Prognostic significance of surgical margin, Ki-67 and cyclin D1 protein expression in grade II canine cutaneous mast cell tumor

The prognosis for canine cutaneous mast cell tumor (CCMT) is thought to be correlated with histopathological grading. However, the wide variety of histopathologic types of grade II is one of the most troublesome and difficult points for prognosis. The objective of this study is to determine the prognostic value of surgical margin, ki-67 and cyclin D1 protein expression in grade II tumor. Surgically resected specimens of solitary grade II CCMT from 48 dogs with follow-up periods over 360 days (median was 1080 days) were used in this study. The expression of cyclin D1 and ki-67 proteins was determined by morphometrically using slides stained immunocytochemically, and the correlations among the results, survival rate, and recurrence and/or metastasis rate of each dog were analyzed statistically. The recurrence and/or metastasis and mortality rate in the incomplete surgical excision group within 30 months postoperatively were higher than that of the complete surgical excision group. In the incomplete surgical excision group, dogs with low positive staining of ki-67 had a significantly better survival, but the recurrence and metastasis rate and ki-67 positivity failed to show a significant correlation. Only a small number of cases showed cyclin D1-positive tumor cells, but most of them had a poor outcome with a high recurrence rate. In grade II CCMT, incomplete excision induced a relatively high metastasis rate and poor prognosis. Ki-67 positivity is a marker for the estimation of overall survival in incomplete surgical excision cases. Cyclin D1 positivity was low and may not have a prognostic role.     

Antihyaluronidase action of ellagic acid effectively prevents polyspermy as a result of suppression of the acrosome reaction induced by sperm-zona interaction during in vitro fertilization of porcine oocytes

The present study was conducted to examine the effects of three tannin relatives (tannic acid, TA; gallic acid, GA; and ellagic acid, EA) on antihyaluronidase and reactive oxygen species (ROS) scavenging activity, in vitro fertilization (IVF) parameters, and the acrosome reaction (AR) induced by sperm-zona interaction. Among the three tannin relatives, TA and EA showed the strongest potency for blocking the hyaluronidase activity of boar sperm, with concentration-dependent inhibition over the range of 2-10 microg/ml. In contrast, ROSs were effectively scavenged by TA and GA, but not EA. When cumulus-free oocytes were inseminated in IVF medium containing 5 microg/ml of the tannin relatives, polyspermy was significantly reduced by TA and EA (32 and 29%, respectively) compared with oocytes treated with or without GA (51 and 69%, respectively) under conditions that maintained a high sperm penetration rate (P<0.05). Interestingly, induction of the AR by treatment of preincubated sperm with progesterone was blocked by TA and GA as a result of their higher levels of ROS scavenging activity, while EA, which possessed weak ROS scavenging activity, did not disturb induction of the AR with progesterone. However, the incidence of AR induced by sperm-zona interaction was significantly decreased by the strong antihyaluronidase actions of TA and EA compared with that in the absence of these compounds. Treatment with the compounds caused neither a protective proteolytic modification of the zona pellucida matrix before fertilization nor a reduction in acrosomal proteolytic activity or the number of zona-bound sperm. These findings suggest that the antihyaluronidase action of EA effectively prevents polyspermy by suppression of AR functionality induced by sperm-zona interaction and that hyaluronidase intervention is therefore required during porcine IVF.     

A case of hyperplastic dermatosis of the West Highland White Terrier controlled by recombinant canine interferon-gamma therapy

A 3.5-year-old, male West Highland White Terrier was diagnosed as having hyperplastic dermatosis by clinical and histopathological findings. Controlling of Malassezia overgrowth by antifungal drugs provided a temporal improvement of the skin lesions, but the disease was deteriorated within the next 2 months despite the negative demonstration of the yeasts. Induction of recombinant canine interferon-gamma (rCaIFN-gamma) therapy resulted in almost complete cure of the skin lesions within 2 months after the initiation of the therapy. No adverse effects were detected during the therapy. Our results suggested that the rCaIFN-gamma therapy is potential to be a novel therapeutic option for controlling the breed-specific hyperplastic skin disease.     

Mast cell MMP-9 production enhanced by bacterial lipopolysaccharide

Although mast cells contribute to host protective immunity against bacterial infections, the exact mechanism of their recruitment at the affected site has been unclear. Recently, we have reported that both mouse and human mast cells are capable of producing matrix metalloproteinase (MMP)-9, a matrix-degrading enzyme necessary for leukocyte transmigration. Here, we demonstrated that bacterial lipopolysaccharide (LPS) enhanced MMP-9 production of mouse bone marrow derived-cultured mast cells. This action of LPS was partially suppressed by the pretreatment of cultured mast cells with a protein kinase C (PKC) inhibitor, indicating the possible involvement of PKC signaling pathways in the production of MMP-9 by LPS. Thus, these suggest the upregulation of mast cell MMP-9 by bacterial components, thereby resulting in their migration at the affected site.     

Ontogeny of testicular inhibin and activin in ducks: An immunocytochemical analysis

The ontogeny of testicular inhibin/activin in ducks was investigated. Testicular localization of three inhibin/activin subunits (α, β_A and β_B) was determined in embryonic and newly hatched ducks from 12 days of incubation to 1 day of age, in immature ducks and in adult ducks. In the duck embryonic testis, positive α‐subunit immunostaining was first detected in the Leydig cells and Sertoli cells on day 15 of incubation, whereas β_A‐subunit and β_B‐subunit immunostaining were found in Sertoli cells and primary germ cells on day 18 of incubation. In 1 month old ducks, intense staining of α‐subunit was present in the seminiferous epithelium consistent with localization in Sertoli cells and primary germ cells, and the immunostaining of the β_A‐ and β_B‐subunit was also present in Sertoli cells and primary germ cells. Specific immunostaining with inhibin/activin α‐, β_A‐ and β_B‐subunits antisera occurred in Sertoli cells in the adult duck testes. In conclusion, it was shown that, in the duck testis, the majority of α‐, β_A‐ and β_B‐subunits are colocalized in Sertoli cells with a certain degree of staining in germ cells and the α‐subunit is present in Leydig cells of embryonic testes before day 18 of incubation. These results indicate that Sertoli cells and possibly germ cells in the embryonic testes of late stage of incubation and newly hatched ducks, immature ducks and mature ducks may produce bioactive inhibin dimers, inhibin A and inhibin B, as a possible regulator of follicle‐stimulating hormone secretion. Free inhibin/activin subunits and their dimers may also play an autocrine/paracrine role in the development of the testis and spermatogenesis. Furthermore, early onset of the α‐subunit in duck testes indicates that it may have an autocrine/paracrine effect on steroid hormones, which is important for sex differentiation.     

Induction of Systemic Resistance in Cucumber against Several Diseases by Plant Growth-promoting Fungi: lignification and Superoxide Generation

Five fungal isolates (Trichoderma, Fusarium, Penicillium, Phoma and a sterile fungus) from zoysiagrass rhizosphere that promote plant growth were tested for their ability to induce systemic resistance in cucumber plants against Colletotrichum orbiculare. Roots of cucumber plants were treated with these fungal isolates using barley grain inocula (BGI), mycelial inocula (MI) or culture filtrate (CF). Most isolate/inoculum form combinations significantly reduced the disease except BGI of Trichoderma. These fungal isolates were also evaluated for induction of systemic resistance against bacterial angular leaf spot and Fusarium wilt by treatment with BGI. Penicillium, Phoma and the sterile fungus significantly reduced the disease incidence of bacterial angular leaf spot. Phoma and sterile fungus protected plants significantly against Fusarium wilt. Roots treated with CFs of these fungal isolates induced lignification at Colletotrichum penetration points indicating the presence of an elicitor in the CFs. The elicitor activity of CFs was evaluated by the chemiluminescence assay using tobacco callus and cucumber fruit disks. The CFs of all isolates elicited conspicuous superoxide generation. The chemiluminescence activity of the CF of Penicillium was extremely high, and its intensity was almost 100-fold higher than that of other isolates. The chemiluminescence activity was not lost following treatment with protease or autoclaving or after removal of lipid. The MW 12,000 dialyzed CF fraction was highly effective in eliciting chemiluminescence activity. Chemiluminescence emission from cucumber fruit disks treated with Penicillium was the same as that obtained from tobacco callus, except that the lipid fraction also showed a high activity. Both the MW 12,000 fraction and the lipid fraction induced lignification in the epidermal tissues of cucumber hypocotyls.     

Development and promotion of laborsaving application technology for paddy herbicides in Japan

Total labor time on paddy rice has been decreasing year by year with the development and introduction of appropriate herbicides, especially ‘one‐shot' herbicides. However, in the case of application of granules, it is still difficult for a farmer to apply herbicides while carrying heavy power backpack sprayers. New formulation recipes and different application technology such as a throw‐in type formulation on jumbo granules or flowable has improved heavy workloads in comparison with granule application by using heavy power backpack sprayers. In addition, other advantageous points such as application volume and elimination of the drift problems of this new application technology were introduced and confirmed. As a result of this introduction, these formulations were used in 830 000 ha and reached 30% of the total treatment area in paddy rice in 2000.     

Biocontrol ofRhizoctonia damping-off of cucumber by non-pathogenic binucleateRhizoctonia

Three isolates of binucleateRhizoctonia (BNR) were tested for biological control of damping-off of cucumber seedlings caused byRhizoctonia solani AG 2-2 and AG 4. BNR isolates L2 (AG Ba) and W1 and W7 (AG A) provided protection of 58 to 71% against virulent isolate C4 of AG 4 and 64 to 75% protection against virulent isolate RH 65 of AG 2-2. Varying protection was provided to the seedlings by the BNR isolates against the virulentR. solani from the two AGs depending on their combination. The BNR isolates did not vary in providing protection to the seedling when tested against virulent C4 when both isolates were inoculated using three different methods,viz. in water agar, combination of water agar and soil and using soil alone. Protection of 58 to 71 % was provided by the isolates when inoculation was done on the hypocotyl using water agar, 62.8 to 75% using the combination of water agar and soil, and 75 to 85% when inoculation of both isolates was done in soil. Pre-incubation of BNR W7 or delayed inoculation of C4 (from 0.5 day to longer duration) using the different methods provided an increased protection to the seedlings to give complete inhibition of damping-off disease. Simultaneous inoculation of both BNR W7 and C4 using the three methods failed to provide protection to the seedlings. Among the BNR isolates, BNR W7 showed plant growth promotion in terms of significant increase in plant height (P=0.01) and fresh weight (P=0.05).