Cyanase-mediated utilization of cyanate in Pseudomonas fluorescens NCIB 11764.

Pseudomonas fluorescens NCIB 11764 was capable of utilizing cyanate (OCN-) as a sole nitrogen source for growth. Crude cell extracts from cells grown on cyanate, but not on ammonium sulfate, were induced for an enzyme catalyzing cyanate conversion to ammonia. Enzymatic activity was shown to be bicarbonate dependent and specific for cyanate as a substrate, suggesting that cyanate utilization in this organism is facilitated by an enzyme resembling cyanase (cyanate amidohydrolase; EC 3.5.5.3), as described previously in Escherichia coli and Flavobacterium sp.     

Oxytocin and V1 vasopressin receptors in rabbit endometrium during pregnancy

Neurohypophysial hormone receptors were identified and characterized in rabbit endometrium and decidua by radioligand binding methods. The results strongly support the presence of a heterogeneity of sites in the decidua of parturient rabbits. The oxytocin site (R1) binds oxytocin and oxytocin analogues ([Thr4, Gly7]oxytocin and OTA) with high affinity, whereas the AVP site (R2) was selective for the V1 AVP analogues, [Phe2, Orn8]VT and d(CH2)5TyrMeAVP. The concentration of oxytocin receptors was low (50-100 fmol/mg protein) at oestrus (Day 0) and on Day 29 of pregnancy, but increased significantly (about 8-fold, P less than 0.05) during parturition. Conversely, V1 AVP receptors were more concentrated than the oxytocin sites at the end of pregnancy (150 fmol/mg protein) but did not change during parturition. These results indicate that neurohypophysial hormones have specific receptors not only in the myometrium but also in the uterine mucosa and we suggest that these receptors may participate in the regulation of uterine activity during pregnancy.     

Sex differences in the response of rats to drugs affecting GABAergic transmission

The administration of diazepam 1.0 mg/kg decreased the level of plasma corticosterone in female but not in male Wistar rats. Picrotoxin, another drug affecting GABAergic transmission, also brought about an increase of plasma corticosterone in both sexes. However, in order to achieve a plasma corticosterone increase of similar magnitude (more than 500%) a threefold higher dose of picrotoxin had to be given to males. When the convulsive properties of picrotoxin were tested, it became evident that the dose of picrotoxin (2.5 mg/kg) which was subconvulsive in male was almost 100% convulsive in female rats. The existing sex differences in the response of rats to drugs affecting GABAergic transmission might have possible implications in the treatment of GABA system dysfunction.     

Degradation of the metal-cyano complex tetracyanonickelate(II) by cyanide-utilizing bacterial isolates

Ten bacterial isolates capable of growth on tetracyanonickelate(II) [K2[Ni(CN)4]] (TCN) as the sole nitrogen source were isolated from soil, freshwater, and sewage sludge enrichments. Seven of the 10 were identified as pseudomonads, while the remaining 3 were classified as Klebsiella species. A detailed investigation of one isolate, Pseudomonas putida BCN3, revealed a rapid growth rate on TCN (generation time, 2 h), with substrate removal and growth occurring in parallel. In addition to TCN, all isolates were able to utilize KCN, although the latter was significantly more toxic; MICs ranged from 0.2 to 0.8 mM for KCN and greater than or equal to 50 mM for TCN. While growth occurred over a wide range of TCN concentrations (0.25 to 16 mM), degradation was most substantial under growth-limiting conditions and did not occur when ammonia was present. In addition, cells grown on TCN were found to accumulate nickel cyanide [Ni(CN)2] as a major biodegradation product. The results show that bacteria capable of growth on TCN can readily be isolated and that degradation (i) appears to parallel the capacity for growth on KCN, (ii) does not occur in the presence of ammonia, and (iii) proceeds via the formation of Ni(CN)2 as a biological metabolite.     

Dystrophin predominantly localizes to the transverse tubule/Z-line regions of single ventricular myocytes and exhibits distinct associations with the membrane

Dystrophin is a high molecular weight protein present at low abundance in skeletal, cardiac and smooth muscle and in trace amounts in brain. In skeletal muscle, dystrophin is uniformly distributed along the inner surface of the plasma membrane. Biochemical fractionation studies have shown that all detectable skeletal muscle dystrophin is tightly associated with a complex of wheat germ agglutinin (WGA)-binding and concanavalin A (Con A) binding sarcolemmal glycoproteins. Absence of dystrophin is the primary biochemical defect in patients with Duchenne muscular dystrophy and leads to segmental necrosis of their skeletal myofibers. Although present in similar amounts in normal cardiac and skeletal muscle, the absence of dystrophin from cardiac muscle has less severe effects on the survival of cardiac cells. We have therefore examined whether there are differences in the properties of cardiac and skeletal dystrophin. We report that in contrast to skeletal muscle, cardiac dystrophin is distributed between distinct pools: a soluble cytoplasmic pool, a membrane-bound pool not associated with WGA-binding glycoproteins and a membrane-bound pool associated with WGA-binding glycoproteins. Cardiac dystrophin was not associated with any Con A binding glycoproteins. Immunohistochemical localization studies in isolated ventricular myocytes reveal a distinct punctate staining pattern for dystrophin, approximating to the level of the transverse tubule/Z-line and contrasting with the uniform sarcolemmal staining reported for skeletal muscle fibers. The distinct properties of cardiac dystrophin suggest unique roles for this protein in cardiac versus skeletal muscle function.Abbreviations Dys Dystrophin - T-tubule Transverse tubule - SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis - WGA Wheat Germ Agglutinin - Con A Concanavalin A - DHP Dihydropyridine receptor - FITC Fluorescein Isothiocyanate Conjugate - NAG N-Acetyl-D-Glucosamine - NP-40 NONIDET P-40 - PBS Phosphate-Buffered Saline - TBST Tris Buffered Saline-Tween     

Ultrastructural observations on the presence of stereocilia in myoblasts of Scyllium stellare

Many Authors described primary 9+0 cilia in different tissue of different animal species. In relation to the study on the ultrastructural architecture of the embryonic heart of a elasmobranch "Scyllium stellare", we saw some primary 9+0 cilia in very undifferentiated heart tissue. The hearts dissected from the Scyllium stellare embryo at 6 months of gestation, were processed for the observations at the transmission electron microscope. The cilia are found in deep invagination of the plasma membrane, near the Golgi complex and in the perinuclear region. Various functions have been postulated for the primary 9+0 cilia.     

Steroid modulation of oxytocin/vasopressin receptors in the uterus

Oxytocin (OT) and V1 vasopressin (VP) receptors are present simultaneously in several tissues, including the uterus. In myometrium these receptors mediate contractility, while in endometrium they mediate the release of other uterotonic substances as endothelin (ET). In rabbit myometrium, estrogens increase, while progesterone blunts neurohypophysial hormone receptors. However, the action of sex steroids on OT and V1 VP receptors differs in terms of the ED₅₀ and maximal effect. Therefore, at parturition, only OT receptors show a dramatic rise, while V1 VP receptors do not change, suggesting a major role for OT in labor. ET is a potent stimulator of uterine activity acting through specific receptors present on myometrial cells. These receptors as well as the endometrial localization of ET are modulated by sex steroids, indicating that ET might represent a paracrine regulator of uterine activity. In humans, OT but not V1 VP receptors increase as pregnancy progresses, confirming the primary relevance of OT in timing delivery.     

THDP17 Decreases Ammonia Production through Glutaminase Inhibition. A New Drug for Hepatic Encephalopathy Therapy

Ammonia production is implicated in the pathogenesis of hepatic encephalopathy (HE), being intestinal glutaminase activity the main source for ammonia. Management of ammonia formation can be effective in HE treatment by lowering intestinal ammonia production. The use of glutaminase inhibitors represents one way to achieve this goal. In this work, we have performed a search for specific inhibitors that could decrease glutaminase activity by screening two different groups of compounds: i) a group integrated by a diverse, highly pure small molecule compounds derived from thiourea ranging from 200 to 800 Daltons; and ii) a group integrated by commonly use compounds in the treatment of HE. Results shown that THDP-17 (10 µM), a thiourea derivate product, could inhibit the intestinal glutaminase activity (57.4±6.7%). Inhibitory effect was tissue dependent, ranging from 40±5.5% to 80±7.8% in an uncompetitive manner, showing V_max and K_m values of 384.62 µmol min⁻¹, 13.62 mM with THDP-17 10 µM, respectively. This compound also decreased the glutaminase activity in Caco-2 cell cultures, showing a reduction of ammonia and glutamate production, compared to control cultures. Therefore, the THDP-17 compound could be a good candidate for HE management, by lowering ammonia production.     

Involvement of caspase-3 and GD3 ganglioside in ceramide-induced apoptosis in Farber disease.

Farber's disease (FD) is a rare genetic disorder caused by ceramidase deficiency, which results in ceramide accumulation in lung, liver, colon, skeletal muscle, cartilage, and bone. Although this disease has been symptomatically characterized, little is known about its molecular pathogenetic process. Because recent studies reported that ceramide accumulation induces GD3 ganglioside formation and apoptosis, we investigated, in tissue obtained via colonoscopy from seriously involved patients, the possible involvement of ceramide in FD colonocyte destruction. Histochemical and TUNEL analyses of paraffin-embedded sections revealed that 45 +/- 4.3% of FD colonocytes showed morphological signs of apoptosis compared with the 8 +/- 2.3% of constitutive epithelial cell death. Importantly, immunohistochemical study for pro-apoptotic factors showed that GD3 accumulation co-localized with active caspase-3 and cleaved K18 in FD colon tissue. These findings provide evidence for a role of the apoptotic ceramide pathway in the pathogenesis of FD.