Search

Background Metal-on-metal (MOM) total hip arthroplasty is associated with unacceptable failure rates secondary to metal ion reactions. Efforts to identify which patients will go on to failure have been limited; recently, there has been a suggestion for a potential genetic basis for the increased risk of revision in MOM hip replacements (MOMHRs). The purpose of this study is to determine whether certain immunologic genotypes are predictive of the need for revision in patients with MOM total hip implants. Methods This is a case-control study of all patients undergoing primary MOMHR between September 2002 and January 2012 with a minimum of 5-year follow-up. Our investigational “case” cohort was comprised of patients who underwent revision for MOMHR for a reason other than infection. A single-nucleotide polymorphism (SNP) array analysis was performed to identify a potential genetic basis for failure. Results Thirty-two patients (15 case and 17 control) were included in our analysis. All patients in the revision group had a chief complain of pain; revision patients were more likely to have a posterior approach (P = .01) and larger head size (P = .04) than nonrevision patients. No patient or implant characteristics were independently associated with revision in a multivariate analysis. Patients with SNP kgp9316441 were identified as having an increased odds of revision for MOM failure (P Conclusion This study identified an SNP, kgp9316441, encoding proteins associated with inflammation and macrophage activation. This SNP was associated with significantly increased odds of revision for MOMHR. Future studies are warranted to validate this gene target both in vitro and in vivo. Level of Evidence III.

Bali cattle (Bos javanicus) are native Indonesian cattle, domesticated from banteng (Bibos banteng). Genes that have an important role in meat quality are calcium-activated neutral protease genes, known as calpains (CAPN). The objective of this study was to evaluate the polymophisms of calpain gene SNP g.232 G>T by PCR-RFLP technique and its influence on growth trait and meat quality of Bali cattle detected by ultrasound imaging of longissimus dorsi thickness (LDT), back fat thickness (BFT), marbling score (MS), and intramuscular fat percentage (PIMF). The polymorphisms of CAPN1 gene were analyzed by PCR-RFLP using BglII restriction enzyme (n=52 cattle). The ultrasound images of longissimus dorsi muscle were carried out transversally and longitudinal between 12th -13th thoracic vertebrae then analyzed by Image-J NIH software. Result showed that SNP g.232 G>T of CAPN1 gene was polymorphic in Bali cattle. SNP g.232 G>T of CAPN1 gene in Bali cattle has higher diversity which was showed of 0.48 heterozygosity value and was in Hardy-Weinberg equilibrium. The polymorphisms of SNP g.232 G>T was associated significantly (P

The activity of calpains, in particular μ-calpain, is associated with several processes occurring in muscle tissue postmortem and influences meat quality parameters. Therefore, the CAPN1 gene coding for large subunit of μ-calpain is considered as a candidate gene associated with meat quality traits. The aim of our study was to identify new polymorphisms in regulatory regions of the porcine CAPN1 gene and to estimate their impact on CAPN1 transcript abundance. In the present study, 7 polymorphisms in the porcine CAPN1 gene were identified, of which 5 were localized in introns (g.1195_1197insCCT; g.1429G>A; g. [4479A>G; 4526A>T; 4529_4530delAG]), one in 3’ untranslated region (g.25676C>T) and one microsatellite sequence in promoter region (c.-155- AGGG [3_5]). The analysed populations (a total of 451 gilts representing three pure breeds: Pietrain, P lish Landrace, Polish Large White and one conservation breed Pu.awska) were not in Hardy-Weinberg equilibrium according to mutation g.25676C>T in 3’ untranslated region (all breeds), g.1429G>A (Pu.awska pigs) and g. [4479A>G; 4526A>T; 4529_4530delAG] (PLW pigs). Furthermore, the analysed SNPs in the porcine CAPN1 gene were in linkage disequilibrium (P≤0.05). The CAPN1 transcript abundance was also estimated in two important muscles (m. longissimus dorsi, m. semimembranosus). In longissimus dorsi muscle, a significant effect of c.-155- AGGG [3_5] polymorphism in promoter region on CAPN1 expression levels was determined. The c.-155AGGG [3/3] pigs showed a statistically higher (P≤0.05) expression level of the CAPN1 gene when compared to c.-155AGGG [4/4] homozygotes. The results obtained suggested that detected SNPs within regulatory regions of the CAPN1 gene could be related to transcript level and activity of μ-calpain. The selected polymorphisms areproposed to be associated with meat production traits.

Two hundred and ten Angus × Simmental steers (initial BW 314 ± 11 kg) were separated into heavy and light BW blocks and allotted evenly by BW to 6 treatments (3 heavy and 2 light pens per treatment) to determine the effect of supplemental vitamin D3: 0 IU (no D), 250,000 IU for 165 d (long-term D), or 5 × 10(6) IU for 10 d (short-term D) on plasma and muscle calcium concentrations and gene expression in steers fed either 0 (NZ) or 8.38 mg/kg (ZH) zilpaterol hydrochloride (ZH) daily for 21 d. Placebo or ZH was added to the diet 24 d, and short-term D was added 13 d before slaughter. Treatments were removed from all diets 3 d before slaughter. Plasma total calcium (Ca(2+)) was determined at study initiation, start of ZH and short-term D feedings, and at vitamin D3 and ZH withdrawal. Both plasma total and ionic Ca(2+) were determined when animals were sent to harvest. Longissimus muscle total and ionic Ca(2+) were determined in meat aged 7 and 4 d postmortem, respectively. When ZH was fed, long-term D decreased plasma total Ca(2+) at slaughter (P 0.28) LM total Ca(2+); however, both long- and short-term D increased LM ionic Ca(2+) when ZH was not fed (P < 0.01). Long-term D reduced LM ionic Ca(2+) when ZH was fed (P < 0.02). Neither long- nor short-term D affected PPARα or δ gene expression (P = 0.19) whether or not ZH was fed. Expression of MYH1 and 2A (P < 0.05) but not 2X (P = 0.21) was decreased in steers fed ZH. Long-term D had no effect on MYH2A expression (P = 0.21). Short-term D increased MYH2A expression when ZH was not fed (P < 0.03). Calpain mRNA tended to be lower in steers fed ZH (P = 0.09), but was not affected by long- or short-term D regardless of whether or not ZH was fed (P = 0.39). Expression of calpastatin did not differ with vitamin D supplementation (P = 0.35). In conclusion, ZH decreased oxidative myosin expression, and when combined with long-term D, ZH decreased LM ionic Ca(2+). Moreover, vitamin D3 supplementation did not increase calpain mRNA. These results help explain why vitamin D3 does not improve tenderness in steers fed ZH.

BACKGROUND: We conducted an in vivo experiment to determine whether vitamin D3 acts as a fat synthesizer and/or meat tenderizer in mice. At 6 weeks of age, 20 male C57BL/6 wild-type mice were randomly divided into two groups (10 mice per group) and fed a modified AIN93G diet with (vitamin D3 diet) or without (basal diet) 10 IU 25-OH-cholecalciferol kg−3 for 3 weeks. RESULTS: When vitamin D3 was fed to mice for 3 weeks, body fat was significantly increased compared to mice fed a basal diet. There was, however, no difference in body weight between the two groups. Vitamin D3 increased the gene expressions of pro-inflammatory cytokines and peroxisome proliferator-activated receptor gamma, but decreased interleukin-15 in adipose tissue through nuclear vitamin D receptor and uncoupling protein-2 signals. The muscle inducible nitrate oxide synthase content of mice fed vitamin D3 was higher than those fed a basal diet, while muscle arginase l showed a reverse phenomenon. longissimuss dorsi muscle of vitamin D3-fed mice showed more severe fat deposition than those fed a basal diet. Vitamin D3 amplified muscle u- and m-calpain protein content and suppressed muscle calpastatin protein content. CONCLUSION: These findings suggest that vitamin D3 can be used as a fat synthesizer and meat tenderizer in meat-producing animals. Copyright © 2011 Society of Chemical Industry

La terneza de la carne está en parte determinada por el sistema proteico calpaína (CAPN1) / calpastatina (CAST). Bolivia posee en los llanos tres biotipos de ganado Criollo: los Yacumeños, los Chaqueños y los Saavedreños. El objetivo del presente trabajo fue determinar la frecuencia alélica y genotípica del gen de la CAPN1 en tres poblaciones de ganado Criollo de Bolivia. Se obtuvieron muestras de sangre de 28 Criollos del Chaco (CCH), 85 Criollos Yacumeños (CYA) y 30 Criollos Saavedreños (CSV). El ADN se extrajo utilizando el kit comercial Wizard® Genomic Purification, y posteriormente se tipificaron dos polimorfismos (CAPN1-316 y CAPN1-4751) del gen CAPN1 mediante el método ARMS-PCR. La frecuencias alélicas y genotípicas, las heterocigosidades esperadas y observadas, así como, el índice FIS y el desequilibrio de ligamiento (LD) fueron calculadas mediante los programas MS-Tools y Genepop. Las frecuencias de los alelos asociados a mayor terneza para las poblaciones de CCH, CYA y CSV fueron: 23%, 22% y 33 % para el alelo C del SNP CAPN1-316 y 75%, 76% y 60% para el alelo C del CAPN1-4751. La heterocigocidad observada en las tres poblaciones se hallan en un rango de 0,30 a 0,46 para el marcador CAPN1-316 y de 0,21 a 0,60 para el polimorfismo CAPN1-4751. Los resultados demuestran que los bovinos criollos en las poblaciones analizadas poseen altas frecuencias alélicas para las variantes asociadas a mayor terneza de la carne. Por otra parte, no se observaron valores significativos de LD (P>0,01) entre los dos polimorfismos tipificados en las poblaciones estudiadas. Sería necesario tipificar ambos polimorfismos en futuros programas de selección asistida por marcadores genéticos., Meat tenderness is in part determined by the calpain (CAPN1) / calpastatin (CAST) genes. In the lowlands of Bolivia, three well differentiated Creole cattle populations can be distinguished: the Yacumeños, Chaqueños and Saavedreños. The main objective of this research was to determine the allelic and genotypic frequencies of two polymorphisms of the calpain gene in three Creole cattle populations in Bolivia. Blood samples of 28 Creole cattle from Chaqueño cattle (CCH), 85 from Yacumeño cattle (CYA) and 30 from Saavadreño cattle (CSV) were collected. Total DNA was extracted using the commercial kit Wizard® Genomic Purification and subsequently two polymorphisms (CAPN1-316 and CAPN1- 4751) of the CAPN1 gene were genotyped by the amplification refractory mutation system (ARMS-PCR) method. Allelic and genotypic frequencies, expected and observed heterozygosities, the FIS index and the magnitude of linkage disequilibrium (LD) were calculated using the software MS-Tools and Genepop. The allelic frequencies of variants associated with tenderness in the three populations CCH, CYA and CSV were 23%, 22% and 23% for the CAPN1- 316 and 75%, 76% and 60% for the CAPN1-4751. The observed heterozygosities in the three populations fluctuated between 0.30 and 0.46 for the CAPN1-316 marker and between 0.21 and 0.60 for the CAPN1-4751 marker. The results showed that the analysed populations of Creole cattle presented high frequencies of the alleles previously associated with tenderness in meat. The analysis of LD, however, did not show evidence of linkage between the two markers. It is necessary to perform a genetic analysis for both polymorphisms if included in future selection programs., Instituto de Genética Veterinaria

We conducted an in vivo experiment to determine whether vitamin D₃ acts as a fat synthesizer and/or meat tenderizer in mice. At 6 weeks of age, 20 male C57BL/6 wild-type mice were randomly divided into two groups (10 mice per group) and fed a modified AIN93G diet with (vitamin D₃ diet) or without (basal diet) 10 IU 25-OH-cholecalciferol kg⁻³ for 3 weeks.When vitamin D₃ was fed to mice for 3 weeks, body fat was significantly increased compared to mice fed a basal diet. There was, however, no difference in body weight between the two groups. Vitamin D₃ increased the gene expressions of pro-inflammatory cytokines and peroxisome proliferator-activated receptor gamma, but decreased interleukin-15 in adipose tissue through nuclear vitamin D receptor and uncoupling protein-2 signals. The muscle inducible nitrate oxide synthase content of mice fed vitamin D₃ was higher than those fed a basal diet, while muscle arginase l showed a reverse phenomenon. longissimuss dorsi muscle of vitamin D₃-fed mice showed more severe fat deposition than those fed a basal diet. Vitamin D₃ amplified muscle u- and m-calpain protein content and suppressed muscle calpastatin protein content.These findings suggest that vitamin D3 can be used as a fat synthesizer and meat tenderizer in meat-producing animals.

Conventional calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. There are two forms of conventional calpains: the mu-calpain, or calpain I, which requires micromolar calcium for half-maximal activation, and the m-calpain, or calpain II, which functions at millimolar calcium concentrations. We evaluated the functional role of the 80-kDa catalytic subunit of mu-calpain by genetic inactivation using homologous recombination in embryonic stem cells. The mu-calpain-deficient mice are viable and fertile. The complete deficiency of mu-calpain causes significant reduction in platelet aggregation and clot retraction but surprisingly the mutant mice display normal bleeding times. No detectable differences were observed in the cleavage pattern and kinetics of calpain substrates such as the beta3 subunit of alphaIIbbeta3 integrin, talin, and ABP-280 (filamin). However, mu-calpain null platelets exhibit impaired tyrosine phosphorylation of several proteins including the beta3 subunit of alphaIIbbeta3 integrin, correlating with the agonist-induced reduction in platelet aggregation. These results provide the first direct evidence that mu-calpain is essential for normal platelet function, not by affecting the cleavage of cytoskeletal proteins but by potentially regulating the state of tyrosine phosphorylation of the platelet proteins.

Calpains play an important role in the postmortem tenderization process of meat and several SNP in the mu-calpain gene (CAPN1) have been reported to be associated with tenderness in beef cattle. Our objectives were to identify the previously reported CAPN1 331GC SNP and to detect new polymorphisms in this gene in Spanish maternal beef breeds. A fragment (exon 8 to 10) of the bovine CAPN1 gene was sequenced and genotyped in a sample of the main Spanish maternal beef breeds including Retinta, Morucha, and Avilenã Negra-Ibérica. These breeds are characterized for their high meat quality, their adaptation to adverse environmental conditions, and their good maternal aptitude. This adaptation makes it possible to rear these breeds in the south and west of Spain, where drought and feed shortages occur frequently. Six SNP in the mu-calpain gene were found, five of which (CAPN1 80CT, 302CG, 310GA, 445CT, 524AC) have not been reported previously. Sequences obtained for these five newly found SNP were submitted to GenBank (Accessions EU386166 to EU386183).

Two hundred and ten Angus × Simmental steers (initial BW 314 ± 11 kg) were separated into heavy and light BW blocks and allotted evenly by BW to 6 treatments (3 heavy and 2 light pens per treatment) to determine the effect of supplemental vitamin D3: 0 IU (no D), 250,000 IU for 165 d (long-term D), or 5 × 10(6) IU for 10 d (short-term D) on plasma and muscle calcium concentrations and gene expression in steers fed either 0 (NZ) or 8.38 mg/kg (ZH) zilpaterol hydrochloride (ZH) daily for 21 d. Placebo or ZH was added to the diet 24 d, and short-term D was added 13 d before slaughter. Treatments were removed from all diets 3 d before slaughter. Plasma total calcium (Ca(2+)) was determined at study initiation, start of ZH and short-term D feedings, and at vitamin D3 and ZH withdrawal. Both plasma total and ionic Ca(2+) were determined when animals were sent to harvest. Longissimus muscle total and ionic Ca(2+) were determined in meat aged 7 and 4 d postmortem, respectively. When ZH was fed, long-term D decreased plasma total Ca(2+) at slaughter (P0.04). Short-term D increased (P0.01) plasma total and ionic Ca(2+) at slaughter regardless of ZH inclusion in the diet. Long- and short-term D, with or without ZH, did not affect (P0.28) LM total Ca(2+); however, both long- and short-term D increased LM ionic Ca(2+) when ZH was not fed (P0.01). Long-term D reduced LM ionic Ca(2+) when ZH was fed (P0.02). Neither long- nor short-term D affected PPARα or δ gene expression (P = 0.19) whether or not ZH was fed. Expression of MYH1 and 2A (P0.05) but not 2X (P = 0.21) was decreased in steers fed ZH. Long-term D had no effect on MYH2A expression (P = 0.21). Short-term D increased MYH2A expression when ZH was not fed (P0.03). Calpain mRNA tended to be lower in steers fed ZH (P = 0.09), but was not affected by long- or short-term D regardless of whether or not ZH was fed (P = 0.39). Expression of calpastatin did not differ with vitamin D supplementation (P = 0.35). In conclusion, ZH decreased oxidative myosin expression, and when combined with long-term D, ZH decreased LM ionic Ca(2+). Moreover, vitamin D3 supplementation did not increase calpain mRNA. These results help explain why vitamin D3 does not improve tenderness in steers fed ZH.

The objective of this study was to determine whether the genetic variants of CAPN1 developed in several cattle populations can be applied for Hanwoo, regarding genetic effects on meat traits. The traits were examined for 286 purebred Hanwoo steers with genotypes classified by restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP) analysis. The nucleotide positions of primers and previously identified genetic variants were based on sequences of the calpain 1 (CAPN1) gene with GenBank accession numbers (AF252504, AF248054, and AY639597). The analysis of genetic distribution estimated levels of minor allele frequencies ranged from 0.165 to 0.392, showing no significant departures from Hardy-Weinberg Equilibrium for all markers. Overall averages of heterozygosites (He) and polymorphic information contents (PICs) for all markers were calculated to 0.503 and 0.429, respectively, and the g.4558G>A marker showed the lowest He (0.425) and PIC (0.367). Animals from 29 months of age were slaughtered to measure Warner-Bratzler shear force (WBSF), cooking loss, water-holding capacity, pH, fat, and moisture. All the CAPN1 markers explained variations of WBSF, showing significant additive effects except g.5709G>A. A significant marginal mean difference in genotypes of g.6545C>T (P=0.046) was found in moisture with additive effects. From the result it may be possible to use three calpain markers (g.4558G>A, g.4685C>T, and g.6545C>T) classified by RFLP and SSCP analysis in marker assisted selection programs to improve WBSF as meat tenderness in Hanwoo.

Aspirin and other nonsteroidal anti-inflammatory drugs show efficacy in the prevention of cancers. It is known that they can inhibit cyclooxygenases, and some studies have shown that they can induce apoptosis. Our objective in this study was to investigate the mechanism by which aspirin exerts its apoptosis effects in human cervical cancer HeLa cells. The effect of aspirin on the gene expression was studied by differential mRNA display RT-PCR. Among the isolated genes, mu-type calpain gene was upregulated by aspirin treatment. To examine whether calpain mediates the antitumor effects, HeLa cells were stably transfected with the mammalian expression vector pCR3.1 containing mu-type calpain cDNA (pCRCAL/HeLa), and tumor formations were measured in nude mice. When tumor burden was measured by day 49, HeLa cells and pCR/HeLa cells (vector control) produced tumors of 2126 mm3and 1638 mm3, respectively, while pCRCAL/HeLa cells produced markedly smaller tumor of 434 mm3in volume. The caspase-3 activity was markedly elevated in pCRCAL/HeLa cells. The increased activity levels of caspase-3 in pCRCAL/HeLa cells, in parallel with the decreased tumor formation, suggest a correlation between caspase-3 activity and calpain protein. Therefore, we conclude that aspirin-induced calpain mediates an antitumor effect via caspase-3 in cervical cancer cells.

Type V collagen is known to be over-deposited in the stroma of ductal infiltrating carcinomas of the breast. When used as a substrate, type V collagen restrains growth and invasion, and affects gene expression of 8701-BC ductal infiltrating carcinomas cells. Here we supplement existing data by demonstrating type V collagen dependent upregulation of capn2 gene expression in 8701-BC cells through differential display-PCR and Western blot assays. Furthermore, we suggest that our data obtained by centrifugal sedimentation and electrophoresis strongly suggest a correlation between calpain overproduction and DNA fragmentation, since the incubation with calpain inhibitor partly reverts the latter.

The effects of dantrolene on serum TNFalpha and corticosterone levels and on muscle calcium, calpain gene expression, and protein breakdown were studied in rats with abdominal sepsis induced by cecal ligation and puncture. Treatment of rats with 10 mg/kg of dantrolene 2 h before and 8 h after induction of sepsis reduced serum TNFalpha and corticosterone, muscle calcium levels, mRNA levels for m- and mu-calpain, and the muscle specific calpain p94, as well as total and myofibrillar protein breakdown rates, determined as release of tyrosine and 3-methylhistidine, respectively, from incubated extensor digitorum longus muscles. The results support the concept that increased calcium concentrations may be an important mechanism of sepsis-induced muscle protein breakdown. The data also indicate that other mechanisms, in addition to reduced muscle calcium concentrations such as decreased levels of TNFalpha and glucocorticoids, may contribute to the anti-catabolic effects of dantrolene during sepsis. The observations are important from a clinical standpoint because they suggest that the catabolic response in skeletal muscle during sepsis may be prevented by treatment with a calcium antagonist.

The proteins nCL-2 and nCL-2' are members of the Ca2+-dependent cysteine protease (calpain) superfamily, with stomach-specific expression. Like other typical calpains, nCL-2 has three distinct domains, a protease, a C2-like, and a 5EF-hand Ca2+-binding domain, as well as the N-terminal propeptide region. On the other hand, nCL-2' lacks the C2-like and 5EF-hand domains but is otherwise identical to nCL-2, except for the three C-terminal residues. To examine the stomach-specific and presumed alternative expression mechanisms of nCL-2 and nCL-2', we have cloned and characterized the mouse gene for nCL-2 and nCL-2'. The mouse nCL-2 gene contains at least 23 exons, spanning more than 50 kb, and possesses an exon specific for nCL-2' in the middle. Therefore, nCL-2 and nCL-2' are generated by alternative splicing of the same gene, Capn8. Capn8 shows the highly conserved gene organization of the other typical calpain large subunit genes, CAPN1, CAPN2, CAPN3, CAPN9, CAPN11, and Capn12, except for the unique exon between exon 9 and exon 10 of Capn8, which encodes the 3' half of the nCL-2' transcript. No such exon in the corresponding regions was found in CAPN1, CAPN2, CAPN3, CAPN9, or CAPN11. Gene and cDNA structures of a presumed human orthologue of mouse nCL-2, CAPN8, were determined, revealing that it overlaps human CAPN2, the gene for the m-calpain large subunit, in head-to-head orientation at 1q32-41. These features of Capn8 and CAPN8 illustrate a process of calpain gene evolution, i.e., the protease, C2-like, and 5EF-hand domains presumably functioned as independent genes, and the calpain superfamily has evolved by ordered fusions of these ancestral gene units, with subsequent amplifications.

1The authors wish to thank K. C. Maddock and B. M. Rickert for their technical assistance. This work was supported in part by Sire Power, Inc. and National Live Stock & Meat Board. Received December 27, 1995. Accepted March 22, 1996. Figure 1. Bovine calpain gene HhaI DNA polymorphism separated on 2% agarose gel after restriction enzyme digestion. Estimated bp sizes of the restriction fragments are shown on the right. U and M on the bottom of the figure represent uncut PCR product and the DNA size marker (HaeIII fx174), respectively. Rapid Communication: A Novel DNA Polymorphism of the Bovine Calpain Gene Detected by PCR-RFLP Analysis1

Endosperm of cereal grains is one of the most important renewable resources for food, feed, and industrial raw material. It consists of four triploid cell types, i.e., aleurone, starchy endosperm, transfer cells, and cells of the embryo surrounding region. In maize, the aleurone layer is one cell layer thick and covers most of the perimeter of the endosperm. Specification of maize aleurone cell fate is proposed to occur through activation of the tumor necrosis factor receptor-like receptor kinase CRINKLY4. A second maize gene essential for aleurone cell development is defective kernel 1 ( dek1 ). Here we show that DEK1 shares high homology with animal calpains. The predicted 2,159-aa DEK1 protein has 21 transmembrane regions, an extracellular loop, and a cysteine proteinase domain that shares high homology with domain II of m -calpain from animals. We propose that DEK1 functions to maintain and restrict the aleurone cell fate imposed by CR4 through activation of its cysteine proteinase by contact with the outer endosperm surface. DEK1 seems to be the only member of the calpain superfamily in plants, Arabidopsis DEK1 sharing 70% overall identity with maize DEK1. The expression of dek1 in most plant tissues in maize and Arabidopsis , as well as its presence in a variety of higher plants, including angiosperms and gymnosperms, suggests that DEK1 plays a conserved role in plant signal transduction.