Age-related changes of aqueous protein profiles in rat fast and slow twitch skeletal muscles

Two-dimensional electrophoresis was used to generate the aqueous protein expression patterns of rat extensor digitorum longus muscle (EDL, fast twitch muscle) and solues muscle (SOL, slow twitch muscle) of different ages. Two specific protein spots, S1 and S3, were identified from EDL muscles at the ages of 12 and 18 months onward respectively. In the EDL muscles of aged rat (24 months) after intensive exercise training, S3 was still detected while S1 disappeared. In addition, diaphragm muscle (DIA, fast twitch muscle), which retains physically active throughout the life span, was used as nondisuse control. The results showed that the expressions of S1 and S3 in 24-month DIA muscle were identical with the trained aged EDL muscle. It is suggested that exercise might delay the onset of S1 expression. However, the expression of S3 over age seemed to be progressive and exercise independent. Another protein spot, S2 was identified to express only in young EDL and SOL muscles, but its expression decreased over age. Furthermore, exercise has no effect on S2 expression since S2 could not be detected in aged DIA as well as trained aged EDL and SOL muscles. These results indicated that aqueous protein expression patterns of skeletal muscle undergo changes during aging. Some of these changes such as S2 and S3 appear progressively, and some such as S1 could be delayed by exercise. S3 was identified as ubiquitin, which might play an important role in protein degradation during skeletal muscle aging process.     

Exercise type and muscle fiber specific induction of caveolin-1 expression for insulin sensitivity of skeletal muscle

It is well known that exercise can have beneficial effects on insulin resistance by activation of glucose transporter. Following up our previous report that caveolin-1 plays an important role in glucose uptake in L6 skeletal muscle cells, we examined whether exercise alters the expression of caveolin-1, and whether exercise-caused changes are muscle fiber and exercise type specific. Fifty week-old Sprague Dawley (SD) rats were trained to climb a ladder and treadmill for 8 weeks and their soleus muscles (SOL) and extensor digitorum longus muscles (EDL) were removed after the last bout of exercise and compared with those from non-exercised animals. We found that the expression of insulin related proteins and caveolins did not change in SOL muscles after exercise. However, in EDL muscles, the expression of insulin receptor beta (IR beta) and glucose transporter-4 (GLUT-4) as well as phosphorylation of AKT and AMPK increased with resistance exercise but not with aerobic exercise. Also, caveolin-1 and caveolin-3 increased along with insulin related proteins only in EDL muscles by resistance exercise. These results suggest that upregulation of caveolin-1 in the skeletal muscle is fiber specific and exercise type specific, implicating the requirement of the specific mode of exercise to improve insulin sensitivity.     

Differential expression of the skeletal muscle proteome in mdx mice at different ages

The mdx mouse is the most commonly used animal model for Duchenne muscular dystrophy (DMD), a disease caused by the absence of dystrophin. Although much has been done to elucidate the structure and function of dystrophin and the dystrophin-associated glycoprotein complex (DGC), little is known about the cascade of molecular events triggered by the absence of dystrophin that lead to muscle degeneration. To study the molecular basis of DMD, we decided to systematically study the skeletal muscle proteome in mdx mice at different ages. By using two-dimensional (2-D) gel electrophoresis, we defined changes in the protein expression pattern between mdx and control muscles. Approximately 46 differentially expressed proteins from the cytosolic fraction of mdx hindlimb muscles at three months of age were detected by 2-D gel analysis, of which 24 were identified by matrix assisted laser desorption/ionization- mass spectrometry. Most of the proteins fell into five groups of functionally related proteins. These functional categories are (i) metabolism and energy production, (ii) serine protease inhibitor family, (iii) growth and differentiation, (iv) calcium homeostasis, and (v) cytoskeletal reorganization and biogenesis. The potential roles of the differentially expressed proteins are discussed in the context of the mdx phenotype. Finally, we analyzed alterations of protein expression in mdx mice at one and six months of age to determine how protein expression changes with disease progression.     

Estimating relative carbonyl levels in muscle microstructures by fluorescence imaging

The increase in the levels of protein carbonyls, biomarkers of oxidative stress, appears to play an important role in aging skeletal muscle. However, the exact distributions of carbonyls among various skeletal muscle microstructures still remain largely unknown, partly owing to the lack of adequate techniques to carry out these measurements. This report describes an immunohistochemical approach to determine the relative abundance of carbonyls in the intermyofibrillar mitochondria (IFM), the subsarcolemmal mitochondria (SSM), the cytoplasm, and the extracellular space of skeletal muscle. These morphological features were defined by labeling the nucleus, the Z-lines, and mitochondria. Carbonyls were detected by derivatization with dinitrophenylhydrazine followed by labeling with an Alexa 488-labeled anti-dinitrophenyl primary antibody. Alexa 488 fluorescence (green) in different fiber microstructures was used to estimate the relative abundance of carbonyls. On the basis of the samples examined, preliminary results suggest that the most dramatic age-related changes in carbonyl levels occur in the extracellular space, followed in a decreasing order by SSM, IFM, and the cytoplasm. These observations were confirmed in the soleus and semimembranosus muscles composed predominantly of type I and type II fibers, respectively. This approach could easily be extended to the investigation of carbonyl levels in other muscles (composed of mixed skeletal muscle fiber types) or other tissues in which protein carbonyls are present. Figure Imaging of Labeled Carbonyls in Rat Skeletal Muscle     

Guanidine affects differentially the twitch response of diaphragm, extensor digitorum longus and soleus nerve-muscle preparations of mice

Guanidine has been used with some success to treat myasthenia gravis and myasthenic syndrome because it increases acetylcholine release at nerve terminals through K?, Na? and Ca2? channels-involving mechanisms. Currently, guanidine derivatives have been proposed for treatment of several diseases. Studies aimed at providing new insights to the drug are relevant. Experimentally, guanidine (10 mM) induces on mouse phrenic nerve-diaphragm (PND) preparations neurotransmission facilitation followed by blockade and a greatest secondary facilitation after its removal from bath. Herein, we hypothesized that this peculiar triphasic response may differ in muscles with distinct twitch/metabolic characteristics. Morphological alterations and contractile response of PND, extensor digitorum longus (EDL) and soleus (SOL) preparations incubated with guanidine (10 mM) for 15, 30, 60 min were analyzed. Guanidine concentrations of 5 mM (for PND and EDL) and 1 mM (for EDL) were also tested. Guanidine triphasic effect was only observed on PND regardless the concentration. The morphological alterations in muscle tissue varied along time but did not impede the PND post-wash facilitation. Higher doses (20-25 mM) did not increase EDL or SOL neurotransmission. The data suggest a complex mechanism likely dependent on the metabolic/contractile muscle phenotype; muscle fiber types and density/type of ion channels, sarcoplasmic reticulum and mitochondria organization may have profound impact on the levels and isoform expression pattern of Ca2? regulatory membrane proteins so reflecting regulation of calcium handling and contractile response in different types of muscle.     

Effects of deoxycorticosterone acetate on muscle electrolytes, resting potential and mitochondria in rats

Deoxycorticosterone acetate, injected daily for 5, 10, 20 or 30 days, reduced the serum potassium levels of rats by about 25%, a decrease that was independent of the treatment period employed. Serum sodium concentrations were unchanged with treatment duration. The potassium concentrations in extensor digitorum longus (EDL) and soleus (SOL) muscles were significantly decreased, and those of sodium increased, after treatment. Accompanying these changes of electrolyte concentration, the resting membrane potentials of treated EDL and SOL were hyperpolarized in vivo, and showed depolarization with the decreases of external potassium concentrations in vitro. The mitochondria in the muscles of treated rats were damaged. The degree of damage was more serious in EDL than in SOL and was dependent on the duration of deoxycorticosterone acetate treatment.     

Absolute quantification of dystrophin protein in human muscle biopsies using parallel reaction monitoring (PRM)

The need for a reliable and accurate method to quantify dystrophin proteins in human skeletal muscle biopsies has become crucial in order to assess the efficacy of dystrophin replacement therapies in Duchenne muscular dystrophy as well as to gain insight into the relationship between dystrophin levels and disease severity in Becker's muscular dystrophy. Current methods to measure dystrophin such as western blot and immunofluorescence, while straightforward and simple, lack precision and sometimes specificity. Here, we standardized a targeted mass spectrometry method to determine the absolute amount of dystrophin in ng/mg of muscle using full‐length ¹³C6–Arg– and ¹³C6,¹⁵N2–Lys–labeled dystrophin and parallel reaction monitoring (PRM). The method was found to be reproducible with a limit of quantification as low as 30 pg of dystrophin protein per mg of total muscle proteins. The method was then tested to measure levels of dystrophin in muscle biopsies from a healthy donor and from Duchenne and Becker's muscular dystrophy patients.     

EFFECTS OF DEOXYCORTICOSTERONE ACETATE ON MUSCLE ELECTROLYTES,RESTING POTENTIAL AND MITOCHONDRIA IN RATS

Deoxycorticosterone acetate, injected daily for 5, 10, 20 or 30 days, reduced the serum potassium levels of rats by about 25%, a decrease that was independent of the treatment period employed. Serum sodium concentrations were unchanged with treatment duration. The potassium concentrations in extensor digitorum longus (EDL) and soleus (SOL) muscles were significantly decreased, and those of sodium increased, after treatment. Accompanying these changes of electrolyte concentration, the resting membrane potentials of treated EDL and SOL were hyperpolarized in vivo, and showed depolarization with the decreases of external potassium concentrations in vitro. The mitochondria in the muscles of treated rats were damaged. The degree of damage was more serious in EDL than in SOL and was dependent on the duration of deoxycorticosterone acetate treatment.     

Diabetes induces compositional, structural and functional alterations on rat skeletal soleus muscle revealed by FTIR spectroscopy: a comparative study with EDL muscle

Diabetes Mellitus (DM) is a metabolic disorder, characterized by abnormally high blood glucose levels due to decreased secretion or effectiveness in function of insulin. Having a role in carbohydrate and lipid metabolism, skeletal muscle is affected by the absence of insulin in diabetic conditions. This current study reports the application of Fourier transform infrared (FTIR) spectroscopy in the determination of macromolecular alterations in streptozotocin (STZ)-induced diabetic rat skeletal Soleus (SOL) muscles, which highlight the promise of this technique in medical research. The results revealed that DM induced several alterations in macromolecular content and structure of slow-contracting SOL muscles. In diabetic SOL muscles, a decrease in the content of lipids, proteins and nucleic acids together with an increase in lipid order was observed. The decrease in the level of unsaturation and acyl chain length of lipids demonstrated the increased lipid peroxidation in DM. There were alterations in protein secondary structure in DM with a decrease in α-helix and β-sheet content of proteins, whereas the content of aggregated β-strands increased, which is generally seen when proteins denature. Besides, the integrity of collagen molecules was found to be decreased, demonstrating the alterations in its triple helical structure in diabetic muscles. Furthermore, the same alterations mentioned above were also observed in diabetic fast-contracting Extensor Digitorum Longus (EDL) muscles. However, having a high content of mitochondria and relying on an oxidative pathway, SOL muscle was found to be more affected by DM.     

Electrophoretic and computer analysis of skeletal muscle used for cardiac assistance.

Skeletal muscle has an inherent plasticity which allows it to undergo fibre type transformation when induced by a specific stimulus. Electrical stimulation has been used here to induce transformation of a predominantly fast type skeletal muscle towards a slow, more fatigue-resistant phenotype, which is more suitable for use in long-term cardiac assistance. Muscle samples from animals electrically stimulated for periods up to 6 months have been analysed by electrophoresis for myosin heavy chain (MHC) and myosin light chain (MLC) fast and slow isoforms. Densitometry and computer analysis have been used to determine the pattern of transformation of the different myosin subunits over this time period. MHC and MLC 2 fast to slow isoform switching preceded that of the alkali light chains (MLC1 and MLC3). After 3 months of stimulation the MHC slow isoform was found to have doubled in concentration relative to the unstimulated control muscle and by 4 months accounted for almost 50% of the total MHC content. The slow isoform accounted for 75% of the MLC2 after 4 months of stimulation. The protein products of mRNA isolated from stimulated muscle samples, translated in vitro and separated by electrophoresis, showed that transformation at the mRNA level preceded that at the protein level. By 2-4 weeks of stimulation MLC2 slow isoform mRNA represented over 60% of the total MLC2 mRNA population. An understanding of the molecular structure of muscle during transformation provides insight into its haemodynamic performance in cardiac assistance.     

Novel cationic carotenoid lipids as delivery vectors of antisense oligonucleotides for exon skipping in Duchenne muscular dystrophy

Duchenne Muscular Dystrophy (DMD) is a common, inherited, incurable, fatal muscle wasting disease caused by deletions that disrupt the reading frame of the DMD gene such that no functional dystrophin protein is produced. Antisense oligonucleotide (AO)-directed exon skipping restores the reading frame of the DMD gene, and truncated, yet functional dystrophin protein is expressed. The aim of this study was to assess the efficiency of two novel rigid, cationic carotenoid lipids, C30-20 and C20-20, in the delivery of a phosphorodiamidate morpholino (PMO) AO, specifically designed for the targeted skipping of exon 45 of DMD mRNA in normal human skeletal muscle primary cells (hSkMCs). The cationic carotenoid lipid/PMO-AO lipoplexes yielded significant exon 45 skipping relative to a known commercial lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC).     

Electrophoretic analysis of electrically trained skeletal muscle.

Sheep latissimus dorsi muscle was electrically trained, thereby inducing fast-to-slow fibre-type transformation. Using a combination of one- and two-dimensional gel electrophoresis techniques with computer analysis, we have analysed altered expression of contractile protein isoforms at protein and mRNA level over a time course of electrical training extending to 5 months. Myosin heavy chain and regulatory myosin light chain analysis showed predominant expression of their slow isoforms (86% and 92%, respectively) after 3 months of training. At the same time point, however, tropomyosin analysis revealed that the slow isoform of the alpha-subunit accounted for 64% of the total alpha expression. Troponin T isoform switching proceeded more slowly over the same time course than tropomyosin and the thick filament proteins studied. Troponin T analysis revealed 5 fast and 2 slow isoforms in the sheep, of which the second slow isoform only became clearly visible after 5 months' training. At this time point the two slow isoforms were more predominant than their fast counterparts. This suggests that a wide heterogeneity of fast and slow isoform combinations are possible in the thin filament of skeletal muscle.     

Electrophoretic and immunochemical evidence showing that marsupial limb muscles express the same fast and slow myosin heavy chains as eutherians

Limb muscles of eutherian (placental) mammals express a slow and three fast isoforms of myosin heavy chain (MyHC), but little is known about marsupial MyHCs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of limb MyHCs from seven marsupial species, spanning two orders, revealed four components, each of which specifically cross-reacted in Western blots with a monoclonal antibody (mAb) against a corresponding eutherian MyHC. For all seven species, the relative mobility of the band identified by each mAb matched that in the rat, suggesting that the four are homologous to eutherian slow, 2B, 2X and 2A MyHCs, respectively, in the order of decreasing mobility. Immunohistochemical analysis of fast marsupial limb muscles identitied four different fiber populations whose relative fiber size spectra (IIA相似文献   

Application of surface-enhanced laser desorption/ionization technology to the detection and identification of urinary parvalbumin-alpha: a biomarker of compound-induced skeletal muscle toxicity in the rat

In toxicity studies, compound-induced changes are typically evaluated using a combination of endpoints and there are often a number of potential markers in biological fluids which can indicate toxic change in tissues and organs. However, some biomarkers are not specific to the organ of injury and therefore there is a continuing search for more sensitive and specific indicators of target organ toxicity. In experiments to assess the potential diagnostic usefulness of surface-enhanced laser desorption/ionization (SELDI) ProteinChip technology, skeletal muscle toxicity was induced in Wistar Han rats by administering 2,3,5,6-tetramethyl-p-phenylenediamine (TMPD). The skeletal muscle toxicity was monitored using established endpoints such as increase in serum aldolase (Aldol), aspartate aminotransferase (AST) and histopathology, and also using SELDI retentate chromatography mass spectrometry of urine samples. Clear differences in urinary protein patterns between control and TMPD-treated animals were observed on the ProteinChip surfaces. Additionally a specific urine marker protein of 11.8 kDa was identified in TMPD-dosed rats, and the detection of the marker was related to the degree of skeletal muscle toxicity assessed by recognized clinical pathology endpoints. The 11.8 kDa protein was identified as parvalbumin-alpha. These experiments demonstrated the potential of urinary parvalbumin-alpha as a specific, noninvasive, and easily detectable biomarker for skeletal muscle toxicity in the rat and the potential of SELDI technology for biomarker detection and identification in toxicology studies.     

Comparative gel‐based proteomic analysis of chemically crosslinked complexes in dystrophic skeletal muscle

Duchenne muscular dystrophy is a highly progressive muscle wasting disease with a complex pathophysiology that is based on primary abnormalities in the dystrophin gene. In order to study potential changes in the oligomerization of high‐molecular‐mass protein complexes in dystrophic skeletal muscle, chemical crosslinking was combined with mass spectrometric analysis. The biochemical stabilization of protein interactions was carried out with the homo‐bifunctional and amine‐reactive agent bis[sulfosuccinimidyl]suberate, followed by protein shift analysis in one‐dimensional gels. The proteomic approach identified 11 and 15 protein species in wild type versus dystrophic microsomal fractions, respectively, as well as eight common proteins, with an electrophoretic mobility shift to very high molecular mass following chemical crosslinking. In dystrophin‐deficient preparations, several protein species with an increased tendency of oligomerisation were identified as components of the sarcolemma and its associated intra‐ and extracellular structures, as well as mitochondria. This included the sarcolemmal proteins myoferlin and caveolin, the cytoskeletal components vimentin and tubulin, extracellular collagen alpha‐1(XII) and the mitochondrial trifunctional enzyme and oxoglutarate dehydrogenase. These changes are probably related to structural and metabolic adaptations, especially cellular repair processes, which agrees with the increased oligomerisation of myosin‐3, myosin‐9 and actin, and their role in cellular regeneration and structural adjustments in dystrophinopathy.     

Chromatographic analysis of tropomyosins from rabbit skeletal, chicken gizzard and earthworm muscle.

Tropomyosins from rabbit skeletal, chicken gizzard and earthworm muscle all exist as dimeric, ca. 100% alpha-helical coiled-coil species in benign media. Two major tropomyosin isoforms from each muscle source have been identified and can be conveniently designated alpha (fast) and beta (slow) based on electrophoretic mobility under denaturing conditions. The ratio of alpha to beta chains is ca. 3-4:1 for rabbit skeletal and ca. 1:1 for chicken gizzard and earthworm tropomyosins. Each chain from the former two muscle sources has been sequenced, thus providing a molecular basis for interpreting the in vivo population of homo- and hetero-dimers. The characteristics of each purified tropomyosin in weak-anion exchange, strong-cation exchange and reversed-phase high-performance liquid chromatography are described. Binding to and/or elution from the reversed-phase matrix results in dissociation into highly helical monomeric chains. This mode of chromatography separates the alpha and beta chains of earthworm and chicken gizzard tropomyosins, but not those of the rabbit protein. Both anion- and cation-exchange chromatography use mild (benign) elution conditions under which the native, in vivo dimer population should be preserved. Only the rabbit protein exhibited peak separation on the anion-exchange resin, with peak assignment corresponding to the known molecular organization of homo- and hetero-dimers. In strong cation-exchange analysis, all three tropomyosins exhibit a chromatographic transition near pH 6.5, possibly the result of histidine(s) titration. Collectively, the chromatographic data confirm the present understanding of the in vivo mixture of dimers for tropomyosin from rabbit skeletal and chicken gizzard. It is concluded that native earthworm tropomyosin exists predominantly as an alpha beta hetero-dimer.     

Agarose isoelectric focusing for the detection of many isoforms and high molecules in muscle protein analysis

Two-dimensional gel electrophoresis with agarose in the first dimension was established. The technique was reported previously and was now much improved without changing the basic procedure, presenting "a protein map" of chicken skeletal muscle, including almost all components such as myosin heavy chains (200 kDa) and dystrofin (400 kDa). Application of this technique for the analysis of all components was also successful with the liver and lens. Since large molecules can get into the agarose in the first dimension, the study on protein interaction was also reported with troponin components as a model system.     

Growth impairment of primary chondrocyte cells by serum of rats with chronic renal failure

Insulin-like growth factor (IGF)/IGF binding protein (IGFBP) abnormalities may be important in the pathogenesis of growth failure in chronic renal failure (CRF). We induced experimental CRF by 5/6 nephrectomy in Sprague Dawley rats (100 g) and observed for 2 weeks comparing with sham-operated pair-fed control rats (Sham- C). CRF rats gained 30% less height than Sham- C rats (P < 0.01). Serum IGFBP profiles by Western ligand blot revealed that IGFBP4 was elevated two fold in CRF rats (P < 0.01 vs. Sham-C). However, IGFBP4 mRNA levels in liver or skeletal muscle were not different in two groups. To determine if the increase of serum IGFBP4 in CRF retarded the growth of cartilage, epiphyseal chondrocytes were isolated from CRF or control rats and cultured in the presence of control or CRF rat sera. Incubation with 10% CRF serum reduced proliferations of normal chondrocytes and L6 rat skeletal muscle cells. In contrast, 10% CRF serum did not inhibit the growth of CRF chondrocytes. Rat sera from two groups were separated into two different fractions, high (>10 kDa, containing IGFBPs) and low (<10 kDa, containing free IGF) molecular weight fractions using a gel filtration column. Both fractions obtained from CRF sera decreased the growth of control chondrocytes up to 40% compared with those from control sera. We suggest that the pathogenesis of growth failure in CRF may be involved in the increase of circulating IGFBP4 as well as the unidentified small molecular weight uremic serum factors which block the growth of chondrocytes in growth plate.     

Human skeletal muscle myosin light chains analyzed by immobilized pH gradients during ontogenesis: identification of new phosphorylatable isoforms of light chain 2

Previous studies using two-dimensional gel electrophoresis have described adult and fetal isoforms of skeletal muscle myosin light chains (MLC). They have also revealed an embryo-specific light chain (LC1emb), apparently absent in most adult skeletal muscles. In order to characterize more thoroughly the MLC family, we have analyzed the MLCs from human skeletal muscle at different developmental stages using a two-dimensional electrophoresis technique with an immobilized pH gradient in the first dimension. The high resolution of this novel technique, resolving components which in isoelectric points are less than or equal to 0.01 pH, combined with sensitive silver staining, has allowed us to identify four phosphorylatable isoforms of MLC2: two slow-myosin light chains (MLC2Sa and b), two fast myosin light chains (MLC2Fa and b), and their phosphorylated counterparts: MLC2SaP and bP, MLC2FaP and bP. The following major modifications during development were observed: (i) The embryonic LC (LC1emb) persists up to at least 26 weeks of fetal life. (ii) The polymorphism of LC2 is already evident at 10 weeks of development but only the nonphosphorylated forms of LC2S and LC2F seem to be present. The LC2Fa form is predominant. As early as 26 weeks of fetal life, the 4 phosphorylated forms are detected. In the adult, LC2Fb is a minor component. (iii) LC3F (fast) is already expressed at an early embryonic stage (10 weeks).     

Influence of ShuJinHuoXue tablets on ischemia reperfusion injury of animals' skeletal muscle

Ischemia-reperfusion (IR) can lead to serious tissue oxidative injury in animals. ShuJinHuoXue tablet (SJHXT) is a Chinese Traditional Medicine which can relax the muscles and stimulate the blood circulation and has been used as a clinical medicine. In the present study, we investigated the effects of SJHXT pretreatment on oxidative injury using an animal model of acute limb IR. Results showed that SJHXT pre-treatment (200, 300 and 400 mg/kg/day) markedly reduced serum endothelin-1 (ET-1), thromboxane B2 (TXB?) levels and thromboxane B2/6-keto- prostaglandin F1α (TXB?/6-Keto-PGF(1α)), wet weight/dried weight (W/D) ratio, myeloperoxidase (MPO), creatine kinase (CK), lactate dehydrogenase (LDH) activities, and increased serum nitric oxide (NO), 6-Keto-PGF(1α) levels and NO/ET-1 ratio in the IR+SJHXT groups. In addition, the SJHXT pre-treatment (200, 300 and 400 mg/kg/day) markedly reduced skeletal muscle Ca2?, malondialdehyde (MDA) levels, increased Na?-K?-ATPase, Ca2?-Mg2?-ATPase, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities. Our results suggest that SJHXT pre-treatment may improve skeletal muscle blood vessel microcirculation, decrease skeletal muscle oxidative injury and enhance antioxidant enzymes activities in IR animals.