Influence of death struggle on the structural changes in chub mackerel muscle during chilled storage

ABSTRACT: Chub mackerel (34–35 cm, approximately 500 g), which were caught by fishing with a rod and line at the Bungo Channel, Oita prefecture, were rested overnight in a fish preserve and either killed by decapitation (control group) or allowed to struggle in air for 30 min (struggled group). Muscle samples were excised every 4 h, and measurements on breaking strength and histological observations were done for both groups. The breaking strength of muscle in the control group was significantly higher than that in the struggled group, whereby a decrease in breaking strength was delayed for 12 h compared to the struggled group. Light microscopy showed space extension among muscle cells in association with a decrease in breaking strength. Especially in the struggled group, the extended area was larger and the difference in area was significant at the time when breaking strength showed a significant difference. Using electron microscopy, the extended area showed cut and/or disappeared collagen fibrils. From these results, it was demonstrated that struggling to death promoted the degradation of collagen fibrils and the weakening of connective tissue and, resultantly, led to the faster softening of muscle of chub mackerel.     

Physiological Races and Vegetative Compatibility Groups of Fusarium oxysporum f. sp. lactucae Isolated from Crisphead Lettuce in Japan

One hundred and sixteen isolates of Fusarium oxysporum f. sp. lactucae obtained from 85 fields in three crisphead lettuce-producing areas in Nagano Prefecture, Japan were typed for races using differential cultivars Patriot, Banchu Red Fire and Costa Rica No. 4. They were also grouped into vegetative compatibility groups (VCGs) using complementation tests with nitrate non-utilizing (nit) mutants. Two California strains reported as F. oxysporum f. sp. lactucum, a type culture of F. oxysporum f. sp. lactucae, and 28 avirulent isolates of F. oxysporum obtained from crisphead lettuce were included for comparison. Among Nagano isolates, 66 isolates were identified as race 1, and 50 as race 2. Race 1 strains derived from Shiojiri and Komoro cities and race 2 from Kawakami village and Komoro city. All isolates of race 2 were biotin auxotrophs, and the race could be distinguished based on its requirement for biotin on minimal nitrate agar medium (MM). Pathogenic isolates were classified into two VCGs and three heterokaryon self-incompatible isolates. Strong correlations were found between race and VCG. All the race 1 strains were assigned to VCG 1 except self-incompatible isolates, and all the race 2 strains to VCG 2. The 28 avirulent isolates of F. oxysporum were incompatible with VCG 1 and VCG 2. California strains was vegetatively compatible with VCG 1, and they were assigned to race 1. Based on vegetative compatibility, these two races of F. oxysporum f. sp. lactucae may be genetically distinct, and F. oxysporum f. sp. lactucae race 1 is identical to F. oxysporum f. sp. lactucum. Received 7 May 2002/ Accepted in revised form 6 September 2002     

The Complete Nucleotide Sequence of a Japanese Isolate of White clover mosaic virus Strain RC

The complete nucleotide sequence was determined for genomic RNA of White clover mosaic virus (WClMV-RC) isolated from red clover (Trifolium pratense) in Japan, It is 5843 nucleotides in length, excluding the poly(A) tail at the 3' terminus. Similar to other potexviruses, it contains five open reading frames (ORFs 1 through 5), which putatively encode an RNA-dependent RNA polymerase (RdRp) (147 kDa), a triple gene block (TGB) (26 kDa/13 kDa/7 kDa), and a coat protein (CP) (22 kDa), respectively. The deduced amino acid sequence of the WClMV-RC CP was identical to that of WClMV-O, one of two New Zealand isolates, but only 85% identical to that of WClMV-M, the other New Zealand isolate, because of heterogeneity in the C-termini of CP amino acid sequences. The implication of this CP heterogeneity is discussed. Received 30 August 2001/ Accepted in revised form 11 January 2002     

Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin‐4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti‐Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c‐erbB‐2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call‐Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.     

Expression dynamics of bovine MX genes in the endometrium and placenta during early to mid pregnancy

MX belongs to a family of type I interferon (IFN)-stimulated genes, and the MX protein has antiviral activity. MX has at least two isoforms, known as MX1 and MX2, in mammals. Moreover, bovine MX1 has been found to have alternative splice variants—namely, MX1-a and MX1B. In ruminants, IFN-τ—a type I IFN—is temporarily produced from the conceptus before implantation and induces MX expression in the endometrium. However, the expression dynamics of MX after implantation are not clear. In the present study, we investigated the expression of MX1-a, MX1B and MX2 in the endometrium and placenta before and after implantation along with the expression of IFN-α, type I receptors (IFNAR1 and IFNAR2) and interferon regulatory factors (IRF3 and IRF9). Pregnant uterine samples were divided into five groups according to pregnancy days 14–18, 25–40, 50–70, 80–100, and 130–150. Tissue samples were collected from the intercaruncular endometrium (IC), caruncular endometrium (C) and fetal placenta (P). Although all the MX expressions were significantly higher in the IC and C at days 14–18, presumably caused by embryo-secreted IFN-τ stimulation, their expressions were also detectable in the IC, C and P after implantation. Furthermore, IFN-α expression was significantly higher in the IC. RT-PCR indicated IFNAR1, IFNAR2, IRF3 and IRF9 mRNA in all the tissues during pregnancy. These results suggest that all the MX genes are affected by the type I IFN pathway during pregnancy and are involved in an immune response to protect the mother and fetus.     

Involvement of a host erythrocyte sialic acid content in Babesia bovis infection

Host sialic acid (SA) has recently been suggested to play an important role in erythrocyte (RBC) infection by Babesia spp. The present study attempted to further determine the specific type of SAs important in the RBC invasion. Bovine RBC was found to bear abundant alpha2-3-linked SA residues but not alpha2-6-linked SA in nature, confirmed by flow cytometric analysis of the neuraminidase (Nm)-treated RBCs. Lectin-blot analyses revealed the removal of alpha2-3-linked SAs from the 97-, 33-, and 31-kDa bands by the Nm treatment. Addition of the Nm-treated RBCs into an in vitro culture of B. bovis resulted in a decreased population of the parasitized RBCs. The thin smear samples from the cultures were then observed under a confocal laser scanning microscope after staining with the alpha2-3-linked SA-specific lectin: a selective invasion of B. bovis was found only in the intact RBCs bearing the SAs, but not in the desialylated RBCs. Furthermore, a significant reduction of the parasitized RBCs was also observed in the culture supplemented with exogenous 3'-sialyllactose containing the alpha2-3-linked SAs. However, the complete inhibition of parasite proliferation was not achieved in the culture. These findings indicate that while the alpha2-3-linked SA-dependent pathway is needed for highly efficient invasion of host RBCs by B. bovis, there might also be other potential alternative pathways.     

Combined effects of treatment with trientine, a copper-chelating agent, and x-irradiation on tumor growth in transplantation model of a murine fibrosarcoma

Combined effects of treatment with trientine, a copper-chelating agent, and X-irradiation on development of fibrosarcoma using a murine transplantation model in vivo and on cellular survival in vitro were examined. Copper contents in the tumors and serum of trientine-treated mice were significantly lower than those of untreated mice. The tumor volumes of mouse fibrosarcoma QRsp-11 cells increased more slowly in the trientine-treated and the X-irradiated mice than in the control mice from 10 to 24 days postinoculation. The extent of inhibition of tumor growth by X-irradiation at 3 Gy was similar to that obtained by treatment with trientine. A combination of trientine and X-irradiation at 3 Gy showed inhibitory effects on tumor growth similar to those obtained by X-irradiation at 6 Gy. The results showed that trientine and X-irradiation interacted additively in inhibition of tumor growth. When QRsp-11 cells and mouse and bovine endothelial cells were treated with trientine after X-irradiation, the surviving fractions of the cells with combined treatments were essentially consistent with the products of the surviving fractions of trientine-treated cells and those of X-irradiated cells. When the cells were pretreated with trientine and X-irradiated, the surviving fractions of the pretreated cells were lower than those of cells without treatment.     

Gene silencing of cyclooxygenase-2 mRNA by RNA interference in bovine cumulus-granulosa cells

Inhibition of specific gene expression using RNA interference (RNAi) is a valuable tool for functional analysis of a target gene. However, there is little information available concerning RNAi for analysis of gene function in relation to the reproductive physiology of follicular cells in ruminants. Thus, the aim of this study was to evaluate the interfering effect of small interference RNA (siRNA) on expression of cyclooxygenase-2 (Cox-2) mRNA and prostagrandin F(2alpha) (PGF(2alpha)) production in bovine cumulus-granulosa (CG) cells. Bovine CG cells were collected from aspirated follicles and cultured. After reaching confluency, two experiments were conducted. In experiment 1, to investigate the effective concentration of siRNA, 0, 100, 250 and 500 pM of Cox-2 siRNA was introduced into the CG cells, respectively. After 24 h, the amount of Cox-2 mRNA expression was measured by RT-PCR and real-time PCR. In experiment 2, to investigate the time required for effective interference of siRNA and Cox-2 activity, 250 pM siRNA was introduced for 0, 3, 6, 12 and 24 h. After culture, the amount of Cox-2 mRNA expression was measured and the culture medium was collected to determine the PGF(2alpha) concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by introduction of 100 pM siRNA into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression. Moreover, the significant suppressive effect of 250 pM siRNA was observed 6 h after introduction, and the reduction of mRNA expression by RNAi became more obvious over 12 h. On the other hand, the PGF(2alpha) concentration in the culture medium was not significantly different 12 h after siRNA introduction; however, the PGF(2alpha) concentration 24 h after siRNA introduction was significantly decreased compared with the control at the same time point. These results suggest that gene silencing of Cox-2 with siRNA is capable of analyzing the function and expression of specific genes in bovine CG cells.