A cell-based reporter assay for the identification of protein kinase C activators and inhibitors

Transmission of extra cellular signals across biological membranes results in the generation of lipid metabolites which in turn influence specific cellular events such as cell growth or differentiation. Many of these lipid messengers can activate protein kinase C (PKC) isozymes of which one function is to perpetuate the extracellular signals to the nucleus by phosphorylating other targets proteins. We have engineered mammalian cell lines to identify and evaluate activators and inhibitors of PKC-dependent and independent signal transduction pathways. The A31 mouse fibroblast cell line, has been stably transfected with a construct containing a triplet repeat of the TPA response element (TRE) upstream of a thymidine kinase promoter fused to the human growth hormone (hGH) gene. A31 cells containing this reporter construct exhibit significant increases in hGH secretion following stimulation by phorbol esters or other mitogens. The levels of hGH secretion are modulated in this system using different pharmacological agents. We demonstrate that this assay can be used to identify specific and general inhibitors as well as activators of the signal transduction pathway mediated by PKC isozymes. (Mol Cell Biochem141: 129–134, 1994)     

BIOCHEMICAL HETEROZYGOSITY AND MORPHOLOGIC VARIATION IN A COLONY OF PAPIO HAMADRYAS HAMADRYAS BABOONS

This analysis examines the association between genetic heterozygosity and individual morphologic variation in a captive population of Papio hamadryas hamadryas consisting of 403 juveniles and adults. The population structure of the colony was artificially generated and maintained and is thus rigorously defined. Subpopulations delimited by age, sex, and degree of inbreeding are also explored. Heterozygosity, as enumerated from six simple Mendelian biochemical loci, is compared with the residual morphologic variation of each individual for each of 20 quantitative traits. Use of a sequential Bonferroni technique nullifies all significant correlations. Principal-components analysis reduces the morphometrics to a single or few significant axes in each population. The first axis of the total population contains 86.07% of the variation in the sample and the absolute values of the factor scores exhibit a significant positive correlation with heterozygosity at P < 0.05. Correcting for age- and sex-related variation in the total population with a linear model subsequently demonstrates that no significant correlation between heterozygosity and morphologic variation exists. No significant relationship is found in the inbred animals or subpopulations when age and sex are controlled. Previous studies have indicated that individuals proximal to the population mean for a specific polygenic trait exhibit a higher biochemical heterozygosity than individuals distant from the mean. The results presented here, which are based on more loci than many studies and a well-defined population, do not support this relationship. Substructuring of a population by age and sex can lead to spurious correlations with univariate or multivariate techniques. Comprehensive indices of genetic variation and rigorous statistical techniques should be used in future analyses. Studies that fail to recognize these design elements should be interpreted with caution.     

A 43 kDa protein inserted at the plasma membrane-cytoskeleton interface in rumen entodiniomorphid ciliates

Summary The P-43 ofEudiplodinium and homologous proteins in three other entodiniomorphid species, free-living ciliates, flagellates, and HeLa cells, were identified at the plasma membrane-cytoskeleton interface. Proteins cross-reacting with MAb B6 were also located at the ciliary inner surface of the plasma membrane. Due to the strong adhesion of the plasma membrane to the underlying cytoskeleton, classical extraction with detergents, urea, NaOH, and PTA, failed to separate the two components completely. However, the extraction properties of P-43, associated with its membrane-cytoskeleton interactive functions, suggest that this unglycosylated protein may present some analogies with proteins of the intermediate filaments. Their ubiquity and localization suggest that P-43 and MAb B6 crossreacting proteins may not be strictly epiplasmic but could be amphitropic proteins, strongly anchored to both the plasma membrane and the underlying microfilament framework, via protein-protein binding or by direct insertion in the lipid bilayer.Abbreviations BSA bovine serum albumin - Con A concanavalin A - EDTA ethylene diamine tetraacetic acid - EM electron microscopy - IF intermediate filaments - MAb monoclonal antibody - MET 2-mercaptoethanol - MW molecular weight - PAb polyclonal antibody - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride - PTA phosphotungstic acid - SDS sodiumdodecyl sulfate - TAME Na-p-tosyl-arginine methyl ester - TLCK Na-p-tosyl-lysine chloromethyl ketone     

The genetic characterisation of novel multi-addition doubled haploid lines derived from triticale x wheat hybrids

Two novel 46-chromosome doubled haploid lines, W66 and M17, derived from separate hexaploid triticale x bread wheat crosses, were characterised using cytological and biochemical markers. Both lines were shown to be relatively stable cytologically, over 11 and 8 generations of selfing, respectively. By examining mitotic and meiotic chromosomes, the stabilities of the two lines were shown to be similar with frequencies of 2n=46 in 74.2–85.5% of cells. However, over selfed generations, the rye chromosomes were shown to have lost some of their heterochromatin, which made it difficult to establish their continued presence using cytological techniques, such as C-banding alone. Cytological evidence from pairing studies, C-banding, and fluorescence in-situ hybridization, showed that both M17 and W66 are wheat/rye multi-addition lines with rye chromosome constitutions of 1R+6R, and 1R+4R, respectively. These conclusions were confirmed by isozyme and storage-protein analysis.     

Automatic Detection of Severely and Mildly Infected COVID-19 Patients with Supervised Machine Learning Models

ObjectivesWhen the prognosis of COVID-19 disease can be detected early, the intense-pressure and loss of workforce in health-services can be partially reduced. The primary-purpose of this article is to determine the feature-dataset consisting of the routine-blood-values (RBV) and demographic-data that affect the prognosis of COVID-19. Second, by applying the feature-dataset to the supervised machine-learning (ML) models, it is to identify severely and mildly infected COVID-19 patients at the time of admission.Material and methodsThe sample of this study consists of severely (n = 192) and mildly (n = 4010) infected-patients hospitalized with the diagnosis of COVID-19 between March-September, 2021. The RBV-data measured at the time of admission and age-gender characteristics of these patients were analyzed retrospectively. For the selection of the features, the minimum-redundancy-maximum-relevance (MRMR) method, principal-components-analysis and forward-multiple-logistics-regression analyzes were used. The features set were statistically compared between mild and severe infected-patients. Then, the performances of various supervised-ML-models were compared in identifying severely and mildly infected-patients using the feature set.ResultsIn this study, 28 RBV-parameters and age-variable were found as the feature-dataset. The effect of features on the prognosis of the disease has been clinically proven. The ML-models with the highest overall-accuracy in identifying patient-groups were found respectively, as follows: local-weighted-learning (LWL)-97.86%, K-star (K*)-96.31%, Naive-Bayes (NB)-95.36% and k-nearest-neighbor (KNN)-94.05%. Also, the most successful models with the highest area-under-the-receiver-operating-characteristic-curve (AUC) values in identifying patient groups were found respectively, as follows: LWL-0.95%, K*-0.91%, NB-0.85% and KNN-0.75%.ConclusionThe findings in this article have significant a motivation for the healthcare professionals to detect at admission severely and mildly infected COVID-19 patients.     

六种鼠苹果酸脱氢酶,血浆蛋白质和血红蛋白的变异分析

本文分析了家鼠属（Ｒａｔｔｕｓ）４种鼠和姬鼠属（Ａｐｏｄｅｍｕｓ）２种鼠的苹果酸脱氢酶、血浆中β１区和β２区蛋白质、血浆清蛋白、前清蛋白以及红细胞中血红蛋白的变异。结果表明,ＭＤＨ在进化上是比较保守的,其中ＭＤＨｍ和ＭＤＨｓ２带在各鼠间位于同一泳动线上,仅ＭＤＨｓ１带的泳动速度出现属间和种间的微小差异;血浆中β１区和β２区的蛋白质带、清蛋白带以及前清蛋白带各鼠间出现明显的变异,表现为黑线姬鼠和社鼠在β１区出现２种多态型区带,褐家鼠在β２区也有２种多态型带;各鼠间的血红蛋白变异比较明显,主要表现在区带数和主成分的泳动度显著不同,而且社鼠的Ｈｂ出现３种多态型,黄毛鼠出现２种多态型,其余各鼠的Ｈｂ皆为单态性。上述１６项生化特性按相似和相异进行配对比较,初步表明黄毛鼠与褐家鼠亲缘上比较相近,白腹巨鼠要比其他３种鼠亲缘上更接近于黑线姬鼠,而社鼠与白腹巨鼠之间进化分歧相对地较大。     

Tachykinin NK-1 receptor probed with constrained analogues of substance P

The action of rotameric probes introduced either in position 7 or 8 in the sequence of substance P (SP) was investigated, i.e. -tetrahydroisoquinoleic acid (Tic), -fluorenylglycine (Flg), -diphenylalanine (Dip), the diastereoisomers of -1-indanylglycine (Ing) and -benz[ƒ]indanylglycine (Bfi), the Z- and E-isomers of dehydrophenylalanine and dehydronaphthylalanine (Δ^ZPhe, Δ^EPhe, Δ^ZNal, Δ^ENal) and (Dmp). The aim of this study was the topographical characterization of the binding subsites of human NK-1 receptor expressed in CHO cells, especially the S₇ and S₈ subsites, corresponding to residues Phe⁷ and Phe⁸ of substance P. According to the binding potencies of these substituted-SP analogues, the S₇ binding subsite is smaller than the S₈ subsite: the S₇ subsite accepts only one aromatic nucleus, while the S₈ can accommodate three coplanar nuclei altogether. These findings are compatible with the idea that the S₈ binding subsite may reside in the extracellular loops of the hNK-1 receptor. NK-1 agonists bind to human NK-1 receptor and activate the production of both inositol phosphates and cyclic AMP. As already quoted for septide, [pGlu⁶, Pro⁹]SP(6–11), discrepancies are observed between affinity (K_i) and activity (EC₅₀) values for IPs production. While a weak correlation between K_i and EC₅₀ values for IPs production could be found (r = 0.70), an excellent correlation could be demonstrated between their affinities (K_i) and their potencies (EC₅₀) for cAMP production (r = 0.97). The high potency (EC₅₀) observed for ‘septide-like’ molecules on PI hydrolysis, compared to their affinity is not an artefact related to the high level of NK-1 receptors expressed on CHO cells since a good correlation was found between EC₅₀ values obtained for PI hydrolysis and those measured for spasmogenic activity in guinea pig ileum bioassay (r = 0.94).

According to the binding potencies of constrained analogues of phenylalanine, the S₇ binding subsite of human NK-1 receptor is small, whereas the S₈, which can accommodate three coplanar nuclei, might probably reside in the extracellular loop. The discrepancies observed between affinity (K_i) and activity (EC₅₀) values for IPs production are not an artefact of CHO cells since a good correlation was found between EC₅₀ for PI hydrolysis and those measured in guinea pig ileum bioassay.     

一类一级饱和反应系统的极限环

本文对一类一级饱和反应系统进行定性分析,得到（1）存在一个唯一环和存在两个（内不稳外稳）环的条件。     

上海四膜虫接合生殖期间皮层细胞骨架蛋白的研究

本文采用生化抽提,结合电泳及显微技术,对上海四膜虫（Ｔｅｔｒａｈｙｍｅｎａｓｈａｎｇｈａｉｅｎｓｉｓ）Ｓ１皮层细胞骨架（ｃｏｒｔｉｃａｌｃｙｔｏｓｋｅｌｅｔｏｎ）的蛋白组份,及其在接合生殖期间的变化进行了一系列的研究,初次探索了Ｓ１株上海四膜虫在接合生殖中皮细胞骨架的蛋白组份及含量,并分析了它们与间期,接合分开时期同类蛋白相互间的差异,发现在接合生殖时期７６－８８ｋＤ蛋白有突出的表现,而９０ｋＤ和