Palaeoceanographic implications of genetic variation in living North Atlantic Neogloboquadrina pachyderma.

The shells of the planktonic foraminifer Neogloboquadrina pachyderma have become a classical tool for reconstructing glacial-interglacial climate conditions in the North Atlantic Ocean. Palaeoceanographers utilize its left- and right-coiling variants, which exhibit a distinctive reciprocal temperature and water mass related shift in faunal abundance both at present and in late Quaternary sediments. Recently discovered cryptic genetic diversity in planktonic foraminifers now poses significant questions for these studies. Here we report genetic evidence demonstrating that the apparent 'single species' shell-based records of right-coiling N. pachyderma used in palaeoceanographic reconstructions contain an alternation in species as environmental factors change. This is reflected in a species-dependent incremental shift in right-coiling N. pachyderma shell calcite delta18O between the Last Glacial Maximum and full Holocene conditions. Guided by the percentage dextral coiling ratio, our findings enhance the use of delta18O records of right-coiling N. pachyderma for future study. They also highlight the need to genetically investigate other important morphospecies to refine their accuracy and reliability as palaeoceanographic proxies.

PALEOCEANOGRAPHY. A warm and poorly ventilated deep Arctic Mediterranean during the last glacial period.

Changes in the formation of dense water in the Arctic Ocean and Nordic Seas [the "Arctic Mediterranean" (AM)] probably contributed to the altered climate of the last glacial period. We examined past changes in AM circulation by reconstructing radiocarbon ventilation ages of the deep Nordic Seas over the past 30,000 years. Our results show that the glacial deep AM was extremely poorly ventilated (ventilation ages of up to 10,000 years). Subsequent episodic overflow of aged water into the mid-depth North Atlantic occurred during deglaciation. Proxy data also suggest that the deep glacial AM was ~2° to 3°C warmer than modern temperatures; deglacial mixing of the deep AM with the upper ocean thus potentially contributed to the melting of sea ice, icebergs, and terminal ice-sheet margins.

Neurothelin: amino acid sequence, cell surface dynamics and actin colocalization.

Neurothelin is a cell surface protein of chicken endothelial cells at the blood-brain barrier and also of pigment epithelial cells forming the blood-eye barrier. Peptide sequencing of affinity-purified neurothelin revealed that it is likely to be identical with the protein called HT7. It belongs to the immunoglobulin superfamily having two extracellular C2-type domains, a membrane spanning region and a cytoplasmic tail. During development neurothelin cell surface localization changed on pigment epithelial cells. In early embryogenesis neurothelin was found preferentially at lateral sites of neighboring epithelial cells, but after hatching, predominantly on basal cell surfaces and on apical microvilli of epithelial cells that contact retinal photoreceptors. Disruption of cell-cell contacts induced a rapid change of neurothelin distribution on the cell surface in vitro as could be shown by confocal laser microscopy. Disintegration of microfilaments by cytochalasin D hampered this specific cell surface rearrangement of neurothelin, whereas depolymerization of microtubules by demecolcine had no effect. In double-labeling experiments neurothelin and F-actin were colocalized. The data suggest that the polarized cell surface distribution of neurothelin is influenced intracellularly by F-actin and extracellularly by cell-cell interactions.

Photoreceptor differentiation analyzed by the novel monoclonal antibody 1G1.

In vertebrates, photoreceptor development has become a key model system to study mechanisms of cell differentiation. A still unresolved question is why photoreceptor maturation is retarded over an extended period of embryogenesis though photoreceptors are among the first cells born in the retina. We have generated the novel monoclonal antibody 1G1 which binds to outer photoreceptor segments of adult retinae of various species including chicken and rat. In the developing chicken retina presumptive photoreceptor cells were labeled by MAb 1G1 at embryonic day 10 (E10). Retinal cell cultures revealed that the corresponding antigen is expressed on the cell surface of rods and cones likewise. Metabolic labeling with bromodeoxyuridine in vitro indicated that 1G1 antigen expression is restricted to postmitotic cells. Comparison of single cell cultures starting from different developmental stages showed that antigen expression can be induced prematurely, if cells are released from their native tissue environment. In order to analyze potential regulatory cell interactions, retinal cells were cultured on cryosections of the eye (cryoculture). The percentage of 1G1+ cells which contacted the pigment epithelium, was significantly lower in comparison to cells located on retinal tissue. The data are consistent with the notion that the pigment epithelial cells which contact retinal photoreceptors in vivo, could be partially inhibitory and consequently delay photoreceptor differentiation.

Tissue-specific neuro-glia interactions determine neurite differentiation in ganglion cells.

Guided formation and extension of axons versus dendrites is considered crucial for structuring the nervous system. In the chick visual system, retinal ganglion cells (RGCs) extend their axons into the tectum opticum, but not into glial somata containing retina layers. We addressed the question whether the different glia of retina and tectum opticum differentially affect axon growth. Glial cells were purified from retina and tectum opticum by complement-mediated cytolysis of non-glial cells. RGCs were purified by enzymatic delayering from flat mounted retina. RGCs were seeded onto retinal versus tectal glia monolayers. Subsequent neuritic differentiation was analysed by immunofluorescence microscopy and scanning electron microscopy. Qualitative and quantitative evaluation revealed that retinal glia somata inhibited axons. Time-lapse video recording indicated that axonal inhibition was based on the collapse of lamellipodia- and filopodia-rich growth cones of axons. In contrast to retinal glia, tectal glia supported axonal extension. Notably, retinal glia were not inhibitory for neurons in general, because in control experiments axon extension of dorsal root ganglia was not hampered. Therefore, the axon inhibition by retinal glia was neuron type-specific. In summary, the data demonstrate that homotopic (retinal) glia somata inhibit axonal outgrowth of RGCs, whereas heterotopic (tectal) glia of the synaptic target area support RGC axon extension. The data underscore the pivotal role of glia in structuring the developing nervous system.

Effects of insulin-induced hypoglycemia on beta 2-adrenoceptor density and proliferative responses of human lymphocytes.

We studied the effects of insulin (0.1 IU/kg BW, iv)-induced hypoglycemia on lymphocyte beta 2-adrenoceptor function, lymphocyte subset distribution, and proliferative response to mitogen stimulation in 10 healthy volunteers. Thirty minutes after insulin injection plasma glucose levels were markedly decreased; concomitantly, plasma epinephrine levels had increased about 10-fold; plasma norepinephrine levels, however, increased only moderately. Lymphocyte beta 2-adrenoceptor density and the cAMP response to 10 mumol/L isoproterenol stimulation were elevated; lymphocyte Ts/c-cells had increased, whereas Th-cells had decreased, resulting in a decrease in the Th-/Ts/c-cell ratio from 1.7 to 1.0. These changes were accompanied by a significantly reduced lymphocyte proliferative response (measured as [3H]thymidine uptake) to mitogen stimulation. Two hours after insulin injection plasma catecholamines, lymphocyte beta 2-adrenoceptor function, lymphocyte subset distribution, and proliferative responses had returned to nearly preinsulin levels. We conclude that acute vigorous increases in endogenous epinephrine evoked by insulin-induced hypoglycemia are associated with increases in lymphocyte beta 2-adrenoceptor function, redistribution of lymphocyte subsets, and an (at least transiently) attenuated in vitro immune responsiveness.

Digital light microscopy: prerequisite for optimum contrast enhancement and increase of resolution.

Since the reproducible production of microscope objectives was enabled by the lens calculations of Ernst Abbe in 1872, various attempts have been made to further increase the resolution of light microscopes. Apart from the improvements on the optical side, especially the introduction of fluorescence methods, the use of digital cameras connected to computers have brought us close to the theoretical limits of optical resolution. Due to improved speed and memory capacity of modern computers mathematical methods can be applied to stored three-dimensional (3D) sequences of digital images which, in addition to just contrast and edge enhancement, may result in the case of real 3D deconvolution, even in the visualisation of structures beyond the theoretical limitations of light optical resolution.

Adrenaline and noradrenaline as possible chemical mediators in the pathogenesis of arteriosclerosis.

We studied the relationships between adrenaline and noradrenaline and factors associated with arteriosclerosis to determine whether catecholamines contribute to the atherogenetic process. We investigated the effects of adrenaline and noradrenaline on cultures of vessel wall cells from rats and analyzed plasma catecholamine levels in humans exposed to atherogenic risk factors, undergoing hemodialysis treatment or following myocardial infarction or stroke. I. Cultured endothelial and smooth muscle cells from vessel walls exhibited enhanced proliferation when exposed to adrenaline or noradrenaline. This indicates that catecholamines trigger the activation of vascular wall cells in vitro. Such activation, the unspecific mesenchymal reaction, is the predominant characteristic change in early atherogenesis. II. In individuals subjected to the atherogenic risk factors smoking, essential hypertension and mental stress, plasma adrenaline concentrations were statistically significantly elevated. Mental stress also caused significantly elevated plasma noradrenaline levels. Plasma noradrenaline concentrations were also elevated in smoking and hypertensive individuals when compared with certain controls, but the differences failed to be statistically significant. III. In dialysis patients, plasma adrenaline and noradrenaline concentrations showed a positive correlation with the activity of the sclerotic process; i.e., plasma catecholamine concentrations increased with the severity of the disease. IV. Patients with persisting arteriosclerotic vascular disease, i.e., patients who had had a myocardial infarction or stroke, had significantly elevated plasma adrenaline and/or noradrenaline levels as late as one year after the event. The results of our investigations suggest that adrenaline and noradrenaline may act as chemical mediators during atherogenesis in man, thus contributing to the development and subsequent complications of arteriosclerosis.

A double antibody sandwich microELISA for measuring human platelet factor 4.

With the aim of investigating patients with a higher risk of thrombosis we developed a method for determining platelet factor 4 (PF4) in human plasma. Using the double antibody sandwich ELISA technique we set up a test system that allows the determination of PF4 concentrations in plasma samples from 1 to 100 ng/ml. This method is more sensitive and as specific as commercially available RIA kits, but has the advantage of being cheaper and less time consuming. Furthermore the ELISA does not require radioactive materials. The complete reaction is carried out in microtiter wells, and an ELISA-reader connected to an Apple computer does all the calculations needed for quantitative measurements.

Axonal versus dendritic outgrowth is differentially affected by radial glia in discrete layers of the retina.

Formation of neural cell polarity defined by oriented extension of axons and dendrites is a crucial event during the development of the nervous system. Ganglion cells of the chicken retina extend axons exclusively into the inner retina, whereas their dendrites grow into the outer retina. To analyze guidance cues for specific neurite extension, novel in vitro systems were established. Ganglion cells were purified by enzymatically facilitated detachment of the ganglion cell layer. A newly developed retrograde labeling technique and the expression analysis of the cell type-specific 2A1 antigen were used to monitor ganglion cell purification. In highly purified ganglion cells explanted onto retinal cryosections (cryoculture), axon formation was induced when the cells were positioned on the inner retina. In contrast, on outer layers of the developing retina dendritic outgrowth was prevalent. Because radial glia have been demonstrated to be instructive in neuritogenesis, distinct glial cell compartments located in inner and outer retina, respectively, were isolated for functional assays. Glial end feet were purified by a physical detachment technique. Glial somata were purified by complement mediated cytolysis of all nonglial cells. When ganglion cells were cultured on different glial compartments, axon formation occurred on end feet but not on glial somata. In striking contrast, on glial somata dendrites were formed. The data support the notion that ganglion cell polarity is affected by the retinal microenvironment, which in turn is possibly influenced by radial glia, being themselves polarized.

[Functional differentiation of the vascular endothelium in high risk pregnancies]. / Funktionelle Differenzierung des vaskulären Endothels bei Hochrisikoschwangerschaften.

Endothelial cell functions regulating fetal hemodynamics and placental perfusion are modulated by metabolites of arachidonic acid, in particular derivatives of cyclooxygenase such as prostaglandins. We utilized an in vitro system to study the possible role of the vascular endothelium in the pathogenesis of chronic placental insufficiency. Cellular growth and prostaglandin metabolism were investigated in cultured umbilical vein endothelial cells from mothers exposed to risk factors such as smoking and diabetes mellitus during pregnancy. The study comprised cells from smoking (n = 18) and diabetic mothers (n = 5) compared to non-smoking, non-diabetic controls (n = 20). Endothelial cells from smoking and diabetic mothers grew less readily than did control cells. The synthesis of the prostaglandins PGI2 and PGE2, both potent vasodilators and platelet aggregation inhibitors, was significantly reduced in endothelial cells from smoking mothers and from mothers suffering from diabetes of various stages. Thus, reduced prostaglandin synthesis may be a cause of impaired placental perfusion in high risk pregnancies. Our data suggest that prostaglandins may play an important role in the etiology and pathogenesis of chronic placental insufficiency.

[N2O-supplemented intravenous anesthesia versus inhalation anesthesia. A comparative study of the sympathoadrenergic reaction and postoperative vigilance]. / N2O-supplementierte, intravenöse Anästhesie versus Inhalationsanästhesie. Vergleichende Untersuchung der sympathoadrenergen Reaktion und der postoperativen Vigilanz.

The aim of the present study was a comparison of the intraoperative sympathoadrenergic response and the postoperative vigilance of a propofol/alfentanil anaesthesia to a conventional isoflurane anaesthesia. 25 patients were admitted to the study undergoing septorhino surgery. Patients with continuous intravenous anaesthesia with propofol/alfentanil combined with nitrous oxide showed better haemodynamic conditions without an increase of blood pressure and catecholamines under laryngoscopy, intubation and surgical stimulation. In contrast to that the patients with isoflurane anesthesia showed a significant increase in haemodynamic parameters and capillary bloodflow. The measured plasma adrenalin levels showed wide intraindividual fluctuation but no significant difference between the groups. The suppression of plasma noradrenaline was more pronounced under intravenous anaesthesia. Recovery was significantly faster and vigilance significantly better in the patients undergoing intravenous anaesthesia. After 30 min patients with i.v. anaesthesia fulfilled all the conditions to be transferred to the regular ward; the other group needed more than one hour. It can be concluded that continuous i.v. anaesthesia with propofol/alfentanil is superior in suppressing the stress response to invasive stimuli and provides faster recovery and better postoperative analgesia.

[A single, rapid, selective and quantitative determination of adrenaline and noradrenaline in the plasma by a combination of solvent extraction, HPLC separation and electrochemical detection]. / Einfache, schnelle, selektive und quantitative Bestimmung von Adrenalin und Noradrenalin im Plasma durch Kombination von Flüssigextraktion, HPLC-Trennung und elektrochemischer Detektion.

A very simple and rapid solvent extraction system for the selective and quantitative isolation of epinephrine and norepinephrine from plasma is described. The extraction system makes use of the complex formation in alkaline medium between diphenylborate and the diol group of the catecholamines in combination with ion-pair formation. The extraction procedure, in conjunction with HPLC-separation and electrochemical detection, allows the quantitative determination of epinephrine and norepinephrine from plasma. This method is a specific as the commonly used adsorption on alumina, but has a much better recovery and even greater precision, especially for the quantitative determination of epinephrine.

A possible role of catecholamines in atherogenesis and subsequent complications of atherosclerosis.

Cultured smooth muscle cells (SMC) from rat aorta and endothelial cells (EC) from pig aorta were used to study the effect of the catecholamines epinephrine and norepinephrine on cell proliferation. Both stimulated growth of SMC and EC when added to the culture medium. Besides epinephrine and norepinephrine, dopamine and some of their metabolites also stimulated proliferation of cultured endothelial cells. Smooth muscle cells originating from rats being exposed to atherosclerotic risk factors, like diabetes, hypertension and balloon-injury, exhibited an increased susceptibility to these catecholamines compared to SMC from control animals. In comparison to normotensive control animals a 10-fold elevated plasma concentration of epinephrine was found in hypertensive rats. In man plasma epinephrine and norepinephrine concentration was determined in a healthy control group and in patients suffering from diabetes mellitus and coronary artery disease. Plasma epinephrine and norepinephrine levels were similar in patients suffering from diabetes mellitus compared to the control group. But in patients with coronary artery disease significantly higher plasma concentrations for epinephrine (p less than 0.001) and norepinephrine (p less than 0.01) were observed. These data support the hypothesis that catecholamines may play a role in the development and subsequent complications of atherosclerosis.

Epithelial folding in vitro: studies on the cellular mechanism underlying evagination of thyrocyte monolayers.

Epithelial monolayers in suspension culture fold in a way which closely resembles epithelial evagination. We have used freshly isolated segments of porcine thyroid follicles to study the mechanism underlying this evagination process. Epithelial folding was accompanied by dramatic changes in cell shape: the cells elongated and apical cell surfaces widened, whereas the basal cell portions were narrowed to about 20% of their original width. Apparently, enzymatic separation of thyroid epithelial cells from their underlying extracellular matrix resulted in an extension of the lateral cell-cell interactions on the expense of the basal cell surface area. Epithelial folding in vitro was Ca2+ dependent and reversibly blocked by cytochalasin D, by which the reorganization of the F-actin network was disturbed. This inhibitory effect was also observed by the action of cAMP analogues known to cause rounding of cells by their effect on cortical F-actin. Moreover, evagination in vitro was reversibly blocked at intracellular pH values of 5.8 and below. Under these conditions, protein phosphorylation was entirely inhibited. Inhibitors of protein kinases, specifically of myosin light chain kinase, were able to disrupt the evagination process, suggesting that protein phosphorylation, presumably of the myosin light chain, was essential for folding. We conclude that enzymatic separation of epithelial monolayers from their extracellular matrix initiated a cascade consisting of extended cell-cell interactions of the lateral plasma membranes and of reorganization of the apical actin-myosin network, finally resulting in profound changes in cell shape characteristic of epithelial evagination.

Norepinephrine and neuropeptide Y increase intracellular Ca2+ in cultured porcine aortic smooth muscle cells.

We used the fluorescent Ca2+ indicator Fura-2 in cultured porcine aortic smooth muscle cells (PASMC) to study effects of the sympathetic neurotransmitters norepinephrine (NE) and neuropeptide Y (NPY) on free intracellular Ca2+ (Cai). Both transmitters transiently increased intracellular Ca2+ in a concentration-dependent manner. Selective agonists and antagonists demonstrated that the NE-stimulated Cai increase is predominantly (if not exclusively) mediated by alpha 2-adrenoceptors, whereas the NPY response appears to be mediated by the peptide YY-insensitive Y3-like receptor subtype. Pretreatment of cells with pertussis toxin abolished NPY and alpha-adrenoceptor agonist-stimulated intracellular Ca2+ elevations (but not those stimulated by angiotensin II) suggesting involvement of a Gi-like G-protein. alpha 2-Adrenoceptor-stimulated Ca2+ increases resulted from mobilization from intracellular stores, whereas Y3-like NPY receptors mobilized Ca2+ from intracellular stores and also promoted Ca2+ influx.