Hind Leg Muscle Research Articles

Contribution of Hindpaw Cutaneous Inputs to the Control of Lateral Stability During Walking in the Cat

To delineate the role of cutaneous feedback from the paws in the regulation of balance during walking, we compared the corrective responses of cats to lateral support surface translation before and after cutaneous denervation of the hindpaws. In addition, we compared characteristics of undisturbed walking before and after denervation. Electromyographic and kinematic data were collected from three cats trained to walk across a walkway, the central portion of which could be unexpectedly translated laterally in either direction. Following denervation, all of the cats changed their step width, lowered their pelvis, and spent more time with the hindlegs in double-support when walking across the walkway. When displaced by lateral support surface translations, the denervated cats made larger lateral steps and required more than a single step to regain balance. However, none of the cats fell following the denervation. The appearance and latency of the responses evoked in the hindleg muscles by the perturbations were unaffected by the denervation. However, the amplitude of these responses was affected by the loss of cutaneous inputs. Responses evoked at paw contact were significantly reduced in most muscles in the absence of cutaneous input, whereas responses evoked at end of stance revealed significant increases in gluteus medius activity with little influence on the activity of other muscles. Therefore the loss of cutaneous inputs leads to instability during gait. Although cutaneous feedback from the hindpaws is not essential for triggering corrective responses to support surface disturbances, it appears that cutaneous inputs are important for scaling the responses initiated by other cues.

Distribution of blood–muscle for clenbuterol in rat using microdialysis

Clenbuterol is clinically used as a bronchodilator, but it is also illegally used to increase lean meat in animal husbandry. To investigate the muscle distribution of protein-unbound clenbuterol, a microdialysis technique coupled to liquid chromatography system was applied to simultaneously monitor clenbuterol in rat blood and muscle. Two microdialysis probes were implanted into the jugular vein/right atrium and hind leg muscle of rat for sampling after clenbuterol administration (10 mg/kg) through the femoral vein. Dialysate samples of clenbuterol were separated by a reversed-phase column (250 mm × 4 mm I.D., particle size 5 μm). The results indicate that the maximum concentration of clenbuterol in muscle was found at 30–45 min after clenbuterol administration (10 mg/kg) and the area under concentration curve (AUC) of clenbuterol in blood and in muscle were 942.75 ± 101.92 and 174.81 ± 13.03 min μg/mL, respectively. The AUC muscle/AUC blood was 0.20 ± 0.03 representing about 20% of the clenbuterol distributing into the muscle. The elimination half-life of clenbuterol in the blood and muscle were about 2 and 6 h, respectively. These results suggest that the protein-unbound concentration of clenbuterol sustained a high level and prolonged elimination in the muscle. The accumulation of clenbuterol might result in some clinical effects when clenbuterol-contaminated meat was consumed.

Identification of candidate genes for congenital splay leg in piglets by alternative analysis of DNA microarray data

The congenital splay leg syndrome in piglets is characterized by a temporarily impaired functionality of the hind leg muscles immediately after birth. Etiology and pathogenetic mechanisms for the disease are still not well understood. We compared genome wide gene expression of three hind leg muscles (M. adductores, M. gracilis and M. sartorius) between affected piglets and their healthy littermates with the GeneChip Porcine Genome Array (Affymetrix) in order to identify candidate genes for the disease. Data analysis with standard algorithms revealed no significant differences between both groups. By application of an alternative approach, we identified 63 transcripts with differences in two muscles and 5 genes differing between the groups in three muscles. The expression of six selected genes (SQSTM1, SSRP1, DDIT4, ENAH, MAF, and PDK4) was investigated with SYBRGreen RT-Real time PCR. The differences obtained with the microarray analysis could be confirmed and demonstrate the validity of the alternative approach to microarray data analysis. Four genes with different expression levels in at least two muscles (SQSTM1, SSRP1, DDIT4, and MAF) are assigned to transcriptional cascades related to cell death and may thus indicate pathways for further investigations on congenital splay leg in piglets.

Optimising ultrasound-mediated gene transfer (sonoporation) in vitro and prolonged expression of a transgene in vivo: Potential applications for gene therapy of cancer

Therapeutic approaches using gene-based medicines promise alternatives or adjuncts to conventional cancer treatment. Because of its non-invasive nature, ultrasound, as a membrane-permeabilising stimulus has the potential to be highly competitive with viral gene delivery and existing non-viral alternatives. In optimising ultrasound-mediated, microbubble-assisted (MB101) gene tranfection in vitro, we demonstrate efficiencies of up to 18% using ultrasound at 1 MHz at a duty cycle of 25% at intensities ranging from 1 to 4 W cm −2. Using ultrasound-mediated transfection together with an episomal plasmid-based gene expression system, we demonstrate prolonged functional gene expression of luciferase in mouse hind leg muscle and in tumours in vivo.

Influence of dexamethasone on masseter muscle wet weight in growing rats

The objective of this study was to investigate the influence of corticosteroids on the masseter wet weight and changes after discontinuation of the administration by comparison with those of hind leg skeletal muscles. Thirty-five male Wister rats, 9 weeks old, were used for the experiment. As a corticosteroids, dexamethasone was administered in drinking water for 14 days in consentrations 0.5 and 3.0 μg/ml administration groups: LD (Low Dose group) and HD (High Dose group), respectively. A 21-day recovery period was set after the discontinuation. Recovery groups: LD-r and HD-r. Non-treated groups: CO (CO-b, CO, CO-r). After the experimental period, the superficial masseter and the rectus femoris and gastrocnemius were excised, and the wet weights were measured. The wet weights of each muscle were significantly lower in the LD and HD than in the CO ( p < 0.05). More significant wet weight losses of hind leg were noted in the HD ( p < 0.05). The wet weight reduction rates of the rectus femoris and gastrocnemius relative to those in the CO were 50 and 44% in the HD, but that of the masseter was only 25%. The reductions recovered to weights similar to those in the CO-r after the discontinuation in the LD-r, but the weights in the HD-r were significantly lower than those in the other 2 groups ( p < 0.05). Dexamethasone reduced the wet weight of the superficial masseter in the growing rats, but the influence was smaller than that on the hind leg muscles, suggesting that the reduction can be recovered by discontinuation when the corticosteroids is not administered at high doses.

Curative effects of extrusive and Fogarty balloon catheter embolectomy in acute femoral vein thrombosis: experiment with rabbits

To compare the influence extrusive and Fogarty balloon catheter embolectomy on the patency rate of bloodstream and the damage of the wall of vein in acute femoral vein thrombosis. Eighty rabbits were randomly divided into 4 groups: Group A (n = 25) undergoing ligation of unilateral femoral to establish acute femoral vein thrombosis model and treated by extrusion of the hind leg muscles 24 h after the operation, Group B (n = 25) treated by Fogarty balloon catheter embolectomy 24 h after establishment of the thrombosis model, Group C (n = 25) undergoing sham operation, and Group D (n = 5) as normal controls.7, 14, and 28 days after the treatment digital subtraction angiography (DSA) was conducted to observe the patency rate of the vessel.1, 4, 7, 14, and 28 days after the treatment specimens of the thrombotic and corresponding sections of the veins were collected from the 4 groups (on days 1 and 7 for Group D) to undergo transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Quantitative reverse transcription and polymerase chain reaction was used to detect the level of tissue thromboplastin (TF). ELISA was used to detect the levels of thromboxane B(2), (TXB(2)) and 6-keto-prostaglandin F1alpha (PGF1alpha). Occlusion was seen in 3 femoral veins of Group B and one femoral vein of Group (P < 0.01), and the other veins were all patent. TEM and SEM showed that the endothelial cell injury was slight in Group A, and aggravated in Group B. TF mRNA expression could be seen 1 day after the treatment in Groups A, B, and C, and peaked on the day 7, and not found in Group D at any time points (all P < 0.01); and the TF mRNA levels at different time points of Group A were all significantly lower than those of Group B (all P < 0.01). The TXB(2) expression levels on days 1 and 7 of Groups A, B, and C were all significantly higher than those of Group D; especially those of Group B (all P < 0.01). The 6-keto-PGF(1)alpha levels on days 1 and 7 of Group D were both significantly higher than those of Groups A, B, and C (all P < 0.01). Compared with Fogarty balloon catheter embolectomy, extrusion embolectomy can discard the thrombus more thoroughly and guarantee the patency rate of bloodstream. Both extrusion embolectomy and Fogarty balloon catheter embolectomy, especially the latter, cause damage to the blood vessel endothelium.

돼지 뒷다리 주요 근육과 등심근육으로 제조된 햄의 품질 특성

돼지 뒷다리 부위 주요 근육 4개와 등심 근육으로 제조 된 햄의 이화학적 특성과 관능적 특성을 비교한 결과, 햄 제조에 사용된 원료육의 pH는 Longissimus 근육과 다른 4개의 뒷다리 부위 근육들 간에 차이가 없었으나 햄 제품의 pH에서는 Longissimus 근육 햄에 비해 Rectus femoris 근육 햄이 유의적으로 높은 pH를 나타내었으며(p<0.05), Longissimus 근육 햄의 pH 값이 가장 낮았다(p<0.05). 일반성분을 비교한 결과, 단백질 함량에서만 Longissimus 햄이 Rectus femoris, Semimembranosus, Gluteus medius 근육으로 제조된 햄보다 유의적으로 높았다(p<0.05). 제품색 특성 중 Hunter L 값은 Longissimus dorsi 근육이 가장 높았으며, Rectus femoris, Gluteus medius 근육이 가장 낮은 수치를 나타내었다(p<0.05). Hunter a값은 Rectus femoris, Biceps femoris, Gluteus medius 근육으로 제조된 햄들이 비슷한 수준을 나타내었으며, Longissimus 근육 햄에서 가장 낮았다(p<0.05). 조직감을 조사한 결과, 경도, 탄력성, 응집성, 검성, 씹힘성 모두 근육에 따른 차이가 발견되지않았다(p>0.05). 근육 햄들의 관능적 특성을 조사한 결과, 육안적 색도, 맛, 조직감은 근육간 차이가 없었으며(p>0.05), 풍미는 Semimembranosus 근육으로 제조된 햄이 가장 높은 점수를 받았고, Gluteus medius 근육으로 제조된 햄이 가장 낮았다(p<0.05). This study was conducted to evaluate the quality attributes of cooked hams made with four hind leg muscles (Biceps femoris, Semimembranosus, Rectus femoris, Gluteus medius) and Longissimus dorsi. Muscles were prepared from three market-weighted crossbreeds (<TEX>$L{\times}Y{\times}D$</TEX>) and the pH, proximate chemical composition, color, texture attributes, and sensory properties of cooked pork muscle hams were evaluated. In the raw state, no significant differences in pH were found among the five muscle hams. However, Rectus femoris ham had the highest pH, while Longissimus dorsi ham had the lowest pH (p<0.05). All muscle hams had similar moisture, fat, and ash contents. The protein content (%) was highest in Longissimus dorsi ham (p<0.05). The Hunter L value was highest for Longissimus dorsi ham while Rectus femoris and Gluteus medius hams had the lowest Hunter L values (p<0.05). The Hunter a values were similar in Rectus femoris, Biceps femoris, and Gluteus medius hams, and lowest for Longissimus dorsi ham (p<0.05). Texture attributes were not significantly different among the five muscle hams (p>0.05). The results of sensory evaluation showed that Semimembranosus hams had the highest flavor score, but there were no significant differences among five muscle hams with regard to color, taste, and texture (p>0.05).

Adeno-associated Virus of a Single-polarity DNA Genome Is Capable of Transduction In Vivo

The adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. AAV vectors with wild-type inverted terminal repeats package either the plus- or the minus-strand DNA genomes with equal frequency. By creating a series of deletions within the, we have developed a genetic approach that can generate an AAV vector that packages its single-stranded DNA genome predominantly in a single polarity (99.4%). This novel reagent efficiently transduced muscle, brain and liver in whole animals. The transduction efficiencies were similar to those of the control mixed-polarity vectors. Our results showed that reannealing of plus- and minus-strand DNA was not required for AAV-mediated transduction in vivo, supporting the hypothesis that second-strand DNA synthesis is a primary pathway in converting the single-stranded AAV genome into double-stranded forms. The availability of the single-polarity AAV vector would aid further studies on the mechanism of AAV transduction as well as the application of AAV vector for gene replacement therapy.

Adenovirus‐mediated interleukin‐13 gene therapy attenuates acute kidney allograft injury

Kidney transplantation is possible by virtue of systemic immunosuppression, which is in turn accompanied by serious side effects. The search for novel therapeutic agents and strategies is ongoing. Here we investigate the effects of adenovirus-mediated gene therapy with interleukin (IL)-13, which is a cytokine with strong immunomodulatory properties, on acute renal allograft injury. In addition, we compare the effects of local (intrarenal) and systemic (intramuscular) IL-13 gene therapy in kidney transplantation. The experiments were performed in a rat Fisher to Lewis acute rejection model of kidney transplantation. An adenovirus-IL-13 or adenovirus-luciferase was injected either into the donor kidney before transplantation (local treatment) or into the hind leg muscle of recipient rats (systemic treatment). A group with no treatment served as control. No additional immunosuppressive therapy was applied. The rats were sacrificed after 8 days and inflammatory markers and renal pre-fibrosis were assessed. Efficient gene transfer was confirmed by ELISA, immunohistochemistry and real-time PCR. IL-13 gene therapy diminished graft infiltration with macrophages and cytotoxic T cells and limited up-regulation of mRNA levels of the adhesion molecule E-selectin and pro-inflammatory cytokines TNF-alpha and IFN-gamma. Moreover, reduced renal interstitial pre-fibrosis was found in the rats receiving IL-13 gene therapy. The effects of local and systemic therapy were similar. This study demonstrates that IL-13 gene therapy in the graft significantly attenuates acute renal allograft damage, suggesting local therapy with IL-13 as a strategy to reduce the need for systemic immunosuppressive medication and thereby its side effects.

Efficacy of Chromium(III) Supplementation on Growth, Body Composition, Serum Parameters, and Tissue Chromium in Rats

Chromium(III) is often claimed to have a positive effect on body composition, while the responses in researches with supplementation of different chemical form of chromium are various and inconsistent. We have studied the effects of 6 weeks of treatment with three different forms of chromium (300 mug/kg) as chromium chloride, chromium tripicolinate, and chromium nanocomposite (CrNano) on growth, body composition, serum parameters, and tissue chromium in rats. The supplementation of CrNano significantly increased average daily gain, food efficiency, and lean body mass and decreased fat mass and body fat proportion and serum levels of glucose, urea nitrogen, triglyceride, and insulin. Chromium contents in liver, kidney, and hind leg muscle were increased significantly with the addition of CrNano in diet. The results indicate that chromium nanocomposite has higher efficacy on growth and body composition compared to the traditional chromium agents.

Effects of chromium nanoparticle dosage on growth, body composition, serum hormones and tissue chromium in Sprague-Dawley rats

This 6-week study was conducted to evaluate the effects of seven different levels of dietary chromium (Cr) (0, 75, 150, 300, 450, 600, and 1 200 ppb Cr) in the form of Cr nanoparticle (CrNano) on growth, body composition, serum hormones and tissue Cr in Sprague-Dawley (SD) rats. Seventy male SD rats (average initial body weight of (83.2+/-4.4) g) were randomly assigned to seven dietary treatments (n=10). At the end of the trial, body composition was assessed via dual energy X-ray absorptiometry (DEXA). All rats were then sacrificed to collect samples of blood, organs and tissues for determination of serum hormones and tissue Cr contents. The results indicated that lean body mass was significantly increased (P<0.05) due to the addition of 300 and 450 ppb Cr from CrNano. Supplementation of 150, 300, 450, and 600 ppb Cr decreased (P<0.05) percent body fat significantly. Average daily gain was increased (P<0.05) by addition of 75, 150, and 300 ppb Cr and feed efficiency was increased (P<0.05) by supplementation of 75, 300, and 450 ppb Cr. Addition of 300 and 450 ppb Cr decreased (P<0.05) the insulin level in serum greatly. Cr contents in liver and kidney were greatly increased (P<0.05) by the addition of Cr as CrNano in the dosage of from 150 ppb to 1 200 ppb. In addition, Supplementation of 300, 450, and 600 ppb Cr significantly increased (P<0.05) Cr content in the hind leg muscle. These results suggest that supplemental CrNano has beneficial effects on growth performance and body composition, and increases tissue Cr concentration in selected muscles.

Creatine uptake in brain and skeletal muscle of mice lacking guanidinoacetate methyltransferase assessed by magnetic resonance spectroscopy

Creatine (Cr) levels in skeletal muscle and brain of a mouse model of Cr deficiency caused by guanidinoacetate methyltransferase absence (GAMT-/-) were studied after Cr supplementation with 2 g.kg body wt-1.day-1 Cr for 35 days. Localized 1H magnetic resonance spectroscopy (MRS) was performed in brain (cerebellum and thalamus/hippocampus) and in hind leg muscle of GAMT-/- mice before and after Cr supplementation and in control (Con) mice. As expected, a signal for Cr was hardly detectable in MR spectra of GAMT-/- mice before Cr supplementation. In the thalamus/hippocampus region of these mice, an increase in N-acetylasparate (NAA) was observed. During Cr administration, Cr levels increased faster in skeletal muscle compared with brain, but this occurred only during the first day of supplementation. Thereafter, Cr levels increased by 0.8 mM/day in all studied locations. After 35 days of Cr supplementation, Cr levels in all locations were higher compared with Con mice on a Cr-free diet and NAA levels normalized. Only because of the repeated MRS measurements performed in this longitudinal Cr supplementation study on GAMT-/- mice were we able to discover the initial faster uptake of Cr in skeletal muscle compared with brain, which may represent muscular Cr uptake independent of Cr transporter expression. Our results can provide the basis for additional experiments to optimize Cr supplementation in GAMT deficiency, as increases in brain Cr are slow in patients after Cr supplementation.

The Molecular Basis for Cyclopiazonic Acid Inhibition of the Sarcoplasmic Reticulum Calcium Pump

The sarcoplasmic reticulum Ca(2+)-ATPase is essential for calcium reuptake in the muscle contraction-relaxation cycle. Here we present structures of a calcium-free state with bound cyclopiazonic acid (CPA) and magnesium fluoride at 2.65 A resolution and a calcium-free state with bound CPA and ADP at 3.4A resolution. In both structures, CPA occupies the calcium access channel delimited by transmembrane segments M1-M4. Inhibition of Ca(2+)-ATPase is stabilized by a polar pocket that surrounds the tetramic acid of CPA and a hydrophobic platform that cradles the inhibitor. The calcium pump residues involved include Gln(56), Leu(61), Val(62), and Asn(101). We conclude that CPA inhibits the calcium pump by blocking the calcium access channel and immobilizing a subset of transmembrane helices. In the E2(CPA) structure, ADP is bound in a distinct orientation within the nucleotide binding pocket. The adenine ring is sandwiched between Arg(489) of the nucleotide-binding domain and Arg(678) of the phosphorylation domain. This mode of binding conforms to an adenine recognition motif commonly found in ATP-dependent proteins.

Discovery of Substituted Human Urocortin 1 Analogs with Improved CRF2 Receptor Selectivity and Increased Efficacy in Preventing Skeletal Muscle Atrophy

We have identified specific amino acid modifications of human Urocortin 1 (hUCN1) that lead to highly potent and selective Corticotropin Releasing Factor Receptor 2 (CRF2R) agonists that are efficacious in preventing skeletal muscle atrophy in animal models. We have demonstrated that the CRF2R versus CRF1R selectivity can be increased by modifying the 40 amino acid hUCN1 at amino acid positions 11, 12, 13, 35 and 39. Further improvement in drug properties, including reduced binding to the CRF binding protein, improved solubility, and improved in vivo potency, were achieved by modifying amino acids at positions 22, 23, 36, and 40. In vivo investigations of selected optimized hUCN1 analogs demonstrated significant anti-atrophy efficacy in a mouse casting model of hind leg muscle disuse atrophy.

Características da carcaça de cordeiros alimentados com dietas contendo diferentes níveis de fibra em detergente neutro

Este estudo foi conduzido com o objetivo de avaliar o efeito de diferentes níveis de FDN na dieta sobre as características de carcaça de cordeiros confinados e abatidos com 30 kg de PV. Foram utilizados 20 cordeiros machos, não-castrados, cruza Ile de France x Texel, distribuídos aleatoriamente em quatro tratamentos (25, 31, 37 e 43% de FDN na dieta). O volumoso utilizado foi silagem de sorgo híbrido (AG 2005 E) e o concentrado foi composto de milho desintegrado, farelo de soja, sal comum, calcário calcítico e fosfato bicálcico. As dietas foram isoprotéicas (17% de PB) e as características avaliadas foram: peso de carcaça quente (PCQ), peso de carcaça fria (PCF), rendimento de carcaça quente (RCQ), rendimento de carcaça fria (RCF), índice de quebra ao resfriamento (IQ), área de olho-de-lombo (AOL), espessura de gordura subcutânea (EG), comprimento da carcaça (CC), largura da perna (LP), profundidade do peito (PPEITO), comprimento de perna (CPERNA), peso e proporção de perna, paleta, costilhar e pescoço e pesos e proporções de osso, músculo e gordura da perna dos cordeiros. O aumento do nível de FDN nas dietas reduziu linearmente o PCQ, PCF, RCQ, RCF e o peso de costilhar. Houve efeito quadrático do nível de FDN sobre o CPERNA. As demais variáveis analisadas não foram influenciadas pelos níveis de FDN das dietas.

In vivo measurements of muscle volume by automatic image analysis of spiral computed tomography scans

AbstractThis study investigates the accuracy of an automatic image analysis method that was developed for spiral computed tomography scans (SCTS), with the objective of calculating the volume of muscle in the hind leg (HLMVCT) and lumbar region (LRMVCT) in lambs. The first step in the image analysis method was the isolation (segmentation) of the muscle regions in each image of the SCTS, using a new program that was implemented in the Sheep Tomogram Analysis Routines software (STAR). Due to the differences of muscle shape in the regions investigated, the new segmentation program applies different segmentation paths in specific subregions. These were automatically identified by the program based on skeletal landmarks. After the segmentation was completed, the muscles areas were automatically measured by counting the pixels representing muscle in each image; the volumes were calculated by adding the muscle areas of each image multiplied by the depth of the image (inter-slice distance). The accuracy of these measures of muscle volume was evaluated, using regression analysis, by comparing HLMVCT and LRMVCT to the hind leg and lumbar region muscle weights measured after dissection (HLMWD, no. =240, and LRMWD, no. =50, respectively) of Texel (TEX) and Scottish Blackface (SBF) female and male lambs slaughtered in 2003-04. The effects of breed, sex and year on the association (SCTS v. dissection) were evaluated. There was a strong association between HLMVCT and HLMWD ( R2=97·4%), which only increased slightly ( R2=97·7%) when breed was included in the model. This indicates that HLMWD can be estimated directly from HLMVCT with a high degree of accuracy. For the lumbar region, the association was high ( R2=83·0% to 88·8% depending on the model) but lower than in the hind leg, probably because the automatic segmentation isolates only the areas of the longissimus lumborum and multifidi muscles. Breed had a significant effect on the prediction of LRMWD from LRMVCT, as well as sex in the case of the TEX lambs. The results indicated that the predictions of LRMWD from LRMVCT require different equations for very divergent breeds such as TEX and SBF.

The Alternative Stimulatory G Protein α-Subunit XLαs Is a Critical Regulator of Energy and Glucose Metabolism and Sympathetic Nerve Activity in Adult Mice

The complex imprinted Gnas locus encodes several gene products including G(s)alpha, the ubiquitously expressed G protein alpha-subunit required for receptor-stimulated cAMP generation, and the neuroendocrine-specific G(s)alpha isoform XLalphas. XLalphas is only expressed from the paternal allele, whereas G(s)alpha is biallelically expressed in most tissues. XLalphas knock-out mice (Gnasxl(m+/p-)) have poor suckling and perinatal lethality, implicating XLalphas as critical for postnatal feeding. We have now examined the metabolic phenotype of adult Gnasxl(m+/p-) mice. Gnasxl(m+/p-) mice had reduced fat mass and lipid accumulation in adipose tissue, with increased food intake and metabolic rates. Gene expression profiling was consistent with increased lipid metabolism in adipose tissue. These changes likely result from increased sympathetic nervous system activity rather than adipose cell-autonomous effects, as we found that XLalphas is not normally expressed in adult adipose tissue, and Gnasxl(m+/p-) mice had increased urinary norepinephrine levels but not increased metabolic responsiveness to a beta3-adrenergic agonist. Gnasxl(m+/p-) mice were hypolipidemic and had increased glucose tolerance and insulin sensitivity. The similar metabolic profile observed in some prior paternal Gnas knock-out models results from XLalphas deficiency (or deficiency of the related alternative truncated protein XLN1). XLalphas (or XLN1) is a negative regulator of sympathetic nervous system activity in mice.

Activity of common anticonvulsant drugs on spinal seizure-induced by sudden cooling

Although some of the clinical signs associated with epilepsy have their origin in supraspinal structures, it is the spinal cord in the end, which is responsible for generating the typical pattern of tonic–clonic contractions associated with a convulsion. Indeed, the spinal cord isolated from influence of the brain is capable of convulsive and paroxysmal activity that exhibits the same stereotyped motor pattern seen in the intact animal. This motor pattern can be reproduced experimentally by cooling the isolated spinal cord of amphibians. The isolated spinal cord–hindleg preparation of toad was used. Convulsive activity was induced by placing the isolated spinal cord into a Ringer's bath kept at 7 °C. The characteristic phases of the convulsion and their intensity were assessed by recording tonic–clonic contractions of hindleg muscles. Two main endpoints were used to assess the anticonvulsive activity of the drugs tested: first, their ability to block only the tonic hind-limb extension (THE) and second, their ability to block all tonic–clonic activity. The ED50 values and its 95% confidence interval estimated for abolition of THE for each drugs was (mg/kg): carbamazepine 8.6 (6.8–10.8), phenytoin 13 (7.1–23.6), diazepam 0.007 (0.004–0.01), MK-801 3.4 (2.0–5.7), valproate 120 (40–400), phenobarbital 17.1 (12.2–23.9), pentobarbital 10 (6–16.4), mephenesin 2–5 and acetazolamide > 500. The ability of some of these drugs to inhibit this kind of seizure activity at doses within therapeutic range suggests a potential use of this isolated preparation as a model in the study and testing of new anticonvulsive drugs.

LIPOSOME-ENCAPSULATED HEMOGLOBIN (TRM645) IMPROVES ENERGY METABOLISM IN SKELETAL MUSCLE ISCHEMIA AND REPERFUSION AS SHOWN ON NUCLEAR MAGNETIC RESONANCE

Background. We tested effects of liposome-encapsulated hemoglobin (TRM645) in skeletal muscle ischemia and reperfusion, which simulates muscular necrosis and metabolic syndrome in humans after limb entrapment. Methods. Intracellular pH and inorganic-phosphate / high energy phosphate ratio (Pi/PCr) were serially monitored using a nuclear magnetic resonance in rodent hind leg muscle undergoing, 1. Ischemia for 70 minutes, 2. Intravenous systemic (10 ml/kg) administration of TRM645, saline, empty liposome or no treatment, 3. Reperfusion for 60 minutes. Results. Intracellular pH decreased precipitously after ischemia and even following reperfusion regardless of treatment. Pi/PCr increased after ischemia until reperfusion, when the ratio returned toward normal or energy status improved in TRM645-treated rats (right panel, n=6) while Pi/PCr remained elevated in animals without treatment (left panel, n=5) as well as in other animals. Conclusions. The results suggest that TRM645 (10 ml/kg) improves intracellular energy metabolism in the skeletal muscle after ischemia and reperfusion. Return of pH may lag behind due to prevailing anaerobic metabolism due to prolonged ischemia.Figure

404. Therapeutic Correction of PKU in a Mouse Model by Ectopic Expression of PAH and Its BH4- Cofactor Genes in Skeletal Muscle by a Recombinant Triple-Cistronic AAV2-Based Pseudotype 1 Vector

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism with a deficiency of the hepatic phenylalanine hydroxylase (PAH) leading to toxic accumulation of circulating phenylalanine (Phe) in blood, resulting in growth failure, microcephaly, seizures, and mental retardation. PAH catalyzes the hydroxylation of Phe to tyrosine and needs oxygen and tetrahydrobiopterin (BH4) as cofactor. BH4 biosynthesis requires the consecutive action of the enzymes GTPCH and PTPS, with dihydroneopterin triphosphate as the first intermediate. Here we aimed at expressing the PAH system in skeletal muscle to degrade serum Phe in PKU patients, as this tissue is abundant, easy accessible, and persistent due to its post-mitotic nuclei. However, BH4 is abundant in liver but scarce in skeletal muscle, as the cofactor- synthesizing enzyme GTPCH is absent in muscle tissue, and PTPS is expressed at low levels. We first demonstrated that transgenic PKU mice that had no liver PAH and expressed coordinately PAH along with GTPCH in skeletal muscle tissue accumulated dihydroneopterin triphosphate and remained hyperphenylalaninemic unless synthetic BH4-cofactor was supplied by intraperitoneal injections. Thus, PTPS activity is definitely limiting in skeletal muscle to synthesize sufficient BH4 and to support Phe hydroxylation. A recombinant triple-cistronic AAV2- based pseudotype 1 vector expressing PAH along with the two cDNA-genes for BH4 biosynthesis, GTPCH and PTPS, was then generated. Upon single injections of at least 3.5x10e12 recombinant triple-cistronic AAV2/1 vector particles into each of the gastrocnemius muscles of the hind legs of the PKU mouse model Pah-enu2 resulted in long-term clearance of blood Phe, including complete phenotypic reversion. A similar therapeutic effect was achieved when a combination of two AAV2/1 vectors expressing individually PAH and GTPCH-PTPS were co-injected into the same hind leg muscles. As a control, an AAV2/1-vector expressing only PAH with GTPCH was not therapeutic. This non-invasive application is the basis to develop an efficient therapy for PKU using a triple-cistronic gene transfer into skeletal muscle.