Search

The study aimed to research the genetic variation of the Awassi and Naimi sheep breeds using the two genes myostatin (MSTN) and calpastatin (CAST). Blood samples were collected from 100 animals of the two breeds, and then DNA was extracted using a commercial kit. We used the PCR and RFLP techniques to determine genotypes and allele frequencies. The results showed that the MSTN and CAST genes are polymorphic. The MSTN gene has allelic frequencies (M and m) of 0.81, 0.19, and 0.76, 0.24 in the Awassi and Naimi breeds, respectively. The frequencies of the genotypes MM, Mm, and mm in the Awassi breed were 0.70, 0.19, and 0.11, but in the Naimi breed, they were 0.67, 0.13, and 0.20, respectively. Moreover, the number of alleles observed (Na), the effective number of alleles (Ne) and observed (Ho), and expected (He) heterozygosity were found to be 3, 2.30, 0.24, and 0.35 in the Awassi breed and 2, 1.62, 0.17, and 0.26 in the Naimi breed, respectively. The allelic frequencies (M and N) of the CAST of the Awassi and Naimi breeds are 0.86, 0.14, and 0.88, 0.12, respectively. The frequencies of the genotypes MM, MN, and NN in the Awassi breed were 0.94, 0.04, and 0.02, respectively, while for the Naimi breed, they were 0.95, 0.02, and 0.03, respectively. Also, the Na, Ne, Ho, and He were found to be 2.8, 1.72, 29.6, and 28.57 in the Awassi breed and 1.10, 1.23, 0.17, and 0.15 in the Naimi breed, respectively. According to the chi-square of MSTN and CAST genes, both breeds were not in Hardy-Weinberg equilibrium balance.

Genes or genetic markers related to meat quality have been studied for many years. Th e CAST gene is one candidate gene aff ecting meat tenderness in cattle. Th is meta-analysis aimed to examine the association of c.2832A>G polymorphism of the CAST gene and meat tenderness in cattle. According to the determined criteria, 17 studies were included in the meta-analysis, and pooled ratios of allele and genotype frequencies were calculated. In addition, the combined Warner-Braztler Shear Force values were calculated for three studies that reported these values according to genotypes for the meat tenderness feature. Heterogeneity between studies and publication bias were also tested. As a result of the meta-analysis, pooled ratios of AA, AG, and GG genotypes are 0.578, 0.342, and 0.080; pooled ratios of A and G alleles are 0.777 and 0.223. Th e highest pooled ratios were obtained for AA genotype and A allele. Th e combined Warner-Braztler shear force values were calculated as 3.707, 3.893, and 5.137 kilogram-force for AA, AG, and GG genotypes. Th e highest mean shear force value was obtained for the GG genotype. In conclusion, the meta-analysis results examined the relationship between c.2832A>G polymorphism of the CAST gene and meat tenderness were closer to the population parameter. Th is study may improve genetic selection and provide new strategies to increase meat quality in cattle.

Simple Summary: This study aimed to evaluate the relationship between the expression levels of the CAST gene and the fatty acid (FA) composition in the longissimus thoracis (LL) muscle and to identify novel variants of CAST and perform association analysis with the FA composition in grazing Sonid sheep. The correlation results showed that high expression levels of CAST are correlated with better FA compositions and classes in LL. Four c.646G>C (G216R), c.1210C>T (R404C), c.1437G>A (479T), and c.2097C>T (699G) mutations were identified in the CAST gene of Sonid sheep. The association studies showed that c.1210C>T is associated with C14:0, C18:0, C18:1n9c, C18:3n3, n3, C12:0, n6, and n6/n3; c.646G>C and c.1437G>A in linkage disequilibrium-Mongolia (r2 = 0.964) were associated with C14:0, C18:0, SFA, C18:1n9c, n3, C10:0, C18:1n9t, and n6/n3; and c.2097C>T was associated with C18:3n3, n3, C10:0, and n6/n3 in the LL of Sonid sheep. Thus, the correlation results and associated mutations were expected to be genetic selection markers for the FA composition and meat quality of Sonid sheep muscle and provide new insight into sheep meat quality traits influenced by the ovine CAST gene. Fatty acid (FA) composition has an important impact on the nutrition and flavor of meat, and on consumer health, and is receiving more attention in the sheep industry. This study aimed to evaluate the relationship between the expression levels of the CAST gene and the FA composition in the longissimus thoracis (LL) muscle, to identify novel variants of CAST, and to perform association analysis with the FA composition in grazing Sonid lambs. The correlation results showed that high expression levels of CAST are correlated with better FA compositions and classes in LL. For association studies, the results showed that c.1210C>T and c.1437G>A in LD-M, and c.2097C>T mutations are associated with some compositions and classes of FA in the LL of grazing Sonid sheep. Two missense c.646G>C (G216R) and c.1210C>T (R404C) mutations were predicted to influence the Calpain_inhib domains of CAST. Thus, the correlation results and associated mutations are expected to be genetic selection markers for the FA composition and meat quality of grazing Sonid lamb muscle and provide new insights into sheep meat quality traits influenced by the ovine CAST gene. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the association of the CAST genotype and growth traits in Batur sheep. Batur lambs were reared under an intensive feeding system. Bodyweight is measured monthly after weaning until six months of age. Blood representing thirty head were collected, genomic DNA was extracted as samples, and then 200 µl of whole blood samples were used. Specific primers were designed to amplify the CAST gene, samples were sequenced, then the researchers used the BioEdit program to identify any mutation. Calculation of genotypes, gene and allele frequencies, heterozygosities, and Chi-square test was performed. The analysis revealed a total of sixteen polymorphic sites in the CAST coding region. There are four alleles observed (A, G, C, and T), trans-versions at c.92TT loci, and transitions at c.214G>A, c.280G>A, c.301CG. One individual disrupted the reading frame in the whole CAST sequenced. The genotype frequency analysis showed the highest predominance of the (TT, GG, CC, and AA) genotypes with frequencies (66.7 %, 62.9 %, 59.3 %, 66.7 %, and 55.6 %) as homozygous. In contrast, the heterozygous (TG, GA, GT, CT, and AG) genotypes were present at lower frequencies (29.6 %, 33.3 %, 37 %, 29.6 %, and 37 %), respectively with four difference alleles (T, G, C, and A). Post-weaning bodyweight till 6-month age of Batur sheep for the heterozygous genotypes at loci (c.92TA, c.280G>A, and c.301CT and c.371A>G) with no significant differences (p > 0.5). The lambs with the heterozygous genotype had a significantly higher muscle percentage as a whole, compared to the other genotypes. Polymorphic sites were present in Batur sheep for the first time about the association with the CAST gene. However, the CAST gene might not be a useful marker for developing future selection programs in Batur sheep unless further investigation of the CAST gene and its interactions with other genes involving muscle growth and carcass traits are analyzed through association studies at a large scale. [ABSTRACT FROM AUTHOR]

Genes or genetic markers related to meat quality have been studied for many years. The CAST gene is one candidate gene affecting meat tenderness in cattle. This meta-analysis aimed to examine the association of c.2832A>G polymorphism of the CAST gene and meat tenderness in cattle. According to the determined criteria, 17 studies were included in the meta-analysis, and pooled ratios of allele and genotype frequencies were calculated. In addition, the combined Warner-Braztler Shear Force values were calculated for three studies that reported these values according to genotypes for the meat tenderness feature. Heterogeneity between studies and publication bias were also tested. As a result of the meta-analysis, pooled ratios of AA, AG, and GG genotypes are 0.578, 0.342, and 0.080; pooled ratios of A and G alleles are 0.777 and 0.223. The highest pooled ratios were obtained for AA genotype and A allele. The combined Warner-Braztler shear force values were calculated as 3.707, 3.893, and 5.137 kilogram-force for AA, AG, and GG genotypes. The highest mean shear force value was obtained for the GG genotype. In conclusion, the meta-analysis results examined the relationship between c.2832A>G polymorphism of the CAST gene and meat tenderness were closer to the population parameter. This study may improve genetic selection and provide new strategies to increase meat quality in cattle. [ABSTRACT FROM AUTHOR]

Fatty acid (FA) composition has an important impact on the nutrition and flavor of meat, and on consumer health, and is receiving more attention in the sheep industry. This study aimed to evaluate the relationship between the expression levels of the CAST gene and the FA composition in the longissimus thoracis (LL) muscle, to identify novel variants of CAST, and to perform association analysis with the FA composition in grazing Sonid lambs. The correlation results showed that high expression levels of CAST are correlated with better FA compositions and classes in LL. For association studies, the results showed that c.1210C>T and c.1437G>A in LD-M, and c.2097C>T mutations are associated with some compositions and classes of FA in the LL of grazing Sonid sheep. Two missense c.646G>C (G216R) and c.1210C>T (R404C) mutations were predicted to influence the Calpain_inhib domains of CAST. Thus, the correlation results and associated mutations are expected to be genetic selection markers for the FA composition and meat quality of grazing Sonid lamb muscle and provide new insights into sheep meat quality traits influenced by the ovine CAST gene.

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Bali cattle are known as native cattle from Indonesia, which commonly utilized as beef-producing animals. Calpastatin gene (CAST) plays essential role in meat quality. The aim of this study was to verify the effect of single-nucleotide polymorphism (SNP) c.100800G>A on the CAST|Cfr13I gene associated with meat characteristics and growth traits in Bali cattle. The meat characteristics, growth traits profile, and blood samples of Bali cattle (n=52 animals) obtained from BPTU Bali Cattle Denpasar, Bali Province. Comparison used were Belgian Blue (n=30 animals), Wagyu (n=7 animals), Limousin (n=14 animals), and Peranakan Onggole (PO) (n=30 animals). Ultrasound measurement was conducted to study the meat characteristics. The examination of the CAST gene polymorphisms used polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method digested by Cfr13I enzyme and the analysis of effect of the CAST|Cfr131 gene on meat characteristics and growth traits in Bali cattle used t test with SAS 9.4 program. Bali cattle revealed polymorphic homozygous genotype (GG) and heterozygous genotype (GA) with allele frequencies of G and A were 0.923 and 0.077, respectively. In comparison, other breeds beef cattle, including Belgian Blue, Limousin, Wagyu, and PO, showed only the GG genotype with allele frequency of G was 1.000. The CAST|Cfr13I gene showed no significant association (P>0.05) with Bali cattle meat characteristics and growth traits. In conclusion, the SNP c.100800G > A may not be purposed as a marker for Bali cattle meat characteristics and growth traits. It was suggested that further study with a higher number of animals would be necessary to validate the effect of the SNP c.100800G > A on the CAST gene with the actual beef cutting of Bali cattle. [ABSTRACT FROM AUTHOR]

The purpose of the present work was to identify polymorphic allelic variants of ovine calpastatin and callipyge genes in Northeast Bulgarian Merino sheep breed. Blood samples were collected from 32 rams, reared in Agricultural Experimental Station - Targovishte. Genomic DNA was extracted using commercial kit for DNA purification from whole blood. After PCR amplification of CAST gene with specific primer set a fragment of 622 bp was obtained. CLPG gene also was amplified with specific primers and a fragment of 426 bp was obtained. Genotypes were determined by restriction fragment length polymorphism (RFLP) method with MspI restriction enzyme for CAST gene and BsmF1 restriction enzyme for CLPG gene. Calpastatin locus was found to be polymorphic, whereas in callipyge gene it was observed uniformity - the presence of genotype AA only. Genotype frequencies established in CAST gene were 0.47% and 0.53% for MM and MN, respectively, whereas genotype NN was not found. [ABSTRACT FROM AUTHOR]

Peeling skin syndrome is a heterogeneous group of rare disorders. Peeling skin, leukonychia, acral punctate keratoses, cheilitis and knuckle pads (PLACK syndrome, OMIM616295) is a newly described form of PSS with an autosomal recessive mode of inheritance. We report a 5.5-year-old boy with features of PLACK syndrome. Additionally, he had mild cerebral atrophy and mild muscle involvements. Whole exome sequencing was performed in genomic DNA of this individual and subsequent analysis revealed a homozygous c.544G > T (p.Glu182*) nonsense mutation in the CAST gene encoding calpastatin. Sanger sequencing confirmed this variant and demonstrated that his affected aunt was also homozygous. Real-time qRT-PCR and immunoblot analysis showed reduced calpastatin expression in skin fibroblasts derived from both affected individuals compared to heterozygous family members. In vitro calpastatin activity assays also showed decreased activity in affected individuals. This study further supports a key role for calpastatin in the tight regulation of proteolytic pathways within the skin. [ABSTRACT FROM AUTHOR]

The aim of study was to estimate genotype frequencies of the polymorphism in the intron 12 of CAST gene of growing lambs, and to evaluate associations between the CAST genotype and carcass and meat traits. A total number of 317 rams representing the synthetic BCP (163) and SCP (154) meaty lines were genotyped by the RFLP/ MspI / NcoI / Hin6I. Three genotypes including "aa", "ae" and "ac" with most frequencies of 0.48, 0.27 and 0.19, were observed in whole population. Lambs with the "aa" CAST genotype showed the highest muscle percentage and the lowest fat percentage in the hind legs. On the other hand, the loins of rams with "ac" genotype had the highest intramuscular fat content, indicative of beneficial health-related properties and technological usefulness. However, differences between CAST genotype stated for the sensory properties, texture and thawing and cooking losses of the meat were insignificant. Breeding efforts aimed at the improvement of lamb meat quality should prioritize the selection of the "ac" genotype. [ABSTRACT FROM AUTHOR]

Objetive. Characterize the genetic polymorphism type SNPs in the calpain (CAPN) and calpastatin (CAST) genes of Colombian creole hair sheep (OPC). Materials and methods. In 300 individuals belonging to two OPC subpopulations from the departments of Sucre (SC) and Valle del Cauca (VC) were genotyped by PCR-RFLP (MspI) for the CAST locus and by PCR-SSCP for the CAPN locus. The allelic and genotypic frequencies, the observed (Ho) and expected heterozygosity (He), the fixation index (F) and the deviations from the Hardy-Weinberg equilibrium (HWE) were calculated and a molecular analysis of variance to estimate the values of FST, FIS and FIT. Results. In the CAST locus, the MM genotype was the most frequent (83.9±1.1%), followed by the other genotypes (MN: 15.5±1.1, NN: 6.0±0.0%) and the allelic frequency of M (91.7±0.4%) exceeded that of N (8.3±0.4%). For the CAPN locus the heterozygous genotype (48.2±0.7%) was the most frequent, the other genotypes presented frequencies TT:44.7 ± 1.9 and CC:7.0 ± 1.4%. The T allele reached a frequency of 68.8±1.5% (C: 31.3±1.5%). Similar percentages of allelic and genotypic frequencies were found in the subpopulations. The He was less than the Ho in both loci, with negative values of F and deviations of EHW only in CAPN. All the variation found was due to differences within the individuals, with nonsignificant values (p>0.05) of FST, FIS, and FIT (0.002, -0.093 and -0.095, respectively). Conclusions. The loci studied has high variability, these results can be used for future gene-assisted selection plans to increase OPC productivity. [ABSTRACT FROM AUTHOR]

Calpastatin (CAST) gene is well known as an inhibitor of muscle protein degradation and relates to muscle growth and meat tenderness. The objective of this study was to determine the association of CAST gene with growth traits and carcass characteristics in Bali cattle. A number of data from 35 Bali bulls were collected from BPTU Bali Cattle to obtain growth traits, carcass characteristics, and blood samples. Polymorphism of CAST gene in Bali bulls was analyzed by using PCR-RFLP and DNA sequencing. The association of CAST gene with growth traits and carcass characteristics were analyzed by using General Linear Model (GLM). The result showed that there were two genotypes (GG and AG) of CAST gene with allele frequencies of 0.857 and 0.143, respectively, for G and A. Notably, mutation A to G occurred in 253 bp CAST fragment gene in Bali Cattle. Genotypes GG and AG of CAST gene significantly affected (P

Abstract. Considered calpastatin as a candidate gene for meat and growth traits in sheep production it is important to understand the genetic variability in this locus. The present work was oriented to identification of calpastatin gene polymorphism and analysis of genetic structure of the populations representing two bulgarian sheep breeds – Local Karnobat and Stara Zagora.The material involved 96 sheep of breeds and genomic DNA was isolated by commersial purified kit and used in order to estimate calpastatin genotypes by PCR-RFLP method. The PCR products were digested with MspI restriction enzyme as a result were detected two different genotypes in the observed locus – homozygous MM and heterozygous MN in Stara Zagora sheep population with frequencies 0.937 and 0.063, respectively. M and N allele frequencies were identified with 0.968 and 0.032. The observed heterozygosity in Stara Zagora sheep population was 0.063 and the chi-square test confirmed the existence of Hardy-Weinberg equilibrium in this population (P>0.05). In the total population of Local Karnobat sheep was detected homozygous MM only. The results presented in this study show polymorphism of the calpastatin gene in the population of Stara Zagora sheep. Therefore,we could be confirm the importance of this gene as a potential DNA marker in marker-assisted selection with respect to meat production.

Recent evidence indicates the involvement of calpains (CAPNs), a family of cysteine proteases, in cancer development and progression, as well as the insufficient response to cancer therapies. The contribution of CAPNs and regulatory calpastatin (CAST) and ERK1/2 kinases to aggressiveness, disease course, and outcome in laryngeal cancer remains elusive. This study was aimed to evaluate the CAPN1/2-CAST-ERK1/2 enzyme system mRNA/protein level and to investigate whether they can promote the dynamic of tumor growth and prognosis. The mRNA expression of marker genes was determined in 106 laryngeal cancer (SCLC) cases and 73 non-cancerous adjacent mucosa (NCLM) controls using quantitative real-time PCR. The level of corresponding proteins was analyzed by Western Blot. SLUG expression, as indicator of pathological advancement was determined using IHC staining. Significant increases of CAPN1/2-CAST-ERK1/2 levels of mRNA/protein were noted in SCLC compared to NCLM ( p < 0.05). As a result, a higher level of CAPN1 and ERK1 genes was related to larger tumor size, more aggressive and deeper growth according to TFG scale and SLUG level ( p < 0.05). There were also relationships of CAPN1/2 and ERK1 with incidences of local/nodal recurrences ( p < 0.05). An inverse association for CAPN1/2, CAST, and ERK1/2 transcripts was determined with regard to overall survival ( p < 0.05). In addition, a higher CAPN1 and phospho-ERK1 protein level was related to higher grade and stage ( p < 0.05) and was found to promote worse prognosis. This is the first study to show that activity of CAPN1/2- CAST-ERK1/2 axis may be an indicator of tumor phenotype and unfavorable outcome in SCLC. [ABSTRACT FROM AUTHOR]

The present study was performed in order to investigate a relationship between polymorphisms on the calpastatin gene (CAST) and pig meat quality traits. The investigation was carried out on 29 gilts and barrows, crosses of Large White x German Landrace randomly selected at slaughter line. Pigs were slaughtered at 130-150 kg of live weight and blood samples were taken for genomic DNA analysis. The following indicators of meat quality and meat chemical composition were evaluated: pH and electric conductivity measured 45 minutes post mortem in m. Longissimus dorsi (LD muscle) and in M. Semimembranosus (SM muscle); electric conductivity measured at the same locations after 24h of cooling; drip loss (determined by “bag method”); colour (measured with Minolta chromameter and expressed as Hunter L, a, b values); instrumental tenderness assessed as Warner-Bratzler (WB) shear force and moisture, fat, protein and collagen content (%) determined on cooked LD muscle after 24h of thawing. The amplification products of the CAST gene were digested with HinfI restriction endonuclease and three genotypes (AA, BB and AB) were revealed. Statistical analysis showed that meat originated from pigs of AB genotype had the lowest WB shear force and the highest protein content of cooked LD muscle. As for the indicators of technological meat quality, statistically significant differences (p

Calpastatin (CAST) is a specific inhibiter of Calpains, playing a role in meat tenderization and myogenesis. In the present study the polymorphism of the CAST gene of Khalkhali goat in Azerbaijan province in Iran was investigated by polymerase chain reaction and restriction fragment length polymorphism technique (PCR-RFLP). Genomic DNA was extracted from whole blood samples collected from 200 Khalkhali goats. A 1552 bp in the region between exons 6 and 7 of the CAST gene was amplified by standard PCR, using the locus specific primers. Two alleles (A and B) and three genotypes (AA, BB and AB) were observed. The frequencies of the observed genotypes were 49.7, 0.09 and 41.4 for AA, BB and AB, respectively. Allele frequencies were 0.705, 0.295 for A and B, respectively. The average heterozygosity for CAST gene was 0.416 and the expected heterozygosity was 0.417. For homozygosity these numbers were 0.91 and 0583, respectively and the chi-square test showed significant (P<0.01) deviation from Hardy-Weinberg equilibrium for this locus in this population. [ABSTRACT FROM AUTHOR]

Calpastatin (CAST) gene is well known as an inhibitor of muscle protein degradation and relates to muscle growth and meat tenderness. The objective of this study was to determine the association of CAST gene with growth traits and carcass characteristics in Bali cattle. A number of data from 35 Bali bulls were collected from BPTU Bali Cattle to obtain growth traits, carcass characteristics, and blood samples. Polymorphism of CAST gene in Bali bulls was analyzed by using PCR-RFLP and DNA sequencing. The association of CAST gene with growth traits and carcass characteristics were analyzed by using General Linear Model (GLM). The result showed that there were two genotypes (GG and AG) of CAST gene with allele frequencies of 0.857 and 0.143, respectively, for G and A. Notably, mutation A to G occurred in 253 bp CAST fragment gene in Bali Cattle. Genotypes GG and AG of CAST gene significantly affected (P<0.05) the back-fat thickness and longissimus dorsi without a significant effect on the growth traits. It could be concluded that CAST gene had a potency as a marker gene for carcass quality in Bali cattle. [ABSTRACT FROM AUTHOR]

Abstract: Calpastatin is associated with the rate of post mortem degradation of structural proteins due to the regulation of calpain activity. In the present research, the associations between polymorphisms within 6th intron of porcine CAST gene and several meat quality traits were analyzed. The CAST gene polymorphisms affected meat colour, pH, water holding-capacity (WHC) and texture parameters (toughness, firmness, cohesiveness, chewiness, and resilience) measured in longissimus dorsi and semimembranosus muscles. The analysis performed on the most numerous breeds maintained in Poland, suggested that the most interesting polymorphisms were CAST/HpaII and CAST/RsaI, which had the greatest effect on WHC regardless of the breed analyzed and had an effect on meat pH, firmness and toughness for most breeds. Interestingly, for almost all breeds, the significant effect of both mutations on intramuscular fat content (IMF) was detected. The provided data confirmed the use of CAST gene as a genetic marker in breeding programmes which allows performing a selection focussed on improving the quality of pork. [Copyright &y& Elsevier]

Calpastatin (CAST) is a specific inhibiter of calpains, playing a role in meat tenderization and myogenesis. In the present study, the polymorphism of the CAST gene of Makoei sheep was investigated by polymerase chain reaction and single strand conformation polymorphism technique (PCR-SSCP). Genomic DNA was extracted from whole blood samples collected from 100 sheep. A 622 bp CAST exon 1 segment was amplified by standard PCR, using the locus specific primers. PCR products were subjected to a non-denaturing gel electrophoresis. Four SSCP patterns, representing four different genotypes, were identified. The frequencies of the observed genotypes were 0.31, 0.04, 0.63 and 0.02 for AA, BB AB and AC, respectively. Allele frequencies were 0.6313, 0.3586 and 0.01 for A, B and C, respectively. The Observed heterozygosity (Hobs) value for CAST gene was 0.4728. The chi-square test showed significant (P< 0.01) deviation from Hardy-Weinberg equilibrium for this locus in Makoei sheep population. [ABSTRACT FROM AUTHOR]

Recent evidence indicates the involvement of calpains (CAPNs), a family of cysteine proteases, in cancer development and progression, as well as the insufficient response to cancer therapies. The contribution of CAPNs and regulatory calpastatin (CAST) and ERK1/2 kinases to aggressiveness, disease course, and outcome in laryngeal cancer remains elusive. This study was aimed to evaluate the CAPN1/2-CAST-ERK1/2 enzyme system mRNA/protein level and to investigate whether they can promote the dynamic of tumor growth and prognosis. The mRNA expression of marker genes was determined in 106 laryngeal cancer (SCLC) cases and 73 non-cancerous adjacent mucosa (NCLM) controls using quantitative real-time PCR. The level of corresponding proteins was analyzed by Western Blot. SLUG expression, as indicator of pathological advancement was determined using IHC staining. Significant increases of CAPN1/2-CAST-ERK1/2 levels of mRNA/protein were noted in SCLC compared to NCLM (p < 0.05). As a result, a higher level of CAPN1 and ERK1 genes was related to larger tumor size, more aggressive and deeper growth according to TFG scale and SLUG level (p < 0.05). There were also relationships of CAPN1/2 and ERK1 with incidences of local/nodal recurrences (p < 0.05). An inverse association for CAPN1/2, CAST, and ERK1/2 transcripts was determined with regard to overall survival (p < 0.05). In addition, a higher CAPN1 and phospho-ERK1 protein level was related to higher grade and stage (p < 0.05) and was found to promote worse prognosis. This is the first study to show that activity of CAPN1/2- CAST-ERK1/2 axis may be an indicator of tumor phenotype and unfavorable outcome in SCLC.

Calpastatin has been introduced as a potential candidate gene for growth and meat quality traits. In this study, genetic variability was investigated in the exon 6 and its intron boundaries of ovine CAST gene by PCR-SSCP analysis and DNA sequencing. Also a protein sequence and structural analysis were performed to predict the possible impact of amino acid substitutions on physicochemical properties and structure of the CAST protein. A total of 487 animals belonging to four ancient Iranian sheep breeds with different fat metabolisms, Lori-Bakhtiari and Chall (fat-tailed), Zel-Atabay cross-bred (medium fat-tailed) and Zel (thin-tailed), were analyzed. Eight unique SSCP patterns, representing eight different sequences or haplotypes, CAST-1, CAST-2 and CAST-6 to CAST-11, were identified. Haplotypes CAST-1 and CAST-2 were most common with frequency of 0.365 and 0.295. The novel haplotype CAST-8 had considerable frequency in Iranian sheep breeds (0.129). All the consensus sequences showed 98-99%, 94-98%, 92-93% and 82-83% similarity to the published ovine, caprine, bovine and porcine CAST locus sequences, respectively. Sequence analysis revealed four SNPs in intron 5 (C24T, G62A, G65T and T69-) and three SNPs in exon 6 (c.197A>T, c.282G>T and c.296C>G). All three SNPs in exon 6 were missense mutations which would result in p.Gln 66 Leu, p.Glu 94 Asp and p.Pro 99 Arg substitutions, respectively, in CAST protein. All three amino acid substitutions affected the physicochemical properties of ovine CAST protein including hydrophobicity, amphiphilicity and net charge and subsequently might influence its structure and effect on the activity of Ca2+ channels; hence, they might regulate calpain activity and afterwards meat tenderness and growth rate. The Lori-Bakhtiari population showed the highest heterozygosity in the ovine CAST locus (0.802). Frequency difference of haplotypes CAST-10 and CAST-8 between Lori-Bakhtiari (fat-tailed) and Zel (thin-tailed) breeds was highly significant (P<0.001), indicating that these two haplotypes might be breed-specific haplotypes that distinguish between fat-tailed and thin-tailed sheep breeds., (Copyright © 2013 Elsevier B.V. All rights reserved.)