The role of thromboxane A2 (TxA2) in allergic cutaneous reactions and the effect of (E)-3-[p-(1H-imidazol-1-ylmethyl) phenyl]-2-propenoic acid hydrochloride (OKY-046), a TxA2 synthetase inhibitor

To study the role of thromboxane A2 (TxA2) in cutaneous allergic reactions, the effect of (E)-3-[p-(1H-Imidazol-1-ylmethyl)phenyl]-2-propenoic acid hydrochloride (OKY-046), a selective TxA2 synthetase inhibitor, on cutaneous reactions in rats and mice was studied. Simultaneously, the effect of 9,11-methanoepoxy-prostaglandin H2 (U-46619), a stable analogue of TxA2, on capillary permeability in mouse and rat skin was investigated. Passive cutaneous anaphylaxis (PCA) in mouse ear was clearly inhibited by OKY-046 but not by indomethacin. The inhibitory action of OKY-046 was not influenced by pretreatment with indomethacin. Moreover, prostaglandin I2, which accumulated as a result of the inhibition of TxA2 synthetase, did not affect the PCA. But, the dye leakages caused by histamine, serotonin and leukotriene C4 in mouse ear were clearly inhibited by OKY-046. In addition, OKY-046 inhibited rat reversed cutaneous anaphylaxis, but its inhibitory action was not affected by pretreatment with indomethacin. Contrary to the above results, rat footpad passive Arthus reaction and mouse footpad tuberculin delayed hypersensitivity reaction were not affected by OKY-046. Additionally, U-46619 did not cause an increase of capillary permeability in either mouse and rat skin. These results suggest a slight role of TxA2 in cutaneous allergic reactions in mice and rats and the efficacy of OKY-046 on Type I and II reactions regardless of the inhibition of TxA2 synthetase activity.     

Primary structure of chicken pituitary prolactin deduced from the cDNA sequence. Conserved and specific amino acid residues in the domains of the prolactins

The perform of chicken prolactin (PRL) deduced from the cDNA sequence contains a signal peptide of 30 amino acid residues followed by a mature PRL of 199 residues. Chicken PRL shows 77, 68, 67, 58, and 31% identity of amino acid sequence with whale, human, ovine, rat, and salmon PRLs, respectively. Elucidation of the primary structure of avian PRL enabled extended analysis of the specific and conserved amino acid residues and domains of the PRL molecules. The mammalian, teleostean, and avian PRLs share 32 common residues, and these conserved residues are observed to cluster in four distinct domains (PD1 to PD4), corresponding to four of five conserved domains of the growth hormones. Of the 32 residues, 8 residues in the PD2 and PD4 domains, including 4 cysteines, are conserved by other members of the growth hormone family, which indicates that these 8 residues may be essential for common structural features of the gene family. On the other hand, 13 other residues distributed among all four domains are conserved almost exclusively in the PRLs, suggesting that these residues are indispensable for specific binding of the PRLs to their receptors.     

Identification of two types of smooth muscle myosin heavy chain isoforms by cDNA cloning and immunoblot analysis

We previously reported the characterization of a rabbit uterus cDNA clone (SMHC29) which encoded part of the light meromyosin of smooth muscle myosin heavy chain (Nagai, R., Larson, D.M., and Periasamy, M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1047-1051). We have now characterized a second cDNA clone (SMHC40) which also encodes part of the light meromyosin but differs from SMHC29 in the following respects. Nucleotide sequence analysis demonstrates that the two myosin heavy chain mRNAs are identical over 1424 nucleotides but differ in part of the 3'-carboxyl coding region and a portion of the 3'-nontranslated sequence. Specifically, SMHC40 cDNA encodes a unique stretch of 43 amino acids at the carboxyl terminus, whereas SMHC29 cDNA contains a shorter carboxyl terminus of 9 unique amino acids which is the result of a 39-nucleotide insertion. Recent peptide mapping of smooth muscle myosin heavy chain identified two isotypes with differences in the light meromyosin fragment that were designated as SM1 (204 kDa) and SM2 (200 kDa) type myosin (Eddinger, T. J., and Murphy, R.A. (1988) Biochemistry 27, 3807-3811). In this study we present direct evidence that SMHC40 and SMHC29 mRNA encode the two smooth muscle myosin heavy chain isoforms, SM1 and SM2, respectively, by immunoblot analysis using antibodies against specific carboxyl terminus sequences deduced from SMHC40 and SMHC29 cDNA clones.     

Diacylglycerol, but not inositol 1,4,5-trisphosphate, accounts for platelet-derived growth factor-stimulated proliferation of BALB 3T3 cells

Recently we found that an intracellular event related to phosphatidylinositol 4,5-bisphosphate (PIP2) is crucial for platelet-derived growth factor (PDGF)-induced mitogenesis in fibroblastic cells (Matuoka, K., et al.: Science 239:640-643, 1988). In the present study we examined the mitogenic effects of PIP2 and its hydrolysis products introduced into the cytoplasm of BALB 3T3 cells by micro-injection to confirm the role of PIP2 hydrolysis in PDGF stimulation of cell proliferation. Injection of 1,2-dioleylglycerol (diolein) into serum-deprived quiescent cells induced DNA synthesis with the same time course as that induced by exposure of the cells to PDGF and, in the presence of PDGF, caused no additional increase in the cell population entering S phase. The injection of PIP2, inositol 1,4,5-trisphosphate, or 1,2-dioleylphosphatidic acid into the cells did not induce mitogenesis. Consistent results were obtained in experiments in which the cells were exposed to 1-oleyl-2-acetylglycerol (OAG) and ionomycin; namely, OAG stimulated proliferation of BALB 3T3 cells, but ionomycin did not induce any mitogenesis. Desensitization of the protein kinase C pathway by prolonged exposure of the cells to phorbol ester abolished the induction of cell proliferation by subsequent injection of diolein or exposure to phorbol ester or OAG as well as by PDGF challenge. These findings strongly suggest that activation of the protein kinase C system following formation of diacylglycerol by PIP2 hydrolysis is mainly responsible for the mitogenic action of PDGF on BALB 3T3 cells.     

Hypervariability of intronic simple (gt)n(ga)m repeats in HLA-DRB genes

We have investigated the extent of DNA variability in intronic simple (gt)_n(ga)_m repeat sequences and correlated this to sequence polymorphisms in the flanking exon 2 of HLA-DRB genes. The polymerase chain reaction (PCR) was used to amplify a DNA fragment containing exon 2 and the repeat region of intron 2. The PCR products were separated on sequencing gels in order to demonstrate length hypervariability of the (gt)_n(ga)_m repeats. In a parallel experiment, the PCR products were cloned and sequenced (each exon 2 plus adjacent simple repeats) to characterize the simple repeats in relation to the HLA-DRB sequences. In a panel of 25 DRB1, DRB4, and DRB5 alleles new sequences were not detected. Restriction fragment length polymorphism (RFLP) subtyping of serologically defined haplotypes corresponds to translated DNA sequences in 85% of the cases, the exceptions involving unusual DR/DQ combinations. Many identical DRB1 alleles can be distinguished on the basis of their adjacent simple repeats. We found group-specific organization of the repeats: the DRw52 supergroup repeats differ from those of DRB1*0101, DRB4*0101, and DRB5*0101 alleles and from those of pseudogenes. Finally, we amplified baboon DNA and found a DRB allele with extensive similarity to DRB1 sequences of the DRw52 supergroup. The simple repeat of the baboon gene, however, resembles that of human pseudogenes. In addition to further subtyping, the parallel study of polymorphic protein and hypervariable DNA alleles may allow conclusions to be drawn on the relationships between the DRB genes and perhaps also on the theory of trans-species evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 34258.     

Quantification of gene expression over a wide range by the polymerase chain reaction.

We investigated the usefulness of the polymerase chain reaction (PCR) method for the relative quantification of gene expression using a simultaneously amplified sequence of beta-actin mRNA as an internal control for the target sequence of tax/rex mRNA of human T-cell leukemia virus type I. The PCR product of the internal control was reduced by delaying the addition of the primers for its sequence. The photostimulated luminescence of the bands was measured with a laser image analyzer, and the values were plotted against the cycle number. The cycle differences between the logarithmic phase of the curves for the target sequence and for beta-actin (delta cycle) showed a linear correlation with the initial concentration of the sample. This method is highly sensitive for evaluating gene expression over a wide range.     

Heterogeneity in smooth muscle cell population accumulating in the neointimas and the media of poststenotic dilatation of the rabbit carotid artery.

Rabbit smooth muscles contain at least three types of myosin heavy chain (MHC) isoforms; SM1, SM2 and SMemb (NMHC-B), the expression of which is developmentally regulated. We have recently reported that smooth muscles with the embryonic phenotype accumulate in the neointimas produced by endothelial denudation or high-cholesterol feeding. In this study, we examined MHC isoform expression in the neointimas and the media of poststenotic dilatation of the rabbit carotid artery, and determined the phenotype of the smooth muscle cell in the dilated segment. We report here that neointimal cells in the dilated segment are smooth muscle cells with the embryonic phenotype as previously reported in our ballooning-injury study. The medial smooth muscles, however, are composed of heterogeneous population of smooth muscles which differ in stage of differentiation as determined by the MHC isoform expression. These results indicate that MHC isoforms are useful molecular markers to identify abnormally proliferating smooth muscles in diseased arteries and to understand the process of atherogenesis occurring following vascular injury.     

Regulation mechanisms of intracellular pH of Xenopus laevis oocyte.

Intracellular pH values (pHi) of Xenopus oocytes were optically measured using a fluorescent dye, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The oocytes were loaded with dye by incubation with a membrane-permeable form (BCECF-AM). Mean pHi of the oocytes in pH 7.6 solution was 7.69. Increasing ambient pCO2 rapidly decreased pHi and estimated buffering power was 23.8 mM/pH unit. Changing ambient HCO3- from 5 to 30 mM did not alter pHi. After incubation in a Na(+)-free solution, Na+ addition to the bath rapidly increased pHi and this response was blocked by amiloride (ED50 2 microM). The addition of NH4Cl to the bath caused an initial transient increase in PHi followed by a secondary decrease. The secondary decrease was greatly inhibited by a histidine specific reagent, diethylpyrocarbonate. It was also slightly inhibited by ouabain, Ba2+ and furosemide, but not by amiloride. These data suggest that (1), fluorescence technique is applicable to PHi measurements of Xenopus oocytes; (2), Xenopus oocytes have an amiloride sensitive Na+/H(+)-exchange, and permeabilities to CO2, NH3, and NH+4. These observation may be useful in studying the relationship between pHi and oocytes development, and the expression of acid/base transporters in Xenopus oocytes.     

An endoprotease homologous to the blood clotting factor X as a determinant of viral tropism in chick embryo.

Host cell proteases responsible for activation of viral fusion glycoproteins are an important determinant for spread and tropism of various animal viruses. Exemplifying such proteases for the first time, we isolated an endoprotease from chick embryo, that activates para- and orthomyxovirus fusion glycoproteins by cleaving their precursor proteins at a specific, single arginine site. The protease is a calcium dependent serine protease consisting of two subunits, the 33 kd catalytic chain and the 23 kd chain possibly required for Ca2+ binding, and was found to be highly homologous, if not identical, to the blood clotting factor X(FX), a member of the prothrombin family. Its high efficiency and specificity in cleavage reactions was attributable to the properties characteristic of FX. Its role in vivo was strongly supported by cleavage inhibition in ovo highly selective for this virus group with a specific peptide inhibitor against FX.