Mitochondrial biogenesis and PGC-1α gene expression in male broilers from ascites-susceptible and -resistant lines.

Ascites is a cardiovascular metabolic disease characterized by accumulation of fluid around the heart and in the abdominal cavity that eventually leads to death. This syndrome is the end-point result of a series of metabolic incidents that are generally caused by impaired oxygen availability. Mitochondria are the major sites of oxygen consumption, therefore major contributors to oxidative stress. Genetic, metabolic and dietary factors can influence variations in mitochondrial biogenesis (mitochondrial size, number and mass) that might have an effect on oxygen consumption and reactive oxygen species production. This study evaluated the effect of genotype on PGC-1α mRNA gene expression and mitochondrial biogenesis. These parameters were examined in male broiler chickens at 22 weeks of age from the SUS and RES lines divergently selected for ascites phenotype. From each line, five birds were sampled for right ventricle and breast muscle. Gene expression and mtDNA copy number were assessed by quantitative PCR. Results showed that birds from SUS had significantly higher PGC-1α mRNA gene (p = .033) and mitochondrial DNA copy number (p = .038) in breast muscle. There was no difference in right ventricle PGC-1α expression or mitochondrial DNA copy number between the two lines. These findings indicate that mitochondrial biogenesis and PGC-1α mRNA gene expression differ between male broiler chickens from RES and SUS lines in a tissue-specific manner.

Multi-generational genome wide association studies identify chromosomal regions associated with ascites phenotype.

Ascites is a multi-faceted disease commonly observed in fast growing broilers, which is initiated when the body is insufficiently oxygenated. A series of events follow, including an increase in pulmonary artery pressure, right ventricle hypertrophy, and accumulation of fluid in the abdominal cavity and pericardium. Advances in management practices along with improved selection programs have decreased ascites incidence in modern broilers. However, ascites syndrome remains an economically important disease throughout the world, causing estimated losses of $100 million per year. In this study, a 60 K Illumina SNP BeadChip was used to perform a series of genome wide association studies (GWAS) on the 16th and 18th generation of our relaxed (REL) line descended from a commercial elite broiler line beginning in 1995. Regions significantly associated with ascites incidence were identified on chromosome 2 around 70 megabase pairs (Mbp) and on chromosome Z around 60 Mbp. Five candidate single nucleotide polymorphisms (SNP) were evaluated as indicators for these 2 regions in order to identify association with ascites and right ventricle to total ventricle weight (RVTV) ratios. Chromosome 2 SNP showed an association with RVTV ratios in males phenotyped as ascites resistant and ascites susceptible (P = 0.02 and P = 0.03, respectively). The chromosome Z region also indicates an association with resistant female RVTV values (P = 0.02). Regions of significance identified on chromosomes 2 and Z described in this study will be used as proposed candidate regions for further investigation into the genetics of ascites. This information will lead to a better understanding of the underlying genetics and gene networks contributing to ascites, and thus advances in ascites reduction through commercial breeding schemes.

Interactive effects of temperature and dietary supplementation of arginine or guanidinoacetic acid on nutritional and physiological responses in male broiler chickens.

1. The aim of this experiment was to study the interactive effect of rearing temperature and dietary supplementation of arginine (Arg) or guanidinoacetic acid (GAA) on performance, gut morphology and ascites indices in broiler chickens raised under the same condition in the first 2 weeks and then reared under normal (23-26°C) or subnormal (17°C) ambient temperatures for the next 3 weeks. 2. This experiment was conducted as a split plot with 900 Ross 308 male broiler chicks that were allocated to two houses (as main plots); each consisted of 5 treatments (as sub-plots) with 6 replicates of 15 birds. The 5 diets were (1) control, (2) control + 0.60 g/kg GAA, (3) control + 1.20 g/kg GAA, (4) control + 0.86 g/kg Arg and (5) control + 1.72 g/kg Arg. 3. Feed intake (0-35 d) of birds fed on a diet containing 1.2 g GAA/kg and reared under normal temperature was reduced compared to control fed birds. Birds fed on a diet containing 1.72 g/kg Arg and reared under subnormal temperature had higher weight gain compared to those fed on control or GAA-added diets in overall study period. 4. Supplementation of diets with Arg alleviated the adverse effect of cold stress as reflected by reduction in blood haematocrit (41% vs. 37%), and right ventricle to total ventricle ratio (0.28 vs. 0.25) at 35 d of age. Addition of Arg to the diet of birds reared under cold stress resulted in a higher jejunal villus surface area compared to those fed on control or GAA-added diets. 5. Findings of this study revealed that Arg or GAA supplementation of diets did not affect performance of birds under normal temperatures, but Arg supplementation of the diet significantly alleviated the adverse effect of cold stress on performance, gut development and ascites syndrome. In addition, GAA supplementation at 1.2 g/kg improved jejunal villus surface area in birds raised under subnormal temperature.

Breeding and Genetics Symposium: a systems biology definition for chicken semen quality.

Rooster semen is an effluent from paired reproductive tracts. Each tract includes a testis, epididymis, and deferent duct. Upon ejaculation, efficacy of sperm propulsion varies among roosters. This phenotype is sperm mobility, that is, the movement of sperm against resistance at body temperature. The present work 1) compares reproductive tract throughput between lines of chickens selected for low and high sperm mobility, 2) demonstrates how semen quality can be defined in terms of an interaction between reproductive tract throughput and the proportion of mobile sperm ejaculated, 3) confirms that phenotype can be linked to genomewide differences in SNPlotype, and 4) shows how breeding can affect semen quality. Sperm mobility phenotype distributions were based on the average of duplicate observations per male (n = 241 and 262 roosters for low and high lines, respectively). Distributions were skewed and normal for low and high lines, respectively. Subsequent analyses used these base populations as sources for test subjects. In the first analysis, 10 males were selected from the mode of each distribution, and sperm mobility data were evaluated by nested ANOVA. Variation was observed between lines (P < 0.0001) but not among males within lines (P = 0.980). Sperm mobility data along with data from paired reproductive tracts were used to estimate combined reproductive tract throughput. Whereas testicular output was 1.2-fold greater in the low line (P = 0.037), the output of mobile sperm per day was 10.5-fold greater in the high line (P < 0.0001). Deferent duct transit differed between tails of the low line (P < 0.0001) but not between the tails of the high line (P = 0.514). Males from the mode and upper tail of the low line were SNPlotyped using a 60k chip by DNA Landmarks. These test subjects were used to associate phenotype with SNPlotype because founder effects and genetic drift could be discounted. Loci of interest were found on multiple chromosomes. Loci on chromosome Z were of particular interest because roosters are homozygous for this sex chromosome and a pronounced maternal effect was observed in a prior heritability study. Midrange phenotypes were produced by crossing low and high sperm mobility lines. Our experimental outcomes demonstrate that genes affect reproductive tract function as well as sperm cell attributes and thereby make semen quality subject to genetic selection.

Pulmonary arterial hypertension (ascites syndrome) in broilers: a review.

Pulmonary arterial hypertension (PAH) syndrome in broilers (also known as ascites syndrome and pulmonary hypertension syndrome) can be attributed to imbalances between cardiac output and the anatomical capacity of the pulmonary vasculature to accommodate ever-increasing rates of blood flow, as well as to an inappropriately elevated tone (degree of constriction) maintained by the pulmonary arterioles. Comparisons of PAH-susceptible and PAH-resistant broilers do not consistently reveal differences in cardiac output, but PAH-susceptible broilers consistently have higher pulmonary arterial pressures and pulmonary vascular resistances compared with PAH-resistant broilers. Efforts clarify the causes of excessive pulmonary vascular resistance have focused on evaluating the roles of chemical mediators of vasoconstriction and vasodilation, as well as on pathological (structural) changes occurring within the pulmonary arterioles (e.g., vascular remodeling and pathology) during the pathogenesis of PAH. The objectives of this review are to (1) summarize the pathophysiological progression initiated by the onset of pulmonary hypertension and culminating in terminal ascites; (2) review recent information regarding the factors contributing to excessively elevated resistance to blood flow through the lungs; (3) assess the role of the immune system during the pathogenesis of PAH; and (4) present new insights into the genetic basis of PAH. The cumulative evidence attributes the elevated pulmonary vascular resistance in PAH-susceptible broilers to an anatomically inadequate pulmonary vascular capacity, to excessive vascular tone reflecting the dominance of pulmonary vasoconstrictors over vasodilators, and to vascular pathology elicited by excessive hemodynamic stress. Emerging evidence also demonstrates that the pathogenesis of PAH includes characteristics of an inflammatory/autoimmune disease involving multifactorial genetic, environmental, and immune system components. Pulmonary arterial hypertension susceptibility appears to be multigenic and may be manifested in aberrant stress sensitivity, function, and regulation of pulmonary vascular tissue components, as well as aberrant activities of innate and adaptive immune system components. Major genetic influences and high heritabilities for PAH susceptibility have been demonstrated by numerous investigators. Selection pressures rigorously focused to challenge the pulmonary vascular capacity readily expose the genetic basis for spontaneous PAH in broilers. Chromosomal mapping continues to identify regions associated with ascites susceptibility, and candidate genes have been identified. Ongoing immunological and genomic investigations are likely to continue generating important new knowledge regarding the fundamental biological bases for the PAH/ascites syndrome.

Validation of a spectrophotometer-based method for estimating daily sperm production and deferent duct transit.

The objectives of the present work were 3-fold. First, a new method for estimating daily sperm production was validated. This method, in turn, was used to evaluate testis output as well as deferent duct throughput. Next, this analytical approach was evaluated in 2 experiments. The first experiment compared left and right reproductive tracts within roosters. The second experiment compared reproductive tract throughput in roosters from low and high sperm mobility lines. Standard curves were constructed from which unknown concentrations of sperm cells and sperm nuclei could be predicted from observed absorbance. In each case, the independent variable was based upon hemacytometer counts, and absorbance was a linear function of concentration. Reproductive tracts were excised, semen recovered from each duct, and the extragonadal sperm reserve determined by multiplying volume by sperm cell concentration. Testicular sperm nuclei were procured by homogenization of a whole testis, overlaying a 20-mL volume of homogenate upon 15% (wt/vol) Accudenz (Accurate Chemical and Scientific Corporation, Westbury, NY), and then washing nuclei by centrifugation through the Accudenz layer. Daily sperm production was determined by dividing the predicted number of sperm nuclei within the homogenate by 4.5 d (i.e., the time sperm with elongated nuclei spend within the testis). Sperm transit through the deferent duct was estimated by dividing the extragonadal reserve by daily sperm production. Neither the efficiency of sperm production (sperm per gram of testicular parenchyma per day) nor deferent duct transit differed between left and right reproductive tracts (P > 0.05). Whereas efficiency of sperm production did not differ (P > 0.05) between low and high sperm mobility lines, deferent duct transit differed between lines (P < 0.001). On average, this process required 2.2 and 1.0 d for low and high lines, respectively. In summary, we developed and then tested a method for quantifying male reproductive tract throughput. This method makes the study of semen production amenable to systems biology.

Physiology and endocrinology symposium: a proteome-based model for sperm mobility phenotype.

Sperm mobility is defined as sperm movement against resistance at body temperature. Although all mobile sperm are motile, not all motile sperm are mobile. Sperm mobility is a primary determinant of male fertility in the chicken. Previous work explained phenotypic variation at the level of the sperm cell and the mitochondrion. The present work was conducted to determine if phenotypic variation could be explained at the level of the proteome using semen donors from lines of chickens selected for low or high sperm mobility. We began by testing the hypothesis that premature mitochondrial failure, and hence sperm immobility, arose from Ca(2+) overloading. The hypothesis was rejected because staining with a cell permeant Ca(2+)-specific dye was not enhanced in the case of low mobility sperm. The likelihood that sperm require little energy before ejaculation and the realization that the mitochondrial permeability transition can be induced by oxidative stress arising from inadequate NADH led to the hypothesis that glycolytic enzymes might differ between lines. This possibility was confirmed by 2-dimensional electrophoresis for aldolase and phosphoglycerate kinase 1. This outcome warranted evaluation of the whole cell proteome by differential detergent fractionation and mass spectrometry. Bioinformatics evaluation of proteins with different expression levels confirmed the likelihood that ATP metabolism and glycolysis differ between lines. This experimental outcome corroborated differences observed between lines in previous work, which include mitochondrial ultrastructure, sperm cell oxygen consumption, and straight line velocity. Although glycolytic proteins were more abundant within highly mobile sperm, quantitative PCR of representative testis RNA, which included mRNA for phosphoglycerate kinase 1, found no difference between lines. In summary, we propose a proteome-based model for sperm mobility phenotype in which a genetic predisposition puts sperm cells at risk of premature mitochondrial failure as they pass through the excurrent ducts of the testis. In other words, we attribute mitochondrial failure to sperm cell and reproductive tract attributes that interact to affect sperm in a stochastic manner before ejaculation. In conclusion, our work provides a starting point for understanding chicken semen quality in terms of gene networks.

Chronic wounds and the medical biofilm paradigm.

There is a growing recognition that biofilms are the principal cause of wound chronicity. The development of treatments for wound biofilms raises the prospect that chronic wounds can be treated, potentially saving many patients' lives.

Bacteriophage therapy of venous leg ulcers in humans: results of a phase I safety trial.

OBJECTIVE: This phase 1 trial set out to examine the safety of a bacteriophage-based preparation for difficult-to-treat wounds. METHOD: The intention-to-treat sample comprised 42 patients with chronic venous leg ulcers (VLUs); 39 patients completed the trial. The ulcers were treated for 12 weeks with either a saline control or bacteriophages targeted against Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli. Follow-up continued until week 24. RESULTS: No adverse events were attributed to the study product. No significant difference (p>0.05) was determined between the test and control groups for frequency of adverse events, rate of healing, or frequency of healing. CONCLUSION: This study found no safety concerns with the bacteriophage treatment. Efficacy of the preparation will need to be evaluated in a phase II efficacy study. DECLARATION OF INTEREST: One of the authors (AS) holds an equity interest in Intralytix. The other authors do not have any interest in commercial activities.

Biofilms in wounds: management strategies.

Biofilms probably induce a chronic and/or 'quiet' inflammation in the chronic wound and so delay healing. This paper reviews current strategies that can be used to suppress biofilms in chronic wounds until better options are available.

Biofilms and chronic wound inflammation.

In contrast to the commonly accepted hypothesis of host-centred pathology, it is possible that surface bacteria, not host dysfunction, cause the chronicity and perpetual inflammation associated with chronic non-healing wounds.

A study of biofilm-based wound management in subjects with critical limb ischaemia.

OBJECTIVE: Bacterial biofilms cause or complicate numerous medical conditions, including chronic wounds. Biofilm-based wound care (BBWC) management strategies that suppress biofilm have been designed and are used extensively at the Southwest Regional Wound Care Center in Lubbock, Texas and are described in this article. This retrospective single-centre study was designed to evaluate the frequency of complete healing in subjects with a chronic wound in a limb with critical limb ischaemia (CLI) when managed using BBWC. METHOD: Of the 4500 subjects admitted with wounds between August 2002 and January 2006, 1400 subjects' TCpO2 levels were measured, and 266 included were identified as having CLI (TCpO2 < 20mmHg). Of these, 190 subjects were considered in the analysis because they received a substantial course of therapy (more than five visits). Each subject was individually managed to reinforce natural healing and suppress bacterial biofilm. Successful healing was defined as complete closure by March 2007. RESULTS: Of the 190 subjects with CLI, 146 (77%) healed completely, and 44 (23%) were categorised as non-healing. The healed group included 47% (68/146) with osteomyelitis and 69% (101/146) with diabetes mellitus. In the non-healed group, 75% (33/44) had osteomyelitis and 77% (34/44) had diabetes mellitus. Ninety-one per cent (30/33) of the subjects without osteomyelitis or diabetes mellitus healed, and 67% (53/79) of the subjects with both osteomyelitis and diabetes mellitus healed. CONCLUSION: When comparing the healing frequency in this study with a previously published study, BBWC strategies significantly improved healing frequency. These findings demonstrate that effectively managing the biofilm in chronic wounds is an important component of consistently transforming 'non-healable' wounds into healable wounds.

An inexpensive, simple protocol for DNA isolation from blood for high-throughput genotyping by polymerase chain reaction or restriction endonuclease digestion.

We describe simple, inexpensive, and reliable methods for isolating DNA from avian blood, semen, or feather pulp. The procedures are readily applicable to high-throughput 96-well plate isolation for genotype analysis of chicken DNA based on restriction endonuclease digestion or PCR. Isolation cost is primarily the cost of a deep-well assay block and a few pipet tips; current price is less than 0.10 dollar per sample, providing a significant cost advantage over commercial kits. The procedure employs inexpensive, nonhazardous reagents and yields intact, double-stranded DNA from as little as 2 to 10 microL of avian blood, suitable for RFLP analysis or hundreds of PCR amplifications. We compared our method to published procedures for alkaline extraction from feather pulp and found our method to be more reliable with the advantage of isolating intact DNA sequences that can be easily quantified. With minor modifications, the method can isolate DNA for PCR genotyping from mammalian whole blood.

An expressed sequence tag analysis of the chicken reproductive tract transcriptome.

Analysis of the chicken reproductive tract transcriptome is important in comparative biology for analysis of reproductive tract development and evolution. In addition, molecular analysis of the reproductive tract is important for identification of genes affecting fertility in the poultry industry. We sampled the chicken reproductive tract (ovary, oviduct, and testis) transcriptome, generating 5,328 expressed sequence tags that assembled into 4,518 contigs. We identified 475 contigs with no match in the current expressed sequence tag databases or in GenBank. The novel contigs included 31 with no match to the current assembly of the chicken genome, 119 representing spliced transcripts, and 309 that were unspliced. More detailed molecular characterization of the 428 novel contigs present in the assembly will be important to gene discovery and annotation of the chicken and other vertebrate genomes.

Regional Population Diversity of Pyricularia grisea in Arkansas and the Influence of Host Selection.

MGR586 DNA fingerprinting has been widely used to characterize population diversity of the rice blast pathogen, Pyricularia grisea. However, the frequency and distribution of particular haplotypes (individuals) within MGR-delimited lineages has not been examined in the United States. MGR586 DNA fingerprinting, mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLPs), and virulence phenotyping were used to examine genetic diversity of P. grisea in Arkansas. A total of 470 monoconidial isolates were recovered from eight rice cultivars in 18 commercial fields in nine counties in Arkansas. All isolates were examined for nuclear DNA RFLPs with the MGR586 DNA fingerprint probe, and both the MGR lineage (isolates with >80% similarity) and the haplotype frequencies were determined. Four distinct MGR586 DNA fingerprint lineages (designated A, B, C, and D) were identified among the 470 field isolates. All four lineages were found in 9 of the 18 locations. Three lineages were found in four locations, two lineages in three locations, and only a single lineage was found at two locations. In all, 10, 19, 16, and 13 haplotypes (isolates which had MGR586 DNA fingerprints which differed by 1 to 20%) were identified within lineages A, B, C, and D, respectively, among the 470 isolates examined. Within each lineage, a single haplotype (clone) predominated, representing 51 to 71% of the isolates collected for each of the four lineages. Overall, 60% of the 470 isolates belonged to one of only four haplotypes (A1, B1, C1, and D1) and these four predominant haplotypes were recovered from between 7 and 14 of the 18 locations sampled, indicating a widespread distribution of these four clones. These data indicate an exceptionally low level of genetic diversity in the regional rice blast pathogen population in Arkansas relative to several other populations of P. grisea examined from tropical environments. In addition, no mtDNA RFLPs were detected among representative haplotypes within each of the lineages, indicating a single mtDNA haplotype was present in the population. Examination of virulence indicated that two races predominated in the regional collection. All 30 isolates in lineages A and C tested had an IB-49 virulence phenotype. Out of 30 isolates in lineages B and D, 29 had an IC-17 virulence phenotype. One isolate in lineage B, isolated from a highly susceptible cultivar (L201), had an IG-1 virulence phenotype. The frequencies of the four lineages varied among the locations sampled and may have been due, in part, to the cultivar from which isolates were recovered. A single lineage was recovered from two cultivars, Mars and Millie. Although only a single field of each of these cultivars was sampled, the data indicate that certain cultivars grown in Arkansas may serve as a "bottleneck", selecting out specific lineages in the regional population. To test this hypothesis, an additional 283 isolates were recovered from replicated plots of cvs. M204 and Mars located within commercial rice fields at two locations during two seasons. All four MGR586 lineages were recovered from each location. However, there was a strong bias for lineage B on cv. M204 (79% of all isolates) and a strong bias for lineage A on cv. Mars (95% of all isolates), indicating some cultivars were effective in excluding certain lineages.

Detection, localization, and sequence analyses of mitochondrial regulatory region RNAs in several mammalian species.

The mitochondrial regulatory region (mrr) located between the tRNAPhe and tRNAPro genes of mitochondrial DNA (mtDNA) is essential for regulation of replication and transcription of the mitochondrial genome. Polyadenylated short RNAs complementary to the L-strand of the mrr in human cells and similar RNAs (polyadenylation status unknown) in rat and mouse cells have been reported. We now report detection of ca. 0.2 kb polyadenylated mrrRNAs in cultured cells of Chinese hamster, African green monkey, mouse, rat, and human. We isolated a cDNA clone to a rat polyadenylated mrrRNA of 158 bp in length excluding the polyadenyl tail, which spans the region from the light strand promoter (LSP) to the origin of heavy strand replication (OriH). This cDNA contains both an open reading frame encoding a 26 amino acid polypeptide and a 12 nucleotide sequence complementary to the 3'-terminus of rat mitochondrial 12S rRNA. A cDNA clone to a human HeLa cell polyadenylated mrrRNA also contains a 12 nucleotide region complementary to the human mitochondrial 12S rRNA. We used a mitochondrial genome-deficient HeLa cell line, rho0 HeLa, and a derived cybrid cell line, HeEB, with a reconstituted mitochondrial genome, to demonstrate that the occurrence of the mrrRNA is dependent on the presence of a mitochondrial genome, and these polyadenylated mrrRNAs are transcribed from the mitochondrial genome. Our results further substantiate the common existence of polyadenylated mrrRNAs among mammals and support previously proposed hypotheses for the multi-functional nature of polyadenylated mrrRNA.

Mapping the Sw-5 locus for tomato spotted wilt virus resistance in tomatoes using RAPD and RFLP analyses.

The Sw-5 locus confers dominant resistance to tomato spotted wilt virus (TSWV). To map the location and facilitate the identification of markers linked to Sw-5 we developed a pair of near-isogenic lines (NILs) and an F2 Lycopersicon esculentum x L. pennellii population segregating for resistance to TSWV. DNA from the NILs was analyzed using 748 random 10-mer oligonucleotides to discern linked molecular markers using a random amplified polymorphic DNA (RAPD) approach. One random primer (GAGCACGGGA) was found to produce a RAPD band of about 2200 bp that demonstrates linkage to Sw-5. Data from co-segregation of resistance and restriction fragment length polymorphisms (RFLPs) in a F2 interspecific population position Sw-5 between the markers CT71 and CT220 near the telomere of the long arm of chromosome 9.

Posterior cruciate function following total knee arthroplasty. A biomechanical study.

The strain developed in the posterior cruciate ligament (PCL) of eight fresh cadaveric knees was measured before and after total knee arthroplasty using a loading technique that simulated stair ascent and descent. Each knee was instrumented with a Hall Effect strain gauge (Micro-Strain, Burlington, VT) in the PCL, a load cell in the quadriceps tendon, an electrogoniometer, and an array of linear displacement transducers to measure femoral rollback. Testing was undertaken with each knee in its normal state with the anterior cruciate cut and with a cruciate-retaining prosthesis, a cruciate-excising prosthesis, and a cruciate-substituting prosthesis. Normal PCL strain levels were produced in only 37% of the trials following implantation of the cruciate-retaining knee arthroplasties. With a cruciate-retaining prosthesis, femoral rollback decreased by an average of 36% and was associated with a 15% loss in extensor efficiency. In the procedures performed with excision of the PCL, rollback decreased by 70% and extensor efficiency by 19%. Cruciate substitution resulted in a 12% loss in rollback and an 11% decrease in extensor efficiency. The strain developed within the PCL during knee flexion was found to be extremely sensitive to the thickness of the polymeric tibial insert. In the majority of cases, it was not possible to restore normal ligament loading with flexion while simultaneously maintaining acceptable varus/valgus stability of the knee joint. Using a range of contemporary knee arthroplasties, the authors were unable to consistently reproduce normal function of the PCL.

Multiple regulatory elements ensure accurate transcription of a human ribosomal protein gene.

Previously we have shown that expression of a cloned human ribosomal protein gene, RPS14, depends upon regulatory sites located within the gene's proximal upstream DNA plus its first intron. In order to identify cis-active sequence motifs within the RPS14 promoter-enhancer complex, we transiently expressed a set of informative deletion clones in cultured Chinese hamster ovary cells. These experiments revealed three DNA sequence motifs that surround the S14 mRNA initiation site and are necessary for accurate transcription. Electrophoretic mobility shift, DNase I footprint, and methylation interference assays resolved two nuclear proteins, NF alpha-1 and NF beta-1, which bind specifically to these regulatory motifs. NF-alpha 1 recognizes a pair of 6-bp target motifs (5'-TTCCGG-3') that flank the 5' end of RPS14 exon I; and NF-beta 1 binds to a 10-bp target sequence (5'-CCGTGGGAAC-3') within the gene's first intron. Site-directed deletion mutations within the NF-alpha 1 and -beta 1 binding sites markedly inhibit S14 mRNA transcription.

The effect of femoral component position on the kinematics of total knee arthroplasty.

In a laboratory study using seven fresh-frozen anatomic specimen knees, the effect of total knee arthroplasty on the three-dimensional kinematics of the patella, femur, and tibia were measured. Experiments were performed in the intact knee, after division of the anterior cruciate ligament (ACL), after total knee arthroplasty, and after 10 degrees internal rotation, 10 degrees external rotation, 5-mm medial shift, and 5-mm lateral shift of the femoral component on the femur. The presence of a high lateral ridge on the anterior surface of the femoral component effectively prevented patellar subluxation or dislocation, but displaced and tilted the patella medially. Internal rotation or medial displacement of the femoral component exaggerated this medial patellar displacement and shift. External rotation of the femoral component corrected it, except at flexion angles greater than 100 degrees, where the femur was shifted medially on the tibia and externally rotated 15 degrees. This combination produced a net 10-mm medial displacement of the patella relative to the tibia at 120 degrees knee flexion. Lateral placement of the femoral component compensated for the effect of the high lateral ridge and allowed more normal patellar tracking while allowing tibiofemoral motions similar to those seen after sectioning of the ACL. The kinematics of the patellofemoral and tibiofemoral joints were not reproduced with a total knee prosthesis that sacrifices the ACL. When using a prosthesis with a high lateral ridge, lateral placement of a femoral component prevented patellar dislocation and allowed patellar tracking patterns similar to those seen in the intact knee without further altering tibiofemoral motions.