Dopamine modulates exocytosis independent of Ca(2+) entry in melanotropic cells

Dopamine is a known inhibitor of pituitary melanotropic cells. It reduces Ca(2+) influx by hyperpolarizing the cell membrane and by modulating high- and low-voltage-activated (HVA and LVA) Ca(2+) channels. As a result, dopamine reduces the hormonal output of the cell. However, it is unknown how dopamine affects each of the four different HVA Ca(2+) channel types individually. Moreover, it is unknown whether dopamine interacts with exocytosis independent of Ca(2+) channels. Here we show that dopamine differentially modulates the HVA Ca(2+) channels and that it affects the stimulus-secretion coupling through a direct effect on the exocytotic machinery. Sustained L- and P-type Ba(2+) currents are reduced in amplitude and inactivating N- and Q-type currents acquire different activation and inactivation kinetics in the presence of dopamine. The Q-type current shows slow activation, which is a hallmark for direct G-protein modulation. We used membrane capacitance measurements to monitor exocytosis. Surprisingly, we find that the amount of exocytosis per step depolarization is not diminished by dopamine despite the reduction in Ca(2+) current. To test whether dopamine affects the release machinery downstream of Ca(2+) entry, we stimulated exocytosis by dialyzing cells with buffered high-Ca(2+) solutions. Dopamine increased the amount and the rate of exocytosis. In the first 90 s, the rate of secretion was increased two- to threefold, but it was normalized again at 180 s, suggesting that predominantly vesicles that fuse early in the exocytotic phase are modulated by dopamine. Thus while Ca(2+) channels are inhibited by dopamine, the exocytotic machinery downstream of Ca(2+) influx is sensitized. As a result, release is more effectively stimulated by Ca(2+) influx during dopamine inhibition.     

At Least Four Percent of the Salmonella typhimurium Genome Is Required for Fatal Infection of Mice

Salmonella typhimurium infection of mice is an established model system for studying typhoid fever in humans. Using this model, we identified S. typhimurium genes which are absolutely required to cause fatal murine infection by testing independently derived transposon insertion mutants for loss of virulence in vivo. Of the 330 mutants tested intraperitoneally and the 197 mutants tested intragastrically, 12 mutants with 50% lethal doses greater than 1,000 times that of the parental strain were identified. These attenuated mutants were characterized by in vitro assays which correlate with known virulence functions. In addition, the corresponding transposon insertions were mapped within the S. typhimurium genome and the nucleotide sequence of the transposon-flanking DNA was obtained. Salmonella spp. and related bacteria were probed with flanking DNA for the presence of these genes. All 12 attenuated mutants had insertions in known genes, although the attenuating effects of only two of these were previously described. Furthermore, the proportion of attenuated mutants obtained in this study suggests that mutations in about 4% of the Salmonella genome lead to 1,000-fold or greater attenuation in the mouse typhoid model of infection. Most of these genes appear to be required during the early stages of a natural infection.     

A gene signature of inhibitory MHC receptors identifies a BDCA3(+) subset of IL-10-induced dendritic cells with reduced allostimulatory capacity in vitro

Dendritic cells (DC) are crucial gatekeepers in regulating immunity. Whereas mature immunostimulatory myeloid DC (DC(ims)) potently promote immune responses, IL-10-induced myeloid DC (DC-IL-10) counteract T cell activation. To elucidate the molecular repertoire by which DC-IL-10 secure reduced T cell activation, comparative gene expression profiling was done using Affymetrix U133A microarrays. Among the genes overexpressed in DC-IL-10, eight immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing inhibitory molecules (ILT2, ILT3, ILT4, ILT5, DCIR, PILRA, FcgammaRIIB, SLAM) were found. Phenotypic analysis of DC-IL-10 defined an ILT(high) DC subset further characterized by expression of CD14, TLR2, DC-SIGN, and CD123 and the lack of lymphocyte, monocyte/macrophage, and plasmacytoid DC markers such as CD3, CD8, and CD68. A unique feature of the ILT(high) DC subset was expression of the novel DC marker BDCA3, which was not detected on monocytes, immature DC, DC(ims), or ILT(low) DC-IL-10. While the allogeneic T cell proliferation induced by the entire DC-IL-10 population was approximately 30% of that stimulated by DC(ims), allogeneic MLR responses driven by the ILT(high) DC subset were reduced to 8% of the allostimulatory capacity of DC(ims), although secretion of the inhibitory cytokine IL-10 and other Th1/Th2-associated cytokines was similar in these cells.     

Hepatocellular Hyperplasia, Plasmacytoma Formation, and Extramedullary Hematopoiesis in Interleukin (IL)-6/Soluble IL-6 Receptor Double-Transgenic Mice

Cytokines interact not only with membrane anchored receptors, but also with specific soluble receptors which circulate in the bloodstream. In general, soluble cytokine receptors such as soluble tumor necrosis factor receptor, soluble interleukin 1 receptor, and soluble interleukin 4 receptor compete with their membrane-bound counterparts for the ligands and therefore act as antagonists. In contrast, soluble receptors for cytokines of the interleukin-6 (IL-6) family complex with their ligands act agonistically. Interestingly, the complex of IL-6 and the soluble interleukin 6 receptor (sIL-6R) activates target cells that do not express the membrane-bound IL-6R and therefore cannot respond to IL-6. To identify cellular responses that are due to IL-6/sIL-6R but not to IL-6 alone, IL-6/sIL-6R double-transgenic mice were generated and compared with IL-6 single-transgenic mice. IL-6/sIL-6R transgenic mice develop a severe phenotype showing 1) marked hepatocellular hyperplasia frequently surrounded by peliosis and necrosis, 2) significant acceleration and aggravation of plasmacytoma formation, and 3) excessive activation of extramedullary hematopoiesis in spleen and liver followed by a subsequent increase of all cellular components in the peripheral blood. These in vivo data suggest that the sIL-6R recruits primarily unresponsive cell populations such as hematopoietic progenitor cells and hepatocytes to IL-6-induced proliferation, but also enhances the known mitogenic effect of IL-6 on plasma cells and thereby contributes to plasmacytoma formation.     

The development of goal-directed reaching in infants: hand trajectory formation and joint torque control

Nine young infants were followed longitudinally from 4 to 15 months of age. We recorded early spontaneous movements and reaching movements to a stationary target. Time-position data of the hand (endpoint), shoulder, and elbow were collected using an optoelectronic measurement system (ELITE). We analyzed the endpoint kinematics and the intersegmental dynamics of the shoulder and elbow joint to investigate how changes in proximal torque control determined the development of hand trajectory formation. Two developmental phases of hand trajectory formation were identified: a first phase of rapid improvements between 16 and 24 weeks of age, the time of reaching onset for all infants. During that time period the number of movement units per reach and movement time decreased dramatically. In a second phase (28–64 weeks), a period of fine-tuning of the sensorimotor system, we saw slower, more gradual changes in the endpoint kinematics. The analysis of the underlying intersegmental joint torques revealed the following results: first, the range of muscular and motiondependent torques (relative to body weight) did not change significantly with age. That is, early reaching was not confined by limitations in producing task-adequate levels of muscular torque. Second, improvements in the endpoint kinematics were not accomplished by minimizing amplitude of muscle and reactive torques. Third, the relative timing of muscular and motion-dependent torque peaks showed a systematic development toward an adult timing profile with increasing age. In conclusion, the development toward invariant characteristics of the hand trajectory is mirrored by concurrent changes in the control of joint forces. The acquisition of stable patterns of intersegmental coordination is not achieved by simply regulating force amplitude, but more so by modulating the correct timing of joint force production and by the system's use of reactive forces. Our findings support the view that development of reaching is a process of unsupervised learning with no external or innate teacher prescribing the desired kinematics or kinetics of the movement.     

Morphometric observations on the rat heart after high-dose treatment with cortisol

Summary Male Wistar rats were treated with high cortisol doses for 1 week. The dose administered daily was 15 mg per animal in group 1 (7 animals) and 30 mg in group 2 (7 animals). 7 rats served as control group. After cortisol treatment the body weights decreased due to skeletal muscle catabolism and the heart weights increased. Morphometric analysis of the left ventricular posterior papillary muscles gave evidence that the increased heart weights resulted from an increased number of mitochondria and an increased volume of the cytoplasm, whereas the myofibrillar mass was not affected. The surface area of inner mitochondrial membranes (+cristae mitochondriales) per myofibrillar unit volume increased from 15.7 ²/³ to 21.3 ²/³ in group 1 and 21.4 ²/³ in group 2. Ultrastructural changes indicating myocardial cell damage were absent. Similar quantitative results have been reported to occur in the early phase of cardiac overload. For elucidating the hemodynamic effects of glucocorticoid a second experiment was performed: 7 Wistar rats were treated with cortisol in the same way as group 1, 7 others of the same body weight served as control. The systolic arterial pressure was significantly elevated in the cortisol group. Though myocardial tissue is known to be able to accumulate large quantities of glucocorticoids our results indicate that the application of high cortisol doses for a short time does not produce myocardial cell damage and does not suppress the myocardial adaption to the glucocorticoid-induced hypertension, i.e. hypertrophy. On the contrary, it seems to be possible that the adaption process is itself facilitated or accelerated by the presence of high cortisol concentrations in the heart. This thesis is supported by the considerably higher relative heart weights in the cortisol groups and is in agreement with observations reported by other authors.Dedicated to Professor Dr. W. Doerr on the occasion of his 65th birthdayThe results have been partially reported in 1977 (cf. G. Mall and H. Reinhard, Verh. Dtsch. Ges. Path. 61, 445)This investigation was supported by the Sonderforschungsbereich 90 of the Deutsche Forschungsgemeinschaft.     

Decreased human semen quality and organochlorine compounds in blood

BACKGROUND: Various studies have been performed in which potential effects of xenoestrogens on fertility or sperm parameters were investigated by comparing groups of subjects exposed to different levels of these chemicals. METHODS: In our study we used an alternative approach, as we selected one group of men with very poor semen quality and another group with normal semen quality and determined the blood organochlorine contents in order to determine whether a difference in these levels could be established. Organochlorine compounds, including polychlorinated biphenyls (PCB) and PCB metabolites, were detected using gas chromatography. The concentrations were compared between both groups, and related to semen parameters. RESULTS: A comparison of both groups did not reveal significant differences in organochlorine levels. Linear relationships were found when PCB and metabolite concentrations were related to the age of the volunteers. Focusing on the subgroup of men with normal semen quality showed that sperm count and sperm progressive motility were inversely related to the concentrations of PCB metabolites within this group. CONCLUSIONS: The finding of a significantly decreased sperm count in relation to an elevated PCB metabolite level within the subgroup of men with normal semen quality is important. This is the first time that a correlation between exposure to environmental pollutants with endocrine-disrupting capacity and human sperm quality has been observed.     

Towards unlimited colors for fluorescence in-situ hybridization (FISH)

We describe a FISH protocol that allows rehybridization of complex DNA probes up to four times to the same specimen. This strategy, which we termed ReFISH, opens a wide range of new applications to conventional band pass filter epifluorescence microscopy. These include M-FISH karyotyping and cross-species color banding that emulate multiplex probe sets labeled with up to 12 fluorochromes in sequential hybridizations to the same specimen. We designed a human 24-color karyotyping probe set in combination with a 29-color cross-species color banding probe set using gibbon painting probes. Applying the ReFISH principle, 53 painting probes on individual metaphases were discriminated. This allowed simultaneous screening for inter- and intrachromosomal rearrangements on normal human diploid cells, a HeLa derived cell line, and highly rearranged gibbon chromosomes. Furthermore, the present ReFISH experiments successfully combine 24-color FISH with laser scanning confocal microscopy to study the 3D organization of all 46 human chromosome territories in individual interphase cell nuclei.     

Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis

Formation of lymphatic metastasis is the initial step of generalized spreading of tumor cells and predicts poor clinical prognosis. Lymphatic vessels generally arise within the peritumoral stroma, although the lymphangiopoietic vascular endothelial growth factors (VEGF)-C and -D are produced by tumor cells. In a carefully selected collection of human cervical cancers (stage pT1b1) we demonstrate by quantitative immunohistochemistry and in situ hybridization that density of lymphatic microvessels is significantly increased in peritumoral stroma, and that a subset of stromal cells express large amounts of VEGF-C and VEGF-D. The density of cells producing these vascular growth factors correlates with peritumoral inflammatory stroma reaction, lymphatic microvessel density, and indirectly with peritumoral carcinomatous lymphangiosis and frequency of lymph node metastasis. The VEGF-C- and VEGF-D-producing stroma cells were identified in situ as a subset of activated tumor-associated macrophages (TAMs) by expression of a panel of macrophage-specific markers, including CD68, CD23, and CD14. These TAMs also expressed the VEGF-C- and VEGF-D-specific tyrosine kinase receptor VEGFR-3. As TAMs are derived from monocytes in the circulation, a search in peripheral blood for candidate precursors of VEGFR-3-expressing TAMs revealed a subfraction of CD14-positive, VEGFR-3-expressing monocytes, that, however, failed to express VEGF-C and VEGF-D. Only after in vitro incubation with tumor necrosis factor-alpha, lipopolysaccharide, or VEGF-D did these monocytes start to synthesize VEGF-C de novo. In conclusion VEGF-C-expressing TAMs play a novel role in peritumoral lymphangiogenesis and subsequent dissemination in human cancer.