Regulation of calcium fluxes in rat pancreatic islets: Quinine mimics the dual effect of glucose on calcium movements

The effects of quinine and 9-aminoacridine, two blockers of potassium conductance in islet cells, on ⁴⁵Ca efflux and insulin release from perifused islets were investigated in order to elucidate the mechanisms by which glucose initially reduces ⁴⁵Ca efflux and later stimulates calcium inflow in islet cells. In the absence of glucose, 100 μM quinine stimulated ⁴⁵Ca net uptake, ⁴⁵Ca outflow rate and insulin release. Quinine also dramatically enhanced the cationic and the secretory response to intermediate concentrations of glucose, but had little effect on ⁴⁵Ca net uptake, ⁴⁵Ca fractional outflow rate and insulin release at a high glucose concentration (16.7 mM). The ability of quinine to stimulate ⁴⁵Ca efflux depended on the presence of extracellular calcium, suggesting that it reflects a stimulation of calcium entry in the islet cells. In the absence of extracellular calcium, quinine provoked a sustained decrease in ⁴⁵Ca efflux. Such an inhibitory effect was not additive to that of glucose, and was reduced at low extracellular Na⁺ concentration. At a low concentration (5 μM), quinine, although reducing ⁸⁶Rb efflux from the islets to the same extent as a non-insulinotropic glucose concentration (4.4 mM), failed to inhibit ⁴⁵Ca efflux. In the presence of extracellular calcium, 9-aminoacridine produced an important but transient increase in ⁴⁵Ca outflow rate and insulin release from islets perifused in the absence of glucose. In the absence of extracellular calcium, 9-aminoacridine, however, failed to reduced ⁴⁵Ca efflux from perifused islets. It is concluded that quinine, by reducing K⁺ conductance, reproduces the effect of glucose to activate voltage-sensitive calcium channels and to stimulate the entry of calcium into the B-cell. However, the glucose-induced inhibition of calcium outflow rate, which may also participate in the intracellular accumulation of calcium, does not appear to be mediated by changes in K⁺ conductance.     

Fasciola hepatica: IgG and IgA levels in the serum and bile of infected cattle

Bile and serum samples were collected from calves with an implanted cannula throughout a 20-week period of infection with Fasciola hepatica. Using indirect fluorescent antibody labelling and plastic-embedded sections of juvenile and adult flukes as antigens, estimates were made of the relative concentrations of IgG and IgA specific for fluke tegumental and gut antigens in the samples of serum and bile. In serum, antibodies against juvenile (t1) tegument and gut antigens reached peak concentrations 4–6 weeks postinfection and declined slowly thereafter as flukes became established in the bile ducts. IgG against adult tegument (t2) antigens appeared in the serum 6 weeks after infection, but no IgA against t2 was detected. In the bile, both IgG and IgA titres against t1 and gut antigens rose to peak values at 4–6 weeks after infection, but there was no activity against t2 antigen. The Ig levels in bile were considerably lower than in serum. Much more IgA relative to IgG occurred in bile as compared to serum (IgG/IgA ratio in serum was 16–32, in bile 1–2) suggesting a role for IgA in defence at mucosal surfaces. Comparison of the antibody profiles in bile and serum suggested that IgG in the bile was derived from circulating IgG whereas IgA may have been preferentially concentrated in the bile.     

Evidence for a triplicate set of glucosephosphate isomerase structural genes in hexaploid wheat

The glucosephosphate isomerase (GPI) zymogram phenotypes of 46 aneuploid derivatives of the cultivar Chinese Spring of hexaploid wheat were determined. Variation was observed among the strains in the relative level of expression of three GPI isozymes. The relationships observed between chromosomal constitution and zymogram phenotype support the hypothesis that the three GPI isozymes are dimers composed of protomers encoded by a minimum of three homoeologous structural genes located one each in the short arms of chromosomes 1A, 1B, and 1D. The relative levels of expression per dose of chromosome arm of the products of the three arms differ in a manner consistent with the presence of a two-fold greater quantity of the product of 1BS than of the product of 1AS and of 1DS, indicating that 1BS may contain duplicate GPI structural genes.     

Metabolic and temporal studies on pancreatic exocrine cells in culture

Summary An approach is described whereby cells with definitive markers are followed from their source through dissociation and fractionation, then during long-term maintenance in vitro. Such sequential studies should enable investigators to define factors regulating proliferation and function of specific cells since ambiguity concerning identity is readily avoided.Pancreatic cells of guinea pigs were isolated by enzymic dissociation, and exocrine cells were enriched by centrifugation with solutions of serum albumin. Resulting populations consisting of up to 95% exocrine cells were then incubated with gyration to produce aggregates, and these were seeded to standard culture plates for further study. Colonial aggregates of exocrine epithelia develop in culture and can be maintained for 20–30 days. The cells exhibit changes with time that are qualitatively similar to those known to occur during serial cultivation of diploid fibroblastlike cells from human and other species. The uptake of tritiated thymidine decreases with maintenance time. Autoradiographic examination indicates that this is due to a reduction in the number of epithelial cells incorporating the isotope. Cell diameters increase from an average of 21 m at day 0 to 44 m by day 26, and a marked increase in heterogeneity of this parameter is also evident. Cellular DNA and protein accumulate during the same interval. Incorporation of tritiated leucine during 24-h exposures increases until about the 10th day in vitro and remains relatively constant for at least 2 weeks thereafter.The data are consistent with the hypothesis that exocrine pancreatic cells like other diploid cells in culture, progress to terminal differentiation under the culture conditions employed. The role of physical, nutritional, and humoral evironmental factors on this process will be the subject of future reports.Supported in part by National Cancer Institute Contracts NO1-CP-43231 and NO1-CP-65751     

Phenice's visual sexing technique for the os pubis: a critique

Innominate bones from 362 California Indians were sexed with Phenice's three non-metrical features of the os pubis. The frequencies of marked, intermediate, and absent cases of these three morphological features were tabulated in males and females to see if unambiguous and reliable distinctions were consistently available. The results suggest that Phenice's technique offered extremely reliable sex evaluations in this material.     

Natural constituents from Cortex Mori Radicis as new pancreatic lipase inhibitors

Pancreatic lipase (PL), a key enzyme responsible for the hydrolysis of triacylglycerides in the gastrointestinal tract, has been identified as the therapeutic target for the regulation of lipid absorption. In the present study, six major constituents from a famous Chinese herbal medicine Cortex Mori Radicis (also named sangbaipi in Chinese), have been collected and their inhibitory effects on PL have been carefully investigated and well characterized by a fluorescence-based assay. The results clearly demonstrated that all tested bioactive constituents from Cortex Mori Radicis including sanggenone C (SC), sanggenone D (SD), kuwanon C (KC), kuwanon G (KG), morin and morusin displayed strong to moderate inhibitory effects towards PL with the IC₅₀ values ranging from 0.77 μM to 20.56 μM. Further investigations on inhibition kinetics demonstrated that SC, SD, KC and KG functioned as potent and mixed inhibitors against PL-mediated 4-MU oleate hydrolysis, with the K_i values less than 5.0 μM. Furthermore, molecular docking simulations demonstrated that SD (the most potent PL inhibitor from Cortex Mori Radicis) could create strong interaction with Ser152 (the key amino acid in the catalytic triad) of PL via hydrogen bonding. All these findings provided a new powerful evidence for explaining the hypolipidemic effect of Cortex Mori Radicis, also suggested that some abundant natural compounds in this herbal medicine could be served as lead compounds for the development of new PL inhibitors.     

A sequential test for assessing observed agreement between raters

Assessing the agreement between two or more raters is an important topic in medical practice. Existing techniques, which deal with categorical data, are based on contingency tables. This is often an obstacle in practice as we have to wait for a long time to collect the appropriate sample size of subjects to construct the contingency table. In this paper, we introduce a nonparametric sequential test for assessing agreement, which can be applied as data accrues, does not require a contingency table, facilitating a rapid assessment of the agreement. The proposed test is based on the cumulative sum of the number of disagreements between the two raters and a suitable statistic representing the waiting time until the cumulative sum exceeds a predefined threshold. We treat the cases of testing two raters' agreement with respect to one or more characteristics and using two or more classification categories, the case where the two raters extremely disagree, and finally the case of testing more than two raters' agreement. The numerical investigation shows that the proposed test has excellent performance. Compared to the existing methods, the proposed method appears to require significantly smaller sample size with equivalent power. Moreover, the proposed method is easily generalizable and brings the problem of assessing the agreement between two or more raters and one or more characteristics under a unified framework, thus providing an easy to use tool to medical practitioners.     

胰腺癌患者血清CEA、CA242、CA199水平变化及联合诊断的ROC曲线分析

目的:探讨胰腺癌患者血清癌胚抗原(CEA)、糖类抗原242(CA242)、糖类抗原199(CA199)水平变化,并分析上述指标对胰腺癌的联合诊断价值,为胰腺癌的临床诊断提供参考。方法:选择2014年2月至2018年2月我院收治的186例胰腺癌患者(胰腺癌组)、89例胰腺炎患者(胰腺炎组)作为研究对象,并取同期来我院检查的268例健康人作为对照组。比较三组受试者的血清CEA、CA242、CA199水平变化,对比分析血清CEA、CA242、CA199的单一以及联合诊断的准确度、特异度以及灵敏度,并绘制ROC曲线以分析上述指标的诊断价值。结果:三组受试者血清CEA、CA242、CA199水平差异具有统计学意义(P<0.05)。且胰腺炎组和胰腺癌组的血清CEA、CA242、CA199水平明显高于对照组,胰腺癌组患者的血清CEA、CA242、CA199水平明显高于胰腺炎组,差异均有统计学意义(P<0.05)。ROC曲线结果显示,CEA诊断价值最大,CA199诊断价值最小。CEA是胰腺癌单项肿瘤标志物中敏感度最高的,为85.48%;特异度最高的为CA242(96.72%);三项肿瘤标志物联合诊断的准确度增加至92.27%,敏感度增加至95.16%,特异度相比略有下降。结论:与单一肿瘤标记物诊断胰腺癌相比,CEA、CA242、CA199联合诊断的敏感度和准确度均明显升高,可以明显改善胰腺癌的漏诊率,提高患者的生存率,具有较好的临床应用价值。     

Next generation sequencing and functional pathway analysis to understand the mechanism of action of copper-tolfenamic acid against pancreatic cancer cells

Anti-cancer activity of tolfenamic acid (TA) in preclinical models for pancreatic cancer (PaCa) is well established. Since the dosage for anti-cancer actions of TA is rather high, we recently demonstrated that IC₅₀ values of Copper-TA are 30–80% less than TA in 12 cancer cell lines. This study elucidates the underlying mechanisms of Copper-TA in PaCa cells. Control and Copper-TA (IC₅₀) treated PaCa cells were processed by next-generation sequencing (NGS) to determine differentially expressed genes using HTG EdgeSeq Oncology Biomarker panel. Ingenuity Pathway Analysis (IPA®) was used to identify functional significance of altered genes. The conformational studies for assessing the expression of key regulators and genes were conducted by Western blot and qPCR. IPA® identified several networks, regulators, as well as molecular and cellular functions associated with cancer. The top 5 molecular and cellular functions affected by Cu-TA treatment were cell death and survival, cellular development, cell growth and proliferation, cell cycle and cellular movement. The expression of top upstream regulators was confirmed by Western blot analysis while qPCR results of selected genes demonstrated that Copper-TA is efficacious at lower doses than TA. Results suggest that Copper-TA alters genes/key regulators associated with cancer and potentially serve as an effective anti-cancer agent.