The activity of matrix metalloproteinases (MMPS) and tissue inhibitors of metalloproteinases (TIMPs) in mammary tumors of dogs and rats

We conducted zymography for detecting the activity of matrix metalloproteinases (MMPs) and reverse zymography for the activity of tissue inhibitors of metalloproteinases (TIMPs) in canine spontaneous and rat 7, 12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumor tissues. The activities of MMPs of canine mammary tumors were quite higher than those of the rat chemically induced tumors. The activities of MMPs were significantly higher in malignant tissues than in benign ones of canine tumors, whereas the activity of only MMP-2 was higher in both benign and malignant rat tumors compared to normal tissues. There were no differences of MMPs activities between benign and malignant rat tumors. The results of reverse zymography indicated that the activities of TIMP-1, -2 and -3 were strikingly higher in rat tumors than in canine tumors. The activities were higher in malignant tissues than in benign ones of dogs, and higher in tumor tissues than in normal mammary tissues of rats. The results of film in situ zymography for tissue localization of gelatinolytic activity showed that the digested area was more extended in malignant tumors than in benign ones of dogs. However, the area was similarly extended in both benign and malignant rat tumors. These results may indicate that the canine spontaneous malignant mammary tumors possess more aggressive nature than the rat chemically induced counterpart, resulting from the high level of MMPs and low level of TIMPs activities of the tumor tissues.     

The relationship between the snowmelt flood and the establishment of non‐native brown trout (Salmo trutta) in streams of the Chitose River,Hokkaido, northern Japan

Flow regime is one of the major determinants of establishment success for non‐native aquatic organisms. Here, we examine the influence of flow variability associated with snowmelt flood on the establishment success of non‐native brown trout in 10 streams in northern Japan. We regarded the presence of Age‐0 brown trout as the index of the successful establishment. The emergence of Age‐0 brown trout in our study region begins in May, a time that overlaps with the occurrence of snowmelt flood. The presence of Age‐0 brown trout was negatively associated with flow variability, and it was also negatively associated with summer water temperature. Our results indicate that the non‐native brown trout tends to establish in the streams with smaller snowmelt floods and lower summer water temperatures. Brown trout is an invasive, non‐native species that is problematic all over the world, and effective management strategies for preventing their further expansion are urgently needed. This study suggests that river managers should recognise that stable streams such as spring‐fed streams (i.e., low flow and summer water temperature) and flow‐regulated streams, have a higher potential risk of brown trout invasion.     

Conversion of dextran to cycloisomaltooligosaccharides using an enzyme-immobilized porous hollow-fiber membrane

This paper describes a cycloisomaltooligosaccharide glucanotransferase (CITase)-multilayer-immobilized porous hollow-fiber membrane used as an enzyme bioreactor. Dextran, a substrate with an average molecular mass of 43000, is converted into seven- to nine-glucose-membered cycloisomaltooligosaccharides, effective as a preventive for dental caries, aided by convective transport of the substrate to the vicinity of the enzyme through the pores. Epoxy-group-containing graft chains were uniformly appended onto the surface of pores throughout a porous hollow-fiber membrane by radiation-induced graft polymerization. Subsequently, a diethylamino group was introduced, as an anion-exchange moiety, to the graft chains, which caused the chains to expand toward the interior of the pores due to mutual electrostatic repulsion. The expanding graft chain provided multilayer binding sites for CITase. Fifty-five milligrams of adsorbed CITase per gram of membrane is equivalent to the degree of multilayer binding of 5. Finally, 80% of the multilayer-adsorbed CITase was immobilized via enzymatic cross-linking with transglutaminase to prevent the leakage of enzymes. CITase, with a degree of multilayer immobilization of 4, produced the target cycloisomaltooligosaccharides at a conversion yield of 55% in weight at 310 K during permeation by the dextran solution at a space velocity defined as the permeation rate divided by membrane volume of 6 per hour.     

Effects of grazing adaptation on herbage intake,milk production,and body weight change in lactating dairy cows after turning out to pasture in early spring

The present study investigated the effects of adaptation to grazing in early spring on the herbage intake, milk production, and body weight changes in lactating dairy cows. The experiment included 12 Holstein lactating cows during early spring. Six cows were allowed to gradually adapt to grazing (ADP) over 10 days. They were allowed to graze on a pasture for 4 h during the first week and for 19 h during the remaining 3 days (adaptation period). During the 10-day period, the remaining six cows were housed in a barn (CON). Cows in both groups received adequate silage, hay, and concentrate during the adaptation period. Subsequently, cows in both groups were allowed to graze together for 19 h for 4 weeks (experimental period). No cow received supplements during the experimental period. In the first week of the experimental period, the ADP cows showed a higher herbage intake than the CON cows. During the subsequent weeks, there were no differences in herbage intake between the two groups. At the start of the experimental period, milk production and body weight of the ADP cows were higher than those of the CON cows, and this difference lasted until the end of the experiment.     

Enzyme activity changes during anthocyanin synthesis in ‘Olympia’ grape berries

Anthocyanin synthesis began in the skin of ‘Olympia’ (Vitis labruscana Bailey) grape when the berry growth reached a late stage in the second sigmoid growth-curve. During berry development, shikimate NADP oxidoreductase (SORase, EC.1.1.1.25), phenylalanine ammonia-lyase (PAL, EC.4.3.1.5), and chalcone flavanone isomerase (CHFI, EC.5.5.1.6) were investigated in the skin. These enzyme activities were high during early development of the young berry, but rapidly decreased thereafter, and were activated again when rapid anthocyanin synthesis subsequently occurred. When PAL from the young berry was analyzed by isoelectricfocusing polyacrylamide gel electrophoresis (IEF-PAGE), it showed a single activity band with a pI value of approximately 5.4, whereas PAL from the ripe berry showed two activity bands with pIs of 5.4 and 6.5. Thus, SORase, PAL, and CHFI seem to be involved in anthocyanin accumulation simultaneously, and newly synthesized isozyme of PAL may play a key role in pigment formation in ‘Olympia’ skin. The function of these enzyme activities in young berry is also discussed in this text.     

A case study of improving milking cow performance and milking system performance with using a flow simulator

Efforts to improve dairy performance have been focused on increasing milk productivity of cows through improved feeding systems and genetic potential. However, methods for evaluating milking system performance based on milk productivity have not yet been established. Milking system performance was evaluated by measuring the claw vacuum at five flow rates (1.9–8.7 kg/min) produced using a flow simulator for a single eight‐swing milking parlor with a high‐line system. Based on these results, a double eight‐parallel milking parlor with a low‐line system was installed and tested. Farmers can take data obtained from evaluations of milking system performance into account for future management decisions, such as renewing the milking system. By renewing the milking system, average milking productivity, somatic cell linear score (LS) of bulk milk, and LS of each cow were significantly improved in the year after installing the new system (p < .01). In addition to checking conventional milking systems, this novel diagnostic method using a flow simulator can be used for checking new installations and also for proposing renovations.     

Molecular cloning, tissue expression, and subcellular localization of porcine peptidoglycan recognition proteins 3 and 4

Peptidoglycan recognition proteins (PGRPs) are innate immune molecules that are present in most invertebrates and vertebrates. Mammals have four PGRPs, PGLYRP1-4. In the present study, we cloned the cDNAs encoding porcine PGLYRP3 and 4 from the esophagus of adult swine. The length of the complete open reading frames of porcine PGLYRP3 and 4 are identical and contain 1125bp encoding 374 amino acid residues. The amino acid sequences of these two proteins were more similar to their human orthologs (78.9% [PGLYRP3] and 73.9% [PGLYRP4]) than to their mouse orthologs (71.3% [PGLYRP3] and 67.9% [PGLYRP4]). Expression analysis revealed that both PGLYRP3 and 4 were more strongly expressed in digestive tract, especially the esophagus, than in immune organs such as spleen or mesenteric lymph nodes in both newborn and adult swine. To analyze the subcellular distribution of porcine PGLYRP1-4, we constructed transfectant cell lines. Western blot and flow cytometric analyses revealed that porcine PGLYRP3 and 4 are not only secreted, but also expressed on the cell surface, unlike PGLYRP1 and 2. These results should help contribute to the understanding of PGLYRP3- and 4-mediated immune responses via their recognition of intestinal microorganisms in newborn and adult swine.