Biochemical differences between distal limb extensor and flexor tendons among equine breeds selected for racing and sport.

Throughout the ages, humans have selected different horse breeds for their locomotor capacities. Consequently, the properties of equine locomotor tissues could have diversified because of the specific requirements of different disciplines. Therefore, this study aimed to compare biochemical properties of tendons in different equine breeds traditionally selected for racing or sports performance. We hypothesised that tendons in racing breeds would have biochemical properties that would increase strength, whereas those in sporting breeds would have more elastic properties. An ex vivo tendon tissue study comparing the common digital extensor tendon (CDET) and superficial digital flexor tendon (SDFT) of sports horses (Friesian horse, Warmblood horse) and racehorses (Thoroughbred horse; the oldest, reference standard breed) was performed. The SDFT and CDET from middle-aged Friesian (n = 12), Warmblood (n = 12) and Thoroughbred horses (n = 8) were harvested, and their biochemical properties were compared. The biochemical analysis demonstrated significantly higher water percentage, lower collagen concentrations/glycosaminoglycan content and higher crosslink concentrations in the SDFT of sports horses compared to racing breed horses (P < 0.05); DNA content was also significantly lower in sports horses than racehorses (P < 0.05). Racehorses had mainly extra fibrillar collagen support, whereas sports horses had mainly extra crosslink collagen support. From a functional perspective, the racing Thoroughbred relied on stronger tendons, while the sporting Friesians and Warmbloods relied on less stiff, more elastic tendons. In conclusion, there were significant biochemical differences in tendon properties between breeds, possibly related to their intended locomotor performance, although this requires further biomechanical and ultimately genetic confirmation.

Effect of strain and nutritional density of the diet on the water-protein ratio, fat and collagen levels in the breast and legs of broilers slaughtered at different ages.

Many requirements are necessary to meet the European Union rules to export poultry, including the amount of physiological water and water-protein ratio (WPR) in carcasses. Therefore, the aim of this study was to identify if strain, nutrition, and age affect the amount of collagen and fat and the WPR in cuts and verify whether the latter meets the international export standards. A total of 3,240 male chicks were housed in a completely randomized design in a 3 × 3 × 5 factorial arrangement, which included 3 nutritional densities (regular, medium, and high), 3 strains (021 Embrapa and 2 commercial strains identified as A and B), and 5 ages. Twelve broilers from each treatment (totaling 540 birds) were slaughtered at 28, 35, 42, 49, and 56 D of age to determine collagen and fat levels and WPR (through the calculation of moisture and protein percentage) in broiler breasts and legs using the near-infrared spectroscopy method. The use of feeds with different nutritional densities presented in this study has no effect on the WPR in the breast and legs of broilers slaughtered between 28 and 56 D of age. However, nutritional density influences liveweight and percentage of fat in the breast and legs. Collagen percentage in the legs decreases with increasing nutritional density. The 021 Embrapa strain cuts present a lower WPR than those of other commercial strains. However, the values found for all strains studied are within the limits of the Europe Union and Brazilian legislations. The liveweight, breast weight, leg weight, and leg fat increases linearly with age. Quite the opposite, water protein ratio, breast fat level, and breast collagen level decrease linearly with age. Leg WPR and leg collagen level are not affected by age. Despite the differences found for strains, nutritional densities and age readers should be aware that these factors may interact with each other depending on the response variable studied.

Effects of feeding flaxseed on performance, carcass trait, and meat fatty acid composition of Guinea pigs (Cavia procellus) under northern Peruvian condition.

A study was conducted to determine the effects of flaxseed supplementation on performance, carcass traits, and hindleg fatty acid composition of guinea pigs. Sixty male and female weaned guinea pigs (1 month old, five animals/cage) were blocked by sex and bodyweight and randomly fed 0 (control) or 100 g/kg flaxseed concentrate diets (15 g/animal) plus ad libitum fresh alfalfa for 30 days. Results showed that flaxseed supplementation had no influence on animal performance. However, final body weight (P = 0.035), total feed intake (P = 0.019), and body weight gain (P < 0.001) were higher in male than female guinea pigs. Similar results were also observed for carcass composition (i.e., hot, chilled, and reference carcass weights). Inclusion of flaxseed reduced saturated (P < 0.001), mono-unsaturated (P = 0.004), and increased (P < 0.001) polyunsaturated (PUFA) fatty acid concentrations in hindlegs. Concentrations of linolenic acid and n-3 PUFA increased (P < 0.001) by 49.7 and 37.1%, respectively as a result of flaxseed inclusion. It was concluded that feeding flaxseed to guinea pigs at 100 g/kg of the concentrate diets improves meat PUFA concentrations with no adverse effects on performance or carcass composition.

Redox regulation of ischemic limb neovascularization - What we have learned from animal studies.

Mouse hindlimb ischemia has been widely used as a model to study peripheral artery disease. Genetic modulation of the enzymatic source of oxidants or components of the antioxidant system reveal that physiological levels of oxidants are essential to promote the process of arteriogenesis and angiogenesis after femoral artery occlusion, although mice with diabetes or atherosclerosis may have higher deleterious levels of oxidants. Therefore, fine control of oxidants is required to stimulate vascularization in the limb muscle. Oxidants transduce cellular signaling through oxidative modifications of redox sensitive cysteine thiols. Of particular importance, the reversible modification with abundant glutathione, called S-glutathionylation (or GSH adducts), is relatively stable and alters protein function including signaling, transcription, and cytoskeletal arrangement. Glutaredoxin-1 (Glrx) is an enzyme which catalyzes reversal of GSH adducts, and does not scavenge oxidants itself. Glrx may control redox signaling under fluctuation of oxidants levels. In ischemic muscle increased GSH adducts through Glrx deletion improves in vivo limb revascularization, indicating endogenous Glrx has anti-angiogenic roles. In accordance, Glrx overexpression attenuates VEGF signaling in vitro and ischemic vascularization in vivo. There are several Glrx targets including HIF-1α which may contribute to inhibition of vascularization by reducing GSH adducts. These animal studies provide a caution that excess antioxidants may be counter-productive for treatment of ischemic limbs, and highlights Glrx as a potential therapeutic target to improve ischemic limb vascularization.

In vivo MRI tracking of iron oxide nanoparticle-labeled human mesenchymal stem cells in limb ischemia.

BACKGROUND: Stem cell transplantation has been investigated for repairing damaged tissues in various injury models. Monitoring the safety and fate of transplanted cells using noninvasive methods is important to advance this technique into clinical applications. METHODS: In this study, lower-limb ischemia models were generated in nude mice by femoral artery ligation. As negative-contrast agents, positively charged magnetic iron oxide nanoparticles (aminopropyltriethoxysilane-coated Fe2O3) were investigated in terms of in vitro labeling efficiency, effects on human mesenchymal stromal cell (hMSC) proliferation, and in vivo magnetic resonance imaging (MRI) visualization. Ultimately, the mice were sacrificed for histological analysis three weeks after transplantation. RESULTS: With efficient labeling, aminopropyltriethoxysilane-modified magnetic iron oxide nanoparticles (APTS-MNPs) did not significantly affect hMSC proliferation. In vivo, APTS-MNP-labeled hMSCs could be monitored by clinical 3 Tesla MRI for at least three weeks. Histological examination detected numerous migrated Prussian blue-positive cells, which was consistent with the magnetic resonance images. Some migrated Prussian blue-positive cells were positive for mature endothelial cell markers of von Willebrand factor and anti-human proliferating cell nuclear antigen. In the test groups, Prussian blue-positive nanoparticles, which could not be found in other organs, were detected in the spleen. CONCLUSION: APTS-MNPs could efficiently label hMSCs, and clinical 3 Tesla MRI could monitor the labeled stem cells in vivo, which may provide a new approach for the in vivo monitoring of implanted cells.

Simultaneous in vivo pH and temperature mapping using a PARACEST-MRI contrast agent.

Altered tissue temperature and/or pH is a common feature in pathological conditions, where metabolic demand exceeds oxygen supply such as in tumors and following stroke. Therefore, in vivo tissue temperature and pH may become valuable biomarkers for disease detection and the monitoring of disease progression or treatment response in conditions with altered metabolic demand. In this study, pH is measured using the amide protons of a thulium (Tm(3+)) complex with a DOTAM-Glycine-Lysine (ligand: Tm(3+)-DOTAM-Gly-Lys). The pH was uniquely determined from the linewidth of the asymmetry curve of the chemical exchange saturation transfer spectrum, independent of contrast agent concentration, or temperature for a given saturation pulse. pH maps with an inter-pixel standard deviation of less than 0.1 pH units were obtained in 10 mM Tm(3+)-DOTAM-Gly-Lys solutions with pH ranging from 6.0 to 8.0 pH units at 37°C. Temperature maps were simultaneously obtained using the chemical shift of the chemical exchange saturation transfer peak. Temperature and pH maps are demonstrated in the mouse leg (N = 3), where the mean and standard deviation for pH was 7.2 ± 0.2 pH unit and temperature was 37.4 ± 0.5°C.

The content of myosin heavy chains in hindlimb muscles of female and male rats.

The aim of the study was to test whether the considerable differences in the hindlimb muscles mass, the number and diameter of muscles fibers were connected with differences in the myosin heavy chain isoform content (expressed as the percentage of the given isoform in respect to total myosin heavy chains). Therefore, the content of myosin heavy chain (MHC) isoforms was studied in four hindlimb muscles: flexor digitorum brevis, soleus, tibialis anterior and gastrocnemius medialis of female and male rats by means of polyacrylamide gel electrophoresis supplemented with densitometric analyses. Muscles were isolated and homogenized prior to electrophoretic analysis. The most interesting result concerned considerably different composition of myosin isoforms for male and female subjects in the slow soleus muscles, which contained predominantly slow MHC isoform (MHC I). However, in the male muscle about 13% of IIa isoform (MHC IIa) was also detected; this isoform was not found in the majority of the studied female muscles (81% of muscle samples). This dimorphic difference was further confirmed by immunofluorescence stainining for slow and fast skeletal myosin isoforms and by assessment of the fiber ATPase activity. For the three remaining fast muscles (flexor digitorum brevis, tibialis anterior and gastrocnemius medialis) all four MHC isoforms were detected with the fast isoforms being dominant ones. However, there were not statistically significant differences observed between males and females, with the exception of IIx isoform, which was more frequent in male tibialis anterior muscle.

MR spectroscopy measurement of the diffusion of dimethyl sulfoxide in articular cartilage and comparison to theoretical predictions.

UNLABELLED: Cartilage cryopreservation requires optimal loading of protective solutes, most commonly dimethyl sulfoxide (DMSO), to maximize chondrocyte survival. Previously, diffusion models have been used to predict the distribution of solutes in tissue samples, but the accuracy of spatiotemporal predictions of these models have not been validated with empirical studies and remains unknown. OBJECTIVE: In this study, magnetic resonance spectroscopic imaging was used to measure the spatial and temporal changes in DMSO and water concentrations in porcine articular cartilage plugs, throughout 1 h of solute loading. DESIGN: A custom NMR spectroscopic imaging pulse sequence provided water and DMSO concentration images with an in-plane spatial resolution of 135 µm and a temporal resolution of 150 s, repeated for 60 min throughout DMSO loading. Delayed gadolinium-enhanced magnetic resonance of cartilage (d-GEMRIC) imaging provided fixed charge density and spin-density imaging provided water density images prior to DMSO loading. RESULTS: The measured spatial and temporal distribution of DMSO in three different samples was compared to independent predictions of Fick's law and the modified triphasic biomechanical model by Abazari et al. (2011) with the empirical data more closely agreeing with the triphasic model. CONCLUSION: Dynamic NMR spectroscopic imaging can measure spatial and temporal changes in water and cryoprotectant concentrations in articular cartilage. The modified triphasic model predictions for the interstitial distribution of DMSO were confirmed and its advantage over the predictions by Fick's law model, which is commonly used in the literature of cryobiology, was demonstrated.

Computed tomography detects changes in contrast agent diffusion after collagen cross-linking typical to natural aging of articular cartilage.

OBJECTIVE: The effect of threose-induced collagen cross-linking on the mechanical and diffusive properties of cartilage was investigated in vitro. In particular, we investigated the potential of Contrast Enhanced Computed Tomography (CECT) to detect changes in articular cartilage after increased collagen cross-linking, which is an age-related phenomenon. METHODS: Osteochondral plugs (Ø=6.0 mm, n=28) were prepared from intact bovine patellae (n=7). Two of the four adjacent samples, prepared from each patella, were treated with threose to increase the collagen cross-linking, while the other two specimen served as paired controls. One sample pair was mechanically tested and then mechanically injured using a material testing device. Contrast agent [ioxaglate (Hexabrix™)] diffusion was imaged in the other specimen pair for 25 h using CECT. Water fraction, collagen and proteoglycan content, collagen network architecture and the amount of cross-links [hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP) and pentosidine (Pent)] of the samples were also determined. RESULTS: Cartilage collagen cross-linking, both Pent and LP, were significantly (P<0.001) increased due to threose treatment. CECT could detect the increased cross-links as the contrast agent penetration and the diffusion flux were significantly (P<0.05) lower in the threose treated than in untreated samples. The equilibrium modulus (+164%, P<0.05) and strain dependent dynamic modulus (+47%, P<0.05) were both significantly greater in the threose treated samples than in reference samples, but there was no association between the initial dynamic modulus and the threose treatment. The water fraction, proteoglycan and collagen contents, as well as collagen architecture, were not significantly altered by the threose treatment. CONCLUSIONS: To conclude, the CECT technique was found to be sensitive at detecting changes in cartilage tissue due to increased collagen cross-linking. This is important since increased cross-linking has been proposed to be related to the increased injury susceptibility of tissue.

MicroRNAs of rat articular cartilage at different developmental stages identified by Solexa sequencing.

BACKGROUND: Expression profiles of microRNAs (miRNAs) can shape the repertoire of proteins expressed in development, differentiation and diseases. This study aimed to identify miRNA profile of articular cartilage at different developmental stages in rats. METHODS: Three small RNA libraries were constructed from the femoral head cartilage of Sprague-Dawley (SD) rats at postnatal day 0, day 21 and day 42 and sequenced by a deep sequencing approach. Then a bioinformatics approach was employed to distinguish genuine miRNAs from small RNAs represented in the mass sequencing data. The expression of indicated miRNAs was determined by stem-loop RT-qPCR to valuate the consistency with Solexa sequencing. RESULTS: Two hundred and fifty-eight of 310 known miRNA and miRNA* genes were organized into 91 compact clusters. Two hundred and forty-six miRNAs were detected in all three small RNA libraries of rat articular cartilage. Forty-six, fifty-two and fifty-six miRNA* genes were identified from three small RNA libraries, respectively, and 86 novel miRNA candidate genes were found simultaneously. In addition, 23 known miRNAs were up-regulated (fold change ≥ 4); six were down-regulated (fold change ≤ -4) during articular cartilage development. The predicted targets of differentially expressed miRNAs were locally secreted factors and transcription factors that regulate proliferation and differentiation of chondrocytes. The same expression tendency of indicated miRNAs during articular cartilage development stages was observed by using Solexa sequencing and stem-loop RT-qPCR. CONCLUSION: Our study provided a unique opportunity to decipher how the elaboration of the miRNA repertoire contributes to the development process of articular cartilage.

Brief communication: oxygen isotopes as a biomarker for sickle-cell disease? Results from transgenic mice expressing human hemoglobin S genes.

The origins of sickle-cell disease (SCD) are well understood, as are its evolutionary pressures on humans and pathological presentation. However, because it has not been possible to identify SCD in archaeological contexts, its biocultural effects on past populations are unknown. Previous research investigating oxygen isotope fractionation during respiration among anemics suggests that oxygen isotopes in bone apatite may provide a biological marker for SCD in skeletal remains. This pilot study reports δ(18) O ratios in bone apatite of transgenic laboratory mice expressing human SCD globins and compares them to healthy control mice. The δ(18) O ratios of sick mice are significantly lower than those of healthy mice (-5.6‰ vs. -4.5‰; P = 0.002), and the sickest mice exhibit the lowest ratios of all (mean δ(18) O = -5.8‰). These preliminary results suggest that this method may be usefully applied to skeletal materials of past human populations whose diets and water sources do not differ substantially.

Novel angiotensin-converting enzyme (ACE) inhibitory peptides derived from boneless chicken leg meat.

Four peptides that inhibit angiotensin-converting enzyme (ACE) were separated from the hydorlysate of boneless chicken leg meat digested with artificial gastric juice (pepsin). Two peptides were identified as the peptides encrypted in myosin heavy chain. The peptide P1 (MNVKHWPWMK) corresponds to the amino acid sequence from amino acids 825 to 834 of myosin heavy chain, and the peptide P4 (VTVNPYKWLP) corresponds to the amino acid sequence from amino acids 125 to 135 of myosin heavy chain. They are novel ACE inhibitory peptides derived from chicken, and IC(50) values of P1 and P4 were determined as 228 and 5.5 microM, respectively. Although these values were much larger than 0.022 microM for captopril, a typical synthetic ACE inhibitor, they are comparable to IC(50) values reported for various ACE inhibitory peptides derived from foods. Because the peptide P4 has a relatively low IC(50) value, it is a good starting substance for designing food supplements for hypertensive patients.

Thyroid-stimulating hormone restores bone volume, microarchitecture, and strength in aged ovariectomized rats.

UNLABELLED: We show the systemic administration of low levels of TSH increases bone volume and improves bone microarchitecture and strength in aged OVX rats. TSH's actions are mediated by its inhibitory effects on RANKL-induced osteoclast formation and bone resorption coupled with stimulatory effects on osteoblast differentiation and bone formation, suggesting TSH directly affects bone remodeling in vivo. INTRODUCTION: Thyroid-stimulating hormone (TSH) receptor haploinsufficient mice with normal circulating thyroid hormone levels have reduced bone mass, suggesting that TSH directly affects bone remodeling. We examined whether systemic TSH administration restored bone volume in aged ovariectomized (OVX) rats and influenced osteoclast formation and osteoblast differentiation in vitro. MATERIALS AND METHODS: Sprague-Dawley rats were OVX at 6 months, and TSH therapy was started immediately after surgery (prevention mode; n = 80) or 7 mo later (restoration mode; n = 152). Hind limbs and lumbar spine BMD was measured at 2- or 4-wk intervals in vivo and ex vivo on termination at 8-16 wk. Long bones were subjected to microCT, histomorphometric, and biomechanical analyses. The direct effect of TSH was examined in osteoclast and osteoblast progenitor cultures and established rat osteosarcoma-derived osteoblastic cells. Data were analyzed by ANOVA Dunnett test. RESULTS: In the prevention mode, low doses (0.1 and 0.3 microg) of native rat TSH prevented the progressive bone loss, and importantly, did not increase serum triiodothyroxine (T3) and thyroxine (T4) levels in aged OVX rats. In restoration mode, animals receiving 0.1 and 0.3 microg TSH had increased BMD (10-11%), trabecular bone volume (100-130%), trabecular number (25-40%), trabecular thickness (45-60%), cortical thickness (5-16%), mineral apposition and bone formation rate (200-300%), and enhanced mechanical strength of the femur (51-60%) compared with control OVX rats. In vitro studies suggest that TSH's action is mediated by its inhibitory effects on RANKL-induced osteoclast formation, as shown in hematopoietic stem cells cultivated from TSH-treated OVX rats. TSH also stimulates osteoblast differentiation, as shown by effects on alkaline phosphatase activity, osteocalcin expression, and mineralization rate. CONCLUSIONS: These results show for the first time that systemically administered TSH prevents bone loss and restores bone mass in aged OVX rats through both antiresorptive and anabolic effects on bone remodeling.

Thiazolidinediones enhance skeletal muscle triacylglycerol synthesis while protecting against fatty acid-induced inflammation and insulin resistance.

In the present investigation, we studied the effects of thiazolidinedione (TZD) treatment on insulin-stimulated fatty acid (FA) and glucose kinetics in perfused muscle from high-fat (HF)-fed rats. We tested the hypothesis that TZDs prevent FA-induced insulin resistance by attenuating proinflammatory signaling independently of myocellular lipid levels. Male Wistar rats were assigned to one of three 3-wk dietary groups: control chow fed (CON), 65% HF diet (HFD), or TZD- (troglitazone or rosiglitazone) enriched HF diet (TZD + HFD). TZD treatment led to a significant increase in plasma membrane content of CD36 protein in muscle (red: P = 0.01, and white: P = 0.001) that correlated with increased FA uptake (45%, P = 0.002) and triacylglycerol (TG) synthesis (46%, P = 0.03) during the perfusion. Importantly, whereas HF feeding caused increased basal TG (P = 0.047), diacylglycerol (P = 0.002), and ceramide (P = 0.01) levels, TZD treatment only prevented the increase in muscle ceramide. In contrast, all of the muscle inflammatory markers altered by HF feeding ( upward arrowNIK protein content, P = 0.009; upward arrowIKKbeta activity, P = 0.006; downward arrowIkappaB-alpha protein, P = 0.03; and upward arrowJNK phosphorylation, P = 0.003) were completely normalized by TZD treatment. Consistent with this, HFD-induced decrements in insulin action were also prevented by TZD treatment. Thus our findings support the notion that TZD treatment causes increased FA uptake and TG accumulation in skeletal muscle under insulin-stimulated conditions. Despite this, TZDs suppress the inflammatory response to dietary lipid overload, and it is this mechanism that correlates strongly with insulin sensitivity.

Interdigital webbing retention in bat wings illustrates genetic changes underlying amniote limb diversification.

Developmentally regulated programmed cell death sculpts the limbs and other embryonic organs in vertebrates. One intriguing example of species-specific differences in apoptotic extent is observed in the tissue between the digits. In chicks and mice, bone morphogenetic proteins (Bmps) trigger apoptosis of the interdigital mesenchyme, leading to freed digits, whereas in ducks, Bmp antagonists inhibit the apoptotic program, resulting in webbed feet. Here, we show that the phyllostomid bat Carollia perspicillata utilizes a distinct mechanism for maintaining interdigit tissue. We find that bat forelimb and hindlimb interdigital tissues express Bmp signaling components but that only bat hindlimbs undergo interdigital apoptosis. Strikingly, the retention of interdigital webbing in the bat forelimb is correlated with a unique pattern of Fgf8 expression in addition to the Bmp inhibitor Gremlin. By using a functional assay, we show that maintenance of interdigit tissue in the bat wing depends on the combined effects of high levels of Fgf signaling and inhibition of Bmp signaling. Our data also indicate that although there is not a conserved mechanism for maintaining interdigit tissue across amniotes, the expression in the bat forelimb interdigits of Gremlin and Fgf8 suggests that these key molecular changes contributed to the evolution of the bat wing.

Inhibition of angiogenesis by interleukin-4 gene therapy in rat adjuvant-induced arthritis.

OBJECTIVE: Interleukin-4 (IL-4) can modulate neovascularization. In this study, we used a gene therapy approach to investigate the role of IL-4 in angiogenesis in rat adjuvant-induced arthritis (AIA), a model for rheumatoid arthritis. METHODS: Rats received an adenovirus producing IL-4 (AxCAIL-4), a control virus without insert, or control vehicle (phosphate buffered saline) intraarticularly before arthritis onset. At peak onset of arthritis, rats were killed. Vascularization was determined in the synovial tissue, and correlations with inflammation were assessed. Ankle homogenates were used in angiogenesis assays in vitro and in vivo, and protein levels of cytokines and growth factors were assessed by enzyme-linked immunosorbent assay. Synovial tissue expression of alphav integrins was determined by immunohistochemistry. RESULTS: IL-4 induced a reduction in synovial tissue vessel density, which was paralleled by a decrease in inflammation. AxCAIL-4 joint homogenates significantly (P < 0.05) inhibited both endothelial cell (EC) migration and tube formation in vitro. Similarly, AxCAIL-4 inhibited capillary sprouting in the rat aortic ring assay, and vessel growth in the in vivo Matrigel plug assay. The angiostatic effect occurred despite high levels of vascular endothelial growth factor (VEGF), and was associated with down-regulation of the proangiogenic cytokines IL-18, CXCL16, and CXCL5 and up-regulation of the angiogenesis inhibitor endostatin. Of interest, AxCAIL-4 also resulted in decreased EC expression of the alphav and beta3 integrin chains. CONCLUSION: In rat AIA, IL-4 reduces synovial tissue vascularization via angiostatic effects, mediates inhibition of angiogenesis via an association with altered pro- and antiangiogenic cytokines, and may inhibit VEGF-mediated angiogenesis and exert its angiostatic role in part via alphavbeta3 integrin. This knowledge of the specific angiostatic effects of IL-4 may help optimize target-oriented treatment of inflammatory arthritis.

Maintenance of slow type I myosin protein and mRNA expression in overwintering prairie dogs (Cynomys leucurus and ludovicianus) and black bears (Ursus americanus).

Hibernating mammals have the remarkable ability to withstand long periods of fasting and reduced activity with dramatic maintenance of skeletal muscle function and protein composition. We investigated several hindlimb muscles of white-tailed prairie dogs (Cynomys leucurus) and black bears (Ursus americanus), two very different hibernators who are dormant and fasting during winter. The black-tailed prairie dog (C. ludovicianus) remains active during winter, but suffers minor skeletal muscle atrophy; nevertheless, they also demonstrate apparent skeletal muscle adaptations. Using SDS-PAGE, we measured myosin protein isoform profiles before and after the hibernation season. All species maintained or increased levels of slow myosin, despite the collective physiological challenges of hypophagia and reduced activity. This contrasts markedly with standard mammalian models of skeletal muscle inactivity and atrophy predicting significant loss of slow myosin. A mechanism for changes in myosin isoforms was investigated using reverse-transcription PCR, following partial sequencing of the adult MHC isoforms in C. leucurus and U. americanus. However, mRNA expression was not well correlated with changes in MHC protein isoforms, and other synthesis and degradation pathways may be involved besides transcriptional control. The muscles of hibernating mammals demonstrate surprising and varied physiological responses to inactivity and atrophy with respect to slow MHC expression.

Expression of Xenopus XlSALL4 during limb development and regeneration.

The multi-C2H2 zinc-finger domain containing transcriptional regulators of the spalt (SAL) family plays important developmental regulatory roles. In a competitive subtractive hybridization screen of genes expressed in Xenopus laevis hindlimb regeneration blastemas, we identified a SAL family member that, by phylogenetic analysis, falls in the same clade as human SALL4 and have designated it as XlSALL4. Mutations of human SALL4 have been linked to Okihiro syndrome, which includes preaxial (anterior) limb defects. The expression pattern of XlSALL4 transcripts during normal forelimb and hindlimb development and during hindlimb regeneration at the regeneration-competent and regeneration-incompetent stages is temporally and regionally dynamic. We show for the first time that a SAL family member (XlSALL4) is expressed at the right place and time to play a role regulating both digit identity along the anterior/posterior axis and epimorphic limb regeneration.

The molecular composition of the sarcomeric M-band correlates with muscle fiber type.

The M-band is the transverse structure that cross-links the thick filaments in the center and provides a perfect alignment of the A-band in the activated sarcomere. The molecular composition of the M-bands in adult mouse skeletal muscle is fiber-type dependent. All M-bands in fast fibers contain M-protein while M-bands in slow fibers contain a significant proportion of the EH-myomesin isoform, previously detected only in embryonic heart muscle. This fiber-type specificity develops during the first postnatal weeks. However, the ratio between the amounts of myosin and of myomesin, taken as sum of both isoforms, remains nearly constant in all studied muscles. Ultrastructural analysis demonstrates that some of the soleus fibers show a diffuse appearance of the M-band, resembling the situation in the embryonic heart. A model is proposed to explain the functional consequence of differential M-band composition for the physiological and morphological properties of sarcomeres in different muscle types.

Age-related changes in bone mineral density, cross-sectional area and the strength of long bones in the hind limbs and first lumbar vertebra in female Wistar rats.

Age-related changes in bone mineral density (BMD) and cross-sectional area and bone strength index (SSI) of the femur, tibia, humerus, and first lumbar vertebra in female Wistar (WM/MsNrs) rats were examined by a quantitative computed tomography (pQCT) method. One hundred and sixteen virgin female Wistar (WM/MsNrs) rats aged 2-33 months were used. The data indicate that the total BMD values of metaphyses and diaphyses of long bones increased until 12 months, then decreased to a varying degree depending on the bone after 15-24 months, but the values of cortical and trabecular BMD with age were not always similar to the total BMD value. Nevertheless, the values for cross-sectional area and SSI in the long bones increased regardless of the total BMD decrease with age, indicating that this increase might have been due to a characteristic of the modeling pattern in rats. The total and cortical BMD values in the first lumbar vertebra decreased after 18 months, and SSI did after 15 months. The data obtained in this study were compared with those obtained from males in a previous study. In conclusion, it was indicated that in this strain the rats over 12 months with the highest total BMD values in the femur and tibia, and before the onset of various tumors, are useful as a model animal for osteoporosis experiments and observation of senile bone change.