Sensory quality characteristics with different beef quality grades and surface texture features assessed by dented area and firmness, and the relation to muscle fiber and bundle characteristics.

The main objective of this study was to compare the eating quality among the groups categorized by the Korean beef quality grade and texture feature of exposed muscle surface assessed by extent of dented areas and firmness. Additionally, this study also investigated the effects of fiber and bundle characteristics on texture feature to establish the cause of differences in muscle surface texture. Significant differences were observed in the sensory quality characteristics among the quality grades mainly determined by marbling score (P < 0.05). However, the coarse texture group with a dented surface required a higher initial force to penetrate meat (P < 0.001), was more difficult to break meat into fragments (P < 0.001), and had a higher amount of perceptible residue in the mouth (P < 0.01) compared to the fine texture group. These differences in the surface texture features between the fine and coarse groups could be explained by bundle area and fiber number per each bundle.

Stretch training induces unequal adaptation in muscle fascicles and thickness in medial and lateral gastrocnemii.

This study compared adaptations in fascicle lengths, pennation angles, and muscle thickness of the lateral and medial gastrocnemii in response to 6 weeks of stretch training. The nondominant plantar flexors of 11 males were stretched five times per week for 6 weeks and compared with the contralateral leg and a nonstretched control group of 10 males. During stretch training, instantaneous electromyography was utilized to ensure passive muscle stretch. At baseline, week three, week six and 1 week after the conclusion of stretch training, ultrasound was used to measure fascicle lengths, pennation angles, muscle thickness of the lateral gastrocnemius and medial gastrocnemius, and Achilles tendon thickness and length. Plantar flexion torque was measured, and voluntary activation was assessed. Muscle thickness increased 5.6% after 6 weeks of stretch training (P=.009). The fascicles in the lateral gastrocnemius lengthened to a greater extent than the medial. Overall, fascicles lengthened 25% (P<.001) in the muscle tendon junction and 5.1% (P<.001) in the muscle belly. Pennation angles were unchanged in the medial gastrocnemius but decreased in the lateral gastrocnemius 7.1% (P=.02). There was no change in maximal voluntary contraction, voluntary activation, tendon length, or thickness. This study demonstrates that stretch training is a viable modality to alter muscle architecture of the human gastrocnemius through lengthening of muscle fascicles, decreasing pennation angles, and increasing muscle thickness, albeit adaptations are unequal between the lateral and medial heads.

Surgical stabilization of flail chest: the impact on postoperative pulmonary function.

OBJECTIVES: Flail chest results in significant morbidity. Controversies continue regarding the optimal management of flail chest. No clear guidelines exist for surgical stabilization. Our aim was to examine the association of bedside spirometry values with operative stabilization of flail chest. METHODS: IRB approval was obtained to identify patients with flail chest who underwent surgical stabilization between August 2009 and May 2011. At our institution, all rib fracture patients underwent routine measurement of their forced vital capacity (FVC) using bedside spirometry. Formal pulmonary function tests were also obtained postoperatively and at three months in patients undergoing stabilization. Both the Synthes and Acute Innovations plating systems were utilized. Data is presented as median (range) or (percentage). RESULTS: Twenty patients (13 male: 65 %) with median age of 60 years (30-83) had a median of four ribs (2-9) in the flail segment. The median Injury Severity Score was 17 (9-41) and the median Trauma and Injury Severity Score was 0.96 (0.04-0.99). Preoperative pneumonia was identified in four patients (20 %) and intubation was required in seven (35 %). Median time from injury to stabilization was four days (1-33). The median number of plates inserted was five (3-11). Postoperative median FVC (1.8 L, range 1.3-4 L) improved significantly as compared to preoperative median value (1 L, range 0.5-2.1 L) (p = 0.003). This improvement continued during the follow-up period at three months (0.9 L, range 0.1-3.0) (p = 0.006). There were three deaths (15 %), none of which were related to the procedure. Subsequent tracheostomy was required in three patients (15 %). The mean hospital stay and ventilator days after stabilization were nine days and three days, respectively. Mean follow-up was 5.6 ± 4.6 months. CONCLUSION: Operative stabilization of flail chest improved pulmonary function compared with preoperative results. This improvement was sustained at three months follow-up.

Characterization and molecular genetic mapping of microsatellite loci in pepper.

Microsatellites or simple sequence repeats are highly variable DNA sequences that can be used as informative markers for the genetic analysis of plants and animals. For the development of microsatellite markers in Capsicum, microsatellites were isolated from two small-insert genomic libraries and the GenBank database. Using five types of oligonucleotides, (AT)(15), (GA)(15), (GT)(15), (ATT)(10) and (TTG)(10), as probes, positive clones were isolated from the genomic libraries, and sequenced. Out of 130 positive clones, 77 clones showed microsatellite motifs, out of which 40 reliable microsatellite markers were developed. (GA)(n) and (GT)(n) sequences were found to occur most frequently in the pepper genome, followed by (TTG)(n) and (AT)(n). Additional 36 microsatellite primers were also developed from GenBank and other published data. To measure the information content of these markers, the polymorphism information contents (PICs) were calculated. Capsicum microsatellite markers from the genomic libraries have shown a high level of PIC value, 0.76, twice the value for markers from GenBank data. Forty six microsatellite loci were placed on the SNU-RFLP linkage map, which had been derived from the interspecific cross between Capsicum annuum "TF68" and Capsicum chinense "Habanero". The current "SNU2" pepper map with 333 markers in 15 linkage groups contains 46 SSR and 287 RFLP markers covering 1,761.5 cM with an average distance of 5.3 cM between markers.

Construction of a deep coverage BAC library from Capsicum annuum, 'CM334'.

A bacterial artificial chromosome (BAC) library consisting of 235,000 clones with an average insert size of 130 kb was constructed from Capsicum annuum, 'CM334'. Based on a pepper haploid genome size of 2,702 Mbp/C, the BAC library is estimated to contain approximately 12 genome equivalents and represents at least 99% of the pepper genome. Screening of the library with mitochondrial DNA probes (coxII, coxIII, atp6 and atp9) and chloroplast DNA probes (atpB, rbcL) indicated that contamination with cytoplasmic DNA was less than 0.5%. To estimate the possibility of isolating a specific clone, the library was screened with single or low-copy gene-specific probes and RFLP probes. Screening of high density BAC filters with RFLP markers linked to L (TMV resistance), y (fruit color), C2 (fruit color) and C (pungency) loci under high stringency conditions revealed that at least three positive BAC clones were found per each probe. This fact indicates that the library is highly reliable and represents a resource for map-based cloning, physical mapping, and characterization of upstream and downstream regulations of the chili pepper genes.

Identification and classification of S haplotypes in Raphanus sativus by PCR-RFLP of the S locus glycoprotein (SLG) gene and the S locus receptor kinase (SRK) gene.

Polymorphism of the S-locus glycoprotein (SLG) and S-locus receptor kinase (SRK) genes in Raphanus sativus was analyzed by PCR-RFLP using SLG- and SRK-specific primers. Twenty four inbred lines of R. sativus could be grouped into nine S haplotypes. DNA fragments of SLG alleles specifically amplified from five S haplotypes by PCR with Class-I SLG-specific primers showed different profiles upon polyacrylamide-gel electrophoresis after digestion with restriction endonucleases. The five R. sativus SLG alleles were determined for their nucleotide sequences of DNA fragments. Comparison of the amino-acid sequences with a reported Brassica SLG (S(6)) showed 77-84% homology. Deduced amino-acid sequences showed 12-conserved cystein residues and three hypervariable regions which are characteristic of Brassicsa SLG. A DNA fragment was also amplified by PCR from two of each S haplotype with Class-II SLG-specific primers, and showed polymorphism when cleaved with restriction endonucleases. The nucleotide sequences of amplified DNA fragments of the Class-II SLG revealed about 60% similarity with those of the Class-I SLG. It is concluded that there exist both Class I and Class II S alleles in R. sativus, as in Brassica campestris and Brassica oleracea. PCR using SRK-specific primers amplified a DNA fragment of about 1.0 kb from seven of each S haplotype out of 24 tested. These DNA fragments showed high polymorphism in polyacrylamide-gel electrophoresis after digestion with restriction endonucleases. Nucleotide sequences of the DNA fragments amplified from the seven S haplotypes showed that the fourth and the fifth exons of SRK are highly conserved, and that there is high variation in the fifth intron, the sixth intron and seventh exon of the SRK which may be responsible for the polymorphic band patterns in PCR-RFLP analysis. The PCR-RFLP method has proven useful for the identification of S alleles in inbred lines and for listing S haplotypes in R. sativus. Phylogenic analysis of the SLG and SRK sequences from Raphanus and Brassica revealed that the Raphanus SLGs and SRKs did not form an independent cluster, but were dispersed in the tree, clustering together with Brassica SLGs and SRKs. Furthermore, SLGs and SRKs from Raphanus were both grouped into Class-I or Class-II S haplotypes. Therefore, these results suggest that the diversification of the SLG and SRK alleles occurred prior to the differentiation of the two genera Brassica and Raphanus.

Molecular cloning of a novel pathogen-inducible cDNA encoding a putative acyl-CoA synthetase from Capsicum annuum L.

By means of differential display, a pool of salicylic acid (SA)-induced mRNAs were identified and subsequently their cDNAs were isolated from a cDNA library prepared from SA-induced leaf tissues of hot pepper. One of these cDNA clones, designated CaSIG4, was 1900 bp and contained an open reading frame encoding 523 amino acids with a calculated molecular mass of 56.3 kDa. The predicted amino acid sequence of CaSIG4 showed high sequence similarity to the AMP-binding protein family of both prokaryotic and eukaryotic acyl-CoA synthetases. CaSIG4 transcripts accumulated rapidly after SA treatment and in response to both incompatible and compatible interactions with Xanthomonas campestris pv. vesicatoria race 1. To investigate the cis-acting elements mediating CaSIG4 expression, the CaSIG4 5'-flanking region was isolated by inverse PCR. Database searches indicated that a potential cis-regulatory element is almost identical to the consensus core sequences ACC(A/T)ACC(A/C) which are conserved among promoters of other phenylpropanoid biosynthetic genes. The subcellular localization of the CaSIG4 protein was studied by using a soluble modified GFP gene fusion delivered into epidermal cells of onion by biolistic bombardment. The CaSIG4-smGFP fusion protein was localized to the plasma membrane. Taken together, CaSIG4 encoding a putative acyl-CoA synthetase could function as a plasma membrane-bound protein with a role in signaling in plant defense.

Construction and characterization of a bacterial artificial chromosome library from chili pepper.

A library of the bacterial artificial chromosome (BAC) that consisted of a total of 78,336 clones with an average insert size of 80 kb was constructed from Capsicum annuum, 'CM334', which is resistant to Phytophthora capsici and PVY. Based on a haploid genome size of pepper of 2,702 Mbp/C, the BAC library was estimated to contain approximately three genome equivalents and represented at least 90% of the pepper genome. In order to determine the percentage of BAC clones that contained mitochondrial DNAs, the entire library was screened with probes of chili pepper mitochondrial DNAs. The result showed that only twenty-five clones, which is 0.03% of the total BAC clones, were hybridized to mitochondrial gene probes. This indicates that the library is comprised predominantly of the nuclear sequences. The library was also tested for isolating specific clones by screening with a few known genes from the chili pepper, phytoene synthase gene, and two MADS genes--HpMADS1 and HpMADS3. The result showed that the three clones for phytoene synthase and the two clones for each MADS gene were positively hybridized to the specific probes. This indicates that the library is highly reliable and represents a resource for initiating map-based cloning and contig mapping in chili pepper.

Production of transgenic male sterile tobacco plants with the cDNA encoding a ribosome inactivating protein in Dianthus sinensis L.

The ribosome inactivating protein (RIP) gene from D. sinensis was used as a cytotoxin gene to induce male sterility in tobacco plants. The TA29 promoter, obtained by PCR amplification from tobacco, was fused to the RIP cDNA, and the chimaeric molecule was then introduced into tobacco plants by Agrobacterium-mediated transformation. Out of twenty-one independent transformants, twenty transgenic tobacco plants exhibited male sterility. Southern blot analysis revealed that four of the transgenic plants contained a single copy of the RIP gene, while the rest of the transgenic tobacco plants had two to four copies of the gene. The transgenic male sterile plants set seeds normally when pollinated with pollens from untransformed control plants, indicating that the RIP gene does not affect the pistil development. Furthermore, the seed yield of the transgenic plant was similar to that of the untransformed, self-pollinated control plant. A light microscopic observation of anther cross sections clearly showed that the tapetal tissue of the anther was selectively and completely destroyed causing male sterility. This study suggests that the RIP gene can be used as a cytotoxin gene for induction of male sterility in the plant.

Controlling self-incompatibility by CO2 gas treatment in Brassica campestris: structural alteration of papillae cell and differential gene expression by increased CO2 gas.

Self-imcompatibility is a controlling genetic mechanism to prevent self-pollination for Chinese cabbage (Brassica campestris), one of the major vegetable crops in Korea. To maintain inbred lines of the crop plant, a method in that high CO2 gas is treated to the pistils to overcome the self-incompatibility and thereby self-pollens can successfully make germination and fertilization has been widely used in seed companies. Despite the common utilization of this method, any molecular and cellular studies on how the self-incompatibility is removed from the Chinese cabbage plant have not been done. In this study, we show that the increased CO2 gas causes a structural alteration of the papillae cell and thereby the self-incompatibility is removed from the Chinese cabbage plant, allowing the self-pollens to germinate and penetrate the papillae cell. Also, gene expression in the pistil treated with CO2 gas was studied by DD/RT-PCR and reverse northern hybridization experiments. The results suggest that the failure in self-incompatible reaction resulted not only from the structural alteration of the papillae cell but also from change in the pistil component production that is either positively or negatively regulated by the environmental stimulation.

Effect of Vitex rotundifolia on immediate-type allergic reaction.

We investigated the effect of aqueous extract of Vitex rotundifolia (L.) (Verbenaceae) fruits (VRFE) on the immediate-type allergic reactions in vivo and in vitro. VRFE (10(-4)-1.0 g/kg) dose-dependently inhibited systemic allergic reaction induced by compound 48/80. When VRFE was employed in a systemic allergic reaction test, the plasma histamine levels were reduced in a dose-dependent manner. VRFE (5x10(-1) and 1.0 g/kg) inhibited passive cutaneous anaphylaxis activated by anti-dinitrophenyl (DNP) IgE. VRFE (10(-3)-1.0 mg/ml) also dose-dependently inhibited the histamine release from the rat peritoneal mast cells (RPMC) by compound 48/80 or anti-DNP IgE. Moreover, VRFE (10(-3) mg/ml) had a significant inhibitory effect on anti-DNP IgE-induced tumor necrosis factor-alpha production from RPMC. These results suggest that VRFE may be beneficial in the regulation of immediate-type allergic reaction.

Isolation and characterization of cDNAs encoding ribosome inactivating protein from Dianthus sinensis L.

To isolate a ribosome inactivating protein (RIP) gene, six plant species were surveyed for antiviral activity. Crude proteins extracted from these plants were tested for the antiviral activity against tobacco mosaic virus (TMV) in Nicotiana glutinosa. All the plants, Spinacia oleracea, Amaranthus lividus, Dianthus superbus, Dianthus sinensis and Celosia cristata, with an exception of Oenanthe stolonifera, presented 70-90% inhibition of viral infectivity. In an attempt to search for the RIP gene from D. sinensis, partial cDNA was obtained by polymerase chain reaction (PCR) of the poly(A)+ RNA from D. sinensis leaves. DNA gel blot analysis showed that D. sinensis has multi-copy RIP genes. The expression of RIP gene was investigated in the flower, leaf, root and stem of D. sinensis, and was found to be most abundant in the leaf. Using the partial cDNA as a probe, seven full-length cDNAs were isolated from a library prepared from D. sinensis leaves. They were divided into three groups on the basis of their nucleotide sequence homology. The three representative clones, cDsRIP1, cDsRIP2 and cDsRIP3 were completely sequenced. They all had an open reading frame of 882 bp. The cDsRIP2 showed 79% homology with dianthin 30 and saporin genes; 59% with PAP and Mirabilis antiviral protein MAP genes. From the analysis of deduced amino acid sequences, it was predicted that D. sinensis RIP cDNAs might have a putative signal peptide of 23 amino acid residues at their N-terminus. When the cDNA was expressed in E. coli, the bacteria was unable to grow upon IPTG induction, suggesting that expression of the gene renders toxicity to E. coli cells.

Cymbidium mosaic virus coat protein gene in antisense confers resistance to transgenic Nicotiana occidentalis.

The nucleotide sequence of the 3'-terminal region of the Korean isolate of cymbidium mosaic virus (CyMV-Ca) from a naturally infected cattleya was determined. The sequence contains an open reading frame (ORF) coding for the viral coat protein (CP) at the 3'-end and three other ORFs (triple gene block or movement protein) of CyMV. The CP gene encodes a polypeptide chain of 220 amino acids with a molecular mass of 23,760 Da. The deduced CP sequence showed a strong homology with those of two CyMVs reported. A construct of the CyMV-Ca CP gene in the antisense orientation in the plant expression vector pMBP1 was transferred via Agrobacterium tumefaciens-mediated transformation into Nicotiana occidentalis which is a propagation host of CyMV. The T1 progeny of the transgenic plants were inoculated with CyMV and found to be highly resistant to CyMV infection.

Satellite-RNA-mediated resistance to cucumber mosaic virus in transgenic plants of hot pepper (Capsicum annuum cv. Golden Tower).

We previously established a system of in vitro regeneration and Agrobacterium-mediated transformation for hot pepper plants. The level of protection against cucumber mosaic virus in the progeny of the transgenic hot pepper plants that express cucumber mosaic virus (CMV) satellite RNA was investigated. The transgenic hot pepper plants were self-fertilized, and their progeny were tested for stable inheritance and expression of the cDNA of CMV satellite RNA. Polymerase chain reaction and RNA gel blot analyses showed that the introduced gene was stably transmitted and expressed in the progeny. Symptom attenuation in the offspring was confirmed upon inoculation with CMV-Y or CMV-Korea (CMV-Kor) strains.

EcoRI variant N199H has enhanced specific activity.

The Asn199 residue of the EcoRI restriction endonuclease has been replaced with other amino acids to investigate whether it mediates nucleotide recognition or catalytic activity. Cassette mutagenesis gave variants of EcoRI: N199D, N199H, N199L, N199R, N199S and N199V. Their relative cleavage rates were found to be in the following order: N199H > EcoRI (wild type; wt) > N199L > N199V > N199S > N199R > N199D. In particular, EcoRI variant N199H showed about a twofold higher specific activity than that of the wt enzyme.

Bovine liver dihydropyrimidine amidohydrolase: purification, properties, and characterization as a zinc metalloenzyme.

Beef liver dihydropyrimidine amidohydrolase has been purified to homogeneity using both an electrophoretic and a hydrophobic chromatographic method. The enzyme is a tetramer with a molecular weight of 226,000 g mol-1, a subunit molecular weight of 56,500 g mol-1, and contains 4 mol of tightly bound (Ks greater than or equal to 1.33 X 10(9) M-1) Zn2+ per mole of active enzyme. The enzyme appears to be a true Zn2+ metalloenzyme because there exists a direct proportionality between enrichment of Zn2+ and active enzyme during purification, there is an almost quantitative relationship between the loss of both enzyme activity and Zn2+ during 8-hydroxyquinoline-5-sulfonic acid treatment to form apoenzyme, Zn2+ and Co2+ reactivate dipicolinic acid-inhibited enzyme, and saturating concentrations of a substrate, dihydrothymine, protect against 8-hydroxyquinoline-5-sulfonic acid inhibition. EDTA does not inhibit the enzyme; however, 8-hydroxyquinoline-5-sulfonic acid, o-phenanthroline, and 2,6-dipicolinic acid cause a time-dependent loss in activity which follows pseudo-first-order kinetics. Analysis of the resulting kinetic data for these three chelators indicates that the reaction pathway involves the formation of an enzyme-Zn2+-chelator ternary complex which then dissociates to form apoenzyme and a Zn2+-chelator complex. Like other Zn2+ metalloenzymes, the enzyme is inhibited by a number of substituted sulfonamides. In the case of p-nitrobenzenesulfonamide, this inhibition is competitive in nature. Using the purified enzyme, kinetic constants were determined for a variety of dihydropyrimidines, ureidocarboxylic acids, and hydantoin substrates. Normal hyperbolic kinetics were observed for the hydrolysis of the cyclic compounds, but the cyclization of the ureidoacids showed biphasic kinetics and different values of Km can be estimated at either high or low concentrations of these substrates.

Cytoplasmic Reversion of cms-S in Maize: Association with a Transpositional Event.

Spontaneous reversion to fertility in S male-sterile cytoplasm of maize is correlated with the disappearance of the mitochondrial plasmid-like DNA's, S-1 and S-2, and changes in the mitochondrial chromosomal DNA. Hybridization data indicate that one of the plasmid-like DNA's, S-2, is prominently involved in the mitochondrial DNA rearrangements.

Dihydropyrimidine amidohydrolase is a zinc metalloenzyme.

Bovine liver dihydropyrimidine amidohydrolase (EC 3.5.2.2) has been subjected to atomic absorption analysis. Three different preparations of homogeneous enzyme indicated that the enzyme contains 4.3 +/- 0.3 g atoms of Zn2+ per mol of enzyme or 1.1 g atoms of Zn2+ per subunit. No Co2+, Mn2+, Mg2+ or Cd2+ was detected. Exhaustive dialysis against either o-phenanthroline or EDTA did not reduce enzyme activity; however, prolonged incubation with dipicolinic acid resulted in inactivation which can be reversed by either Zn2+ or Co2+ but not Mg2+.

Role of enzymatically catalyzed 5-iodo-5,6-dihydrouracil ring hydrolysis on the dehalogenation of 5-iodouracil.

Incubation of 5-iodo-5,6-dihydrouracil (IH2Ura) with soluble rat liver enzymes at 37 degrees, pH 8.2, results in the rapid release of iodide ion. The second product resulting from the carbon skeleton of the dihydropyrimidine ring system is 2-amino-2-oxazoline-5-carboxylic acid (I). Ultraviolet absorbance measurements at 225 nm, where both IH2Ura and iodide ion absorb, indicate that IH2Ura dehalogenation is a two-step process. The first step, which is enzyme-dependent, involves dihydropyrimidine amidohydrolase (EC 3.5.2.2.)-catalyzed hydrolysis of the IH2Ura ring system presumably to yield 2-iodo-3-ureidopropionate. The enzyme preparations also catalyze the hydrolysis of 5-bromo-5,6-dihydrouracil, 5,6-dihydrouracil, and 5,6-dihydrothymine, the latter two of which are the natural substrates for dihydropyrimidine amidohydrolase. The second step in IH2Ura dehalogenation involves the nonenzymatically catalyzed, pH-independent intramolecular cyclization of 2-iodo-3-ureidopropionate via nucleophilic attack of the ureido oxygen atom on carbon-2 resulting in iodide ion and the oxazoline (I) as final products. The results are discussed relative to the role of pyrimidine catabolizing enzymes in 5-halopyrimidine dehalogenation.