De novo transcriptome assembly of sugarcane leaves submitted to prolonged water-deficit stress.

Sugarcane production is strongly influenced by drought, which is a limiting factor for agricultural productivity in the world. In this study, the gene expression profiles obtained by de novo assembly of the leaf transcriptome of two sugarcane cultivars that differ in their physiological response to water deficit were evaluated by the RNA-Seq method: drought-tolerant cultivar (SP81-3250) and drought-sensitive cultivar (RB855453). For this purpose, plants were grown in a greenhouse for 60 days and were then submitted to three treatments: control (-0.01 to -0.015 MPa), moderate water deficit (-0.05 to -0.055 MPa), and severe water deficit (-0.075 to -0.08 MPa). The plants were evaluated 30, 60, and 90 days after the beginning of treatment. Sequencing on an Illumina platform (RNA-Seq) generated more than one billion sequences, resulting in 177,509 and 185,153 transcripts for the tolerant and sensitive cultivar, respectively. These transcripts were aligned with sequences from Saccharum spp, Sorghum bicolor, Miscanthus giganteus, and Arabidopsis thaliana available in public databases. The differentially expressed genes detected during the prolonged period of water deficit permit to increase our understanding of the molecular patterns involved in the physiological response of the two cultivars. The tolerant cultivar differentially expressed a larger number of genes at 90 days, while in the sensitive cultivar the number of differentially expressed genes was higher in 30 days. Both cultivars perceived the lack of water, but the tolerant cultivar responded more slowly than the sensitive cultivar. The latter requires rapid activation of different water-deficit stress response mechanisms for its survival. This rapid activation of metabolic pathways in response to water stress does not appear to be the key mechanism of drought tolerance in sugarcane. There is still much to clarify on the molecular and physiological pattern of plants in response to drought.

Actividad antioxidante y marcha fitoquímica de los capítulos de Tagetes filifolia Lag. "pacha anís" / Antioxidant activity and phytochemical screening of capitula of Tagetes filifolia Lag. or "pacha anís"

Objetivo: Estudiar la actividad antioxidante y marcha fitoquímica de los capítulos de Tagetes filifolia Lag. "pacha anís".Materiales y métodos: Estudio de tipo experimental en el cual se empleó 5 kg de los capítulos de la planta medicinal Tagetes filifolia lag., provenientes de Junín. Se usó el método de cribado fitoquímico de Olga Lock para la marcha fitoquímica y el método DPPH para la determinación de la actividad antioxidante. Se dividió la muestra en 3 grupos: etéreo, alcohol etílico y agua destilada a concentraciones de 100, 50 y 5 µg/ml.Resultados: Se encontró fenoles en cantidades abundantes tanto en el extracto en agua destilada como en el extracto en alcohol etílico, además este último tuvo cantidades moderadas de quinonas. Por otro lado, el extracto en alcohol etílico fue el que presentó el mayor porcentaje de captación de radicales libres (91.26%) a una concentración de 100 µg/ml, similares resultados se encontró con el extracto etéreo (88.94%) y el extracto en agua destilada (75.58%).Conclusiones: Los principales componentes químicos fueron fenoles y quinonas. El mayor efecto antioxidante se obtuvo del extracto etanólico de la planta Tagetes filifolia a una concentración de 100 µg/ml.

Effects of chronic treatment with corticosterone and imipramine on fos immunoreactivity and adult hippocampal neurogenesis.

In a previous study we showed that rats chronically treated with corticosterone (CORT) display anxiogenic behavior, evidenced by facilitation of avoidance responses in the elevated T-maze (ETM) model of anxiety. Treatment with the tricyclic antidepressant imipramine significantly reversed the anxiogenic effects of CORT, while inhibiting ETM escape, a response related to panic disorder. To better understand the neurobiological mechanisms underlying these behavioral effects, analysis of c-fos protein immunoreactivity (fos-ir) was used here to map areas activated by chronic CORT (200 mg pellets, 21-day release) and imipramine (15 mg/kg, IP) administration. We also evaluated the number of cells expressing the neurogenesis marker doublecortin (DCX) in the hippocampus and measured plasma CORT levels on the 21st day of treatment. Results showed that CORT increased fos-ir in the ventrolateral septum, medial amygdala and paraventricular hypothalamic nucleus and decreased fos-ir in the lateral periaqueductal gray. Imipramine, on the other hand, increased fos-ir in the medial amygdala and decreased fos-ir in the anterior hypothalamus. CORT also decreased the number of DCX-positive cells in the ventral and dorsal hippocampus, an effect antagonized by imipramine. CORT levels were significantly higher after treatment. These data suggest that the behavioral effects of CORT and imipramine are mediated through specific, at times overlapping, neuronal circuits, which might be of relevance to a better understanding of the physiopathology of generalized anxiety and panic disorder.

Effects of chronic corticosterone and imipramine administration on panic and anxiety-related responses

It is known that chronic high levels of corticosterone (CORT) enhance aversive responses such as avoidance and contextual freezing. In contrast, chronic CORT does not alter defensive behavior induced by the exposure to a predator odor. Since different defense-related responses have been associated with specific anxiety disorders found in clinical settings, the observation that chronic CORT alters some defensive behaviors but not others might be relevant to the understanding of the neurobiology of anxiety. In the present study, we investigated the effects of chronic CORT administration (through surgical implantation of a 21-day release 200 mg pellet) on avoidance acquisition and escape expression by male Wistar rats (200 g in weight at the beginning of the experiments, N = 6-10/group) tested in the elevated T-maze (ETM). These defensive behaviors have been associated with generalized anxiety and panic disorder, respectively. Since the tricyclic antidepressant imipramine is successfully used to treat both conditions, the effects of combined treatment with chronic imipramine (15 mg, ip) and CORT were also investigated. Results showed that chronic CORT facilitated avoidance performance, an anxiogenic-like effect (P < 0.05), without changing escape responses. Imipramine significantly reversed the anxiogenic effect of CORT (P < 0.05), although the drug did not exhibit anxiolytic effects by itself. Confirming previous observations, imipramine inhibited escape responses, a panicolytic-like effect. Unlike chronic CORT, imipramine also decreased locomotor activity in an open field. These data suggest that chronic CORT specifically altered ETM avoidance, a fact that should be relevant to a better understanding of the physiopathology of generalized anxiety and panic disorder.

Effects of chronic corticosterone and imipramine administration on panic and anxiety-related responses.

It is known that chronic high levels of corticosterone (CORT) enhance aversive responses such as avoidance and contextual freezing. In contrast, chronic CORT does not alter defensive behavior induced by the exposure to a predator odor. Since different defense-related responses have been associated with specific anxiety disorders found in clinical settings, the observation that chronic CORT alters some defensive behaviors but not others might be relevant to the understanding of the neurobiology of anxiety. In the present study, we investigated the effects of chronic CORT administration (through surgical implantation of a 21-day release 200 mg pellet) on avoidance acquisition and escape expression by male Wistar rats (200 g in weight at the beginning of the experiments, N = 6-10/group) tested in the elevated T-maze (ETM). These defensive behaviors have been associated with generalized anxiety and panic disorder, respectively. Since the tricyclic antidepressant imipramine is successfully used to treat both conditions, the effects of combined treatment with chronic imipramine (15 mg, ip) and CORT were also investigated. Results showed that chronic CORT facilitated avoidance performance, an anxiogenic-like effect (P < 0.05), without changing escape responses. Imipramine significantly reversed the anxiogenic effect of CORT (P < 0.05), although the drug did not exhibit anxiolytic effects by itself. Confirming previous observations, imipramine inhibited escape responses, a panicolytic-like effect. Unlike chronic CORT, imipramine also decreased locomotor activity in an open field. These data suggest that chronic CORT specifically altered ETM avoidance, a fact that should be relevant to a better understanding of the physiopathology of generalized anxiety and panic disorder.

[Natural history of localized prostate cancer. Preliminary data on progression and mortality]. / Historia natural del cáncer de próstata localizado. Datos preliminares de progresión y mortalidad.

OBJECTIVE: To address the effect of therapy options and other factors on the natural history of localized prostate cancer (PCa). METHODS: Men with diagnosed clinically localized PCa who underwent radical prostatectomy (RP), radiotherapy (RT) or watchful waiting (WW). Rates of biochemical progression (BQP) and clinical progression (CLP) were calculated. The effects of therapy, initial PSA, presence of palpable tumor and Gleason score were assessed with Kaplan-Meier analysis and log-rank test. Similar methods were used to study overall and disease-specific survival. RESULTS: A total of 228 patients were studied (135 underwent RP, 46 RT, and 47 WW). Median followup time was 2.5 years. Forty patients presented with BQP. The probability of being free from BQP after 2 and 5 years was 76.8% and 57.9% respectively for the whole population, 70.9% and 57.6% for RP patients, 100% and 100% for RT, and 87.1% and 47.2% for WW (p = 0.031). Nineteen patients presented with CLP, with no significant differences with regard to therapy option. A poorly differentiated Gleason score favoured the probability of presenting with CLP (p = 0.022) and shift to metastatic disease (p < 0.001). No cancer-specific mortality was recorded in the studied population. CONCLUSIONS: Short and medium-term prognosis is excellent for localized prostate cancer in terms of survival. Nevertheless, some patients show a higher risk of progressing to metastatic disease (poorly differentiated Gleason score).

Historia natural del cáncer de próstata localizado: Datos preliminares de progresión y mortalidad / Natural history of localized prostate cancer: Preliminary data on progression and mortality

OBJETIVO: Conocer el impacto de la alternativa terapéutica y de otros factores sobre la historia natural del cáncer de próstata (CaP) localizado. MÉTODOS: Pacientes con CaP clínicamente localizado sometidos a prostatectomía radical (PR), radioterapia (RT) u observación (OBS). Se calcularon las tasas de progresión bioquímica (PBQ) y clínica (PCL). Se evaluaron los efectos del tratamiento, del PSA al diagnóstico, de la presencia de tumor palpable y del score de Gleason mediante análisis Kaplan- Meier y test log-rank. Del mismo modo se estudiaron la mortalidad global y la cáncer específica. RESULTADOS: Se estudiaron 228 pacientes (135 sometidos a PR, 46 a RT, y 47 a OBS). La mediana del tiempo de seguimiento fue de 2,5 años. Cuarenta pacientes presentaron PBQ. La probabilidad de permanecer libre de PBQ a los 2 y 5 años fue de 76,8% y 57,9% respectivamente para la serie completa, 70,9% y 57,6% para PR, 100% y 100% para RT, y 87,1% y 47,2% para OBS (p=0,031). Diecinueve pacientes presentaron PCL, no observándose diferencia significativa respecto del tratamiento efectuado. Un score de Gleason pobremente diferenciado influyó en la probabilidad de presentar PCL (p=0,022) y en la evolución a enfermedad metastásica (p<0,001). No se registró mortalidad cáncer-específica en la población estudiada. CONCLUSIONES: El pronóstico a corto y medio plazo del cáncer de próstata localizado es, en términos de supervivencia, excelente. No obstante, algunos enfermos presentan un mayor riesgo de desarrollar enfermedad metastásica (Gleason pobremente diferenciado) (AU)

OBJECTIVE: To address the effect of therapy options and other factors on the natural history of localized prostate cancer (PCa). METHODS: Men with diagnosed clinically localized PCa who underwent radical prostatectomy (RP), radiotherapy (RT) or watchful waiting (WW). Rates of biochemical progression (BQP) and clinical progression (CLP) were calculated. The effects of therapy, initial PSA, presence of palpable tumor and Gleason score were assessed with Kaplan-Meier analysis and log-rank test. Similar methods were used to study overall and disease-specific survival. RESULTS: A total of 228 patients were studied (135 underwent RP, 46 RT, and 47 WW). Median followup time was 2.5 years. Forty patients presented with BQP. The probability of being free from BQP after 2 and 5 years was 76.8% and 57.9% respectively for the whole population, 70.9% and 57.6% for RP patients, 100% and 100% for RT, and 87.1% and 47.2% for WW (p=0.031). Nineteen patients presented with CLP, with no significant differences with regard to therapy option. A poorly differentiated Gleason score favoured the probability of presenting with CLP (p=0.022) and shift to metastatic disease (p<0.001). No cancer-specific mortality was recorded in the studied population. CONCLUSIONS: Short and medium term prognosis is excellent for localized prostate cancer in terms of survival. Nevertheless, some patients show a higher risk of progressing to metastatic disease (poorly differentiated Gleason score) (AU)

[Surgical treatment of stress urinary incontinence in our setting: analysis of results]. / Tratamiento quirúrgico de la incontinencia urinaria de esfuerzo en nuestro medio: análisis de resultados.

INTRODUCTION AND OBJECTIVE: Surgical repair is the most effective option for the treatment of stress urinary incontinence (SUI) between the different therapeutical options available at present. The main objective of our study is to compare the outcome of the different techniques employed in the treatment of SUI in our setting. METHOD: We have performed a retrospective analysis of the patients who underwent surgical intervention for SUI between 1991 to 1999 (213 surgical interventions in 194 patients) clustering the surgical procedures into three groups: abdominal, abdomino-vaginal, and sling procedures. The results of the treatment were defined as follows: total continence, significant improvement and insufficient improvement. Comparison of continence rates was performed with chi 2 test and Fisher's exact test. Association between qualitative variables was also evaluated by means of chi 2 test. Multivariate analysis of predictive factors was performed with a Cox model. The outcome was also evaluated by Kaplan-Meier's curves, and comparisons made with log-rank test. Statistical significance level was established for p < 0.05. RESULTS: Global cure rate at 24 months was 54.5% (116 patients). Significant improvement was observed in 33 patients (15.5%), and insufficient improvement was seen in 64 patients (30%). The most frequent postoperative complications were suprapubic pain (33%), acute urinary retention (26%), significant postvoiding residual urine (24%) and wound seroma or infection (20%). None of the analyzed factors (age, weight, number of births, preoperative pads, postoperative acute urinary retention, and need for postoperative bladder clean intermittent catheterization were independent predictive factors for postoperative continence. The actuarial analysis with Kaplan-Meier curves shows no statistical differences between the studied techniques (log rank p = 0.41). Sling techniques presented with a superior rate of most postoperative complications. CONCLUSIONS: The cure rate of our serie was 54.5% at 24 months, with a 70% of clinically satisfactory responses. With regard to continence status, it seems that there is not a better surgical technique in our hands, presenting sling techniques with a higher rate of postoperative complications. We could not find no pre o postoperative independent factors as predictors of postoperative continence.

Tratamiento quirúrgico de la incontinencia urinaria de esfuerzo en nuestro medio: análisis de resultados / Outcome analysis for surgical treatment of stress urinary incontinence in our setting

INTRODUCCIÓN: La corrección quirúrgica es actualmente el método más efectivo de tratamiento para la incontinencia urinaria de esfuerzo (IUE) de entre las diferentes opciones terapéuticas de las que se dispone en la actualidad. El objetivo de este trabajo consiste en comparar la eficacia de los distintos grupos de técnicas empleadas para el tratamiento de la IUE en nuestras pacientes. MATERIAL Y MÉTODO: Hemos llevado a cabo un análisis retrospectivo de las pacientes intervenidas en nuestro Servicio por IUE durante el periodo comprendido entre abril de 1991 y julio de 1999 (213 intervenciones en 194 pacientes), agrupando los procedimientos en 3 grupos: técnicas abdominales, técnicas abdomino-vaginales, y técnicas de cabestrillo. La tasa de éxito de la intervención fue valorada mediante tres categorías: continencia total, mejoría clínicamente significativa, y ausencia de mejoría. La comparación bivariante de proporciones se realizó mediante la prueba exacta de Fisher y el test de Chi-cuadrado. La asociación de variables cualitativas se evaluó mediante el test de Chi-cuadrado: se utilizó un modelo de Cox para el análisis multivariante de los factores predictores de continencia, y curvas de Kaplan-Meier para la evaluación de la supervivencia de la continencia post-operatoria. Para todos ellos se tomó como nivel de significación estadística una p<0,05. RESULTADOS: La tasa de continencia total de nuestra serie fue del 54,5 por ciento (116 pacientes continentes) a los 2 años. En el grupo de mejoría clínica significativa fueron incluidas 33 pacientes (15,5 por ciento) y en el grupo de pacientes sin mejoría 64 pacientes (30 por ciento) (Figs. 3 y 4). Las complicaciones más frecuentes fueron la presencia de dolor suprapúbico (33 por ciento), la infección de la herida (20 por ciento), la retención de orina post-operatoria (26 por ciento), y la presencia de residuo post-miccional significativo (24 por ciento). Ninguno de los múltiples factores analizados, se mostraron como factores predictivos independientes para la continencia post-operatoria. En el análisis actuarial, tras la comparación de las curvas de Kaplan-Meier correspondientes a cada técnica, objetivamos que no existen diferencias entre las distintas técnicas (logrank; p=0,41) presentando la técnica de sling un mayor número de complicaciones. CONCLUSIONES: La tasa de continencia total post-quirúrgica de nuestra serie es de un 54,5 por ciento a los dos años, encontrando un total de 70 por ciento de respuestas clínicamente satisfactorias. No parece existir una técnica claramente superior a las demás en cuanto a eficacia para nuestras pacientes, presentando la técnica de sling un porcentaje superior de complicaciones. No hemos encontrado factores pre o post-operatorios que influyan de modo independiente en la predicción de la continencia post-quirúrgica (AU)

Kinetics of TNF-alpha and IFN-gamma mRNA expression in islets and spleen of NOD mice.

Insulin-dependent diabetes mellitus is caused by autoimmune destruction of pancreatic beta cells. Non-obese diabetic (NOD) mice spontaneously develop diabetes similar to the human disease. Cytokines produced by islet-infiltrating mononuclear cells may be directly cytotoxic and can be involved in islet destruction coordinated by CD4+ and CD8+ cells. We utilized a semiquantitative RT-PCR assay to analyze in vitro the mRNA expression of TNF-alpha and IFN-gamma cytokine genes in isolated islets (N = 100) and spleen cells (5 x 10(5) cells) from female NOD mice during the development of diabetes and from female CBA-j mice as a related control strain that does not develop diabetes. Cytokine mRNAs were measured at 2, 4, 8, 14 and 28 weeks of age from the onset of insulitis to the development of overt diabetes. An increase in IFN-gamma expression in islets was observed for females aged 28 weeks (149 +/- 29 arbitrary units (AU), P<0.05, Student t-test) with advanced destructive insulitis when compared with CBA-j mice, while TNF-alpha was expressed in both NOD and CBA-j female islets at the same level at all ages studied. In contrast, TNF-alpha in spleen was expressed at higher levels in NOD females at 14 weeks (99 +/- 8 AU, P<0.05) and 28 weeks (144 +/- 17 AU, P<0.05) of age when compared to CBA-j mice. The data suggest that IFN-gamma and TNF-alpha expression in pancreatic islets of female NOD mice is associated with beta cell destruction and overt diabetes.

Kinetics of TNF-alpha and IFN-gamma mRNA expression in islets and spleen of NOD mice

Insulin-dependent diabetes mellitus is caused by autoimmune destruction of pancreatic ß cells. Non-obese diabetic (NOD) mice spontaneously develop diabetes similar to the human disease. Cytokines produced by islet-infiltrating mononuclear cells may be directly cytotoxic and can be involved in islet destruction coordinated by CD4+ and CD8+ cells. We utilized a semiquantitative RT-PCR assay to analyze in vitro the mRNA expression of TNF-alpha and IFN-gamma cytokine genes in isolated islets (N = 100) and spleen cells (5 x 10(5) cells) from female NOD mice during the development of diabetes and from female CBA-j mice as a related control strain that does not develop diabetes. Cytokine mRNAs were measured at 2, 4, 8, 14 and 28 weeks of age from the onset of insulitis to the development of overt diabetes. An increase in IFN-gamma expression in islets was observed for females aged 28 weeks (149 ± 29 arbitrary units (AU), P<0.05, Student t-test) with advanced destructive insulitis when compared with CBA-j mice, while TNF-alpha was expressed in both NOD and CBA-j female islets at the same level at all ages studied. In contrast, TNF-alpha in spleen was expressed at higher levels in NOD females at 14 weeks (99 ± 8 AU, P<0.05) and 28 weeks (144 ± 17 AU, P<0.05) of age when compared to CBA-j mice. The data suggest that IFN-gamma and TNF-alpha expression in pancreatic islets of female NOD mice is associated with ß cell destruction and overt diabetes

Modulation of the severity of experimental autoimmune encephalomyelitis by gammadelta T lymphocytes activated by mycobacterial antigens.

Immunity to mycobacterial antigens may contribute to the maintenance of self-tolerance. Exposure of the immune system to mycobacterial antigen might well stimulate the immune system to exert control over unwanted self-reactive clones. We demonstrated that in vivo administration of Mycobacterium tuberculosis, PPD, and PPD peptide (180-196) prior to immunization with Myelin Basic Protein (MBP) led to a moderate increase of gammadelta T cells, suppression of the immune response, and reduction in the severity of Experimental Autoimmune Encephalomyelitis. The immunosuppression observed is due, at least in part, to the production of Transforming growth factor-beta (TGFbeta) by the gammadelta T lymphocytes.

Outward potassium current oscillations in macrophage polykaryons: extracellular calcium entry and calcium-induced calcium release

Outward current oscillations associated with transient membrane hyperpolarizations were induced in murine macrophage polykaryons by membrane depolarization in the absence of external Na+. Oscillations corresponded to a cyclic activation of Ca2+ -dependent K+ currents (IKCa) probably correlated with variations in intracellular Ca2+ concentration. Addition of external Na+ (8mM) immediately abolished the outward current oscillations, suggesting that the absence of the cation is necessary not only for their induction but also for their maintenance. Oscillations were completely blocked by nisoldipine. Ruthenium red and ryanodine reduced the number of outward current cycles in each episode, whereas quercetin prolonged the hyperpolarization 2- to 15-fold. Neither low molecular weight heparin nor the absence of a Na+ gradient across the membrane had any influence on oscillations. The evidence suggests that Ca+ entry through a pathway sensitive to Ca2+ channel blockers is elicited by membrane depolarization in Na+ -free medium and is essential to initiate oscillations, which are also dependent on the cyclic release of Ca2+ from intracellular Ca2+ -sensitive stores; Ca2+ ATPase acts by reducing intracellular Ca2+, thus allowing slow deactivation of IKCa. Evidence is presented that neither a Na+/Ca2+ antiporter nor Ca2+ release from IP3 -sensitive Ca2+ stores participate directly in the mechanism of oscillation.

Outward potassium current oscillations in macrophage polykaryons: extracellular calcium entry and calcium-induced calcium release.

Outward current oscillations associated with transient membrane hyperpolarizations were induced in murine macrophage polykaryons by membrane depolarization in the absence of external Na+. Oscillations corresponded to a cyclic activation of Ca(2+)-dependent K+ currents (IKCa) probably correlated with variations in intracellular Ca2+ concentration. Addition of external Na+ (8 mM) immediately abolished the outward current oscillations, suggesting that the absence of the cation is necessary not only for their induction but also for their maintenance. Oscillations were completely blocked by nisoldipine. Ruthenium red and ryanodine reduced the number of outward current cycles in each episode, whereas quercetin prolonged the hyperpolarization 2- to 15-fold. Neither low molecular weight heparin nor the absence of a Na+ gradient across the membrane had any influence on oscillations. The evidence suggests that Ca2+ entry through a pathway sensitive to Ca2+ channel blockers is elicited by membrane depolarization in Na(+)-free medium and is essential to initiate oscillations, which are also dependent on the cyclic release of Ca2+ from intracellular Ca(2+)-sensitive stores; Ca2+ ATPase acts by reducing intracellular Ca2+, thus allowing slow deactivation of IKCa. Evidence is presented that neither a Na+/Ca2+ antiporter nor Ca2+ release from IP3-sensitive Ca2+ stores participate directly in the mechanism of oscillation.

ATP- and UTP-induced currents in macrophages and macrophage polykaryons.

We have investigated the currents induced by extracellular ATP (ATPo), extra-cellular UTP, and other related compounds in macrophages. At potentials of -20 to -60 mV, a typical response to ATPo puffs consists of a fast-activating inward current followed by a transient outward current. The phenomenon lasts 5-20 s, but for sustained exposure to ATP the inward current persists for up to 10 min (our longest recording time). Both currents are inhibited by Mg2+, suggesting that the phenomenon is mediated by ATP4-. The outward current can be ascribed to a Ca(2+)-dependent K+ conductance, and release of Ca2+ from intracellular stores is at least in part responsible for this current. The inward current has a reversal potential of approximately 0 mV, and it is nonspecific for monovalent cations. UTP, a nucleotide that induces an increase in the cytoplasmic concentration of free Ca2+ but does not permeabilize macrophages, and ATP-gamma-S can also induce inward and outward current similar to those described for ATP, but higher doses are required. Adenosine and AMP produce no detectable effect, whereas ADP induces a small outward current. The implications of these results to the phenomenon of ATPo-induced permeabilization of macrophage membranes to large molecules are discussed.

Electrophysiology of phagocytic membranes: intracellular K+ activity and K+ equilibrium potential in macrophage polykaryons.

The role of K+ as current carrier during the slow membrane hyperpolarizations (SH) elicited by iontophoretic Ca2+ injections into macrophage polykaryons is studied. The intracellular K+ activity (ak) and the K+ equilibrium potential (Ek) are measured using ion-sensitive microelectrodes. The mean value of ak is 84 +/- 5 mM in a culture medium containing 5.3 mM K+, but increases to 100 +/- 8 mM when the extracellular K+ concentration is raised to 30.3 mM. Under the same conditions the values of Ek obtained from the Nernst equation are -81 +/- 2 mV and -40 +/- 2 mV, respectively. The reversal potentials (ER) of the SH are calculated from changes observed in transmembrane potential and input resistance, according to an equivalent model based only on passive ionic fluxes. The mean ER values obtained are -74 +/- 8 mV in the presence of low K+ concentration and -37 +/- 3 mV for the high K+ medium. These values are significantly smaller than the estimated Ek for the corresponding situations. Evidence for the existence of an electrogenic (Na+ + K+)-ATPase activity is also presented. The evidence indicates that an increase in the membrane potassium permeability can account for about 90% of the total permeability change occurring during the SH.

Reception-mediated phagocytosis of yeast by macrophage polykarions.

1. The phagocytic capacity of polynuclear macrophages (PNM) produced by intraperitoneal implants in mice was analyzed and compared with that of the mononuclear macrophage (M luminal diameter). The ability of PNM and M luminal diameter to interiorize aldehyde-fixed Saccharomyces cerevisiae is described. 2. PNM containing two to five nuclei and M luminal diameter exhibited a similar time course of phagocytosis. The phagocytic index reached a plateau from 30 to 60 min after exposure to yeast. 3. Mannose, mannan extracted from yeast and horseradish peroxidase inhibited yeast phagocytosis by PNM and M luminal diameter subpopulations to a similar extent. 4. Amino sugars (glucosamine, N-acetylglucosamine, galactosamine and N-acetylgalactosamine) caused similar reductions in the phagocytic indices of PNM and M luminal diameter. 5. The presence of monosaccharides (glucose, galactose, fructose and mannitol) did not alter the phagocytic capacity of either type of phagocyte. 6. Neither opsonin-dependent ingestion of sheep red blood cells nor ingestion of aldehyde-fixed cells by PNM or M luminal diameter is affected by mannose, suggesting that these processes involve internalization pathways different from the mannose-hexosamine recognition system. 7. Trypsin sensitivity, temperature dependence and divalent cation requirements for PNM phagocytosis were comparable to those of the M luminal diameter. 8. The similarities of the PNM to their mononuclear precursors suggest that the mannose-hexosamine receptor was preserved after polykaryon formation.

Electrophysiology of phagocytic membranes. Role of divalent cations in membrane hyperpolarizations of macrophage polykaryons.

The electrophysiological properties of the membrane of mouse peritoneal macrophage polykaryons are studied. Slow hyperpolarizations can be elicited by iontophoretic injections of either Ca2+ or Sr2+ into the cytoplasm. The effect of both cations is identical, since: it is invariably triggered by the cation injection, the amplitude is dependent on the K+ gradient, quinine blocks reversibly the response to both cation injections. Mg2+, Ba2+ and Mn2+ did not elicit responses when injected into the cytoplasm. Ca2+ induced slow hyperpolarizations were reversibly blocked by the addition of Ba2+ to the external saline, but were not affected by the presence of external tetraethylammonium chloride. Cells maintained in saline containing high concentrations of Ca2+, Sr2+ or Mn2+ exhibited sustained hyperpolarizations. Quinine blocked the hyperpolarization induced by high Ca2+ or Sr2+, but was ineffective for the case of Mn2+. Cells hyperpolarized by external Mn2+ frequently exhibited nonlinear, voltage-current characteristics. Similar patterns could also be observed in a small fraction (less than 10%) of the cells in control conditions. Current-induced shifts between two stable membrane potentials were seen either in high Ca2+ or normal medium. The great variability of the responses described for this phagocytic membrane is discussed. The evidence supports the assumption that Ca2+ and Sr2+ can induce transient or persistent hyperpolarized states by activating a potassium permeability. External Mn2+ may act in part by reducing impalement-related current leakage from the phagocytic membrane.