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A cell line (PL38PB) was established from blood samples of a 6-month-old pig that was diagnosed with lymphoma with CD5 expression. Histopathological examination revealed neoplastic lesions in the spleen, liver and lymph nodes. Tumor cells were immunohistochemically positive for CD20 and immunoglobulin heavy chains (μ, γ and α). Membranous CD5 and cytoplasmic Immunoglobulin M (IgM), ​Immunoglobulin G (IgG) and ​Immunoglobulin A (IgA) were detected in PL38PB cells by flow cytometry. In addition, the cytoplasm of PL38PB cells were positive for IgM, IgG and IgA by immunofluorescent. However, no Ig secretion was detected in culture supernatant by Ouchterlony gel diffusion method. Results suggest that PL38PB cells express three Ig isotypes that are produced but not secreted.

For broad detection of pestivirus A (bovine viral diarrhea virus 1: BVDV1) and pestivirus B (BVDV2) by a reverse transcription loop-mediated isothermal amplification (RT-LAMP) test, the P25 primer set was designed using nucleotide sequences of 5’-UTR region of 1454 BVDVs. The base coverage of each primer against diverse BVDVs were more than 99% in each base position. The one step LAMP test with the P25 primer set could detect both BVDV1 (TK) and BVDV2 (KZ), but did not amplify 5 other bovine viruses. Detection limit of the LAMP test was 103 copies of synthesized DNAs, and 10−3 and 10−4 dilutions of viral RNAs of TK and KZ strains, respectively, whereas that with current Aebischer’s primer set was 10−2 dilution and negative of these RNAs, respectively. All of the 63 viral RNA samples of persistently infected (PI) cattle, consisting of the 1a (12), 1b (31), 1c (11), and 2a (9) subgenotypes, were broadly detected with the P25, while only 65% of them were positive with Aebischer’s primer set. The validation study showed that the RT-LAMP test with the P25 had 100% sensitivity and 100% specificity against that with updated Vilcek’s PCR primers. Also, by using the P26 primer set which contained 3 species-specific primers, all 63 RNA samples were clearly distinguished from BVDV1 or BVDV2 by the typing RT-LAMP test. These results indicate that the one step RT-LAMP test using P25 or P26 primer sets would be useful for broad detection and rapid differentiation of BVDV1 and BVDV2.

Approximately 20 major-to-ultratrace elements in each organ (mantle, muscle, liver, kidney) of a giant clam, Tridacna crocea, and 16 elements in symbiotic zooxanthellae were determined by ICP-AES, ICP-MS, and CHN coder. The biogeochemical properties of T. crocea were then investigated using multi-element profiling analyses. The values for most elements were the highest in the kidney among all organs. However, the concentrations of Mn, Co, Ni, Zn, Se, Ag, Cd, and Pb in the mantle were higher than those in the muscle and liver. Therefore, these results indicate that most trace elements taken in by T. crocea through zooxanthellae may finally accumulate in the kidney. Moreover, 14 trace elements in zooxanthellae were present in the concentration range from 181 µg g−1 for Zn to 0.58 µg g−1 for U based on dry weight after isolating zooxanthellae from the mantle. The concentrations of some trace metals in zooxanthellae were relatively higher than those in other organs except for the kidney. These results suggest...

Loop-mediated isothermal amplification (LAMP) is a potential screening test for avian influenza (AI), but its narrow detection spectrum limits its applications. To improve this narrow detection spectrum, 3 types of primers were compared for detection of diverse H5 subtype hemagglutinin (HA) genes. Four and 6 genes, of 10 genetically different H5 HA genes tested, were detected with S primers specific for A/duck/Tsukuba/9/2005 (H5N2) and with M primers (which contained mixed bases), respectively. In contrast, all 10 HA genes became positive with population primers (P primers) (a mixture of primers designed for each subpopulation of 2,202 HA genes). Our study indicated that the P primers for the forward inner primer (FIP) and backward inner primer (BIP) sites were essential for exhaustive detection, whereas those for the F3, forward loop (FL), backward loop (BL), and B3 sites were exchangeable with M primers. A base mismatch experiment demonstrated that HA genes with ≤2 base mismatches per primer site and ≤10 base mismatches per HA gene were amplifiable. Reverse transcription-LAMP was broadly reactive, specific for H5 subtype HA genes, and applicable to field samples, with the sensitivity of real-time PCR. The in silico analysis suggested that most H5 HA genes (2,586 positive genes/2,588 genes tested) registered in the GenBank database might be amplifiable. These results indicate that the use of subpopulation primers in LAMP allows exhaustive detection of diverse HA genes and H5 LAMP can be used as a reliable AI screening test in general diagnostic laboratories.

Targeted genome editing is a widely applicable approach for efficiently modifying any sequence of interest in animals. It is very difficult to generate knock-out and knock-in animals except for mice up to now. Very recently, a method of genome editing using zinc-finger nucleases (ZFNs) has been developed to produce knockout rats. Since only injection of ZFNs into the pronuclear (PN) embryo is required, it seems to be useful for generating gene-targeted animals, including domestic species. However, no one has reported the successful production of knockout pigs by direct injection of ZFNs into PN embryos. We examined whether ZFN works on editing the genome of porcine growth hormone receptor in two kinds of cell lines (ST and PT-K75) derived from the pig as a preliminary study. Our data showed that pZFN1/2 vectors were efficiently transfected into both ST and PT-K75 cells. In both cell lines, results from Cel-I assay showed that modification of the targeted gene was confirmed. We injected ZFN1/2 mRNAs into the nucleus of PN stage embryos and then they were transferred to the recipients. However, pups were not delivered. Taken together, ZFN can be an available technology of genome editing even in the pig but further improvement will be required for generating genome-modified pigs.

PCBs are persistent environmental agents that induce multiple impairments in living beings. In this study we used a transgenic mouse model (Muta(TM) Mouse), carrying bacterial lacZ genes for mutation assays and for assessment of the genotoxic effect of PCB126 on fetal mice. Mothers of experimental groups were subjected to a single oral dose of PCB126 (125, 250 and 500 microg/kg) on the 10th day of pregnancy, respectively. Fetuses were autopsied on the 18th day of gestation. Cleft palate was observed in 2 out of 11 fetuses from 3 litters in 500 microg/kg treated group. Other external malformations were not observed. The DNA mutation frequencies (MF) of fetuses in each group were 1.15 +/- 0.24 x 10(-5), 0.90 +/- 0.20 x 10(-5) and 1.08 +/- 0.24 x 10(-5) in fetuses of 125, 250 and 500 microg/kg treated groups, respectively. The MF of controls was 0.81 +/- 0.22 x 10(-5). There were no significant differences among the groups. However, the MF of each treated group was a little highter than that of control group. Possible relationships between PCB and its mutagenic effects in the offspring of mice are discussed.

We investigated the membrane transport of Na and K ions in red blood cells (RBCs) of northern fur seal (Callorhinus ursinus) by measurement of unidirectional fluxes. Like red blood cells of other carnivores, those of northern fur seal contain high Na and low K concentrations, which result from the lack of Na–K ATPase activity on their membranes. In physiological conditions, activities of bumetanide-sensitive Na, K–Cl cotransport and amiloride-sensitive Na/H exchange were measured. K–Cl cotransport and Na–Cl cotransport were not detected. Hypertonicity activated only Na/H exchange. We further examined the ion transport systems for regulatory volume increase (RVI) in red blood cells. In the hyperosmotic condition, shrunken RBCs restored their original cell volume in Na medium but not in Na-free medium, and this restoration with Na medium was inhibited by amiloride. From these results, it is suggested that RVI in northern fur seal RBCs are performed by amiloride-sensitive Na/H exchanger but not Na, K–Cl cotransporter.

This study assessed a technique for the isolation and differentiation of dog erythroid progenitors. Mononuclear cells from peripheral blood were cultured in a two-phase liquid culture system. The colony burst was confirmed after the first-phase culture by using a semi-solid culture containing methylcellulose. By immunofluorescent observation or reverse transcriptase-polymerase chain reaction analysis, the cells which proliferated during the second-phase culture were found to express glycophorin, globin, and glutamate/aspartate transporter, which are markers of dog erythrocytes. This system is beneficial for the analysis of dog erythrocytes as a reasonable amount of well-matured cells (0.5×106/ml) are obtainable.

As the first step in investigating the possiblity of applying ribozyme technology to artificial control of the sex ratios at birth in farm animals, where the demand for females exceeds that for males, we designed a hammerhead ribozyme (HHRz) and 2 tRNA(val)-hammerhead ribozyme complexes (tRNARz3 and tRNARz4), and examined their effects upon murine Sry mRNA in vitro and in cells. We demonstrated that HHRz and tRNARz3 could effectively cleave the target Sry mRNA in vitro. For the purpose of experiments in vivo, HHRz was cloned into the highly efficient pUC-CAGGS mammalian expression vector (pCAG/HHRz), and the tRNA ribozyme complexes were cloned into the pol III promoter-driven pPUR-KE vector (pPUR/tRNARz3 and pPUR/tRNARz4); the ribozyme vectors were co-transfected with the target vector (pCAG/Sry). A suppressive action (up to approx. 60%) was confirmed for pCAG/HHRz and pPUR/tRNARz3 upon the transiently expressed exogenously introduced Sry in M15 cultured cells.

K-Cl cotransport plays a crucial role in regulatory volume decrease of erythrocytes. K-Cl cotransport activities in dog erythrocytes with an inherited high Na-K pump activity (HK) and normal erythrocytes (LK) were compared. Nitrite (NO(2)) stimulated K-Cl cotransport activity in HK cells around 14-fold at 2.4 mM, and it also increased the Km value of this cotransporter. Real-time PCR and western blot analysis revealed that K-Cl cotransporter 1 was dominant, and that the quantity of K-Cl cotransporter 1 protein was comparable between HK and LK erythrocytes. These results suggest that the difference in cotransport activity was not caused by the amount of K-Cl cotransport protein but by a difference in the regulation system, which is susceptible to oxidant.

The rabbit is considered to be a valuable laboratory animal. We compared glycerol, lactamide, acetamide, and dimethylsulfoxide (DMSO) as cryoprotectants in egg-yolk diluent of ejaculated Japanese white rabbit spermatozoa for improvement of sperm cryopreservation methods. Rabbit semen was frozen with 1.0 M glycerol, lactamide, acetamide, or DMSO in plastic straws. Forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rate of forward progressive motile spermatozoa in lactamide (37.8 +/- 3.0%) was significantly (P

The present study investigated the effect of separation of spermatozoa by sonication or Piezo-pulse on in vitro development of oocytes injected with sperm heads in the rat. We also examined development to term of rat oocytes injected with sperm heads. Rat frozen-thawed spermatozoa were separated into heads and tails by sonication for 10 sec or Piezo-pulse in KRB medium, and each treated sperm head was injected into an ooplasm. The oocytes were observed for formation of two pronuclei and development to 2-cell embryos. The percentages of formation of two pronuclei and development to the 2-cell stage did not significantly (P>0.05) differ between the two groups. Oocytes injected with sonicated sperm heads that reached the pronuclear stage at 10 h after injection of sperm heads were transferred into 7 recipients. Five recipients became pregnant, and 8 living pups were obtained. The results indicate that rat oocytes injected with sonicated sperm heads can develop to term in vivo. Furthermore, no differ...

The levels of verotoxin-1 and verotoxin-2 released by verotoxigenic Escherichia coli O157:H7 treated in vitro with sodium nitrite, sodium chloride and several antibiotics were evaluated. Of the three strains of E. coli O157:H7 used in this study, two strains produced both verotoxin-1 and verotoxin-2, and one strain produced only verotoxin-2. Treatment of E. coli O157:H7 with sodium nitrite (6000 mg/l, minimum inhibitory concentration) did not increase the levels of verotoxin-1 and verotoxin-2 compared with a treatment by sodium chloride or antibiotics. When the electron paramagnetic resonance spectrum of sodium nitrite-treated bacterial cells was examined at 77 K to clarify the mechanism for the anti-bacterial activity of nitric oxide derived from sodium nitrite, electron paramagnetic resonance signals with g-values of 2.035 and 2.010 were observed. These were identified as being derived from iron-nitric oxide complexes. It appears that the dinitrosyl iron complexes in the E. coli O157:H7 cells were generated from the reaction of iron-sulfur proteins (enzymes) with nitric oxide formed by the reduction of sodium nitrite. The amount of ATP was decreased by the presence of sodium nitrite in the cell suspension. These findings indicate that nitric oxide derived from sodium nitrite penetrated the cells and inactivated enzymes related to the respiratory chain.

Enterococcus faecalis TH10 is a lactic acid bacterial strain isolated from the Malaysian traditional fermented food, “tempeh”, and has antibacterial activity against various pathogens. To identify the antibacterial substance, the butanol extract of the culture supernatant of E. faecalis TH10 was fractionated by HPLC equipped with a reversed-phase partition column, and the elutes were subjected to antibacterial assay. As the activity was observed in a fraction eluted by ca 80% methanol, the fraction was analyzed by gas chromatography/mass spectrometry and 3-phenyllactic acid was identified as the major compound. Fractionation with an optical isomer separation column showed that the preparation contained D-and L-forms of 3-phenyllactic acid at a ratio of 2: 1. Authentic 3-phenyllactic acid showed antibacterial activity against various bacteria such as Staphylococcus aureus and Escherichia coil. These results suggest the possibility that 3-phenyllactic acid is a biopreservative.

Targeted genome editing is a widely applicable approach for efficiently modifying any sequence of interest in animals. It is very difficult to generate knock-out and knock-in animals except for mice up to now. Very recently, a method of genome editing using zinc-finger nucleases (ZFNs) has been developed to produce knockout rats. Since only injection of ZFNs into the pronuclear (PN) embryo is required, it seems to be useful for generating gene-targeted animals, including domestic species. However, no one has reported the successful production of knockout pigs by direct injection of ZFNs into PN embryos. We examined whether ZFN works on editing the genome of porcine growth hormone receptor in two kinds of cell lines (ST and PT-K75) derived from the pig as a preliminary study. Our data showed that pZFN1/2 vectors were efficiently transfected into both ST and PT-K75 cells. In both cell lines, results from Cel-I assay showed that modification of the targeted gene was confirmed. We injected ZFN1/2 mRNAs into the nucleus of PN stage embryos and then they were transferred to the recipients. However, pups were not delivered. Taken together, ZFN can be an available technology of genome editing even in the pig but further improvement will be required for generating genome-modified pigs.

A novel method to determine the content of 5-methylcytosine in DNA precisely, sensitively and simply has been developed using liquid chromatography (LC) with fluorescence detection. DNA was mildly digested by cooperation of nuclease P1 and DNase I, then the generated 2′-deoxynucleoside 5′-monophosphates (dNmp) were specifically derivatized fluorometrically at the position of the phosphoric acid moiety with 5-dimethylaminonaphthalene-1-[ N -(2-aminoethyl)]sulfonamide (dansylEDA), followed by the separation of fluorescent dansylEDA derivatives of dNmp on the reversed-phase partition column. The method was more than 10 times as sensitive as the conventional ultraviolet detection method.

We designed three hairpin ribozymes to cleave Escherichia coli beta-glucuronidase (GUS) mRNA and tested those activities in vitro. One of the ribozymes was designed to form 9 base pairs in total with the target GUS mRNA, and the other two ribozymes had longer substrate binding sites. All ribozymes cleaved the model substrate (100 bases long) at the predicted target site. Two ribozymes containing longer substrate binding sites cleaved the substrate much more efficiently than the other ribozyme containing shorter substrate binding site. Also, the ribozymes with long substrate binding sites had high activity against the full-length GUS mRNA (1.9 kilobases) and maintained the activity even at a low temperature, 26 degrees C, a general growth condition of plant cells. Effects of the substrate binding site length of the ribozyme on cleavage activity are discussed.

To detect the predominant lactobacilli in the intestinal flora of healthy thoroughbreds, we isolated lactobacilli from the feces of nine thoroughbreds (five males and four females; 0-15-year-old). The isolated lactobacilli comprise 17 species (37 strains), and they were classified into five groups: Lactobacillus salivarius (6 species), L. reuteri (6 species), Lactobacillus delbrueckii (3 species), L. buchneri (1 species) and L. vitulinus (1 species). On the basis of 16S rRNA gene sequences, we identified 3 other phylogenetic relatives belonging to the genus Lactobacillus. These results suggest that the intestinal flora of thoroughbreds may comprise many species of the genus Lactobacillus. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analyses of the 340-bp fragments of the 16S rRNA genes from the same nine fecal samples showed that L. hayakitensis, L. equigenerosi and L. equi are contained in all the samples, suggesting that these species are predominant lactobacilli in the intestinal flora of thoroughbreds.

The white-rot fungus T. versicolor UAMH 8272 produced two groups of laccases, each of which included several isoforms showing different isoelectric points (pI). Group 1 and group 2 laccases, respectively, displayed higher pI 5–6 and lower pI 3–4. Of the four cloned full-length laccase cDNAs, Lac 1 and Lac 4 were expressed in the heterologous protein expression system using Aspergillus oryzae. The measured pI of each Lac 1 and Lac 4 expressed in A. oryzae was lower than that of pI predicted from the amino acid composition. With this regard, isoelectric focusing of Lac 1 showed the presence of multiple protein bands in the 3.0–4.0 pI range, although the predicted pI value of Lac 1 was 4.7. Similarly, Lac 4 exhibited a pI value which was lower than that predicted (3.6 vs. 4.3, respectively). In all tested hydroxyPCBs, higher chlorinated hydroxyPCBs were less susceptible to in vitro degradation by laccase than lower chlorinated hydroxyPCBs. Although Lac 4 showed a generally higher activity than Lac 1, the two laccases were characterized by quite different substrate specificity toward two hydroxy-tetrachlorobiphenyl congeners. Two metabolites were obtained from the metabolism of hydroxy-pentachlorobiphenyl: a ten chlorine-substituted dimer with a C–O bond, and one with a C–C bond.

The behavior of heavy metals in wood during its carbonization process was examined. Cadmium in wood samples was found to be volatile when the samples were carbonized at 600 degrees C or higher, which demonstrated that removal of cadmium was feasible. Meanwhile, lead was found to be barely volatile even if the wood samples were carbonized at 1,000 degrees C or higher, which demonstrated that lead was difficult to remove and recover. The possibility of removing/recovering lead contained in wood by energization was then examined. By examining the concentration of sulfuric acid used as an electrolyte as well as load voltage, approximately 10% of lead was found to be recoverable.

Lactobacillus reuteri is a heterofermentative lactic acid bacterium that naturally inhabits the gut of humans and other animals. The probiotic effects of L. reuteri have been proposed to be largely associated with the production of the broad-spectrum antimicrobial compound reuterin during anaerobic metabolism of glycerol. We determined the complete genome sequences of the reuterin-producing L. reuteri JCM 1112(T) and its closely related species Lactobacillus fermentum IFO 3956. Both are in the same phylogenetic group within the genus Lactobacillus. Comparative genome analysis revealed that L. reuteri JCM 1112(T) has a unique cluster of 58 genes for the biosynthesis of reuterin and cobalamin (vitamin B(12)). The 58-gene cluster has a lower GC content and is apparently inserted into the conserved region, suggesting that the cluster represents a genomic island acquired from an anomalous source. Two-dimensional nuclear magnetic resonance (2D-NMR) with (13)C(3)-glycerol demonstrated that L. reuteri JCM 1112(T) could convert glycerol to reuterin in vivo, substantiating the potential of L. reuteri JCM 1112(T) to produce reuterin in the intestine. Given that glycerol is shown to be naturally present in feces, the acquired ability to produce reuterin and cobalamin is an adaptive evolutionary response that likely contributes to the probiotic properties of L. reuteri.

The rabbit is considered to be a valuable laboratory animal. We compared 2% acetamide and glycerol as cryoprotectants in egg-yolk diluent for ejaculated Japanese white rabbit spermatozoa to improve sperm cryopreservation methods. Fertility through artificial insemination, forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rates of forward progressive motility and plasma membrane integrity of the spermatozoa frozen with acetamide (27.1 +/- 8.3% and 24.5 +/- 6.5%) were significantly (P0.05) higher than those of the spermatozoa frozen with glycerol (16.3 +/- 10.9% and 14.3 +/- 7.6%). Though there was no significant difference in the kindling rates, the litter size of females inseminated with spermatozoa frozen with acetamide (6.0 +/- 1.1) were significantly (P0.05) higher than those of spermatozoa frozen with glycerol (3.0 +/- 0.4). The results indicate that 2% acetamide has a higher cryoprotective effect than 2% glycerol for sperm cryopreservation in the Japanese white rabbit.

The transport system responsible for glutamine, alanine and glutamate in MOLT4 human T4 leukemia cell line were characterized. Kinetic studies of sodium-dependent glutamine and alanine transport exhibited a single saturable high-affinity carrier with a Michaelis constant of 152 +/- 26 microm and 203 +/- 36 microm and a maximal transport velocity of 960 +/- 165 and 1096 +/- 208 nmol/10(9)cells/min, respectively. Glutamate uptake was less than one-tenth of glutamine and alanine, and linearly increased with glutamate concentration which was mediated by diffusion. 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), known as anion channel blockers, inhibited the sodium-dependent glutamine and alanine transport by 40% at 10 microm. Cellular contents of these amino acids in MOLT4 cells revealed glutamate to be the highest among them despite low glutamate influx. A glutamine metabolism study using whole cells indicated this high conversion rate from glutamine to glutamate, but no conversion to another amino acid. Based on these results, the high glutamate concentration in MOLT4 was speculated to be synthesized from transported glutamine by active glutaminase.

We examined motility, plasma membrane integrity, and binding capacity to homologous zona pellucidae (ZP) of frozen/thawed epididymal cat sperm as a model species for endangered felines. Epididymal spermatozoa from 20 domestic cats were frozen with freezing egg-yolk extender containing 3.0% glycerol in 0.25-ml straws. Post-thaw motility and plasma membrane integrity of the frozen/thawed spermatozoa were 31.8 +/- 2.4% and 32.2 +/- 4.2%, respectively. The frozen/thawed spermatozoa were co-cultured with frozen/thawed immature homologous oocytes with intact ZP for 3 h to examine their ability to bind to the ZP. Sixteen of the 20 frozen/thawed sperm samples demonstrated the ability to bind to ZP. These results indicated that the freezing system for epididymal sperm used in the present study gives appropriate information for banking the genetic resources of wild felid species.

Treatment with excessive amounts of Vitamin A during maternity induces fetal malformations. However, it is unclear whether these malformations are due to gene mutations or not. Using transgenic mice (containing lacZ gene showing beta-galactosidase enzymatic activity), we planned to observe whether gene mutations occur in the fetal tissues after treatment during maternity with Vitamin A (retinol palmitate). On the 11th day of pregnancy, mothers were given 30 mg (group 2), 150 mg (group 3) and 300 mg (group 4) of Vitamin A/kg body weight orally. Fetuses obtained on the 18th day of gestation showed malformations, such as cleft palate, origodactyly, brachydactyly and ectromeria. Most notably, cleft palate occurred dose dependently. The incidental rates were 100% in group 4, 58% in group 3 and 6% in group 2. The number of dead and absorbed fetuses also increased dose dependently with the treatments. DNA (integrated vectors containing lacZ genes) extracted from each fetus showed Vitamin A-induced lacZ mutations, especially in the malformed fetuses. The mutation frequencies were 4.99x10(-5) in group 4, 5.28x10(-5) in group 3 and 4.26x10(-5) in group 2. The frequencies of group 3 were significantly higher (p

A novel PCR assay was developed in order to examine the prevalence of Haemobartonella felis (H. felis) in Japanese domestic cats and which was able to differentiate of the Ohio strain and the California strain of H. felis. Blood samples from a total of 21 cats suspected of having haemobartonellosis were examined employing a novel PCR assay and demonstrated positive results in 18 cats which was confirmed by cytological examination of blood smears. Four out of 18 positive cats (22%) were infected with the California strain, whilst the other 12 cats (67%) were infected with the Ohio strain and two animals (11%) were infected with both strains. As most of the cats with moderate to severe anemia were infected with the Ohio strain, it is suggested that the most prevalent strain of H. felis in Japanese domestic cats might be the Ohio strain. In the present study, it was thought that molecular detection and characterization of H. felis may provide valuable information regarding the severity and prognosis of this illness.

A simple tubing stent was attempted to test a model for sutureless gastrointestinal anastomosis in 6 male rats at the age of 15 weeks. In the 3rd and 4th weeks after the operation, X-ray examination demonstrated that the gastrointestinal passage in the anastomotic site was quite satisfactory. There was no incidence of anastomotic leakage. In the 6th week after the operation, there were no macroscopic or microscopic ruptures, nor were there any obstructions at the anastomotic site. This simple sutureless method was effective at preparing anastomosis in the gastrointestinal tract in the rat and could be applied to other small experimental animals.

We investigated the expression of aquaporin 1 (AQP1) in tissues from canines with an inherited anomaly that causes their erythrocytes to have high K(+). Northern blot analysis revealed abundant AQP1 expression in lung and kidney, though little expression was found in spleen. Using anti-C-terminus for dog AQP1, abundant expression was shown in kidney, trachea, and eye, but little expression was shown in pancreas and cerebrum, indicating that AQP1 expression in canine tissues is similar to that noted in other mammals.

We have previously reported successful cryopreservation of epididymal rat spermatozoa (Nakatsukasa et al. 2001 Reproduction 122, 463). However, the procedure for cryopreservation of rat spermatozoa has a disadvantage; a male has to be euthanized for collection of spermatozoa from its epididymides. Obtaining ejaculated spermatozoa repeatedly from the same male could be useful for cryopreservation of invaluable spermatozoa which carry mutations including transgenes. The objective of the present study was to develop a reliable system for cryopreservation of ejaculated rat spermatozoa and efficient production of offspring from the cryopreserved spermatozoa. Matured Wistar females were mated with three males of the same strain, and killed by cervical dislocation after formation of vaginal plugs. The uteri of mated females were excised and flushed with freezing medium containing 23.0% egg yolk, 8.0% lactose, and 0.7% Equex STM to recover ejaculated spermatozoa. The semen samples were loaded into 0.25-mL straws. The straws were cooled to 5.0�C at 0.5�C/min in a programmable freezer and then exposed to liquid nitrogen (LN) vapor at 4 cm (-150�C) above the LN level for 15 min. The straws were then plunged into LN and stored for at least a week. The straws were thawed in a 37.0�C water bath for 10 min. The thawed samples were diluted to 0.5-1.5 � 106 sperm/mL into a 200-�L droplet of R1ECM and then pre-incubated for 5 h. Ovulated oocytes collected from superovulated females were introduced into the droplet and co-cultured for 10 h for in vitro fertilization (IVF). The oocytes were denuded and examined for the presence of two pronuclei (2PN) microscopically. The denuded oocytes with 2PN were transferred into the oviducts of pseudo-pregnant females. The rates of sperm motility at recovery, post-thaw, and the initiation of IVF (after pre-incubation) were 57 � 6%, 24 � 5%, and 18 � 3%, respectively. After co-culture, 46 (14%) of the total 329 co-cultured oocytes were confirmed to contain 2PN. A total of the 44 putative zygotes were transferred to five recipients, and a total of 21 live young (48%) were born from all of the transferred recipients. We were able to produce zygotes and offspring derived from cryopreserved ejaculated spermatozoa of all three males used in the present study. In conclusion, the cryopreservation system for ejaculated rat spermatozoa used in the present study is a workable protocol for banking of valuable genetic resources of laboratory rats. Further studies on the IVF procedure with cryopreserved ejaculated spermatozoa in the rat are needed to improve the fertilization rate.

The aim of the present study was to examine transient expression of transgene injected into nuclei of rat 2-cell stage embryos. We also investigated the relationship between expression in both blastomeres and tail position of penetrated spermatozoa in rat 2-cell stage embryos. Rat 2-cell stage embryos were recovered from superovulated Wistar females mated with same strain mature males at 48 h after hCG injection. DNA fragments, as the transgene containing the EGFP (enhanced green fluorescent protein) gene controlled under the CMV-IE promoter, were microinjected into one nucleus of 2-cell stage embryos. After microinjection, embryos were cultured in KRB at 37.0°C in a 5% CO2 and 95% humidified air until observation. First, transient EGFP expression in 151 injected embryos was observed using a fluorescence microscope at 6 h intervals until 48 h after injection. At 6 h after microinjection fluorescent embryos were detected, and the proportion of fluorescent embryos increased over time. The rate reached maximum (84%, 52/62) at 24 h after microinjection, and several fluorescent patterns of fluorescent blastomeres in the embryos were observed. There were blastomeres with the same or different fluorescence levels and a single fluorescent blastomere. Second, to assess tail position of the penetrated sperm in the fluorescent embryos, 75 whole mount specimens were observed by inverted phase-contrast microscopy at 24 h after the injection. Also, parthenogenetic 2-cell stage embryos that never contained sperm tail were microinjected with the transgene and observed in the same manner. To obtain parthenogenetic 2-cell embryos, 80 ovulated ova were collected from non-mated females, and incubated with 2 mM 6-DMAP for 4 h. The ova were additionally cultured for 20 h in KRB at 37.0°C in a 5% CO2 and 95% humidified air. In embryos with both blastomeres fluorescent (94%, 33/35), the sperm tail existed in both blastomeres like a bridge between blastomeres. In contrast, in one embryo with a single fluorescent blastomere (4%, 1/24), the sperm tail existed in both blastomeres, and in other embryos with a single fluorescent blastomere (75%, 18/24), the sperm tail was positioned in the one blastomere. On the other hand, in 63 parthenogenic rat 2-cell embryos in which there was no sperm tail, most embryos (86%, 54/63) had a single fluorescent blastomere at 24 h after microinjection. The results indicated that the sperm tail position in the 2-cell embryos makes the protein migration variable. In conclusion, when the CMV-IE/EGFP gene was microinjected into nuclei of rat 2-cell embryos, at 24 h after the microinjection the EGFP was detected in most embryos; however, fluorescent patterns in blastomeres varied. It seems that EGFP derived from the transgene injected into one blastomere may move into another blastomere in rat 2-cell stage embryos, and that the presence of a sperm tail in both blastomeres may influence EGFP distribution.

RNA interference (RNAi) using double-stranded RNA (dsRNA) is a useful tool to inhibit posttranscriptional specific gene expression in mammalian cells. We investigated the effects of cytoplasmic injection of dsRNAs on transient gene expression in rat embryos when the enhanced green fluorescence protein (EGFP) gene was injected into pronuclei of zygotes. Wistar strain females were superovulated by injections of eCG and hCG and mated with males of the same strain. Pronuclear stage embryos were collected 29 h after hCG injection. The construction of the transgene, the EGFP gene controlled under the CMV-IE promoter, was dissolved at a concentration of 5 μg/mL in DNA injecting buffer (10 mM Tris-HCl, 0.1 mM EDTA), and it (3–5 pL) was microinjected into the pronuclei of embryos. After injection, embryos were cultured in KRB (Toyoda Y and Chang M C. 1974. J. Reprod. Fertil. 36, 9–22) at 37.0°C in 5% CO2 and 95% humidified air until observation. In Experiment I, transient expression of the EGFP gene after microinjection into pronuclei of zygotes was examined. The EGFP expression in the embryos was observed at 6 h intervals until 48 h after the injection using fluorescence microscopy. In Experiment II, the inhibitory effect of dsRNAs targeting the EGFP mRNA was investigated. The dsRNAs for the EGFP were synthesized with T7 RNA polymerase from PCR product amplified with a pair of hybrid primers for the EGFP and T7 RNA polymerase priming site. Then the dsRNAs (1 μg/μL) solution (3–5 pL) was injected into the cytoplasm of the DNA-microinjected embryos 1 h after microinjection. As a control, the TE buffer solution (3–5 pL) was injected into the cytoplasm of the DNA-microinjected embryos. These embryos were cultured and then observed by fluorescence microscopy 48 h after DNA injection. Fluorescent intensity of the embryos was captured and analyzed with image analyzing software (Scion Image, Scion Corporation, Frederick, MD, USA), and all data were analyzed by χ2 test. In Experiment I, the initiation of the fluorescent embryos was observed 24 h after DNA microinjection, and the proportion of fluorescent embryos in five replicates reached maximum (67%, 106/159) at 48 h after DNA injection. As shown in Table 1, there was a difference (P < 0.05) in the rate of fluorescent embryos between the dsRNA-injected group (18.4%, 33/179; range of 3.0–42.3%) and controls (58.8%, 77/131; range of 42.9–100%) in five replicates of Experiment II. The results of the present study suggest that injection of dsRNAs targeting the EGFP mRNA into the cytoplasm had an inhibitory effect on the transient expression of the EGFP gene microinjected into pronuclei of rat embryos. The inhibition system of the present study provides a powerful tool to study specific gene silencing, gene function, and development of early embryos. Table 1. Effect of cytoplasmic injection of dsRNA on transient expression of the EGFP gene microinjected into rat embryos

The use of chloroacetaldehyde (CAA) as a potential precolumn fluorimetric labelling reagent for adenine compounds was examined in detail. The reaction kinetics was greatly influenced by parameters such as the pH, temperature and CAA concentration. These parameters were optimized with regard to the reaction yield. The resulting procedure for CAA derivatization of adenine compounds was found to be excellent for quantitative analysis. Because the CAA derivatives of the adenine compounds studied were markedly stable, precise quantitative results were easily obtained using high-performance liquid chromatography. CAA derivatives of the five adenine compounds, i.e., 1,N6-etheno-adenine, -adenosine, -adenosine 5′-monophosphate, -adenosine 5′-diphosphate and -adenosine 5′-triphosphate were separated. The detection limits for the 1,N6-etheno derivatives were 0.5—1.7 pmol per 10-μl injection. Due to its simplicity, speed, sensitivity and selectivity, this procedure is recommended for use in studies on the metabolism and biology of adenine compounds.