Identification and characterization of insulin-like growth factor receptors on adult rat cardiac myocytes: linkage to inositol 1,4,5-trisphosphate formation.

Cultured cardiac myocytes from adult Sprague-Dawley rats express both insulin-like growth factor-I (IGF-I) receptors and insulin-like growth factor-II/mannose 6-phosphate (IGF-II/Man6P) receptors and respond to IGF-I with a dose-dependent accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)P2]. Specific binding of [125I]IGF-I to isolated membranes from cultured cardiac myocytes amounted to 1-1.2%. Binding of [125I]IGF-I was inhibited by unlabeled IGF-I at nanomolar concentrations and insulin at much higher concentrations. These data suggest that IGF-I binds to its own receptor on rat cardiac myocytes. Competitive binding studies using isolated membranes from cardiac myocytes and [125I]IGF-II showed 2-4% specific binding. Binding of [125I]IGF-II was inhibited by IGF-II and much less potently by IGF-I and insulin. Immunoglobulin G (IgG) 3637 (an IgG directed against the IGF-II/Man6P receptor) partially inhibited binding of [125I]IGF-II whereas nonimmune IgG did not. Affinity cross-linking studies with [125I]IGF-II and cardiac myocyte membranes and subsequent analysis of the ligand-receptor complex using SDS-PAGE and autoradiography showed a radiolabeled band of approximately 250 kilodalton (kDa). The formation of the [125I]IGF-II-receptor complex was inhibited by incubation with IGF-II and IgG 3637 but not by insulin or nonimmune IgG. Western blotting of protein extracts from cultured cardiac myocytes was performed using IgG 3637 and an immunoperoxidase technique for the visualization of the IGF-II/Man6P receptor protein. A specific band at 220 kDa under nonreducing conditions was detected on the blots, providing further evidence for the expression of the IGF-II/Man6P receptor by cardiac myocytes. The effect of IGFs on the accumulation of inositol phosphates was measured by HPLC analysis of perchloric acid extracts from myo-[3H]inositol-labeled cultured cardiac myocytes. IGF-I (50 ng/ml) stimulated the accumulation both of Ins(1,4,5)P3 and Ins(1,4)P2 after 30 sec by 43% and 63%. IGF-II (up to 500 ng/ml) had no significant effect on inositol phosphate accumulation under the same conditions. However, in the presence of millimolar concentrations of Man6P, IGF-II (500 ng/ml) also increased Ins(1,4,5)P3 accumulation by 59%. We conclude that cardiac myocytes from adult rats express IGF receptors and respond to IGFs with the accumulation of Ins(1,4,5)P3 and Ins(1,4)P2. This effect seems to be mediated by an IGF-I receptor-specific pathway

Identification and characterization of Ca2+-activated K+ channels in granulosa cells of the human ovary

<p>Abstract</p> <p>Background</p> <p>Granulosa cells (GCs) represent a major endocrine compartment of the ovary producing sex steroid hormones. Recently, we identified in human GCs a Ca²⁺-activated K⁺channel (K_Ca) of big conductance (BK_Ca), which is involved in steroidogenesis. This channel is activated by intraovarian signalling molecules (e.g. acetylcholine) via raised intracellular Ca²⁺levels. In this study, we aimed at characterizing 1. expression and functions of K_Cachannels (including BK_Cabeta-subunits), and 2. biophysical properties of BK_Cachannels.</p> <p>Methods</p> <p>GCs were obtained from in vitro-fertilization patients and cultured. Expression of mRNA was determined by standard RT-PCR and protein expression in human ovarian slices was detected by immunohistochemistry. Progesterone production was measured in cell culture supernatants using ELISAs. Single channels were recorded in the inside-out configuration of the patch-clamp technique.</p> <p>Results</p> <p>We identified two K_Catypes in human GCs, the intermediate- (IK) and the small-conductance K_Ca(SK). Their functionality was concluded from attenuation of human chorionic gonadotropin-stimulated progesterone production by K_Cablockers (TRAM-34, apamin). Functional IK channels were also demonstrated by electrophysiological recording of single K_Cachannels with distinctive features. Both, IK and BK_Cachannels were found to be simultaneously active in individual GCs. In agreement with functional data, we identified mRNAs encoding IK, SK1, SK2 and SK3 in human GCs and proteins of IK and SK2 in corresponding human ovarian cells. Molecular characterization of the BK_Cachannel revealed the presence of mRNAs encoding several BK_Cabeta-subunits (beta2, beta3, beta4) in human GCs. The multitude of beta-subunits detected might contribute to variations in Ca²⁺dependence of individual BK_Cachannels which we observed in electrophysiological recordings.</p> <p>Conclusion</p> <p>Functional and molecular studies indicate the presence of active IK and SK channels in human GCs. Considering the already described BK_Ca, they express all three K_Catypes known. We suggest that the plurality and co-expression of different K_Cachannels and BK_Cabeta-subunits might allow differentiated responses to Ca²⁺signals over a wide range caused by various intraovarian signalling molecules (e.g. acetylcholine, ATP, dopamine). The knowledge of ovarian K_Cachannel properties and functions should help to understand the link between endocrine and paracrine/autocrine control in the human ovary.</p

Dopamine receptor repertoire of human granulosa cells

<p>Abstract</p> <p>Background</p> <p>High levels of dopamine (DA) were described in human ovary and recently evidence for DA receptors in granulosa and luteal cells has been provided, as well. However, neither the full repertoire of ovarian receptors for DA, nor their specific role, is established. Human granulosa cells (GCs) derived from women undergoing in vitro fertilization (IVF) are an adequate model for endocrine cells of the follicle and the corpus luteum and were therefore employed in an attempt to decipher their DA receptor repertoire and functionality.</p> <p>Methods</p> <p>Cells were obtained from patients undergoing IVF and examined using cDNA-array, RT-PCR, Western blotting and immunocytochemistry. In addition, calcium measurements (with FLUO-4) were employed. Expression of two DA receptors was also examined by in-situ hybridization in rat ovary. Effects of DA on cell viability and cell volume were studied by using an ATP assay and an electronic cell counter system.</p> <p>Results</p> <p>We found members of the two DA receptor families (D₁- and D₂ -like) associated with different signaling pathways in human GCs, namely D₁ (as expected) and D₅ (both are Gs coupled and linked to cAMP increase) and D₂, D₄ (Gi/Gq coupled and linked to IP3/DAG). D₃ was not found. The presence of the trophic hormone hCG (10 IU/ml) in the culture medium for several days did not alter mRNA (semiquantitative RT-PCR) or protein levels (immunocytochemistry/Western blotting) of D_1,2,4,5 DA receptors. Expression of prototype receptors for the two families, D₁ and D₂, was furthermore shown in rat granulosa and luteal cells by in situ hybridization. Among the DA receptors found in human GCs, D₂ expression was marked both at mRNA and protein levels and it was therefore further studied. Results of additional RT-PCR and Western blots showed two splice variants (D₂L, D₂S). Irrespective of these variants, D₂ proved to be functional, as DA raised intracellular calcium levels. This calcium mobilizing effect of DA was observed in the absence of extracellular calcium and was abolished by a D₂ blocker (L-741,626). DA treatment (48 h) of human GCs resulted in slightly, but significantly enlarged, viable cells.</p> <p>Conclusion</p> <p>A previous study showed D₂ in human GCs, which are linked to cAMP, and the present study reveals the full spectrum of DA receptors present in these endocrine cells, which also includes D₂-like receptors, linked to calcium. Ovarian DA can act thus via D_1,2,4,5, which are co-expressed by endocrine cells of the follicle and the corpus luteum and are linked to different signaling pathways. This suggests a complex role of DA in the regulation of ovarian processes.</p

A rapid and robust method for the cryopreservation of human granulosa cells

Human primary granulosa cells (GCs) derived from women undergoing oocyte retrieval can be cultured and used as a cellular model for the study of human ovarian function. In vitro, they change rapidly, initially resembling cells of the preovulatory follicle and then cells of the corpus luteum. They are derived from individual patients, whose different medical history, lifestyle and age lead to heterogeneity. Thus, cells can rarely be ideally matched for cellular experiments or, if available, only in small quantities. We reasoned that cryopreservation of human GCs may be helpful to improve this situation. Previous studies indicated the feasibility of such an approach, but low survival of human GCs was reported, and effects on human GC functionality were only partially evaluated. We tested a slow freezing protocol (employing FCS and DMSO) for human GCs upon isolation from follicular fluid. We compared cryopreserved and subsequently thawed cells with fresh, non-cryopreserved cells from the same patients. About 80% of human GCs survived freezing/thawing. No differences were found in cell morphology, survival rate in culture, or transcript~levels of mitochondrial (COX4, OPA1, TOMM20), steroidogenic (CYP11A1, CYP19A1) or cell-cell contact genes (GJA1) between the two groups in cells cultured for 1-5~days. A proteomic analysis revealed no statistically significant change in the abundance of a total of 5962 proteins. The two groups produced comparable basal levels of progesterone and responded similarly to hCG with elevation of progesterone. Taken together, our results show this to be a rapid and readily available method for the cryopreservation of human GCs. We anticipate that it will allow future large-scale experiments and may thereby improve cellular studies with human ovarian cells

Entwicklung eines Bewertungsverfahrens zur erfolgreichen Anwendung einer Kalzitaufspülung für die interne Restaurierung eutropher Baggerseen in Baden-Württemberg

Zusammenfassung Laboruntersuchungen haben gezeigt, daß Kalzit grundsätzlich zur Phosphat-Fixierung und Phosphat-Retention in eutrophierten Seesedimenten geeignet ist. Freilanduntersuchungen im Kirchentellinsfurter Baggersee bei Tübingen (Epplesee) machten jedoch deutlich, daß die Barrierewirkung einer Kalzitschicht durch biologische und hydrodynamische Parameter drastisch herabgesetzt werden kann. Dies wurde in Ergebnissen von Laboruntersuchungen bestätigt, die zeigten, daß die Intaktheit der Schicht eine wesentliche Voraussetzung für die längerfristige Effizienz einer Kalzitbarriere zur Seenrestaurierung ist. Hinsichtlich der Erarbeitung objektiver Bewertungskriterien zur Nachhaltigkeit des Verfahrens wurden Laborversuche durchgeführt, um die Einflußparameter Bioturbation und Resuspension auf die Stabilität der Kalzitbarriere am Beispiel des Epplesees zu quantifizieren. Es konnte gezeigt werden, daß in einem Fall eine Kalzitbarriere mit einer Mächtigkeit von 1 cm innerhalb von drei bis vier Monaten durch die Bioturbation der Larven von Chironomus plumosus bei einer Larvendichte, die den natürlichen Verhältnissen im Baggersee entspricht, fast vollständig ins Sediment eingearbeitet wird. Auch eine höhere Kalzitauflage von 2,5 cm wurde im anderen Fall in einer etwas größeren Zeitspanne von sechs Monaten in das Oberflächensediment eingemischt. In flachen Seen besteht zusätzlich die Gefahr, daß die Kalzitschicht durch windinduzierte Wellen erodiert werden kann. Die kritischen Schubspannungen von drei potentiellen Barrierematerialien wie Socal® U1-R, Kalzitmehl und Quarzsand K12 betragen 0.16 N/m2, 0.31 N/m2 bzw. 0.32 N/m2, so daß die Auflagen z. B. im durchschnittlich 4 m tiefen Epplesee nach einer Modellrechnung erst ab kritischen Windgeschwindigkeiten von ca. 15 m/s, 31 m/s bzw. 32 m/s verdriftet werden könnten. Bei entsprechender Morphometrie (Oberfläche/Tiefen-Verhältnis) und Windexposition kann jedoch eine windinduzierte Resuspension einer Kalzitbarriere in Flachseen nicht ausgeschlossen werden. The Development of Instrumental Valuation Criteria for a Successful Application of Calcite as an Internal Restoration Method for Eutrophic Gravel-pit Lakes in Baden-Württemberg (BWC 20002) Summary Laboratory experiments showed that calcite is in principle suitable for phosphorus fixation and phosphorus retention in eutrophic lake sediments. However, field studies in the gravel-pit lake of Kirchentellinsfurt near Tübingen (Epplesee) clearly pointed to the fact that the efficiency of a calcite barrier can be dramatically reduced by biological and hydrodynamic influences. This was confirmed in laboratory tests, which showed that the completeness of the calcite layer is essential to achieve a successful lake restoration over a longer time range. In view of the elaboration of objective valuation criteria for the sustainability of the method, laboratory investigations were carried out to quantify the influence of bioturbation and resuspension on the stability of the calcite barrier in Lake Epplesee. The experiments showed that a calcite barrier of 1 cm thickness in one case was almost entirely burrowed within 3 to 4 months by the bioturbation of the larvae of Chironomus plumosus. The number of the larvae corresponded to the natural occurrence in the gravel-pit lake. Even a thicker calcite layer of 2.5 cm in the other case was nearly completely mixed into the deeper sediment in a slightly longer time period of about 6 months. Wind-generated waves can erode the calcite layer, especially in shallow lakes. Therefore, the critical shear stress of three potential barrier materials (Socal® U1-R, calcite powder and sand K12) was measured. In shallow lakes like Epplesee with a mean depth of 4 m, the calculated critical wind velocities by about 16 m/s, 31 m/s and 32 m/s exceed the critical shear stresses of the barrier materials (0.15 N/m2, 0.31 N/m2 and 0.32 N/m2, respectively) and can cause erosion of these layers. According to morphometry (ratio surface/depth) and wind exposition of shallow lakes wind-induced resuspension cannot be excluded

Frequency of secondary dyslipidemia in obese children

Ulrike Korsten-Reck1, Katrin Kromeyer-Hauschild2, Katrin Korsten1, Manfred W Baumstark1, Hans-H Dickhuth1, Aloys Berg11Department of Rehabilitative and Preventive Sports Medicine, University Medical Center, University of Freiburg, 79106 Freiburg, Germany; 2Institute of Human Genetics and Anthropology, Friedrich-Schiller-University Jena, 07740 Jena, GermanyObjective: This paper reports the frequency, type, and degree of dyslipidemia in obese children before therapeutic intervention. The relationships between lipid values and weight status, as well as lipid values and physical fitness, of these children were also investigated.Design and methods: The initial examination of the Freiburg Intervention Trial for Obese Children (FITOC) measured the values of triglycerides (TG), total cholesterol (C), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in 546 obese children aged 7&ndash;12 (body mass index [BMI]&nbsp;&gt; 97th percentile), and compared these values with those of the age- and sex-specific reference group in the Lipid Research Clinics Population Studies Data Book (LRC). Four groups were selected according to the following scheme: A, Normolipidemia; B, Hyper-LDL-cholesterolemia alone; C, Hypo-HDL-C + hypertriglyceridemia; D, Combined hyperlipidemia = Hyper-LDL-C + hypertriglyceridemia. Body mass index, BMI-SDS (corrected BMI), and physical performance in watt/kg body weight were measured.Results: A total of 45.8% of the overweight children showed an abnormal lipid profile. Ten percent of the children had high LDL-C levels (group B), while 15% had increased LDL-C and increased TG (group D) (higher prevalence in boys). In 18.9% we found increased TG, combined with decreased HDL-C values (group C).Conclusion: Obese children are at risk of dyslipoproteinemia and related diseases. Children with the highest BMI-SDS and lowest physical fitness have the lowest HDL-C values and increased TG, indicating a higher risk for the metabolic syndrome.Keywords: atherosclerotic risk, childhood, dyslipidemia, obesit

Prognostic impact of Claudin 18.2 in gastric and esophageal adenocarcinomas

Introduction: The tight junction molecule Claudin 18.2 is selectively expressed in healthy and malignant gastric epithelial tissue and is a promising therapy target for high Claudin 18.2 expressing adenocarcinomas of the esophagogastric junction and stomach (AEG/S). Methods: This study analyzed the prevalence, characteristics and prognostic impact of Claudin 18.2 expression in primary tumor, lymph node and distant metastasis in a large Caucasian AGE/S cohort with 414 patients. Results: Claudin 18.2 was highly expressed in 17.1% of primary tumors, 26.7% of lymph node metastasis and 16.7% of distant metastasis. High Claudin 18.2 expression in lymph node metastasis and primary tumors correlated significantly (p < 0.001). High expression of Claudin 18.2 was neither associated with histomorphogical subtype, or tumor state, nor with overall survival. Conclusion: In Caucasian AEG/S patients, 17.1% appeared to be eligible for an anti-Claudin 18.2 therapy. Claudin 18.2 expression itself has no impact on prognosis and is not related to any tumor subtype

Sediment budgeting of short‐term backfilling processes: The erosional collapse of a Carolingian canal construction

Sediment budgeting concepts serve as quantification tools to decipher the erosion and accumulation processes within a catchment and help to understand these relocation processes through time. While sediment budgets are widely used in geomorphological catchment-based studies, such quantification approaches are rarely applied in geoarchaeological studies. The case of Charlemagne's summit canal (also known as Fossa Carolina) and its erosional collapse provides an example for which we can use this geomorphological concept and understand the abandonment of the Carolingian construction site. The Fossa Carolina is one of the largest hydro-engineering projects in Medieval Europe. It is situated in Southern Franconia (48.9876°N, 10.9267°E; Bavaria, southern Germany) between the Altmühl and Swabian Rezat rivers. It should have bridged the Central European watershed and connected the Rhine–Main and Danube river systems. According to our dendrochronological analyses and historical sources, the excavation and construction of the Carolingian canal took place in AD 792 and 793. Contemporary written sources describe an intense backfill of excavated sediment in autumn AD 793. This short-term erosion event has been proposed as the principal reason for the collapse and abandonment of the hydro-engineering project. We use subsurface data (drillings, archaeological excavations, and direct-push sensing) and geospatial data (a LiDAR digital terrain model (DTM), a pre-modern DTM, and a 3D model of the Fossa Carolina] for the identification and sediment budgeting of the backfills. Dendrochronological findings and radiocarbon ages of macro remains within the backfills give clear evidence for the erosional collapse of the canal project during or directly after the construction period. Moreover, our quantification approach allows the detection of the major sedimentary collapse zone. The exceedance of the manpower tipping point may have caused the abandonment of the entire construction site. The spatial distribution of the dendrochronological results indicates a north–south direction of the early medieval construction progress