Activities of calpastatin, μ-calpain and m-calpain are stable during frozen storage of meat

The stability of μ-calpain, m-calpain and calpastatin activity during frozen storage of pork was studied in two experiments. In experiment 1, pork longissimus muscle was stored at either -20 or -80°C, and the samples were assayed at 2-3 weeks interval for calpain activity and calpastatin activity using a m-calpain stock solution stored at 4°C. No effects on calpain activity at either temperature were observed for up to 123 days of storage. Calpastatin activity was stable the first few weeks of storage, where after it decreased up to 143 days of storage independently of meat storage temperature. At day 143, calpastatin activity was also assayed using a newly purified stock solution of m-calpain giving a calpastatin activity equal to the activity measured day 0 using the original m-calpain stock solution. The m-calpain stock solution was unstable during storage at 4°C and the activity decreased in a linear manner and was highly related to the observed decrease in calpastatin activity during storage. In experiment 2, meat was stored as in experiment 1 and was assayed at 2-3week intervals for calpastatin activity using a m-calpain stock solution stored at either 4 or -80°C. As in experiment 1, the measured activity of calpastatin decreased during storage using m-calpain stock solution stored at 4°C and this decrease was highly correlated to the decrease in the activity of the m-calpain stock solution. The activity of the m-calpain stock solution stored at -80°C was constant during storage period of 153 days and likewise was the calpastatin activity measured using this stock solution. The relation between measured calpastatin activity and storage time of m-calpain stock solution was tested by adding, to a calpastatin assay, up to 10μL of a partly inactivated m-calpain solution. A negative relationship was observed between added inactivated m-calpain and measured calpastatin activity which suggests that the inactive m-calpain molecules mask some of the binding sites on calpastatin and thereby prevent some of the active m-calpain molecules from binding to calpastatin. This would underestimate the measured calpastatin activity. In conclusion, the calpains as well as calpastatin are stable during frozen storage of meat, and the observed decreased in calpastatin activity is due to instability of the m-calpain stock solution used in the calpastatin assay.     

Effects of nutritional level on pork quality and gene expression of μ-calpain and calpastatin in muscle of finishing pigs

The study was designed to investigate the effects of nutritional level (control diet (CD), 14.19% crude protein, 13.81 MJ of DE/kg; low nutritional level diet (LND), 11.08% crude protein, 12.55 MJ of DE/kg) on pork quality and gene expression of μ-calpain and calpastatin in muscle of finishing pigs. The LND treatment increased drip loss (P < 0.05), had a trend to increase intramuscular fat (IMF) content (P = 0.09), decreased Warner–Bratzler shear force (WBSF) of pork (P < 0.05), improved mRNA level of μ-calpain (P < 0.05) in skeletal muscle, but had no effect on gene expression of calpastatin, compared with the CD treatment. These data suggest that a moderately reduced energy and protein diet increased pork tenderness and intramuscular fat. The increase in tenderness by LND treatment may be partly due to increased gene expression of μ-calpain in muscle.     

The effect of temperature on the activity of μ- and m-calpain and calpastatin during post-mortem storage of porcine longissimus muscle

The experiment was conducted to determine the effect of temperature during post-mortem muscle storage on the activity of the calpain system, the myofibril fragmentation and the free calcium concentration. Porcine longissimus muscle were incubated from 2h post-mortem at temperatures of 2, 15, 25 and 30 °C and sampling times were at 2, 6, 24, 48 and 120 h post-mortem. After 120 h at 30 °C the free calcium concentration increased to 530 μM from 440 μM at 2 °C. Incubation at temperatures higher than 2 °C resulted in the appearance of autolyzed m-calpain activity and a decrease of native m-calpain activity. Native m-calpain decreased more slowly than native μ-calpain, and the autolysis process started later. Myofibril fragmentation increased with storage time and incubation temperature, while calpastatin activity decreased. The study showed that high temperature incubation not only rapidly activated μ-calpain but at higher temperatures and later time points also m-calpain.     

Effect of ageing and μ-calpain markers on meat quality from Brangus steers finished on pasture

Brangus steers (n = 247) finished on pasture were used to evaluate the effects of post-mortem ageing and polymorphism CAPN1 316 and CAPN1 4751 markers on meat tenderness and objective colour measurements (CIEL*a*b*) of m. Longissimus dorsi. Ageing meat for 7 days decreased shear force (SF) by 13.7% and improved a* (8.4%) and b* (10%) compared to ageing for 1 day. No difference between 7 and 14 days of ageing was found for SF, a* and b*. However, L* increased markedly with ageing. Fitting both markers simultaneously, CAPN1 316 showed association with SF and L* and CAPN1 4751 with a* and b*. Fitting the markers individually, CAPN1 4751 affected all traits and CAPN1 316 showed association with SF and L*. Post-mortem ageing and the use of markers represent two independent and alternative tools that could be used for improving quality of meat from Brangus cattle.     

In vitro study to evaluate the degradation of bovine muscle proteins post-mortem by proteasome and μ-calpain

The degradation of bovine muscle proteins by proteasome and ubiquitous calpains was explored via 2D gel proteome analysis by inhibition of the physiological level of the proteases by specific inhibitors. The inhibition of the proteasome chymotrypsin- and trypsin-like activity results in the lack of degradation of several fragments of structural proteins such as actin, troponin T, myosin light chain and nebulin. In addition the degradation of several sarcoplasmatic proteins was eliminated when proteasome was inhibited. The inhibition of the ubiquitous calpain only resulted in minor changes in the degradation pattern, which might indicate that p94, which is not inhibited by calpastatin, is involved in the degradation post-mortem. The results of the present study indicate a sequential degradation of the structural proteins post-mortem, where calpain initiates the disruption and destabilisation of the myofibrillar structure, and thereby allows the proteasome to act.     

Identification of myofibrillar substrates for μ-calpain

To identify myofibrillar substrates of μ-calpain under post-mortem conditions, a combination of SDS–PAGE, two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) was used. Purified myofibrils were incubated with μ-calpain under post-mortem-simulated conditions for two or four days at 4 °C. The resulting protein changes were analyzed by SDS–PAGE and 2DE. The μ-calpain-mediated protein changes were identified by peptide-mass mapping using MALDI-TOF MS and revealed that desmin, actin, myosin heavy chain, myosin light chain I, troponin T, tropomyosin 1, tropomyosin 4, thioredoxin and CapZ are all degraded in vitro by μ-calpain. The findings that actin and myosin heavy chain are substrates of μ-calpain were rather surprising, as it has previously been reported that these proteins are resistant to μ-calpain degradation. However, both actin and myosin heavy chain are poor substrates compared with desmin.     

Contribution of postmortem changes of integrin, desmin and μ-calpain to variation in water holding capacity of pork

The purpose of this study was to examine the relationship between integrin, desmin, μ-calpain and water holding capacity in fresh pork. High levels of intact integrin at one day postmortem were negatively correlated with day 1 (P<0.05) and days 1-5 (cumulative) drip loss (P<0.05). High levels of intact integrin at five days postmortem were negatively correlated with days 1-7 (cumulative) purge loss (P<0.05). Intensity of intact desmin at one day postmortem was positively correlated with days 1-7 purge loss (P<0.01). There were positive correlations between intensity of intact desmin at day 7 and day 1 (P<0.01), days 1-5 drip loss (P<0.01) and days 1-7 purge loss (P<0.05). Autolysis of μ-calpain was associated with the degradation of desmin and drip or purge loss postmortem. Our results indicate that low levels of degradation of integrin and high levels of desmin degradation were associated with low drip loss values in fresh pork.     

Identification of calpastatin, μ-calpain and m-calpain in Atlantic salmon (Salmo salar L.) muscle

A soft fish muscle is generally considered as a poor quality trait among consumers and producers. This degradation and softening of post mortem muscle is thought to be partly caused by proteolytic enzymes such as the calpain system. Separation and identification of μ-calpain and m-calpain and their inhibitor – calpastatin, from Atlantic salmon (Salmo salar) muscle were for the first time assessed in this study. A two-step chromatography approach was used, starting with a hydrophobic interaction column and followed by an anion exchange column. Calpastatin was successfully separated from calpain by hydrophobic interaction chromatography, and following the anion exchange chromatography, two forms of calpastatin (I and II) and two forms of calpain (micro (μ) and milli (m)) were revealed. The proteolytic activity of μ-calpain was detectable with column chromatography, but not consistently detected with casein zymography, and m-calpain was detected with both chromatography and casein zymogram. The proteolytic activity of m-calpain per g muscle was 15 times higher than that of μ-calpain. μ-Calpain had a temperature optimum of 15 °C and a maximum calcium requirement at 0.2 mM, while m-calpain had temperature optimum at 25 °C and a maximum calcium requirement of 0.6 mM. The two forms of calpastatin differed in inhibitory activity with calpastatin II having the highest activity. Both calpastatins tolerated heat treatment, as previously seen for mammals, and they kept their activity when stored at −80 °C, but not at −20 °C. The calpain to calpastatin ratio was 1:4.5 as observed for beef muscle. This study provides evidence that two calpain isoforms, likely to be μ- and m-calpain, in addition to two forms of calpastatin exist in Atlantic salmon muscle.     

Effect of added μ-calpain and post-mortem storage on the mechanical properties of bovine single muscle fibres extended to fracture

The effects of μ-calpain and post-mortem storage on the strength of single muscle fibres were investigated. During the 10 min of incubation at pH 7.5, μ-calpain became evenly distributed throughout the fibre. μ-Calpain-incubation resulted in thinner (P <0.001) Z-lines and reduced (P <0.001) the strength of the fibres compared to controls. These results demonstrate that μ-calpain is capable of mechanically weakening the muscle fibres. Post-mortem storage of meat for 10 days at 2?°C weakened (P <0.001) the muscle fibres compared to 24-h fibres. The presence or absence of Ca(2+) affected fibre stiffness. Fibres incubated at pH 7.5 in 100 μM Ca(2+) were less stiff than fibres incubated in 200 μM EGTA. Breaking stress and strain were not affected by Ca(2+). We hypothesise that Ca(2+) causes conformational changes in some of the load-bearing proteins, which alters their initial resistance to extension, but does not affect the breaking strength of the fibres.     

Characterization of peptides released from rabbit skeletal muscle troponin-T by μ-calpain under conditions of low temperature and high ionic strength

Rabbit skeletal muscle troponin-T (200 μg ml(-1)) was incubated with μ-calpain (2 μl ml(-1)) under conditions of low temperature and high ionic strength for 180 min at 4°C and the peptides released analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Troponin-T was hydrolyzed rapidly with the simultaneous appearance of eight peptides with masses of less than 14 up to 26 kDa. Two peptides produced by 10 min of incubation were electroblotted and analysis of these peptides by N-terminal sequencing and mass spectrometry showed that the principal cleavage sites of μ-calpain on troponin-T were at Thr(45)-Ala(46), Leu(69)-Met(70), Glu(220)-Lys(221) and Asn(231)-Val(232). The peptides present in insufficient quantity for electroblotting were isolated by reverse-phase high performance liquid chromatography (RP-HPLC). Cleavage sites were also identified at Met(151)-Gly(152), Asn(188)-Ile(189), Lys(223)-Arg(224), Arg(233)-Ala(234) and Ala(240)-Lys(241). In general, μ-calpain cleaved bonds containing one hydrophobic amino acid residue and mainly towards the C-terminus of troponin-T.     

Mutations in calpastatin and μ-calpain are associated with meat tenderness,flavor and juiciness in Hanwoo (Korean cattle): Molecular modeling of the effects of substitutions in the calpastatin/μ-calpain complex

The objective of this study was to evaluate the effects of seven single nucleotide polymorphisms (SNPs) in Calpain 1 and Calpastatin genes previously associated with meat tenderness attributes in other cattle breeds in Korean Hanwoo cattle. The Hanwoo resource population was used to study association of 7 SNPs with beef tenderness, flavor, juiciness, intramuscular fat and shear force. In this association study, CAST:c.182A > G (+ 0.14, P = 0.04) and CAST:c.1985G > C (− 0.12, P = 0.02) had significant effects on juiciness, but no effects on other traits. In contrast, CAPN1:c.1589G > A was associated with meat tenderness (P = 0.01) and juiciness (P = 0.04). The CAPN1:c.1589G > A (Val530Ile) SNP marker displayed significant effect on the meat tenderness score which is strongly supported by molecular modeling of the CAPN1:c.1589G > A (Val530Ile) variant that inhibits CAST protein from binding more strongly than the wild-type protein, which may explain its effect on meat tenderness.     

基于KeilC51 μVision4平台的倒车语音警示系统的设计

本设计是基于AT89S52为控制核心的倒车语音警示系统。系统采用了超声波传感器采集信号,通过AT89S52对采集到的信号进行处理,并通过LCD1602将距离实时的显示出来,通过语音把距离报出来,当距离小于某个值时,提醒驾驶员采取必要的措施。     

The cardinality of μM,D orthogonal exponentials of self-affine measures with two-elements digit set

对于由M=pIN(|p|＞1,p∈Z),D={0,l1 e1+l2 e2+…+lN eN}(∈)ZN (l21+22+…+l2N≠0,lj∈Z,j=1,2,…,N)决定的自仿测度μM,D,支撑在吸引子T(M,D)上.证明当p为奇数时,L2(μM,D)空间中的正交指数函数系最多有2个元素,而且2是最好的估计;当p为偶数时,L2(μM,D)空间中存在含有无限个元素的正交指数函数系.     

Frequency and behavior of Salmonella and Escherichia coli on whole and sliced jalapeño and serrano peppers

The frequencies of coliform bacteria (CB), thermotolerant coliforms (TC), Escherichia coli, and Salmonella were determined for jalape?o and serrano peppers. In addition, the behavior of four serotypes of Salmonella and three E. coli strains on whole and sliced jalape?o and serrano peppers as well as in blended sauce at 25 ± 2°C and 3 to 5°C was investigated. Chili peppers were collected from markets in the city of Pachuca, Hidalgo, Mexico. CB, TC, E. coli, and Salmonella were detected on serrano peppers in 100, 90, 50, and 10 % of the samples, and on jalape?o peppers in 100, 86, 32, and 12 % of the samples. Concentrations of CB ranged from 3.8 to 7.9 log CFU per serrano sample and from 5.3 to 8.2 log CFU per jalape?o sample, whereas concentrations of TC and E. coli were between < 3 and 1,100 most probable number per serrano and jalape?o samples. On whole serrano and jalape?o peppers stored at 25 ± 2°C or 3 to 5°C, no growth was observed for rifampin-resistant strains of Salmonella and E. coli. After 6 days at 25 ± 2°C, the tested Salmonella serotypes and E. coli strains had decreased from an initial inoculum level of 5 log CFU to 1 and 2.5 log on serrano and jalape?o peppers, respectively, and at 3 to 5°C they decreased to approximately 1.8 and 1.2 log, respectively, on serrano and jalape?o. Both the Salmonella serotypes and E. coli grew on sliced chili peppers and in blended sauce; after 24 h at 25 ± 2°C, both bacteria types had grown to approximately 4 and 5 log CFU on pepper slices and in sauce, respectively. At 3 to 5°C the bacterial growth was inhibited.     

Legal limit for reducing sugars in prefabricates targeting 50 μg/kg acrylamide in French fries

A legal limit for the reducing sugars in the prefabricates for French fries is a simple and efficient measure to reduce the exposure to acrylamide from the predominant source for many consumers. The acrylamide content of French fries of comparable crispiness is approximately proportional to the concentration of the reducing sugars glucose and fructose in the potato sticks. On average, optimally prepared French fries from prefabricates with a (moderately low) sugar content of 0.3 g/kg contained 32 g/kg acrylamide. With 0.15 g/kg reducing sugar even severe overfrying at 170 °C only resulted in 90 g/kg acrylamide, i.e. a low sugar content keeps acrylamide low even under inappropriate frying conditions. In the prefabricates, the sugar content is about 10% lower than in the raw potato (resulting from the effects of blanching and prefrying). It is similar to that in the finished French fries, which enables one to distinguish whether a high acrylamide content in French fries results from high sugars in the raw material or unsuitable frying conditions. An average concentration of 50 g/kg acrylamide in French fries could be targeted by limiting the reducing sugars in the prefabricates to 0.7 g/kg and the frying temperature to 170 °C. Even considerable overfrying in terms of duration can be tolerated then.     

Synthesis and structure–activity relationships and effects of phenylpropanoid amides of octopamine and dopamine on tyrosinase inhibition and antioxidation

Phenylpropanoid amides of octopamine (OA) 1a–1e and dopamine (DA) 2a–2e were synthesised and the structure–activity relationships (SARs) for antioxidant and tyrosinase inhibition activities were analysed. Among synthesised compounds, 2c, which contains two catechol moieties, exhibited the most DPPH radical-scavenging activity (EC₅₀ = 16.2 ± 2.4 μM), and 1d exhibited significant tyrosinase inhibitory activity (IC₅₀ = 5.3 ± 1.8 μM). Interestingly, with the same acid moiety, OA derivatives showed more inhibitory effect on tyrosinase than did compounds derived from DA, whereas DA derivatives were found to have higher antioxidant activity than compounds derived from OA. The relationship between their structures and their potencies, demonstrated in the current study, will be useful for the design of optimal agents.