Successful suppression of endogenous α-1,3-galactosyltransferase expression by RNA interference in pig embryos generated in vitro

RNA interference (RNAi) technology using small interfering RNAs (siRNA) has been widely used as a powerful tool to knock down gene expression in various organisms. In pig preimplantation embryos, no attempt to suppress the target gene expression with such technology has been made. The purpose of this study is to demonstrate that the RNAi technology is useful for suppression of endogenous target gene expression at an early stage of development in pigs. Alpha-1,3-Galactosyltransferase (α-GalT) is an enzyme that creates the Galα1-3Gal (α-Gal) epitope on the cell surface in some mammalian species, and removal of the epitope is considered to be a prerequisite for pig-to-human xenotransplantation. We decided to suppress the endogenous α-GalT mRNA expression in pig early embryos, since reduction of α-GalT synthesis is easily monitored by cytochemical staining with Bandeiraea simplicifolia isolectin-B(4), a lectin that specifically binds to the α-Gal epitope, and by RT-PCR analysis. Cytoplasmic microinjection of double-stranded RNA and pronuclear injection of an siRNA expression vector into the embryos generated in vitro resulted in a significant reduction in expression of the α-GalT gene and α-Gal epitope in blastocysts, at which stage the α-Gal epitope is abundantly expressed. Somatic cell nuclear transfer of embryonic fibroblasts stably transfected with an siRNA expression vector also led to a significant reduction in the level of α-GalT mRNA synthesis together with decreased amounts of the α-Gal epitope at the blastocyst stage. These results indicate that the RNAi technology is useful for efficient suppression of a target gene expression during embryogenesis in pigs and suggest the possibility of production of siRNA-expressing pigs for use in xenotransplantation.     

A comparative study of chronic kidney disease in dogs and cats: induction of cyclooxygenases

The present study investigated whether renal cyclooxygenase (COX) induction is associated with the severity of chronic kidney disease (CKD) in dogs and cats. The collected kidneys were examined histopathologically and immunohistochemically. The immunoreactivities of COX-1 and COX-2 were evaluated quantitatively, and the correlations to the plasma creatinine concentrations, glomerular size, glomerulosclerosis, interstitial fibrosis, and interstitial cell infiltration were evaluated statistically. Immunoreactivities for COX-1 were heterogeneously observed in the medullary distal tubules and collecting ducts; no correlations with the severity of renal damage were detected. Immunoreactivities for COX-2 were heterogeneously observed in the macula densa (MD) regions. In dogs, the percentage of COX-2-positive MD was significantly correlated with the glomerular size. In cats, glomeruli with COX-2-positive MD had significantly higher sclerosis scores than those with COX-2-negative MD. In conclusion, renal COX-2 is induced in canine and feline CKD, especially in relation to the glomerular changes.     

Influence of moisture content on estimating Young’s modulus of full-scale timber using stress wave velocity

The effect of moisture content on the stress wave propagation velocity was investigated in order to estimate the Young’s modulus of full-scale timbers in an air-drying state using the measurement of stress wave propagation velocity above the fiber saturation point. Using Japanese cedar lumber, the velocity and the density under high-moisture condition and air-drying states were measured respectively; after measuring the modulus of elasticity in an air-drying state, the moisture content of each condition was measured. By performing numerical analysis on these data, the relationship between the moisture content and the rate of change of velocity of full-scale timbers was derived. This relationship was used to estimate the Young’s modulus of the timber in the air-drying state from the velocity in high-moisture condition. First, the velocity and the Young’s modulus in an air-drying state were estimated accurately from its density, moisture content and velocity under high-moisture condition. In cases where the density could not be measured, using the database of mechanical properties with the Monte Carlo simulation method, the Young’s modulus of the full-scale timber in an air-drying state might be estimated within 20% accuracy from its moisture content and velocity under high-moisture condition.     

Lignan content of selected foods from Japan

Lignans constitute a group of phytochemicals widely distributed in the human diet. Once ingested, most dietary lignans are metabolized by the gut microflora to enterolactone and enterodiol, also known as enterolignans. Together with isoflavones and coumestrol, enterolignans have been traditionally classified as phytoestrogens, plant-derived compounds that exhibit estrogen-like activity. In addition to a higher intake of vegetables, many studies have suggested that the presence of phytoestrogens in the Japanese diet is one of the factors that might explain the reduced incidence of certain chronic diseases in Japanese populations compared with Western countries. Being a vegetable-based diet, exposure to dietary lignans should be high, but to assess this exposure, a lignan food database is required. Stable isotope-dilution gas chromatography coupled with mass spectrometry was used to quantify six plant lignans in 86 food items commonly consumed in Japan. These data will complement the previous databases and most importantly expand the knowledge of occurrence of lignans in food to Eastern diets.     

Screening of wood-rotting fungi for kraft pulp bleaching by the Poly R decolorization test and biobleaching of hardwood kraft pulp byPhanerochaete crassa WD1694

From among 419 wood-rotting fungi 10 were selected by the Poly R decolorization test, and their ability to bleach hardwood kraft pulp was assayed. Of the 10 selected, 6 fungi (i.e.,Phanerochaete crassa WD1694 and F150;Pleurotus pulmonarius PSC-H, PSC-M, and PSC-T;and Pleurotus species A119) showed much higher bleaching ability thanPhanerochaete chrysosponum BKMF1767 orTrametes versicolor WD1670, both of which are well-known high ligninolytic fungi.P. crassa WD1694 had the highest bleaching ability among the selected strains, and it increased the pulp brightness from 28 to 54, with a corresponding decrease in kappa number from 16 to 6 after 10 days of cultivation and alkali extraction. MnP was a predominant ligninolytic enzyme ofP. crassa WD1694 during the biobleaching.This research was presented in part at the 46th Annual Meeting of the Japan Wood Research Society, Kumamoto, April 1996     

Extracellular peroxidase activity at the hyphal tips of the white-rot fungus Phanerochaete crassa WD1694

The white-rot fungus Phanerochaete crassa WD1694 was cultivated and peroxidase activity staining was performed to determine the sites at which the extracellular peroxidase reaction actually occurs in vivo. Although the ligninolytic peroxidases were found in the culture filtrates, the culture medium did not show a color reaction. However, a particularly strong color reaction was observed on the hyphal tips. Visible spectra and absorbance of the staining were analyzed by microspectrophotometry, and the catalytic rates of the peroxidase reaction at the hyphal tips were calculated. The estimated catalytic rate of the peroxidase reaction at the hyphal tips peaked at 794 μM/min, expressed as the consumption rate of H₂O₂, on day 3 of the cultivation. Analysis of the extracellular enzyme eluted with 0.1% Tween 80 from the mycelium revealed that manganese peroxidase accounted for 89% of all the peroxidase activity measured. The results clearly showed the existence of the concentrated manganese peroxidase reaction around the hyphal tips of the organism.     

Root temperature drives winter acclimation of shoot water relations in Cryptomeria japonica seedlings

In many temperate evergreen plant species, reductions in turgor loss point of leaves (Psi(tlp)) and leaf osmotic potential at full turgor (pi(sat)) occur from late summer to winter. To test the hypothesis that this seasonal change in leaf water relations is driven by root temperature, we manipulated the temperature of the roots and shoots of Cryptomeria japonica D. Don seedlings separately. Whole-plant warming diminished the seasonal changes in shoot water relations observed in the control plants, whereas shoot warming did not. Compared with the controls, root warming diminished the change in Psi(tlp) but not in pi(sat), whereas cooling accelerated the seasonal changes in shoot water relations. These results indicate that: (1) temperature responses of roots are involved in the seasonal changes in Psi(tlp) from late summer to winter; and (2) root temperature is partly responsible for the simultaneous changes in pi(sat). Whole-plant cooling caused increased root hydraulic resistance, suggesting that seasonal changes in shoot water relations represent adaptive responses to increased root hydraulic resistance at low root temperatures.     

The first report of the ante-mortem diagnosis of Ollulanus tricuspis infection in two dogs

Ollulanus tricuspis is a small nematode parasite of the stomach, and its infection has been reported worldwide in cats but only one report in dogs as post-mortem diagnosis. Two dogs, kept in the Tokyo area, were presented for chronic vomiting. Chronic gastritis was diagnosed histologically, and many nematodes were detected in endoscopically-biopsied gastric samples and in the mucus of vomitus in both dogs. The parasites were small (<1 mm), and their morphological characteristics were consistent with those previously reported for O. tricuspis. The symptoms in one dog completely disappeared after anthelmintic therapy. To our knowledge, this is the first report describing ante-mortem diagnosis of spontaneous gastric O. tricuspis infection in dogs in which infectivity and pathogenicity of the nematode are suggested.     

Effects of vertically transferred 3,3',4,4',5-pentachlorobiphenyl on gene expression in the ovaries of immature Sprague-Dawley rats

We have previously shown that 3,3',4,4',5-pentachlorobiphenyl (PCB-126) vertically transferred from dams potentially exerts a direct effect on the ovaries of offspring and adversely affects female puberty. To investigate its toxicological targets in ovarian tissues, mRNAs encoding representative peptides that regulate follicular development in granulosa cells, theca cells, and oocytes were quantified using ovaries collected on postnatal days (PND) 5, 15, and 24 from the offspring of dams administered oral doses of 0, 1 or 3 microg/kg PCB-126 starting 2 weeks prior to mating and continuing until 20 days after delivery. Quantification using the real-time RT-PCR method revealed that PCB-126 lowered the amounts of mRNAs that encoded the inhibin alpha- and inhibin/activin beta A-subunits from PND 15 onwards; the amounts of mRNAs for inhibin/activin beta B-subunit, follicle-stimulating hormone (FSH) receptor, and aromatase on PND 15; and the amounts of luteinizing hormone receptor mRNA on PND 24 compared with those of the age-matched controls. In contrast, no differences were noted for mRNAs encoding c-kit, growth differentiation factor-9, bone morphogenetic protein-15, or kit ligand for any of the age groups examined. The serum FSH level on PND 24 was higher than that in the control. Since the earliest effects on the mRNAs in the rat ovaries were observed in those expressed in the granulosa cells of the growing follicles after the antral follicles had developed, molecules in granulosa cells but not in oocytes during the early stages of the antral follicles might be the primary targets of vertically transferred PCB-126.     

Removal of sperm tail using trypsin and pre-activation of oocyte facilitates intracytoplasmic sperm injection in mice and rats

We examined various methods to enhance the accessibility of intracytoplasmic sperm injection (ICSI) technology to more users by making the technique easier, more efficient, and practical. First, the methods for artificially removing the mouse sperm tail were evaluated. Trypsin treatment was found to efficiently remove the sperm tails. The resultant sperm cells had a lower oocyte activation capacity; however, the use of activated oocytes resulted in the same fecundity as that of fresh, untreated sperm. Pre-activated oocytes were more resistant to physical damage, showed higher survival rates, and required less time per injection. Testing this method in rats yielded similar results, although the oocyte activation method was different. Remarkably, this method resulted in higher birth rates of rat progeny than with conventional methods of rat ICSI. Our method thereby streamlines mouse and rat ICSI, making it more accessible to laboratories across many disciplines.