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In this study, gas chromatography-mass spectrometry (GC-MS) and gas chromatography-ion mobility spectrometry (GC-IMS) were used to analyze the volatiles of fish cakes obtained using five cooking methods, namely, steaming, baking, air frying, pan frying and deep frying. The odor activity value (OAV) and relative odor activity value (ROAV) were used to screen for the major aroma compounds. Orthogonal partial least squares discriminant analysis (OPLS-DA) and the variable influence on projection (VIP) were used to determine the characteristic flavor compounds in the fish cakes. A total of 72 volatile compounds were identified by GC-MS, and 41 volatile compounds were detected by GC-IMS. 3-ethyl-2,5-dimethylpyrazine and 2,5-dimethylpyrazine were not detected in either CK or SS. The OPLS-DA models for GC-MS and GC-IMS analyses were constructed based on VIP values, and 8 and 7 compounds, respectively, were screened as characteristic aroma compounds. The results of this study provide new insights into the dynamics of flavor formation in reheated fish cakes and provide a theoretical basis for the optimization of the production process of this food product.
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The cooking method is extremely important for the production of low-salt, wet-marinated, fermented golden pomfret because it strongly influences its flavor components and organoleptic quality. There are also significant differences in flavor preferences in different populations. The present study analyzed differences in the aroma characteristics of wet-marinated fermented golden pomfret after boiling, steaming, microwaving, air-frying, and baking using a combination of an electronic nose, GC-IMS, and SPME-GC-MS. Electronic nose PCA showed that the flavors of the boiled (A), steamed (B), and microwaved (C) treatment groups were similar, and the flavors of the baking (D) and air-frying (E) groups were similar. A total of 72 flavor compounds were detected in the GC-IMS analysis, and the comparative analysis of the cooked wet-marinated and fermented golden pomfret yielded a greater abundance of flavor compounds. SPME-GC-MS analysis detected 108 flavor compounds, and the results were similar for baking and air-frying. Twelve key flavor substances, including hexanal, isovaleraldehyde, and (E)-2-dodecenal, were identified by orthogonal partial least-squares discriminant analysis (OPLS-DA) and VIP analysis. These results showed that the cooking method could be a key factor in the flavor distribution of wet-marinated fermented golden pomfret, and consumers can choose the appropriate cooking method accordingly. The results can provide theoretical guidance for the more effective processing of fish products and the development of subsequent food products.
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The effect of temperature fluctuations on the freshness of shrimp in simulated trays was investigated by setting a freeze-thaw (F-T) cycle of 12 h after freezing at -20 °C and thawing at 1 °C under refrigeration. The results showed that the shrimp's physicochemical properties deteriorated to different extents with the increase in F-T cycles. The total colony count of shrimp was 6.07 lg CFU/g after 21 cycles, and the volatile saline nitrogen content reached 30.36 mg/100 g, which exceeded the edible standard. In addition, the sensory quality and textural properties (hardness, elasticity, chewiness, and adhesion) declined to different degrees with increased F-T cycles. LF-NMR and protein property measurements showed that F-T cycles resulted in reduced water holding capacity and protein denaturation, which were the main factors leading to the deterioration of shrimp quality. Furthermore, flavor changes were analyzed using an electronic nose sensor to establish a freshness model. The W1W, W1S, W2S, and W5S sensors were correlated with the quality changes in shrimp and used as the main sensors for detecting the freshness of Penaeus vannamei. As a result, to better maintain the overall freshness, temperature fluctuations should be minimized in sales and storage, and fewer than 8 F-T cycles should be performed.
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Antifreeze peptides have become effective antifreeze agents for frozen products, but their low quantity of active ingredients and high cost limit large-scale application. This study used the glycosylation of fish collagen peptides with glucosamine hydrochloride catalyzed by transglutaminase to obtain a transglutaminase-catalyzed glycosylation product (TGP) and investigate its antifreeze effect on tilapia. Compared with the blank group, the freshness (pH value of 6.31, TVB-N value of 21.7 mg/100 g, whiteness of 46.28), textural properties (especially hardness and elasticity), and rheological properties of the TGP groups were significantly improved. In addition, the protein structures of the samples were investigated using UV absorption and fluorescence spectroscopy. The results showed that the tertiary structure of the TGP groups changed to form a dense polymer. Therefore, this approach can reduce the denaturation and decomposition of muscle fibers and proteins in fish meat more effectively and has a better protective effect on muscle structure and protein aggregation, improving the stability of fish meat. This study reveals an innovative method for generating antifreeze peptides by enzymatic glycosylation, and glycosylated fish collagen peptide products can be used as new and effective green antifreeze agents in frozen foods.
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In this experiment, the changes in protein hydrolysis, protein oxidation, and flavor of low-salt wet-marinated fermented golden pomfret were studied during processing. During processing, a decrease in sulfhydryl content (P < 0.05), a significant increase in protein surface hydrophobicity (P < 0.05), a significant increase in carbonyl content and TCA-soluble peptide (P < 0.05), an increase in TVB-N and amino acid nitrogen (P < 0.05), and a significant increase in the content of free amino acids (P < 0.05), indicating that proteins were gradually oxidized and degraded to small molecules and flavor precursors under the action of bacterial reduction pretreatment, deodorization, marination and fermentation processes, small molecules and aroma precursors was generated by gradual oxidative decomposition. In the course of processing, a total of 113 volatile flavor compounds were identified using GC-MS analysis, while OPLS-DA analysis and VIP value determination led to the identification of 10 characteristic flavor compounds. The results showed that an abundance of flavor compounds was generated during the processing, thereby imparting a more pronounced taste profile to the low-salt wet-marinated fermented golden carp. The results showed that a large number of flavor substances were generated during the processing to give a richer flavor to low-salt wet-marinated fermented golden pomfret that could provide data and theoretical support for the subsequent processing industry of golden pomfret.
Assuntos
Fermentação , Paladar , Compostos Orgânicos Voláteis , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/metabolismo , Animais , Aromatizantes/química , Aromatizantes/metabolismo , Manipulação de Alimentos , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Produtos Pesqueiros/análise , HumanosRESUMO
Low molecular weight sulfate glycosaminoglycan has attracted more attention recently for its great bioactivity. In the present study, a degraded sulfate glycosaminoglycan (named D-SBSG) was prepared from swimming bladder by enzymatic depolymerization, the structure characteristics of D-SBSG and its effects on blood coagulation and inflammation in vitro was investigated. HPGPC analysis showed that the molecular weight (Mw) of SBSG was 115.84 kDa, while the Mw of D-SBSG was 4.96 kDa. The bioactivities had arose dramatic differences, though its main molecule structure had little change after enzymatic degradation. Compared with heparin sodium, relatively milder anticoagulant activity in vitro, which were positively associated with molecular weight, were found in SBSG and D-SBSG. In contrast, the results of anti-inflammatory assays indicated that D-SBSG with the lower molecular weight possessed higher bioactivity than SBSG. Additionally, the D-SBSG inhibited the LPS-induced inflammatory in RAW264.7 macrophages by down-regulation of inflammatory mediators, both of NF-κB (including p65) and MAPK (including p38) signaling pathways to exert its anti-inflammatory function. These results indicated that enzymolysis is a viable strategy for degradation of sulfate glycosaminoglycan, and D-SBSG could be a promising ingredient for inflammation management.
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Anticoagulantes , Glicosaminoglicanos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Anticoagulantes/farmacologia , Glicosaminoglicanos/farmacologia , Glicosaminoglicanos/uso terapêutico , Humanos , Inflamação/tratamento farmacológico , Sulfatos , Natação , Bexiga UrináriaRESUMO
T cell-engaging bispecific antibodies (TCB) are highly potent therapeutics that can recruit and activate cytotoxic T cells to stimulate an antitumor immune response. However, the development of TCBs against solid tumors has been limited by significant on-target toxicity to normal tissues. Probody therapeutics have been developed as a novel class of recombinant, protease-activated antibody prodrugs that are "masked" to reduce antigen binding in healthy tissues but can become conditionally unmasked by proteases that are preferentially active in the tumor microenvironment (TME). Here, we describe the preclinical efficacy and safety of CI107, a Probody TCB targeting EGFR and CD3. In vitro, the protease-activated, unmasked CI107 effectively bound EGFR and CD3 expressed on the surface of cells and induced T-cell activation, cytokine release, and cytotoxicity toward tumor cells. In contrast, dually masked CI107 displayed a >500-fold reduction in antigen binding and >15,000-fold reduction in cytotoxic activity. In vivo, CI107 potently induced dose-dependent tumor regression of established colon cancer xenografts in mice engrafted with human peripheral blood mononuclear cells. Furthermore, the MTD of CI107 in cynomolgus monkeys was more than 60-fold higher than that of the unmasked TCB, and much lower levels of toxicity were observed in animals receiving CI107. Therefore, by localizing activity to the TME and thus limiting toxicity to normal tissues, this Probody TCB demonstrates the potential to expand clinical opportunities for TCBs as effective anticancer therapies for solid tumor indications. SIGNIFICANCE: A conditionally active EGFR-CD3 T cell-engaging Probody therapeutic expands the safety window of bispecific antibodies while maintaining efficacy in preclinical solid tumor settings.
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Anticorpos Biespecíficos , Complexo CD3 , Neoplasias do Colo , Receptores ErbB , Animais , Humanos , Camundongos , Anticorpos Biespecíficos/uso terapêutico , Complexo CD3/antagonistas & inibidores , Neoplasias do Colo/terapia , Receptores ErbB/antagonistas & inibidores , Leucócitos Mononucleares/metabolismo , Peptídeo Hidrolases/metabolismo , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Probody therapeutics (Pb-Txs) are conditionally activated antibody-drug conjugates (ADCs) designed to remain inactive until proteolytically activated in the tumor microenvironment, enabling safer targeting of antigens expressed in both tumor and normal tissue. Previous attempts to target CD71, a highly expressed tumor antigen, have failed to establish an acceptable therapeutic window due to widespread normal tissue expression. This study evaluated whether a probody-drug conjugate targeting CD71 can demonstrate a favorable efficacy and tolerability profile in preclinical studies for the treatment of cancer. CX-2029, a Pb-Tx conjugated to maleimido-caproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E, was developed as a novel cancer therapeutic targeting CD71. Preclinical studies were performed to evaluate the efficacy and safety of this anti-CD71 PDC in patient-derived xenograft (PDX) mouse models and cynomolgus monkeys, respectively. CD71 expression was detected at high levels by IHC across a broad range of tumor and normal tissues. In vitro, the masked Pb-Tx form of the anti-CD71 PDC displayed a >50-fold reduced affinity for binding to CD71 on cells compared with protease-activated, unmasked anti-CD71 PDC. Potent in vivo tumor growth inhibition (stasis or regression) was observed in >80% of PDX models (28/34) at 3 or 6 mg/kg. Anti-CD71 PDC remained mostly masked (>80%) in circulation throughout dosing in cynomolgus monkeys at 2, 6, and 12 mg/kg and displayed a 10-fold improvement in tolerability compared with an anti-CD71 ADC, which was lethal. Preclinically, anti-CD71 PDC exhibits a highly efficacious and acceptable safety profile that demonstrates the utility of the Pb-Tx platform to target CD71, an otherwise undruggable target. These data support further clinical development of the anti-CD71 PDC CX-2029 as a novel cancer therapeutic.
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Antineoplásicos , Imunoconjugados , Neoplasias , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Chumbo , Macaca fascicularis/metabolismo , Camundongos , Neoplasias/tratamento farmacológico , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Immune-checkpoint blockade has revolutionized cancer treatment. However, most patients do not respond to single-agent therapy. Combining checkpoint inhibitors with other immune-stimulating agents increases both efficacy and toxicity due to systemic T-cell activation. Protease-activatable antibody prodrugs, known as Probody therapeutics (Pb-Tx), localize antibody activity by attenuating capacity to bind antigen until protease activation in the tumor microenvironment. Herein, we show that systemic administration of anti-programmed cell death ligand 1 (anti-PD-L1) and anti-programmed cell death protein 1 (anti-PD-1) Pb-Tx to tumor-bearing mice elicited antitumor activity similar to that of traditional PD-1/PD-L1-targeted antibodies. Pb-Tx exhibited reduced systemic activity and an improved nonclinical safety profile, with markedly reduced target occupancy on peripheral T cells and reduced incidence of early-onset autoimmune diabetes in nonobese diabetic mice. Our results confirm that localized PD-1/PD-L1 inhibition by Pb-Tx can elicit robust antitumor immunity and minimize systemic immune-mediated toxicity. These data provide further preclinical rationale to support the ongoing development of the anti-PD-L1 Pb-Tx CX-072, which is currently in clinical trials.
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Anticorpos Monoclonais/uso terapêutico , Antígeno B7-H1/uso terapêutico , Imunoterapia/métodos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos , Microambiente TumoralRESUMO
4-Nonylphenol (NP) and bisphenol A (BPA) are high-production and high-volume chemicals used to manufacture various commercial products. They are also ubiquitous contaminants that disrupt endocrine systems in wildlife and humans. We collected, from Taiwan cities with the highest food production, and analyzed, using high performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS), 278 food samples for NP and BPA from 11 categories. We found background levels of 100% for NP and 72% for BPA in total samples. High levels of contamination (up to 918 and 49.4⯵g/kg) were found in some foods of seafood and animal origin. We used a probabilistic approach to calculate daily dietary dose (Monte Carlo-estimated 95th percentile dietary exposure [MCS 95]) from the Taiwan National Food Consumption database for each sex- and age-specified population. For NP and BPA, the highest average daily dose (ADDs) were in the 4- to 6-year-old group (MCS 95â¯=â¯1.57/1.28 and 0.157/0.147 [Male/Female] µg/kgâ¯bw/day, respectively), and the lowest ADDs were in the ≥65-year-old group (MCS 95â¯=â¯0.674/0.581 and 0.054/0.045 [M/F] µg/kgâ¯bw/day, respectively). Based on the European Food Safety Authority (4⯵g/kg bw/day for BPA) and Danish Institute of Safety and Toxicology guidelines (5⯵g/kg bw/day for NP), the 95th percentile HQ of NP and BPA intake in different sex- and age-specified groups in Taiwan posed no risks through dietary exposure. The intake quantity and concentrations of grains, livestock, and seafood are important variables for the integrated risk of NP and BPA. In conclusion, a combination of multiple and long-term exposure via food consumption should be considered rather than individual endocrine-disrupting chemicals during dietary risk assessment in specific populations. SUMMARY: The 95th percentile HQ of NP and BPA intake in different age and sex groups in Taiwan posed no risks through dietary exposure based on probabilistic and sensitive approach.
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Compostos Benzidrílicos/análise , Exposição Dietética/estatística & dados numéricos , Contaminação de Alimentos/análise , Carne/análise , Fenóis/análise , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Cidades , Disruptores Endócrinos/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Medição de Risco , Alimentos Marinhos/análise , Taiwan , Adulto JovemRESUMO
Aromatase is a crucial enzyme for the biosynthesis of estrogens and is involved in the process of breast carcinogenesis. Concerns have been raised regarding the effects of environmental estrogens as potential regulators of aromatase expression in human breast cells. Zeranol is a nonsteroidal agent with potent estrogenic activity, which is widely used as a growth promoter for cattle in certain countries. The present study hypothesized that aromatase expression and activity may be elevated by low dose zeranol exposure, providing a source of estrogens that may stimulate cell proliferation. In the present study, primary cultured human breast preadipocytes were used as an in vitro model. The effects of zeranol on cell proliferation were measured using the MTS assay, aromatase expression levels were determined by immunocytochemical staining and reverse transcriptionpolymerase chain reaction, and aromatase enzyme activity and estrogen production were analyzed using corresponding assay kits. The results demonstrated that low dose zeranol (250 nM) was able to significantly promote cell proliferation, aromatase mRNA expression, aromatase activity and estrogen production in primary cultured human breast preadipocytes, thus suggesting that zeranol may act as an aromatase activator. The findings of the present study suggest that zeranol promotes breast cancer cell growth by stimulating aromatase activation and increasing estrogen biosynthesis in adipose tissue.
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Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Aromatase/metabolismo , Glândulas Mamárias Humanas/citologia , Zeranol/farmacologia , Adipócitos/citologia , Tecido Adiposo/citologia , Adulto , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Estradiol/biossíntese , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies. To redirect the activity of antibodies recognizing widely distributed targets to the site of disease, we have applied a prodrug strategy to create an epidermal growth factor receptor (EGFR)-directed Probody therapeutic-an antibody that remains masked against antigen binding until activated locally by proteases commonly active in the tumor microenvironment. In vitro, the masked Probody showed diminished antigen binding and cell-based activities, but when activated by appropriate proteases, it regained full activity compared to the parental anti-EGFR antibody cetuximab. In vivo, the Probody was largely inert in the systemic circulation of mice, but was activated within tumor tissue and showed antitumor efficacy that was similar to that of cetuximab. The Probody demonstrated markedly improved safety and increased half-life in nonhuman primates, enabling it to be dosed safely at much higher levels than cetuximab. In addition, we found that both Probody-responsive xenograft tumors and primary tumor samples from patients were capable of activating the Probody ex vivo. Probodies may therefore improve the safety profile of therapeutic antibodies without compromising efficacy of the parental antibody and may enable the wider use of empowered antibody formats such as antibody-drug conjugates and bispecifics.
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Anticorpos Antineoplásicos/uso terapêutico , Receptores ErbB/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Pró-Fármacos/uso terapêutico , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Cetuximab , Humanos , Imuno-Histoquímica , Macaca fascicularis , Camundongos , Camundongos Nus , Pró-Fármacos/toxicidade , Pele/efeitos dos fármacos , Pele/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The Wnt coreceptor LRP6 is required for canonical Wnt signaling. To understand the molecular regulation of LRP6 function, we generated a series of monoclonal antibodies against the extra cellular domain (ECD) of LRP6 and selected a high-affinity mAb (mAb135) that recognizes cell surface expression of endogenous LRP6. mAb135 enhanced Wnt dependent TCF reporter activation and antagonized DKK1 dependent inhibition of Wnt3A signaling, suggesting a role in modulation of LRP6 function. Detailed analysis of LRP6 domain mutants identified Ser 243 in the first propeller domain of LRP6 as a critical residue for mAb135 binding, implicating this domain in regulating the sensitivity of LRP6 to DKK1. In agreement with this notion, mAb135 directly disrupted the interaction of DKK1 with recombinant ECD LRP6 and a truncated form of the LRP6 ECD containing only repeats 1 and 2. Finally, we found that mAb135 completely protected LRP6 from DKK1 dependent internalization. Together, these results identify the first propeller domain as a novel regulatory domain for DKK1 binding to LRP6 and show that mAb against the first propeller domain of LRP6 can be used to modulate this interaction.
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Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Western Blotting , Linhagem Celular , Endocitose/efeitos dos fármacos , Citometria de Fluxo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/imunologia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Mutação , Ligação Proteica/efeitos dos fármacos , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt3 , Proteína Wnt3ARESUMO
The R-Spondin (RSpo) family of secreted proteins is implicated in the activation of the Wnt signaling pathway. Despite the high structural homology between the four members, expression patterns and phenotypes in knockout mice have demonstrated striking differences. Here we dissected and compared the molecular and cellular function of all RSpo family members. Although all four RSpo proteins activate the canonical Wnt pathway, RSpo2 and 3 are more potent than RSpo1, whereas RSpo4 is relatively inactive. All RSpo members require Wnt ligands and LRP6 for activity and amplify signaling of Wnt3A, Wnt1, and Wnt7A, suggesting that RSpo proteins are general regulators of canonical Wnt signaling. Like RSpo1, RSpo2-4 antagonize DKK1 activity by interfering with DKK1 mediated LRP6 and Kremen association. Analysis of RSpo deletion mutants indicates that the cysteine-rich furin domains are sufficient and essential for the amplification of Wnt signaling and inhibition of DKK1, suggesting that Wnt amplification by RSpo proteins may be a direct consequence of DKK1 inhibition. Together, these findings indicate that RSpo proteins modulate the Wnt pathway by a common mechanism and suggest that coexpression with specific Wnt ligands and DKK1 may determine their biological specificity in vivo.
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Transdução de Sinais , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Animais , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Ligantes , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Estrutura Terciária de Proteína , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição TCF/metabolismo , Trombospondinas/química , beta Catenina/metabolismoRESUMO
NTB-A is a CD2-related cell surface protein expressed primarily on lymphoid cells including B-lymphocytes from chronic lymphocytic leukaemia (CLL) and lymphoma patients. We have generated a series of monoclonal antibodies (mAbs) against NTB-A and assessed their therapeutic potential for CLL. Selective mAbs to NTB-A were further tested in functional complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicty (ADCC) assays in cell lines and B lymphocytes freshly isolated from CLL patients. While lower levels of NTB-A were detected in T and natural killer (NK) cells, CDC activity was demonstrated primarily in B cells isolated from CLL patients and B lymphoma cell lines. Knockdown of NTB-A by small interfering RNA in target cells results in lower cytotoxicity, demonstrating the specificity of the mAbs. Furthermore, anti NTB-A mAbs demonstrated anti-tumour activity against CA46 human lymphoma xenografts in nude mice and against systemically disseminated Raji human lymphoma cells in severe combined immunodeficient mice. Taken together, these results demonstrate NTB-A as a potential new target for immunotherapy of leukaemia and lymphomas.
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Anticorpos Monoclonais/uso terapêutico , Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Linfócitos B/imunologia , Imunização Passiva/métodos , Leucemia Linfocítica Crônica de Células B/terapia , Receptores de Superfície Celular/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Reações Antígeno-Anticorpo , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Citometria de Fluxo , Humanos , Hibridomas , Leucemia Linfocítica Crônica de Células B/imunologia , Ativação Linfocitária , Camundongos , Camundongos Nus , Camundongos SCID , Transplante de Neoplasias , Interferência de RNA , Família de Moléculas de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Transplante HeterólogoRESUMO
Alfimeprase is a recombinant, direct-acting fibrinolytic zinc metalloprotease. Alfimeprase has direct proteolytic activity primarily against the fibrin(ogen) Aalpha chain. Alfimeprase is covalently bound and neutralised by serum alpha(2)-macroglobulin, a prevalent mammalian protease inhibitor. Preclinical pharmacology studies have shown that fibrinolysis with alfimeprase is up to sixfold more rapid than with select plasminogen activators, such as tissue-type plasminogen activator and urokinase. Alfimeprase directly delivered to a site of thrombosis has the potential to be a fast and effective fibrinolytic, which does not generate the systemic lytic state seen with plasminogen activators that is associated with major bleeding, including intracerebral haemorrhage. Phase I and II studies in individuals with arterial or venous thrombotic events indicate that alfimeprase is active and generally well tolerated.
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Drogas em Investigação/uso terapêutico , Fibrinolíticos/uso terapêutico , Metaloendopeptidases/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Animais , Drogas em Investigação/farmacologia , Fibrinolíticos/farmacologia , Humanos , Metaloendopeptidases/farmacologia , Proteínas Recombinantes/farmacologiaRESUMO
Regulated changes in the affinity of integrin adhesion receptors ("activation") play an important role in numerous biological functions including hemostasis, the immune response, and cell migration. Physiological integrin activation is the result of conformational changes in the extracellular domain initiated by the binding of cytoplasmic proteins to integrin cytoplasmic domains. The conformational changes in the extracellular domain are likely caused by disruption of intersubunit interactions between the alpha and beta transmembrane (TM) and cytoplasmic domains. Here, we reasoned that mutation of residues contributing to alpha/beta interactions that stabilize the low affinity state should lead to integrin activation. Thus, we subjected the entire intracellular domain of the beta3 integrin subunit to unbiased random mutagenesis and selected it for activated mutants. 25 unique activating mutations were identified in the TM and membrane-proximal cytoplasmic domain. In contrast, no activating mutations were identified in the more distal cytoplasmic tail, suggesting that this region is dispensable for the maintenance of the inactive state. Among the 13 novel TM domain mutations that lead to integrin activation were several informative point mutations that, in combination with computational modeling, suggested the existence of a specific TM helix-helix packing interface that maintains the low affinity state. The interactions predicted by the model were used to identify additional activating mutations in both the alpha and beta TM domains. Therefore, we propose that helical packing of the alpha and beta TM domains forms a clasp that regulates integrin activation.
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Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Citoplasma , Dimerização , Modelos Moleculares , Mutagênese , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Ligação Proteica , Estrutura Terciária de Proteína , TransfecçãoRESUMO
Engagement of very late Ag-4 (integrin alpha(4)beta(1)) by ligands such as VCAM-1 markedly stimulates leukocyte migration mediated by LFA-1 (integrin alpha(L)beta(2)). This form of integrin trans-regulation in T cells requires the binding of paxillin to the alpha(4) integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by an alpha(4) mutation (alpha(4)(Y991A)) that disrupts paxillin binding. Furthermore, cellular expression of an alpha(4)-binding fragment of paxillin that blocks the alpha(4)-paxillin interaction, selectively blocked VCAM-1 stimulation of alpha(L)beta(2)-dependent cell migration. The alpha(4)-paxillin association mediates trans-regulation by enhancing the activation of tyrosine kinases, focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase-2 (Pyk2), based on two lines of evidence. First, disruption of the paxillin-binding site in the alpha(4) tail resulted in much less alpha(4)beta(1)-mediated phosphorylation of Pyk2 and FAK. Second, transfection with cDNAs encoding C-terminal fragments of Pyk2 and FAK, which block the function of the intact kinases, blocked alpha(4)beta(1) stimulation of alpha(L)beta(2)-dependent migration. These results define a proximal protein-protein interaction of an integrin cytoplasmic domain required for trans-regulation between integrins, and establish that augmented activation of Pyk2 and/or FAK is an immediate signaling event required for the trans-regulation of integrin alpha(L)beta(2) by alpha(4)beta(1).
Assuntos
Movimento Celular/imunologia , Proteínas do Citoesqueleto/metabolismo , Integrina alfa4/metabolismo , Antígeno-1 Associado à Função Linfocitária/fisiologia , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Substituição de Aminoácidos/genética , Sítios de Ligação/genética , Movimento Celular/genética , Citoplasma/metabolismo , Proteínas do Citoesqueleto/fisiologia , Ativação Enzimática/genética , Quinase 1 de Adesão Focal , Quinase 2 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Integrina alfa4/genética , Integrina alfa4/fisiologia , Integrina alfa4beta1/antagonistas & inibidores , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Integrina alfa4beta1/fisiologia , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/metabolismo , Paxilina , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/fisiologia , Fosfoproteínas/fisiologia , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Subunidades Proteicas/fisiologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/fisiologia , Receptor Cross-Talk/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/enzimologia , Transfecção , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
The alpha(4) integrins play important roles in embryogenesis, hematopoiesis, cardiac development, and the immune responses. The alpha(4) integrin subunit is indispensable for these biological processes, possibly because the alpha(4) subunit regulates cellular functions differently from other integrin alpha subunits. We have previously reported that the alpha(4) cytoplasmic domain directly and tightly binds paxillin, an intracellular signaling adaptor molecule, and this interaction accounts for some of the unusual functional responses to alpha(4) integrin-mediated cell adhesion. We also have identified a conserved 9-amino acid region (Glu(983)-Tyr(991)) in the alpha(4) cytoplasmic domain that is sufficient for paxillin binding, and an alanine substitution at either Glu(983) or Tyr(991) within this region disrupted the alpha(4)-paxillin interaction and reversed the effects of the alpha(4) cytoplasmic domain on cell spreading and migration. In the current study, we have mapped the alpha(4)-binding site within paxillin using mutational analysis, and examined its effects on the alpha(4) tail-mediated functional responses. Here we report that sequences between residues Ala(176) and Asp(275) of paxillin are sufficient for binding to the alpha(4) tail. We found that the alpha(4) tail, paxillin, and FAT, the focal adhesion targeting domain of pp125(FAK), could form a ternary complex and that the alpha(4)-binding paxillin fragment, P(Ala(176)-Asp(275)), specifically blocked paxillin binding to the alpha(4) tail more efficiently than it blocked binding to FAT. Furthermore, when expressed in cells, this alpha(4)-binding paxillin fragment specifically inhibited the alpha(4) tail-stimulated cell migration. Thus, paxillin binding to the alpha(4) tail leads to enhanced cell migration and inhibition of the alpha(4)-paxillin interaction selectively blocks the alpha4-dependent cellular responses.