Solution structure of neuronal bungarotoxin determined by two-dimensional NMR spectroscopy: sequence-specific assignments, secondary structure, and dimer formation

The solution structure of neuronal bungarotoxin (nBgt) has been studied by using two-dimensional 1H NMR spectroscopy. Sequence-specific assignments for over 95% of the backbone resonances and 85% of the side-chain resonances have been made by using a series of two-dimensional spectra at four temperatures. From these assignments over 75% of the NOESY spectrum has been assigned, which has in turn provided 582 distance constraints. Twenty-seven coupling constants (NH-alpha CH) were determined from the COSY spectra, which have provided dihedral angle constraints. In addition, hydrogen exchange experiments have suggested the probable position of hydrogen bonds. The NOE constraints, dihedral angle constraints, and the rates of amide proton exchange suggest that a triple-stranded antiparallel beta sheet is the major component of secondary structure, which includes 25% of the amino acid residues. A number of NOE peaks were observed that were inconsistent with the antiparallel beta-sheet structure. Because we have confirmed by sedimentation equilibrium that nBgt exists as a dimer, we have reinterpreted these NOE constraints as intermolecular interactions. These constraints suggest that the dimer consists of a six-stranded antiparallel beta sheet (three from each monomer), with residues 55-59 forming the dimer interface.     

Abdominal muscle use during quiet breathing and hyperpnea in uninformed subjects

Although there is electromyographic evidence for abdominal muscle activity during quiet breathing in standing subjects, several studies have shown, or assumed, that subjects normally breathe on their relaxation characteristics. This latter observation would by itself suggest that abdominal muscles do not contract during quiet breathing. To test this assumption we observed abdominal and rib cage displacements with magnetometers in 17 uninformed subjects. During quiet breathing most subjects showed evidence of tonic or phasic abdominal muscle contraction while standing and sitting but not supine. Subjects studied during hyperpnea immediately following exercise-showed evidence of greater abdominal muscle contraction than at rest. We conclude that most subjects standing at rest normally contract their abdominal muscles.     

Analysis of developmentally homogeneous neural crest cell populations in vitro: I. Formation,morphology and differentiative behavior

When early embryonic quail neural tubes are dissected free from surrounding tissues and placed in culture, small stellate neural crest cells usually migrate from the explant onto the substratum. This outgrowth has been reported to consist of a mixture of cells, some of which undergo melanogenesis, while the rest remain unpigmented. We have, in contrast to earlier observations, obtained a spatial separation of the two phenotypes. In these cultures the primary outgrowth of migrating cells remained almost free of pigment-forming cells, whereas small spherical clusters containing several hundred pigment-forming cells appeared on the explanted neural tubes. Whether the clusters remained with the tube explants or were subcultured, all cluster cells differentiated into melanocytes. Prior to melanogenesis, the appearance of the cultured cells from a cluster was indistinguishable from the cells in the outgrowth. The clusters provide a source of neural crest cells, that (1) can be easily obtained in comparatively large numbers, (2) is not contaminated with any other cell type, (3) can be isolated before the onset of differentiation, and (4) is developmentally homogeneous. Thus, the cluster population is well suited for many types of experiments, such as the identification of specific environmental factors that might control neural crest cell differentiation.     

Are the immune responses different in middle-aged and young mice following bone fracture, tissue trauma and hemorrhage?

Although immune responses following soft-tissue trauma-hemorrhage are markedly different in young (6-8 weeks) and aged (18-20 months) mice, it remains unknown if there are any differences in immune responses in middle-aged and young mice following bone fracture, soft-tissue trauma-hemorrhage (Fx-TH). To study this, young (6-8 weeks) and middle-aged (approximately 12 months) C3H/HeN male mice were subjected to sham operation or Fx-TH followed by resuscitation with Ringer's lactate. The mice were sacrificed 2 h thereafter and splenocytes, bone marrow cells (BM) and Kupffer cells (KC) were harvested, purified and stimulated with ConA (for splenocytes) or LPS (for BM and KC) in vitro. Splenocyte release of Th1 (IL-2 and IFN-gamma) cytokines was decreased and Th2 (IL-4 and IL-10) cytokine release was increased following Fx-TH in both young and middle-aged mice. However, the decrease in IL-2 and increase in IL-10 were significantly more in middle-aged mice compared to young mice (p < 0.05). Furthermore, splenocyte proliferation was decreased more in middle-aged mice compared to young mice following Fx-TH (p < 0.05). Additionally, TNF-alpha production was more in BM from middle-aged compared to BM from young mice after Fx-TH (p < 0.05). The production of IL-6 and IL-10 was also significantly higher in KC from middle-aged mice compared to young ones following Fx-TH. These results suggest that at middle age, the immune responses to Fx-TH are significantly different from those observed in young mice in different compartments of the body. Although the mechanism of the difference in various compartments in middle-aged vs. young mice following Fx-TH remains unknown, the decreased IL-2 production along with other altered T cell and macrophage functions may contribute to an increased susceptibility to sepsis in middle-aged vs. young individuals.     

Decreased 5alpha-dihydrotestosterone catabolism suppresses T lymphocyte functions in males after trauma-hemorrhage

Trauma-hemorrhage producesprofound immunosuppression in males but not in proestrus females.Prior castration or flutamide treatment of males followingtrauma-hemorrhage prevents immunosuppression, implicating5-dihydrotestosterone for the immunosuppressive effects. 5-Dihydrotestosterone, a high-affinity androgen receptor-binding steroid, is synthesized in tissues as needed and seldom accumulates. The presence of steroidogenic enzymes in T lymphocytes suggests bothsynthesis and catabolism of 5-dihydrotestosterone. We hypothesized, therefore, that the basis for high 5-dihydrotestosterone activity inT lymphocytes of males following trauma-hemorrhage is due to decreasedcatabolism. Accordingly, catabolism of 5-dihydrotestosterone wasassessed in splenic T lymphocytes by examining the activity andexpression of enzymes involved. Analysis showed increased synthesis anddecreased catabolism of 5-dihydrotestosterone in intact male Tlymphocytes following trauma-hemorrhage. In contrast, reduced5-reductase activity and increased expression of17-hydroxysteroid dehydrogenase oxidative isomers suggestinactivation of 5-dihydrotestosterone in precastrated males. Thusour study suggests increased synthesis and decreased catabolism of5-dihydrotestosterone as a reason for loss of T lymphocyte functionsin intact males following trauma-hemorrhage, as evidenced by decreasedrelease of interleukin-2 and -6.

     

The cholinergic system is involved in regulation of the development of the hematopoietic system

Gene expression profiling demonstrated that components of the cholinergic system, including choline acetyltransferase, acetylcholinesterase and nicotinic acetylcholine receptors (nAChRs), are expressed in embryonic stem cells and differentiating embryoid bodies (EBs). Triggering of nAChRs expressed in EBs by nicotine resulted in activation of MAPK and shifts of spontaneous differentiation toward hemangioblast. In vivo, non-neural nAChRs are detected early during development in fetal sites of hematopoiesis. Similarly, in vivo exposure of the developing embryo to nicotine resulted in higher numbers of hematopoietic progenitors in fetal liver. However postpartum, the number of hematopoietic stem/progenitor cells (HSPC) was decreased, suggesting an impaired colonization of the fetal bone marrow with HSPCs. This correlated with increased number of circulating HSPC and decreased expression of CXCR4 that mediates migration of circulating cells into the bone marrow regulatory niche. In addition, protein microarrays demonstrated that nicotine changed the profile of cytokines produced in the niche. While the levels of IL1alpha, IL1beta, IL2, IL9 and IL10 were not changed, the production of hematopoiesis-supportive cytokines including G-CSF, GM-CSF, IL3, IL6 and IGFBP-3 was decreased. This correlated with the decreased repopulating ability of HSPC in vivo and diminished hematopoietic activity in bone marrow cultures treated with nicotine. Interestingly, nicotine stimulated the production of IL4 and IL5, implying a possible role of the cholinergic system in pathogenesis of allergic diseases. Our data provide evidence that the nicotine-induced imbalance of the cholinergic system during gestation interferes with normal development and provides the basis for negative health outcomes postpartum in active and passive smokers.     

Normal human pluripotent stem cell lines exhibit pervasive mosaic aneuploidy

Human pluripotent stem cell (hPSC) lines have been considered to be homogeneously euploid. Here we report that normal hPSC--including induced pluripotent--lines are karyotypic mosaics of euploid cells intermixed with many cells showing non-clonal aneuploidies as identified by chromosome counting, spectral karyotyping (SKY) and fluorescent in situ hybridization (FISH) of interphase/non-mitotic cells. This mosaic aneuploidy resembles that observed in progenitor cells of the developing brain and preimplantation embryos, suggesting that it is a normal, rather than pathological, feature of stem cell lines. The karyotypic heterogeneity generated by mosaic aneuploidy may contribute to the reported functional and phenotypic heterogeneity of hPSCs lines, as well as their therapeutic efficacy and safety following transplantation.