Enhancement of Drug Resistance by Lysophosphatidic Acid Receptor-3 in Mouse Mammary Tumor FM3A Cells

Lysophosphatidic acid (LPA) acts as a simple phospholipid that interacts with G protein-coupled transmembrane LPA receptors. Recently, it has been reported that each LPA receptor plays different biological roles in acquisition of the malignant property of tumor cells. In this study, to assess the involvement of LPA receptor-3 (LPA₃) in cell survival after treatment with anticancer drugs, we generated Lpar3-expressing FM3A-a3A9 cells from mouse mammary tumor FM3A cells and examined the cell survival rate after treatment with anticancer drugs compared with Lpar3-unexpressing cells. Cells were treated with 0.005 to 10 μM of cisplatin (CDDP) or doxorubicin (DOX) for 3 days. For the CDDP and DOX treatments, the cell survival rate of FM3A-a3A9 cells was significantly higher than that of Lpar3-unexpressing cells. The expression level of the Mdr1a gene in FM3A-a3A9 cells was higher than that of Lpar3-unexpressing cells, whereas no significant difference in multidrug resistance 1b (Mdr1b) and glutathione S-transferase mu1 (Gstm1) expressions was found. These results suggest that LPA₃ may enhance the cell survival rate after treatment with anticancer drugs in mouse mammary tumor cells, correlating with increased expression of the Mdr1 gene.     

Endoscopic transcervical intrauterine artificial insemination in Labrador Retriever bitches

Besides post-thawing reduced semen quality, there are some difficulties in the execution of the endoscopic transcervical intrauterine artificial insemination (AI) with frozen-thawed semen in bitches. Therefore, the aims of the present study were to evaluate behavioral and reproductive parameters (i.e., vaginal cytology and serum progesterone level) to determine time of insemination and to investigate the particularities and difficulties of this technique in bitches using fresh semen. Ten Labrador Retriever bitches were submitted to three endoscopic transcervical intrauterine AIs (with 48 h intervals). Oestrus and ovulation period were established by behaviour evaluation, progesterone assays and vaginal cytology, enabling optimal timing for AI during oestrus. During AI, the following aspects were evaluated: cervical os catheterization difficulty, semen deposition resistance, occurrence of semen backflow, and time required to perform the AI. In this study, it was possible to catheterize the cervical os in all bitches, with different degrees of difficulty, by manipulating the equipment to allow cervical visualization and catheter introduction in the cervical canal. Serial serum progesterone assays enabled estimation of LH surge day, and thus of ovulation. The pregnancy rate was 90%, with a litter size of 5.0 ± 2.6 puppies per bitch. It was concluded that the difficulties in the execution of the endoscopic transcervical intrauterine AI technique in Labrador Retriever bitches were minimized by the equipment manipulation and practical experience.     

Molecular cloning and expression analysis of the canine chemokine receptor CCR9

The chemokine receptor CCR9, which interacts with the thymus-expressed chemokine TECK/CCL25, contributes to the localization of lymphocytes to the small intestine, and is implicated in the development of human inflammatory bowel disease (IBD); however, their role in canine IBD is unknown. The objective of this study was to isolate cDNA encoding CCR9 and to investigate CCR9 expression in normal canine tissues and lymphoid cell lines. The complete open reading frame contained 1104 bp, encoding 367 amino acids, with 85% and 81% identity to human and mouse homologs, respectively. CCR9 mRNA was detected in all tissues investigated with the highest expression level in the small intestine. CCR9 mRNA was also expressed in GL-1, a canine B cell leukemia cell line, but not in CLBL-1, a canine B cell lymphoma cell line. Immunoblot and flow cytometry analyses of these cell lines using an anti-human CCR9 monoclonal antibody revealed that CCR9 protein expression was detected only in GL-1, indicating the cross-reactivity of the antibody. Using the antibody, flow cytometry showed that the proportions of CCR9(+) cells were small (mean, 4.88%; SD, 2.15%) in the normal canine PBMCs. This study will be useful in understanding canine intestinal immunity and the immunopathogenesis of canine IBD.     

Relations between plasma IGF-I concentrations during treatment with CIDR-based or Ovsynch protocol for timed AI and conception in early postpartum Japanese black beef cows

We examined the relations between plasma insulin-like growth factor (IGF) -I concentrations during treatment with CIDR-based or Ovsynch protocol for timed AI and conception and plasma steroid concentrations in early postpartum Japanese Black beef cows. Cows in the control group (Ovsynch; n = 21) underwent Ovsynch protocol (GnRH analogue on Day 0, PGF(2alpha) analogue on Day 7, and GnRH analogue on Day 9), with AI on Day 10, approximately 20 h after the second GnRH treatment. Cows in the Ovsynch+CIDR group (n = 22) received Ovsynch protocol plus a CIDR for 7 days (starting on Day 0). Cows in the further treatment group (EB+CIDR+GnRH; n = 22) received 2 mg of estradiol benzoate (EB) on Day 0 in lieu of the first GnRH treatment, followed by the same treatment as in the Ovsynch+CIDR protocol. Plasma IGF-I concentrations were determined on Days -7, 0, 7, 9 and 17. Conception rates were improved in the CIDR-combined groups (both CIDR-treated groups were combined) relative to Ovsynch group (P < 0.05) for cows with low IGF-I concentrations (<1,000 ng/ml) on Days -7, 0, and 7, but improved conception rate produced by the CIDR-based protocols did not occur in cows with a high IGF-I concentration (> or =1,000 ng/ml). Plasma estradiol-17beta concentrations increased from Day 0 to 7 (P < 0.05) and were unchanged from Day 7 to 9 in the Ovsynch group with low IGF-I concentrations on Day 0, while they were unchanged from Day 0 to 7 and increased from Day 7 to 9 (P < 0.05) in the Ovsynch group with high IGF-I concentrations on Day 0 and in the CIDR-combined group. Plasma progesterone concentrations in the Ovsynch group with low IGF-I concentrations on Day 0 were higher on Day 14 than in the Ovsynch group with high IGF-I concentrations on Day 0 and in the CIDR-combined group (P < 0.05). In conclusion, CIDR-based protocols may improve conception relative to Ovsynch in early postpartum beef cows with lower plasma IGF-I concentrations at the start of the protocols. This improvement is probably due to prevention of premature increases of estradiol-17beta and progesterone concentrations, which occurred in cows with low IGF-I concentrations treated with Ovsynch, by the CIDR treatment.     

Rheological properties of fast skeletal myosin rod and light meromyosin from walleye pollack and white croaker: contribution of myosin fragments to thermal gel formation

Myosin rod and light meromyosin (LMM) of walleye pollack and white croaker were examined for their rheological properties by measuring dynamic viscoelastic parameters. Rods from walleye pollack and white croaker increased their storage moduli (G') in the ranges of 29-43 degrees C and 31-38 degrees C, respectively, in temperature sweep analysis. Walleye pollack LMM showed no peak of G' upon heating, whereas the white croaker counterpart exhibited a single sharp peak of G' at 35 degrees C. Loss modulus (G") showed similar temperature-dependent changes for the two fish species as the case of G', irrespective of rod and LMM, although G" values were lower than those of G'. Thus, rheological properties of rod and LMM were different between walleye pollack and white croaker. Taken together with data previously reported for myosin, it was considered that both myosin rods from walleye pollack and white croaker are attributed to thermal gel formation of myosin in a low-temperature range, though in a species-specific manner.     

Neuropathogenicity of Equine Herpesvirus 9 in Cattle

The pathogenicity of equine herpesvirus 9 (EHV-9), a neurotropic equine herpesvirus isolated from a herd of Gazella thomsoni, was studied in cattle. Seven calves were inoculated intranasally with 10⁵ and 10⁷ plaque-forming units of the EHV-9 P19. Three animals showed brain lesions consisting of glial reactions and perivascular cuffings in the olfactory bulb and the frontal and temporal lobes. Additionally, the animal that was inoculated with 10⁷ plaque-forming units showed neuronal degeneration and loss, as well as nuclear inclusions compatible with herpesvirus. EHV-9 was isolated from the brain of calf 6 and the lungs of calves 1 and 2. The results suggested that cattle are susceptible to experimental infection with EHV-9 and at risk from natural infection from reservoir hosts.     

Detection of Chlamydophila psittaci by using SYBR green real-time PCR

Chlamydophila psittaci is the causative agent of human psittacosis and avian chlamydiosis. This zoonotic pathogen is frequently transmitted from infected birds to humans. Therefore proper and rapid detection of C. psittaci in birds is important to control this disease. We developed a method for detecting C. psittaci by using SYBR Green Real-time PCR based on targeting the cysteine-rich protein gene (envB) of C. psittaci. This one step procedure was highly sensitive and rapid for detection and quantification of C. psittaci from fecal samples. This assay was also able to detect other zoonotic Chlamydophila species such as C. abortus and C. felis. The assay is well suited for use as a routine detection method in veterinary medicine.