The impact of the association between Val16Ala-SOD2 SNP and SOD2 immunohistochemistry expression in the prognosis of patients with esophageal cancer.

INTRODUCTION: Esophageal cancer is an extensive public health issue worldwide, warranting the search for biomarkers related to its risk and progression. Previous studies have indicated an association between Val16AlaSOD2 single nucleotide polymorphism in the gene encoding the enzyme superoxide dismutase 2 and esophageal cancer. However, further investigations are needed to clarify its role in disease risk and progression. OBJECTIVE: To investigate the role of Val16AlaSOD2-SNP in esophageal cancer progression and in the survival of patients METHODS: Tumor samples were utilized for Val16Ala-SNP genotyping, while SOD2 expression levels in tissue were assessed using immunohistochemistry. A SOD2 Val16Ala-SNP database was used to obtain information on the genotype of healthy individuals. Risk and overall survival analyzes were performed. RESULTS: The Val16Ala SNP was associated with an increased risk of esophageal cancer (RR 2.18, 95%CI 1.23-3.86), regardless of age and gender, but did not have a significant effect on patient survival. In contrast, weak SOD2 expression demonstrated a significantly associated with poor overall survival after treatment, independent of other clinicopathological variables (HR, 0.41; 95% CI, 0.22-0.79 P = 0.007). CONCLUSIONS: Val16Ala SNP was positively associated with esophageal cancer, and the expression of SOD2 was an independent prognostic marker.

Spatial patterns of the leaf crown borer Eupalamides cyparissias (Cramer, 1775) (Lepidoptera: Castiniidae) in coconut (Cocos nucifera L.) tropical region.

The leaf crown borer Eupalamides cyparissias (Cramer, 1775) is an important pest of coconut (Cocos nucifera L.) and other palms (Arecaceae) of economic importance, attacking the base of leaves, inflorescences, and infructescences, increasing fruit abortion. The objective of this study was to evaluate the spatial correlation of the infestation rate of E. cyparissias in coconut plantation blocks in the Brazilian Amazon, from January to December 2019, in the city of Santa Izabel, Pará, Brazil. The study area is a farm subdivided into 157 blocks of a commercial plantation of the green dwarf coconut. The Local Moran's Index was used to evaluate the existence of spatial autocorrelation of the E. cyparissias infestation rate in the 157 blocks with neighboring blocks. The infestation rate was calculated by the ratio between the number of plants attacked by the borer and the total number of plants in a block. There is a significant correlation of the symptomatology of the attack by E. cyparissias in the blocks of the experimental area, which indicates an aggregated pattern of distribution. There is no significant correlation between the attack by the borer and the age of the coconut tree; however there is a significant correlation between the attack by the borer and proximity to forest areas. These results indicate that forest regions are foci of infestation by the borer in coconut plantations.

Epidemiología y factores predisponentes de los hallazgos dermatológicos en neonatos de Uruguay / Neonatal Dermatologic Findings in Uruguay: Epidemiology and Predisposing Factors

Introducción: La piel es fundamental en la transición de la vida intrauterina a la extrauterina. El recién nacido presenta cambios fisiológicos, siendo frecuente observar hallazgos cutáneos benignos y transitorios que varían según las características maternas, neonatales y de la gestación. Objetivos: Estimar la frecuencia de distintos hallazgos dermatológicos en neonatos en las primeras 72h de vida e identificar la existencia de diferencias de proporciones estadísticamente significativas con factores neonatales, maternos y de la gestación. Métodos Se realizó un estudio descriptivo, observacional de corte transversal entre abril-julio 2015 y julio-noviembre de 2017 en la maternidad del Centro Hospitalario Pereira Rossell. Se incluyeron neonatos de hasta 72h de vida, a los que se les practicó un examen dermatológico completo. Se presenta la proporción de cada uno de los hallazgos con el intervalo de confianza (IC) correspondiente. Se analiza la relación entre los hallazgos y determinados factores asociados. Resultado Se incluyeron 2811 neonatos. El 100% presentó al menos un hallazgo a nivel de la piel. La mediana fue de 8 lesiones por paciente (rango intercuartílico: 6-9/mínimo-máximo: 1-16). De los 46 hallazgos explorados clínicamente se encontraron 42. Las lesiones benignas y transitorias correspondieron al 99,9%. Entre ellas se observó: lanugo 98% (IC: 97,7-98,7), descamación fisiológica 79,7% (IC: 78,2-81,1) e hiperplasia sebácea 73,3% (IC: 71,6-74,9). El lanugo (p=0,001), la descamación fisiológica (p<0,001) y el eritema tóxico neonatal (p=0,001) se observaron con más frecuencia en los neonatos de término y postérmino. La hiperplasia sebácea (p=0,001) y la hiperpigmentación transitoria neonatal (p<0,001) se encontraron con mayor frecuencia en varones (AU)

Background: The skin is a fundamental organ in the transition from intrauterine to extrauterine life. The newborn infant experiences physiological changes and often presents benign, transient skin characteristics that vary according to maternal, gestational, and neonatal factors. Objectives: To estimate the frequency of various dermatologic findings during the first 72hours of life and to identify their association with maternal, gestational, or neonatal factors. Methods Descriptive, observational, cross-sectional study from April to July 2015 and July to November 2017 in the maternity ward of Centro Hospitalario Pereira Rossell. We examined the skin of neonates within 72hours of birth. Proportions and 95% CI were calculated for all findings. Associations between findings and factors were analyzed. Results A total of 2811 neonates were included. We observed at least one neonatal skin finding in all of the neonates and found a median (interquartile range) of 8 (6-9) findings (minimum–maximum, 1-16). We observed 42 of the 46 possible characteristics we looked for; 99.9% of the findings were benign and transient. Among the findings were lanugo (98%; 95% CI, 97.7%-98.7%), physiological scaling (79.7%; 95% CI: 78.2%-81.1%), and sebaceous hyperplasia (73.3%; 95% CI: 71.6%-74.9%). Lanugo (P=.001), physiological scaling (P<.001), and erythema toxicum (P=.001) were observed significantly more often in full- and late-term neonates. Sebaceous hyperplasia (P=.001) and transient hyperpigmentation (P<.001) were found more often in newborn males. Erythema toxicum was more common after vaginal births (P=.008). Transient hyperpigmentation (P<.001) and dermal melanocytosis (P<.001) were seen more often in neonates of African descent (AU)

Neonatal Dermatologic Findings in Uruguay: Epidemiology and Predisposing Factors. / Epidemiología y factores predisponentes de los hallazgos dermatológicos en neonatos de Uruguay.

BACKGROUND: The skin is a fundamental organ in the transition from intrauterine to extrauterine life. The newborn infant experiences physiological changes and often presents benign, transient skin characteristics that vary according to maternal, gestational, and neonatal factors. OBJECTIVES: To estimate the frequency of various dermatologic findings during the first 72hours of life and to identify their association with maternal, gestational, or neonatal factors. METHODS: Descriptive, observational, cross-sectional study from April to July 2015 and July to November 2017 in the maternity ward of Centro Hospitalario Pereira Rossell. We examined the skin of neonates within 72hours of birth. Proportions and 95% CI were calculated for all findings. Associations between findings and factors were analyzed. RESULTS: A total of 2811 neonates were included. We observed at least one neonatal skin finding in all of the neonates and found a median (interquartile range) of 8 (6-9) findings (minimum-maximum, 1-16). We observed 42 of the 46 possible characteristics we looked for; 99.9% of the findings were benign and transient. Among the findings were lanugo (98%; 95% CI, 97.7%-98.7%), physiological scaling (79.7%; 95% CI: 78.2%-81.1%), and sebaceous hyperplasia (73.3%; 95% CI: 71.6%-74.9%). Lanugo (P=.001), physiological scaling (P<.001), and erythema toxicum (P=.001) were observed significantly more often in full- and late-term neonates. Sebaceous hyperplasia (P=.001) and transient hyperpigmentation (P<.001) were found more often in newborn males. Erythema toxicum was more common after vaginal births (P=.008). Transient hyperpigmentation (P<.001) and dermal melanocytosis (P<.001) were seen more often in neonates of African descent. CONCLUSIONS: All neonates have skin characteristics that are part of their adaptation to extrauterine life. Most are benign and transient. Maternal age, type of delivery, and certain neonatal factors such as gestational age, birth weight, sex, and ethnicity are associated with specific findings.

Quantitative and qualitative traits of carcass and meat of goats fed diets with cactus meal replacing corn.

The purpose of this study was to evaluate the quantitative and qualitative characteristics of the carcass and meat of goats fed diets containing cactus meal (pectin source) replacing corn (starch source). Twenty-eight goats with an average initial weight of 16 ± 2.02 kg were confined in a completely randomized design with four treatments (the replacement levels of 0, 330, 660, and 1000 g kg-1 of dry matter) and seven replicates. The productive performance of the animals was not affected by the replacement of corn by cactus meal. The carcass commercial yield and the dressing percentage decreased with the addition of cactus meal levels in the diets. The commercial cuts, however, especially prime cuts like hind limbs and loin, were not changed by the use of cactus meal. Muscle:bone and fat:bone ratios and muscularity index of hind limbs were influenced by the substitution. The protein and ash contents of the longissimus lumborum muscle decreased while cholesterol levels increased with the presence of cactus meal. Sensory traits of goats' meat fed cactus meal in the diets were not affected. The substitution of corn for cactus meal reduced carcass yield but did not change the yield of commercial cuts or the qualitative characteristics of the meat.

Genotoxic potential of leaf extracts of Jatropha gossypiifolia L.

Jatropha gossypiifolia L. (Euphorbiaceae) is widely used in popular medicine. However, further toxicological studies are necessary for its reliable use. The present study aimed to evaluate the cytotoxic, genotoxic, and mutagenic effects of ethanolic and aqueous leaf extracts of J. gossypiifolia, using the test system Allium cepa. In addition, the phytochemical profile of the extracts was also obtained. Seeds of A. cepa were subjected to different concentrations of the two extracts (0.001, 0.01, 0.1, 1, and 10 mg/mL). Distilled water was used for the negative control and methyl methanesulfonate (4 x 10(-4) M) and trifluralin (0.84 ppm) for the positive controls. The values of mitotic index at all concentrations of ethanolic extract and at 0.1, 1, and 10 mg/mL aqueous extract showed a significant decrease. Alterations, such as chromosome adherence, C-metaphases, chromosome bridges, nuclear buds, and micronuclei were verified in both extracts but chromosome loss indicating genotoxic activity was observed only in the ethanolic extract. Presence of micronuclei on administration of the extracts, also indicated mutagenic action at the chromosome level. In the ethanolic extract, aneugenicity seemed to be the main activity, probably as a result of the action of terpenes and/or flavonoids, whereas in the aqueous extract, clastogenic action appeared to be the principal activity, presumably as a consequence of the effect of flavonoids and/or saponins. Thus, we suggest that the extracts of this species should be used with great caution for medicinal purpose.

Identification of C-type isolectins in the venom of the scorpionfish Scorpaena plumieri.

Chemical analyses of the hemagglutinating fraction from Scorpaena plumieri venom revealed that it contains five components (Sp-CL 1-5) with similar chromatographic elution profiles (35-38% of acetonitrile), molecular masses (16,800-17,000 Da) and N-terminal sequences, suggesting that they are isoforms of the same protein. The amino acid sequence of Sp-CL4 was determined and shown to have homology with fish C-type lectins. These data demonstrate for the first time the presence of C-type isolectins in a scorpionfish venom.

PP100. Pre-eclampsia: Risk factors and outcomes - A two-year study.

INTRODUCTION: Pre-eclampsia (PE) is a hypertensive disorder responsible for major morbidity and mortality in both mother and fetus. There are some risk factors associated with this entity, but it remains very difficult to predict. OBJECTIVES: Study the incidence of PE and the related risk factors, as well as the maternal and fetal outcome. METHODS: We reviewed the clinical records of pregnant women admitted to Prof. Fernando Fonseca's Hospital from January 2008 to December 2009, with the diagnosis of pre-eclampsia. The statistic analysis was based on Excel 2007. RESULTS: There were 90 cases of PE, among the 308 hypertensive disorders reviewed, with an incidence of 1,1% in overall population of pregnant women. Risk factors with higher association were Chronic Hypertension before pregnancy (24,4%), maternal age above 35 years old (16,67%), maternal age under 20 years old (14,44%), and previous episode of pre-eclampsia (8,89%). Major maternal complications that determined Intensive Care Unit admission were recorded in 17 cases (18,89%), with 3 HELLP syndromes (Hemolysis, elevated liver enzymes, and low platelets)(3,33%). No maternal death was recorded. Preterm delivery (PTD) was seen in 61,1%, 32% before 34weeks and 6,67% before 28weeks. There were 19 cases of 1st minute Apgar Index below 7 and 5 cases of 5th minute Apgar Index below 7. There was one in utero death and two interruptions of pregnancy below 24 weeks due to serious PE. Three twin pregnancies. CONCLUSIONS: PE is a form of hypertensive pregnancy disorder, with a risk of recurrence in subsequent pregnancies. It has a catastrophic potential, mainly associated to PTD, and also with significant morbidity to the pregnant women, reflected in the incidence of admissions to ICU, HELLP syndrome and end-organ failure. In our study we confirmed the adverse outcomes related to this entity, and the risk factors associated.

PP145. Hypertensive disorders of pregnancy: A two year study (2008-2009).

INTRODUCTION: The hypertensive disorders of pregnancy are a leading cause of maternal mortality and morbidity. According to the group studies of NHPBEB 2000 four entities are defined: Chronic Hypertension (CH) previous to pregnancy, Gestational Hypertension (GH), Preeclampsia/Eclampsia (PE/E) and superimposed Preeclampisa/Eclampsia in Chronic Hypertension (PE/E CH). All this entities have different outcomes and require adequate follow-up and specific attitude. OBJECTIVES: Review all cases of hypertensive disorders in a two-year period, its incidences, and related maternal and fetal complications. METHODS: In a retrospective study, from January 2008 to December 2009, all files related with hypertensive disorders, seen in our department, were reviewed. The statistic analysis was based on Excel 2007. RESULTS: The global incidence of hypertensive disorders was 3.8% (309 cases), with each entity with an incidence of: 40% in CH, 40% GH, 25% PE/E and 7% PE/E CH. In terms of demographic characteristics the majority of the population were caucasian (46%) and black (40%), the mean age was of 31years (minimum of 12-maximum of 47), and mainly previous Chronic Hypertension and endocrinologic disorders as co-morbidities (Diabetes Mellitus, obesity and thyroid pathology). The fetal/maternal complications were mainly preterm delivery (26.2%), with a low percentage of Abruptio Placentae (1%). Maternal complications were analysed in terms of ICU admissions of 7%, cardiovascular/renal disorders of 1% and maternal bleeding 1%. No maternal death was described. Fetal outcomes were also studied, specifically in terms of birth weight, with an average of 2794 (500-5480g), apgar index in 1st and 5th minute below seven in respectively, 14% and 3.5%. CONCLUSION: The incidence of maternal complications in our analysis was lower than described in literature. The incidence of preterm delivery was similar to that reported in other studies, mostly due to late pre-terms (>32w).

PP151. Hypertensive complications in pregnancy - Two year study (2008-2009).

INTRODUCTION: Hypertension affects 10% of all pregnancies and accounts for approximately a quarter of all antenatal admissions. Hypertension in pregnancy includes a wide spectrum of conditions, including pre-eclampsia, eclampsia, pre-eclampsia superimposed on chronic hypertension, chronic hypertension, and gestational hypertension. Pregnancies complicated by hypertension are associated with increased risk of adverse fetal, neonatal and maternal outcomes, including preterm birth, fetal growth restriction, perinatal death, acute renal or hepatic failure, ante partum haemorrhage, postpartum haemorrhage and maternal death. Overall pre-eclampsia complicates 5-6% of pregnancies and eclampsia complicates 1-2% of pre-eclamptic pregnancies. OBJECTIVES: Analysis of the maternal complications (incidence of ICU admissions, preeclampsia/eclampsia, renal or cardiovascular acute dysfunction, HELLP syndrome, placental abruption, maternal death) and fetal/neonatal outcome. METHODS: In a retrospective study, from January 2008 to December 2009, all files related with complications of hypertensive disorders, seen in our institution, were analysed. The statistic analysis was based on Excel 2007. RESULTS: Of 309 cases, 123 patients (40%) were found to have gestational hypertension while 121 (40%) suffer chronic hypertension. Ninety patients (29%) have preeclampsia or eclampsia (4 cases). 22 patients with chronic hypertension had a superimposed preeclampsia. Fetal growth restriction, HELLP syndrome and placental abruption were the obstetric complications in 4%, 1% and 1% of the cases, respectively. Additionally, multiple pregnancy and gestational diabetes were noted in 2.6% and 10.7% of the patients. Delivery route was vaginal in 90 patients while 68.9% underwent caesarean section. 6.5% of the patients were admitted to ICU and no woman has died. Preterm delivery occurred in 26.2% of the cases and 2 interruptions of pregnancy before 24weeks were performed due to maternal complications. Intrauterine fetal demise was recorded in 2 cases on admission. CONCLUSION: Women with hypertensive disorders of pregnancy are more likely to have received medical or obstetric interventions such as caesarean section operations. Pregnancies complicated by preeclampsia and eclampsia may be associated with life-threatening complications for both the mother and infant.

Proteomic analysis of human mesenchymal stromal cells derived from adipose tissue undergoing osteoblast differentiation.

BACKGROUND AIMS: Stem cells derived from human adipose tissue (ASC) have the capacity for renewal, are easily obtained and have plasticity properties that allow them to differentiate into several cell types, including osteoblast cells. With the aim of understanding the issue of the osteogenic process and finding reliable biomarkers in cells undergoing the osteogeneic differentiation process, this work took advantage of a proteomic approach to identify proteins involved in osteogenesis. METHODS: For this purpose, ASC were analyzed under three conditions: S0, in the absence of stimulation; S1, with 2 weeks of osteogenic medium stimulation; and S2, with 4 weeks of osteogenic medium stimulation. The identification of ASC was carried out by flow cytometry using antibodies specific to known undifferentiated stem cell-surface markers. Cell viability, enzymatic activity, mineral deposition, collagen structure and production and gene analyzes were evaluated for each condition. RESULTS: Phenotypic modifications were observed during the in vitro osteogenic differentiation process by two-dimensional (2-D) differential image gel electrophoresis (DIGE). The proteins were identified by mass espectrometry in tandem (MS/MS) analyzes using Matrix-assisted laser desorption/ionization with TOF/TOF is a tandem mass spectrometry method where two time-of-flight mass spectrometers are used consecutively (MALDI-TOF/TOF). A total of 51 differentially expressed proteins was identified when comparing the three observed conditions. Sixteen different spots were identified in the S0 stage compared with S2, while 28 different spots were found in S2 compared with S0. S1 expressed seven different spots compared with S0 and S2. CONCLUSIONS: These findings suggest the involvement of several proteins directly related to the osteogenic pathway, which can be used to improve understanding of the osteogenic process.

MR imaging features of brain stem hypoplasia in familial horizontal gaze palsy and scoliosis.

We report the case of a child with horizontal gaze palsy, pendular nystagmus, and discrete thoracolumbar scoliosis. MR imaging of the brain depicted pons hypoplasia with an absence of the facial colliculi, hypoplasia, butterfly configuration of the medulla, and the presence of a deep midline pontine cleft (split pons sign). These MR imaging findings suggest familial horizontal gaze palsy with progressive kyphoscoliosis, a rare congenital disorder. To the best of our knowledge, MR imaging findings of only 4 similar cases, with or without progressive idiopathic scoliosis, have been reported. We discuss the pathogenesis substratum of this entity. Early recognition of this rare entity is important if supportive therapeutic measures in progressive scoliosis are to be applied.

Thrombin is a novel regulator of hexokinase activity in mesangial cells.

BACKGROUND: Hexokinase (HK) activity is fundamentally important to cellular glucose uptake and metabolism. Phorbol esters increase both HK activity and glucose utilization in cultured mesangial cells via a protein kinase C (PKC)- and extracellular signal-regulated kinases 1 and 2 (ERK1/2)-dependent mechanism. In adult kidneys, increased HK activity has been reported in both glomerular injury and in diabetes, but the mechanisms responsible for these changes are unknown. Thrombin, a known activator of both PKC and ERK1/2, is increased in the settings of renal injury and diabetes. Thus, thrombin may contribute to the observed changes in HK activity in vivo. METHODS: Thrombin and thrombin receptor agonists were tested for the ability to increase HK activity and glucose metabolism in murine mesangial (SV40 MES 13) cells. ERK1/2 activation was also evaluated in parallel. Thrombin inhibition (hirudins), PKC depletion, Ser-Thr kinase inhibition (H-7), MEK1/2 inhibition (PD98059), pertussis toxin (PTX), and general inhibitors of transcription or translation were then tested for the ability to attenuate these effects. RESULTS: Thrombin (>/=0.01 U/mL) mimicked the effect of phorbol esters, increasing HK activity> 50% within 12 to 24 hours (P < 0.05). This effect was inhibited by hirudins, mimicked by thrombin receptor agonists, and accompanied by increased Glc utilization. H-7, PD98059, and general inhibitors of transcription or translation-but not PTX-prevented thrombin-induced HK activity at 24 hours. PKC depletion and PD98059 also blocked the associated phosphorylation and activation of ERK1/2. CONCLUSIONS: Thrombin increases mesangial cell HK activity via a PTX-insensitive mechanism involving thrombin receptor activation, PKC-dependent activation of ERK1/2, and both ongoing gene transcription and de novo protein synthesis. As such, thrombin is a novel regulator of HK activity in mesangial cells and may play a role in coupling renal injury to metabolism.

Regulation of mesangial cell hexokinase activity by PKC and the classic MAPK pathway.

Phorbol esters increase glucose (Glc) uptake and utilization in a variety of cell types, and, in some cells, these changes have been attributed to increased Glc phosphorylation and better functional coupling of hexokinases (HKs) to facilitative Glc transporters. Phorbol esters are potent mesangial cell mitogens, but their effects on HK-catalyzed Glc phosphorylation and metabolism are unknown. When examined in murine mesangial cells, active, but not inactive, phorbol esters increased HK activity in a time- and dose-dependent manner. Maximal induction of HK activity at 12-24 h was accompanied by parallel increases in both Glc utilization and lactate production and was blocked by the specific MEK1/2 inhibitor PD-98059 (IC(50) approximately 3 microM). This effect involved early activation of protein kinase C (PKC), MEK1/2, and ERK1/2, and the prolonged time course of subsequent HK induction was attributable, in part, to requirements for ongoing gene transcription and de novo protein synthesis. Mesangial cell HK activity thus exhibits novel regulatory behavior involving both PKC and classic MAPK pathway activation, suggesting specific mechanisms whereby PKC activation may influence Glc metabolism.

Basolateral Na(+)/HCO(3)(-) cotransport activity is regulated by the dissociable Na(+)/H(+) exchanger regulatory factor.

In the renal proximal tubule, the activities of the basolateral Na(+)/HCO(3)(-) cotransporter (NBC) and the apical Na(+)/H(+) exchanger (NHE3) uniformly vary in parallel, suggesting that they are coordinately regulated. PKA-mediated inhibition of NHE3 is mediated by a PDZ motif-containing protein, the Na(+)/H(+) exchanger regulatory factor (NHE-RF). Given the common inhibition of these transporters after protein kinase A (PKA) activation, we sought to determine whether NHE-RF also plays a role in PKA-regulated NBC activity. Renal cortex immunoblot analysis using anti-peptide antibodies directed against rabbit NHE-RF demonstrated the presence of this regulatory factor in both brush-border membranes (BBMs) and basolateral membranes (BLMs). Using a reconstitution assay, we found that limited trypsin digestion of detergent solubilized rabbit renal BLM preparations resulted in NBC activity that was unaffected by PKA activation. Co-reconstitution of these trypsinized preparations with a recombinant protein corresponding to wild-type rabbit NHE-RF restored the inhibitory effect of PKA on NBC activity in a concentration-dependent manner. NBC activity was inhibited 60% by 10(-8)M NHE-RF; this effect was not observed in the absence of PKA. Reconstitution with heat-denatured NHE-RF also failed to attenuate NBC activity. To establish further a physiologic role for NHE-RF in NBC regulation, the renal epithelial cell line B-SC-1, which lacks detectable endogenous NHE-RF expression, was engineered to express stably an NHE-RF transgene. NHE-RF-expressing B-SC-1 cells (B-SC-RF) exhibited markedly lower basal levels of NBC activity than did wild-type controls. Inhibition of NBC activity in B-SC-RF cells was enhanced after 10 microM of forskolin treatment, consistent with a postulated role for NHE-RF in mediating the inhibition of NBC activity by PKA. These findings not only suggest NHE-RF involvement in PKA-regulated NBC activity, but also provide a unique molecular mechanism whereby basolateral NBC and apical NHE3 activities may be coordinately regulated in renal proximal tubule cells.

A prospective study of protein-specific assays used to investigate idiopathic thrombocytopenic purpura.

Idiopathic thrombocytopenic purpura (ITP) is a disorder in which platelets, sensitized by autoantibodies, are destroyed by the reticuloendothelial system. The diagnosis of ITP has been a clinical one because assays measuring platelet-associated IgG (PAIgG) have low specificity. The recently introduced assays that measure antibodies against specific platelet glycoproteins (GP) offer the possibility of improved specificity. In this report we describe two prospective studies. In the first study we compared two protein-specific assays (AC and MAIPA) looking for the presence of autoantibodies against GP IIb/IIIa in 81 patient samples. These results were compared with an immunoradiometric assay for PAIgG. The second study investigated the enhanced sensitivity of measuring anti-GP Ib/IX autoantibodies in 76 patient samples. The protein-specific assays were able to differentiate immune from non-immune thrombocytopenia (specificity 91%, sensitivity 39%), whereas the PAIgG assay could not (specificity 19%, sensitivity 78%). The addition of the Ib/IX AC assay maintained a specificity of 92% while increasing the diagnostic sensitivity to 66%. In contrast to the PAIgG assay, there was no correlation between the platelet count and the likelihood or degree of positivity within the control samples using the glycoprotein assays. These studies confirm that glycoprotein assays can be used as diagnostic tests for ITP.

pH-dependent fluorescence of a heterologously expressed Aequorea green fluorescent protein mutant: in situ spectral characteristics and applicability to intracellular pH estimation.

The green fluorescent protein of Aequorea victoria (GFP) is a natural peptide chromophore without substrate or cofactor requirements for fluorescence. In vitro, a recombinant F64L/S65T GFP mutant (GFPmut1) exhibited pH sensitive fluorescence within the physiologic range. When heterologously expressed in BS-C-1 cells or rabbit proximal tubule cells, uniform cytosolic and nuclear fluorescence was observed. Cytosolic fluorescence constituted over 80% of the total. Excitation scanning of transfected cells revealed two GFPmut1-specific regions that were pH-sensitive over the physiologic range, and each region exhibited a unique pH "bias" in fluorescence emission. Excitation at or near the expected maximum of 488 nm (region II) uniformly resulted in fluorescence that was preferentially altered at acidic pH. In contrast, a novel "wild-type" excitation peak at 400 nm (region I) resulted in alkaline-biased fluorescence similar to that described for the wild-type chromophore in vitro, suggesting that wild-type spectral features disrupted in vitro by mutagenesis may be recovered in intact cells. Calibration of intracellular pH (pHi) with in situ fluorescence following excitation in either region revealed a semilogarithmic relationship between fluorescence intensity and pH within the physiologic range. We therefore measured pHi changes attributable to altered Na/HCO3 cotransport (NBC) activity both in GFPmut1-expressing cells and in paired untransfected cells loaded with BCECF. Basal NBC activity was the same in each group, as was the stimulation of activity by 10% CO2, thus validating the utility of GFPmut1 as a fluorescent probe for pHi and establishing a novel, useful, and practical application for GFPmut1 in monitoring pHi in real time.

Single standard calibration and data processing in flow injection titration based on concentration gradients.

This paper describes use of gradients of concentration generated in flow injection (FI) systems to perform determinations based on points where the concentration of titrant and analyte are at stoichiometric ratio. Two procedures were developed. In one procedure the titrant is injected in a FI manifold and merges with the sample which is continuously pumped towards the detector. In the other procedure the sample is injected and merged with the titrant which is continuously pumped. Both techniques make use of concentration gradients of the sample or titrant generated in FI manifolds that contain a mixing chamber. This gradient is calibrated employing only one standard solution (usually the titrant) in order to convert any detector signal, obtained in the elapsed time after injection, to instantaneous concentration values. The flow system is microcomputer controlled and data are treated to locate points where the concentration of titrant and analyte are at the stoichiometric ratio. These points are found in abrupt changes of the signal x concentration curves obtained in the presence of the reaction. The method has been evaluated for determination of Fe(II) and acetic acid by spectrophotometric and conductimetric detection, respectively. Results show a mean relative standard deviation lower than 1%, an average accuracy of 1% and a high sampling processing capability (40 to 60 samples per hour).

Defective expression of adhesion molecules on human bladder tumour and human tumour cell lines.

The pattern of expression of cell adhesion molecules, i.e., leucocyte function associated antigen 3 (LFA-3) and intercellular adhesion molecule 1 (ICAM-1) on human bladder tumour biopsy specimens was investigated. Attempts were also made to study the pattern of induction of these molecules by established human cell lines in response to cytokines. The results indicated that 15 of 25 tumour biopsy specimens were negative for ICAM-1, and amongst the remaining 10, only 1 showed strong positivity, whilst LFA-3 was expressed in 21 of 23 cases. Unlike LFA-3, the pattern of ICAM-1 expression on established tumour cell lines was different in that there were 7 of 21 cases showing positive staining. The parallel investigation of ICAM-1 and major histocompatibility complex class II antigen expression on bladder tumours showed that in 11 of 18 cases, there was a concomitant expression or complete absence of these molecules. In the remaining 7, there were 6 cases where only class II expression was observed. Exposure of cell lines to interferons alpha or gamma had no effects on LFA-3 expression. In contrast, interferon gamma induced ICAM-1 on all the eight lines with constitutive ICAM-1 expression, whereas interferon alpha upregulated only 2 of these 8 lines. The mean +/- SD values for ICAM-1 expression on the eight inducible lines were 617 +/- 406 cpm before and 943 +/- 471 cpm (p = 0.001) after interferon gamma stimulation. The pattern of ICAM-1 inducibility of a bladder cell line Fen to interferon remained unchanged following transfection of a beta2-microglobulin gene and correction of cell surface HLA class I antigens. These results indicate that there was a significant minority of bladder tumours and tumour cell lines with abnormal cell adhesion molecule expression. In some cases, the abnormality in cell lines could not be corrected by cytokine stimulation. It is possible that these abnormalities may play a critical role in the overall tumour strategy for escape from immunological detection.

Super-sensitive epithelial cell line and colorimetric assay to replace the conventional K562 target and chromium release assay for assessment of non-MHC-restricted cytotoxicity.

Using colorimetric MTT assay the susceptibility of a newly established bladder epithelial cell line, Fen cells was compared with conventional target cells, i.e., K562 and Daudi and other epithelial lines for investigation of non-specific killing activity (NK/LAK) of effector cells previously activated with interleukin-2 (IL-2). The results showed that Fen cell line was more sensitive than K562 and Daudi targets and this was seen whether the effector cells were IL-2-activated or not. The percent killing of effector cells from nine normal donors against Fen, K562 and Daudi targets at effector/target (E/T) ratio of 10/1 after IL-2 activation were 63.4 +/- 7.3, 42.6 +/- 4.3 (p = 0.0001) and 38.6 +/- 5.1 (p = 0.0001) respectively. The corresponding values for inactivated effector cells at 50/1, E/T ratio were 44.8 +/- 9.0, 25.1 +/- 8.3 (p = 0.0001) and 24.4 +/- 9.4 (p = 0.0001) indicating exquisite sensitivity of Fen cells to NK/LAK killing. The susceptibility of Fen cells was found to increase by pre-treatment of target cells with interferons (IFN). Thus the percent killing of untreated, IFN-alpha (1000 U/ml), beta (2000 U/ml) and gamma (100 U/ml) treated cells were 52%, 64% (p = 0.005), 70% (p = 0.001) and 67% (p = 0.001) respectively. These results indicated that Fen cells were more susceptible to NK/LAK killing than the conventional K562 and Daudi target cells. These results and the epithelial origin of Fen cells indicate that this cell line might prove to be a more realistic system to replace the conventional approach for assessment of NK/LAK activity in patients with cancer of epithelial origin.