RESUMO
Phosphotyrosine (pTyr) signaling has evolved into a key cell-to-cell communication system. Activated receptor tyrosine kinases (RTKs) initiate several pTyr-dependent signaling networks by creating the docking sites required for the assembly of protein complexes. However, the mechanisms leading to network disassembly and its consequence on signal transduction remain essentially unknown. We show that activated RTKs terminate downstream signaling via the direct phosphorylation of an evolutionarily conserved Tyr present in most SRC homology (SH) 3 domains, which are often part of key hub proteins for RTK-dependent signaling. We demonstrate that the direct EPHA4 RTK phosphorylation of adaptor protein NCK SH3s at these sites results in the collapse of signaling networks and abrogates their function. We also reveal that this negative regulation mechanism is shared by other RTKs. Our findings uncover a conserved mechanism through which RTKs rapidly and reversibly terminate downstream signaling while remaining in a catalytically active state on the plasma membrane.
Assuntos
Receptores Proteína Tirosina Quinases/fisiologia , Receptor EphA4/metabolismo , Domínios de Homologia de src/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Comunicação Celular , Drosophila/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligantes , Proteínas Oncogênicas/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Tirosina/metabolismoRESUMO
BACKGROUND: Senescent cells (SCs) are involved in proliferative disorders, but their role in pulmonary hypertension remains undefined. We investigated SCs in patients with pulmonary arterial hypertension and the role of SCs in animal pulmonary hypertension models. METHODS: We investigated senescence (p16, p21) and DNA damage (γ-H2AX, 53BP1) markers in patients with pulmonary arterial hypertension and murine models. We monitored p16 activation by luminescence imaging in p16-luciferase (p16LUC/+) knock-in mice. SC clearance was obtained by a suicide gene (p16 promoter-driven killer gene construct in p16-ATTAC mice), senolytic drugs (ABT263 and cell-permeable FOXO4-p53 interfering peptide [FOXO4-DRI]), and p16 inactivation in p16LUC/LUC mice. We investigated pulmonary hypertension in mice exposed to normoxia, chronic hypoxia, or hypoxia+Sugen, mice overexpressing the serotonin transporter (SM22-5-HTT+), and rats given monocrotaline. RESULTS: Patients with pulmonary arterial hypertension compared with controls exhibited high lung p16, p21, and γ-H2AX protein levels, with abundant vascular cells costained for p16, γ-H2AX, and 53BP1. Hypoxia increased thoracic bioluminescence in p16LUC/+ mice. In wild-type mice, hypoxia increased lung levels of senescence and DNA-damage markers, senescence-associated secretory phenotype components, and p16 staining of pulmonary endothelial cells (P-ECs, 30% of lung SCs in normoxia), and pulmonary artery smooth muscle cells. SC elimination by suicide gene or ABT263 increased the right ventricular systolic pressure and hypertrophy index, increased vessel remodeling (higher dividing proliferating cell nuclear antigen-stained vascular cell counts during both normoxia and hypoxia), and markedly decreased lung P-ECs. Pulmonary hemodynamic alterations and lung P-EC loss occurred in older p16LUC/LUC mice, wild-type mice exposed to Sugen or hypoxia+Sugen, and SM22-5-HTT+ mice given either ABT263 or FOXO4-DRI, compared with relevant controls. The severity of monocrotaline-induced pulmonary hypertension in rats was decreased slightly by ABT263 for 1 week but was aggravated at 3 weeks, with loss of P-ECs. CONCLUSIONS: Elimination of senescent P-ECs by senolytic interventions may worsen pulmonary hemodynamics. These results invite consideration of the potential impact on pulmonary vessels of strategies aimed at controlling cell senescence in various contexts.
Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Camundongos , Ratos , Animais , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Células Endoteliais/metabolismo , Monocrotalina/metabolismo , Senoterapia , Artéria Pulmonar , Hipertensão Pulmonar Primária Familiar/metabolismo , Hipóxia/metabolismo , Senescência Celular , Fatores de Transcrição Forkhead/metabolismoRESUMO
Choline contributes to the biogenesis of methyl groups, neurotransmitters, and cell membranes. Our genome-wide association study (GWAS) of circulating choline in 2228 college students found that alleles in SLC25A48 (rs6596270) influence choline concentrations in men (p = 9.6 × 10-8), but not women. Previously, the subcellular location and function of SLC25A48 were unknown. Using super-resolution immunofluorescence microscopy, we localized SLC25A48 to the inner mitochondrial membrane. Our results suggest that SLC25A48 transports choline across the inner mitochondrial membrane.
RESUMO
Proximal fifth metatarsal fractures are the most common foot fractures in children. Attempts to classify these injuries are misapplied and inadequately predict outcomes. This is the first study to identify factors associated with healing in pediatric fifth metatarsal fractures. In this retrospective cohort study (N = 305), proximal fifth metatarsal fractures were classified on radiographs by location on the bone, alignment (transverse or oblique), displacement (>2 mm), and completion through the bone. Based on the literature, they were secondarily sorted by category: apophyseal, intra-articular metaphyseal, extra-articular metaphyseal, and diaphyseal. Primary outcomes included times to healing, indicated by clinical symptoms, immobilization, and return to sports, as well as radiographic callus formation, bridging, and remodeling. Healing times were compared by ANOVA and linear regression. Location had a significant effect on times of immobilization and return to sports, but alignment, displacement, and completion were not associated with healing. When re-classified, the categories were also associated with immobilization and return to sports. Apophyseal fractures healed fastest and diaphyseal fractures required the most time to heal. There was no difference between extra- and intra-articular fractures. For every year of age, symptoms resolved about 2 days sooner. Neither gender nor body mass index (BMI) was positively or negatively associated with healing times. In conclusion, classifying fractures by apophyseal, metaphyseal, and diaphyseal is the most concise, accurate, and useful system. This is the largest series of nonoperatively treated proximal fifth metatarsal fractures in children and a robust standard to which surgical management can be compared.
Assuntos
Traumatismos do Pé , Fraturas Ósseas , Ossos do Metatarso , Humanos , Criança , Recém-Nascido , Ossos do Metatarso/cirurgia , Estudos Retrospectivos , Fraturas Ósseas/diagnóstico por imagem , Fraturas Ósseas/terapia , Pé , Traumatismos do Pé/terapia , Traumatismos do Pé/cirurgiaRESUMO
Cells enter senescence, a state of stable proliferative arrest, in response to a variety of cellular stresses, including telomere erosion, DNA damage, and oncogenic signaling, which acts as a barrier against malignant transformation in vivo. To identify genes controlling senescence, we conducted an unbiased screen for small hairpin RNAs that extend the life span of primary human fibroblasts. Here, we report that knocking down the chemokine receptor CXCR2 (IL8RB) alleviates both replicative and oncogene-induced senescence (OIS) and diminishes the DNA-damage response. Conversely, ectopic expression of CXCR2 results in premature senescence via a p53-dependent mechanism. Cells undergoing OIS secrete multiple CXCR2-binding chemokines in a program that is regulated by the NF-kappaB and C/EBPbeta transcription factors and coordinately induce CXCR2 expression. CXCR2 upregulation is also observed in preneoplastic lesions in vivo. These results suggest that senescent cells activate a self-amplifying secretory network in which CXCR2-binding chemokines reinforce growth arrest.
Assuntos
Senescência Celular , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Transdução de Sinais , Adenocarcinoma/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Dano ao DNA , Regulação para Baixo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Ligantes , Neoplasias Pulmonares/metabolismo , Camundongos , NF-kappa B/metabolismo , Lesões Pré-Cancerosas/metabolismo , Interferência de RNA , Receptores de Interleucina-8A/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Coronavirus disease 2019 (COVID-19) convalescent plasma has emerged as a promising therapy and has been granted Emergency Use Authorization by the US Food and Drug Administration for hospitalized COVID-19 patients. We recently reported results from interim analysis of a propensity score-matched study suggesting that early treatment of COVID-19 patients with convalescent plasma containing high-titer anti-spike protein receptor binding domain (RBD) IgG significantly decreases mortality. We herein present results from a 60-day follow-up of a cohort of 351 transfused hospitalized patients. Prospective determination of enzyme-linked immunosorbent assay anti-RBD IgG titer facilitated selection and transfusion of the highest titer units available. Retrospective analysis by the Ortho VITROS IgG assay revealed a median signal/cutoff ratio of 24.0 for transfused units, a value far exceeding the recent US Food and Drug Administration-required cutoff of 12.0 for designation of high-titer convalescent plasma. With respect to altering mortality, our analysis identified an optimal window of 44 hours after hospitalization for transfusing COVID-19 patients with high-titer convalescent plasma. In the aggregate, the analysis confirms and extends our previous preliminary finding that transfusion of COVID-19 patients soon after hospitalization with high-titer anti-spike protein RBD IgG present in convalescent plasma significantly reduces mortality.
Assuntos
COVID-19/mortalidade , COVID-19/terapia , Imunoglobulina G/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Feminino , Seguimentos , Hospitalização , Humanos , Imunização Passiva , Estimativa de Kaplan-Meier , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Pontuação de Propensão , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Risco , SARS-CoV-2 , Resultado do Tratamento , Soroterapia para COVID-19RESUMO
The biological and clinical significance of the p.E88del variant in the transcobalamin receptor, CD320, is unknown. This allele is annotated in ClinVar as likely benign, pathogenic, and of uncertain significance. To determine functional consequence and clinical relevance of this allele, we employed cell culture and genetic association studies. Fibroblasts from 16 CD320 p.E88del homozygotes exhibited reduced binding and uptake of cobalamin. Complete ascertainment of newborns with transiently elevated C3 (propionylcarnitine) in New York State demonstrated that homozygosity for CD320 p.E88del was over-represented (7/348, p < 6 × 10-5 ). Using population data, we estimate that ~85% of the p.E88del homozygotes born in the same period did not have elevated C3, suggesting that cobalamin metabolism in the majority of these infants with this genotype is unaffected. Clinical follow-up of 4/9 homozygous individuals uncovered neuropsychological findings, mostly in speech and language development. None of these nine individuals exhibited perturbation of cobalamin metabolism beyond the newborn stage even during periods of acute illness. Newborns homozygous for this allele in the absence of other factors are at low risk of requiring clinical intervention, although more studies are required to clarify the natural history of various CD320 variants across patient populations.
Assuntos
Receptores de Superfície Celular , Transcobalaminas , Antígenos CD , Estudos de Associação Genética , Humanos , Lactente , Recém-Nascido , Receptores de Superfície Celular/genética , Transcobalaminas/genética , Transcobalaminas/metabolismo , Vitamina B 12/metabolismoRESUMO
BACKGROUND: Mitochondrial nucleoside diphosphate kinase (NDPK-D, NME4, NM23-H4) is a multifunctional enzyme mainly localized in the intermembrane space, bound to the inner membrane. RESULTS: We constructed loss-of-function mutants of NDPK-D, lacking either NDP kinase activity or membrane interaction and expressed mutants or wild-type protein in cancer cells. In a complementary approach, we performed depletion of NDPK-D by RNA interference. Both loss-of-function mutations and NDPK-D depletion promoted epithelial-mesenchymal transition and increased migratory and invasive potential. Immunocompromised mice developed more metastases when injected with cells expressing mutant NDPK-D as compared to wild-type. This metastatic reprogramming is a consequence of mitochondrial alterations, including fragmentation and loss of mitochondria, a metabolic switch from respiration to glycolysis, increased ROS generation, and further metabolic changes in mitochondria, all of which can trigger pro-metastatic protein expression and signaling cascades. In human cancer, NME4 expression is negatively associated with markers of epithelial-mesenchymal transition and tumor aggressiveness and a good prognosis factor for beneficial clinical outcome. CONCLUSIONS: These data demonstrate NME4 as a novel metastasis suppressor gene, the first localizing to mitochondria, pointing to a role of mitochondria in metastatic dissemination.
Assuntos
Neoplasias , Núcleosídeo-Difosfato Quinase , Animais , Membranas Intracelulares , Camundongos , Mitocôndrias , Nucleosídeo NM23 Difosfato Quinases/genética , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Nucleosídeo Difosfato Quinase D/metabolismo , Núcleosídeo-Difosfato Quinase/genética , Núcleosídeo-Difosfato Quinase/metabolismoRESUMO
In response to many stresses, such as oncogene activation or DNA damage, cells can enter cellular senescence, a state of proliferation arrest accompanied by a senescence-associated secretory phenotype (SASP). Cellular senescence plays a key role in many physiopathological contexts, including cancer, aging and aging-associated diseases, therefore, it is critical to understand how senescence is regulated. Calcium ions (Ca2+) recently emerged as pivotal regulators of cellular senescence. However, how Ca2+ levels are controlled during this process is barely known. Here, we report that intracellular Ca2+ contents increase in response to many senescence inducers in immortalized human mammary epithelial cells (HMECs) and that expression of calbindin 1 (CALB1), a Ca2+-binding protein, is upregulated in this context, through the Ca2+-dependent calcineurin/NFAT pathway. We further show that overexpression of CALB1 buffers the rise in intracellular Ca2+ levels observed in senescent cells. Finally, we suggest that increased expression of Ca2+-binding proteins calbindins is a frequent mark of senescent cells. This work thus supports that, together with Ca2+channels, Ca2+-binding proteins modulate Ca2+ levels and flux during cellular senescence. This opens potential avenues of research to better understand the role of Ca2+ and of Ca2+-binding proteins in regulating cellular senescence.
Assuntos
Envelhecimento , Calbindina 1 , Cálcio , Senescência Celular , Calbindina 1/metabolismo , Cálcio/metabolismo , Dano ao DNA , Células Epiteliais/metabolismo , HumanosRESUMO
BACKGROUND: Cell senescence is a key process in age-associated dysfunction and diseases, notably chronic obstructive pulmonary disease (COPD). We previously identified phospholipase A2 receptor 1 (PLA2R1) as a positive regulator of cell senescence acting via Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling. Its role in pathology, however, remains unknown. Here, we assessed PLA2R1-induced senescence in COPD and lung emphysema pathogenesis. METHODS: We assessed cell senescence in lungs and cultured lung cells from patients with COPD and controls subjected to PLA2R1 knockdown, PLA2R1 gene transduction and treatment with the JAK1/2 inhibitor ruxolitinib. To assess whether PLA2R1 upregulation caused lung lesions, we developed transgenic mice overexpressing PLA2R1 (PLA2R1-TG) and intratracheally injected wild-type mice with a lentiviral vector carrying the Pla2r1 gene (LV-PLA2R1 mice). RESULTS: We found that PLA2R1 was overexpressed in various cell types exhibiting senescence characteristics in COPD lungs. PLA2R1 knockdown extended the population doubling capacity of these cells and inhibited their pro-inflammatory senescence-associated secretory phenotype (SASP). PLA2R1-mediated cell senescence in COPD was largely reversed by treatment with the potent JAK1/2 inhibitor ruxolitinib. Five-month-old PLA2R1-TG mice exhibited lung cell senescence, and developed lung emphysema and lung fibrosis together with pulmonary hypertension. Treatment with ruxolitinib induced reversal of lung emphysema and fibrosis. LV-PLA2R1-treated mice developed lung emphysema within 4â weeks and this was markedly attenuated by concomitant ruxolitinib treatment. CONCLUSIONS: Our data support a major role for PLA2R1 activation in driving lung cell senescence and lung alterations in COPD. Targeting JAK1/2 may represent a promising therapeutic approach for COPD.
Assuntos
Enfisema , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Animais , Senescência Celular , Humanos , Pulmão , Camundongos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Receptores da Fosfolipase A2RESUMO
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has spread globally, and proven treatments are limited. Transfusion of convalescent plasma collected from donors who have recovered from COVID-19 is among many approaches being studied as potentially efficacious therapy. We are conducting a prospective, propensity score-matched study assessing the efficacy of COVID-19 convalescent plasma transfusion versus standard of care as treatment for severe and/or critical COVID-19. We present herein the results of an interim analysis of 316 patients enrolled at Houston Methodist hospitals from March 28 to July 6, 2020. Of the 316 transfused patients, 136 met a 28-day outcome and were matched to 251 non-transfused control COVID-19 patients. Matching criteria included age, sex, body mass index, comorbidities, and baseline ventilation requirement 48 hours from admission, and in a second matching analysis, ventilation status at day 0. Variability in the timing of transfusion relative to admission and titer of antibodies of plasma transfused allowed for analysis in specific matched cohorts. The analysis showed a significant reduction (P = 0.047) in mortality within 28 days, specifically in patients transfused within 72 hours of admission with plasma with an anti-spike protein receptor binding domain titer of ≥1:1350. These data suggest that treatment of COVID-19 with high anti-receptor binding domain IgG titer convalescent plasma is efficacious in early-disease patients.
Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/mortalidade , Plasma/imunologia , Pneumonia Viral/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Transfusão de Componentes Sanguíneos/métodos , COVID-19 , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/terapia , Infecções por Coronavirus/virologia , Feminino , Humanos , Imunização Passiva/mortalidade , Masculino , Pessoa de Meia-Idade , Pandemias , Plasma/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Estudos Prospectivos , SARS-CoV-2 , Soroterapia para COVID-19RESUMO
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used with varying degrees of success to treat severe microbial infections for >100 years. Patients (n = 25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28, 2020, to April 14, 2020. Patients were transfused with convalescent plasma, obtained from donors with confirmed severe acute respiratory syndrome coronavirus 2 infection who had recovered. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 after transfusion. Clinical improvement was assessed on the basis of a modified World Health Organization six-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains. At day 7 after transfusion with convalescent plasma, nine patients had at least a one-point improvement in clinical scale, and seven of those were discharged. By day 14 after transfusion, 19 (76%) patients had at least a one-point improvement in clinical status, and 11 were discharged. No adverse events as a result of plasma transfusion were observed. Whole genome sequencing data did not identify a strain genotype-disease severity correlation. The data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease.
Assuntos
Infecções por Coronavirus/terapia , Pneumonia Viral/terapia , Adulto , Idoso , Betacoronavirus/genética , COVID-19 , Feminino , Humanos , Imunização Passiva , Aplicação de Novas Drogas em Teste , Masculino , Pessoa de Meia-Idade , Pandemias , SARS-CoV-2 , Texas , Sequenciamento Completo do Genoma , Adulto Jovem , Soroterapia para COVID-19RESUMO
The proper tissue-specific regulation of gene expression is essential for development and homeostasis in metazoans. However, the illegitimate expression of normally tissue-restricted genes-like testis- or placenta-specific genes-is frequently observed in tumors; this promotes transformation, but also allows immunotherapy. Two important questions are: how is the expression of these genes controlled in healthy cells? And how is this altered in cancer? To address these questions, we used an unbiased approach to test the ability of 350 distinct genetic or epigenetic perturbations to induce the illegitimate expression of over 40 tissue-restricted genes in primary human cells. We find that almost all of these genes are remarkably resistant to reactivation by a single alteration in signaling pathways or chromatin regulation. However, a few genes differ and are more readily activated; one is the placenta-expressed gene ADAM12, which promotes invasion. Using cellular systems, an animal model, and bioinformatics, we find that a non-canonical but druggable TGF-ß/KAT2A/TAK1 axis controls ADAM12 induction in normal and cancer cells. More broadly, our data show that illegitimate gene expression in cancer is an heterogeneous phenomenon, with a few genes activatable by simple events, and most genes likely requiring a combination of events to become reactivated.
Assuntos
Regulação da Expressão Gênica/genética , Neoplasias/genética , Especificidade de Órgãos/genética , Transcrição Gênica/genética , Proteína ADAM12/genética , Proteína ADAM12/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Histona Acetiltransferases/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Transdução de Sinais/genética , Fator de Crescimento Transformador beta1/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Epithelial-mesenchymal transition (EMT) is important for the initial steps of metastasis. Although it is well accepted that the nucleoside diphosphate kinase NME1 is a metastasis suppressor, its effect on EMT remains poorly documented, as does that of its closely related isoform, NME2. Here, by using gene silencing, inactivation and overexpression strategies in a variety of cellular models of cancer, we show that NME1 is a powerful inhibitor of EMT. Genetic manipulation of NME2, by contrast, had no effect on the EMT phenotype of cancer cells, indicating a specific function of NME1 in EMT regulation. Loss of NME1 in epithelial cancer cells resulted in a hybrid phenotype intermediate between epithelial and mesenchymal cells, which is known to be associated with cells with a highly metastatic character. Conversely, overexpression of NME1 in mesenchymal cancer cells resulted in a more epithelial phenotype. We found that NME1 expression was negatively associated with EMT markers in many human cancers and was reduced in human breast tumor cell lines with the aggressive 'triple-negative' phenotype when compared to human breast tumor cell lines positive for estrogen receptor. We show that NME1, but not NME2, is an inhibitor of essential concerted intracellular signaling pathways involved in inducing EMT, including the AKT and MAPK (ERK, p38, and JNK) pathways. Additionally, NME1 depletion considerably altered the distribution of E-cadherin, a gatekeeper of the epithelial phenotype, shifting it from the plasma membrane to the cytosol and resulting in less E-cadherin on the cell surface than in control cells. Functional aggregation and dispersion assays demonstrated that inactivation of NME1 decreases E-cadherin-mediated cell-cell adhesion. We conclude that NME1, but not NME2, acts specifically to inhibit EMT and prevent the earliest stages of metastasis.
Assuntos
Transição Epitelial-Mesenquimal , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Animais , Caderinas/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Feminino , Edição de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos Nus , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
This review addresses current views on prevention of infectious endocarditis (IE). History of establishing the concept of antibacterial prophylaxis (ABP), major approaches, and substantiation of changes in ABP in recent years are described. Recent international and national guidelines are highlighted, specifically, guidelines of the European Society of Cardiologists, American Heart Association/American College of Cardiology, and the Japanese Circulation Society. The review presents critical evaluation of previously approved international guidelines, including analysis of the effect of partial or complete ABP restriction on IE morbidity and incidence of complications. Special attention is paid to awareness of practitioners, particularly dentists, about ABP issues in their practice. Aspects of validity and key features of preventive approaches in implanting cardiac electronic devices and transcatheter aortic valve implantation are discussed.
Assuntos
Endocardite Bacteriana , Endocardite , American Heart Association , Antibacterianos/uso terapêutico , Antibioticoprofilaxia , Endocardite/prevenção & controle , Endocardite Bacteriana/prevenção & controle , Humanos , Estados UnidosRESUMO
The selective targeting of protein-protein interactions remains a significant determinant for the proper modulation and regulation of cell apoptosis. Prototypic galectins such as human galectin-7 (GAL-7) are characterized by their ability to form homodimers that control the molecular fate of a cell by mediating subtle yet critical glycan-dependent interactions between pro- and anti-apoptotic molecular partners. Altering the structural architecture of GAL-7 can therefore result in resistance to apoptosis in various human cancer cells, further illustrating its importance in cell survival. In this study, we used a combination of biophysical and cellular assays to illustrate that binding of a water-soluble meso-tetraarylporphyrin molecule to GAL-7 induces protein oligomerization and modulation of GAL-7-induced apoptosis in human Jurkat T cells. Our results suggest that the integrity of the GAL-7 homodimer architecture is essential for its molecular function, in addition to providing an interesting porphyrin binding modulator for controlling apoptosis in mammalian cells.
Assuntos
Galectinas/química , Galectinas/metabolismo , Mesoporfirinas/química , Mesoporfirinas/metabolismo , Apoptose/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Galectinas/farmacologia , Humanos , Técnicas In Vitro , Células Jurkat , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Espalhamento a Baixo Ângulo , Solubilidade , Difração de Raios XRESUMO
Ribonuclease 6 (RNase 6) is one of eight catalytically active human pancreatic-type RNases that belong to a superfamily of rapidly evolving enzymes. Like some of its human homologues, RNase 6 exhibits host defense properties such as antiviral and antibacterial activities. Recently solved crystal structures of this enzyme in its nucleotide-free form show the conservation of the prototypical kidney-shaped fold preserved among vertebrate RNases, in addition to revealing the presence of a unique secondary active site. In this study, we determine the structural and conformational properties experienced by RNase 6 upon binding to substrate and product analogues. We present the first crystal structures of RNase 6 bound to a nucleotide ligand (adenosine 5'-monophosphate), in addition to RNase 6 bound to phosphate ions. While the enzyme preserves B2 subsite ligand preferences, our results show a lack of typical B2 subsite interactions normally observed in homologous ligand-bound RNases. A comparison of the dynamical properties of RNase 6 in its apo-, substrate-, and product-bound states highlight the unique dynamical properties experienced on time scales ranging from nano- to milliseconds. Overall, our results confirm the specific evolutionary adaptation of RNase 6 relative to its unique catalytic and biological activities.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Ribonucleases/química , Ribonucleases/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Sítios de Ligação/fisiologia , Humanos , Ligantes , Estrutura Secundária de ProteínaRESUMO
In humans, poor nutrition, malabsorption and variation in cobalamin (vitamin B12) metabolic genes are associated with hematological, neurological and developmental pathologies. Cobalamin is transported from blood into tissues via the transcobalamin (TC) receptor encoded by the CD320 gene. We created mice carrying a targeted deletion of the mouse ortholog, Cd320. Knockout (KO) mice lacking this TC receptor have elevated levels of plasma methylmalonic acid and homocysteine but are otherwise healthy, viable, fertile and not anemic. To challenge the Cd320 KO mice we maintained them on a vitamin B12-deficient diet. After 5 weeks on this diet, reproductive failure develops in Cd320 KO females but not males. In vitro, homozygous Cd320 KO embryos from cobalamin-deficient Cd320 KO dams develop normally to embryonic day (E) 3.5, while in vivo, few uterine decidual implantation sites are observed at E7.5, suggesting that embryos perish around the time of implantation. Dietary restriction of vitamin B12 induces a severe macrocytic anemia in Cd320 KO mice after 10-12 months while control mice on this diet are anemia-free up to 2 years. Despite the severe anemia, cobalamin-deficient KO mice do not exhibit obvious neurological symptoms. Our results with Cd320 KO mice suggest that an alternative mechanism exists for mice to transport cobalamin independent of the Cd320 encoded receptor. Our findings with deficient diet are consistent with historical and epidemiological data suggesting that low vitamin B12 levels in humans are associated with infertility and developmental abnormalities. Our Cd320 KO mouse model is an ideal model system for studying vitamin B12 deficiency.