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1.
J Neurointerv Surg ; 2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34475254

RESUMO

This case report shows that real-time MRI may aid in the precision of intra-arterial delivery of bevacizumab to butterfly glioblastoma. Fast clinical improvement, decrease of contrast enhancing status, and no serious adverse effects were observed at discharge from hospital. The patient regained pre-recurrent neurological status for 2 months with a subsequent fast clinical decline and an increase in tumor volume. The patient underwent a second procedure of intra-arterial delivery of bevacizumab to the brain, with substantial clinical and radiological improvement, but not the level of improvement observed after the first procedure. Another clinical decline occurred with an increase in tumor size and the patient was treated 2 months later with a third intra-arterial infusion of bevacizumab. While another positive effect was achieved, it was less pronounced than before, and the patient died 1.5 months later. There were no technical, ischemic or other complications during the procedures. The patient survived 218 days from the first symptoms of tumor recurrence, 190 days from the first MRI, and 175 days from the first intra-arterial treatment of bevacizumab.

2.
J Agric Food Chem ; 69(35): 10358-10370, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34428040

RESUMO

The advancement of mass spectrometry provides advantages for transgenic protein characterization in support of safety assessments of genetically modified crops. Here, we describe how matrix-assisted laser desorption ionization in-source decay (ISD) mass spectrometry (MS) in combination with intact mass and bottom-up analyses can be applied to achieve high confidence in the sequences of transgenic proteins expressed in plants and establish the biochemical equivalence of microbially produced protein surrogates. ISD confirmed 40-60 near terminal residues regardless of the protein size, including the improvement of the coverage of cysteine-rich proteins by the reduction/alkylation of disulfide bonds. Negative ISD significantly improved spectral quality and sequence coverage of acidic proteins. Various post-translational modifications, such as terminal truncations and N-terminal methionine excision and acetylation, were identified in plant-produced proteins by top-down MS. Finally, we demonstrated that a combination of top-down and bottom-up analyses provides high confidence in sequence equivalence of plant and microbially produced proteins.


Assuntos
Produtos Agrícolas , Proteínas , Produtos Agrícolas/genética , Plantas Geneticamente Modificadas/genética , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
3.
Sci Rep ; 11(1): 6581, 2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33753789

RESUMO

Stem cell transplantation proved promising in animal models of neurological diseases; however, in conditions with disseminated pathology such as ALS, delivery of cells and their broad distribution is challenging. To address this problem, we explored intra-arterial (IA) delivery route, of stem cells. The goal of this study was to investigate the feasibility and safety of MRI-guided transplantation of glial restricted precursors (GRPs) and mesenchymal stem cells (MSCs) in dogs suffering from ALS-like disease, degenerative myelopathy (DM). Canine GRP transplantation in dogs resulted in rather poor retention in the brain, so MSCs were used in subsequent experiments. To evaluate the safety of MSC intraarterial transplantation, naïve pigs (n = 3) were used as a pre-treatment control before transplantation in dogs. Cells were labeled with iron oxide nanoparticles. For IA transplantation a 1.2-French microcatheter was advanced into the middle cerebral artery under roadmap guidance. Then, the cells were transplanted under real-time MRI with the acquisition of dynamic T2*-weighted images. The procedure in pigs has proven to be safe and histopathology has demonstrated the successful and predictable placement of transplanted porcine MSCs. Transplantation of canine MSCs in DM dogs resulted in their accumulation in the brain. Interventional and follow-up MRI proved the procedure was feasible and safe. Analysis of gene expression after transplantation revealed a reduction of inflammatory factors, which may indicate a promising therapeutic strategy in the treatment of neurodegenerative diseases.

4.
J Clin Med ; 10(4)2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673251

RESUMO

BACKGROUND: Although pregnancy has been identified as one of the risk factors for venous disease, the mechanism of this interaction remains unclear. Possibly, pregnancy results in overstrain and vein dilatation, which exceed their durability and persist after pregnancy. The aim of this study was the assessment of the relationship between the number of pregnancies in women with venous disease and the selected parameters of their venous systems. PATIENTS AND METHODS: The retrospective assessment concerned 518 patients subjected to the diagnostics of the venous system in the lower limbs and the abdomen/pelvis using ultrasound scan and magnetic resonance or computed tomography. RESULTS: We found that the occurrence of pelvic venous symptoms increases proportionally to the number of pregnancies and is correlated with ovarian and parauterine vein dilatation/incompetence (e.g., 13.5% of nulliparous women reported pelvic pain, and reflux in left ovarian veins was detected in 21.4% of the patients from that group, whereas in women after two pregnancies, pain and reflux concerned 22.8% and 90.6% of patients, respectively). In the nulliparous group, the development of venous disease resulted from the presence of anatomic abnormalities in abdominal/pelvic veins. CONCLUSIONS: Our report proved that the number of pregnancies is correlated with the incidence of pelvic vein insufficiency. Although not specifically addressed in this study, some correlation was found with saphenous disease as well. However, further studies are necessary to provide more evidence about the role of pelvic vein insufficiency in chronic venous disease of the lower limbs.

5.
J Neurosurg ; : 1-9, 2020 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-31978881

RESUMO

OBJECTIVE: The goal of this study was to evaluate the effectiveness and safety of a new noncompletely occlusive net-assisted remodeling technique in which the Cascade net device is used for temporary bridging of intracranial aneurysms. METHODS: Between July 2018 and May 2019, patients underwent coil embolization with the Cascade net device within 4 centers in Europe. Analysis of angiographic (modified Raymond-Roy classification [MRRC]) and clinical outcomes data was conducted immediately following treatment and at the 6-month follow-up. RESULTS: Fifteen patients were included in the study (mean age 58 ± 13 years, 11/15 [73.3%] female). Ten patients had unruptured aneurysms, and 5 presented with ruptured aneurysms with acute subarachnoid hemorrhage. The mean aneurysm dome length was 6.27 ± 2.33 mm and the mean neck width was 3.64 ± 1.19 mm. Immediately postprocedure, MRRC type I (complete obliteration) was achieved in 11 patients (73.3%), whereas a type II (residual neck) was achieved in 4 patients (26.7%). Follow-up examination was performed in 7/15 patients and showed stabilization of aneurysm closure with no thromboembolic complications and only 1 patient with an increased MRRC score (from I to II) due to coil compression. CONCLUSIONS: Initial experience shows that the use of a new noncompletely occlusive net-assisted remodeling technique with the Cascade net device may be safe and effective for endovascular coil embolization of intracranial aneurysms.

6.
Eur J Nucl Med Mol Imaging ; 46(9): 1940-1951, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31161257

RESUMO

INTRODUCTION: We have recently shown that intracerebral delivery of an anti-VEGF monoclonal antibody bevacizumab using an intra-arterial (IA) infusion is more effective than intravenous administration. While antibodies are quickly emerging as therapeutics, their disadvantages such as large size, production logistics and immunogenicity motivate search for alternatives. Thus we have studied brain uptake of nanobodies and polyamidoamine (PAMAM) dendrimers. METHODS: Nanobodies were conjugated with deferoxamine (DFO) to generate NB(DFO)2. Generation-4 PAMAM dendrimers were conjugated with DFO, and subsequently primary amines were capped with butane-1,2-diol functionalities to generate G4(DFO)3(Bdiol)110. Resulting conjugates were radiolabeled with zirconium-89. Brain uptake of 89ZrNB(DFO)2 and 89ZrG4(DFO)3(Bdiol)110 upon carotid artery vs tail vein infusions with intact BBB or osmotic blood-brain barrier opening (OBBBO) with mannitol in mice was monitored by dynamic positron emission tomography (PET) over 30 min to assess brain uptake and clearance, followed by whole-body PET-CT (computed tomography) imaging at 1 h and 24 h post-infusion (pi). Imaging results were subsequently validated by ex-vivo biodistribution. RESULTS: Intravenous administration of 89ZrNB(DFO)2 and 89ZrG4(DFO)3(Bdiol)110 resulted in their negligible brain accumulation regardless of BBB status and timing of OBBBO. Intra-arterial (IA) administration of 89ZrNB(DFO)2 dramatically increased its brain uptake, which was further potentiated with prior OBBBO. Half of the initial brain uptake was retained after 24 h. In contrast, IA infusion of 89ZrG4(DFO)3(Bdiol)110 resulted in poor initial accumulation in the brain, with complete clearance within 1 h of administration. Ex-vivo biodistribution results reflected those on PET-CT. CONCLUSIONS: IA delivery of nanobodies might be an attractive therapeutic platform for CNS disorders where prolonged intracranial retention is necessary.


Assuntos
Artérias , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Dendrímeros/metabolismo , Nylons/metabolismo , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Anticorpos de Domínio Único/metabolismo , Animais , Dendrímeros/química , Processamento de Imagem Assistida por Computador , Camundongos , Nylons/química , Transporte Proteico , Radioisótopos , Distribuição Tecidual , Zircônio
7.
J Neurointerv Surg ; 11(8): e3, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31123186

RESUMO

Patients suffering from malignant brain tumors are burdened with a grim prognosis. The blood brain barrier is considered a primary obstacle in therapeutic drug delivery to the brain. Intra-arterial (IA) delivery of therapeutic agents following osmotic BBB opening has been attempted for years, but high variability has limited its widespread implementation. It has been recently shown in animal studies that MRI is superior to X-ray for guiding IA infusions, as it allows direct visualization of the brain parenchyma perfused and facilitates predictable drug targeting. Moreover, PET imaging has revealed that IA, not intravenous, delivery of bevacizumab results in brain accumulation, providing strong rationale for utilizing the IA route. Here, we present our experience in a patient with recurrent butterfly glioblastoma enrolled in first-in-man MRI-guided neurointervention for targeted IA drug delivery.


Assuntos
Bevacizumab/administração & dosagem , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/diagnóstico por imagem , Glioblastoma/tratamento farmacológico , Imageamento por Ressonância Magnética/métodos , Angiografia Digital/métodos , Antineoplásicos Imunológicos/administração & dosagem , Barreira Hematoencefálica/diagnóstico por imagem , Barreira Hematoencefálica/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Humanos , Infusões Intra-Arteriais/métodos , Injeções Intra-Arteriais , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/diagnóstico por imagem , Recidiva Local de Neoplasia/tratamento farmacológico
8.
Vasc Endovascular Surg ; 53(3): 259-263, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30630394

RESUMO

Pseudoaneurysm formation is a rare but dangerous complication of carotid artery dissection. It can result from trauma, spontaneous artery dissection, or iatrogenic causes. Presence of symptoms and ineffective medical therapy are one of the indications for invasive treatment. We present the case of 3 symptomatic patients with dissecting pseudoaneurysms-2 traumatic and 1 spontaneous. They were treated with 3 different endovascular procedures: the use of covered stentgraft, trans-stent coil embolization, and carotid stenting. After invasive and dual antiplatelet therapy, complete resolution of symptoms was achieved in all patients.


Assuntos
Falso Aneurisma/etiologia , Dissecação da Artéria Carótida Interna/etiologia , Lesões do Sistema Vascular/etiologia , Acidentes de Trânsito , Adulto , Falso Aneurisma/diagnóstico por imagem , Falso Aneurisma/fisiopatologia , Falso Aneurisma/cirurgia , Prótese Vascular , Implante de Prótese Vascular/instrumentação , Dissecação da Artéria Carótida Interna/diagnóstico por imagem , Dissecação da Artéria Carótida Interna/fisiopatologia , Dissecação da Artéria Carótida Interna/cirurgia , Angiografia por Tomografia Computadorizada , Embolização Terapêutica/instrumentação , Procedimentos Endovasculares/instrumentação , Feminino , Humanos , Masculino , Inibidores da Agregação Plaquetária/administração & dosagem , Stents , Resultado do Tratamento , Lesões do Sistema Vascular/diagnóstico por imagem , Lesões do Sistema Vascular/fisiopatologia , Lesões do Sistema Vascular/cirurgia , Adulto Jovem
9.
BMJ Case Rep ; 12(1)2019 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-30679227

RESUMO

Patients with malignant brain tumors have a poor prognosis. The blood-brain barrier (BBB) is considered a primary obstacle in therapeutic drug delivery to the brain. Intra-arterial (IA) delivery of therapeutic agents following osmotic BBB opening has been attempted for years, but high variability has limited its widespread implementation. It has recently been shown in animal studies that MRI is superior to X-ray for guiding IA infusions, as it allows direct visualization of the brain parenchyma supplied by the catheter and facilitates predictable drug targeting. Moreover, PET imaging has shown that IA rather than intravenous delivery of bevacizumab results in accumulation in the brain, providing a strong rationale for using the IA route. We present a patient with recurrent butterfly glioblastoma enrolled in a first-in-man MRI-guided neurointervention for targeted IA drug delivery.


Assuntos
Antineoplásicos Imunológicos/administração & dosagem , Bevacizumab/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Barreira Hematoencefálica/diagnóstico por imagem , Barreira Hematoencefálica/cirurgia , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Humanos , Imageamento Tridimensional , Infusões Intra-Arteriais/métodos , Angiografia por Ressonância Magnética/métodos , Imagem por Ressonância Magnética Intervencionista/métodos , Pessoa de Meia-Idade
10.
Front Neurol ; 9: 807, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30319536

RESUMO

Spinal dural arteriovenous fistula (SDAVF) is the most common vascular malformation of the spine in adults. However, the coincidence of tethered cord syndrome, lipoma, and SDAVF on the sacral level is exceptionally rare. We describe two patients, probably the fifth and sixth ever reported. The first was a 33 year-old female who underwent surgical cord de-tethering. Surprisingly, a sacral SDAVF was discovered intraoperatively, despite negative digital subtraction angiography (DSA). The second patient was a 30 year-old male with similar pathologies. After three failed embolizations, the fistula was surgically disconnected. Both patients recovered well. A review of patients with sacral SDAVF coexisting with spinal dysraphism, with an emphasis on the basis of symptoms was done. As a rule, in these coincident disorders, the SDAVF was the direct cause of increasing symptoms. Previous reports and our findings reveal that surgery might be superior to endovascular embolization for treating sacral SDAVFs with coexisting entities, because surgery offers a one-step treatment.

11.
Pol J Radiol ; 82: 203-208, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28469736

RESUMO

BACKGROUND: Hydrogel coils were created to improve the chances of an effective endovascular treatment of cerebral aneurysms. Achieving a high packing density of coils in the lumen of aneurysms can decrease the risk of recurrence. The aim of the present study is to report our initial experience on the effectiveness and safety of endovascular treatment of intracranial aneurysms with the use hydrogel coils. MATERIAL/METHODS: Sixty patients (age: 28-72 years) (45 women, 15 men) were treated. In 18 patients (30%), subarachnoid hemorrhage was present. Digital subtraction angiography (DSA) of cerebral vessels with rotational scanning was performed. Image analysis was performed by the Philips Integris 3D RA device,which is a specialized workstation (Three-Dimensional Rotational Angiography). 3D reconstructions of cerebral arteries were created based on the data. Sixty-six cerebral aneurysms were embolized with hydrogel coils, which expand in contact with blood. reaching the maximum diameter in about 20 minutes. In 29 aneurysms (43.9%), the effect of the procedure was confirmed on a follow-up DSA after 8.0±4.1 months from the initial treatment. RESULTS: A complete embolization was performed in 55 aneurysms (83.3%), and partial embolization in 11 aneurysms (16.7%). In 6 aneurysms (9.1%), re-embolization was necessary and it resulted in a complete embolization of 5 aneurysms. On a follow-up DSA, complete embolization was present in 25 aneurysms (86.2%), and partial embolization in 4 aneurysms (13.8%), respectively. CONCLUSIONS: Endovascular embolization with hydrogel coils is an effective and safe treatment method for cerebral aneurysms, although it carries the risk of some complications.

12.
Int J Mol Sci ; 18(2)2017 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-28146070

RESUMO

Primary biliary cholangitis (PBC) is an immune-mediated cholestatic disease. Vitamin D receptor (VDR)-dependent signaling constrains an inflammatory response by targeting the miRNA155-SOCS1 (suppressor of cytokine signaling 1) axis. The VDR-miRNA155-SOCS1 pathway was investigated in the context of the autoimmune response associated with PBC. Human liver tissues from non-cirrhotic PBC (n = 22), cirrhotic PBC (n = 22), cirrhotic primary sclerosing cholangitis (PSC, n=13), controls (n = 23), and peripheral blood mononuclear cells (PBMC) obtained from PBC (n = 16) and PSC (n = 10) patients and healthy subjects (n = 11) were used for molecular analyses. VDR mRNA and protein expressions were substantially reduced in PBC livers (51% and 59%, respectively). Correspondingly, the decrease of SOCS1 protein expression in PBC livers, after normalization to a marker of lymphocytes and forkhead family transcriptional regulator box P3 (FOXP3, marker of Treg), was observed, and this phenomenon was accompanied by enhanced miRNA155 expression. In PSC livers, protein expressions of VDR and SOCS1 were comparable to the controls. However, in PBM cells, protein expressions of VDR and SOCS1 were considerably decreased in both PBC and PSC. We demonstrated that VDR/miRNA155-modulated SOCS1 expression is decreased in PBC which may lead to insufficient negative regulation of cytokine signaling. These findings suggest that the decreased VDR signaling in PBC could be of importance in the pathogenesis of PBC.


Assuntos
Colangite Esclerosante/etiologia , Colangite Esclerosante/metabolismo , Imunomodulação , MicroRNAs/genética , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Proteína 1 Supressora da Sinalização de Citocina/genética , Colangite Esclerosante/patologia , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Cirrose Hepática Biliar/genética , Cirrose Hepática Biliar/metabolismo , Cirrose Hepática Biliar/patologia , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina/metabolismo
13.
Pol J Radiol ; 82: 19-23, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28144386

RESUMO

BACKGROUND: Radial artery is now the most frequent access for coronary angiography and intervention. Despite the common opinion that it is safer than femoral access, it has the potential for serious complications. One of them is upper limb ischemia caused by radial artery thrombosis. CASE REPORT: We are presenting a case of critical hand ischemia after coronary angiography performed through radial access despite existing risk factors, which may be considered as relative contraindications. CONCLUSIONS: In the presented case, decision was made to use radial access despite several risk factors of upper limb ischemia - diabetes, end-stage renal failure, hyperparathyroidism, or even symptoms of left upper limb ischemia. Furthermore, for diagnostic coronary angiography 5F instead of 4F introducer was used.

14.
Pol J Radiol ; 77(4): 22-9, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23269933

RESUMO

The goal of this work was to assess the effectiveness of endovascular treatment of common and external iliac artery stenosis/occlusion classified according to TASC using a self-expanding stent Jaguar SM. The study group included 95 patients (61 men and 34 women) who underwent treatment for stenosis or occlusion of lower limb arteries at the Department of Radiology of the University Hospital in Bialystok and the Diagnostic Radiology Department of the Central Clinical Hospital of the Ministry of Interior (MSWiA) in Warsaw between 2005 and 2007. All arterial lesions were of atherosclerotic etiology. The shortest stenotic fragment was 10 mm long and the longest occluded arterial fragment did not exceed 90 mm. Morphological classification of iliac artery lesions in treated patients was performed according to TASC II classification and included 10 patients with type A, 39 cases of type B, 36 with type C and 10 patients with type D lesions. Endovascular procedure failed to restore flow in five patients with TASC type D lesions, who were later referred for surgery. One patient suffered a complication - vessel perforation during predilatation, and had a stentgraft implanted. In 95% of patients stents were expanded using a balloon after implantation. Good results were achieved in practically all patients who underwent stent implantation. Patients were subjected to follow-up clinical and imaging evaluation during next 1-24 months. Success rate of the performed procedures as well as in a 30-day observation period was 100% in case of stenosis and 80% in case of vessel occlusion. A follow-up after 12 and 24 months showed patency of treated vessels in 84% and 76% of patients, respectively.

15.
J Struct Funct Genomics ; 11(2): 167-80, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20419351

RESUMO

The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology.


Assuntos
Laboratórios/organização & administração , Proteínas/química , Proteínas/metabolismo , Proteômica/organização & administração , Biologia Computacional , Cristalização , Humanos , Proteínas/genética , Escócia
16.
Artigo em Inglês | MEDLINE | ID: mdl-17565195

RESUMO

Sar2028, an aspartate/tyrosine/phenylalanine pyridoxal-5'-phosphate-dependent aminotransferase with a molecular weight of 48,168 Da, was overexpressed in methicillin-resistant Staphylococcus aureus compared with a methicillin-sensitive strain. The protein was expressed in Escherichia coli, purified and crystallized. The protein crystallized in a primitive orthorhombic Laue group with unit-cell parameters a = 83.6, b = 91.3, c = 106.0 A, alpha = beta = gamma = 90 degrees. Analysis of the systematic absences along the three principal axes indicated the space group to be P2(1)2(1)2(1). A complete data set was collected to 2.5 A resolution.


Assuntos
Proteínas de Bactérias/química , Resistência a Meticilina , Staphylococcus aureus/química , Transaminases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Cromatografia de Afinidade , Clonagem Molecular , Cristalização , Conformação Proteica
17.
Artigo em Inglês | MEDLINE | ID: mdl-17554169

RESUMO

Sar2676, a pantothenate synthetase with a molecular weight of 31 419 Da from methicillin-resistant Staphylococcus aureus, has been expressed, purified and crystallized at 293 K. The protein crystallizes in a primitive triclinic lattice, with unit-cell parameters a = 45.3, b = 60.5, c = 117.6 A, alpha = 87.2, beta = 81.2, gamma = 68.4 degrees . A complete data set has been collected to 2.3 A resolution at the ESRF. Consideration of the likely solvent content suggested the asymmetric unit to contain four molecules. This has been confirmed by molecular-replacement phasing calculations, which give a solution with four monomers using a monomer of pantothenate synthetase from Escherichia coli (PDB code 1iho), which is 41% identical to Sar2676, as a search model.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Resistência a Meticilina , Peptídeo Sintases/isolamento & purificação , Staphylococcus aureus/isolamento & purificação , Proteínas de Bactérias/química , Cristalização , Cristalografia por Raios X , Peptídeo Sintases/química , Staphylococcus aureus/química
18.
Acta Crystallogr D Biol Crystallogr ; 63(Pt 5): 636-45, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17452789

RESUMO

A strategy of rationally engineering protein surfaces with the aim of obtaining mutants that are distinctly more susceptible to crystallization than the wild-type protein has previously been suggested. The strategy relies on replacing small clusters of two to three surface residues characterized by high conformational entropy with alanines. This surface entropy reduction (or SER) method has proven to be an effective salvage pathway for proteins that are difficult to crystallize. Here, a systematic comparison of the efficacy of using Ala, His, Ser, Thr and Tyr to replace high-entropy residues is reported. A total of 40 mutants were generated and screened using two different procedures. The results reaffirm that alanine is a particularly good choice for a replacement residue and identify tyrosines and threonines as additional candidates that have considerable potential to mediate crystal contacts. The propensity of these mutants to form crystals in alternative screens in which the normal crystallization reservoir solutions were replaced with 1.5 M NaCl was also examined. The results were impressive: more than half of the mutants yielded a larger number of crystals with salt as the reservoir solution. This method greatly increased the variety of conditions that yielded crystals. Taken together, these results suggest a powerful crystallization strategy that combines surface engineering with efficient screening using standard and alternate reservoir solutions.


Assuntos
Proteínas/química , Cristalização , Mutagênese , Propriedades de Superfície
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