Innervation of holothurian body wall muscle: inhibitory effects and localization of 5-HT

We investigated innervation to body wall muscles as well as distribution of 5-HT (serotonin) and its effects on longitudinal muscles of body wall (LMBW) of the sea cucumber Apostichopus japonicus. With serial sections we found neural branches and fibers extending from hyponeural part of radial nerve towards LMBW and circular muscles of body wall. With the aqueous aldehyde (Faglu) method yellow fluorescence indicating indolamines was observed in LMBW and in the mesentery connecting LMBW to the body wall. With indirect immunohistochemistry 5-HT-like immunoreactivity was observed in LMBW and in mesentery. These results strongly suggested that both LMBW and mesentery contained 5-HT. The effects of monoamine neurotransmitters were studied in LMBW. Putative neurotransmitters tested were 5-HT, adrenaline, noradrenaline, dopamine, and DOPA at the concentration of 10(-6) M. The application of 5-HT caused no contraction or relaxation, but it inhibited the contraction induced by 10(-6)-10(-5) M acetylcholine (ACh). Catecholamines were ineffective by themselves and had no effects on the contraction induced by ACh. The present histological, histochemical, and pharmacological studies strongly suggested that holothurian LMBW was innervated by inhibitory serotonergic neurons of the hyponeural nervous system.     

Monitoring oxygen consumption of single mouse embryos using an integrated electrochemical microdevice

Oxygen consumption (respiration activity) has been found to be the most remarkable criterion for determining the viability of an embryo produced in vitro. In this study, we propose an accurate, simple, and user-friendly device for measurement of the oxygen consumption of single mammalian embryos. An integrated electrode array was fabricated to determine the oxygen consumption of a single embryo, including the blastocyst stage, which has an inhomogeneous oxygen consumption rate, using a single measurement procedure. A single mouse embryo was positioned in a microwell at the center of an integrated electrode array, using a mouthpiece pipette, and immobilized by a cylindrical micropit with good reproducibility. The oxygen consumption of two-cell, morula, and blastocyst stages was measured amperometrically using the device. The recorded current profile was corrected to take into consideration transient background current during the measurement. A calculation method for oxygen consumption based on spherical diffusion centered on the defined point of the device was developed. This procedure is quite simple because it is not necessary to estimate the radius of the embryo being measured. The calculated values of oxygen consumption for two-cell, morula, and blastocyst stages were 1.36 ± 0.33 × 10⁻¹⁵ mol s⁻¹, 1.38 ± 0.58 × 10⁻¹⁵ mol s⁻¹, and 3.44 ± 2.07 × 10⁻¹⁵ mol s⁻¹, respectively. The increasing pattern of oxygen consumption from morula to blastocyst agreed well with measurements obtained using conventional scanning electrochemical microscopy (SECM).     

Secretory and continuous expression of Aspergillus niger glucose oxidase gene in Pichia pastoris

We proposed a yeast transformant cell incorporating the Aspergillus niger glucose oxidase gene (GOX gene), which is capable of constitutively as well as secretory expression. The GOX gene has been cloned in this study. This conclusion is based on the following: first, the ligated DNA determined by electrophoresis, was a 1489-1882bp fragment, close to the size of glucose oxidase (GOD), which is 1818bp. Secondly, the single open reading frame encoded a protein of 605 amino acids. Thirdly, secreted GOD recombinant proteins in the culture supernatants of the GOX gene transformant migrated as a single band in SDS-PAGE with an apparent molecular mass of between 75,000 and 100,000 Da, which is glycosylated GOD by the Pichia pastoris X-33 host machinery during the secretion process. Finally, the clones were cultured and secreted a protein, which possessed the GOD activity of catalyzing beta-d-glucose oxidation. With regard to the pH characteristics, the activity was more than 80% of the maximum activity in the range between pH 5 and pH 7. As for the temperature characteristics, the activity was not less than 92% of the maximum in the temperature range between 10 and 45 degrees C. The GOX gene transformant was able to maintain the GOD enzyme activity and produce recombinant GOD continuously for at least 2 weeks.     

Immunohistochemical Localization of Arginase II and Other Enzymes of Arginine Metabolism in Rat Kidney and Liver

Arginine is a precursor for the synthesis of urea, polyamines, creatine phosphate, nitric oxide and proteins. It is synthesized from ornithine by argininosuccinate synthetase and argininosuccinate lyase and is degraded by arginase, which consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Recently, cDNAs for human and rat arginase II have been isolated. In this study, immunocytochemical analysis showed that human arginase II expressed in COS-7 cells was localized in the mitochondria. Arginase II mRNA was abundant in the rat small intestine and kidney. In the kidney, argininosuccinate synthetase and lyase were immunostained in the cortex, intensely in proximal tubules and much less intensely in distal tubules. In contrast, arginase II was stained intensely in the outer stripes of the outer medulla, presumably in the proximal straight tubules, and in a subpopulation of the proximal tubules in the cortex. Immunostaining of serial sections of the kidney showed that argininosuccinate synthetase and arginase II were collocalized in a subpopulation of proximal tubules in the cortex, whereas only the synthetase, but not arginase II, was present in another subpopulation of proximal tubules. In the liver, all the enzymes of the urea cycle, i.e. carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase and lyase and arginase I, showed similar zonation patterns with staining more intense in periportal hepatocytes than in pericentral hepatocytes, although zonation of ornithine transcarbamylase was much less prominent. The implications of these results are discussed.     

Comparisons of the transferability of plasmids pCAR1, pB10, R388, and NAH7 among Pseudomonas putida at different cell densities

The transferability of plasmids pCAR1, pB10, R388, and NAH7 was compared using the same donor-recipient system at different cell density combinations in liquid or on a solid surface. pCAR1 was efficiently transferred in liquid, whereas the other plasmids were preferentially transferred on a solid surface. Difference of liquid or solid affected the transfer frequency especially at lower cell densities.     

Histological studies on the therapeutic effect of sustained-release microspheres of a potent LHRH agonist (leuprorelin acetate) in an experimental endometriosis model in rats

The therapeutic effect of sustained-release microspheres of a potent LHRH agonist (leuprorelin acetate) on experimental endometriosis in female rats was examined histologically. Endometriosis was produced in rats by autotransplantation of endometrial tissue obtained from the left uterine horn into the renal subcapsular space. In the nontreated rats, the transplants were well established and had formed large cysts containing fluid. The walls of the cysts were composed of epithelium and stroma resembling that of normal endometrium. In the rats which received the microspheres of leuprorelin acetate, growth of the transplant was markedly suppressed as evidenced by the reduced size of the cystic cavity and the flattened and pyknotic epithelium. Also, the uterine and ovarian weight decreased significantly. In the ovariectomized rats, growth of the transplant was also markedly suppressed, and the uterine weight decreased. The present results clearly indicate that a single injection of the sustained-release microspheres of leuprorelin acetate markedly suppresses growth of the transplant and produces uterine and ovarian atrophy in the rats.     

Molecular cloning and expression of the novel fungal beta-glucosidase genes from Humicola grisea and Trichoderma reesei

A novel fungal beta-glucosidase gene (bgl4) and its homologue (bgl2) were cloned from the cellulolytic fungi Humicola grisea and Trichoderma reesei, respectively. The deduced amino acid sequences of H. grisea BGL4 and T. reesei BGL2 comprise 476 and 466 amino acids, respectively, and share 73.1% identity. These beta-glucosidases show significant homology to plant beta-glucosidases belonging to the beta-glucosidase A (BGA) family. Both genes were expressed in Aspergillus oryzae, and the recombinant beta-glucosidases were purified. Recombinant H. grisea BGL4 is a thermostable enzyme compared with recombinant T. reesei BGL2. In addition to beta-glucosidase activity, recombinant H. grisea BGL4 showed a significant level of beta-galactosidase activity, while recombinant T. reesei BGL2 showed weak beta-galactosidase activity. Cellulose saccharification by Trichoderma cellulases was improved by the addition of recombinant H. grisea BGL4.     

Mitogenomic phylogeny of the Percichthyidae and Centrarchiformes (Percomorphaceae): comparison with recent nuclear gene-based studies and simultaneous analysis

Delineation of the fish family Percichthyidae (Percomorphaceae) has a long and convoluted history, with recent morphological-based studies restricting species members to South American and Australian freshwater and catadromous temperate perches. Four recent nuclear gene-based phylogenetic studies, however, found that the Percichthyidae was not monophyletic and was nested within a newly discovered inter-familial clade of Percomorphaceae, the Centrarchiformes, which comprises the Centrarchidae and 12 other families. Here, we reexamined the systematics of the Percichthyidae and Centrarchiformes based on new mitogenomic information. Our mitogenomic results are globally congruent with the recent nuclear gene-based studies although the overall amount of phylogenetic signal of the mitogenome is lower. They do not support the monophyly of the Percichthyidae, because the catadromous genus Percalates is not exclusively related to the freshwater percichthyids. The Percichthyidae (minus Percalates) and Percalates belong to a larger clade, equivalent to the Centrarchiformes, but their respective sister groups are unresolved. Because all recent analyses recover a monophyletic Centrarchiformes but with substantially different intra-relationships, we performed a simultaneous analysis for a character set combining the mitogenome and 19 nuclear genes previously published, for 22 centrarchiform taxa. This analysis furthermore indicates that the Centrarchiformes are divided into three lineages and the superfamily Cirrhitoidea is monophyletic as well as the temperate and freshwater centrarchiform perch-like fishes. It also clarifies some of the relationships within the freshwater Percichthyidae.     

Arabinosylnucleoside 5'-triphosphate inhibits DNA primase of calf thymus

It has been shown that DNA primase activity is tightly associated with 10S DNA polymerase alpha from calf thymus and that the ribonucleotide-dependent DNA synthesis is more sensitive to araCTP than DNA-primed DNA synthesis (Yoshida, S., et al. (1983) Biochim. Biophys. Acta 741, 348-357). Here we measured DNA primase activity using poly(dT) template or M13 bacteriophage single-stranded DNA template and primer RNA synthesis was coupled to the reaction by Escherichia coli DNA polymerase I Klenow fragment. By this method, the primer RNA synthesis can be measured independently of the associating DNA polymerase alpha. Using poly(dT) template, it was found that arabinosyladenine 5'-triphosphate (araATP) strongly inhibited DNA primase in competition with rATP. The apparent Ki for araATP was 21 microM and the ratio of Ki/Km (for rATP) was as low as 0.015. With poly(dI, dT) or M13 DNA, it was shown that araCTP also inhibited DNA primase in the similar manner. Product analysis using [alpha-32P]rATP showed that araATP inhibited the elongation of primer RNA. However, it is not likely that arabinosylnucleotides act as chain-terminators, since incubation of primer RNA with araATP did not abolish its priming activity. From these results, it is suggested that arabinosylnucleotide inhibits the initiation as well as elongation of Okazaki fragments in mammalian cells.