Occurrence of neurotensin-immunoreactive cells in the digestive tract of lower vertebrates and deuterostomian invertebrates

Summary In the mucosal epithelium of the digestive tract of two marine teleost bony fish, one cartilaginous fish, one cyclostome, and in that of two of three representatives of deuterostomian invertebrates studied, endocrine cells of open type were found, exhibiting immunoreactivity with antisera against C-terminal sequences of mammalian neurotensin and of the structurally closely related amphibian neurohormonal peptide xenopsin.From these observations, and from those of previous studies, it is suggested that neurotensin cells do not occur in the digestive tract mucosa until at the evolutionary level of the more highly developed deuterostomian invertebrates. Three evolutionary stages seem to exist in the distribution pattern. The first stage, characterized by few, widely scattered cells, is found in the uro- and cephalochordates, the cyclostomes, the cartilaginous fish, and the stomachless bony fish. In the second stage, comprising the remaining submammalian classes, including more highly developed bony fish, the typical distribution pattern is that of numerous neurotensin immunoreactive cells in the antrum, pylorus, and duodenum. The final stage of neurotensin evolution is found in higher mammals and is characterized by a great density of neurotensin immunoreactive cells in the ileum.Dedicated to Prof. Dr. J. Staubesand on the occasion of this 60th birthday     

Distinct organ-specific up- and down-regulation of IGF-I and IGF-II mRNA in various organs of a GH-overexpressing transgenic Nile tilapia

Several lines of GH-overexpressing fish have been produced and characterized concerning organ integrity, growth, fertility and health but few and contradictory data are available on IGF-I that mediates most effects of GH. Furthermore, nothing is known on IGF-II. Thus, the expression of both IGFs in liver and various extrahepatic sites of adult transgenic (GH-overexpressing) tilapia and age-matched wild-type fish was determined by real-time PCR. Both IGF-I and IGF-II mRNA were found in all organs investigated and were increased in gills, kidney, intestine, heart, testes, skeletal muscle and brain of the transgenics (IGF-I: 1.4–4-fold; IGF-II: 1.7–4.2-fold). Except for liver, brain and testis the increase in IGF-I mRNA was higher than that in IGF-II mRNA. In pituitary, no significant change in IGF-I or IGF-II mRNA was detected. In spleen, however, IGF-I and IGF-II mRNA were both decreased in the transgenics, IGF-I mRNA even by the 19-fold. In agreement, in situ hybridisation revealed a largely reduced number of IGF-I mRNA-containing leukocytes and macrophages when compared to wild-type. These observations may contribute to better understanding the reported impaired health of GH-transgenic fish. Growth enhancement of the transgenics may be due to the increased expression of both IGF-I and IGF-II in extrahepatic sites. It is also reasonable that the markedly enhanced expression of liver IGF-II mRNA that may mimick an early developmental stage is a further reason for increased growth.     

Flow Injection Analysis for Simultaneous Quantification of Prolactin Concentration and Glycosylation Macroheterogeneity in Cell Culture Samples

A flow injection analysis (FIA) system is presented for a twostep immunoassay-based determination of the total humanprolactin (hPRL) concentration along with its degree ofglycosylation. Separate measurement of total hPRL and nonglysosylated human prolactin (nG-hPRL) were made using twoflow-through cartridges each containing immobilized antibodiesof different specificity. The antibodies are immobilized on thesurface of a carrier. Glycosylated hPRL (G-hPRL) and, thus, thedegree of glycosylation were calculated by the differencebetween the two specific determinations. Enhanced specificityfor the determination of nG-hPRL was obtained using unfavorablebinding conditions through incorporation of alkaline pH andchaotropic agents into the carrier/dispersion buffer. The assayfor total hPRL and nG-hPRL were each found to be linear withinthe relevant concentration range. The results of the two-stepFIA method were found to agree with those obtained by thestandard methods of ELISA and western blotting while offeringthe advantage of minimal analysis time (10 min) and eliminationof manual manipulations.     

Reaction of an insect cell line to ecdysterone

Ecdysterone applied above the 0.1 μg/ml level to Grace's Antheraea eucalypti Scott [Austrocaligula eucalypti (Scott)] cell line increased cell volume, cell motion, the breakdown of thymidine, and the uptake and breakdown of uridine and decreased the rate of growth of the cell population and the uptake of thymidine. Time-lapse photomicrography, spectrophotometry, and liquid scintillation spectrometry techniques were used in these analyses.     

Purification and properties of hamamelosekinase

Hamamelosekinase (ATP:hamamelose 2(1)-phosphotransferase) was purified from a crude extract of Kluyvera citrophila 627 (Enterobacteriaeceae) which has been grown on D-hamamelose. Ammonium-sulfate fractionation and twofold chromatography on DEAE-cellulose resulted in a 51-fold purification of the enzyme. Neither glucosekinase nor significant ATPase activity could be detected in the pure preparation. Besides D-hamamelose only D-hamamelitol was utilized as a substrate; however, the latter was phosphorylated at a very low rate. The molecular weight of the enzyme as estimated by gel chromatography is 21 000. The Km values for hamamelose and ATP were 3 mM nd 2.5 mM, respectively. The pH optimum was found at 7.5. In contrast to hexokinase, purified hamamelosekinase is very labile and could only be stabilized by addition of its substrate D-hamamelose. The most unusual property with respect to yeast hexokinase is a pronounced substrate inhibiton by hamamelose (> 5mM) and ATP (> 7mM), respectively, which could be interpreted as due to an economic utilization of the nutrient. Hamamelosekinase as well as glucosekinase are inducible by growing the microorganisms on the corresponding monosaccharides.     

Automated imunoanalysis systems for monitoring mammalian cell cultivation processes

Two different automated immunoanalysis systems are presented. Both are based on the principles of flow-injection analysis and were developed to provide reliable, rapid monitoring of relevant proteins in animal cell cultivation processes. One system uses a turbidimetric analysis, and the other employs a heterogeneous chemistry with immobilized immunocomponents. For both systems, the analysis time is in the range of a few minutes, and a complete analysis cycle, including triplicate analyses and various washing steps, is in the range of 20–30 minutes. Samples from cultivation processes can be analyzed directly without dilution. Quantitation of proteins such as rt-PA or monoclonal antibodies can be performed over an analyte concentration range of 1–1000 mg/L. Both systems were compared to conventional ELISA assays on microtiter plates. The turbidimetric analysis system also included a biosensor for simultaneous glucose determination.     

Metabolomics of urinary organic acids in respiratory chain deficiencies in children

Metabolomic analysis of the urinary organic acids from 39 selected children with defined respiratory chain deficiencies (RCDs) was performed using untargeted gas chromatography–mass spectrometry, revealing the presence of 255 endogenous and 46 exogenous substances. Variable reduction identified 92 variables from the endogenous substances, which could be analysed by univariate and multivariate statistical methods. Using these methods, no characteristic organic acid biomarker profile could be defined of practical value for diagnostic purposes for complex I (CI), complex III (CIII) and multiple complex (CM) deficiencies. The statistical procedures used did, however, disclose 24 metabolites that were practical highly (d > 0.75) and statistically (P < 0.05) significant for the combined and clinically closely related group of RCDs. Several of these metabolites occur in single enzyme inherited metabolic diseases, but most were not previously reported to be linked to the metabolic perturbations that are due to RCDs. Ultimately, we constructed a global metabolic profile of carbohydrate, amino acid and fatty acid catabolism, illuminating the diverse and complex biochemical consequences of these disorders. This metabolomics investigation disclosed a metabolite profile that has the potential to define an extended and characteristic biosignature for RCDs and the development of a non-invasive screening procedure for these disorders.     

Immunohistochemical evidence for the presence, localization and partial coexistence of insulin, insulin-like growth factor I and relaxin in the protochordate Ciona intestinalis

The occurrence and coexistence of peptides of the insulin-like growth factor (IGF)/insulin superfamily were investigated in the ovary and gastro-intestinal tract of the protochordate Ciona intestinalis. Antisera specific for mammalian IGF-I, insulin and relaxin were used in a double-immunofluorescence method on paraffin sections and with an immunogold technique on consecutive semi-thin sections. IGF-I and relaxin immunoreactions but no insulin immunoreactions occurred in the ovary and were confined to medium-sized and mature follicle cells. Two subpopulations of reacting follicular cells were present: those containing only IGF-I immunoreactivity (5%) and those containing IGF-I and relaxin immunoreactivities (95%). In the gastro-intestinal tract, IGF-I and insulin immunoreactions coexisted, whereas no relaxin immunoreactions were obtained. Gel chromatography and radioimmunoassay in Ciona ovary revealed IGF-I immunoreactivity in two peaks with apparent molecular masses of approximately 16 kDa and 3 kDa. The present results indicate that (1) the same IGF-I-related peptide probably occurs in gastro-intestinal tract and ovary, (2) three different members of the insulin/IGF family of peptides are probably present in protochordates, (3) different types of coexistence of these peptides seem to exist in protochordates, i.e. an IGF-I-related peptide and an insulin-related peptide in the digestive tract and, as shown previously, in central nervous system, and the IGF-I-related peptide and relaxin in the ovary, (4) an IGF-I-related peptide and relaxin may be involved in oocyte maturation in the protochordate ovary.