A simple method for preparing physiologically active mitochondria from plant leaves rich in oils and phenolics

Amberlite XAD-7, a nonionic polyacrylate adsorbent, was found to be a very effective protectant for isolating mitochondria from tissues rich in oils and phenolics. Physiologically active, well-coupled mitochondria were successfully prepared from young green leaf tissues of citrus, apple, pear and tobacco.Abbreviations DNP 2,4-dinitrophenol - CCCP carbonyl cyanide m-chlorophenylhydrazone - BSA bovine serum albumin - PVP polyvinylpyrrolidone     

Activation of quiescent ovaries by administration of PMSG after LH-RH analogue treatment in heifers

The effect of pregnant mare serum gonadotrophin (PMSG) treatment on activation of quiescent ovaries was examined in heifers. Groups of thirteen, twenty and twelve heifers which showed ovulation within 2 d and corpus luteum (CL) development after injection with a luteinizing hormone releasing hormone analogue (LH-RH-A) were supplementally injected with 500 IU of PMSG (Group I); 500 IU of PMSG and 500 mug of Prostaglandin F(2alpha) analogue (PGF(2alpha)-A; Group II); and 500 mug of PGF(2alpha)-A (Group III) on Day 6 after the injection of 200 mug of LH-RH-A (Day 0), respectively. Estrus appeared in 33.3 to 45.0% of the heifers of the respective groups after the treatment. Ovulation occurred at a significantly (P<0.01) higher rate in Groups I (100%) and II (90.0%) than in Group III (41.7%). The ovarian cyclic activity was initiated in all the heifers that ovulated. Plasma progesterone levels decreased significantly (P<0.05) to about 1 ng/ml on Day 8 and Day 7 in Group I and Groups II and III, respectively. Plasma estradiol-17beta (E(z)) levels increased significantly (P<0.05), reaching a peak on Days 7 to 7.5 in Groups I and II but not in Group III. It is concluded that PMSG treatment stimulates maturation and E(z) secretion of a follicle, thus promoting ovulation and the onset of ovarian cyclic activity.     

The evidence for post-meiotic expression of a testis-specific isoform of a regulatory subunit of calcineurin using a monoclonal antibody.

The expression of a regulatory subunit of calcineurin (CaN beta) during rat spermatogenesis was examined in rat testes using a monoclonal antibody Va1. Results showed that a testis-specific isoform of CaN beta was expressed only 3 weeks after birth, when meiosis begins, and increased in amount depending on the maturation of spermatogenesis. The matured sperm, which consists of only post-meiotic cells, is most likely to have only the testis-specific isoform of CaN beta. The brain type isoform of CaN beta was not detected in rat sperm. Immunoblot analysis of testes from different rodent species by a monoclonal antibody Va1 showed that all rodent species examined had their own homologues corresponding to a testis-specific isoform of CaN beta in rats, although they showed distinctively different molecular weights on SDS-PAGE compared to the testis-specific isoform in rats. Each homologue was shown to be specifically expressed in post-meiotic phase of spermatogenesis, as was seen in rats.     

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.     

Enzymatic synthesis of alkyl xylobioside and xyloside from xylan and alcohol

Summary Alkyl -D-xylobioside and alkyl -D-xyloside were prepared by the one-pot reaction of xylan and a fatty alcohol, such as 1-octanol, 1-decanol, 2-octanol and 2-ethylhexanol using the cell-free culture filtrate of the xylan-assimilating strain, Aureobasidium pullulans KK415. Using this strain, a novel surfactant, alkyl -D-xylobioside, was produced as the main product when the alcohol and xylan was incubated at a temperature of 65 °C and pH 4.5.     

Immunocytochemical localization of phenylalanine ammonia-lyase in tissues of Populus kitakamiensis

The polypeptide encoded by the partial fragment of cDNA of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), PALcDNAl (Osakabe et al., 1995, Plant Sci. 105: 217–226), isolated from Populus kitakamiensis (P. sieboldii x P. grandidentata), was expressed in Escherichia coli cells. The polypeptide was purified and an antiserum raised against it. The antiserum recognized a protein of 77 kDa on nitrocellulose blots after sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of total protein and the partially purified PAL protein from P. kitakamiensis. Moreover,the antiserum recognized a protein on the blot after non-denaturing polyacrylamide gel electrophoresis of P. kitakamiensis proteins and this protein had PAL activity. Furthermore, the antibody inhibited PAL activity of extracts from stem tissues. These results showed that the antiserum against the partial PAL peptide recognized only the PAL subunits in extracts of P. kitakamiensis. Immunolocalization studies of P. kitakamiensis tissues revealed that the PAL protein was specifically localized in the xylem and the phloem fibers and no immunogold signal was found in the epidermis, the cortex, the pith, or the cambium of either stems or leaves.Abbreviations IgG immunoglobulin G - IPTG isopropylthio--d-galactoside - PAL phenylalanine ammonia-lyase The authors thank Dr. Kunio Hata of Nippon Paper Industries Co., Ltd. (Japan) for supplying P. kitakamiensis. This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 07406008).     

A cis-acting element and a trans-acting factor involved in the wound-induced expression of a horseradish peroxidase gene

The mechanisms that control the wound-induced expression of the prxC2 gene for horseradish peroxidase (HRP) have been investigated. Analysis of the regulatory properties of 5′-deleted promoters showed that a positive element involved in the response to wounding was located between −307 and −99 bp from the site of initiation of translation. In in vitro binding assays of tobacco nuclear proteins and DNA fragments of prxC2 promoter, the binding site was the Box 1 from −296 to −283 containing the CACGTG motif. To identify the functional role of Box 1, the prxC2 promoter that has been digested from the 5′ end to −289 with a disrupted Box 1 was fused to a reporter gene for β-glucuronidase (GUS). No induction of GUS activity was observed in transgenic tobacco plants with the prxC2(−289)/GUS construct. These data indicated that the expression of prxC2 in response to wounding required the Box 1 sequence from −296 to −283. Furthermore, a tobacco cDNA expression library was screened and a cDNA clone for a protein, designated TFHP-1, that bound specifically to the Box 1 sequence was identified. The putative TFHP-1 protein contains a basic region and leucine zipper (bZip) motif and a helix—loop—helix (HLH) motif. The mRNA for TFHP-1 was abundant in roots and stems, and it was not induced by wounding in leaves. In tobacco protoplasts, antisense TFHP-1 suppressed the expression of prxC2 (−529)/GUS.     

Suppression of lymphocyte spontaneous proliferative response by proteolipid protein peptide in patients with HAM/TSP

To understand the immune mechanism suggested in HTLV-I-associated myelopathy (HAM/TSP), we investigated T cell response to proteolipid protein (PLP). Because of high autologous proliferative response (APR) of peripheral blood mononuclear cells (PBMC) in culture, the lymphocyte proliferation assay was not useful in this disease. Unexpectedly, however, APR was profoundly (70–98%) suppressed in 6 of 9 cases when PLP peptide 105-124 was added in the culture. PLP peptide 85-104 or 145-159 also suppressed APR in a few cases. Time course study showed that the peptide-mediated suppression became apparent after day 4 in culture. The results can be interpreted as that suppressor cells recognizing the PLP peptides were present in the PBMC of HAM/TSP patients and suppressed the APR as the consequence of antigen specific response. This may indicate that a T cell response to certain PLP determinants is involved in the pathomechanism of HAM/TSP at least in part. Molecular mimicry between PLP and HTLV-I mayaccount for the T cell sensitization to PLP in HAM/TSP.Special issue dedicated to Dr. Marjorie B. Lees.     

Factor in urinary extracts from pregnant women that inhibit mouse oocyte maturation in vitro

Mouse oocyte maturation inhibitory factors, on the basis of inhibitory activity of spontaneous germinal vesicle breakdown (GVBD) of denuded mouse oocytes in culture, were extracted and partially purified by reversed-phase resin adsorption and Sephadex G-100 and G-50 column chromatographies from the urine of pregnant women. Denuded oocytes obtained from ovaries of ICR mice underwent spontaneous GVBD by cultivation for 3 h in modified Krebs–Ringer's buffered solution, while this spontaneous GVBD was found to be inhibited by adding the final preparation (U-D-4) of urine. The inhibition was dose dependent, ranging from 0.6 to 10 μg protein/ml medium. Oocytes treated with U-D-4 and resuspended in control medium resumed GVBD. The molecular mass of U-D-4 was estimated to be less than 2,000 Da with gel filtration. Ether treatment failed to extract inhibitory factor(s) from U-D-4 and pepsin treatment inactivated U-D-4, indicating that inhibitory factor(s) in U-D-4 are peptide-like substances. The inhibitory effect of U-D-4 on spontaneous GVBD was partially reversed in the presence of naloxone, a potent opioid antagonist. U-D-4s obtained from urine samples of pregnant women, nonpregnant women, and men showed the inhibitory effect on spontaneous GVBD; however, the activity of U-D-4 obtained from pregnancy urine was significantly more potent than those of the other urine samples. © 1993 Wiley-Liss, Inc.