RESUMO
Recent studies have demonstrated that scorpion venom contains unique two-domain peptides with the peculiarity of possessing different functions, i.e. neurotoxic and cytolytic activities. Here we report systematic characterization of a new two-domain peptide (named MeuTXKbeta1) belonging to the TsTXKbeta molecular subfamily from the scorpion Mesobuthus eupeus by molecular cloning, biochemical purification, recombinant expression, functional assays, CD and NMR studies. Its full-length bioactive form as well as 1-21 and 22-72 fragments (named N(1-21) and C(22-72), respectively) was produced in Escherichia coli by an on-column refolding approach. Recombinant peptide (rMeuTXKbeta1) exhibited a low affinity for K(+) channels and cytolytic effects against bacteria and several eukaryotic cells. N(1-21) was found to preserve anti-Plasmodium activity in contrast to haemolytic activity, whereas C(22-72) retains these two activities. Circular dichroism analysis demonstrates that rMeuTXKbeta1 presents a typical scorpion toxin scaffold in water and its alpha-helical content largely increases in a membrane-mimicking environment, consistent with the NMR structure of N(1-21) and an ab initio structure model of MeuTXKbeta1 predicted using I-TASSER algorithm. Our structural and functional data clearly indicate an evolutionary link between TsTXKbeta-related peptides and antiparasitic scorpines which both comprise the betaSPN (beta-KTxs and scorpines) family.
Assuntos
Neurotoxinas/química , Neurotoxinas/toxicidade , Canais de Potássio/química , Venenos de Escorpião/química , Venenos de Escorpião/toxicidade , Sequência de Aminoácidos , Animais , Bactérias/efeitos dos fármacos , Sequência de Bases , Primers do DNA/genética , Hemólise/efeitos dos fármacos , Técnicas In Vitro , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Neurotoxinas/genética , Neurotoxinas/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Plasmodium berghei/efeitos dos fármacos , Canais de Potássio/genética , Canais de Potássio/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Venenos de Escorpião/genética , Venenos de Escorpião/metabolismo , Escorpiões/química , Escorpiões/genética , Homologia de Sequência de Aminoácidos , Sinaptossomos/metabolismoRESUMO
Potassium channel tetramerization domain (KCTD) proteins contain a bric-a-brac, tramtrak and broad complex (BTB) domain that is most similar to the tetramerization domain (T1) of voltage-gated potassium channels. Some BTB-domain-containing proteins have been shown recently to participate as substrate-specific adaptors in multimeric cullin E3 ligase reactions by recruiting proteins for ubiquitination and subsequent degradation by the proteasome. Twenty-two KCTD proteins have been found in the human genome, but their functions are largely unknown. In this study, we have characterized KCTD5, a new KCTD protein found in the cytosol of cultured cell lines. The expression of KCTD5 was upregulated post-transcriptionally in peripheral blood lymphocytes stimulated through the T-cell receptor. KCTD5 interacted specifically with cullin3, bound ubiquitinated proteins, and formed oligomers through its BTB domain. Analysis of the interaction with cullin3 showed that, in addition to the BTB domain, some amino acids in the N-terminus of KCTD5 are required for binding to cullin3. These findings suggest that KCTD5 is a substrate-specific adaptor for cullin3-based E3 ligases.
Assuntos
Proteínas Culina/metabolismo , Canais de Potássio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Dados de Sequência Molecular , Canais de Potássio/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
In addition to its wide role in metabolism, iron in insects has been implicated in vitellogenesis and the immune response. The NRAMP family comprises a well-conserved family of divalent cation transporters in metazoans. To gain insight on the role of NRAMP in Anopheles albimanus, we cloned a cDNA encoding a 571-residue protein (AnaNRAMP) with the structural features defining the NRAMP family. AnaNRAMP mRNA induced (59)Fe(2+) incorporation when injected into Xenopus oocytes. Western blot analysis revealed that AnaNRAMP is expressed in the head, midgut and at high levels in Malpighian tubules of unfed female mosquito. Upon blood feeding, AnaNRAMP levels were reduced in the midgut whereas they increased in the Malpighian tubules. Using immuno-localization by transmission electron microscopy, AnaNRAMP was localized in the membrane of the intra-cellular concretions or spherites of the Malpighian tubule principal cells. Taken together, our results suggest an important role of AnaNRAMP in iron transport and indicate a role of the mosquito Malpighian tubule as an important organ for iron homeostasis.
Assuntos
Anopheles/química , Proteínas de Transporte de Cátions/genética , Ferro/metabolismo , Sequência de Aminoácidos , Animais , Anopheles/genética , Anopheles/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Clonagem Molecular , DNA Complementar , Sistema Digestório/metabolismo , Feminino , Masculino , Túbulos de Malpighi/metabolismo , Dados de Sequência Molecular , Oócitos/metabolismo , Filogenia , Alinhamento de Sequência , XenopusRESUMO
Penile squamous cell carcinoma (SCC) is primarily treated by surgical resection. Locally advanced and metastatic diseases require a multidisciplinary treatment approach. However, mortality and morbidity remain high, and novel molecular and immunotherapeutic targets are actively being sought. We investigated the expression of immune-checkpoint markers in penile cancers. Fifty-three invasive penile SCCs diagnosed between 1985 and 2013 were retrieved from our surgical pathology archives. Representative formalin-fixed, paraffin-embedded archival blocks were used for the construction of 2 high-density tissue microarrays. Tissue microarrays were stained with immunohistochemistry for PD-L1, FOXP3, CD8, and Ki-67. PD-L1 was investigated using rabbit monoclonal anti-PD-L1 antibody (Cell Signaling, Boston, MA; E1L3N, 1:100). Overall, 21 (40%) of 53 penile SCCs had positive PD-L1 expression. PD-L1 was expressed by a significant proportion of advanced penile SCC. Forty-four percent (15/34) of stage pT2 or more SCC and 38% (6/16) of tumors with lymph node metastasis were positive for PD-L1. PD-L1 expression did not correlate with patient age, tumor location, histologic subtype, tumor stage, anatomic depth of invasion, or tumor grade. FOXP3 expression in tumoral immune cells was found in 26 (49%) of 53 cases. FOXP3 expression in stromal immune cells correlated with tumor thickness (P = .0086). The ratio of CD8/FOXP3 was greater than 1 in 62% of cases in tumor-infiltrating immune cells and 34% of cases in stromal immune cells. Our current study is the largest to assess expression of PD-L1 in a clinically well-annotated North American cohort of penile SCC. Our findings support a rationale for targeting immune-checkpoint inhibitor pathways in advanced penile SCC.
Assuntos
Antígeno B7-H1/análise , Biomarcadores Tumorais/análise , Linfócitos T CD8-Positivos/imunologia , Carcinoma de Células Escamosas/imunologia , Fatores de Transcrição Forkhead/análise , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Penianas/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Baltimore , Biópsia , Linfócitos T CD8-Positivos/patologia , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/secundário , Carcinoma de Células Escamosas/terapia , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Metástase Linfática , Linfócitos do Interstício Tumoral/patologia , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Neoplasias Penianas/mortalidade , Neoplasias Penianas/patologia , Neoplasias Penianas/terapia , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Células Estromais/imunologia , Células Estromais/patologia , Análise Serial de Tecidos , Microambiente TumoralRESUMO
Inflammatory myofibroblastic tumor (IMT) is a rare neoplasm. Approximately 50 % of IMTs show an anaplastic lymphoma kinase (ALK) gene fusion resulting in ALK overexpression on immunohistochemistry (IHC). A novel anti-ALK monoclonal antibody (D5F3) has been suggested to be of superior sensitivity to the ALK1 antibody which is currently used. We compared the performance of D5F3 in detecting ALK protein expression in IMTs from various anatomic sites compared to the currently utilized ALK1. We selected 25 IMTs from our surgical pathology files (2005-2015). The novel rabbit monoclonal anti-human CD246 (clone D5F3) and the currently used mouse monoclonal anti-human CD246 (clone ALK1) were used for immunohistochemical staining (IHC) in an automated slide stainer. The percentage of immunoreactive tumor cells (0, <5 %, 5-50 %, >50 %) and cytoplasmic staining intensity (graded 0-3) were assessed and compared between the two antibodies. Fluorescence in situ hybridization (FISH) studies for ALK gene rearrangement were performed on 11 tumors. D5F3 antibody stained 76 % and ALK1 antibody stained 72 % of IMTs (p = 0.747). Compared to staining with ALK1, D5F3 stained a higher proportion of cases extensively (>50 % cells) (76 vs. 28 %, p < 0.001) and with high intensity (grade 3 76 % vs 0; p < 0.001). FISH and IHC findings (for both antibodies) were concordant in 9/10 (90 %) IMTs, in which results were informative. The novel anti-ALK rabbit monoclonal antibody (D5F3 clone) demonstrates superior overall performance in term of intensity and extent of staining of ALK protein in IMT. We found IHC staining with both antibody clones to correlate equally well with FISH results for detection of ALK rearrangement.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/metabolismo , Receptores Proteína Tirosina Quinases/imunologia , Adulto , Quinase do Linfoma Anaplásico , Anticorpos/imunologia , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Feminino , Fusão Gênica/genética , Rearranjo Gênico/genética , Humanos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
Extended-spectrum Beta (beta)-lactamases (ESBLs) have emerged as an important mechanism of resistance to B-lactam antibiotics in gram-negative bacteria (GNB). They are enzymes that hydrolyze older B-lactam antibiotics as well as broad-spectrum cephalosporins and monobactams. ESBL producers have been reported in many bacteria but special attention has been paid to the ones in E.coli and Klebsiella spp. Detection of the ESBLs by the clinical laboratory is a special challenge. Surveillance to monitor resistance is important to decide when detection of ESBLs must be started. This study determined the prevalence of ESBL producers in the strains E.coli and K.pneumoniae at the San Juan VA Medical Center, and characterized their phenotypes to evaluate the importance to identify these bacteria as a standard routine procedure in the institution. All E.coli and K.pneumoniae isolated from Jan 1 to Mar 31, 2003 were evaluated according to National Committee for Clinical Laboratory Standards (NCCLS) screening criteria for suspected ESBL producers. Phenotypic confirmation of the ESBL production was performed using the Etest method. A total of 112/253 (44%) E.coli and 72/137 (53%) K.pneumoniae were identified as suspected ESBL producers. Etest was performed in 60% of the E.coli and 57% of the K.pneumoniae suspected to be ESBL producers. The overall ESBL prevalence for E.coli was 25% and in K.pneumoniae was 26%. Most E.coli ESBL-producers were from urine while the K.pneumoniae were from sputum. ESBL-producers were isolated from different sources including pleural and synovial fluids, blood, and skin besides urine and sputum. According to susceptibility results, the most reliable antibiotic in predicting a negative ESBL was cefpodoxime (CPD), and in the strains studied, the ESBL producers were consistently resistant to aztreonam (ATM). A large proportion (95%) of ESBL producing K.pneumoniae were susceptible to cefepime (CEP). Of the ESBL producing E.coli, 24% were susceptible. In the case of E.coli ESBLproducers, Cefepime can be considered as a therapeutic option if susceptibilities are available. Automated identification and sensitivity systems are valid alternatives for routine evaluation of B-lactam resistance but when increased resistance is documented in GNB and/or ESBL prevalence is high, ESBL detection should be performed. All confirmed ESBL producers should be reported resistant to all penicillins, cephalosporins, and aztreonam in spite of having susceptible ranges with routine susceptibility tests. Inappropriate antibiotic selection in infections caused by these organisms is associated with treatment failures, poor clinical outcomes, increased mortality and longer hospital stays.
Assuntos
Infecções por Escherichia coli/microbiologia , Escherichia coli/enzimologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/enzimologia , beta-Lactamases/análise , Antibacterianos/uso terapêutico , Técnicas de Tipagem Bacteriana , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/tratamento farmacológico , Hospitais de Veteranos/estatística & dados numéricos , Humanos , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Fenótipo , Porto Rico , Resistência beta-Lactâmica , beta-Lactamas/uso terapêuticoRESUMO
YopH is an exceptionally active tyrosine phosphatase that is essential for virulence of Yersinia pestis, the bacterium causing plague. YopH breaks down signal transduction mechanisms in immune cells and inhibits the immune response. Only a few substrates for YopH have been characterized so far, for instance p130Cas and Fyb, but in view of YopH potency and the great number of proteins involved in signalling pathways it is quite likely that more proteins are substrates of this phosphatase. In this respect, we show here YopH interaction with several proteins not shown before, such as Gab1, Gab2, p85, and Vav and analyse the domains of YopH involved in these interactions. Furthermore, we show that Gab1, Gab2 and Vav are not dephosphorylated by YopH, in contrast to Fyb, Lck, or p85, which are readily dephosphorylated by the phosphatase. These data suggests that YopH might exert its actions by interacting with adaptors involved in signal transduction pathways, what allows the phosphatase to reach and dephosphorylate its susbstrates.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Yersinia pestis/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Linhagem Celular , Humanos , Ativação Linfocitária , Ligação Proteica , Proteínas Tirosina Fosfatases/genética , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Transdução de Sinais/fisiologia , Especificidade por Substrato , Linfócitos T/imunologia , Yersinia pestis/genética , Yersinia pestis/patogenicidadeRESUMO
In this work, we describe the original characterization of peptides and proteins present in the skin secretions of the Mexican amphibian Hyla eximia. To this purpose, a novel water/dark extraction method, as well as the classic electrical stimulation procedure, was applied in order to extract the skin secretion. Two novel antimicrobial peptides He-1 and He-2 were sequenced. In addition, a molecular mass fingerprint revealed more than one hundred different molecules. Eight peptides in homogeneous form were assayed against five species of bacteria. Thereafter, the peptide He-2 demonstrated high antiparasitic activity against ookinete forms of malaria parasites at low concentration.
Assuntos
Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Anuros , Secreções Corporais/química , Pele/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias , Proliferação de Células/efeitos dos fármacos , Fracionamento Químico/métodos , México , Testes de Sensibilidade Microbiana , Plasmodium berghei , Análise de Sequência de ProteínaRESUMO
We studied effects of dehydration and rehydration on germination of seeds of six mature tropical rain forest species-Cupania glabra Swartz, Cymbopetalum baillonii Fries, Poulsenia armata (Miq.) Standl., Stemmadenia donnell-smithii (Rose) Woodson, Rheedia edulis Triana & Planch. and an understory palm Chamaedorea alternans H. Wendl.-from Veracruz, México. Before the seeds were sown, their water content was reduced by 0 (control), 30, 54 and 72% of their original water content. Dehydration affects the ability of seeds to rehydrate, as well as the rate and final percentage of germination when seeds are subsequently rehydrated. Seed survival and germination behavior showed three patterns: (1) C. baillonii, P. armata and S. donnell-smithii had greater tolerance to seed dehydration than C. glabra, C. alternans and R. edulis; (2) partial dehydration enhanced germinability of C. glabra and C. baillonii seeds; and (3) partial dehydration of C. alternans and R. edulis seeds resulted in delayed or sporadic germination. A relationship was found between the effects of dehydration on germination and the seasonality of seed production.
RESUMO
We address the specific role of cytoplasmic Ca(2+) overload as a cell death trigger by expressing a receptor-operated specific Ca(2+) channel, vanilloid receptor subtype 1 (VR1), in Jurkat cells. Ca(2+) uptake through the VR1 channel, but not capacitative Ca(2+) influx stimulated by the muscarinic type 1 receptor, induced sustained intracellular [Ca(2+)] rises, exposure of phosphatidylserine, and cell death. Ca(2+) influx was necessary and sufficient to induce mitochondrial damage, as assessed by opening of the permeability transition pore and collapse of the mitochondrial membrane potential. Ca(2+)-induced cell death was inhibited by ruthenium red, protonophore carbonyl cyanide m-chlorophenylhydrazone, or cyclosporin A treatment, as well as by Bcl-2 expression, indicating that this process requires mitochondrial calcium uptake and permeability transition pore opening. Cell death occurred without caspase activation, oligonucleosomal/50-kilobase pair DNA cleavage, or release of cytochrome c or apoptosis inducer factor from mitochondria, but it required oxidative/nitrative stress. Thus, Ca(2+) influx triggers a distinct program of mitochondrial dysfunction leading to paraptotic cell death, which does not fulfill the criteria for either apoptosis or necrosis.