulations that differ in basal secretory activity: small-plaque (SP) cells and large-plaque (LP) cells. Analysis of Ba2+ currents indicates that both SP and LP lactotropes express two types of Ca channels: low-threshold, inactivating, slowly deactivating (SD) channels and high-threshold, noninactivating, fast deactivating (FD) channels. Ba2+ current amplitude is smaller in SP cells than in LP cells. Plaque area, and thus prolactin release, is positively correlated with the density of Ba2+ current through FD channels, but not with that through SD channels. The results suggest that the surface density of functional FD Ca channels in the plasma membrane is a major factor that determines the rate of basal prolactin secretion in single lactotropes.

Modification of sodium channel gating by lanthanum. Some effects that cannot be explained by surface charge theory.

In clonal pituitary (GH3) cells we studied the changes in sodium channel gating caused by substitution of La3+ for Ca2+ ion. Gating of sodium channels was simplified by using intracellular papain to remove inactivation. To quantify La effects, we empirically fitted closing and the late phase of opening of the channels with single exponentials, determined the opening (a) and closing (b) rate, and plotted these rates as a function of Vm (membrane voltage). The midpoint of the fraction open-Vm curve was also determined. Changing from Ca to La shifted the curves for these three measures of Na channel gating along the voltage axis and changed their shape somewhat. Surface charge theory, in the form usually presented, predicts equal shifts of all three curves, with no change in shape. We found, however, that the shift for each of the measurements was different. 2 mM La, for example, shifted opening kinetics by +52 mV (i.e., 52 mV must be added to the depolarization to make activation in 2 mM La as fast as in 2 mM Ca), the fraction open voltage curve by +42.5 mV, and the closing rate curve by +28 mV. The shift was an almost linear function of log [La] for each of the measures. The main finding is that changing from 2 mM Ca to 10 microM La causes a positive shift of the opening rate and fraction open curves, but a negative shift of the closing rate curve. The opposite signs of the two effects cannot be explained in terms of surface charge theory. We briefly discuss some alternatives to this theory.

Voltage-dependent inactivation of slow calcium channels in intact twitch muscle fibers of the frog.

Inactivation of slow Ca2+ channels was studied in intact twitch skeletal muscle fibers of the frog by using the three-microelectrode voltage-clamp technique. Hypertonic sucrose solutions were used to abolish contraction. The rate constant of decay of the slow Ca2+ current (ICa) remained practically unchanged when the recording solution containing 10 mM Ca2+ was replaced by a Ca2+-buffered solution (126 mM Ca-maleate). The rate constant of decay of ICa monotonically increased with depolarization although the corresponding time integral of ICa followed a bell-shaped function. The replacement of Ca2+ by Ba2+ did not result in a slowing of the rate of decay of the inward current nor did it reduce the degree of steady-state inactivation. The voltage dependence of the steady-state inactivation curve was steeper in the presence of Ba2+. In two-pulse experiments with large conditioning depolarizations ICa inactivation remained unchanged although Ca2+ influx during the prepulse greatly decreased. Dantrolene (12 microM) increased mechanical threshold at all pulse durations tested, the effect being more prominent for short pulses. Dantrolene did not significantly modify ICa decay and the voltage dependence of inactivation. These results indicate that in intact muscle fibers Ca2+ channels inactivate in a voltage-dependent manner through a mechanism that does not require Ca2+ entry into the cell.