Staining pattern of the brush border and detection of cytoplasmic granules in the uriniferous tubules of female DBA/2Cr mouse kidney: comparison among various fixations and stains.

The proximal straight tubules of the female mouse kidney exhibit heavy periodic acid Schiff (PAS) staining in their brush borders and numerous cytoplasmic granules. In the present study, the female DBA/2Cr mouse kidney was examined, using various fixatives (formalin, PFA, PLP, Zamboni's, Bouin, or Carnoy solution) and various staining methods (HE, PAS, alcian blue, periodic acid methenamine-silver (PAM), toluidine blue, azan, or Congo red). Under azan and PAM, the staining pattern of the brush border was similar to that of PAS, and few effects of the fixative were observed. Cytoplasmic granules were clearly detected with PAM as well as PAS. However, these granules were not detected with Carnoy solution. Furthermore, distribution of granules differed between PAS and PAM.     

Alternative Route of Infection for Bacterial Seedling Blight of Rice Caused by Burkholderia plantarii

Burkholderia plantarii , the pathogen of bacterial seedling blight of rice, was detected in paddy water. Its concentration rose in July and August. The bacterial concentration in the paddy water was always higher along levees than at distances more than 5 m from levees. Confirmed to be released into water when graminaceous weeds were immersed, B. plantarii survived for at least 4 days at 30°C. B. plantarii was splashed at least 30 cm upward by rain splash in the field. Harvested seeds, which had been sprayed with B. plantarii released from graminaceous weeds at the flowering stage, retained the bacteria. Bacterial seedling blight occurred when the seeds were then sown in nursery boxes. These results indicated that graminaceous weeds growing on levees of paddy fields are a source of infection of the disease and that rice seeds are infected through the paddy water. Received 23 May 2002/ Accepted in revised form 1 September 2002     

Lipid-rich bovine serum albumin improves the viability and hatching ability of porcine blastocysts produced in vitro

The effects of lipid-rich bovine serum albumin (LR-BSA) on the development of porcine blastocysts produced in vitro were examined. Addition of 0.5 to 5 mg/ml LR-BSA to porcine blastocyst medium (PBM) from Day 5 (Day 0 = in vitro fertilization) significantly increased the hatching rates of blastocysts on Day 7 and the total cell numbers in Day-7 blastocysts. When Day-5 blastocysts were cultured with PBM alone, PBM containing LR-BSA, recombinant human serum albumin or fatty acid-free BSA, addition of LR-BSA significantly enhanced hatching rates and the cell number in blastocysts that survived compared with other treatments. The diameter, ATP content and numbers of both inner cell mass and total cells in Day-6 and Day-7 blastocysts cultured with PBM containing LR-BSA were significantly higher than in blastocysts cultured with PBM alone, whereas LR-BSA had no effect on mitochondrial membrane potential. The mRNA levels of enzymes involved in fatty acid metabolism and β-oxidation (ACSL1, ACSL3, CPT1, CPT2 and KAT) in Day-7 blastocysts were significantly upregulated by the addition of LR-BSA. The results indicated that LR-BSA enhanced hatching ability and quality of porcine blastocysts produced in vitro, as determined by ATP content, blastocyst diameter and expression levels of the specific genes, suggesting that the stimulatory effects of LR-BSA arise from lipids bound to albumin.     

Comparison of dendritic cell-mediated immune responses among canine malignant cells

Dendritic cell (DC) vaccination is one of the most attractive immunotherapies for malignancies in dogs. To examine the differences in DC-mediated immune responses from different types of malignancies in dogs, we vaccinated dogs using autologous DCs pulsed with keyhole limpet hemocyanin (KLH) and cell lysate prepared from squamous cell carcinoma SCC2/88 (SCC-KLH-DC), histiocytic sarcoma CHS-5 (CHS-KLH-DC), or B cell leukemia GL-1 (GL-KLH-DC) in vitro. In vivo inductions of immune responses against these tumor cells were compared by the delayed-type hypersensitivity (DTH) skin test. The DTH response against SCC2/88 cells were observed in dogs vaccinated with autologous SCC-KLH-DC, while the response was undetectable against CHS-5 and GL-1 cells in dogs vaccinated with autologous CHS-KLH-DC and GL-KLH-DC. Skin biopsies taken from DTH challenge sites were then examined for immunohistochemistry, and recruitment of CD8 and CD4 T cells was detected at the site where SCC2/88 cells were inoculated in dogs vaccinated with SCC-KLH-DC. By contrast, neither CD8 nor CD4 T cell infiltration was found at the DTH challenge site in the dogs vaccinated with CHS-KLH-DC or GL-KLH-DC. These findings may reflect that the efficacy of immune induction by DC vaccination varies among tumor types and that immune responses could be inducible in squamous cell carcinoma. Our results encouraged further investigation of therapeutic vaccination for dogs with advanced squamous cell carcinoma in clinical trials.     

Effects of estrous cycle on the mouse kidney morphology

Although mice kidney morphology shows various sexual dimorphisms, the effect of the estrous cycle has not previously been discussed. In this study, we investigated the effects of the estrous cycle on kidney morphology, including renin-positive areas, of female DBA/2 mice. No effects were confirmed in most of the histometrical parameters, however, the percentage of the renal corpuscles in which cuboidal epithelium covered under 50% of the parietal layer was significantly higher during estrus compared to that during anestrus.     

Rhizome-induction activity of chiral 2-α-methylbenzylamino-4-alkylamino-6-chloro-1, 3, 5-triazines in Cyperus serotinus Rottb.

A cytokinin-like effect of chiral 2-α-methylbenzylamino-4-alkylamino-6-chloro-1,3,5-triazines was found using a rhizome-inducing assay with Cyperus serotinus Rottb. tubers. C. serotinus tubers germinated in distilled water yielded plantlets with roots and leaves. Secondary rhizomes were normally not observed within the regular 14-day incubation time in water culture, whereas after increasing incubation periods a very short rhizome appeared (controls). 6-Benzylaminopurine (BA) significantly stimulated rhizome induction, while other plant hormones were inactive. The (R)-isomers of the 1, 3, 5-triazine compounds also stimulated induction of the rhizomes, whereas the (S)-isomers did not. The described rhizome induction system seems to be suitable as a cytokinin bioassay. The (R)-1, 3, 5-triazine compounds showing rhizome-inducing activity (RI activity) inhibited root formation and plant growth at high concentrations with symptoms which were very similar to those of BA. Therefore, the (R)-isomers appear to act as cytokinins in the rhizome induction assay.     

Evaluation of bovine embryos produced in high performance serum-free media

This review evaluates the quality of bovine embryos developed from in vitro-matured (IVM) and -fertilized (IVF) oocytes cultured in either serum-free or serum-containing media. Bovine embryos cultured in serum-supplemented medium contain numerous cytoplasmic lipid droplets and immature mitochondria compared to those cultured in serum-free medium. The accumulation of cytoplasmic lipids in embryos developed in serum-containing medium may be a result of incorporation of lipoproteins from the serum and may result in impaired function of mitochondria. The improved serum-free media (IVMD101 and IVD101) offer several advantages over culture in serum-containing medium, including increased rates of blastocyst formation and higher cell numbers. Additionally, the survival and hatching rates of embryos produced in serum-free media after post-thaw culture were superior to those of embryos produced in the serum-containing medium, suggesting that the abnormal accumulation of cytoplasmic lipids in embryos may have a negative effect on the sensitivity of embryos to chilling and freezing. These serum-free culture systems have proven to be beneficial for the production of good quality embryos from IVM-IVF bovine oocytes. Furthermore, recent studies have shown a correlation between mitochondrial function (oxygen consumption) and embryo quality. A new method using scanning electrochemical microscopy may be capable of assessing the viability and developmental potential of bovine embryos.     

Validation and application of real-time polymerase chain reaction assays for representative rumen bacteria

Real‐time polymerase chain reaction (PCR) assays for 11 representative rumen bacterial species were validated. The sensitivity was tested by using the serially diluted target 16S rDNA from respective bacterial species. The recovery of the target DNA and the assay reproducibility were determined using DNA from rumen fluid spiked with different quantities of the target. Minimum detection levels for the target were 10–100 copies in pure culture. The recovery of the added target ranged from 82.4 to 116.6%. The intra‐ and inter‐assay variations of each assay were <9.4 and <12.6%, respectively. Therefore, the real‐time PCR assays evaluated in the present study are considered to be sufficiently reliable for monitoring all 11 bacterial species in the rumen. The assays were then applied to the monitoring of the bacterial species attached to ruminally incubated rice straw. Among the monitored fibrolytic species, Fibrobacter succinogenes was found to be the most dominant, accounting for 2.61% of total bacteria after 24 h incubation. Selenomonas ruminantium and Streptococcus bovis, non‐fibrolytics, were detected on the rice straw at 8.96% and 1.16% of total bacteria, respectively. Such high levels of non‐fibrolytics on the plant fiber suggest a synergistic relationship between fibrolytics and non‐fibrolytics.     

A Practical Technique for Electron Microscopy of Buffy Coats in Dogs and Cats

Plastic hematocrit tubes (PHTs) are convenient tools for electron microscopy (EM) of peripheral blood buffy coats, and the PHT‐EM technique is expected to be a practical method for veterinary clinical medicine. In this study, fixatives composed of various concentrations of sucrose, glutaraldehyde, and phosphate buffer (PB) were tested for preparing canine and feline buffy coats. The highest quality images were obtained using a fixative consisting of 2.5% glutaraldehyde in 0.1 m PB, and it was concluded that this method allows clinicians who are inexperienced in histological techniques can conveniently transport buffy coat samples to diagnostic laboratories for analysis by EM.