Identification and endothelin-induced activation of multiple extracellular signal-regulated kinases in aortic smooth muscle cells.

In order to explore intracellular signaling pathways of the mitogenic action of endothelin (ET), we examined the effect of ET on activities of extracellular signal-regulated kinases (ERKs) in rat aortic smooth muscle cells (SMCs). Treatment of rat aortic SMCs with ET-1 increased kinase activities toward myelin basic protein (MBP). Both 43- and 41-kDa proteins were activated when kinase assays were done in MBP-containing polyacrylamide gels after SDS-PAGE. These proteins were identified as ERK1 and ERK2 with immunoprecipitation and immunoblotting using antipeptide antibodies, respectively. These results indicate that ERKs mediate signal transduction by ET.     

Time-related changes in the labeling pattern of motor and sensory neurons innervating the gastrocnemius muscle,as revealed by the retrograde transport of the cholera toxin B subunit

Summary Morphological changes in the motor and sensory neurons in the lumbar spinal cord and the dorsal root ganglia were investigated at different survival times following the injection of the B subunit of cholera toxin (CTB) into the medial gastrocnemius muscle. Unconjugated CTB, visualized immunohistochemically, was found to be retrogradely transported through ventral and dorsal roots to motor neurons in the anterior horn, each lamina in the posterior horn, and ganglion cells in the dorsal root ganglia at L₃–L₆. The largest numbers of labeled motor neurons and ganglion cells were observed 72 h after the injection of CTB. Thereafter, labeled ganglion cells were significantly decreased in number, whereas the amount of labeled motor neurons showed a slight reduction. Motor neurons had extensive dendritic trees filled with CTB, reaching lamina VII and even the pia mater of the lateral funiculus. Labeling was also seen in the posterior horn, but the central and medial parts of laminae II and III had the most extensively labeled varicose fibers, the origin of which was the dorsal root ganglion cells. The results indicate that CTB is taken up by nerve terminals and can serve as a sensitive retrogradely transported marker for identifying neurons that innervate a specific muscle.     

Preferential distribution of group-II-like phospholipase A2 in mononuclear phagocytic cells in rat spleen and liver

The localization of calcium-dependent phospholipase A2, (PLA2) immunochemically closely related to the enzyme of the viperid and crotalid type (group II), in cells isolated from rat spleen and liver was examined using a polyclonal antibody directed against rat spleen group II, PLA2 (PLA2M). In isolated spleen cells, the monocyte/macrophage fraction had the highest PLA2 activity (1.28 +/- 0.35.min-1.10(6) cells-1) which was almost completely inhibited by the anti-PLA2M antibody. An immunoblot analysis confirmed the presence of the enzyme in this fraction. An immunocytochemical study revealed that the PLA2 was present in spleen macrophages. In the isolated liver cells, Kupffer cells (0.92 +/- 0.22 nmol.min-1.10(6) cells-1) contained higher anti-PLA2M-antibody-inhibitable PLA2 activity than parenchymal cells (0.26 +/- 0.06.min-1.10(6) cells-1). The immunocytochemical study showed that cells immunopositive with anti PLA2M antibody were Kupffer cells. These results suggest that the mononuclear phagocytic cells in rat spleen and liver have relatively high activity of group-II-like PLA2. Subcellular distribution patterns of the anti-PLA2M-antibody-inhibitable phospholipase A2 activity in different cell populations from spleen and liver were compared. A mode of the distribution of the enzyme in the spleen macrophages was essentially similar to that in the spleen lymphocytes. The distribution in Kupffer cells was similar to that in parenchymal cells.     

Conformation and disulfide bond formation of the constant fragment of an immunoglobulin light chain: effect of truncation at the C-terminal region

Constant fragments with different carboxyl terminals, CL(109-211), CL(109-207), and CL-(109-200), were prepared by limited carboxypeptidase P or Y proteolysis of the constant fragment, CL-(109-214), of a type lambda immunoglobulin light chain, and their conformations and stabilities, and formation of the disulfide bond from the reduced fragments, were studied. No change in conformation or stability was observed on removal of three residues from the C-terminal end. Removal of seven or more residues from the C-terminal end destabilized the CL fragment. The rate of disulfide bond formation from reduced CL(109-207) was about 7 times faster than that for CL(109-214). These findings suggest that elongation of the polypeptide chain at least beyond the 207th residue is necessary for folding of the CL fragment into a definite conformation.     

Altered phosphorylation of Bacillus subtilis DegU caused by single amino acid changes in DegS.

The Bacillus subtilis sacU locus consists of the degS and degU genes, which play a major role in controlling the production of degradative enzymes including extracellular proteases. DegS has been shown to be autophosphorylated and to transfer the phosphoryl group to DegU. In this study, we partially purified the DegS proteins which carry amino acid changes resulting from various mutations and examined the phosphorylation reaction. The mutations used were degS42, causing a reduction in exoprotease production, and degS100(Hy) and degS200(Hy), causing overproduction of the enzymes. The following results were obtained. The DegS protein derived from degS42 was deficient in both autophosphorylation and subsequent phosphate transfer to DegU. Compared with wild-type DegS, the DegS proteins derived from the overproduction mutations, degS100(Hy) and degS200(Hy), were less active in the autophosphorylation and phosphorylation of DegU. However, the DegU phosphates produced by the mutant DegS proteins were more stable than that produced by the wild-type DegS. These results suggest that phosphorylation is tightly linked to exoprotease production and that the prolonged retention of the phosphoryl moiety on DegU activates the genes for the extracellular proteases. It was also shown that the rate of dephosphorylation of DegU-phosphate was increased as the amount of DegS was increased. All of these results suggest that DegS is involved in the dephosphorylation of DegU-phosphate.     

Receptor binding affinity and biological activity of C-terminal elongated forms of endothelin-1

Endothelin-1 (21 amino acids; ET-21) is considered to be derived from a precursor, proendothelin (38 amino acids; ET-38). In order to make the physiological significance of this conversion clear, we synthesized various C-terminal elongated derivatives of ET-21, such as ET-22, ET-23, ET-25, ET-31, ET-36 and ET-38 (each number implies the number of amino acid residues), and measured their receptor binding affinities and biological activities. When inhibition of [¹²⁵I]ET-21 binding to cultured rat smooth muscle cells (A10 cells) was measured, ET-21 inhibited with the highest affinity (IC₅₀ = 1.6 × 10⁻¹⁰ M) and the affinity of ET-38 was 30-fold less than that of ET-21. The binding affinities of the C-terminal elongated peptides were reduced with increasing number of amino acid residues, except for ET-22 whose affinity was lower than those of other peptides (IC₅₀ = 1.6 × 10⁻⁸ M). When contractions of rat aortic segments induced by these peptides were measured, ET-21 was the most potent (EC₅₀ = 2.8 × 10⁻¹⁰ M). All C-terminal elongated peptides, including ET-38, were more than 100-fold less active. It is noteworthy that ET-22 was the least potent peptide (EC₅₀ = 1.2 × 10⁻⁷ M). When bolus doses of C-terminal elongated peptides were administered to chemically denervated rats, the time-dependent change in blood pressure induced by each peptide was different from that induced by ET-21. Although ET-21 elicited a three phase depressor/pressor blood pressure response (an initial rapid hypotension, then a rapid transient hypertension followed by a slowly developing long-lasting hypertensive effect), the C-terminal elongated peptides, including ET-38, did not cause the initial transient hypotensive response. Very interestingly, the ability of the peptides to induce the rapid phase of hypertension in vivo does not seem to be correlated with the affinity of each peptide for the smooth muscle cell receptor, since the peptides with lower affinities for the smooth muscle receptor, such as ET-22, ET-23 and ET-25, showed more potent hypertensive effects. On the other hand, the slow and long-lasting hypertensive effect is likely to be related to the affinity of the compounds. The maximal hypertensive effects of cumulatively administered ET-21 derivatives were similar to those of ET-21. These results suggest that ET-21 is the most potent vasoconstrictor among the peptides and that the conversion from ET-38 to ET-21 may be important as an activation process.     

Target-specific innervation by autonomic and sensory nerve fibers in hairy fetal skin transplanted into the anterior eye chamber of adult rat

Summary Pieces of hairy skin tissue of fetal rat were transplanted into the anterior eye chamber of adult rats. The ability of autonomic and sensory nerve fibers from the host iris to innervate the grafted skin tissue was immunohistochemically and enzyme-histochemically examined using antisera against tyrosine hydroxylase (TH), substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP), and a reaction medium for acetylcholinesterase (AchE). The grafted tissue was successfully implanted and connected with the host iris. Epidermis, dermis, subcutaneous tissue, hairs, hair follicles, sebaceous glands, and piloerector muscles developed in the graft. Two weeks after transplantation, TH-, SP-, and CGRP-immunoreactive fibers were observed in association with the blood vessels in the graft. Four weeks after transplantation, TH-immunoreactive fibers were distributed in the piloerector muscles, whereas SP-and CGRP-immunoreactive fibers were present around the hair follicles. VIP-immunoreactive and AchE-positive fibers were restricted to the host iris at all survival times. These results suggest that the outgrowth of autonomic and sensory nerve fibers from the host iris show target specificity for the grafted skin tissue.     

Vibrio fluvialis andV. furnissii serotyping scheme for international use

A combination of two serotyping systems forVibrio fluvialis andV. furnissii, which were developed independently at the National Institute of Health (Tokyo) and Tokyo Metropolitan Research Laboratory of Public Health (Tokyo), was established as a single serotyping scheme comprising 35 O-antigen groups for international use.