Production of ceramides causes apoptosis during early neural differentiation in vitro.
(17/324)
To investigate signal transduction pathways leading to apoptosis during the early phase of neurogenesis, we employed PCC7-Mz1 cells, which cease to proliferate and begin to differentiate into a stable pattern of neurons, astroglial cells, and fibroblasts upon incubation with retinoic acid (RA). As part of lineage determination, a sizable fraction of RA-treated cultures die by apoptosis. Applying natural long-chain C(16)-ceramides as well as membrane-permeable C(2)/C(6)-ceramide analogs caused apoptosis, whereas the biologically nonactive C(2)-dihydroceramide did not. Treating PCC7-Mz1 stem cells with a neutral sphingomyelinase or with the ceramidase inhibitor N-oleoylethanolamine elevated the endogenous ceramide levels and concomitantly induced apoptosis. Addition of RA caused an increase in ceramide levels within 3-5 h, which reached a maximum (up to 3.5-fold of control) between days 1 and 3 of differentiation. Differentiated PCC7-Mz1 cells did not respond with ceramide formation and apoptosis to RA treatment. The acidic sphingomyelinase contributed only weakly and the neutral Mg(2+)-dependent and Mg(2+)-independent sphingomyelinases not at all to the RA-mediated production of ceramides. However, ceramide increase was sensitive to the ceramide synthase inhibitor fumonisin B(1), suggesting a crucial role for the de novo synthesis pathway. Enzymatic assays revealed that ceramide synthase activity remained unaltered, whereas serine palmitoyltransferase (SPT), a key enzyme in ceramide synthesis, was activated approximately 2.5-fold by RA treatment. Activation of SPT seemed to be mediated via a post-translational mechanism because levels of the mRNAs coding for the two SPT subunits were unaffected. Expression of marker proteins shows that ceramide regulates apoptosis, rather than differentiation, during early neural differentiation. (+info)
Monopolar protrusive activity: a new morphogenic cell behavior in the neural plate dependent on vertical interactions with the mesoderm in Xenopus.
(18/324)
We compared the type and patterning of morphogenic cell behaviors driving convergent extension of the Xenopus neural plate in the presence and absence of persistent vertical signals from the mesoderm by videorecording explants of deep neural tissue with involuted mesoderm attached and of deep neural tissue alone. In deep neural-over-mesoderm explants, neural plate cells express monopolar medially directed motility and notoplate cells express randomly oriented motility, two new morphogenic cell behaviors. In contrast, in deep neural explants (without notoplate), all cells express bipolar mediolateral cell motility. Deep neural-over-mesoderm and deep neural explants also differ in degree of neighbor exchange during mediolateral cell intercalation. In deep neural-over-mesoderm explants, cells intercalate conservatively, whereas in deep neural explants cells intercalate more promiscuously. Last, in both deep neural-over-mesoderm and deep neural explants, morphogenic cell behaviors differentiate in an anterior-to-posterior and lateral-to-medial progression. However, in deep neural-over-mesoderm explants, morphogenic behaviors first differentiate in intervals along the anteroposterior axis, whereas in deep neural explants, morphogenic behaviors differentiate continuously from the anterior end of the tissue posteriorly. These results describe new morphogenic cell behaviors driving neural convergent extension and also define roles for signals from the mesoderm, up to and beyond late gastrulation, in patterning these cell behaviors. (+info)
Evidence that adenosine 5'-triphosphate is the third inhibitory non-adrenergic non-cholinergic neurotransmitter in the rat gastric fundus.
(19/324)
In the rat gastric fundus, non-adrenergic, non-cholinergic (NANC) relaxations are mediated by nitric oxide (NO), vasoactive intestinal polypeptide (VIP), and a third, as yet unidentified, neurotransmitter. The possible involvement of adenosine 5'-triphosphate (ATP) in the NANC relaxations was examined using pyridoxalphosphate-6-azophenyl-2',5'-disulphonic acid (PPADS), apamin and desensitization to alpha,beta-methylene ATP. NANC responses were studied in the absence and presence of N(G)-nitro-L-arginine methyl ester (NAME; 100 microM) and alpha-chymotrypsin (1 u ml(-1)), to inhibit responses to NO and VIP, respectively. PPADS (100 microM), apamin (1 microM) and desensitization to alpha,beta-methylene ATP (10 microM, three additions) all significantly (P<0.05) reduced NANC relaxations to electrical field stimulation (0.5 - 4 Hz, 30 s trains) in longitudinal strips of rat gastric fundus and almost abolished the residual relaxation remaining in the presence of NAME and alpha-chymotrypsin. PPADS had no effect on responses to the NO-donor, sodium nitroprusside (SNP), or VIP. Apamin slightly reduced relaxations to SNP, but did not affect those to VIP, whereas desensitization to alpha,beta-methylene ATP markedly reduced responses to both SNP and VIP. The effects of PPADS and apamin in this study provide strong evidence that the third inhibitory NANC neurotransmitter in the rat gastric fundus is ATP. (+info)
Effects of a selective neuropeptide Y Y(1) receptor antagonist BIBP 3226 on double peaked vasoconstrictor responses to periarterial nerve stimulation in canine splenic arteries.
(20/324)
The periarterial electrical nerve stimulation (30 s trains of pulses at a frequency of 1, 4 or 10 Hz) induced a double peaked vasoconstriction consisting of an initial transient constriction (first peak) followed by a prolonged response (second peak) in the isolated, perfused canine splenic artery. At low frequencies (1 and 4 Hz), a neuropeptide Y (NPY) Y(1) receptor antagonist BIBP 3226 (0.1-1 microM) produced a dose-dependent inhibitory effect on the second peak, but did not modify the first peak. At a high frequency (10 Hz), 1 microM BIBP 3226 induced a slight, but significant inhibition on both the first and second peaked responses. At a low frequency (1 Hz), the first peak was not influenced by blockade of alpha(1)-adrenoceptors or NPY Y(1) receptors with prazosin (0.1 microM) or BIBP 3226 (1 microM), respectively, but abolished by P2X receptor desensitization with alpha,beta-methylene ATP (alphabeta-m ATP, 1 microM). At a high frequency (10 Hz), the first peak was mostly inhibited by alphabeta-m ATP and partially by prazosin and BIBP 3226. On the other hand, the second peak at a low frequency was largely decreased by BIBP 3226 and partially by prazosin and alphabeta-m ATP, whereas at a high frequency, it was largely attenuated by prazosin and partially by alphabeta-m ATP and BIBP 3226. The results suggest that at a low frequency, the firstly transient constriction of double peaked responses is mainly induced via an activation of P2X-receptors, whereas at a high frequency, it is mostly mediated by the P2X-receptors, and partially by alpha(1)-receptors and NPY Y(1)-receptors. The secondary prolonged vasoconstriction at frequencies used is predominantly mediated via both alpha(1)-receptor and NPY Y(1) receptor activations, and in part by P2X-receptors. Furthermore, an activation of NPY Y(1) receptors may play an important role in evoking the prolonged vasoconstrictor response to longer pulse trains of stimulation at a low frequency, whereas an alpha(1)-adrenoceptor activation exerts a main vasomotor effect for the prolonged response at a high frequency. (+info)
Neurovascular plasticity in the knee joint of an arthritic mouse model.
(21/324)
Lower numbers of neuropeptide-containing fibers in arthritic joints have been found as compared to control joints. This may be the result of fiber depletion, necrosis of fibers, or proliferation of soft tissues without neural sprouting. To discriminate between these possibilities, we studied the relationships between soft tissue proliferation, changes in vascularity of synovial tissues, and changes in joint innervation during arthritis. Arthritis was induced in the knee joint of mice by a single subpatellar injection of methylated bovine serum albumin after previous immunization. Antibodies to protein gene product 9.5, S-100, and growth-associated protein-43 (GAP-43) were used to study the general innervation pattern. Antibodies to calcitonin gene-related peptide (CGRP), vasointestinal polypeptide (VIP), substance P (SP), and tyrosine hydroxylase (TH) were used to localize sensory (SP, CGRP, VIP) and sympathetic (TH) fibers. Blood vessels of the joint were studied with ink perfusion, GAP-43, and a vascular marker (LF1). Directly after the induction of arthritis, the synovial cavity was enlarged and filled with leukocytes. From day 4 onward, small sprouting blood vessels penetrated the avascular mass of cells in the joint cavity. After 1 week, the vascular sprouting activity and GAP-43 immunoreactivity were maximal, and after 2 weeks, vascular sprouting activity diminished. In the subsequent period, the synovia slowly regained their prearthritic appearance and thickness. The most pronounced changes in the general staining pattern of CGRP, SP, VIP, and TH were found in the periosteum. From 2 days to 4 weeks after the induction of arthritis, the layer of SP, CGRP, and VIP fibers in the femoral periosteum was thicker and more irregular. GAP-43 staining showed many terminal varicosities, which suggested sprouting of nerve fibers. From 2 days to 2 weeks after the induction of arthritis, the SP and CGRP fibers in the periosteum showed gradual depletion. In the thickened subsynovial tissues that were revascularized, no ingrowth of neural elements was found. As the total number of nerve fibers in the synovial tissue did not change, large parts of the synovia directly facing the joint cavity were not innervated at 1 week after the induction of arthritis. These results strongly suggest that periosteal SP and CGRP fibers were depleted during arthritis. Synovial proliferation without concomitant fiber growth is the main cause of the reduced number of immunocytochemically detectable fibers in the mouse arthritic knee joint. (+info)
Frog lim-1-like protein is expressed predominantly in the nervous tissue, gonads, and early embryos of the bivalve mollusc Mytilus galloprovincialis.
(22/324)
In a few well-known cases, the biological consequences of the disruption of lim-1 homeodomain (HD) genes have demonstrated the important roles of these genes in vertebrate development, especially in the nervous tissue, kidney, and gonads. Functional assay approaches require information not only about lim-1 gene organization, but also about properties and tissue localization of Lim-1 proteins. Although lim-1 genes have been identified in certain phyla of invertebrates, no information is available on Lim-1 proteins and genes in bivalve molluscs. Our study represents the beginning stage of identification of the Lim-1-related proteins in marine bivalves. Using antibodies against the C-terminal region of the Xenopus laevis Lim-1 protein, we describe cross-reactive antigen patterns in adults and early embryos of the mussel Mytilus galloprovincialis, as well as in sea urchin and chick embryos. In adult mussels, nervous ganglia and gonads display the most prominent Lim-1 immunoreactivity. Further, the antibodies verified the prediction that mussel Lim-1 antigens, like Lim-1 HD proteins in general, can be localized in the nucleus. Moreover, antibody detection allowed us to identify the Lim-1-like antigens in unfertilized mature eggs, as well as in very early embryos of bivalve molluscs and sea urchins (Strongylocentrotus purpuratus). In mussel eggs and embryos, Lim-1 antigens are expressed in multiple forms (40, 45, and 65 kDa), as detected by SDS-PAGE followed by Western blot. Taken together, the observations emphasize the conservation of the Lim-1 protein expression pattern in the nervous tissue and gonads of different animal groups, and demonstrate that Lim-1-like polypeptides can be maternally accumulated in eggs and, therefore, are present in very early embryos before zygotic expression of the genes begins. (+info)
The forkhead transcription factor UNC-130 is required for the graded spatial expression of the UNC-129 TGF-beta guidance factor in C. elegans.
(23/324)
Secreted proteins required for cellular movements along the circumference of the body wall in Caenorhabditis elegans include UNC-6/netrin and the novel TGF-beta UNC-129. Expression of these proteins is graded along the dorsoventral (D/V) axis, providing polarity information to guide migrations. Here we show that the graded expression of UNC-129 in dorsal but not ventral body muscles depends on unc-130, which encodes a Forkhead transcription factor. The phenotype of unc-130 mutants closely mimics the reported effects of ectopically expressing unc-129 in both dorsal and ventral body muscles (). This fits our present finding that unc-130 cell autonomously represses unc-129 expression in the ventral body muscles. Thus the cell-specific effects of unc-130 on ventral, but not dorsal, body muscle expression of unc-129 accounts for the D/V polarity information required for UNC-129-mediated guidance. Genetic interactions between unc-130 and other guidance genes show that several molecular pathways function in parallel to guide the ventral to dorsal migration of distal tip cells (DTCs) and axonal growth cones in C. elegans. Genetic interactions confirm that UNC-129 does not require the only known type II TGF-beta receptor in C. elegans (DAF-4) for its guidance functions. Also, unc-130 is partially required for male tail morphogenesis and for embryogenesis. (+info)
Muscular innervation of the proximal duodenum of the guinea pig.
(24/324)
We investigated the muscular structure and innervation of the gastroduodenal junction in the guinea pig. In the gastroduodenal junction, the innermost layer of the circular muscle contained numerous nerve fibers and terminals. Since this nerve network continued onto the deep muscular plexus (DMP) of the duodenum, we surmised that the numerous nerve fibers in the gastroduodenal junction were specialized DMP in the most proximal part of the duodenum. The innermost layer containing many nerve fibers was about 1,000 microm in length and 100 microm in thickness in the proximal duodenum. This layer contained numerous connective tissue fibers composed of collagen and elastic fibers. Five to 30 smooth muscle cells lay in contact with each other and were surrounded by fine connective tissue. The nerve fibers in the proximal duodenum contained nerve terminals immunoreactive for choline acetyltransferase, dynorphin, enkephalin, galanin, gastrin-releasing peptide, nitric oxide synthase, substance P, and vasoactive intestinal polypeptide. Adrenergic fibers which contained tyrosine hydroxylase immunoreactivity were rare in the proximal duodenum. In the innermost layer of the proximal duodenum, there were numerous c-Kit immunopositive cells that were in contact with nerve terminals. This study allowed us to clarify the specific architecture of the most proximal portion of the duodenum. The functional significance of the proximal duodenum in relation to the electrical connection and neural cooperation of the musculature between the antrum and the duodenum is also discussed. (+info)