In order to identify and characterize genetic polymorphism of the swine major histocompatibility complex ( Mhc: SLA) class I genes, RT-PCR products of the second and third exons of the three SLA classical class I genes, SLA-1, SLA-2 and SLA-3 were subjected to nucleotide determination. These analyses allowed the identification of four, eight and seven alleles at the SLA-1, SLA-2 and SLA-3 loci, respectively, from three different breeds of miniature swine and one mixed breed. Among them, 12 alleles were novel. Construction of a phylogenetic tree using the nucleotide sequences of those 19 alleles indicated that the SLA-1 and -2 genes are more closely related to each other than to SLA-3. Selective forces operating at single amino acid sites of the SLA class I molecules were analyzed by the Adaptsite Package program. Ten positive selection sites were found at the putative antigen recognition sites (ARSs). Among the 14 positively selected sites observed in the human MHC ( HLA) classical class I molecules, eight corresponding positions in the SLA class I molecules were inferred as positively selected. On the other hand, four amino acids at the putative ARSs were identified as negatively selected in the SLA class I molecules. These results suggest that selective forces operating in the SLA class I molecules are almost similar to those of the HLA class I molecules, although several functional sites for antigen and cytotoxic T-lymphocyte recognition by the SLA class I molecules may be different from those of the HLA class I molecules.
The combination of PCR-SSP and PCR-RFLP methods facilitate the rapid identification of the three haplotypes and SLA class I and II homozygotes in individual Clawn miniature swine.
We investigated the role of 20 kDa myosin light chain (MLC20) phosphorylation in contractions following protein kinase C (PKC) activation by 12-deoxyphorbol-13-isobutyrate (DPB) in rabbit aortae. DPB induced a sustained contraction and phosphorylation of MLC20 independent of a change in cytosolic Ca2+ ([Ca2+]i). Phosphorylation on Ser19 of MLC20, which is a target site of MLC kinase (MLCK), was 9.2 +/- 5.1% and 22.3 +/- 4.9% of the phosphorylation caused by KCl, at 5 and 30 min of application of DPB, respectively. When KCl-precontracted muscles were rinsed with Ca2+-free, EGTA solution, [Ca2+]i rapidly declined, MLC20 was dephosphorylated and the tension decreased. If DPB was present in the Ca2+-free solution, the relaxation and the dephosphorylation of either total MLC20 or Ser19 were inhibited. The phospholipase A2 inhibitor ONO-RS-082 partially antagonized the effects of DPB on the tension and the MLC20 dephosphorylation. In Ca2+-free solution, DPB induced a contraction smaller than that in normal solution without an increase in MLC20 phosphorylation, and the contraction was also sensitive to ONO-RS-082. These results suggest that a part of MLC20 phosphorylation following PKC activation is due to inhibition of MLC20 phosphatase and the phosphorylation is responsible for the contraction. Furthermore, a mechanism independent of [Ca2+]i and phosphorylation may play a significant role in the PKC-dependent contraction. The involvement arachidonic acid is suggested, not only in the inhibition of dephosphorylation but also in the Ca2+-independent regulation of contractile proteins.
Protein kinase C (PKC) activation by a phorbol ester increases myosin light chain (MLC(20)) phosphorylation through inhibition of MLC phosphatase (MLCP) and enhances contraction of vascular smooth muscle. We investigated whether Rho kinase, which is known to inhibit MLCP, is involved in the MLC(20) phosphorylation caused by a phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), in rabbit aortas. DPB (1 microM) increased MLC(20) phosphorylation and tension. The Rho kinase inhibitor fasudil (10 microM) inhibited the DPB-induced contraction and decreased the MLC(20) phosphorylation at Ser19, a site phosphorylated by MLC kinase, although it did not affect the phosphorylation of total MLC(20). Rinsing a 65.4 mM KCl-contracted aorta with Ca(2+)-free, EGTA solution caused rapid dephosphorylation of MLC(20) and relaxation. When DPB was present in the rinsing solution, the MLC(20) dephosphorylation and the relaxation were inhibited. In this protocol, Ro31-8220 (10 microM), a PKC inhibitor, suppressed the phosphorylation of total MLC(20) and Ser19 induced by DPB. Fasudil also inhibited the Ser19 phosphorylation to a degree similar to Ro31-8220 and accelerated relaxation, which was less than the relaxation caused by Ro31-8220. The phospholipase A(2) inhibitor ONO-RS-082 (5 microM) inhibited the DPB-induced Ser19 phosphorylation but only transiently decreased the tension, suggesting the involvement of arachidonic acid in the phosphorylation and the existence of a MLC(20) phosphorylation-independent mechanism. When fasudil was combined with ONO-RS-082, fasudil exerted additional inhibition of the tension without further inhibition of the Ser19 phosphorylation. DPB phosphorylated the 130 kDa myosin binding subunit (MBS) of MLCP and fasudil inhibited the phosphorylation. These data suggest that the inhibition by fasudil of DPB-induced contraction and phosphorylation of MLC(20) at the MLC kinase-targeted site is a result of inhibition of Rho kinase. Thus, the PKC-dependent Ca(2+)-sensitization of vascular smooth muscle involves Rho kinase. A MLC(20) phosphorylation-independent mechanism is also involved in the Ca(2+)-sensitization.
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