Cyclic AMP-dependent phosphorylation of tyrosine residues in a protein with a molecular mass of 15-kDa is thought to play a key role in the initiation of sperm motility in salmonid fishes (7). In this study, flagellar proteins were solubilized with 7 M urea and fractionated by sucrose densitygradient isoelectrie focusing. A band relevant to 15-kDa was detected in the fraction at pH 5 on electrophoresis and analyzed using phosphotyrosine antibody. Analysis of the band by reversed phase chromatography showed a single peak, suggesting that the 15-kDa protein was completely purified. Amino acid sequence of His—Ile-Phe at the N-terminus was detected by analysis using peptide sequencer, and the fourth base could not be determined. Further analysis of the molecular structure of the protein by LC—MS/MS showed that partial amino acid sequence of the 15-kDa protein from tryptic fragments were similar to parts of the amino acid sequence of tubulin. The protein was not detected in inununoblots using anti-tubulin polyclonal antibody, suggesting that the 15-kDa protein may be not debris of the tubulin but a novel protein with a common partial sequence of tubulin.
We examined whether the glutamate and glycine levels in the central nervous system (CNS) were related to bladder activity and their serum levels in rats after spinal cord injuly (SCI). Female rats were anesthetized with halothane, and the spinal cord was completely transected at the lower thoracic level. At 1 day to 8 weeks after SCI, bladder activity and the glutamate and glycine levels in the CNS and serum were measured. Urinary retention was observed in the acute period after SCI. Iscvolumetric cystometry showed no bladder contractions at 1 and 3 days after SCI, but contractions were seen after 2 to 8 weeks. The glycine level was increased in the lumbosacral cord at 1 day after SCI, but it was decreased at 2 to 8 weeks. The serum glycine level was also increased at 1 week after SCI, and it was decreased gradually over 4 to 8 weeks. The glutamate level in each CNS region and serum of SCI rats did not differ from those of control rats and sham-operated rats at 1 day to 8 weeks after surgery. Therefore, the change of the glycine level in the lumbosacral cord may influence bladder contractions and its serum level after SCI.
We examined the relationship between bladder activity and amino acid levels in the central nervous system (CNS) after cerebral infarction (Cl) in rats. Female rats were anesthetized with halothane, and a nylon thread was inserted into the right middle cerebral artery to cause CI. At 1 day to 8 weeks after CI, bladder activity and glutamate and glycine levels in the CNS and serum were measured. The frequency of isovolumetric bladder contractions was increased at 1 day to 1 week after CI, but it returned to baseline at 2 weeks. The glutamate level of the cerebrum and the glycine levels of the brainstem and the cervicothoracic cord were decreased at 1 day to 8 weeks after CI. The glycine level of the lumbosacral cord was also decreased at 1 day to 2 weeks after CI, but it returned to baseline at 4 weeks. Serum glutamate and glycine levels of CI rats did not differ from those of control rats and sham-operated rats at 1 day to 8 weeks after surgery. Therefore, the change of bladder activity after CI may be partly dependent on the activity of glycinergic neurons in the lumbosacral cord.
Gastrin-releasing peptide (GRP) was originally isolated from porcine upper gastric tissue. Its structure was found to contain a C-terminal region with striking homology toward bombesin. Recently, GRP has been suggested as an important novel regulatoiy peptide in the female reproductive tract of mammals, including the cow. However, the nature of GRP in the uterus of cows remains to be determined, and there are no reports on the nucleotide sequence of cDNA encoding bovine GRP. In this study, we have determined the GRP cDNA from bovine endometrial tissues. The GRP cDNA consists of 973 bp, comprising a short 5'-untranslated region (108 bp), an open reading frame (543 bp), a stop codon (TGA), and a 3'-untranslated region (319 bp), including a polyadenylation signal (AATAAA) and a poly (A) tail. The deduced amino acid sequence shows that bovine GRP is part of a 182 amino acids precursor. The putative mature bovine GRP is composed of 27 amino acids after 28 amino acid signal peptide. At the C-terminus, the GRP sequence is flanked by one Gly and two basic residues (Lys-Lys), indicative of C-terminal amidation and cleavage site.
Effects of pentoxifylline (PTX; 20 mg/kg/day) on mortality, blood pressure, acidosis, and inflammatory mediators were studied in rats subjected to cecal ligation and puncture (CLP). PTX was in travenously given once 30 min after CLP. Compared with controls with CLP but no drug, the PTX group showed improved survival ever 10 days of observation. PTX treatment significantly attenuated the decrease in blood pressure and increase in serum lactate concentrations in comparison to controls administrated with normal saline at 24 h after operation. Tumor necrosis factor (TNF)-α concentrations were reduced significantly by PTX in serum measured by radioimmunoassays. According to the semi-quantitative and quantitative (TaqMan) reverse-transcription polymerase chain reactions, PTX had significantly decreased TNF-α but not IL-1β gene expression in lung and liver (both p＜0.05) at 24 h postoperatively. Thus, PTX attenuated mortality, TNF-α, and lactate in this sepsis model, in association with down-regulation of TNF gene transcription. Moreover, PTX may have other various benefitical actions on sepsis. Clinical trials of PTX given early in sepsis should be considered.
Carbon foams are suitable for scaffolds used in tissue engineering that have a porosity ratio of more than 90% with a three-dimensional honeycomb structure. Titanium films were deposited isotropically using an electron cyclotron resonance (ECR) sputtering system on 5 mm-thick carbon foams. SEM observation and electron probe microanalysis revealed that all the surfaces of the carbon foam in the center of the specimens were coated with an approximately 5 μm-thick titanium film, indicating that the ECR sputtering method enables titanium coating into both the surrounding and deep regions of carbon foams. The flexural strength and elastic modulus of the coated specimens increased by approximately 1.5 and 2.5 times, respectively, than those of the uncoated specimens. Cell culture tests with mouse fibroblasts (L-929) and rat bone marrow osteoblast-like cells demonstrated the presence of cells not only on the surface but also inside the foam. No toxic effect on both cells on the titanium-coated carbon foams was apparent. A small amount of extracellular matrices appeared on the foam struts, but those matrices did not grow enough to occupy the spaces between them. These results indicate that carbon foams coated with titanium are useful for skeletons used for tissue engineering.
Enamelysin (MMP-20) is a matrix metalloproteinase isolated from the cDNA library of the enamel organ. Although enamelysin is also expressed in the tooth pulp, especially in the odontoblast, the functional significance of enamelysin protein in dentinogenesis has not been elucidated. Thus, the present study was designed to clarify the characteristics of enamelysin in the pulp from the porcine deciduous molar tooth germ and developing teeth. Western blot analysis of proteins extracted from the pulp of the tooth germ revealed that immunoreactive enamelysin occurred as 54 kDa and 51 kDa proteins. None of the protein bands lower than 51 kDa was stained. Light and electron microscopic immunohistochemistry of tooth germs and erupting teeth demonstrated positive reactions over the cytoplasmic matrix of odontoblast cell bodies and processes (dentinal fibers). The secretoty machinery of the odontoblast was shown to be devoid of immunoreactivity. Intensity of the immunoreaction in the odontoblast was variable but essentially dependent upon the position and differentiation stage of the odontoblast. The highest immunoreactivity was found in some odontoblasts located at the cuspal region of the tooth germ. During tooth eruption, cell bodies of odontoblasts in the crown showed faint or no immunoreactivity. No immunoreaction was detected in the odontoblast facing the root dentin, although dentinal fibers of the root dentin showed faint immunoreactivity. RT-PCR and cDNA sequence analysis failed to find any differences between enamelysin mRNA extracted from the enamel organ and that from the pulp. These results suggest that odontoblasts transiently express enamelysin during tooth formation and that the enamelysin in the odontoblast is not secreted from the cell.