RESUMEN
The intermolt crustacean Y-organ (YO) maintains a basal state mediated by pulsatile release of molt inhibiting hormone (MIH), a neuropeptide produced in the eyestalk ganglia, inhibiting YO ecdysteroidogenesis. Reduction of MIH results in YO activation and the animal enters premolt. In the crab, Gecarcinus lateralis, molting was induced by eyestalk ablation (ESA). ESA animals were injected with either rapamycin, an mTOR inhibitor, or DMSO vehicle at Day 0. YOs were harvested at 1, 3, and 7 days post-ESA and processed for high throughput RNA sequencing. ESA-induced increases in mRNA levels of mTOR signaling genes (e.g., mTOR, Rheb, TSC1/2, Raptor, Akt, and S6 kinase) declined following rapamycin treatment. In concert with mTOR inhibition, mRNA levels of ecdysteroid biosynthesis genes (e.g., Nvd, Spo, Sad, Dib, and Phm) were decreased and accompanied by a decrease in hemolymph ecdysteroid titer. By contrast, rapamycin increased the mRNA level of FKBP12, the rapamycin-binding protein, as well as the mRNA levels of genes associated with Wnt and insulin-like growth factor signaling pathways. Many MIH and transforming growth factor-ß signaling genes were down regulated in ESA animals. These results indicate that mTOR activity either directly or indirectly controls transcription of genes that drive activation of the YO.
Asunto(s)
Braquiuros/citología , Braquiuros/genética , Perfilación de la Expresión Génica , Muda/genética , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/metabolismo , Animales , Braquiuros/efectos de los fármacos , Braquiuros/crecimiento & desarrollo , Ecdisteroides/biosíntesis , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Muda/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacologíaRESUMEN
In decapod crustaceans, claw muscle undergoes atrophy in response to elevated ecdysteroids while thoracic muscle undergoes atrophy in response to unweighting. The signaling pathways that regulate muscle atrophy in crustaceans are largely unknown. Myostatin is a negative regulator of muscle growth in mammals, and a myostatin-like cDNA is preferentially expressed in muscle of the land crab, Gecarcinus lateralis (Gl-Mstn). Contrary to prediction, levels of Gl-Mstn mRNA decreased dramatically in both the claw closer and weighted thoracic muscles when molting was induced by either eyestalk ablation (ESA) or multiple limb autotomy (MLA). However, the effect of molt induction was greater in the claw muscle. By late premolt, Gl-Mstn mRNA in the claw muscle decreased 81% and 94% in ESA and MLA animals, respectively, and was negatively correlated with ecdysteroids. Gl-Mstn mRNA in thoracic muscle decreased 68% and 82% in ESA and MLA animals, respectively, but was only weakly correlated with ecdysteroid. Claw and thoracic muscles also differed to varying extents in the expression of ecdysteroid receptor (Gl-EcR and Gl-RXR), elongation factor-2 (Gl-EF-2), and calpain T (Gl-CalpT) in response to molt induction, but levels of the four transcripts were not correlated with ecdysteroid. The downregulation of Gl-Mstn expression in premolt claw muscle coincided with 11- and 13-fold increases in protein synthesis in the myofibrillar and soluble protein fractions, respectively. Furthermore, the rate of the increase in the synthesis of soluble proteins was greater than that of myofibrillar proteins during early premolt (1.4:1, soluble:myofibrillar), but the two were equivalent during late premolt. By contrast, Gl-Mstn mRNA increased 3-fold and Gl-CalpT mRNA decreased 40% in unweighted thoracic muscle; there was little or no effect on Gl-EF-2, Gl-EcR, and Gl-RXR mRNA levels. These data indicate that Gl-Mstn expression is negatively regulated by both ecdysteroids and load-bearing contractile activity. The downregulation of Gl-Mstn in claw muscle may induce the elevated protein turnover associated with remodeling of the contractile apparatus during molt-induced atrophy. The upregulation of Gl-Mstn in unweighted thoracic muscle suggests that this factor is also involved in disuse atrophy when hemolymph ecdysteroid levels are low.
Asunto(s)
Braquiuros/metabolismo , Muda , Músculo Esquelético/metabolismo , Miostatina/genética , Animales , Ecdisteroides/sangre , Hemolinfa/química , Pezuñas y Garras/metabolismo , Masculino , Miostatina/metabolismo , Técnicas de Cultivo de Órganos , Biosíntesis de Proteínas , ARN Mensajero/análisis , ARN Mensajero/genéticaRESUMEN
Crustacean muscle has four calpain-like proteinase activities (CDP I, IIa, IIb and III) that are involved in molt-induced claw muscle atrophy, as they degrade myofibrillar proteins in vitro and in situ. Using PCR cloning techniques, three full-length calpain cDNAs (Gl-CalpB, Gl-CalpM and Gl-CalpT) were isolated from limb regenerates of the tropical land crab Gecarcinus lateralis. All three had highly conserved catalytic (dII) and C2-like (dIII) domains. Gl-CalpB was classified as a typical, or EF-hand, calpain, as the deduced amino acid sequence had a calmodulin-like domain IV in the C-terminus and was most similar to Drosophila calpains A and B. Based on its estimated mass (approximately 88.9 kDa) and cross-immunoreactivity with a polyclonal antibody raised against Dm-CalpA, Gl-CalpB may encode CDP IIb, which is a homodimer of a 95-kDa subunit. It was expressed in all tissues examined, including skeletal muscle, heart, integument, gill, digestive gland, hindgut, nerve ganglia, gonads and Y-organ (molting gland). Both Gl-CalpM and Gl-CalpT were classified as atypical, or non-EF-hand, calpains, as they lacked a domain IV sequence. Gl-CalpM was a homolog of Ha-CalpM from lobster, based on similarities in deduced amino acid sequence, estimated mass (approximately 65.2 kDa) and structural organization (both were truncated at the C-terminal end of dIII). It was expressed at varying levels in most tissues, except Y-organ. Gl-CalpT (approximately 74.6 kDa) was similar to TRA-3 in the nematode Caenorhabditis elegans; domain IV was replaced by a unique ;T domain' sequence. It was expressed in most tissues, except eyestalk ganglia and Y-organ. The effects of elevated ecdysteroid, induced by eyestalk ablation, on calpain and ecdysone receptor (Gl-EcR) mRNA levels in skeletal muscles were quantified by real-time PCR. At 1 day after eyestalk ablation, Gl-EcR and Gl-CalpT mRNA levels increased 15- and 19.3-fold, respectively, in claw muscle but not in thoracic muscle. At 3 days after eyestalk ablation, Gl-EcR and Gl-CalpT mRNA levels in claw muscle had decreased to 2.8-fold and 4.3-fold higher than those in intact controls, respectively, suggesting a feedback inhibition by ecdysteroid. There was no significant effect of eyestalk ablation on Gl-CalpB and Gl-CalpM mRNA levels. Gl-CalpT and Gl-EcR mRNA levels were significantly correlated in both claw and thoracic muscles from intact and eyestalk-ablated animals. The data suggest that Gl-CalpT is involved in initiation of claw muscle atrophy by ecdysteroids. Premolt reduction in claw muscle mass and concomitant remodeling of the sarcomere probably result from post-transcriptional regulation of calpains.
Asunto(s)
Braquiuros/genética , Calpaína/genética , Ecdisteroides/metabolismo , Regulación de la Expresión Génica , Receptores de Esteroides/genética , Secuencia de Aminoácidos , Análisis de Varianza , Animales , Secuencia de Bases , Braquiuros/metabolismo , Calpaína/metabolismo , Clonación Molecular , Análisis por Conglomerados , Cartilla de ADN , Componentes del Gen , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Puerto Rico , Receptores de Esteroides/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
The cumulative cardiotoxicity that occurs as a result of doxorubicin chemotherapy is irreversible and can affect both quality and quantity of life for the cancer patient. Cardiac troponin I (cTnI) is a sensitive and specific marker of cardiomyocyte death. The purpose of this retrospective study was to evaluate serum concentrations of cTnI in dogs with lymphoma or osteosarcoma given doxorubicin chemotherapy, and with known cardiac outcome, based on a minimum assessment by physical examination and thoracic radiography. Serum samples were also available for cTnI measurement from seven healthy dogs given intracoronary doxorubicin. Serial serum samples obtained before, during and after doxorubicin chemotherapy showed increased cTnI concentrations in some clinical patients following chemotherapy (P = 0.0083 compared to baseline), but this did not correlate with clinical signs of cardiomyopathy. In dogs that subsequently developed cardiomyopathy however, serum cTnI concentrations were elevated before clinical signs became evident (confirmed with echocardiography).
RESUMEN
In the isopod Idotea emarginata, the neuropeptide proctolin is contained in a single pair of motoneurones located in pereion ganglion 4. The two neurones supply dorsal extensor muscle fibres of all segments. Proctolin (1 micromoll(-1)) potentiates the amplitude of contractures of single extensor muscle fibres elicited by 10 mmoll(-1) caffeine. In western blots of myofibrillar proteins isolated from single muscle fibres and treated with an anti-phosphoserine antibody, a protein with an apparent molecular mass of 30 kDa was consistently found. The phosphorylation of this protein was significantly increased by treating the fibres with proctolin. After separation of myofibrillar filaments, a 30 kDa protein was found only in the thin filament fraction. This protein is phosphorylated and detected by an antiserum against crustacean troponin I.
Asunto(s)
Crustáceos/metabolismo , Proteínas Musculares/metabolismo , Músculos/química , Neuropéptidos , Oligopéptidos/farmacología , Fosfoproteínas/metabolismo , Animales , Western Blotting , Cafeína/farmacología , Sinergismo Farmacológico , Sueros Inmunes , Inmunohistoquímica , Masculino , Peso Molecular , Contracción Muscular/efectos de los fármacos , Músculos/inervación , Fosforilación , Troponina I/análisis , Troponina I/inmunologíaAsunto(s)
Endopeptidasas , Inhibidores de Proteasas , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Endopeptidasas/clasificación , Endopeptidasas/genética , Endopeptidasas/metabolismo , Internet , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Especificidad por SustratoRESUMEN
The anatomy, physiology, and biochemistry of the dorsal membrane muscle (DMA) and the superficial extensor muscle accessory head (SEAcc) in the abdomen of the crayfish, Procambarus clarkii and lobster, Homarus americanus, are reported. These muscles have not been previously characterized physiologically or biochemically. The anatomy was originally described by Pilgrim and Wiersma (1963. J Morph 113:453-587). The arrangement of these muscles varies depending on the abdominal segment. The function of the dorsal membrane muscle is to retract the thin articulating membrane joining the cuticular segments so that the dorsal membrane does not evert during extension of the abdomen. Consequently, the articular membrane does not protrude, and thus potential damage to the membrane is minimized. Examination of nerve terminal morphology revealed strings of varicosities, usually only associated with tonic terminals. The electrophysiological data indicate that there are at least four tonic excitatory and one inhibitory motor neuron innervating these muscles. Facilitation indices and fatigue-resistance indicate physiologically the tonic nature of innervation. Anti-GABA antibodies demonstrate the anatomical presence of an inhibitor motor neuron. The SDS electrophoretic analysis of myofibrillar proteins and Western blots of key protein isoforms for these muscles in crayfish and lobsters also indicate that the DMA and SEAcc muscles are tonic phenotype. J. Exp. Zool. 287:353-377, 2000.
Asunto(s)
Astacoidea/anatomía & histología , Neuronas Motoras/fisiología , Músculo Esquelético/anatomía & histología , Nephropidae/anatomía & histología , Abdomen/anatomía & histología , Abdomen/inervación , Animales , Electrofisiología , Músculo Esquelético/fisiologíaAsunto(s)
Calpaína/aislamiento & purificación , Crustáceos/enzimología , Cisteína Endopeptidasas/aislamiento & purificación , Animales , Resinas de Intercambio Aniónico , Calpaína/química , Cromatografía en Agarosa/métodos , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Cisteína Endopeptidasas/química , Peso Molecular , Músculo Esquelético/enzimología , Nephropidae/enzimología , Compuestos Organomercuriales , Resinas SintéticasRESUMEN
The crustacean integument consists of the exoskeleton and underlying epithelium and associated tissues. The epithelium, which is composed of a single layer of cells, is responsible for the cyclical breakdown and synthesis of the exoskeleton associated with molting (ecdysis). During premolt (proecdysis) the epithelial cells lengthen and secrete the two outermost layers (epicuticle and exocuticle) of the new exoskeleton while partially degrading the two innermost layers (endocuticle and membranous layer) of the overlying old exoskeleton. This increased cellular activity is associated with increased protein synthesis and a change in cell shape from cuboidal to columnar. The cytoskeleton, composed of microfilaments (actin) and microtubules (tubulin), plays important roles in the intracellular organization and motility of eukaryotic cells. Immunoblot analysis shows that the land crab exoskeleton contains actin, tubulin, and actin-related proteins (Varadaraj et al. 1996. Gene 171:177-184). In the present study, immunocytochemistry of land crab and lobster integument showed that both proteins were localized in various cell types, including epithelia, connective tissue, tendinal cells, and blood vessels. Muscle immunostained for actin and myosin, but not for tubulin. The membranous layer of land crab (the other layers of the exoskeleton were not examined) and membranous layer and endocuticle of lobster also reacted specifically with anti-beta-actin and anti-alpha-tubulin monoclonal antibodies, but not with an anti-myosin heavy chain antibody. During proecdysis immunolabeling of the membranous layer decreased probably due to protein degradation. The staining intensity for actin and tubulin in the proecdysial epithelium was similar to that in the intermolt (anecdysial) epithelium, suggesting that there was a net accumulation of both proteins proportional to the increase in cellular volume. These results support the previous biochemical analyses and, more specifically, localize actin and tubulin in exoskeletal structures, suggesting that they may serve both intracellular and extracellular functions in crustaceans. J. Exp. Zool. 286:329-342, 2000.
Asunto(s)
Actinas/química , Braquiuros/química , Nephropidae/química , Tubulina (Proteína)/química , Actinas/análisis , Actinas/inmunología , Animales , Braquiuros/inmunología , Inmunohistoquímica , Muda , Nephropidae/inmunología , Tubulina (Proteína)/análisis , Tubulina (Proteína)/inmunologíaRESUMEN
Two dominant temperature-sensitive (DTS) Drosophila mutants are missense mutations of proteasome genes encoding beta-type subunits beta6/C5 (DTS5) and beta2/Z (DTS7). At nonpermissive temperature (29 degrees C), heterozygotes (DTS5/+ and DTS7/+) develop normally until metamorphosis; pupae fail to mature and die before eclosion. Proteasomes were purified from wild-type (WT) and heterozygous adult flies raised at permissive temperature (25 degrees C). Two-dimensional gel electrophoresis separated at least 28 proteins, 13 of which were identified with monospecific antibodies to alpha6/C2 (five species), alpha2/C3 (three species), alpha7/C8 (three species), alpha5/zeta, and beta1/Y subunits. Both quantitative and qualitative differences were observed between WT and DTS/+ proteasomes, with DTS5/+ deviating more from WT than DTS7/+ proteasomes. In DTS5/+ there was a shift to more acidic species of C2 and C3 and a shift to less acidic species of 32-kDa subunits (#3-#7) recognized by an anti-alpha subunit monoclonal antibody (MCP222) and were losses of two 32-kDa subunits (#2 and #3), decreases in Y (25 kDa; 2-fold) and 31-kDa (#9; 2-fold) subunits, and increases in 52-kDa (#1; 1.9-fold) and 24-kDa (#13; 2.3-fold) subunits. In DTS7/+ there was a less pronounced shift to acidic species of C3 and no pI shift in C2 species and subunits #3-#7 and were decreases in #9 (2.5-fold) and #14 (3-fold) and a loss of #2. The three C8 species were similar between WT, DTS5/+, and DTS7/+ proteasomes. Qualitatively, the most dramatic difference was the appearance of a new 24-kDa subunit (#16) in DTS/+ preparations, with about a 14-fold greater amount of #16 in DTS7/+ than in DTS5/+ proteasomes. Catalytically, WT and DTS/+ proteasomes had similar peptidase activities, although the DTS/+ proteasomes were slightly more sensitive to SDS and elevated temperatures in vitro. The incorporation of DTS subunits apparently altered proteasome assembly and/or processing at permissive temperature with little effect on catalytic activities. These data suggest that at nonpermissive temperature, assembly/processing is more severely affected, producing DTS-containing complexes that lack functions essential for cellular proliferation and differentiation at metamorphosis.
Asunto(s)
Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Animales , Cisteína Endopeptidasas/genética , Drosophila melanogaster/crecimiento & desarrollo , Electroforesis en Gel Bidimensional , Heterocigoto , Immunoblotting , Punto Isoeléctrico , Modelos Biológicos , Peso Molecular , Complejos Multienzimáticos/genética , Mutación , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal , Conformación Proteica , TemperaturaRESUMEN
Recent work on structural/functional relationships in arthropod proteasomes is reviewed. Taking advantage of our ability to induce a stable, proteolytically-active conformation of the lobster proteasome, the structures of basal and heat-activated complexes were probed with exogenous proteases. Increased sensitivity to chymotrypsin and trypsin showed that heat activation induced a more 'open' conformation, allowing entry of large substrates into the catalytic chamber. In Drosophila, the effects of two developmental mutant alleles (DTS-7 and DTS-5) encoding proteasome subunits (Z and C5, respectively) on the subunit composition and catalytic activities of the enzyme were examined. Both qualitative and quantitative differences in compositions between wild-type (+/+) and heterozygotes (+/DTS) indicated that incorporation of mutant subunits alters post-translational modifications of the complex. Catalytic activities, however, were similar, which suggests that the developmental defect involves other proteasome properties, such as intracellular localization and/or interactions with endogenous regulators. A hypothetical model in which DTS subunits act as poison subunits is presented.
Asunto(s)
Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Drosophila/enzimología , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Nephropidae/enzimología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Animales , Complejo de la Endopetidasa Proteasomal , Relación Estructura-ActividadRESUMEN
Cytosolic proteinases carry out a variety of regulatory functions by controlling protein levels and/or activities within cells. Calcium-dependent and ubiquitin/proteasome-dependent pathways are common to all eukaryotes. The former pathway consists of a diverse group of Ca(2+)-dependent cysteine proteinases (CDPs; calpains in vertebrate tissues). The latter pathway is highly conserved and consists of ubiquitin, ubiquitin-conjugating enzymes, deubiquitinases, and the proteasome. This review summarizes the biochemical properties and genetics of invertebrate CDPs and proteasomes and their roles in programmed cell death, stress responses (heat shock and anoxia), skeletal muscle atrophy, gametogenesis and fertilization, development and pattern formation, cell-cell recognition, signal transduction and learning, and photoreceptor light adaptation. These pathways carry out bulk protein degradation in the programmed death of the intersegmental and flight muscles of insects and of individuals in a colonial ascidian; molt-induced atrophy of crustacean claw muscle; and responses of brine shrimp, mussels, and insects to environmental stress. Selective proteolysis occurs in response to specific signals, such as in modulating protein kinase A activity in sea hare and fruit fly associated with learning; gametogenesis, differentiation, and development in sponge, echinoderms, nematode, ascidian, and insects; and in light adaptation of photoreceptors in the eyes of squid, insects, and crustaceans. Proteolytic activities and specificities are regulated through proteinase gene expression (CDP isozymes and proteasomal subunits), allosteric regulators, and posttranslational modifications, as well as through specific targeting of protein substrates by a diverse assemblage of ubiquitin-conjugases and deubiquitinases. Thus, the regulation of intracellular proteolysis approaches the complexity and versatility of transcriptional and translational mechanisms.
Asunto(s)
Calcio/metabolismo , Cisteína Endopeptidasas/metabolismo , Invertebrados/enzimología , Complejos Multienzimáticos/metabolismo , Ubiquitinas/metabolismo , Animales , Complejo de la Endopetidasa ProteasomalRESUMEN
Crustaceans are a major cause of seafood allergy. Recent studies have identified tropomyosin as the major allergen in shrimp. However, such data are lacking in other crustaceans. In the present study lobster allergens were identified and characterized by molecular cloning, sequencing, and expression. An IgE-reactive complementary DNA clone of 2 kilobase pairs (kb) was identified by screening an expression library of the spiny lobster Panulirus stimpsoni using sera from subjects with crustacean allergy. Expression and sequencing of this clone showed that it has an opening reading frame of 274 amino acids, coding for a 34-kDa protein designated as Pan s I. In addition, we expressed the fast muscle tropomyosin from the American lobster Homarus americanus and found that this protein, coined Hom a I, was also recognized by IgE from patients with crustacean allergies. The deduced amino acid sequences of Pan s I and Hom a I, which are the first identified lobster allergens, show significant homology to shrimp tropomyosin. Sera from subjects with crustacean allergies, when preabsorbed with recombinant proteins Pan s I or Hom a I, lost their IgE reactivity to muscle extract of P. stimpsoni and H. americanus. Preincubation of crustacean allergy sera with the recombinant shrimp tropomyosin Met e I also removed their IgE reactivity to lobster muscle extracts. The results suggest that patients with allergic reactions to crustaceans have common and possibly cross-reactive IgE-reactive epitopes in lobster and shrimp.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad a los Alimentos/etiología , Nephropidae/inmunología , Mariscos , Tropomiosina/inmunología , Alérgenos/biosíntesis , Alérgenos/genética , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , ADN Complementario/genética , Decápodos/genética , Decápodos/inmunología , Hipersensibilidad a los Alimentos/inmunología , Biblioteca de Genes , Humanos , Inmunoglobulina E/inmunología , Datos de Secuencia Molecular , Nephropidae/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Tropomiosina/biosíntesis , Tropomiosina/genéticaRESUMEN
Complementary DNAs encoding fibre-type-specific isoforms of tropomyosin (Tm) have been isolated from lobster (Homarus americanus) striated muscle expression libraries made from poly(A)+ RNA purified from deep abdominal (fast-type) and crusher-claw closer (slow-type) muscles. A cDNA of slow-muscle Tm (sTm1), containing a complete open reading frame (ORF) and portions of the 5' and 3' untranslated regions (UTRs), encodes a protein of 284 amino acid residues with a predicted mass of 32,950, assuming acetylation of the amino terminus. The nucleotide sequence of a fast-muscle tropomyosin (fTm cDNA), which includes the entire ORF and part of the 3' UTR, is identical to that of sTm1 cDNA, except in the region encoding amino acid residues 39-80 (equivalent to exon 2 of mammalian and Drosophila muscle tropomyosin genes). The deduced amino acid sequences, which display the heptameric repeats of nonpolar and charged amino acids characteristic of alpha-helical coiled-coils, are highly homologous to tropomyosins from rabbit, Drosophila, and shrimp (57% to 99% identities, depending on species). Northern blot analysis showed that two transcripts (1.1 and 2.1 kb) are present in both fibre types. Mass spectrometry indicated that fast muscle contains one major isoform (fTm: 32,903), while slow muscle contains two major isoforms (sTm1 and sTm2: 32,950 and 32,884 respectively). Both Tm preparations contained minor species with a mass of about 32,830. Sequences of peptides derived from purified slow and fast Tms were identical to the deduced amino acid sequences of the sTm1 and fTm cDNAs, respectively, except in the C-terminal region of fTm. The difference in mass between that predicted by the deduced sequence (32,880) and that measured by mass spectrometry (32,903) suggests that fTm is posttranslationally modified, in addition to acetylation of the N-terminal methionine. These data are consistent with the hypothesis that the fTm and sTm1 are generated by alternative splicing of two mutually-exclusive exons near the 5' end of the same gene.
Asunto(s)
ADN Complementario/genética , Músculo Esquelético/química , Nephropidae , Transcripción Genética/genética , Tropomiosina/química , Músculos Abdominales/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/síntesis química , Inmunoglobulina G , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Conejos , Tropomiosina/genéticaRESUMEN
A BRIC (Biological Research In a Canister) experiment to investigate the effects of reduced gravity at the molecular level using Arabidopsis has been initiated. In preparation for a space flight experiment, a series of ground-based studies were conducted. Results from these studies indicate that: 1) up to 20,000 seeds can be germinated on a 100 mm diameter Petri plate, 2) nylon membrane is the best surface for recovery of plant material after freezing, 3) depending on the age of the seedlings at the time of freezing, 20 to 40 g of tissue can be obtained from Petri plates that fit in a single canister; 4) tissue from one canister yields adequate amounts of RNA to perform differential display to isolate gravity-regulated genes. Our results indicate that the proposed BRIC experiment is feasible and can provide valuable information on the possible effects of microgravity on gene regulation.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , ARN de Planta , Estudios de Factibilidad , Germinación/fisiología , Hipergravedad , Semillas , Vuelo Espacial , Vibración , IngravidezRESUMEN
Ca2+, an important intracellular messenger in plants, is implicated in controlling diverse cellular functions by regulating the activity of several enzymes. Here we report the presence of a Ca(2+)-dependent proteinase (CDP) activity in roots of Arabidopsis using in-gel assays (zymograms). The CDP activity showed absolute Ca2+ requirement for its activation; other divalent ions such as Mg2+, Sr2+, and Zn2+ did not substitute for Ca2+ in stimulating protease activity. The CDP activity was inhibited by the proteinase inhibitors leupeptin, E-64, and N-ethylmaleimide, whereas pepstatin A and phenylmethylsulfonyl fluoride were without effect. These data indicate that the enzyme is likely to be a cysteine proteinase. The CDP activity was partially purified from root cultures using ammonium sulfate precipitation, DE-52, Mono-Q, and Superdex 200 column chromatography. This purification scheme resulted in about 40-fold purification of the CDP activity. Based on the elution of Arabidopsis CDP (ACDP) activity on gel filtration column the molecular mass of CDP was estimated to be about 75 kDa. Isoelectric focusing showed that the enzyme had a pI between 5.2 and 5.4. SDS-polyacrylamide gel analysis showed that activity was associated with a 45-kDa polypeptide, suggesting that the native ACDP is a homodimer. Five different antibodies raised to animal CDPs did not cross-react with the partially purified protein. These data suggest that the plant CDP differs from the known CDPs characterized from animals and is likely to be a new CDP that is unique to plants.
Asunto(s)
Arabidopsis/enzimología , Calcio/farmacología , Cisteína Endopeptidasas/metabolismo , Animales , Cationes Bivalentes/farmacología , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Cisteína Endopeptidasas/aislamiento & purificación , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Cinética , Nephropidae , Raíces de Plantas/enzimología , Inhibidores de Proteasas/farmacologíaRESUMEN
Two crustacean models for understanding molecular mechanisms of muscle plasticity are reviewed. Metabolic changes underlying muscle protein synthesis and degradation have been examined in the Bermuda land crab, Gecarcinus lateralis. During proecdysis, the claw closer muscle undergoes a programmed atrophy, which results from a highly controlled breakdown of myofibrillar proteins by Ca(2+)-dependent and, possibly, ATP/ubiquitin-dependent proteolytic enzymes. The advantage of this model is that there is neither fiber degeneration nor contractile-type switching, which often occurs in mammalian skeletal muscles. The second model uses American lobster, Homarus americanus, to understand the genetic regulation of fiber-type switching. Fibers in the claw closer muscles undergo a developmentally-regulated transformation as the isomorphic claws of larvae and juveniles differentiate into the heteromorphic cutter and crusher claws of adults. This switching occurs at the boundary between fast- and slow-fiber regions, and thus the transformation of a specific fiber is determined by its position within the muscle. The ability to predict fiber switching can be exploited to isolate and identify putative master regulatory factors that initiate and coordinate the expression of contractile proteins.
Asunto(s)
Crustáceos/fisiología , Contracción Muscular/fisiología , Músculo Esquelético/fisiología , Animales , Crustáceos/crecimiento & desarrollo , Muda/fisiología , Desarrollo de Músculos , Fibras Musculares Esqueléticas/fisiología , Proteínas Musculares/genética , Proteínas Musculares/fisiología , Músculo Esquelético/crecimiento & desarrollo , Miofibrillas/fisiologíaRESUMEN
Lobster skeletal muscles contain four Ca2+-dependent cysteine proteinases (CDPs I, IIa, IIb, and III) that degrade myofibrillar proteins. Lobster CDPs share many properties with calpains from vertebrate tissues, but differ in native mass and subunit composition. Recently, cDNAs encoding a calpain-like protein (Dm-calpain; 91.5 or 94 kDa) have been isolated from fruit fly, Drosophila melanogaster. To further clarify the relationship between invertebrate CDPs and mammalian calpains, antibodies specific for mu-, m-, p94 (nCL-1), and Dm-calpains and lobster CDP IIb (native M(r) 195,000, subunit M(r) 95,000) were used in immunoblots to test for antigenic cross-reactivity. No common epitopes were found between CDP IIb and vertebrate calpains. However, polyclonal antibodies to CDP IIb cross-reacted strongly with a C-terminal 70-kDa portion of Dm-calpain expressed in Escherichia coli. Conversely, polyclonal antibodies to Dm-calpain recognized CDP IIb. A second CDP, CDP IIa (native M(r) 125,000), was partially purified from lobster muscle; enzyme activity coeluted with a 60-kDa polypeptide using anion-exchange chromatography. The 60-kDa protein reacted with a polyclonal antibody raised against a 20-amino acid peptide sequence found around the catalytic cysteine residue of mu- and m-calpains, but not with antibodies raised against other regions of mu- or m-calpain or with the anti-CDP IIb antibody. These results suggest that (1) the CDP IIb is the homolog of Drosophila calpain in crustaceans and (2) the active site regions of CDP IIa and mu- and m-calpains are similar.