TIMP3: a physiological regulator of adult myogenesis

Myogenic differentiation in adult muscle is normally suppressed and can be activated by myogenic cues in a subset of activated satellite cells. The switch mechanism that turns myogenesis on and off is not defined. In the present study, we demonstrate that tissue inhibitor of metalloproteinase 3 (TIMP3), the endogenous inhibitor of TNF alpha-converting enzyme (TACE), acts as an on-off switch for myogenic differentiation by regulating autocrine TNF alpha release. We observed that constitutively expressed TIMP3 is transiently downregulated in the satellite cells of regenerating mouse hindlimb muscles and differentiating C2C12 myoblasts. In C2C12 myoblasts, perturbing TIMP3 downregulation by overexpressing TIMP3 blocks TNF alpha release, p38 MAPK activation, myogenic gene expression and myotube formation. TNF alpha supplementation at a physiological concentration rescues myoblast differentiation. Similarly, in the regenerating soleus, overexpression of TIMP3 impairs release of TNF alpha and myogenic gene expression, and delays the formation of new fibers. In addition, downregulation of TIMP3 is mediated by the myogenesis-promoting microRNA miR-206. Thus, TIMP3 is a physiological regulator of myogenic differentiation

Regional accretion of gelatinase B in mammary gland during gradual and acute involution of dairy animals

The level of gelatinases in surrounding body fluids of actively remodelling tissue is indicative of basement membrane and extracellular matrix degradation under various physiological and pathological circumstances. To elucidate the association of gelatinase with mammary tissue remodelling during gradual or acute involution, in the first trial, goats milked twice daily (lactation) and goats receiving decreased milking frequency (involution) served to provide a total of 12 milk samples and 11 mammary secretion samples, respectively. In the second trial, 6 cows served to provide samples of dry secretion in 3 consecutive weeks immediately following milk stasis. Gelatin zymography was applied for gelatinase phenotyping and quantification on milk, plasma and the degranulation medium/lysate of milk somatic cells. Results indicated that the most prevalent gelatinase subtype switched from gelatinase A in milk to gelatinase B in involution secretion. Mammary secretion of goats during involution contained marginally higher protein level, significantly lower casein ratio and greater specific capacity of gelatinase B compared with those of milk during lactation. Specific capacities of gelatinases A and B in plasma of goats were similar during lactation and involution, while gelatinase B capacity in degranulation medium/lysates based on unit number of goat somatic cell was significantly higher during involution than during lactation. Milk stasis of cows induced a significant increase in specific capacity of gelatinase B, but not gelatinase A, of dry secretion up to the third week. Results of both trials agree that regional selective accretion of gelatinase B in milk might have played a role in mammary tissue remodelling during involution induced by either decreasing milking frequency or milk stasis. It is suggested that infiltrated polymorphonuclear neutrophils are one of the potential contributors responsible for the accumulation of gelatinase B during involution

Calcium homeostasis and its relationship to superoxide production in blood and milk neutrophils of lactating goats

Polymorphonuclear neutrophils (PMN), which comprise over 70% of the somatic cells in goat milk, are a major cellular component of innate immunity in the goat mammary gland. However, the function of milk PMNs is modified after diapedesis compared to PMNs in blood. As many aspects of PMN activity depend directly on intracellular Ca(2+) concentration ((Ca(2+))(i)), the present study aimed to determine the changes in Ca(2+) homeostasis of milk PMNs from lactating goats compared to autologous blood PMNs, and to examine the significance of these variations to the immuno-competency of milk PMNs. The intracellular Ca(2+) store of freshly prepared milk cells was estimated from the elevation of (Ca(2+))(i) after ionomycin treatment, which was found to be significantly less than blood PMNs. Replenishment of the intracellular Ca(2+) store in milk cells after intracellular Ca(2+) depletion by Bapta-AM followed by spiking with 2.5 mM Ca(2+) for 20 min was also compared to that of blood PMNs, showing that after depletion/spiking the intracellular Ca2+ store in milk cells was much less than blood PMNs. The production of superoxide anion (O(2)(-)) in vitro in response to (Ca(2+))(i)-dependent or (Ca(2+))(i)-independent modulators was used to evaluate the relevance of altered Ca(2+) homeostasis on the immunocompetency of milk cells compared to blood PMNs. The results indicated that milk cells produced similarly low levels Of O(2)(-) as blood PMNs when treated with ionomycin. However, the amount Of O(2)(-) produced by milk cells in response to phorbol 12-myristate 13-acetate (PMA) stimulation, although greater than ionomycin treatment, was significantly less than that of blood, PMNs. The capacity for O(2)(-) production by both cell types in response to PMA reverted to the resting state with use of the protein kinase C (PKC) inhibitor, staurosporine. In conclusion, the current study demonstrated an irreversible shortage of intracellular Ca(2+) in the milk PMNs of lactating goats compared to blood PMNs. It also showed that preliminary O(2)(-) production, primed by ionomycin treatment, remained unchanged in milk PMNs, despite the shortage in intracellular Ca(2+), but decreased O(2)(-) production capacity, mediated via the PKC pathway, in milk PMN. It is suggested that the defects in Ca(2+) homeostasis in milk PMNs of lactating goats is partially attributable for the post-diapedesis functionality modifications. (C) 2009 Elsevier B.V. All rights reserved

TNF alpha-mediated plasminogen activation on neutrophils is involved in the high plasmin activity in mammary secretion of drying-off cows

Interactions between inflammatory cytokines and plasminogen (Pg) activation system on immune cells are yet to be established. In previous studies we reported a somatic cell-associated elevation of proteolytic activity in mammary secretion of drying-off goats and cows. The purposes of the present study were to examine the role of TNF-alpha in polymorphonuclear neutrophil (PMN)-associated Pg activation, and the significance of this activation pathway for overall plasmin (I'm) activity in mammary secretion of drying-off cows. Results of experiments in vitro showed that the spontaneous Pg activation observed on fresh preparations of bovine blood PMN was completely blocked by anti bovine TNF-alpha antibody, and was further up-regulated by exogenous bovine TNF-alpha. Monitoring the parameters of mammary secretion of drying-off cows revealed that both somatic cell counts and differential PMN ratio was significantly elevated at weeks 1, 2 and 3 of milk stasis. Nevertheless, specific activity of soluble Pm in mammary secretion increased and the level of 17-kDa TNF-alpha decreased immediately following milk stasis. limmunoblotting revealed that although both 26-kDa pro-TNF-alpha. and 17-kDa TNF-alpha were consistently present in somatic cells of mammary secretion collected at weeks 0, 1, 2 and 3 of milk stasis, only 26-kDa pro-TNF-alpha was present in somatic cells of milk during lactation. In-vitro assay indicated that cell-free mammary secretion of drying-off cows exerted no Pg activation bioactivity towards bovine blood PMN. Altogether, the current study suggests the existence of an active TNF-alpha-Pg-Pm autocrine/paracrine loop on the massively infiltrated PMN inside udders of drying-off cows, which involves extensive binding and internalization of 17-kDa TNF-alpha on PMN and consequently activation of Pg, resulting in high Pm activity and low 17-kDa TNF-alpha. level in mammary secretion. These coordinated mechanisms may play a role in the defence of drying-off mammary gland

奈米微粒對動物生理之影響

Nanoparticles are structures sized between 1 to 100 nm. Due to their extremely microscopic size, the value of ratio of activity to weight is enormously high. In other words, nanoparticles have the same or even better efficacy by using relatively much lighter dosages than those of the larger particles under the same condition. So far, nanoparticles have been widely applied in variable aspects, such as inhibition of microbial growth, medical applications, or feed additive of weaning piglets to reduce the population of intestine coliforms and ameliorate food intake and growth performance. Despite the broad applications to the products for daily use, little is known about the impact of nanoparticles on the livestock while the animals are administered the microscopic particles. Recent studies have demonstrated that the nanoparticles could penetrate capillaries and distribute throughout the body, which would affect physiology of animal cells. It has been shown that not only cytotoxicity and immunoreactions, but also the changes of organ weights, cell morphology, serum hormone concentrations and gene expression would be possibly caused by the administration of nanoparticles reached to a certain level. Hence, the further and thorough investigations are required to understand the effects of nanoparticles on the animal health and environment.顆粒大小介於1-100nm時稱之為奈米微粒，由於其體積微小，故活性與重量比相當高。於相同條件下，奈米微粒可較其他大粒子以相對較少之劑量達到相同效果，甚至更為優越。奈米微粒應用範圍廣泛，可應用於抑制微生物生長、醫學用途或作為離乳仔豬飼料添加物，以降低其腸道內總大腸菌(coliform)之數量，並改善離乳仔豬之採食量與生長性能。儘管奈米微粒應用廣泛，然當其使用於禽畜，是否會造成負面影響仍不明瞭。研究指出，奈米微粒可穿過微血管而遍佈於體內，進而影響動物細胞之生理，且其濃度若達一定量，不但具有細胞毒性並可能引起免疫反應，造成動物器官重量、細胞形態、血漿中激素濃度與基因表現之變化。因此，奈米微粒之應用對於動物健康與環境影響之相關資訊，仍需更進一步之深入研究

Contribution of somatic cell-associated activation of plasminogen to caseinolysis within the goat mammary gland

Functional regression of the mammary gland is partly reflected by proteolysis of milk protein and tissue protein. The involvement of the plasminogen activation system in degradation of milk protein and mammary tissue damage has been demonstrated under inflammatory conditions. In this study, mammary secretion from 23 dairy goats primarily grouped as lactation (milking twice daily) or involution (milking once daily or less) was used to determine the ratio of gravity-precipitated casein to total milk protein (casein ratio) as an index of caseinolysis, and activities of components of plasminogen activation system as well as their expressions on somatic cells. Based on the casein ratio, lactation goats were subcategorized as very active (71.8 +/- 1.0%) or less active (29.9 +/- 1.0%) in mammary function; involution goats were subcategorized as gradual (21.7 +/- 1.0%) or acute (5.9 +/- 0.2%) involution. This result suggests that caseinolysis occurred during regular lactation as well as during involution. On the other hand, activities of components of the plasminogen activation system in mammary secretion were increased along with the decreasing casein ratio, in contrast to the similar activities of their counterparts in circulation throughout various mammary statuses. Correlation analysis between casein ratio and activities of plasminogen activation system of goat milk indicated a significant negative relationship for plasmin (r = -0.64), plasminogen (r = -0.69), and urokinase-type plasminogen activator (uPA; r = -0.78) during involution but not during lactation. As for the cellular components of plasminogen activation system, there was an increase in immunoreactivity on somatic cells toward both monoclonal antibodies of human uPA and human uPA receptor under involution conditions suggesting their upregulation relative to lactation condition. Collectively, these results suggest that plasminogen activation system within the mammary gland differentially contribute to milk caseinolysis along the various stages of goat lactation. Meanwhile, a somatic cell-mediated local elevation of plasmin activity may be committed to extensive caseinolysis during involution

Obesity-associated cardiac pathogenesis in broiler breeder hens: Pathological adaption of cardiac hypertrophy

Broiler hens consuming feed to appetite (ad libitum; AL) show increased mortality. Feed restriction (R) typically improves reproductive performance and livability of hens. Rapidly growing broilers can exhibit increased mortality due to cardiac insufficiency but it is unknown whether the increased mortality of non-R broiler hens is also due to cardiac compromise. To assess cardiac growth and physiology in fully mature birds, 45-week-old hens were either continued on R rations or assigned to AL feeding for 7 or 21 days. AL hens exhibited increased bodyweight, adiposity, absolute and relative heart weight, ventricular hypertrophy, and cardiac protein/DNA ratio by d 21 (P < 0.05). Increased heart weights due to hypertrophic growth was attributed to enhanced IGF-1-Akt-FoxO1 signaling and its downstream target, translation initiation factor 4E-BP1 in conjunction with down-regulation of ubiquitin ligase atrogin-1/MAFbx (P < 0.05). Reduced activation of cardiac AMPK and downstream activation of ACC-1 in parallel with increased cardiac nitric oxide levels, calcineurin activity, and MAPK activation in AL hens (P < 0.05) suggested that metabolic derangement develops along the cardiovascular remodeling. These indictors of cardiac maladaptive hypertrophic growth were further supported by uregulation of heart failure markers, BNP and MHC-β (P < 0.05). Hens allowed AL feeding for 70 d exhibited a higher incidence of mortality (40% vs. 10%) in association with ascites, pericardial effusion, and ventricle dilation. A higher incidence of irregular ECG patterns and rhythmicity consistent with persistently elevated systolic blood pressure and ventricle fibrosis were observed in AL hens (P < 0.05). These observations support the conclusion that AL feeding in broiler hens results in maladaptive cardiac hypertrophy that progresses to overt pathogenesis in contractility and thereby increases mortality. Feed restriction provides clear physiological benefit to heart function of adult broiler hens

Profile of gelatinolytic capacity of raw goat milk and the implications for milk quality

Both endogenous and exogenous proteinases occur in milk, and they can have beneficial or detrimental effects on dairy production. Because the lactation length of dairy goats is shorter and the somatic cell count (SCC) of goat milk is generally greater compared with dairy cows, the objectives of the present study were to investigate the prevalence of major proteinases in raw goat milk, their association with SCC and production stage, and their effects on milk quality. Milk samples were collected from individual goats in consecutive weeks for different durations, covering regular lactation, late lactation, and post-milk stasis. Long-term (monthly) or short-term (weekly) fluctuations of milk fibrinolytic and gelatinolytic capacities of individual goats were revealed chronologically on fibrin and gelatin zymograms, respectively. In a separate trial involving milk samples from 23 goats at random production stages, the percentage of ultracentrifuge force-precipitable casein of total milk protein was calculated to represent milk quality and was assessed to evaluate its correlation with the corresponding proteolytic capacities. The results for regular milk indicate that gelatinase B was more abundant than gelatinase A when they first appeared at SCC of similar to 1 x 10(6)/mL. During the last month before milk stasis, both gelatinases A and B were found to be prevalent and prominent in milk regardless of the broad SCC range recorded there. Fibrinolytic activity and the active form of gelatinase A were only regularly detected in post-stasis secretions and were scarce before stasis. The results of the milk quality trial indicate that milk of relatively high proteinase capacity tended to have a low casein ratio. Correlation analysis confirmed a significant relationship between gelatinase capacity of goat milk and production stage, SCC, or casein ratio. It is suggested that an elevation of gelatinolytic capacity of goat milk coincides with an increase in somatic cell number accompanying the extension of lactation length, which is unfavorable for the production of a more desirable quality of goat milk