In order to study the phosphorylated proteins in the resting Xenopus laevis oocytes, the proteins detected by Western blotting using phospho-(Ser/Thr) PKA substrate antibody (PKA substrate antibody) in forskolin-stimulated oocytes were purified and identified by mass spectrometry. Several proteins (ribosomal S6 protein, elongation factor-2 (EF-2), poly A binding protein, releasing factor 1) were identified, and the phosphorylation of EF-2 was further studied. Partially purified Xenopus EF-2 (xEF-2) was phosphorylated by PKA in vitro and this phosphorylation was detected by Western blotting using PKA substrate antibody. The phosphorylation of Thr-57 in xEF-2 (corresponding to Thr-56 of the mammalian enzyme) was detected in the partially purified xEF-2 from the resting oocytes, but this xEF-2 did not react with the PKA substrate antibody. These results suggest that Thr-57 in xEF-2 was phosphorylated, but xEF-2 does not seem to be phosphorylated by PKA in resting oocytes although PKA can phosphorylate xEF-2 in vitro and probably in forskolin-treated oocytes.
Bicarbonate secretion occurs in almost all segments of the gastrointestinal tract. This study examined HCO3- secretion in the ileum, since it is less understood than HCO3- secretion in other intestinal segments. Mouse ileal mucosa was mounted in vitro in Ussing chambers, and the mucosal alkalinization rate (JOH) was determined by pH stat titration, while the mucosal side was bathed with a buffer-free solution (100% O2) and the serosal side with a HCO3-/CO2-buffered solution. The transmural potential difference (PD) was recorded. The mucosal alkalinization rate (JOH) was higher in the presence of mucosal Cl- than in its absence. Forskolin, an activator of adenylate cyclase, enhanced JOH and PD in both the presence and absence of mucosal Cl-. Mucosal SO42- also caused an increase in JOH, although the magnitude was smaller than that induced by Cl-. Mucosal Cl--dependent JOH was partially inhibited by acetazolamide, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), tenidap and probably also by niflumic acid, but not by glibenclamide, DIDS or bumetanide. The forskolin-induced JOH value and PD were both inhibited by NPPB and probably also by tenidap. It is concluded that HCO3-- secretion in the ileum follows a mucosal Cl--dependent pathway and a cAMP-activated pathway, each being distinct from each other. The Cl--dependent pathway is probably mediated by the slc26a6 anion exchanger, and possibly also by the slc26a3 anion exchanger. The cAMP-activated HCO3- secretion is probably mediated by the cystic fibrosis transmembrane conductance regulator.
Leukocyte-cell-derived chemotaxin 2 (LECT2) was first isolated from the culture fluid of phytohemagglutinin-activated human T-cell leukemia SKW-3 cells and was found to be expressed in the human, bovine and murine livers. To further analyze the role of LECT2 in the liver, we investigated the expression of mouse LECT2 (mLECT2) during liver regeneration after partial hepatectomy (PHx) using immunohistochemical and in situ hybridization techniques. Mouse LECT2 protein and mRNA were detected in most hepatocytes in normal mouse; however, at 30 min after PHx, they were not detected in liver tissue. At 2 h after PHx, expression of mLECT2 protein was seen in hepatocytes surrounding the central vein, although mRNA expression levels were still low. At 6 h after PHx, a marked number of hepatocytes expressing mLECT2 protein and mRNA were seen throughout the liver, and at 12 h after PHx, hepatocytes expressing mLECT2 protein and mRNA further increased in number. However, expression levels of mLECT2 protein and mRNA at 24 h after PHx were significantly lower when compared with levels after 12 h. These results indicate that LECT2 triggers the early events of regeneration with concomitant suppression of hepatocyte proliferation.
We explored capsaicin pretreatment, prior to spinal trauma, as a method to prevent the development of neurogenic detrusor overactivity (NDO) and urethral-bladder dyssynergia reflex after spinal cord injury (SCI). In addition, the duration of effect of capsaicin therapy on NDO in a rat model of SCI was investigated. Two sets of experiments were performed on female Sprague Dawley rats transected at the T9-T10 spinal level. First, SCI rats received capsaicin (125 mg/kg s.q.) 3-4 days before and 4-5 days after SCI. Cystometrograms (CMG) was performed 4 weeks after injury. In the second set of experiments, serial CMG in the same SCI animal was performed after one time injection of capsaicin (125 mg/kg s.q.) 4 weeks after spinalization. There were no differences in intercontraction intervals, voiding efficiency, or voiding pressure between the capsaicin pretreated and control SCI rats. However, the number of uninhibited detrusor contractions decreased 4 weeks after injury. We found that a single dose of capsaicin suppressed uninhibited detrusor contractions for 34 days in the chronic SCI animals. Early therapy with capsaicin was able to prevent/reduce detrusor hyperreflexia in spinal cord injured animals 4 weeks after injury. Early vanilloid therapy may prevent development of urologic sequelae after SCI.
Neural stem/progenitor cells (NSCs) proliferate vigorously as neurospheres in medium containing basic fibroblast growth factor (FGF-2), but start differentiating into neurons, astrocytes or oligodendrocytes in FGF-2-free medium. An extract of royal jelly (RJ) significantly increased the percentage in the total cell population of not only neurons immunoreactive for class III β-tubulin (Tuj1) but also astrocytes immunoreactive for glial fibrillary acidic protein (GFAP), and oligodendrocytes immunoreactive for 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) generated from NSCs, but decreased that of nestin-positive NSCs. These results highlight a novel and outstanding property of the RJ, i.e., that it facilitates the differentiation of all types of brain cells (neurons, astrocytes, and oligodendrocytes). On the other hand, 10-hydroxy-trans-2-decenoic acid (HDEA), an unsaturated fatty acid characteristic of RJ, increased the generation of neurons and decreased that of astrocytes from NSCs. These observations suggest that RJ contains plural components that differently influence neuronal and/or glial lineages and that HDEA is one of such components of RJ that facilitates neurogenesis by NSCs.
We evaluated the effects of N-hexacosanol, a cyclohexenonic long-chain fatty alcohol, on muscarinic receptors in diabetic rat ileal dysfunction. Eight-week-old male SD rats were divided into four groups. After induction of diabetes (streptozotocin 50 mg/kg, i.p.), three groups were maintained for eight weeks with treatment by N-hexacosanol (0, 2 or 8 mg/kg, s.c. every day). Ileum function was investigated by organ bath studies using carbachol and KCl, and the expression levels of muscarinic M2 and M3 receptors were investigated by real-time polymerase chain reaction. Various concentrations of subtype-selective muscarinic antagonists, i.e., atropine (non-selective), pirenzepine (M1 selective), methoctramine (M2 selective), and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, M1/M3 selective), were used in this study. In the presence and absence of these antagonists, contractile response curves to increasing concentrations of carbachol were investigated. Treatment with N-hexacosanol did not alter the diabetic status of the rats, but did significantly prevent the carbachol-induced hypercontractility in diabetic rat ileum. Estimation of the pA2 values for atropine, pirenzepine, methoctramine, and 4-DAMP indicated that the carbacholinduced contractile response in the ileum is mainly mediated through the muscarinic M3 receptor subtype in all groups. Furthermore, N-hexacosanol significantly prevented the diabetes-induced up-regulation of intestinal muscarinic M2 and M3 receptor mRNAs in streptozotocin-diabetic rats. Our data indicated that N-hexacosanol exerts preventive effects with respect to carbachol-induced hypercontractility in the diabetic rat ileum without qualitative alteration of the muscarinic receptor system.
We attempted to increase bladder contraction by bone marrow cell transplantation in rats with underactive bladder due to bladder outlet obstruction (BOO). Twelve female rats were anesthetized with halothane to create BOO. After 1 month, the urethral obstruction was removed and they were divided into a transplant group and a sham-operated group (n = 6 each). Bone marrow cells (1 × 107 / 0.2 mL) isolated from green fluorescent protein transgenic rats were injected into the bladder wall of the transplant group. Rats from the sham-operated group received injection of culture medium alone. One month after transplantation, isovolumetric cystometry parameters and histological features of bladder were observed as well as intact control rats (n = 6). The amplitude of bladder contractions was larger and the interval between contractions was shorter in the transplant group than the sham-operated group, and there were no differences in these parameters between the transplant group and the control group. Some green fluorescent muscle layers were found in the bladder wall of the transplant group, and these layers were also labeled by anti alpha-smooth muscle actin antibody. These results suggest that transplanted bone marrow cells may improve bladder contractility by differentiating into smooth muscle-like cells.