Role of a Ca2+-activated K+ current in the maintenance of resting membrane potential of isolated, human, saphenous vein smooth muscle cells

 Calcium-activated potassium currents were studied in dissociated smooth muscle cells from human saphenous vein (HSV) using the patch-clamp technique in the whole-cell configuration. The average measured resting membrane potential (V _m) was –41±2 mV (n=39), when the cells were dialysed with an intracellular pipette solution (IPS) containing 0.1 mM ethyleneglycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (IPS-0.1 mM EGTA). When the EGTA concentration was increased to 10 mM (IPS-10 mM EGTA) V _m became significantly less negative: –13±2 mV (n=23, P<0.05). These results suggest that 10 mM EGTA reduces a calcium-dependent current involved in the maintenance of V _m. Depolarizing voltage steps up to +60 mV from holding potentials of –60 mV resulted in large (1–10 nA) time- and voltage-dependent outward currents. The amplitudes of total whole-cell current densities measured at voltages above –20 mV were significantly greater in the cells dialysed with IPS-0.1 mM EGTA than in those dialysed with IPS-10 mM EGTA. In the cells dialysed with IPS-0.1 mM EGTA, 0.1 mM tetraethylammonium chloride (TEA) and 50 nM iberiotoxin (IBTX), which selectively block large conductance Ca²⁺-activated potassium channels (BK_Ca), diminished the total current recorded at +60 mV by 45±14% (P<0.05, n=5) and 50±6% (n=8, P<0.05), respectively. These blockers at the same concentrations did not affect the total current in cells dialysed with IPS-10 mM EGTA. When tested on intact HSV rings, both 0.1 mM TEA and 50 nM IBTX elicited vessel contraction. We conclude that BK_Ca channels present in HSV smooth muscle cells contribute to the maintenance of the V _m and sustain a significant portion of the total voltage-activated, outward current. Finally, BK_Ca channels appear to play a significant role in the regulation of HSV smooth muscle contractile activity. Received: 3 April 1998 / Received after revision: 23 September 1998 / Accepted: 13 October 1998     

Enrichment of human ESC-derived multipotent mesenchymal stem cells with immunosuppressive and anti-inflammatory properties capable to protect against experimental inflammatory bowel disease

Human ESCs provide access to the earliest stages of human development and may serve as an unlimited source of functional cells for future cell therapies. The optimization of methods directing the differentiation of human embryonic stem cells (hESCs) into tissue-specific precursors becomes crucial. We report an efficient enrichment of mesenchymal stem cells (MSCs) from hESCs through specific inhibition of SMAD-2/3 signaling. Human ESC-derived MSCs (hESC-MSCs) emerged as a population of fibroblastoid cells expressing a MSC phenotype: CD73+ CD90+ CD105+ CD44+ CD166+ CD45- CD34- CD14- CD19- human leucocyte antigen-DR (HLA-DR)-. After 28 days of SMAD-2/3 inhibition, hESC cultures were enriched (>42%) in multipotent MSCs. CD73+CD90+ hESC-MSCs were fluorescence activated cell sorting (FACS)-isolated and long-term cultures were established and maintained for many passages displaying a faster growth than somatic tissue-derived MSCs while maintaining MSC morphology and phenotype. They displayed osteogenic, adipogenic, and chondrocytic differentiation potential and exhibited potent immunosuppressive and anti-inflammatory properties in vitro and in vivo, where hESC-MSCs were capable of protecting against an experimental model of inflammatory bowel disease. Interestingly, the efficient enrichment of hESCs into MSCs through inhibition of SMAD-2/3 signaling was not reproducible with distinct induced pluripotent stem cell lines. Our findings provide mechanistic insights into the differentiation of hESCs into immunosuppressive and anti-inflammatory multipotent MSCs with potential future clinical applications.     

Skeletal development of Kinosternon scorpioides limbs (Chelonia: Kinosternidae)

The morphological study of limbs is important for the understanding of tetrapod biology, where it can be applied to taxonomy and phylogeny, as well ecology and behavior. In this study area, osteogenesis is a subject in Kinosternidae, which has been little researched. The main aim of this study was to characterize the skeletogenesis of Kinosternon scorpioides limbs. Samples were histologically processed, and the embryos were cleared with potassium hydroxide and stained with alcian blue and alizarin red. It was observed that the limbs arose in embryonic Stage 10 as mesenchymal condensate cells. The first stylopodium chondrification centers were noted at Stage 14. Zeugopodium chondrification centers appeared at Stage 15; carpal, metacarpal, tarsal, and metatarsal regions were observed at Stage 16, and the cartilage molds of all bones limbs were present at Stage 18. Ossification began in the humerus and femur at Stage 20, and continued into the radius, ulna, tibia, and fibula bones. By Stage 23, it was already effectively directed toward the bone epiphyses in both limbs. At Stage 26 and hatching, only articular cartilages remained, and in the majority of samples the carpal region showed no affinity for alcian blue or alizarin red staining. This study acts as an indicative parameter of the taxon's normal development and can contribute to the phylogenetic understanding of this group.     

PDX1-MODY: A rare missense mutation as a cause of monogenic diabetes

Maturity-Onset Diabetes of the Young type 4 is a rare form of diabetes mellitus, caused by mutations in the PDX1 gene. However, only a few mutations in this gene have been associated as a cause of monogenic diabetes up to date. It makes difficult to create a clinical manifestation profile of this disease and, consequently, to improve the therapeutic management for these patients. Here we report a normal weight woman, diagnosed with diabetes mellitus at 27 years old, during her first pregnancy. At the time of the recruitment, she was 40 years old and had a body mass index of 23.9 kg/m², glycated hemoglobin level of 9.6%, and fasting plasma glucose (FPG) of 254 mg/dL. She presented no diabetic complications and she was being treated with insulin. She reported a family history of diabetes mellitus characteristic of an autosomal dominant mode of inheritance. Molecular analysis of the PDX1 gene revealed the missense variant c.532G > A (p.(Glu178Lys)) segregating from the patient to her son, reported as diabetic. It was absent in her healthy daughter. The c.532G > A seems to be a rare variant, absent in human variants databases, and among 86 normoglycemic controls. Eight in silico algorithms classified this variant as probably pathogenic. Additionally, analysis of the evolutionary conservation showed the glutamic acid in the position 178 of PDX-1 protein as conserved among several species. Our findings reinforce the importance of screening rare MODY genes among families with suspicion of monogenic diabetes to help better understand the clinical manifestations of this disease.     

Confirmatory factor analysis and psychometric properties of the Emotion Regulation Questionnaire in participants with personality disorders

Emotional dysregulation is a key symptom in participants with personality disorders. The Emotional Regulation Questionnaire (ERQ) has been studied with nonclinical samples; however, it is necessary to confirm the factorial structure of the ERQ in participants with personality disorders. The aims of the present study were to confirm the factorial structure of the Spanish version of the ERQ and analyse its psychometric properties as well as the association between the ERQ and the Borderline Symptoms List (BSL-23) and the Difficulties in Emotion Regulation Scale (DERS). The overall sample was composed of 250 patients with personality disorders, of whom 195 met the criteria for borderline personality disorder. Confirmatory factor analysis was conducted. The two-factor model showed an acceptable fit, similar to the original structure, in the participants with personality disorders and with borderline personality disorder. Cognitive reappraisal was negatively correlated with the DERS and BSL-23, and expressive suppression was positively correlated with the BSL-23. The ERQ is a reliable and valid instrument to evaluate emotional dysregulation in participants with personality disorders and participants with borderline personality disorder.     

Effect of etoposide on experimental testicular teratoma in 129/SvJ mice

To study the effects of etoposide on experimental testicular teratoma in 129/SvJ mouse we analysed the tumour growth, differentiation, apoptosis and the localisation of mdr₁ P-glycoprotein (mdr₁-Pgp). In this model the implanted gonadal ridges developed into testicular teratomas in 17 out of 56 implanted testes (30%) and in 14 out of 28 mice (50%). The tumour-bearing mice were treated with etoposide on 4 successive days either 4 weeks or 6 weeks after implantation, and killed 7 days after the last dose. The mice in the control groups did not receive etoposide. The teratomas consisted mainly of neural tissue. The etoposide-treated 4-week teratomas, but not the 6-week teratomas, were significantly smaller than those in the corresponding control groups. The density of apoptotic cells and the distribution of the mdr₁-Pgp were not altered by etoposide. The decreased proportion of immature neuroectodermal tissue components was observed in all treated teratomas, converting the histology towards that of a mature teratoma. In addition, a low proportion of immature tissue components was frequently combined with a low density of apoptotic cells. In conclusion, etoposide decreased the immature tissue components of teratomas, while mature tissues remained unaffected. These results may have clinical relevance in man, since they confirm that postchemotherapy mature teratomas cannot be treated with chemotherapy. Despite benign histology, the human residual tumours have a significant malignant potential and require complete surgical excision and close surveillance. Received: 20 August 1999 / Accepted: 20 January 2000     

Low-Intensity physical activity beneficially alters the ultrastructural renal morphology of spontaneously hypertensive rats

INTRODUCTION AND OBJECTIVE:

Kidney disorders can cause essential hypertension, which can subsequently cause renal disease. High blood pressure is also common among those with chronic kidney disease; moreover, it is a well-known risk factor for a more rapid progression to kidney failure. Because hypertension and kidney function are closely linked, the present study aimed to observe the beneficial effects of low-intensity physical activity on structural and ultrastructural renal morphology and blood pressure in normotensive and spontaneously hypertensive rats.

METHOD:

Male Wistar-Kyoto rats and spontaneously hypertensive rats were randomly allocated into four groups: sedentary or exercised Wistar-Kyoto and sedentary or exercised spontaneously hypertensive rats. The exercise lasted 20 weeks and consisted of treadmill training for 1 hour/day, 5 days/week.

RESULTS:

The exercised, spontaneously hypertensive rats showed a significant blood pressure reduction of 26%. The body masses of the Wistar-Kyoto and spontaneously hypertensive strains were significantly different. There were improvements in some of the renal structures of the animals treated with physical activity: (i) the interdigitations of the proximal and distal convoluted tubules; (ii) the basal membrane of the proximal and distal convoluted tubules; and (iii) in the basal membrane, slit diaphragm and pedicels of the glomerular filtration barrier. The spontaneously hypertensive rats also showed a decreased expression of connexin-43.

CONCLUSION:

Physical exercise could be a therapeutic tool for improving kidney ultrastructure and, consequently, renal function in hypertensive individuals.     

Distribution of filamentous fungi causing invasive fungal disease at the Haematological Unit,Hospital de Clínicas de Porto Alegre,Brazil

Very limited data are available in the literature to elucidate the aetiology of invasive mould infections in Latin America. Here we report that Aspergillus species caused only half of such cases in a cohort study conducted over 21 months in a university hospital in Porto Alegre, Southern Brazil. Fusarium spp. were the second most prevalent moulds (20.7%), followed by Zygomycetes (13.8%). The importance of obtaining local epidemiological data for adequately guiding empirical antifungal therapy is reinforced.     

Caspase-3 gene expression in human luteinized granulosa cells is inversely correlated with the number of oocytes retrieved after controlled ovarian stimulation

Granulosa cells control oocyte maturation through paracrine signalling and changes to the microenvironment around the oocyte. Apoptosis occurs as a physiological mechanism of granulosa cell renewal, but how it relates with the ovarian response to induced ovulation is still unclear. Therefore, this study evaluated apoptosis-related gene expression levels in granulosa cells of patients undergoing controlled ovarian stimulation. We enrolled prospectively 59 consecutive IVF patients referred to a tertiary academic hospital for couple infertility treatment. Luteinized granulosa cells were isolated from follicular fluid and the RNA was extracted, reverse-transcribed and the gene expression of apoptosis inducers (caspase-3, caspase-8 and bax) and inhibitor (Bcl-2) was quantified by real-time polymerase chain reaction. Caspase-3 gene expression correlated negatively with the number of pre-ovulatory follicles (Spearman’s r?= ?0.308), the number of collected oocytes (r?= ?0.451), the number of mature oocytes (r?= ?0.526), the number of fertilized oocytes (r?= ?0.439) and the number of viable embryos (r?= ?0.443, all statistically significant at p?相似文献