Effects of synchronization of donor cell cycle on embryonic development and DNA synthesis in porcine nuclear transfer embryos

The relationship between donor cell cycle and the developmental ability of somatic cell nuclear transfer (SCNT) embryos has not fully been elucidated. Donor cells that are usually prepared by serum starvation or confluent-cell culture for SCNT represent a heterogeneous population that includes mainly G0 phase cells, other cells in different phases of the cell cycle and apoptotic cells. In this study, we compared the developmental ability of porcine SCNT embryos reconstructed from G0 phase cells (G0-SCNT embryos) and strictly synchronized-G1 phase cells (G1-SCNT embryos), and examined the developmental rates and timing of first DNA synthesis. The G0 phase cells were synchronized by confluent culture, and the G1 phase cells were prepared from actively dividing M phase cells. The G1-SCNT embryos showed a significantly higher (P<0.05) developmental rate to the blastocyst stage per cleaved embryo (59%) than the G0-SCNT embryos (43%). Moreover, initiation of first DNA synthesis and cleavage occurred significantly earlier in the G1-SCNT embryos than in the G0-SCNT embryos. Delay of initiation of first DNA synthesis in the SCNT embryos by aphidicolin resulted in decreased developmental rates to the blastocyst stage without any effect on cleavage rates. Our data demonstrates that synchronized-G1 phase cells can be used as donor cells for SCNT embryos and that earlier initiation of first DNA synthesis may be important for subsequent development of SCNT embryos. The SCNT system using G1-synchronized cells, in terms of their highly uniform and viable cell states, can be useful for studying the reprogramming processes and embryonic development of SCNT embryos.     

Age- and sex-related changes in susceptibility of Wistar rats to Strongyloides venezuelensis infection

The effects of host age and sex on susceptibility to Strongyloides venezuelensis in Wistar rats were examined by counting larvae recovered from the lungs of animals 3 days after infection. The susceptibility of female rats to S. venezuelensis rapidly decreased with age and elevated estrogen. Resistance in female rats inoculated at 6 and 10 weeks of age was nine and twenty-fold higher, respectively than that in the youngest group (3 weeks). In contrast, the susceptibility of male animals was lowest in the youngest group, then increased with age and elevated testosterone. Sex differences in susceptibility were not evident in the youngest group, but became apparent with age.     

Androgen- and estrogen-dependent sex differences in host resistance to Strongyloides venezuelensis infection in Wistar rats

The effects of male and female sex hormones on the protective capacity of Wistar rats against infection with Strongyloides venezuelensis were investigated. Male rats were more susceptible than females in terms of worm recovery from the lungs. Orchidectomy of male animals significantly reduced the plasma testosterone concentration and increased host resistance to the migratory stages of S. venezuelensis larvae. In contrast, ovariectomy of female animals significantly decreased host resistance in association with a significant reduction of estrogen levels. To examine the direct effect of sex hormones, exogenous testosterone and estrogen were implanted into animals. Susceptibility significantly increased or decreased in ovariectomized females given testosterone or estrogen, respectively. These results suggest that male and female sex hormones are important in the down- and up-regulation of host resistance against S. venezuelensis in Wistar rats.     

Comparative Analysis of mRNA Expression of Surface Antigens between Histiocytic and Nonhistiocytic Sarcoma in Dogs

Background

Definitive diagnosis of histiocytic sarcoma (HS) in dogs is relatively difficult by conventional histopathological examination because objective features of HS are not well defined.

Hypothesis

Quantitative analysis of mRNA expression of selected cellular surface antigens (SAs) specific to HS in dogs can facilitate objective and rapid diagnosis.

Animals

Dogs with HS (n = 30) and dogs without HS (n = 36), including those with other forms of lymphoma (n = 4), inflammatory diseases (n = 6), and other malignant neoplasias (n = 26).

Methods

Retrospective clinical observational study. Specimens were collected by excisional biopsy, needle core biopsy, or fine needle aspiration. To determine HS detection efficacy, mRNA expression levels of selected SAs specific to HS in dogs, including MHC class IIα, CD11b, CD11c, and CD86, were quantitatively analyzed using real‐time quantitative polymerase chain reaction.

Results

Each SA mRNA expression level was significantly higher in HS dogs than in non‐HS dogs (P = .0082). Cutoff values for discriminating between HS and non‐HS dogs based on these expression levels were calculated on the basis of receiver‐operating characteristic analysis. Accuracy of the cutoff values, including MHC class IIα, CD11b, CD11c, and CD86, was 87.9, 86.4, 86.4, and 84.8%, respectively.

Conclusions and Clinical Importance

Our results suggest that quantitative analysis of mRNA expression of the selected SAs could be an adjunctive diagnostic technique with high diagnostic accuracy for HS in dogs. Substantial investigation is required for exclusion of diseases with similar cell types of origin to lymphoma.     

Analysis of KIT expression and KIT exon 11 mutations in canine oral malignant melanomas

KIT, a transmembrane receptor tyrosine kinase, is one of the specific targets for anti-cancer therapy. In humans, its expression and mutations have been identified in malignant melanomas and therapies using molecular-targeted agents have been promising in these tumours. As human malignant melanoma, canine malignant melanoma is a fatal disease with metastases and the poor response has been observed with all standard protocols. In our study, KIT expression and exon 11 mutations in dogs with histologically confirmed malignant oral melanomas were evaluated. Although 20 of 39 cases were positive for KIT protein, there was no significant difference between KIT expression and overall survival. Moreover, polymerase chain reaction amplification and sequencing of KIT exon 11 in 17 samples did not detect any mutations and proved disappointing. For several reasons, however, KIT expression and mutations of various exons including exon 11 should be investigated in more cases.     

Improvement of rice (Oryza sativa L.) growth in upland conditions with deep tillage and mulch

The productivity of upland rice in Japan as well as in the world is low and unstable owing to scarce and unpredictable rainfall. The objective of this study was to examine whether agronomic methods could enhance grain yield of upland rice. Four field experiments were conducted from 2001 to 2003 in Nishitokyo, Japan, under upland conditions with different water supplies, in order to quantify the effects of deep tillage combined with deep placement of manure (50-cm depth), straw mulch (6 t ha⁻¹), or their combinations on the growth and grain yield of rice. Mulch kept surface soil moisture higher than without mulch even at reproductive stage, and it increased yield to the greatest extent under the most favourable conditions with much rainfall before heading (i.e., 2003). Deep tillage with deep placement of manure induced deep root proliferation and higher nitrogen uptake, increasing biomass production, and panicle number, and consequently grain yield was enhanced under the two lowest yielding environments with less rainfall before heading. Rice plants with deep tillage with deep manure application without mulch tended to have lower leaf water potential and higher diffusion resistance during drought, and negative effects on grain filling and harvest index in some experiments compared with the control. When deep tillage with deep placement of manure was combined with mulching in two experiments in 2002 and 2003, grain yield always enhanced compared with the control (P < 0.10, 6.0 t ha⁻¹ versus 5.4 t ha⁻¹ on average), suggesting their synergetic mechanisms for yield increase and stabilization. The results showed that deep tillage or mulching can improve grain yield of rice under drought-prone rainfed upland conditions in a temperate climate on an Andosol, and their combination had more consistent and greater positive effects.     

In vitro study on the increase of trypsin resistance in the rat testicular sperm head.

The alteration of trypsin resistance in rat sperm head was investigated after the sperm were incubated in the culture media (199-Earele, 1% BSA) containing the inhibitors of protein synthesis. The head of non-incubated testicular sperm was easily digested by trypsin (1 mg/ml) while the head of the incubated sperm showed resistance to trypsin digestion after 3 hr-incubation. Trypsin resistance in epididymal sperm head did not change after the sperm was incubated. Neither cycloheximide (100 micrograms/ml) nor chloramphenicol (100 micrograms/ml) affected the trypsin resistance in the testicular sperm head.     

Molecular cloning, chromosomal location, and biological activity of porcine interleukin-21

A pig interleukin-21 (IL-21) cDNA was successfully cloned and sequenced from porcine peripheral blood lymphocytes (PBL) stimulated with 10 microg/ml concanavalin A (ConA), 10 microg/ml phytohemagglutinin P (PHA), 50 ng/ml phorbol 12-myristate 13-acetate (PMA), and 0.5 microg/ml anti-porcine CD3 antibody for 48 hr. The open reading frame of the porcine IL-21 cDNA is 459 base pairs in length and encodes 152 amino acids. The predicted amino acid sequence of the porcine IL-21 shows 86.2%, 77.7%, and 58.4% identity to the bovine, human, and murine IL-21, respectively. The porcine IL-21 gene was mapped to porcine chromosome 8 (8q22-->q23) by means of fluorescence in situ hybridization and radiation hybrid mapping, where the porcine IL-2 gene had been mapped nearby. The recombinant porcine mature IL-21 expressed by E. coli induced dose-dependent proliferation and IFN-gamma production from a human NK cell line, NK0. The porcine IL-21 identified in this study will be helpful for the enhancement of innate immune responses of pigs.