Evolution of duplicate genes in a tetraploid animal, Xenopus laevis

To understand the evolution of duplicate genes, we compared rates of nucleotide substitution between 17 pairs of nonallelic duplicated genes in the tetraploid frog Xenopus laevis with rates between the orthologous loci of human and rodent. For all duplicated X. laevis genes, the number of synonymous substitutions per site (dS) was greater than the number of nonsynonymous substitutions per site (dN), indicating that these genes are subject to purifying selection. There was also a significant positive correlation (r = 0.915) between dN for the X. laevis genes and dN for the mammalian genes, suggesting that, at the amino acid level, the X. laevis genes and the mammalian genes are under similar constraints. Results of relative-rate tests showed nearly equal rates of nonsynonymous substitution in each copy of the X. laevis genes; apparently there are similar constraints on both copies. No correlation was found between dS for the X. laevis genes and dS for the mammalian genes. There was a significant positive correlation both between members of pairs of duplicated X. laevis genes (r = 0.951) and between human and rodent orthologues (r = 0.854) with respect to third- position G+C content but no such relationship between the X. laevis genes and either of their mammalian orthologues. The results indicate that both copies of a duplicate gene can be subject to purifying selection and thus support the hypothesis of selection against all genotypes containing a null allele at either of two duplicate loci.      

Immunochemical identity of peroxisomal enoyl-CoA hydratase with the peroxisome-proliferation -associated 80,000 mol wt polypeptide in rat liver

Peroxisome proliferators, which induce proliferation of hepatic peroxisomes, have been shown previously to cause a marked increase in an 80,000 mol wt polypeptide predominantly in the light mitochondrial and microsomal fractions of liver of rodents. We now present evidence to show that this hepatic peroxisome-proliferation-associated polypeptide, referred to as polypeptide PPA-80, is immunochemically identical with the multifunctional peroxisome protein displaying heat-labile enoyl-CoA hydratase activity. This conclusion is based on the following observations: (a) the purified polypeptide PPA-80 and the heat- labile enoyl-CoA hydratase from livers of rats treated with the peroxisome proliferators Wy-14,643 {[4-chloro-6(2,3-xylidino)-2-pyrimidinylthio]acetic acid} exhibit identical minimum molecular weights of approximately 80,000 on SDS polyacrylamide gel electrophoresis; (b) these two proteins are immunochemically identical on the basis of ouchterlony double diffusion, immunotitration, rocket immunoelectrophoresis, and crossed immunoelectrophoresis analysis; and (c) the immunoprecipitates formed by antibodies to polypeptide PPA-80 when dissociated on a sephadex G-200 column yield enoyl-CoA hydratase activity. Whether the polypeptide PPA-80 exhibits the activity of other enzyme(s) of the peroxisomal β-oxidation system such as fatty acyl-CoA oxidase activity or displays immunochemical identity with such enzymes remains to be determined. The availability of antibodies to polypeptide PPA-80 and enoyl-CoA hydratase facilitated immunofluorescent and immunocytochemical localization of the polypeptide PPA- 80 and enoyl-CoA hydratase in the rat liver. The indirect immunofluorescent studies with these antibodies provided direct visual evidence for the marked induction of polypeptide PPA-80 and enoyl-CoA hydratase in the livers of rats treated with Wy-14,643. The present studies also provide immunocytochemical evidence for the localization of polypeptide PPA- 80 and the heat-labile enoyl-CoA hydratase in the peroxisome, but not in the mitochondria, of hepatic parenchymal cells. These studies, therefore, provide morphological evidence for the existence of fatty acyl-CoA oxidizing system in peroxisomes. An increase of polypeptide PPA-80 on SDS polyacrylamide gel electrophoretic analysis of the subcellular fractions of liver of rodents treated with lipid-lowering drugs should serve as a reliable and sensitive indicator of enhanced peroxisomal β- oxidation system.     

A scaffoldless technique for self-generation of three-dimensional keratinospheroids on liquid crystal surfaces

We describe a new scaffold-free three-dimensional (3D) cell culture model using cholesteryl ester based lyotropic liquid crystal (LC) substrates. Keratinocytes were deposited randomly on the LC surface where they self-assembled into 3D microtissues or keratinospheroids. The cell density required to form spheroids was optimized. We investigated cell viability using dead/live cell assays. The adhesion characteristics of cells within the microtissues were determined using histological sectioning and immunofluorescence staining. Fourier transform infrared spectroscopy (FTIR) was used to characterize the biochemistry of the keratinospheroids. We found that both cells and microtissues could migrate on the LC surface. The viability study indicated approximately 80% viability of cells in the microtissues up to 20 days of culture. Strong intercellular adhesion was observed in the stratification of the multi-layered microspheroids using field emission-scanning electron microscopy (FE-SEM) and histochemical staining. The cytoskeleton and vinculins of the cells in the microtissues were expressed diffusely, but the microtissues were enriched with lipids and nucleic acids, which indicates close resemblance to the conditions in vivo. The basic 3D culture model based on LC may be used for cell and microtissue migration studies in response to cytochemical treatment.     

Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp,Ctenopharyngodon idellus

Background  

Hypoxia-inducible factors (HIFs) are involved in adaptive and survival responses to hypoxic stress in mammals. In fish, very little is known about the functions of HIFs.     

The influence of foot position on body dynamics

During locomotion, the human body exhibits inherent dynamic properties such as mass (M), stiffness (K) and damping (B). During the gait cycle, foot contact with the ground progresses from the heel to the toe. Contact forces between the foot and ground are defined as ground reaction forces (GRF). It is unclear how body dynamics are affected by foot landing position. If the shape of GRF is indicative of body dynamics, our understanding of gait patterns in normal and pathologic conditions may improve. The aims of this study were to determine:(1) whether foot landing position affects the inherent dynamics of the human body and (2) the extent to which the GRF curve reflects the response of inherent body dynamics to sudden loading.Eight non-disabled control volunteers performed a series of small jumps and landed on one leg with a fully extended knee in three foot landing positions: heel, mid-foot, and toe. They then walked at self-paced velocity over force plates. For each foot landing position, values of K, B and the dimensionless damping coefficient, ξ, were calculated from the period of vertical body oscillations, T, and compared with an ANOVA test. In addition, the time between the two peaks of the vertical GRF, T_GRF, was compared with T. We found that that K and B decreased and ξ did not change (p<0.01) between heel to toe-landing positions. T_GRF was not different than T for the toe-landing position, which suggests that the dynamic body response has major impact on the shape of GRF.     

The kinematic consequences of invariant dynamics in children 6-18 years of age

The effect of limb dynamics on trajectory formation is unclear. The natural frequency of a limb is the major factor in its dynamics. It has previously been shown with an indirect measurement method that the natural frequency of body segments is invariant during human growth from the age of 6 to 18. The aim of our study was to determine, using a direct measurement method, whether human growth affects: (1) lower limb dynamics (i.e. the natural frequency of the lower leg) and (2) the maximum velocities of the knee during selected motor tasks. In 20 non-disabled children, 6-18 years of age, measurements were taken of the natural frequency of the lower leg (including the foot), and the maximum velocities of knee flexion and extension during voluntary movement (MVV) and at initial and terminal swing phases of self-paced walking (WAL). The velocities were also estimated using a dynamic model and the results were compared to the measured velocities with a paired t-test. Correlations among the frequencies, velocities, and body height (an indicator of growth) were calculated. The natural frequency of the lower leg (mean+/-standard deviation, omega(0)=6.58+/-0.54s(-1)), maximum velocities of knee extension and flexion during voluntary movement (MVV(e)=10.1+/-1.8rads(-1) and MVV(f)=7.8+/-1.3rads(-1), respectively), and maximum velocities of knee flexion and extension during the swing phase of walking (WAL(f)=5.4+/-0.6rads(-1) and WAL(e)=6.3+/-0.87rads(-1), respectively) were each found to be independent of body height. The MVV measured velocities were 22% larger and WAL(f) measured velocities were 25% smaller than the velocities predicted from the dynamic model (p<0.05). The study found that a segment's dynamic properties, as well as selected kinematics, may be considered invariant with human growth.     

Gastrocnemius fascicle length changes with two-joint passive movements

Predicting muscle fascicle length changes during passive movements may lead to a better understanding of muscle function. The purpose of this study was to experimentally compare fascicle length changes in the gastrocnemius during two-joint passive movements with a previously derived kinematic model based on anatomical measures from a cadaver. The ratio of passive ankle to knee motion was manipulated to generate medial gastrocnemius fascicle elongation and lateral gastrocnemius fascicle shortening. Ultrasound images from both heads of the gastrocnemius fascicles were acquired at 10 degrees knee flexion increments and compared with this kinematic model. Our results suggest that the two-joint kinematic model from which we originally based our knee and ankle movements did not adequately reflect fascicle length changes during any of the movement conditions in this study. From our data, we propose that for every degree of ankle motion the medial and lateral gastrocnemius changes 0.42 mm and 0.96 mm, respectively, whereas changes of 0.14 mm and 0.22 mm are observed for the medial and lateral gastrocnemius, respectively, during knee movements.     

Non-invasive measurements of exhaled NO and CO associated with methacholine responses in mice

Background

Nitric oxide (NO) and carbon monoxide (CO) in exhaled breath are considered obtainable biomarkers of physiologic mechanisms. Therefore, obtaining their measures simply, non-invasively, and repeatedly, is of interest, and was the purpose of the current study.

Methods

Expired NO (E_NO) and CO (E_CO) were measured non-invasively using a gas micro-analyzer on several strains of mice (C57Bl6, IL-10^-/-, A/J, MKK3^-/-, JNK1^-/-, NOS-2^-/- and NOS-3^-/-) with and without allergic airway inflammation (AI) induced by ovalbumin systemic sensitization and aerosol challenge, compared using independent-sample t-tests between groups, and repeated measures analysis of variance (ANOVA) within groups over time of inflammation induction. E_NO and E_CO were also measured in C57Bl6 and IL-10-/- mice, ages 8–58 weeks old, the relationship of which was determined by regression analysis. S-methionyl-L-thiocitrulline (SMTC), and tin protoporphyrin (SnPP) were used to inhibit neuronal/constitutive NOS-1 and heme-oxygenase, respectively, and alter NO and CO production, respectively, as assessed by paired t-tests. Methacholine-associated airway responses (AR) were measured by the enhanced pause method, with comparisons by repeated measures ANOVA and post-hoc testing.

Results

E_NO was significantly elevated in naïve IL-10^-/- (9–14 ppb) and NOS-2^-/- (16 ppb) mice as compared to others (average: 5–8 ppb), whereas E_CO was significantly higher in naïve A/J, NOS-3^-/- (3–4 ppm), and MKK3^-/- (4–5 ppm) mice, as compared to others (average: 2.5 ppm). As compared to C57Bl6 mice, AR of IL-10^-/-, JNK1^-/-, NOS-2^-/-, and NOS-3^-/- mice were decreased, whereas they were greater for A/J and MKK3^-/- mice. SMTC significantly decreased E_NO by ~30%, but did not change AR in NOS-2^-/- mice. SnPP reduced E_CO in C57Bl6 and IL-10^-/- mice, and increased AR in NOS-2^-/- mice. E_NO decreased as a function of age in IL-10^-/- mice, remaining unchanged in C57Bl6 mice.

Conclusion

These results are consistent with the ideas that: 1) E_NO is associated with mouse strain and knockout differences in NO production and AR, 2) alterations of E_NO and E_CO can be measured non-invasively with induction of allergic AI or inhibition of key gas-producing enzymes, and 3) alterations in AR may be dependent on the relative balance of NO and CO in the airway.     

Biomarkers in osteoarthritis

Biomarkers aid the study of osteoarthritis (OA) in a number of different ways. In this article we summarise briefly their multiple uses and reflect on how the study reported in a previous edition of Arthritis Research & Therapy should promote further investigation of cartilage oligomeric matrix protein (COMP). COMP is foremost among hitherto investigated biomarkers and is most consistently shown to predict knee OA progression. Precisely what role it plays in OA pathogenesis remains unclear and elucidating this may be key to defining, and then targeting, the cellular pathways involved in OA.