(1/1306) Regulation of body length and male tail ray pattern formation of Caenorhabditis elegans by a member of TGF-beta family.
We have identified a new member of the TGF-beta superfamily, CET-1, from Caenorhabditis elegans, which is expressed in the ventral nerve cord and other neurons. cet-1 null mutants have shortened bodies and male tail abnormal phenotype resembling sma mutants, suggesting cet-1, sma-2, sma-3 and sma-4 share a common pathway. Overexpression experiments demonstrated that cet-1 function requires wild-type sma genes. Interestingly, CET-1 appears to affect body length in a dose-dependent manner. Heterozygotes for cet-1 displayed body lengths ranging between null mutant and wild type, and overexpression of CET-1 in wild-type worms elongated body length close to lon mutants. In male sensory ray patterning, lack of cet-1 function results in ray fusions. Epistasis analysis revealed that mab-21 lies downstream and is negatively regulated by the cet-1/sma pathway in the male tail. Our results show that cet-1 controls diverse biological processes during C. elegans development probably through different target genes. (+info)
(2/1306) Spinal antinociceptive synergism between morphine and clonidine persists in mice made acutely or chronically tolerant to morphine.
Morphine (Mor) tolerance has been attributed to a reduction of opioid-adrenergic antinociceptive synergy at the spinal level. The present experiments tested the interaction of intrathecally (i.t.) administered Mor-clonidine (Clon) combinations in mice made acutely or chronically tolerant to Mor. ICR mice were pretreated with Mor either acutely (40 nmol i.t., 8 h; 100 mg/kg s.c., 4 h) or chronically (3 mg/kg s.c. every 6 h days 1 and 2; 5 mg/kg s.c. every 6 h days 3 and 4). Antinociception was detected via the hot water (52.5 degrees C) tail-flick test. After the tail-flick latencies returned to baseline levels, dose-response curves were generated to Mor, Clon, and Mor-Clon combinations in tolerant and control mice. Development of tolerance was confirmed by significant rightward shifts of the Mor dose-response curves in tolerant mice compared with controls. Isobolographic analysis was conducted; the experimental combined ED50 values were compared statistically against their respective theoretical additive ED50 values. In all Mor-pretreated groups, the combination of Mor and Clon resulted in significant leftward shifts in the dose-response curves compared with those of each agonist administered separately. In all tolerant and control groups, the combination of Mor and Clon produced an ED50 value significantly less than the corresponding theoretical additive ED50 value. Mor and Clon synergized in Mor-tolerant as well as in control mice. Spinally administered adrenergic/opioid synergistic combinations may be effective therapeutic strategies to manage pain in patients apparently tolerant to the analgesic effects of Mor. (+info)
(3/1306) Morphogenesis of the Caenorhabditis elegans male tail tip.
Using electron microscopy and immunofluorescent labeling of adherens junctions, we have reconstructed the changes in cell architecture and intercellular associations that occur during morphogenesis of the nematode male tail tip. During late postembryonic development, the Caenorhabditis elegans male tail is reshaped to form a copulatory structure. The most posterior hypodermal cells in the tail define a specialized, sexually dimorphic compartment in which cells fuse and retract in the male, changing their shape from a tapered cone to a blunt dome. Developmental profiles using electron microscopy and immunofluorescent staining suggest that cell fusions are initiated at or adjacent to adherens junctions. Anterior portions of the tail tip cells show the first evidence of retractions and fusions, consistent with our hypothesis that an anterior event triggers these morphogenetic events. Available mutations that interfere with morphogenesis implicate particular regulatory pathways and suggest loci at which evolutionary changes could have produced morphological diversity. (+info)
(4/1306) Facilitation and depression of ATP and noradrenaline release from sympathetic nerves of rat tail artery.
1. Excitatory junction currents (EJCs) were used to measure ATP release; noradrenaline (NA) oxidation currents and fractional overflow of labelled NA, [3H]NA, were used to monitor the release of endogenous and exogenous NA, respectively, from post-ganglionic sympathetic nerves of rat tail artery. 2. During nerve stimulation with 100 pulses at 5-20 Hz the EJCs initially grew in size (maximally by 23 %, at 2-10 Hz), and then depressed, maximally by 68 % at 20 Hz. 3. The peak amplitude of NA oxidation currents in response to nerve stimulation with 100 pulses at 2-20 Hz grew in size with frequency, while the area was independent of frequency and roughly constant. 4. The size of the NA oxidation currents evoked by nerve stimulation with 4-100 pulses at 20 Hz grew linearly with train length between pulses 4-16. Between pulses 20-100 there was a train length-dependent depression of the signal. 5. Fractional overflow of [3H]NA in response to nerve stimulation with 5-100 pulses at 20 Hz behaved similarly to the EJCs. It initially grew roughly linearly between pulses 5-25, and then showed a dramatic depression similar to that of the EJCs. 6. The alpha2-adrenoceptor antagonists rauwolscine and yohimbine increased the overflow of [3H]NA and the amplitude of NA oxidation currents, but not that of the EJCs. 7. It is concluded that during high-frequency stimulation (i) the release of ATP and NA is first briefly facilitated then markedly depressed, (ii) facilitation and depression of the two transmitters are similar in magnitude and time course, and (iii) alpha2-adrenoceptor antagonists differentially modify EJCs and the NA signals. The results obtained in the absence of drugs are compatible with the hypothesis that ATP and NA are released in parallel, while the effects of alpha2-adrenoceptor antagonists seem to suggest dissociated release. (+info)
(5/1306) Effects of Ca2+ concentration and Ca2+ channel blockers on noradrenaline release and purinergic neuroeffector transmission in rat tail artery.
1. The effects of Ca2+ concentration and Ca2+ channel blockers on noradrenaline (NA) and adenosine 5'-triphosphate (ATP) release from postganglionic sympathetic nerves have been investigated in rat tail arteries in vitro. Intracellularly recorded excitatory junction potentials (e.j.ps) were used as a measure of ATP release and continuous amperometry was used to measure NA release. 2. Varying the extracellular Ca2+ concentration similarly affected the amplitudes of e.j.ps and NA-induced oxidation currents evoked by trains of ten stimuli at 1 Hz. 3. The N-type Ca2+ blocker, omega-conotoxin GVIA (omega-CTX GVIA, 0.1 microM) reduced the amplitudes of both e.j.ps (evoked by trains of ten stimuli at 1 Hz) and NA-induced oxidation currents (evoked by trains of ten stimuli at 1 Hz and 50 stimuli at 10 Hz) by about 90%. 4. The omega-CTX GVIA resistant e.j.ps and NA-induced oxidation currents evoked by trains of 50 stimuli at 10 Hz were abolished by the non-selective Ca2+ channel blocker, Cd2+ (0.1 mM), and were reduced by omega-conotoxin MVIIC (0.5 microM) and omega-agatoxin IVA (40 nM). 5. Nifedipine (10 microm) had no inhibitory effect on omega-CTX GVIA resistant e.j.ps and NA-induced oxidation currents. 6. Thus both varying Ca2+ concentration and applying Ca2+ channel blockers results in similar effects on NA and ATP release from postganglionic sympathetic nerves. These findings are consistent with the hypothesis that NA and ATP are co-released together from the sympathetic nerve terminals. (+info)
(6/1306) Protein kinase C reduces the KCa current of rat tail artery smooth muscle cells.
The hypothesis that protein kinase C (PKC) is able to regulate the whole cell Ca-activated K (KCa) current independently of PKC effects on local Ca release events was tested using the patch-clamp technique and freshly isolated rat tail artery smooth muscle cells dialyzed with a strongly buffered low-Ca solution. The active diacylglycerol analog 1,2-dioctanoyl-sn-glycerol (DOG) at 10 microM attenuated the current-voltage (I-V) relationship of the KCa current significantly and reduced the KCa current at +70 mV by 70 +/- 4% (n = 14). In contrast, 10 microM DOG after pretreatment of the cells with 1 microM calphostin C or 1 microM PKC inhibitor peptide, selective PKC inhibitors, and 10 microM 1,3-dioctanoyl-sn-glycerol, an inactive diacylglycerol analog, did not significantly alter the KCa current. Furthermore, the catalytic subunit of PKC (PKCC) at 0.1 U/ml attenuated the I-V relationship of the KCa current significantly, reduced the KCa current at +70 mV by 44 +/- 3% (n = 17), and inhibited the activity of single KCa channels at 0 mV by 79 +/- 9% (n = 6). In contrast, 0.1 U/ml heat-inactivated PKCC did not significantly alter the KCa current or the activity of single KCa channels. Thus these results suggest that PKC is able to considerably attenuate the KCa current of freshly isolated rat tail artery smooth muscle cells independently of effects of PKC on local Ca release events, most likely by a direct effect on the KCa channel. (+info)
(7/1306) A novel BMP expressed in developing mouse limb, spinal cord, and tail bud is a potent mesoderm inducer in Xenopus embryos.
The bone morphogenetic proteins (BMPs) play critical roles in patterning the early embryo and in the development of many organs and tissues. We have identified a new member of this multifunctional gene family, BMP-11, which is most closely related to GDF-8/myostatin. During mouse embryogenesis, BMP-11 is first detected at 9.5 dpc in the tail bud with expression becoming stronger as development proceeds. At 10.0 dpc, BMP-11 is expressed in the distal and posterior region of the limb bud and later localizes to the mesenchyme between the skeletal elements. BMP-11 is also expressed in the developing nervous system, in the dorsal root ganglia, and dorsal lateral region of the spinal cord. To assess the biological activity of BMP-11, we tested the protein in the Xenopus ectodermal explant (animal cap) assay. BMP-11 induced axial mesodermal tissue (muscle and notochord) in a dose-dependent fashion. At higher concentrations, BMP-11 also induced neural tissue. Interestingly, the activin antagonist, follistatin, but not noggin, an antagonist of BMPs 2 and 4, inhibited BMP-11 activity on animal caps. Our data suggest that in Xenopus embryos, BMP-11 acts more like activin, inducing dorsal mesoderm and neural tissue, and less like other family members such as BMPs 2, 4, and 7, which are ventralizing and anti-neuralizing signals. Taken together, these data suggest that during vertebrate embryogenesis, BMP-11 plays a unique role in patterning both mesodermal and neural tissues. (+info)
(8/1306) A developmental pathway controlling outgrowth of the Xenopus tail bud.
We have developed a new assay to identify factors promoting formation and outgrowth of the tail bud. A piece of animal cap filled with the test mRNAs is grafted into the posterior region of the neural plate of a host embryo. With this assay we show that expression of a constitutively active Notch (Notch ICD) in the posterior neural plate is sufficient to produce an ectopic tail consisting of neural tube and fin. The ectopic tails express the evenskipped homologue Xhox3, a marker for the distal tail tip. Xhox3 will also induce formation of an ectopic tail in our assay. We show that an antimorphic version of Xhox3, Xhox3VP16, will prevent tail formation by Notch ICD, showing that Xhox3 is downstream of Notch signalling. An inducible version of this reagent, Xhox3VP16GR, specifically blocks tail formation when induced in tailbud stage embryos, comfirming the importance of Xhox3 for tail bud outgrowth in normal development. Grafts containing Notch ICD will only form tails if placed in the posterior part of the neural plate. However, if Xwnt3a is also present in the grafts they can form tails at any anteroposterior level. Since Xwnt3a expression is localised appropriately in the posterior at the time of tail bud formation it is likely to be responsible for restricting tail forming competence to the posterior neural plate in our assay. Combined expression of Xwnt3a and active Notch in animal cap explants is sufficient to induce Xhox3, provoke elongation and form neural tubes. Conservation of gene expression in the tail bud of other vertebrates suggests that this pathway may describe a general mechanism controlling tail outgrowth and secondary neurulation. (+info)