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1.
J Med Life ; 15(6): 768-771, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35928367

RESUMO

Staphylokinase (SAK), also known as staphylococcal fibrinolysin, is a protein with a molecular mass of about 15 kDa produced by Staphylococcus aureus. Staphylokinase is synthesized in the late exponential phase, similar to streptokinase. The current study identified and predicted the protein SAK from Staphylococcus aureus. SAK is a fibrinolytic enzyme of the third generation that acts as an indirect activator of plasminogen. The current study cloned and expressed SAK protein isolated from Staphylococcus aureus and used in the form of a grid for enhancement of SAK Catalyst with PCR, disengagement, and change into articulation vector PET24b(+). The recombinant plasmid was changed into E. coli strain BL21 (codon additionally to 440) acceptance with isopropyl ß-D-1-thiogalactopyranoside (IPTG).


Assuntos
Escherichia coli , Staphylococcus aureus , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Metaloendopeptidases , Plasminogênio/metabolismo , Staphylococcus aureus/genética
2.
Int J Mol Sci ; 23(15)2022 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-35955687

RESUMO

Leishmanolysin, also known as major promastigote protease (PSP) or gp63, is the most abundant surface glycoprotein of Leishmania spp., and has been extensively studied and recognized as the main parasite virulence factor. Characterized as a metalloprotease, gp63 can be powerfully inactivated in the presence of a metal chelator. In this study, we first used the structural parameters of a 7-hydroxycoumarin derivative, L1 compound, to evaluate the theoretical-computational experiments against gp63, comparing it with an available metal chelator already described. The methodology followed was (i) analysis of the three-dimensional structure of gp63 as well as its active site, and searching the literature and molecular databases for possible inhibitors; (ii) molecular docking simulations and investigation of the interactions in the generated protein-ligand complexes; and (iii) the individual energy of the gp63 amino acids that interacted most with the ligands of interest was quantified by ab initio calculations using Molecular Fraction with Conjugated Caps (MFCC). MFCC still allowed the final quantum balance calculations of the protein interaction to be obtained with each inhibitor candidate binder. L1 obtained the best energy quantum balance result with -2 eV, followed by DETC (-1.4 eV), doxycycline (-1.3 eV), and 4-terpineol (-0.6 eV), and showed evidence of covalent binding in the enzyme active site. In vitro experiments confirmed L1 as highly effective against L. amazonensis parasites. The compound also exhibited a low cytotoxicity profile against mammalian RAW and 3T3 cells lines, presenting a selective index of 149.19 and 380.64 µM, respectively. L1 induced promastigote forms' death by necrosis and the ultrastructural analysis revealed disruption in membrane integrity. Furthermore, leakage of the contents and destruction of the parasite were confirmed by Spectroscopy Dispersion analysis. These results together suggested L1 has a potential effect against L. amazonensis, the etiologic agent of diffuse leishmaniasis, and the only one that currently does not have a satisfactory treatment.


Assuntos
Leishmania , Animais , Quelantes , Leishmania/metabolismo , Mamíferos/metabolismo , Metaloendopeptidases/metabolismo , Metaloproteases , Camundongos , Simulação de Acoplamento Molecular , Fagocitose
3.
BMC Genomics ; 23(1): 503, 2022 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-35831784

RESUMO

BACKGROUND: The filamentous temperature-sensitive H protease (ftsH) gene family plays an important role in plant growth and development. FtsH proteins belong to the AAA protease family. Studies have shown that it is a key gene for plant chloroplast development and photosynthesis regulation. In addition, the ftsH gene is also involved in plant response to stress. At present, the research and analysis of the ftsH gene family are conducted in microorganisms such as Escherichia coli and Oenococcus and various plants such as Arabidopsis, pear, rice, and corn. However, analysis reports on ftsH genes from tobacco (Nicotiana tabacum L.), an important model plant, are still lacking. Since ftsH genes regulate plant growth and development, it has become necessary to systematically study this gene in an economically important plant like tobacco. RESULTS: This is the first study to analyze the ftsH gene from Nicotiana tabacum L. K326 (NtftsH). We identified 20 ftsH genes from the whole genome sequence, renamed them according to their chromosomal locations, and divided them into eight subfamilies. These 20 NtftsH genes were unevenly distributed across the 24 chromosomes. We found four pairs of fragment duplications. We further investigated the collinearity between these genes and related genes in five other species. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis identified differential expression patterns of NtftsH in different tissues and under various abiotic stress conditions. CONCLUSIONS: This study provides a comprehensive analysis of the NtftsH gene family. The exon-intron structure and motif composition are highly similar in NtftsH genes that belong to the same evolutionary tree branch. Homology analysis and phylogenetic comparison of ftsH genes from several different plants provide valuable clues for studying the evolutionary characteristics of NtftsH genes. The NtftsH genes play important roles in plant growth and development, revealed by their expression levels in different tissues as well as under different stress conditions. Gene expression and phylogenetic analyses will provide the basis for the functional analysis of NtftsH genes. These results provide a valuable resource for a better understanding of the biological role of the ftsH genes in the tobacco plant.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Evolução Molecular , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Metaloendopeptidases/genética , Família Multigênica , Peptídeo Hidrolases/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética , Temperatura , Tabaco/metabolismo
4.
Int J Mol Sci ; 23(13)2022 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-35806299

RESUMO

Thimet oligopeptidase (TOP) is a metallopeptidase involved in the metabolism of oligopeptides inside and outside cells of various tissues. It has been proposed that substrate or inhibitor binding in the TOP active site induces a large hinge-bending movement leading to a closed structure, in which the bound ligand is enclosed. The main goal of the present work was to study this conformational change, and fluorescence techniques were used. Four active TOP mutants were created, each equipped with a single-Trp residue (fluorescence donor) and a p-nitro-phenylalanine (pNF) residue as fluorescence acceptor at opposite sides of the active site. pNF was biosynthetically incorporated with high efficiency using the amber codon suppression technology. Inhibitor binding induced shorter Donor-Acceptor (D-A) distances in all mutants, supporting the view that a hinge-like movement is operative in TOP. The activity of TOP is known to be dependent on the ionic strength of the assay buffer and D-A distances were measured at different ionic strengths. Interestingly, a correlation between the D-A distance and the catalytic activity of TOP was observed: the highest activities corresponded to the shortest D-A distances. In this study for the first time the hinge-bending motion of a metallopeptidase in solution could be studied, yielding insight about the position of the equilibrium between the open and closed conformation. This information will contribute to a more detailed understanding of the mode of action of these enzymes, including therapeutic targets like neurolysin and angiotensin-converting enzyme 2 (ACE2).


Assuntos
Metaloendopeptidases , Oligopeptídeos , Domínio Catalítico , Ligantes , Metaloendopeptidases/química , Oligopeptídeos/metabolismo , Especificidade por Substrato
5.
Immunohematology ; 38(2): 51-54, 2022 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-35852066

RESUMO

The importance of identifying variant alleles among blood donors is significant to the safety of transfusion for recipients. Molecular methods have become more prominent in the routine process of antigen typing donor units. Some variant antigens cannot be detected using only serologic methods. Molecular testing allows the determination of nucleotide sequences that are used to predict a phenotype. Antigens of the Kell blood group system are known for being highly immunogenic and causing adverse reactions upon antibody formation. A female white blood donor who typed Kp(b-) using serologic methods on multiple donations since 2005 was the subject of a typing discrepancy investigation. Routine genotyping using a commercial genotyping kit (HemoID DQS Panel; Agena Bioscience, San Diego, CA) predicted the donor to type Kp(a+b+). Investigation of the discrepancy between these two results identified a rare single nucleotide variant in the KEL gene at nucleotide position c.948G>T that alters amino acid residue 316 from tryptophan (Trp) to cysteine (Cys). After discovery of the novel allele, adsorption and elution studies were performed to see if there was weakened Kpb expression. The elution studies yielded negative results, which indicated that Kpb is not expressed. The KEL transcripts expressed by the donor were determined using cDNA analysis, and the predicted amino acid sequence of the novel allele was modeled to investigate the impact of the amino acid sequence on the structure of the KEL polypeptide. Both SWISS-MODEL and Robetta software were used to evaluate the impact of the p.Trp316Cys on the three-dimensional protein structure. There was no conformational change noted with SWISS-MODEL, whereas the Robetta software showed a significant conformational change compared with the normal Kp(b+) reference sequence. Because the donor is homozygous for variants associated with k and Jsb expression, it was not possible to determine whether the novel allele is associated with loss of Kpb only or loss of all Kell antigens.


Assuntos
Doadores de Sangue , Sistema do Grupo Sanguíneo de Kell , Alelos , Feminino , Humanos , Sistema do Grupo Sanguíneo de Kell/genética , Sistema do Grupo Sanguíneo de Kell/metabolismo , Glicoproteínas de Membrana , Metaloendopeptidases/genética , Nucleotídeos , Fenótipo
6.
Artigo em Russo | MEDLINE | ID: mdl-35904293

RESUMO

AIM OF THE STUDY: To investigate the efficacy and safety of non-immunogenic staphylokinase (NS) compared with alteplase (A) in patients with acute ischemic stroke (AIS) within 4.5 h after symptom onset. MATERIAL AND METHODS: 336 patients with IS within 4.5 h after symptom onset were included in a randomized, open-label, multicenter, parallel-group, non-inferiority comparative trial of NS vs A (168 patients in each group). NS was administered as an intravenous bolus in a dose of 10 mg, regardless of body weight, over 10 s, A was administered as a bolus infusion in a dose of 0.9 mg/kg, maximum 90 mg over 1 hour. The primary efficacy endpoint was a favorable outcome, defined as a modified Rankin scale (mRS) score of 0-1 on day 90. Safety endpoints included all-cause mortality on day 90, symptomatic intracranial haemorrhage, and other serious adverse events (SAEs). RESULTS: At day 90, 84 (50%) patients reached the primary endpoint (mRS 0-1) in the NS group, 68 (41%) patients - in the A group (p=0.10, OR=1.47, 95% CI=0.93-2.32). The difference between groups NS and A was 9.5% (95% CI= -1.7-20.7) and the lower limit of the 95% CI did not cross the margin of non-inferiority (pnon-inferiority<0.0001). There were no significant differences in the frequency of deaths between the groups: on day 90, 17 (10%) patients in the NS group and 24 (14%) in the A group had died (p=0.32). There was a trend towards significant differences in the frequency of symptomatic intracranial haemorrhage: NS group - 5 (3%) patients, A group - 13 (8%) patients (p=0.087, OR=0.37, 95% CI=0.1-1.13). There were significant differences in the number of patients with SAEs: in the NS group - 22 (13%) patients, in the A group - 37 (22%) patients (p=0.044, OR=0.53, 95% CI=0.28-0.98). CONCLUSION: The presented results of the FRIDA trial are the first in the world to use a drug based on NS in patients with IS. It has been shown that a single bolus (within 10 s) administration of NS at a standard dose of 10 mg, regardless of body weight, allows to conduct fast, effective and safe thrombolytic therapy in patients with IS within 4.5 h after symptom onset. In further clinical tials of NS, it is planned to expand the therapeutic window beyond 4.5 h after symptom onset in patients with IS.


Assuntos
Isquemia Encefálica , AVC Isquêmico , Metaloendopeptidases , Acidente Vascular Cerebral , Peso Corporal , Isquemia Encefálica/complicações , Isquemia Encefálica/tratamento farmacológico , Fibrinolíticos/uso terapêutico , Humanos , Hemorragias Intracranianas/induzido quimicamente , Hemorragias Intracranianas/complicações , Metaloendopeptidases/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/etiologia , Terapia Trombolítica , Resultado do Tratamento
7.
Toxins (Basel) ; 14(7)2022 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-35878210

RESUMO

Small RNAs (sRNAs) and microRNAs (miRNAs) are small endogenous noncoding single-stranded RNAs that regulate gene expression in eukaryotes. Experiments in mice and humans have revealed that a typical small RNA can affect the expression of a wide range of genes, implying that small RNAs function as global regulators. Here, we used small RNA deep sequencing to investigate how jararhagin, a metalloproteinase toxin produced from the venom of Bothrops jararaca, affected mmu-miRNAs expression in mice 2 hours (Jar 2hrs) and 24 hours (Jar 24hrs) after injection compared to PBS control. The findings revealed that seven mmu-miRNAs were substantially differentially expressed (p value (p (Corr) cut-off 0.05, fold change ≥ 2) at 2 hrs after jararhagin exposure and that the majority of them were upregulated when compared to PBS. In contrast to these findings, a comparison of Jar 24hrs vs. PBS 24hrs demonstrated that the majority of identified mmu-miRNAs were downregulated. Furthermore, the studies demonstrated that mmu-miRNAs can target the expression of several genes involved in the MAPK signaling pathway. The steady antithetical regulation of mmu-miRNAs may correlate with the expression of genes that trigger apoptosis via MAPK in the early stages, and this effect intensifies with time. The findings expand our understanding of the effects of jararhagin on local tissue lesions at the molecular level.


Assuntos
Bothrops , Venenos de Crotalídeos , MicroRNAs , Animais , Bothrops/metabolismo , Venenos de Crotalídeos/metabolismo , Humanos , Metaloendopeptidases/metabolismo , Metaloproteases/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo
8.
Int J Mol Sci ; 23(14)2022 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-35887004

RESUMO

Leishmania tarentolae is a non-pathogenic trypanosomatid isolated from lizards widely used for heterologous protein expression and extensively studied to understand the pathogenic mechanisms of leishmaniasis. The repertoire of leishmanolysin genes was reported to be expanded in L. tarentolae genome, but no proteolytic activity was detected. Here, we analyzed L. tarentolae leishmanolysin proteins from the genome to the structural levels and evaluated the enzymatic activity of the wild-type and overexpressing mutants of leishmanolysin. A total of 61 leishmanolysin sequences were retrieved from the L. tarentolae genome. Five of them were selected for phylogenetic analysis, and for three of them, we built 3D models based on the crystallographic structure of L. major ortholog. Molecular dynamics simulations of these models disclosed a less negative electrostatic potential compared to the template. Subsequently, L. major LmjF.10.0460 and L. tarentolae LtaP10.0650 leishmanolysins were cloned in a pLEXSY expression system into L. tarentolae. Proteins from the wild-type and the overexpressing parasites were submitted to enzymatic analysis. Our results revealed that L. tarentolae leishmanolysins harbor a weak enzymatic activity about three times less abundant than L. major leishmanolysin. Our findings strongly suggest that the less negative electrostatic potential of L. tarentolae leishmanolysin can be the reason for the reduced proteolytic activity detected in this parasite.


Assuntos
Leishmania , Leishmaniose , Parasitos , Animais , Leishmania/genética , Leishmania/metabolismo , Leishmaniose/parasitologia , Metaloendopeptidases/metabolismo , Filogenia
9.
Biomolecules ; 12(7)2022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35883515

RESUMO

Osteoarthritis (OA) is a principal cause of aches and disability worldwide. It is characterized by the inflammation of the bone leading to degeneration and loss of cartilage function. Factors, including diet, age, and obesity, impact and/or lead to osteoarthritis. In the past few years, OA has received considerable scholarly attention owing to its increasing prevalence, resulting in a cumbersome burden. At present, most of the interventions only relieve short-term symptoms, and some treatments and drugs can aggravate the disease in the long run. There is a pressing need to address the safety problems due to osteoarthritis. A disintegrin-like and metalloprotease domain with thrombospondin type 1 repeats (ADAMTS) metalloproteinase is a kind of secretory zinc endopeptidase, comprising 19 kinds of zinc endopeptidases. ADAMTS has been implicated in several human diseases, including OA. For example, aggrecanases, ADAMTS-4 and ADAMTS-5, participate in the cleavage of aggrecan in the extracellular matrix (ECM); ADAMTS-7 and ADAMTS-12 participate in the fission of Cartilage Oligomeric Matrix Protein (COMP) into COMP lyase, and ADAMTS-2, ADAMTS-3, and ADAMTS-14 promote the formation of collagen fibers. In this article, we principally review the role of ADAMTS metalloproteinases in osteoarthritis. From three different dimensions, we explain how ADAMTS participates in all the following aspects of osteoarthritis: ECM, cartilage degeneration, and synovial inflammation. Thus, ADAMTS may be a potential therapeutic target in osteoarthritis, and this article may render a theoretical basis for the study of new therapeutic methods for osteoarthritis.


Assuntos
Proteínas ADAMTS , Osteoartrite , Proteínas ADAMTS/genética , Proteínas ADAMTS/metabolismo , Humanos , Inflamação , Metaloendopeptidases , Osteoartrite/metabolismo , Zinco
10.
Commun Biol ; 5(1): 618, 2022 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-35739262

RESUMO

Pneumonia caused by community-associated Staphylococcus aureus (CA-SA) has high morbidity and mortality, but its pathogenic mechanism remains to be further investigated. Herein, we identify that staphylokinase (SAK) is significantly induced in CA-SA and inhibits biofilm formation in a plasminogen-dependent manner. Importantly, SAK can enhance CA-SA-mediated pneumonia in both wild-type and cathelicidins-related antimicrobial peptide knockout (CRAMP-/-) mice, suggesting that SAK exacerbates pneumonia in a CRAMP-independent manner. Mechanistically, SAK induces pro-inflammatory effects, especially in the priming step of NLRP3 inflammasome activation. Moreover, we demonstrate that SAK can increase K+ efflux, production of reactive oxygen species production, and activation of NF-κB signaling. Furthermore, the NLRP3 inflammasome inhibitor can counteract the effective of SAK induced CA-SA lung infection in mice. Taken together, we speculate that SAK exacerbates CA-SA-induced pneumonia by promoting NLRP3 inflammasome activation, providing new insights into the pathogenesis of highly virulent CA-SA and emphasizes the importance of controlling inflammation in acute pneumonia.


Assuntos
Pneumonia Estafilocócica , Staphylococcus aureus , Animais , Inflamassomos , Metaloendopeptidases , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Pneumonia Estafilocócica/tratamento farmacológico , Staphylococcus aureus/fisiologia
11.
Biomed Pharmacother ; 150: 112970, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35658218

RESUMO

The coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, is a current pandemic that has resulted in nearly 250 million cases and over 5 million deaths. While vaccines have been developed to prevent infection, and most COVID-19 cases end up being fairly light, there are severe cases of COVID-19 that may end up in death, even with adequate healthcare treatment. New options to combat this disease's effects, therefore, could prove to be invaluable in saving lives. Adamalysins are proteins that have several roles in regulating different functions in the human body but are also known to have functions in inflammation. They are also known to have roles in several different diseases, including COVID-19, where ADAM17, in particular, is now well-known to have a prominent role, but also several diseases which include comorbidities that may worsen cases of COVID-19. Therefore, investigating the functions of adamalysins in disease may give us clues to the molecular workings of COVID-19 as well as potentially new therapeutic targets. Understanding these molecular mechanisms may also allow for an understanding of the mechanisms behind the rare severe side effects that occur in response to current COVID-19 vaccines, which may lead to better monitoring measures for people who may be more at risk of developing these side effects. This review investigates the known roles and functions of adamalysins in disease, including what is currently known of their involvement in COVID-19, and how these functions might be involved.


Assuntos
COVID-19 , Vacinas contra COVID-19 , Humanos , Metaloendopeptidases , Pandemias/prevenção & controle , SARS-CoV-2
12.
Artigo em Inglês | MEDLINE | ID: mdl-35772642

RESUMO

Fish embryonic hatching glands (HGs) secrete choriolysin-zymogens, which when activated degrade the chorion, allowing larval exit. Thus, hatching encompasses two dissimilar choriolysin-processes: pre-choriolysis including activated choriolysins accessing the perivitelline space (PVS), followed by choriolysis. Discovery of serine-proteolytic zonase in Atlantic salmon hatching fluid (HF) raises questions concerning its participation in hatching. This work aims to identify salmon choriolysins, and evaluate their role and that of zonase during hatching. Precocious salmon hatching occurs under alkaline conditions, producing an HF containing similar metallo- and serine- proteolytic activities. Purified zonase is selectively inhibited by peFabloc, whose MW (580 Da) allows diffusion through the chorion into the PVS. Without apparent toxicity, brief peFabloc-pretreatment (2 mM) of salmon eggs reduced precocious hatching substantially, compatible with a zonase-relevance for hatching. Atlantic salmon differed from other fishes since their HGs were not immuno-stained by polyclonal antibodies against pike choriolysins. However, cloning and sequencing experiments revealed a single low choriolytic enzyme (LCE) of 69% identity to pike LCE. Similar experiments with high choriolytic enzymes (HCEs) revealed two types (HCE-1 and HCE-2) with respectively 71% and 91% identity to pike HCE-1 & HCE-2. In situ hybridization revealed typical HGs. However, zebrafish-choriolysis is achieved by HCE-class choriolysins alone. Another zebrafish choriolysin (HE2) was not expressed. Peptide-bond hydrolysis by non-choriolytic zonase mimicks HCE-action generating hydrophilic sites for LCE-choriolytic catalysis. Ultimately, precise definitions of choriolytic and pre-choriolytic catalysis requires developmental genetics. Our data are compatible with a complex pre-choriolytic hatching-stage in Atlantic salmon, before LCE-choriolysis degrades the chorion.


Assuntos
Oryzias , Salmo salar , Sequência de Aminoácidos , Animais , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Oryzias/metabolismo , Peptídeo Hidrolases/metabolismo , Salmo salar/metabolismo , Serina/metabolismo , Peixe-Zebra/metabolismo
13.
Artigo em Russo | MEDLINE | ID: mdl-35758961

RESUMO

Currently, five oral anticoagulants have been shown to be effective in preventing recurrent ischemic stroke and/or systemic embolism in patients with non-valvular atrial fibrillation. However, 1.1-2.2% of patients taking oral anticoagulants develop ischemic strokes. The use of oral anticoagulants limits the possibility of systemic thrombolytic therapy, as this is associated with an increased risk of symptomatic hemorrhagic transformation. The exception is cases when, with the help of a specific antagonist, it is possible to neutralize the effect of the anticoagulant in the shortest possible time and achieve normocoagulation. Currently, the Russian Federation allows two drugs for systemic thrombolytic therapy in patients with ischemic stroke in the «therapeutic window¼ up to 4.5 hours from the onset of the disease - recombinant tissue plasminogen activator and non-immunogenic staphylokinase, which showed no less efficacy and safety in the FRIDA study compared to recombinant tissue plasminogen activator. This article describes a clinical case of the first systemic thrombolytic therapy with a non-immunogenic staphylokinase after the use of idarucizumab in a patient taking dabigatran etexilate, followed by thrombectomy.


Assuntos
Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Anticoagulantes/uso terapêutico , Isquemia Encefálica/complicações , Isquemia Encefálica/tratamento farmacológico , Dabigatrana , Fibrinolíticos/uso terapêutico , Humanos , Metaloendopeptidases , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/etiologia , Trombectomia , Terapia Trombolítica/efeitos adversos , Ativador de Plasminogênio Tecidual/uso terapêutico , Resultado do Tratamento
14.
J Food Sci ; 87(7): 3107-3116, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35638323

RESUMO

Enzymatic hydrolysis is the most commonly used method to reduce the antigenicity of milk protein, but free protease is unstable and difficult to recycle after application. In this study, alcalase and neutrase were selected for immobilization on the modified magnetic Fe3 O4 nanoparticles. The reusability of the immobilized enzyme was 68.23% of the total starting activity after 5 recycling batches. The optimal hydrolysis conditions were an enzyme to substrate ratio of 6000 U/g and reaction at 50℃ and pH 8.5 for 3 h. Under these conditions, 22.76% hydrolysis of hydrolysate was achieved, and the antigenicity reduction rates of ß-lactoglobulin and casein were 21.34% and 30.89%, respectively. In addition, 82.75% of the hydrolysate had a molecular weight less than 1 kDa, and free amino acids represented 13.65% of the sample. This result showed that the hydrolysis with immobilized enzyme was similar to that with free enzyme and the immobilized enzyme could be applied to produce hypoallergenic hydrolysate. PRACTICAL APPLICATION: Reduces milk protein allergenicity.


Assuntos
Nanopartículas de Magnetita , Hidrolisados de Proteína , Enzimas Imobilizadas/metabolismo , Hidrólise , Nanopartículas de Magnetita/química , Metaloendopeptidases/metabolismo , Proteínas do Leite/metabolismo , Subtilisinas/metabolismo
15.
BMC Neurol ; 22(1): 200, 2022 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-35637455

RESUMO

BACKGROUND: Hereditary spastic paraplegia 7 (SPG7) is one of the subtypes of autosomal-recessive hereditary spastic paraplegia, which is a clinically heterogeneous neurodegenerative disorder. SPG7 often displays a complicated phenotype, including optic atrophy, ophthalmoparesis, and impaired emotional communication. In the Chinese population, sporadic cases of SPG7 variant-associated spastic ataxia are rarely reported. CASE PRESENTATION: We carefully analysed the clinical features, imaging and genetic tests of two sporadic patients with SPG7, both from the Hebei region of China. One patient presented with progressive bilateral lower limb weakness, spastic-ataxia and no cognitive impairment. Brain MRI revealed mild cerebellar atrophy. Genetic analysis revealed c.1150_1151insCTAC (p.G384Afs*13) frameshift variant and exon1-3 heterozygous deletion. The other patient presented with progressive bilateral lower limb weakness, ataxia, dysarthria and a mild psychosis associated with persecutory delusions, which drew almost no attention, in addition to mild cognitive impairments characterized by a decrease in verbal memory and executive function. Genetic analysis identified two heterozygous variants in the SPG7 gene: c.1150_1151insCTAC (p.G384Afs*13) and c.1496delC (p.Q500Sfs*13). CONCLUSIONS: The c.1496delC (p.Q500Sfs*13) variant in exon 11 has not been reported before. The c.1150_1151insCTAC variant is speculated to be a hotspot variant in the Chinese population. Patients with SPG7 may have cognitive impairments and psychosis, displaying specific characteristics, which should be of concern.


Assuntos
Ataxia Cerebelar , Atrofia Óptica , Paraplegia Espástica Hereditária , Ataxias Espinocerebelares , ATPases Associadas a Diversas Atividades Celulares/genética , Ataxia Cerebelar/genética , Delusões , Humanos , Deficiência Intelectual , Metaloendopeptidases/genética , Espasticidade Muscular , Mutação , Atrofia Óptica/genética , Paraplegia Espástica Hereditária/genética , Ataxias Espinocerebelares/genética
16.
J Microbiol ; 60(7): 727-734, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35614378

RESUMO

Three major proteases, elastase B (LasB), protease IV (PIV), and elastase A (LasA) expressed in Pseudomonas aeruginosa play important roles in infections and pathogeneses. These are activated by a proteolytic cascade initiated by the activation of LasB. In this study, we investigated whether LasB could be inhibited using its propeptide (LasBpp). Although LasA and PIV were inhibited by their propeptides, LasB was not inhibited by purified LasBpp because LasB degraded LasBpp. To address this problem, mutant LasBpp variants were constructed to obtain a mutant LasBpp resistant to LasB degradation. A C-terminal deletion series of LasBpp was tested in vivo, and two positive candidates, T2 and T2-1, were selected. However, both caused growth retardation and were unstably expressed in vivo. Since deleting the C-terminal end of LasBpp significantly affected its stable expression, substitution mutations were introduced at the two amino acids near the truncation site of T2-1. The resulting mutants, LasBppE172D, LasBppG173A, and LasBppE172DG173A, significantly diminished LasB activity when overexpressed in vivo and were stably expressed in MW1, a quorum sensing mutant that does not produce LasB. In vitro analysis showed that purified LasBppE172DG173A inhibited LasB activity to a small extent. Summarizing, C-terminal modification of LasBpp profoundly affected the stable expression of LasBpp, and little enhanced the ability of LasBpp to resist degradation by LasB.


Assuntos
Metaloendopeptidases , Pseudomonas aeruginosa , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Elastase Pancreática/genética , Elastase Pancreática/metabolismo , Peptídeos/antagonistas & inibidores , Peptídeos/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum/genética
19.
Cell ; 185(12): 2148-2163.e27, 2022 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-35584702

RESUMO

Zinc (Zn) is an essential micronutrient and cofactor for up to 10% of proteins in living organisms. During Zn limitation, specialized enzymes called metallochaperones are predicted to allocate Zn to specific metalloproteins. This function has been putatively assigned to G3E GTPase COG0523 proteins, yet no Zn metallochaperone has been experimentally identified in any organism. Here, we functionally characterize a family of COG0523 proteins that is conserved across vertebrates. We identify Zn metalloprotease methionine aminopeptidase 1 (METAP1) as a COG0523 client, leading to the redesignation of this group of COG0523 proteins as the Zn-regulated GTPase metalloprotein activator (ZNG1) family. Using biochemical, structural, genetic, and pharmacological approaches across evolutionarily divergent models, including zebrafish and mice, we demonstrate a critical role for ZNG1 proteins in regulating cellular Zn homeostasis. Collectively, these data reveal the existence of a family of Zn metallochaperones and assign ZNG1 an important role for intracellular Zn trafficking.


Assuntos
Metaloendopeptidases/metabolismo , Zinco , Animais , GTP Fosfo-Hidrolases/metabolismo , Homeostase , Metalochaperonas/metabolismo , Metaloproteínas/genética , Camundongos , Peixe-Zebra/metabolismo , Zinco/metabolismo
20.
AIDS ; 36(10): 1345-1354, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35608117

RESUMO

OBJECTIVE: Investigate the presence of inflammation, endothelial dysfunction and complement activation in patients with HIV-associated thrombotic thrombocytopenic purpura (HIV-TTP) to support the hypothesis that these processes probably contribute to the development of this thrombotic microangiopathy. DESIGN: A prospective, investigational cohort study of 35 consecutive patients diagnosed with HIV-associated TTP presenting to three academic, tertiary care hospitals in Johannesburg, South Africa over 2 years. METHODS: The patients with HIV-TTP received therapeutic plasma therapy and supportive treatment. Demographic data, the results of routine investigations and patient outcomes were recorded. Peripheral blood samples were collected prior to and on completion of plasma therapy and the following additional parameters were assessed at both time points: activity of the von Willebrand factor (VWF) cleaving protease, a-disintegrin-and-metalloproteinase-with-thrombospondin-motifs 13 (ADAMTS-13) and the presence of ADAMTS-13 autoantibodies, levels of pro-inflammatory cytokines, interleukin-6 and tumour necrosis factor-alpha, and two endothelial cell adhesion molecules. Complement activation was assessed by sequential measurement of C3 and C4 as well as levels of the complement inhibitor, factor H. RESULTS: The inflammatory and endothelial activation markers were significantly ( P  < 0.001) elevated in the cohort of patients prior to plasma therapy compared with levels on discharge. Complement was activated and normalized with therapy. The ADAMTS-13 levels were reduced with significant auto-antibodies to this protease at presentation. CONCLUSION: Inflammation in HIV mediates endothelial damage and complement activation. This study proposes that these processes are probably contributory to the development of HIV-TTP, which can therefore be characterized in part as a complementopathy, resembling TTP-like syndrome.


Assuntos
Infecções por HIV , Púrpura Trombocitopênica Trombótica , Proteína ADAMTS13 , Estudos de Coortes , Infecções por HIV/complicações , Humanos , Inflamação , Metaloendopeptidases/metabolismo , Estudos Prospectivos , Púrpura Trombocitopênica Trombótica/complicações , Púrpura Trombocitopênica Trombótica/terapia , África do Sul , Fator de von Willebrand
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