[Construction of TRAF6 ubiquitin site 331 mutant colorectal cancer cell stable line and its effect on biological behavior of colorectal cancer cells].

Objective: To investigate the effect of ubiquitin mutation at position 331 of tumor necrosis factor receptor related factor 6 (TRAF6) on the biological characteristics of colorectal cancer cells and its mechanism. Methods: lentivirus wild type (pCDH-3×FLAG-TRAF6) and mutation (pCDH-3×FLAG-TRAF6-331mut) of TRAF6 gene expression plasmid with green fluorescent protein tag were used to infect colorectal cancer cells SW480 and HCT116, respectively. The infection was observed by fluorescence microscope, and the expressions of TRAF6 and TRAF6-331mut in cells was detected by western blot. Cell counting kit-8 (CCK-8) and plate cloning test were used to detect the proliferation ability of colorectal cancer cells in TRAF6 group and TRAF6-331mut group, cell scratch test to detect cell migration, Transwell chamber test to detect cell migration and invasion, immunoprecipitation to detect the ubiquitination of TRAF6 and TRAF6-331mut with ubiquitinof lysine binding sites K48 and K63. Western blot was used to detect the effects of TRAF6 and TRAF6-331mut over expression on the nuclear factor kappa-B (NF-κB) and mitogen activated protein kinase mitogen-activated protein kinase (MAPK)/activating protein-1(AP-1) signal pathway. Results: The successful infection of colorectal cancer cells was observed under fluorescence microscope. Western blot detection showed that TRAF6 and TRAF6-331mut were successfully expressed in colorectal cancer cells. The results of CCK-8 assay showed that on the fourth day, the absorbance values of HCT116 and SW480 cells in TRAF6-331mut group were 1.89±0.39 and 1.88±0.24 respectively, which were lower than those in TRAF6 group (2.09±0.12 and 2.17±0.45, P=0.036 and P=0.011, respectively). The results of plate colony formation assay showed that the number of clones of HCT116 and SW480 cells in TRAF6-331mut group was 120±14 and 85±14 respectively, which was lower than those in TRAF6 group (190±21 and 125±13, P=0.001 and P=0.002, respectively). The results of cell scratch test showed that after 48 hours, the percentage of wound healing distance of HCT116 and SW480 cells in TRAF6-331mut group was (31±12)% and (33±14)%, respectively, which was lower than those in TRAF6 group [(43±13)% and (43±7)%, P=0.005 and 0.009, respectively]. The results of Transwell migration assay showed that the migration numbers of HCT116 and SW480 cells in TRAF6-331mut group were significantly lower than those in TRAF6 group (P<0.001 and P<0.002, respectively). The results of Transwell invasion assay showed that the number of membrane penetration of HCT116 and SW480 cells in TRAF6-331mut group was significantly lower than those in TRAF6 group (P=0.008 and P=0.009, respectively). The results of immunoprecipitation detection showed that the ubiquitin protein of K48 chain pulled by TRAF6-331mut was lower than that of wild type TRAF6 in 293T cells co-transfected with K48 (0.57±0.19), and the ubiquitin protein of K63 chain pulled down by TRAF6-331mut in 293T cells co-transfected with K63 was lower than that of wild type TRAF6 (0.89±0.08, P<0.001). Western blot assay showed that the protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-HCT116 cells were 0.63±0.08, 0.42±0.08 and 0.60±0.07 respectively, which were lower than those in TRAF6-HCT116 cells (P=0.002, P<0.001 and P<0.001, respectively). The expression level of AP-1 protein in TRAF6-HCT116 cells was 0.89±0.06, compared with that in TRAF6-HCT116 cells. The difference was not statistically significant (P>0.05). The protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-SW480 cells were 0.50±0.06, 0.51±0.04, 0.48±0.02, respectively, which were lower than those in TRAF6-SW480 cells (all P<0.001). There was no significant difference in AP-1 protein expression between TRAF6-331mut-SW480 cells and TRAF6-SW480 cells. Conclusion: The ubiquitin site mutation of TRAF6 gene at 331 may prevent the binding of TRAF6 and ubiquitin lysine sites K48 and K63, and then affect the expressions of proteins related to downstream NF-κB and MAPK/AP-1 signal pathways, and inhibit the proliferation, migration and invasion of colorectal cancer cells.

Comparison of genetic diversity between wild-caught broodstock and hatchery-produced offspring populations of the vulnerable Korean kelp grouper (Epinephelus bruneus) by microsatellites.

The kelp grouper Epinephelus bruneus (Perciformes: Haemulidae), is one of the most economically important fishery resources in Korea. This fish is regarded as a target for prospective aquaculture diversification; therefore, maintenance of stock quality is important. To investigate the effects of current artificial reproduction in a hatchery facility, genetic variation in wild-caught broodstock and hatchery-produced offspring of kelp grouper was analyzed using eight polymorphic nuclear microsatellite DNA loci; 77 alleles were identified. Allelic variability ranged from 2 to 22 in the broodstock and from 1 to 10 in the offspring. The average observed and expected heterozygosities were 0.620 and 0.623 in the broodstock and 0.600 and 0.513 in the offspring, respectively. The possibility of a recent genetic bottleneck was suggested in both populations of E. bruneus. The minor, but significant, genetic differentiation (FST = 0.047, P < 0.05) observed was mainly due to statistically significant reductions in the number of alleles in the offspring compared with the broodstock, suggesting that these genetic changes could be related to genetic drift. Our results demonstrate the usefulness of microsatellite markers to monitor genetic variation and raise concerns about potential harmful genetic effects of inappropriate hatchery procedures. Therefore, genetic variation between broodstock and offspring in a hatchery should be monitored in both breeding and release programs as a routine hatchery operation, and inbreeding should ideally be controlled to improve kelp grouper hatchery management. Our data provide a useful genetic basis for future planning of sustainable culture and management of E. bruneus in fisheries.

Rnh1 promotes differentiation and myelination via RhoA in oligodendrocytes.

Increases in Rattus norvegicus ribonuclease/angiogenin inhibitor 1 (Rnh1) are observed in rat primary neuron injury and/or the regeneration process and in differentiated oligodendrocytes. However, the roles of Rnh1 in the central nervous system are still largely unexplored. RhoA is an important signaling protein that has been implicated in oligodendrocyte differentiation and myelination. We demonstrate enhanced differentiation and myelination of oligodendrocytes mediated by Rnh1 in vitro. We further show that Rnh1 is expressed in oligodendrocyte precursors and oligodendrocytes. Importantly, Rnh1 strongly affects oligodendrocyte differentiation through RhoA-ROCK signaling. Moreover, changes in Rnh1 expression in oligodendrocytes regulates the expression and phosphorylation of Fyn, a regulator of RhoA activity. Finally, Rnh1 promotes myelination in vitro. These results show that Rnh1-mediated RhoA inactivation enhances the differentiation and myelination in oligodendrocytes. Overall, Rnh1 might contribute to oligodendrocyte differentiation and myelination processes in vitro.

Development of microsatellite markers for the kelp grouper Epinephelus bruneus by 454 pyrosequencing and transfer to related species.

The kelp or longtooth grouper (Epinephelus bruneus), which inhabits Eastern Asia, is the most economically important of 11 grouper species that inhabit the Southern Sea near Jeju Island in Korea. This species is listed as vulnerable by the International Union for the Conservation of Nature and Natural Resources because of a rapid decrease in its resources. We developed microsatellite markers for E. bruneus using the pyrosequencing technique for applications in resource management and aquaculture. In addition, we tested the cross-species transferability of the microsatellite markers in four species belonging to the Epinephelus genus. Among 66,452 simple sequence repeats, 64 loci containing more than eight CA or TG repeats were randomly selected for primer synthesis; 45 primer sets (75.0%) produced polymerase chain reaction (PCR) products of 100-300 bp and were selected as candidates. After primary testing with four E. bruneus fish, 28 polymorphic loci were selected as the final microsatellite markers, and 23 sets showing clear amplification of polymorphic loci were used to analyze 71 fish. These loci have allele numbers ranging from 2 to 23. Null alleles were detected at three loci, and three loci showed an excess of homozygotes in the Hardy-Weinberg equilibrium test. Of the three species used for cross-species transfer of these markers, Epinephelus moara showed the highest transferability (92.9%) and polymorphism (67.9%), followed by Epinephelus fuscoguttatus (75.0 and 67.9%, respectively) and Epinephelus septemfasciatus (57.1 and 46.4%, respectively). These results suggested that these microsatellite loci should be valuable tools for population genetic studies of the species Epinephelus.

Interleukin-27 as a negative regulator of human neutrophil function.

Interleukin-27 (IL-27) is a novel cytokine of the IL-6/12 family with a broad range of immune regulation properties, which has been considered as a potential therapeutic agent for immune diseases and cancers. However, little is known about the effect of IL-27 on human neutrophils before its clinical administration. In this study, we investigated the effects of IL-27 on human neutrophil functions including adhesion, reactive oxygen species (ROS)/cytotoxic granule components production, inflammatory cytokines production, major histocompatibility complex (MHC) molecules expression and neutrophils' survival. We showed that IL-27 receptor complex, WSX-1/TCCR and gp130, is constitutively expressed on human neutrophils. In vitro, IL-27 suppressed neutrophil adhesion in response to fMLP, which might depend on the down-regulation of Mac-1. IL-27 also suppressed lipopolysaccharide-induced ROS production and attenuated cytotoxic granule components production in the cytoplasm of human neutrophils. In addition, IL-27 enhanced the production of IL-1ß but not TNF-α from neutrophils. However, IL-27 failed to regulate the expression of MHC molecules and the survival of human neutrophils. In conclusion, our data demonstrate that IL-27 mainly down-modulates human neutrophil function, which might extend our understanding of the role of IL-27 in the innate immune response.

New insights into application of cardiac monophasic action potential.

Monophasic action potential (MAP) recording plays an important role in a more direct view of human myocardial electrophysiology under both physiological and pathological conditions. The procedure of MAP measuring can be simply performed using the Seldinger technique, when MAP catheter is inserted through femoral vein into the right ventricle or through femoral artery to the left ventricle. The MAP method represents a very useful tool for electrophysiological research in cardiology. Its crucial importance is based upon the fact that it enables the study of the action potential (AP) of myocardial cell in vivo and, therefore, the study of the dynamic relation of this potential with all the organism variables. This can be particularly helpful in the case of arrhythmias. There are no doubts that physiological MAP recording accuracy is almost the same as transmembrane AP as was recently confirmed by anisotropic bidomain model of the cardiac tissue. MAP recording devices provide precise information not only on the local activation time but also on the entire local repolarization time course. Although the MAP does not reflect the absolute amplitude or upstroke velocity of transmembrane APs, it delivers highly accurate information on AP duration and configuration, including early afterdepolarizations as well as relative changes in transmembrane diastolic and systolic potential changes. Based on available data, the MAP probably reflects the transmembrane voltage of cells within a few millimeters of the exploring electrode. Thus MAP recordings offer the opportunity to study a variety of electrophysiological phenomena in the in situ heart (including effects of cycle length changes and antiarrhythmic drugs on AP duration).

Decreased urinary secretion of belotecan in folic acid-induced acute renal failure rats due to down-regulation of Oat1 and Bcrp.

The effects of folic acid-induced acute renal failure on the renal excretion of belotecan were investigated in rats after intravenous administration. Both glomeruli and renal tubules were seriously damaged by folic acid-induced acute renal failure. The renal excretion clearance, CLr, of belotecan was significantly decreased by folic acid-induced acute renal failure. Furthermore, glomerular filtration rate and secretion clearance of the drug were dramatically decreased by folic acid-induced acute renal failure. In vivo renal uptake of belotecan was inhibited by p-aminohippurate, whereas renal excretion was inhibited by GF120918, but not by verapamil and bromosulphalein. This indicates that Oat1/3 and Bcrp are involved in the renal uptake and urinary excretion of belotecan, respectively. Both mRNA and protein levels of Oat1, Oat3 and Bcrp were significantly decreased in folic acid-induced acute renal failure rats. Based on the finding that belotecan is a substrate of OAT1 but not of OAT3, the decrease in CLr of belotecan in folic acid-induced acute renal failure could, therefore, mainly be attributed to the down-regulation of Oat1 and Bcrp, in addition to the decrease in glomerular filtration rate.

MiR-592 functions as a tumor suppressor in acute myeloid leukemia by targeting ROCK1 and predicts patients' prognosis.

OBJECTIVE: Dysregulation of miR-592 has been reported in several tumors. However, its role in acute myeloid leukemia (AML) remains unknown. The present study aimed at investigating the expression pattern and biological function of miR-592 in AML and to elucidate the mechanism involved. PATIENTS AND METHODS: qRT-PCR was used to analyze the expression of miR-592 in bone marrow and serum obtained from AML patients and healthy controls. The associations between serum miR-592 expression and clinical features and prognosis of AML patients were statistically analyzed. Then we detected the effect of miR-592 on proliferation, metastasis and apoptosis by CCK-8 assay, Transwell assays and flow cytometry, respectively. Dual-luciferase reporter assays were performed to validate the regulation of a putative target of miR-592. Rescue experiments were performed to confirm whether ROCK1 was a direct and functional target of miR-592 in AML. RESULTS: We found that the expression level of miR-592 was significantly lower in AML patients and AML cell lines. Low expression of serum miR-592 was associated with advanced French-American-British classification, cytogenetics and poor prognosis. Multivariate analysis confirmed that serum miR-592 expression was an independent prognostic factor for AML patients. Functionally, overexpression of miR-592 suppressed AML cell growth and metastasis, and promoted apoptosis. Further mechanistic investigation showed ROCK1 was a direct target gene of miR-592. Finally, ROCK1 overexpression rescued the effect of miR-592-mediated AML cell proliferation and metastasis. CONCLUSIONS: These findings suggest that miR-592 acted as a tumor suppressor by targeting ROCK1 and may serve as a potential biomarker in AML.

The curative effect of ALSS on 1-month mortality in AoCLF patients after 72 to 120 hours.

BACKGROUND: Hepatitis B virus (HBV)-related acute-on-chronic liver failure (AoCLF) is associated with a high mortality rate. An artificial liver support system (ALSS) creates a better environment for the self-regeneration of retained hepatocytes. AIM AND PATIENTS: We investigated the curative effect of ALSS on 1-month mortality at 72-120 h post-ALSS in 289 AoCLF patients. METHODS: Of the 289 patients, 117 who survived for at least 1 month post-ALSS comprised the survival group; the remaining cases who died within 1 month served as controls. The improvements in laboratory data and clinical syndromes at 72-120 h post-ALSS were compared with those at 24 h. RESULTS: Total bilirubin, international normalized ratio, and creatinine levels, and encephalopathy were significantly improved at 24 h post-ALSS in both the groups (p<0.05); however, these variables showed deterioration at 72-120 h; a rebound occurred in the nonsurvivors (p>0.05). The improvements in these variables in the nonsurvivors were considerably smaller than those in the survivors (p<0.05), particularly at 72-120 h. One-month mortality was more accurately predicted by the logistic regression model at 72-120 h than at 24 h. CONCLUSIONS: The prognosis of AoCLF patients was highly dependent on the improvement in encephalopathy, total bilirubin, international normalized ratio, and creatinine levels at 72-120 h post-ALSS. These variables are useful, therefore, as disease severity indexes to determine organ allocation priorities for liver transplant.

Stable bioavailability of cyclosporin A, regardless of food intake, from soft gelatin capsules containing a new self-nanoemulsifying formulation.

AIM: We recently succeeded in preparing soft gelatin capsules containing a new self-nanoemulsifying formulation consisting of cyclosporin A (CsA), triacetin, polyoxyl 40 hydrogenated castor oil, polysorbate 20, medium chain triglycerides and medium chain mono- and diglycerides. The soft capsules containing the new formulation exhibited a significantly improved physical stability in terms of the appearance of the gelatin capsule shells and the composition of the fill mass during long-term storage, compared to commercially available soft capsules containing CsA, in which ethanol was employed as a cosolvent of CsA. In the present study, the influence of a fat-rich meal on the bioavailability of CsA from the soft capsule containing the new formulation (test drug) was evaluated and the results compared to those obtained with a representative soft capsule of CsA. VOLUNTEERS AND METHODS: A randomized, open-label, 3-way crossover study was performed in the test capsules and reference soft capsules, in a fasted state or after a fat-rich breakfast. 18 healthy male volunteers received a single dose of the reference formulation (Neoral, Novartis AG, Basel, Switzerland) or test formulation (2 capsules each, 200 mg as CsA) with 240 ml of water with a 1-week washout period between the treatments, after a fat-rich (670 kcal, 45 g fat) breakfast (for the test drug, Treatment A; for the reference drug, Treatment B) or a 12-h fasting (for the test drug, Treatment C). Serial blood samples, collected over a 24-h period after the administration, were assayed for blood CsA concentrations using a specific monoclonal radioimmunoassay. RESULTS: The differences in bioavailability parameters (i.e., AUC(0-24h), AUC(0-infinity) and C(max)) between the treatments were within the range of 80-125% of the reference treatment. An analysis of variance (ANOVA) revealed no significant differences (p > 0.05) between subjects, formulations or periods. The 90% confidence intervals (CI) indicated that the differences between the treatments (Treatments A and B, Treatments A and C) were also within the criteria. CONCLUSION: These results indicate that the bioavailability of CsA from the test drug is equivalent to reference in the fed state, and is likely to be less influenced by a fat-rich meal. Therefore, the new formulation of CsA using triacetin appears to have an advantage over the commercial soft capsules of CsA using a volatile cosolvent such as ethanol.

Preparation and characterization of straight and zigzag AlN nanowires.

Hexagonal single-crystal AlN nanowires with straight or zigzag morphologies were successfully synthesized by the reaction of aluminum alloy in an ammonia/nitrogen atmosphere at 1100 degrees C. It is found that the crystal tropism of the nanowires is along [0001], whereas the growth directions of the zigzag nanowires shift between [2111] and [2111].

Perfil hematológico e bioquímico sérico de capivaras (Hydrochoerus hydrochaeris) de vida livre nos biomas Mata Atlântica e Caatinga / Hematological and serum biochemical profile of free-living capybaras (Hydrochoerus hydrochaeris) in Atlantic Forest and Caatinga biomes

Devido à ausência de estudos sobre capivaras na região Nordeste do Brasil, o objetivo deste estudo foi avaliar a sanidade desses roedores de vida livre em três áreas dos biomas Mata Atlântica (2) e Caatinga (1) do estado de Pernambuco, por meio da determinação de parâmetros da hematologia e bioquímica sérica. De novembro de 2016 a dezembro de 2017, foram capturados 21 animais, dos quais foram coletadas amostras de sangue para avaliação hematológica (eritrograma, leucograma e plaquetometria) e bioquímica sérica (atividade enzimática, perfil proteico, energético e mineral). A maioria dos parâmetros esteve dentro dos valores de normalidade para a espécie, embora alguns apresentassem diferenças estatisticamente significativas de acordo com a área de estudo (hemoglobina, hematócrito, VCM, CHCM, eosinófilos, fosfatase alcalina, proteína total, albumina, ácido úrico, creatinina, lactato, sódio e magnésio) e o sexo dos animais (ácido úrico). Os parâmetros obtidos são apresentados como referência e atestam a sanidade e o bom estado nutricional de populações de capivaras nos biomas Mata Atlântica e Caatinga da região Nordeste do Brasil. As informações aportadas neste estudo pioneiro na região Nordeste contribuem para aumentar o conhecimento sobre a ecofisiologia e a conservação in situ de capivaras.(AU)

Due to the lack of studies about capybaras in the northeast region of Brazil, the objective of this study was to evaluate the health status of free-ranging capybaras in three areas of Atlantic Forest (2) and Caatinga (1) biomes in Pernambuco state, through the determination of hematological and serum biochemical parameters. From November 2016 to December 2017, 21 animals were captured and blood samples were collected for the hematological (erythrogram, leukogram and platelet counts) and serum biochemistry (enzymatic activity, protein, energy and mineral profile) evaluation. Hematological and serum biochemical parameters were within the normal range for the species, but some presented statistically significant variations according to the study area (hemoglobin, hematocrit, MCV, MCHC, eosinophils count, alkaline phosphatase, total proteins, albumin, uric acid, creatinine, lactate, sodium and magnesium) and sex of the animals (uric acid). The parameters obtained are presented as reference and attest to the health and good nutritional status of populations of capybaras in the Atlantic Forest and Caatinga biomes of northeastern Brazil. The information provided in this pioneering study in the northeast region contributes to increased knowledge about the ecophysiology and in situ conservation of capybaras.(AU)

Recurrent arch obstruction after repair of isolated coarctation of the aorta in neonates and young infants: is low weight a risk factor?

BACKGROUND: Repair of aortic coarctation is often delayed in small infants because of the belief that such patients are at risk of recurrent arch obstruction and that growth will decrease this risk. To determine whether low weight was a risk factor for recurrent arch obstruction, we reviewed our experience with coarctation repair via left thoracotomy in infants less than 3 months of age. METHODS: From 1990 to 1999, 103 patients less than 3 months of age underwent repair of aortic coarctation through a left thoracotomy. Median age was 18 days (1-90 days), with 45 patients less than 2 weeks. Median weight was 3.3 kg (1.0-6.4 kg) and 14 patients were less than 2 kg. The method of repair was resection and end-to-end anastomosis in 64 patients, subclavian flap angioplasty in 34, and patch augmentation of the arch in 5. Demographic, echocardiographic, and operative variables were analyzed for correlation with recurrent arch obstruction. RESULTS: One early and 1 late death occurred, both in patients who had complications but no evidence of recoarctation. At median follow-up of 24 months, reinterventions for recurrent arch obstruction were performed in 15 patients. The median time to reintervention was 5 months and was less than 1 year in 12 patients. Kaplan-Meier freedom from arch reintervention was 88% at 1 year (95% confidence intervals: 82%-94%) and 82% at 5 years (95% confidence intervals: 72%-92%). Factors associated with shorter duration to arch reintervention by univariable Cox regression included younger age (continuous, P =.01; <2 weeks, P =.005), smaller transverse arch (absolute diameter, P <.001; indexed to weight, P =.03; indexed to ascending aortic diameter, P =.02), and smaller ascending aorta (absolute diameter, P =.02). Smaller absolute transverse arch diameter and younger age were the only independent predictors of shorter time to arch reintervention by multivariable Cox regression analysis. Weight and type of repair did not correlate with risk of recoarctation. CONCLUSIONS: Low weight is not a risk factor for recurrent obstruction after repair of coarctation of the aorta in infants less than 3 months of age. Rather, risk of recoarctation is more a function of the anatomy of the arch. Thus, it is not indicated to delay repair in low weight infants with the goal of achieving growth.

Contribution of the C-terminal dipeptide of transforming growth factor-alpha to its activity: biochemical and pharmacologic profiles.

We have used a matrix of biological (two distinct guinea-pig stomach contractile smooth muscle preparations) and biochemical (human placental membrane receptor binding and phosphorylation) assays to evaluate the activity profiles of epidermal growth factor-urogastrone (EGF-URO, mouse and human), transforming growth factor-alpha (TGF-alpha, human) and the TGF-alpha derivative lacking the C-terminal dipeptide, Leu49-Ala50, TGF-alpha-(1-48). In the longitudinal muscle (LM) bioassay, the relative potencies of the peptides were: EGF-URO greater than TGF-alpha greater than TGF-alpha-(1-48), with relative activity ratios (EC50S) of approximately 1:3:16. In the LM assay system, TGF-alpha-(1-48) was a partial agonist. In the circular muscle (CM) bioassay, the relative order of potencies was: TGF-alpha- greater than EGF-URO greater than TGF-alpha-(1-48), with EC50S of about 1:2:7. In the CM assay, all three peptides were full agonists, even though EGF-URO caused an intense desensitization of the tissue whereas TGF-alpha and TGF-alpha-(1-48) did not. The relative affinities of the peptides in the placenta membrane binding assay, EGF-URO greater than TGF-alpha greater than TGF-alpha-(1-48), were in good qualitative and quantitative agreement with the LM (but not the CM) bioassay, with relative KDS in the proportions of about 1:3:17. In the phosphorylation assay, using either the phosphorylated EGF-URO receptor or calpactin-II as an index of receptor kinase activation, the relative potencies of the peptides, EGF-URO greater than TGF-alpha greater than TGF-alpha-(1-48), were also qualitatively in accord with the relative potencies measured in the LM and ligand binding assays (but not in the CM preparation); however, quantitatively, the relative potency ratios (EC50S) observed in the phosphorylation assay (1:2:3) were somewhat out of keeping with the relative values observed in the LM and ligand binding assays. All three peptides were full agonists in the phosphorylation assay. Our data point to the importance of the C-terminal dipeptide, Leu49-Ala50 of TGF-alpha in terms of the binding affinity and intrinsic activity of this polypeptide; and our work provides further evidence for the distinct nature of the EGF-URO/TGF-alpha receptor system present in the CM bioassay preparation. The biological/biochemical activity profiles documented for the three polypeptides can serve as a basis for the further evaluation of other synthetic and naturally occurring members of the EGF-URO/TGF-alpha family of polypeptides.

Diacylglycerol lipase and the contractile action of epidermal growth factor-urogastrone: evidence for distinct signal pathways in a single strip of gastric smooth muscle.

In guinea pig gastric longitudinal muscle preparations, wherein epidermal growth factor-urogastrone (EGF-URO) causes contraction via the generation of arachidonate-derived prostaglandins, the specific diacylglycerol lipase (DG lipase) inhibitor, U57,908 (formerly designated RHC 80267) completely blocked EGF-URO and transforming growth factor-alpha (TGF-alpha)-mediated contraction, without affecting contractions caused by other agonists such as bradykinin, prostaglandin F2 alpha or arachidonic acid (AA). In contrast, the contractile actions of EGF-URO and TGF-alpha on the gastric circular muscle component, present in the same tissue strip as the longitudinal muscle preparation, were unaffected by concentrations of U57,908 that maximally inhibited contraction in the longitudinal muscle preparation. We conclude that in the longitudinal muscle preparation, EGF-URO acts not by the activation of phospholipase A2, but rather via the metabolism of diacylglycerol by DG lipase, thereby liberating arachidonic acid for the synthesis of contractile prostanoids. We also conclude that, even in the same tissue, the effects of EGF-URO on anatomically different components (longitudinal muscle versus circular muscle) can be mediated via two quite distinct signal transduction pathways.

Actions of thrombin and thrombin receptor peptide analogues in gastric and aortic smooth muscle: development of bioassays for structure-activity studies.

We have examined the biological activities of thrombin and the thrombin-receptor-related polypeptides, S42FLLRNPNDKYEPF55(TRP42-55), S42FLLRNPND50(TRP42-50), and A42FLLRNPND50(A42-TRP42-50) as well as an arginine-containing basic peptide beginning with the SF motif (SFRGHITR), in rat aortic (RA) rings and in a gastric guinea pig longitudinal (LM) smooth muscle preparation. In the RA preparation, thrombin, as well as the three receptor-related peptides caused a relaxation in tissue that was precontracted with noradrenaline; the basic peptide, SFRGHITR, was inactive either as an agonist or as an antagonist to TRP42-55. In the LM bioassay, which unlike the RA preparation did not persistently desensitize in response to thrombin, all three receptor-related peptides, like thrombin, caused a prompt phasic reproducible contraction. The basic peptide, SFRGHITR, was inactive. In the LM assay, TRP42-55, TRP42-50 and A42-TRP42-55 all caused comparable contractile responses. We conclude that the gastric LM smooth muscle possesses a thrombin receptor and provides a convenient and reliable assay for the activities of thrombin receptor-related peptides. Our data also demonstrated that neither the C-terminal hirudin-related pentapeptide nor the N-terminal serine hydroxyl group are required for the biological activity of the thrombin receptor-derived peptide previously described (TRP42-55). Based on our findings we suggest that only a small portion of the N-terminal sequence of TRP42-55 may be required for thrombin-like biological activity.

Inflammatory nodules of the lower legs: a clinical and histological analysis of 134 cases in Korea.

We analysed 134 Korean cases with inflammatory nodules of the lower legs on the basis of clinicopathological findings, responsiveness to various therapeutic agents, and clinical course. There were 53 cases of erythema induratum (EI), 18 of erythema nodosum (EN), 40 of EN-like lesions of Behcet's disease, 15 of other entities, including superficial migratory thrombophlebitis, cutaneous periarteritis nodosa, sarcoidosis, malignant lymphoma, Churg-Strauss syndrome, and parasitosis, and eight unclassified cases. The unclassified group was composed of a spectrum of diseases with clinicopathologic features ranging between those typical of EN and EI. The present study revealed that the profiles of diseases featuring inflammatory nodules of the lower legs in Korea differed from those found in other areas. These geographic and demographic differences should be kept in mind when managing a patient with inflammatory nodules of the lower legs.

[Improvement of amplification method for Cryptosporidium parvum oocysts from mice].

OBJECTIVE: To establish a simplified amplification method for obtaining a large number of purified Cryptosporidium parvum oocysts from infected C57BL/6N mice. METHODS: All mice in the experimental groups were immunosuppressed by given different concentrations of dexamethasone phosphate added in drinking water throughout the experiment. The recovery and purity of the oocysts obtained using different purification methods was compared. The infectivity of the oocysts obtained from the same origin but different animals and different purification methods in a bovine fallopian tube epithelial cell culture system was studied. RESULTS: 4.16 x 10(9) oocysts were obtained in 30 mice in the 3rd group with dexamethasone of 20 micrograms/ml in drinking water. No significant difference in the oocyst recovery, purity and infectivity was found between methods using saturated saline floatation and sucrose density gradient centrifugation. The infectivity of the oocysts obtained from the same origin but different animals was similar. CONCLUSION: A simplified amplification method for obtaining a large number of purified Cryptosporidium parvum oocysts from the infected mice was established.