RESUMO
Since 1961, L-asparaginase has been used to treat patients with acute lymphocytic leukemia. It rapidly depletes the plasma asparagine and deprives the blood cells of this circulating amino acid, essential for the metabolic cycles of cells. In the search for viable alternatives to produce L-asparaginase, this work aimed to produce this enzyme from Escherichia coli in a shaker and in a 3 L bioreactor. Three culture media were tested: defined, semi-defined and complex medium. L-asparaginase activity was quantified using the ß-hydroxamate aspartic acid method. The defined medium provided the highest L-asparaginase activity. In induction studies, two inducers, lactose and its analog IPTG, were compared. Lactose was chosen as an inducer for the experiments conducted in the bioreactor due to its natural source, lower cost and lower toxicity. Batch and fed-batch cultures were carried out to reach high cell density and then start the induction. Batch cultivation provided a final cell concentration of 11 g L-1 and fed-batch cultivation produced 69.90 g L-1 of cells, which produced a volumetric activity of 43,954.79 U L-1 after lactose induction. L-asparaginase was produced in a shaker and scaled up to a bioreactor, increasing 23-fold the cell concentration and thus, the enzyme productivity.
RESUMO
Recently, Conorfamide-Sr3 (CNF-Sr3) was isolated from the venom of Conus spurius and was demonstrated to have an inhibitory concentration-dependent effect on the Shaker K+ channel. The voltage-gated potassium channels play critical functions on cellular signaling, from the regeneration of action potentials in neurons to the regulation of insulin secretion in pancreatic cells, among others. In mammals, there are at least 40 genes encoding voltage-gated K+ channels and the process of expression of some of them may include alternative splicing. Given the enormous variety of these channels and the proven use of conotoxins as tools to distinguish different ligand- and voltage-gated ion channels, in this work, we explored the possible effect of CNF-Sr3 on four human voltage-gated K+ channel subtypes homologous to the Shaker channel. CNF-Sr3 showed a 10 times higher affinity for the Kv1.6 subtype with respect to Kv1.3 (IC50 = 2.7 and 24 µM, respectively) and no significant effect on Kv1.4 and Kv1.5 at 10 µM. Thus, CNF-Sr3 might become a novel molecular probe to study diverse aspects of human Kv1.3 and Kv1.6 channels.
Assuntos
Venenos de Moluscos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Animais , Caramujo Conus , Ativação do Canal Iônico , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/metabolismo , Canal de Potássio Kv1.4/antagonistas & inibidores , Canal de Potássio Kv1.4/genética , Canal de Potássio Kv1.4/metabolismo , Canal de Potássio Kv1.5/antagonistas & inibidores , Canal de Potássio Kv1.5/genética , Canal de Potássio Kv1.5/metabolismo , Canal de Potássio Kv1.6/antagonistas & inibidores , Canal de Potássio Kv1.6/genética , Canal de Potássio Kv1.6/metabolismo , Potenciais da Membrana , Oócitos , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismo , Xenopus laevisRESUMO
Enzymes are becoming tools in industrial processes because of several advantages, including activity in mild environmental conditions, and high specificity. Peroxidase, for one, stably oxidizes several substrates. The present study aimed to develop advanced oxidation processes (AOP), using non-commercial rice bran peroxidase to remove color and toxicity of synthetic textile wastewater. Using a microwave and shaker system, we obtained 38.9% and 100% of effluent color removal after peroxidase treatment, respectively. In addition, the shaker system decants residual dye particles through filtration, providing the textile industry with an economical and environmentally viable alternative to effluent treatment. In toxicity tests results, both treatment systems damaged the used genetic material. This damage occurs because of industrial discharge of wastewater into water bodies; effluent dilution reduced this damage. The data suggest that peroxidase as a textile effluent treatment has potential uses in industrial processes, because rice bran peroxidase has demonstrated affinity with dyes.
Assuntos
Corantes/química , Oryza/enzimologia , Peroxidase/química , Proteínas de Plantas/química , Têxteis , Purificação da Água , Oxirredução , Indústria Têxtil , Águas Residuárias/químicaRESUMO
In silico and in vitro studies have made progress in understanding protein-protein complex formation; however, the molecular mechanisms for their dissociation are unclear. Protein-protein complexes, lasting from microseconds to years, often involve induced-fit, challenging computational or kinetic analysis. Charybdotoxin (CTX), a peptide from the Leiurus scorpion venom, blocks voltage-gated K+-channels in a unique example of binding/unbinding simplicity. CTX plugs the external mouth of K+-channels pore, stopping K+-ion conduction, without inducing conformational changes. Conflicting with a tight binding, we show that external permeant ions enhance CTX-dissociation, implying a path connecting the pore, in the toxin-bound channel, with the external solution. This sensitivity is explained if CTX wobbles between several bound conformations, producing transient events that restore the electrical and ionic trans-pore gradients. Wobbling may originate from a network of contacts in the interaction interface that are in dynamic stochastic equilibria. These partially-bound intermediates could lead to distinct, and potentially manipulable, dissociation pathways.
Assuntos
Charibdotoxina/metabolismo , Íons/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Potássio/metabolismo , Animais , Aracnídeos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Ligação Proteica , Conformação ProteicaRESUMO
Conorfamides (CNFs) are toxins initially characterized from the venom duct of the venomous marine snail Conus spurius from the Gulf of Mexico; at their C-termini, these toxins are amidated and have high sequence similarity with the molluskan cardioexcitatory tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide or FMRFa) and other FMRFa-related peptides (FaRPs) found in the five molluskan classes, and in other invertebrate and vertebrate phyla. These peptides were the first FaRPs found to be present in any venom, and they are biologically active in mice, limpets, and/or freshwater snails. However, the molecular targets of the known CNFs (CNF-Sr1 and CNF-Sr2 from C. spurius, and CNF-Vc1 from C. victoriae) remain unidentified. Very recently, three FaRPs from C. textile have been found to potentiate the currents of acid-sensing ion channels. In this work, we characterized a novel conorfamide, CNF-Sr3 (ATSGPMGWLPVFYRF-NH2), comprised of 15 amino acid residues, and with a specific blocking activity for the Shaker subtype of the voltage-gated potassium channels, without significant effect on the Shab, Shaw, Shal and Eag channels. This peptide is the third type of disulfide-free conotoxins that has been discovered to target K+ channels.
Assuntos
Caramujo Conus/química , Venenos de Moluscos/química , Neuropeptídeos/farmacologia , Peptídeos/farmacologia , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Baculoviridae , Células HEK293 , Humanos , Venenos de Moluscos/síntese química , Venenos de Moluscos/farmacologia , Neuropeptídeos/síntese química , Neuropeptídeos/química , Peptídeos/química , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/farmacologia , Células Sf9/virologiaRESUMO
Background: Syndrome responsive to corticosteroid tremor (SRCT) is an immune-mediated cerebellitis commonly observed among young adults small dogs. The main clinical sign of this disorder is a tremor of high frequency and low intensity of head, trunk and limbs. The diagnosis is based on the signalment, the historical, and examinations, physical, neurological and laboratory. The aim of this study was to report four cases of SRCT in dog, stressing the importance of the neurological examination and anamnesis data for location of the lesion in the central nervous system, to establish a list of differential diagnoses and treatment to be instituted. Cases: Two mixed breed dogs, one Shih tzu and one Poodle, being three females and one male, aged between four and six years, with a history of generalized tremors between one and seven days, which ceased when they slept, were presented to a veterinary neurologist at the Veterinary Medical teaching Hospital of the UFSM. The onset of clinical signs was acute, with no progressive evolution. The dogs had no history of intoxication, trauma, or use of medications. Clinical and neurological examination was performed complete, the latter consisting of: observation of mental status (level of consciousness and behavior), evaluation of gait and posture, postural reactions, spinal segmental reflexes, cranial nerves and nociception (pain deep)...
Tremores musculares constituem um distúrbio de movimento comum e importante em cães e pode indicar enfermidades neurológicas de diversas etiologias. A principal causa de tremores em cães está relacionada a doenças que afetam o cerebelo, o qual é responsável pela frequência e amplitude dos movimentos [7]. Desta forma, os sinais clínicos mais característicos de doença cerebelar são ataxia, tremor intencional e dismetria [1,3,5,6,8,9].A síndrome do tremor responsivo ao corticosteroide (STRC) foi observada inicialmente em cães de raças pequenas de pelagem branca, como Maltês, Poodle e West highland white terrier, e por isso, foi denominada de síndrome do tremor do cão branco. Como pode afetar cães de todas as colorações, passou a ser chamada também de síndrome do cão tremedor ou síndrome do tremor responsivo ao corticosteroide [1,3,5,6,8,9]. O principal sinal clínico desse distúrbio é um tremor de alta frequência e baixa intensidade de cabeça, tronco e membros. O diagnóstico baseia-se na resenha, no histórico, e nos exames, físico, neuroló- gico e laboratorial (análise de líquido cerebroespinhal [LCE]) e o prognóstico é favorável para cães tratados com corticosteroides em doses imunossupressoras [1,3,5,8,9].Os dados sobre a STRC são encontrados basicamente na literatura internacional [8,9]. Na literatura nacional, no entanto, poucos são os relatos sobre o assunto [5]. Diante disso...
Assuntos
Animais , Cães , Corticosteroides/análise , Corticosteroides/uso terapêutico , Imunossupressores/uso terapêutico , Tremor/patologia , Tremor/veterinária , Doenças do Sistema Nervoso/veterináriaRESUMO
Background: Syndrome responsive to corticosteroid tremor (SRCT) is an immune-mediated cerebellitis commonly observed among young adults small dogs. The main clinical sign of this disorder is a tremor of high frequency and low intensity of head, trunk and limbs. The diagnosis is based on the signalment, the historical, and examinations, physical, neurological and laboratory. The aim of this study was to report four cases of SRCT in dog, stressing the importance of the neurological examination and anamnesis data for location of the lesion in the central nervous system, to establish a list of differential diagnoses and treatment to be instituted. Cases: Two mixed breed dogs, one Shih tzu and one Poodle, being three females and one male, aged between four and six years, with a history of generalized tremors between one and seven days, which ceased when they slept, were presented to a veterinary neurologist at the Veterinary Medical teaching Hospital of the UFSM. The onset of clinical signs was acute, with no progressive evolution. The dogs had no history of intoxication, trauma, or use of medications. Clinical and neurological examination was performed complete, the latter consisting of: observation of mental status (level of consciousness and behavior), evaluation of gait and posture, postural reactions, spinal segmental reflexes, cranial nerves and nociception (pain deep)...(AU)
Tremores musculares constituem um distúrbio de movimento comum e importante em cães e pode indicar enfermidades neurológicas de diversas etiologias. A principal causa de tremores em cães está relacionada a doenças que afetam o cerebelo, o qual é responsável pela frequência e amplitude dos movimentos [7]. Desta forma, os sinais clínicos mais característicos de doença cerebelar são ataxia, tremor intencional e dismetria [1,3,5,6,8,9].A síndrome do tremor responsivo ao corticosteroide (STRC) foi observada inicialmente em cães de raças pequenas de pelagem branca, como Maltês, Poodle e West highland white terrier, e por isso, foi denominada de síndrome do tremor do cão branco. Como pode afetar cães de todas as colorações, passou a ser chamada também de síndrome do cão tremedor ou síndrome do tremor responsivo ao corticosteroide [1,3,5,6,8,9]. O principal sinal clínico desse distúrbio é um tremor de alta frequência e baixa intensidade de cabeça, tronco e membros. O diagnóstico baseia-se na resenha, no histórico, e nos exames, físico, neuroló- gico e laboratorial (análise de líquido cerebroespinhal [LCE]) e o prognóstico é favorável para cães tratados com corticosteroides em doses imunossupressoras [1,3,5,8,9].Os dados sobre a STRC são encontrados basicamente na literatura internacional [8,9]. Na literatura nacional, no entanto, poucos são os relatos sobre o assunto [5]. Diante disso...(AU)
Assuntos
Animais , Cães , Corticosteroides/análise , Corticosteroides/uso terapêutico , Tremor/patologia , Tremor/veterinária , Imunossupressores/uso terapêutico , Doenças do Sistema Nervoso/veterináriaRESUMO
Opisthacanthus cayaporum belongs to the Liochelidae family, and the scorpions from this genus occur in southern Africa, Central America and South America and, therefore, can be considered a true Gondwana heritage. In this communication, the isolation, primary structure characterization, and Kâº-channel blocking activity of new peptide from this scorpion venom are reported. OcyKTx2 is a 34 amino acid long peptide with four disulfide bridges and molecular mass of 3807 Da. Electrophysiological assays conducted with pure OcyKTx2 showed that this toxin reversibly blocks Shaker B Kâº-channels with a Kd of 82 nM, and presents an even better affinity toward hKv1.3, blocking it with a Kd of â¼18 nM. OcyKTx2 shares high sequence identity with peptides belonging to subfamily 6 of α-KTxs that clustered very closely in the phylogenetic tree included here. Sequence comparison, chain length and number of disulfide bridges analysis classify OcyKTx2 into subfamily 6 of the α-KTx scorpion toxins (systematic name, α-KTx6.17).