Evolution of rbcL group IA introns and intron open reading frames within the colonial Volvocales (Chlorophyceae)

Mobile group I introns sometimes contain an open reading frame (ORF) possibly encoding a site-specific DNA endonuclease. However, previous phylogenetic studies have not clearly deduced the evolutionary roles of the group I intron ORFs. In this paper, we examined the phylogeny of group IA2 introns inserted in the position identical to that of the chloroplast-encoded rbcL coding region (rbcL-462 introns) and their ORFs from 13 strains of five genera (Volvox, Pleodorina, Volvulina, Astrephomene, and Gonium) of the colonial Volvocales (Chlorophyceae) and a related unicellular green alga, Vitreochlamys. The rbcL-462 introns contained an intact or degenerate ORF of various sizes except for the Gonium multicoccum rbcL-462 intron. Partial amino acid sequences of some rbcL-462 intron ORFs exhibited possible homology to the endo/excinuclease amino acid terminal domain. The distribution of the rbcL-462 introns is sporadic in the phylogenetic trees of the colonial Volvocales based on the five chloroplast exon sequences (6021 bp). Phylogenetic analyses of the conserved intron sequences resolved that the G. multicoccum rbcL-462 intron had a phylogenetic position separate from those of other colonial volvocalean rbcL-462 introns, indicating the recent horizontal transmission of the intron in the G. multicoccum lineage. However, the combined data set from conserved intron sequences and ORFs from most of the rbcL-462 introns resolved robust phylogenetic relationships of the introns that were consistent with those of the host organisms. Therefore, most of the extant rbcL-462 introns may have been vertically inherited from the common ancestor of their host organisms, whereas such introns may have been lost in other lineages during evolution of the colonial Volvocales. In addition, apparently higher synonymous substitutions than nonsynonymous substitutions in the rbcL-462 intron ORFs indicated that the ORFs might evolve under functional constraint, which could result in homing of the rbcL-462 intron in cases of spontaneous intron loss. On the other hand, the presence of intact to largely degenerate ORFs of the rbcL-462 introns within the three isolates of Gonium viridistellatum and the rare occurrence of the ORF-lacking rbcL-462 intron suggested that the ORFs might degenerate to result in the spontaneous intron loss during a very short evolutionary time following the loss of the ORF function. Thus, the sporadic distribution of the rbcL-462 introns within the colonial Volvocales can be largely explained by an equilibrium between maintenance of the introns by the intron ORF and spontaneous loss of introns when the introns do not have a functional ORF.     

Actin-Bundling Protein Isolated from Pollen Tubes of Lily : Biochemical and Immunocytochemical Characterization

A 135-kD actin-bundling protein was purified from pollen tubes of lily (Lilium longiflorum) using its affinity to F-actin. From a crude extract of the pollen tubes, this protein was coprecipitated with exogenously added F-actin and then dissociated from F-actin by treating it with high-ionic-strength solution. The protein was further purified sequentially by chromatography on a hydroxylapatite column, a gel-filtration column, and a diethylaminoethyl-cellulose ion-exchange column. In the present study, this protein is tentatively referred to as P-135-ABP (Plant 135-kD Actin-Bundling Protein). By the elution position from a gel-filtration column, we estimated the native molecular mass of purified P-135-ABP to be 260 kD, indicating that it existed in a dimeric form under physiological conditions. This protein bound to and bundled F-actin prepared from chicken breast muscle in a Ca²⁺-independent manner. The binding of 135-P-ABP to actin was saturated at an approximate stoichiometry of 26 actin monomers to 1 dimer of P-135-ABP. By transmission electron microscopy of thin sections, we observed cross-bridges between F-actins with a longitudinal periodicity of 31 nm. Immunofluorescence microscopy using rhodamine-phalloidin and antibodies against the 135-kD polypeptide showed that P-135-ABP was colocalized with bundles of actin filaments in lily pollen tubes, leading us to conclude that it is the factor responsible for bundling the filaments.Actin filaments, one of the major components of the cytoskeleton, are organized into a highly ordered architecture and are involved in various kinds of cell motility. Their architecture is regulated by several kinds of actin-binding proteins, including cross-linking proteins, severing proteins, end-capping proteins, and monomer-sequestering proteins in animal, protozoan, and yeast cells (Stossel et al., 1985; Pollard and Cooper, 1986; Vandekerckhove and Vancompernolle, 1992). In plant cells the organization of the actin cytoskeleton also changes remarkably during the cell cycle or during developmental processes, and it is suggested that actin-binding proteins are involved in their dynamic change. However, little is known about actin-binding proteins in plant cells.Only a low-M_r actin-binding and -depolymerizing protein, profilin, in white birch (Betula verrucosa; Valenta et al., 1991), maize (Zea mays; Staiger et al., 1993; Ruhlandt et al., 1994), bean (Phaseolus vulgaris; Vidali et al., 1995), tobacco (Nicotiana tabacum; Mittermann et al., 1995), tomato (Lycopersicon esculentum; Darnowski et al., 1996), Arabidopsis (Arabidopsis thaliana; Huang et al., 1996), and lily (Lilium longiflorum; Vidali and Hepler, 1997), and an ADF in lily (Kim et al., 1993), rapeseed (Brassica napus; Kim et al., 1993), and maize (Rozycka et al., 1995; Lopez et al., 1996), have been identified by biochemical or molecular biological means.The native and recombinant forms of these proteins are capable of binding to animal or plant actin (Valenta et al., 1993; Giehl et al., 1994; Ruhlandt et al., 1994; Lopez et al., 1996; Perelroizen et al., 1996; Carlier et al., 1997). Plant profilin expressed in mammalian BHK-21 cells (Rothkegel et al., 1996) or profilin-deficient Dictyostelium discoideum cells (Karakesisoglou et al., 1996) was able to functionally substitute for endogenous profilin in these cells. The introduction of plant profilin into living stamen hair cells by microinjection caused the rapid reduction of the number of actin filaments (Staiger et al., 1994; Karakesisoglou et al., 1996; Ren et al., 1997). These results indicate that plant profilin and ADF share many functional similarities with other eukaryote profilins and ADFs.It is well known that the actin cytoskeleton undergoes dynamic changes in organization during hydration and activation of the vegetative cells of pollen grains (Pierson and Cresti, 1992). Before hydration actin filaments exist as fusiform or spiculate structures (a storage form), but they are rearranged to form a network upon hydration (Heslop-Harrison et al., 1986; Tiwari and Polito, 1988). In the growing pollen tube there are strands or bundles of actin filaments parallel to the long axis (Perdue et al., 1985; Pierson et al., 1986; Miller et al., 1996) that are involved in cytoplasmic streaming (Franke et al., 1972; Mascarenhas and Lafountain, 1972) and transport of vegetative nuclei and generative cells to the growing tip (Heslop-Harrison et al., 1988; Heslop-Harrison and Heslop-Harrison, 1989). Characterization of the function of actin-binding proteins is essential to understanding the regulation of actin organization during the developmental process of pollen. Since only a small number of vacuoles containing proteases develop in pollen grains and pollen tubes at a younger stage, pollen tubes are suitable materials for isolating and biochemically studying actin-binding proteins responsible for organizing actin filaments into various forms.In a previous paper we reported that several components in a crude extract prepared from lily pollen tubes, including a 170-kD myosin heavy chain and 175-, 135-, and 110-kD polypeptides, could be coprecipitated with exogenously added F-actin (Yokota and Shimmen, 1994). We also found that rhodamine-labeled F-actin was tightly bound to the glass surface treated with the fraction containing the 135- and 110-kD polypeptides (Yokota and Shimmen, 1994). These results suggested that either one or both of the 135- and 110-kD polypeptides possesses an F-actin-binding activity. In the present study, we purified the 135-kD polypeptide from lily pollen tubes by biochemical procedures and then characterized its F-actin-binding properties and distribution in the pollen tubes. This protein was able to bundle F-actin isolated from chicken breast muscle and colocalized with actin-filament bundles in pollen tubes. We refer to this protein as P-135-ABP (Plant 135-kD Actin-Bundling Protein).     

Purification and characterization of NADPH-cytochrome P-450 reductase from rat epidermis

NADPH-cytochrome P-450 oxidoreductase (P-450 red) transfers reducing equivalents from NADPH to cytochrome P-450 (P-450) in the monooxygenase system. Detergent solubilized proteins from the membrane fraction of neonatal rat epidermis were purified by 2′,5′-ADP-agarose affinity column chromatography. The purified protein showed an apparent homogeneity on sodium dodecylsulfate-polyacrylamide gel electrophoresis and molecular weight was estimated to be 78 kDa. NADPH-cytochrome c reductase activity increased by 95-fold in the purified enzyme. Epidermal P-450 red in vitro reconstituted benzo(a)pyrene hydroxylase activity in a dose dependent manner with P-450 purified from either rat liver or epidermis. Western blot analysis demonstrated that epidermal P-450 red immunologically cross reacts to liver P-450 red. Immunohistochemical staining showed that the enzyme was predominantly localized in the epidermis. The intensity of immunohistochemical staining of rat skin sections and tissue distribution did not change in the skin treated with β-naphtoflavone, which results in a substantial increase in P-450 1A1 activity. Quantitative assessment of P-450 red in treated and untreated epidermis also showed no change. These findings indicate that constitutive P-450 red, fully capable of supporting P-450, exists in rat epidermis, and can function in metabolism of endogenous and exogenous compounds.     

Inhibitory spectrum of mouse contrapsin and alpha-1-antitrypsin against mouse serine proteases

Contrapsin and alpha-1-antitrypsin have been recently characterized as major protease inhibitors in mouse plasma (Takahara, H. & Sinohara, H. (1982) J. Biol. Chem. 257, 2438-2446). We have studied the effects of the two inhibitors upon various serine proteases prepared from mouse tissues. Trypsin, plasmin and trypsin-like proteases of the submaxillary gland were inhibited by contrapsin but not by alpha-1-antitrypsin. On the other hand, chymotrypsin, elastase, and thrombin were inactivated by alpha-1-antitrypsin but not by contrapsin. Thus, their inhibitory spectra did not overlap each other in spite of their broad specificities. The inhibition of trypsin, chymotrypsin, and elastase was rapid and stoichiometric, whereas the inhibition of the other proteases was relatively slow. Contrapsin accounted for almost the total capacities of mouse plasma to inhibit both trypsin and submaxillary gland trypsin-like proteases, whereas alpha-1-antitrypsin was responsible for nearly all the capacities of plasma to inhibit both chymotrypsin and elastase.     

Purification and characterization of peptidylarginine deiminase from rabbit skeletal muscle

The preceding paper described the identification and some properties of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle, kidney, brain, and lung. In the present work we purified peptidylarginine deiminase from rabbit skeletal muscle with a 16% yield by 7 steps. The purification involved ion-exchange chromatography on DEAE-Sephacel, gel filtration on Bio-Gel A-0.5 m, and affinity chromatography on soybean trypsin inhibitor-Sepharose 4B and aminohexyl-Sepharose 4B. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be about 83,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 130,000-140,000 by gel filtration on Sephadex G-200. The isoelectric point was 5.3 and the amino acid composition was also determined. The enzyme preferably catalyzed the formation of citrulline derivatives from arginine derivatives in which both the amino and carboxyl groups were substituted and showed the highest activity towards Bz-L-Arg-O-Et among the arginine derivatives tested. The Km value for Bz-L-Arg-O-Et was found to be 0.50 X 10(-3) M. The enzyme also showed marked activities towards native protein substrates, such as protamine sulfate, soybean trypsin inhibitor, histone and bovine serum albumin.     

Effects of 2-bromo-α-ergocryptine on β-endorphin-induced growth hormone,prolactin and luteining hormone release in urethane anesthetized rats

Adult male rats were injected intraperitoneally either with saline or 2-Br- α-ergocryptine(CB-154)(10 ng/0.5 ml/rat) 30 min prior to an intraventricular injection of saline or β-endorphin (1 μg/10 μl or 5 μg/10 μl) and 30 min after β-endorphin, they were sacrificed by decapitation. Intraventricular injection of β-endorphin elicited significant increases in serum GH, prolactin and LH levels in a dose-related manner. Pretreatment with CB-154 inhibited the release of GH, prolactin and LH induced by β-endorphin. These results indicate that the stimulatory effects of β-endorphin on GH, prolactin and LH may be involved in an inhibition of dopaminergic mechanism in the central nervous system.     

Evidence for immunoreactive relaxin in boar seminal vesicles using combined light and electron microscope immunocytochemistry.

Light-microscope immunocytochemistry using the peroxidase-antiperoxidase technique and a polyclonal rabbit antiserum raised against purified porcine relaxin showed that cytoplasmic immunostaining for relaxin could be visualized in the epithelial cells of the seminal vesicle. No relaxin immunoreactivity was seen in the testis, epididymis, ductus deferens, prostate or bulbo-urethral gland. A ten times higher concentration of porcine relaxin antiserum was necessary to achieve immunostaining in the seminal vesicle comparable to that in the corpora lutea of pregnant sows. Ultrastructural examination showed that the epithelial cells of the boar seminal vesicle resembled typical protein-secreting cells with prominent rough endoplasmic reticulum and well-developed Golgi apparatus. The most striking feature of these cells was the accumulation of granules with a limiting membrane, which ranged from 200 to 600 nm in diameter and contained flocculent material of moderate electron density. Electron-microscope immunocytochemistry using the protein A-gold technique and relaxin antiserum demonstrated that the granules were the only intracellular organelles that showed immunoreactivity for relaxin. These results indicate that a relaxin-like substance is present in boar seminal vesicles and that the subcellular site of its localization is the granules, suggesting that the seminal vesicle produces and stores a relaxin-like substance, but that it is present at much lower concentrations than in the corpora lutea of pregnant sows.     

Cross fertilization between sea urchin eggs and oyster spermatozoa

Interphylum crossing was examined between sea urchin eggs (Temnopleurus hardwicki) and oyster sperm (Crassostrea gigas). The eggs could receive the spermatozoa with or without cortical change. The fertilized eggs that elevated the fertilization envelope began their embryogenesis. Electron microscopy revealed that oyster spermatozoa underwent acrosome reaction on the sea urchin vitelline coat, and their acrosomal membrane fused with the egg plasma membrane after the appearance of an intricate membranous structure in the boundary between the acrosomal process and the egg cytoplasm. Oyster spermatozoa penetrated sometimes into sea urchin eggs without stimulating cortical granule discharge and consequently without fertilization envelope formation. The organelles derived from oyster spermatozoa seemed to be functionally inactive in the eggs whose cortex remained unchanged.     

The effect of serotonin agonist 1-(trifluoromethylphenyl)-piperazine on corticotropin releasing factor and arginine vasopressin in rat hypothalamic nuclei

Effects of 1-(m-trifluoromethylphenyl)-piperazine, a serotonin agonist, were examined on rat plasma levels of adrenocorticotropin (ACTH) and arginine vasopressin (AVP), and on hypothalamic contents of corticotropin releasing factor (CRF) and AVP, to investigate the role of brain serotonin in ACTH regulation. Both plasma ACTH and AVP levels increased markedly 30 min after injection of the compound and were still elevated at 80 min. CRF and AVP contents in the median eminence decreased 30 min after injection but returned to the basal levels by 80 min. The AVP content in the supraoptic nucleus was elevated 80 min after injection. The CRF and aVP content did not significantly change in the paraventricular, suprachiasmatic and arcuate nuclei. Serotonin or 1-(m-trifluoromethylphenyl)-piperazine did not stimulate the release of ACTH in pituitary cell cultures. These results suggest that both CRF and AVP were secreted into the portal vessels by 1-(m-trifluoromethylphenyl)-piperazine to release ACTH from the anterior pituitary and that both the ACTH and AVP release were stimulated via the brain serotonergic mechanism.     

Two types of ftsZ genes isolated from the unicellular primitive red alga Galdieria sulphuraria.

FtsZ plays a crucial role in bacterial cell division, and may be involved in plastid division in eukaryotes. To investigate the evolution of the dividing apparatus from prokaryotes to eukaryotes, the ftsZ genes were isolated from the unicellular primitive red alga Galdieria sulphuraria. Two ftsZ genes (GsftsZ1 and GsftsZ2) were isolated. This suggests that duplication and divergence of the ftsZ gene occurred in an early stage of plant evolution. A comparison of the FtsZs of G. sulphuraria and other organisms shows that FtsZ is highly and universally conserved among prokaryotes, primitive eukaryotic algae, and higher plants. The GsftsZ2 gene seems to contain an intron. Southern hybridization analysis of the G. sulphuraria chromosomes separated by CHEF revealed that each ftsZ gene and its flanking region may be duplicated.