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1.
J Hand Surg Am ; 45(5): 389-398, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31733980

RESUMO

PURPOSE: Studies have shown the effects of surgical treatments for trapeziometacarpal osteoarthritis on thumb biomechanics; however, the biomechanical effects on the wrist have not been reported. This study aimed to quantify alterations in wrist muscle forces following trapeziectomy with or without ligament reconstruction and replacement. METHODS: A validated physiological wrist simulator replicated cyclic wrist motions in cadaveric specimens by applying tensile loads to 6 muscles. Muscle forces required to move the intact wrist were compared with those required after performing trapeziectomy, suture suspension arthroplasty, prosthetic replacement, and ligament reconstruction with tendon interposition (LRTI). RESULTS: Trapeziectomy required higher abductor pollicis longus forces in flexion and higher flexor carpi radialis forces coupled with lower extensor carpi ulnaris forces in radial deviation. Of the 3 surgical reconstructions tested post-trapeziectomy, wrist muscle forces following LRTI were closest to those observed in the intact case throughout the range of all simulated motions. CONCLUSIONS: This study shows that wrist biomechanics were significantly altered following trapeziectomy, and of the reconstructions tested, LRTI most closely resembled the intact biomechanics in this cadaveric model. CLINICAL RELEVANCE: Trapeziectomy, as a standalone procedure in the treatment of trapeziometacarpal osteoarthritis, may result in the formation of a potentially unfilled trapezial gap, leading to higher wrist muscle forces. This biomechanical alteration could be associated with clinically important outcomes, such as pain and/or joint instability.


Assuntos
Osteoartrite , Trapézio , Fenômenos Biomecânicos , Cadáver , Humanos , Osteoartrite/cirurgia , Polegar , Trapézio/cirurgia , Punho , Articulação do Punho/cirurgia
2.
J Interprof Care ; 32(1): 24-32, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29083250

RESUMO

To practice interprofessional collaboration (IPC), understanding the roles of each profession in the team is key. Anesthesia assistants (AAs) are a relatively new addition to the Canadian healthcare system. As a result, its role in the delivery of anesthesia care can be misunderstood by other healthcare professionals. Using an exploratory multiple case study design, this article explores healthcare professionals' perceptions of the AA role and its impact on patients and IPC. Sixteen semi-structured, in-depth interviews were conducted with a purposive sample of nine healthcare professions from inside and outside the peri-operative care unit in two urban, acute care, university-affiliated teaching research hospitals in Ontario. A thematic analysis of the interview transcripts identified five overarching themes: limited understanding of the AA role, improved patient-centred care, improve IPC and interprofessional education, ongoing challenges, and the future direction for professional growth. Results indicated that despite regular clinical practice collaboration, participants have a limited understanding of AAs in terms of their educational prerequisites, scope of practice, and roles. One reason for this lack of understanding is that there is a high variability of titles and clinical duties for non-physician anesthesia providers. The diverse range of anesthesia services provided by AAs can also become a barrier to the full understanding of their scope of practice and roles. The limited understanding of the AA role was reported as one of AAs' ongoing challenges. It prevents AAs from realising their full scope of practice. Participants suggested that AAs' professional growth should focus on promoting and expanding their role. Understanding other healthcare professionals' perceptions of AAs will assist them to become better ambassadors for their role, and to more effectively promote and practice IPC. Ultimately, this will result in improved interprofessional teamwork to deliver effective and efficient patient care.


Assuntos
Anestesistas/organização & administração , Atitude do Pessoal de Saúde , Pessoal de Saúde/psicologia , Relações Interprofissionais , Equipe de Assistência ao Paciente/organização & administração , Comportamento Cooperativo , Hospitais de Ensino , Humanos , Entrevistas como Assunto , Ontário , Assistência Centrada no Paciente/organização & administração , Percepção , Papel Profissional , Pesquisa Qualitativa
3.
Soft Matter ; 13(35): 5842-5848, 2017 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-28815232

RESUMO

We report on electrically-induced, large magnitude (>300 nm), and reversible tuning of the selective reflection in polymer stabilized cholesteric liquid crystals (PSCLCs) prepared from negative dielectric anisotropy nematic liquid crystal hosts. The electrically-induced blue shift in the selective reflection of the PSCLCs is distinguished from our prior reports of bandwidth broadening and red-shifting tuning of the selective reflection in PSCLCs. The dominant factor in delineating the electro-optic response of the PSCLCs detailed here are the preparation conditions. Specifically, long exposure to UV intensity exceeding 250 mW cm-2. Other factors are shown to contribute to the response, including the type and concentration of photoinitiator.

6.
J Shoulder Elbow Surg ; 23(3): 401-8, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24090978

RESUMO

BACKGROUND: Late complications after humeral head replacement (HHR) for comminuted proximal humeral fractures are common and may necessitate revision surgery. This study evaluated the outcome of revision surgery of failed post-traumatic HHR with a less medialized reverse shoulder prosthesis. METHODS: Thirty-three patients with failed post-traumatic HHR due to rotator-cuff insufficiency and glenoid erosion, but with sufficient preservation of the glenoid bone stock to permit primary stability of an inverted glenoid implant, underwent revision using the Bayley-Walker reverse shoulder prosthesis (Stanmore Implants, Elstree, UK) and were monitored up for a mean of 31 months. Outcome measures included the Oxford Shoulder Score, subjective shoulder value, pain rating, active range of motion, and shoulder radiographs. RESULTS: The average postrevision Oxford Shoulder Score and subjective shoulder value improved from 50 ± 6 to 29 ± 11 and from 23 ± 19 to 51 ± 23, respectively (P < .001). Pain level decreased from 6.2 ± 2.1 to 1.4 ± 2.0 (P < .001). Active forward flexion increased from 34° ± 22° to 63° ± 30° and external rotation from 11° ± 14° to 20° ± 16°(P < .01). More patients were able to use their affected arm to reach a functional triangle consisting the mouth, opposite armpit, and ipsilateral buttock after revision (24% vs 73%; P < .001). Seven patients (21%) had postrevision complications. No glenoid loosening or scapular notching occurred. CONCLUSION: Revision of failed post-traumatic HHR with the Bayley-Walker shoulder offers reliable pain relief and improvement in shoulder function with a complication rate similar to other reverse prostheses. Nevertheless, revision shoulder arthroplasty remains challenging with a high rate of complications.


Assuntos
Artroplastia de Substituição/efeitos adversos , Artroplastia de Substituição/métodos , Cabeça do Úmero/lesões , Prótese Articular/efeitos adversos , Fraturas do Ombro/cirurgia , Idoso , Idoso de 80 Anos ou mais , Artralgia/diagnóstico , Artralgia/etiologia , Feminino , Humanos , Cabeça do Úmero/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Desenho de Prótese , Falha de Prótese , Radiografia , Amplitude de Movimento Articular , Reoperação , Rotação , Fraturas do Ombro/fisiopatologia , Articulação do Ombro/diagnóstico por imagem , Articulação do Ombro/fisiopatologia , Articulação do Ombro/cirurgia , Resultado do Tratamento
7.
Cureus ; 14(9): e29238, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36262938

RESUMO

INTRODUCTION: The mismanagement of an occult scaphoid fracture is a significant concern in patients presenting with anatomical snuffbox tenderness and no radiographic signs of injury. AIM: This study investigated whether a virtual fracture clinic (VFC) could improve care efficiency and expedite management decisions surrounding suspected pediatric scaphoid fractures. METHOD: Data was reviewed for patients referred via the VFC for suspected scaphoid fractures at a local trauma unit over 19 months. Patients received an "appointment" in VFC. Based on their notes and imaging, patients were referred to an outpatient clinic for repeat radiographs within two weeks (if initial radiographs demonstrated no fracture). Patients with unremarkable second x-rays were contacted and informed to mobilize and return if the pain persisted at four weeks. RESULTS: The pathway received 175 referrals; 114 male, 61 female, mean age 14 years, range 9-17) with 42 scaphoid fractures diagnosed, 35 (83.3%) on first x-ray, and 7 (16.7%) occult fractures. The pathway managed all patients as intended; 71 patients were seen face-to-face in the clinic due to age or pathology picked up on the first x-ray, and 104 required repeat radiographs. Following the second radiograph, 78 patients were discharged directly. Twenty-six patients required further review in a face-to-face clinic after their second radiograph. CONCLUSION: VFC appears to be a safe and efficient method of managing patients with suspected scaphoid fractures on short-term follow-up analysis. This cohort presents no 'missed' injuries and therefore appears safe compared to conventional treatment pathways.

8.
JCI Insight ; 7(2)2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-34874915

RESUMO

We performed next-generation sequencing in patients with familial steroid-sensitive nephrotic syndrome (SSNS) and identified a homozygous segregating variant (p.H310Y) in the gene encoding clavesin-1 (CLVS1) in a consanguineous family with 3 affected individuals. Knockdown of the clavesin gene in zebrafish (clvs2) produced edema phenotypes due to disruption of podocyte structure and loss of glomerular filtration barrier integrity that could be rescued by WT CLVS1 but not the p.H310Y variant. Analysis of cultured human podocytes with CRISPR/Cas9-mediated CLVS1 knockout or homozygous H310Y knockin revealed deficits in clathrin-mediated endocytosis and increased susceptibility to apoptosis that could be rescued with corticosteroid treatment, mimicking the steroid responsiveness observed in patients with SSNS. The p.H310Y variant also disrupted binding of clavesin-1 to α-tocopherol transfer protein, resulting in increased reactive oxygen species (ROS) accumulation in CLVS1-deficient podocytes. Treatment of CLVS1-knockout or homozygous H310Y-knockin podocytes with pharmacological ROS inhibitors restored viability to control levels. Taken together, these data identify CLVS1 as a candidate gene for SSNS, provide insight into therapeutic effects of corticosteroids on podocyte cellular dynamics, and add to the growing evidence of the importance of endocytosis and oxidative stress regulation to podocyte function.


Assuntos
Proteínas de Transporte/genética , Endocitose , Síndrome Nefrótica , Estresse Oxidativo , Podócitos , Corticosteroides , Animais , Apoptose/efeitos dos fármacos , Sistemas CRISPR-Cas/genética , Células Cultivadas , Endocitose/efeitos dos fármacos , Endocitose/genética , Técnicas de Inativação de Genes , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Síndrome Nefrótica/tratamento farmacológico , Síndrome Nefrótica/genética , Síndrome Nefrótica/metabolismo , Síndrome Nefrótica/patologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Peixe-Zebra , Proteínas de Peixe-Zebra
9.
Crim Behav Ment Health ; 20(1): 39-50, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20104476

RESUMO

BACKGROUND: There is a need for research to promote an understanding among service developers on why people with intellectual disabilities (ID) are referred to offender services in order for them to receive appropriate assessment and treatment. Previous studies investigating referrals into forensic ID services have concentrated on referral sources and administrative variables such as legal status. AIMS: To construct a predictive model for choice of service referral based on a comprehensive range of information about the clientele. METHOD: We conducted a case record study of 336 people referred to community services and 141 to secure provision. We gathered information on referral source, demographics, diagnosis, index behaviour, prior problem behaviours and history of abuse. RESULTS: Comparisons revealed 19 candidate variables which were then entered into multivariate logistic regression. The resulting model retained six variables: community living at time of referral, physical aggression, being charged, referral from tertiary health care, diverse problem behaviour and IQ < 50, which correctly predicted the referral pathway for 85.7% of cases. CONCLUSIONS: An index act of physical aggression and a history of diversity of problem behaviours as predictors against the likelihood of community service referral suggest that professionals have similar concerns about people with ID as they do about their more average offending peers; however, the more severe levels of ID mitigated in favour of community referral, regardless. Offenders with ID tend to be referred within levels of service rather than between them, for example, form tertiary services into generic community services.


Assuntos
Criminosos/estatística & dados numéricos , Desinstitucionalização/estatística & dados numéricos , Pessoas com Deficiência Mental/estatística & dados numéricos , Encaminhamento e Consulta/estatística & dados numéricos , Seguridade Social/estatística & dados numéricos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criminosos/psicologia , Desinstitucionalização/métodos , Feminino , Humanos , Modelos Logísticos , Masculino , Transtornos Mentais/epidemiologia , Pessoa de Meia-Idade , Pessoas com Deficiência Mental/psicologia , Medição de Risco , Reino Unido/epidemiologia , Adulto Jovem
10.
World J Plast Surg ; 9(2): 135-140, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32934923

RESUMO

BACKGROUND: National Institute for Health and Care Excellence (NICE) CG74 has set out evidence-based guidance on which types of surgery require antibiotic prophylaxis. Our aim was to establish what the current practice for antibiotic prophylaxis in Dupuytren's surgery is amongst hand surgeons in the United Kingdom, through the British Society for Surgery of the Hand (BSSH). METHODS: Permission was granted for our online survey to be distributed to BSSH hand surgeons via consecutive BSSH e-bulletins. Hand surgeons who did not perform fasciectomy or dermofasciectomy were excluded from the study. RESULTS: There were 64 respondents, represented an estimated 7.4-7.8% of membership. Eleven percent of respondents used antibiotics for fasciectomy, with an increasing trend towards revision surgery and dermofasciectomy. Over 30% prescribed them for revision dermofasciectomy. Dupuytren's surgery was classified as clean, non-prosthetic and uncomplicated which NICE CG74 suggestions did not require antibiotic prophylaxis. CONCLUSION: This study highlighted variation in practice amongst hand surgeons in the United Kingdom. Further consultation to create guidelines for hand surgery may help guide members and reduce potentially unnecessary prophylactic antibiotic use.

11.
J Biomech ; 77: 218-222, 2018 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30054091

RESUMO

The abductor pollicis longus (APL) is one of the primary radial deviators of the wrist, owing to its insertion at the base of the first metacarpal and its large moment arm about the radioulnar deviation axis. Although it plays a vital role in surgical reconstructions of the wrist and hand, it is often neglected while simulating wrist motions in vitro. The aim of this study was to observe the effects of the absence of APL on the distribution of muscle forces during wrist motions. A validated physiological wrist simulator was used to replicate cyclic planar and complex wrist motions in cadaveric specimens by applying tensile loads to six wrist muscles - flexor carpi radialis (FCR), flexor carpi ulnaris, extensor carpi radialis longus (ECRL), extensor carpi radialis brevis, extensor carpi ulnaris (ECU) and APL. Resultant muscle forces for active wrist motions with and without actuating the APL were compared. The absence of APL resulted in higher forces in FCR and ECRL - the synergists of APL - and lower forces in ECU - the antagonist of APL. The altered distribution of wrist muscle forces observed in the absence of active APL control could significantly alter the efficacy of in vitro experiments conducted on wrist simulators, in particular when investigating those surgical reconstructions or rehabilitation of the wrist heavily reliant on the APL, such as treatments for basal thumb osteoarthritis.


Assuntos
Modelos Biológicos , Movimento , Punho/fisiologia , Fenômenos Biomecânicos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/fisiologia
12.
J Biomech ; 60: 232-237, 2017 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-28669547

RESUMO

Although the orientations of the hand and forearm vary for different wrist rehabilitation protocols, their effect on muscle forces has not been quantified. Physiologic simulators enable a biomechanical evaluation of the joint by recreating functional motions in cadaveric specimens. Control strategies used to actuate joints in physiologic simulators usually employ position or force feedback alone to achieve optimum load distribution across the muscles. After successful tests on a phantom limb, unique combinations of position and force feedback - hybrid control and cascade control - were used to simulate multiple cyclic wrist motions of flexion-extension, radioulnar deviation, dart thrower's motion, and circumduction using six muscles in ten cadaveric specimens. Low kinematic errors and coefficients of variation of muscle forces were observed for planar and complex wrist motions using both novel control strategies. The effect of gravity was most pronounced when the hand was in the horizontal orientation, resulting in higher extensor forces (p<0.017) and higher out-of-plane kinematic errors (p<0.007), as compared to the vertically upward or downward orientations. Muscle forces were also affected by the direction of rotation during circumduction. The peak force of flexor carpi radialis was higher in clockwise circumduction (p=0.017), while that of flexor carpi ulnaris was higher in anticlockwise circumduction (p=0.013). Thus, the physiologic wrist simulator accurately replicated cyclic planar and complex motions in cadaveric specimens. Moreover, the dependence of muscle forces on the hand orientation and the direction of circumduction could be vital in the specification of such parameters during wrist rehabilitation.


Assuntos
Músculo Esquelético/fisiologia , Punho/fisiologia , Fenômenos Biomecânicos , Feminino , Antebraço/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Movimento/fisiologia , Rotação
13.
Nucleic Acids Res ; 32(18): 5442-51, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15479781

RESUMO

Two archaeal Holliday junction resolving enzymes, Holliday junction cleavage (Hjc) and Holliday junction endonuclease (Hje), have been characterized. Both are members of a nuclease superfamily that includes the type II restriction enzymes, although their DNA cleaving activity is highly specific for four-way junction structure and not nucleic acid sequence. Despite 28% sequence identity, Hje and Hjc cleave junctions with distinct cutting patterns--they cut different strands of a four-way junction, at different distances from the junction centre. We report the high-resolution crystal structure of Hje from Sulfolobus solfataricus. The structure provides a basis to explain the differences in substrate specificity of Hje and Hjc, which result from changes in dimer organization, and suggests a viral origin for the Hje gene. Structural and biochemical data support the modelling of an Hje:DNA junction complex, highlighting a flexible loop that interacts intimately with the junction centre. A highly conserved serine residue on this loop is shown to be essential for the enzyme's activity, suggesting a novel variation of the nuclease active site. The loop may act as a conformational switch, ensuring that the active site is completed only on binding a four-way junction, thus explaining the exquisite specificity of these enzymes.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , DNA Cruciforme/metabolismo , Resolvases de Junção Holliday/química , Resolvases de Junção Holliday/metabolismo , Proteínas Arqueais/genética , Sequência de Bases , Sítios de Ligação , Catálise , DNA Cruciforme/química , Evolução Molecular , Resolvases de Junção Holliday/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Serina/química , Especificidade por Substrato , Sulfatos/metabolismo , Sulfolobus/enzimologia , Proteínas Virais/genética
14.
ACS Macro Lett ; 4(9): 942-946, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-35596461

RESUMO

The spontaneous conversion of a flat film into a 3-D shape requires local programming of the mechanical response. Historically, the ability to locally program the mechanical response of high strain (>30%) liquid crystalline elastomers (LCEs) has been limited to magnetic or mechanical alignment techniques, which limits spatial resolution. Recently, we reported on the preparation of LCEs capable of 55% strain with spatial control of the mechanical response at scales as small as 0.01 mm2. Here, we report a distinct formulation strategy to realize programmable stimulus-response in LCEs. Photopolymerization of thiol-ene/acrylate formulations yields materials that exhibit large reversible strain up to 150%. The photopolymerization reaction is extremely rapid, reducing preparation time from days to minutes. The mechanical behavior of these materials can be tuned by varying cross-link density. Spatial and hierarchical programming of the director profile is demonstrated, enabling 3-D shape change, including twisting ribbons and localized Gaussian curvature.

16.
J Biomed Mater Res A ; 94(2): 586-93, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20198691

RESUMO

Percutaneous implants require a seal at the skin interface. Laminin (L-332) is a component of the basement membrane, integral to epidermal attachment. To enhance the attachment of keratinocytes onto the surface of titanium alloy (Ti(6)Al(4)V), we attached L-332 onto the surface using silanization (L-332==Ti(6)Al(4)V). Iodinated L-332 was used to investigate protein attachment kinetics. L-332==Ti(6)Al(4)V remained attached after immersion in serum compared with adsorbed L-332. Cells from a keratinocyte line (HaCaT) grown on L-332==Ti(6)Al(4)V were significantly smaller (p < 0.05) with over a 20-fold increase in the number of adhesion plaques compared with nontreated Ti(6)Al(4)V control discs or with adsorbed L-332 surfaces. We have shown that it is possible to augment the surface of Ti(6)Al(4)V with L-332 and that this significantly increases the attachment of keratinocytes.


Assuntos
Ligas/química , Laminina/química , Próteses e Implantes , Titânio/química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Adesão Celular , Linhagem Celular , Proliferação de Células , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Laminina/metabolismo , Teste de Materiais , Proteínas/metabolismo , Propriedades de Superfície , Vinculina/metabolismo
17.
J Biomed Mater Res A ; 92(3): 1028-37, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19296546

RESUMO

The success of intraosseous transcutaneous amputation prostheses (ITAP) relies on soft tissue attachment to prevent infection which leads to implant failure. Fibronectin (Fn) has been shown to enhance dermal fibroblast attachment in vitro, however measurement of cell attachment strength has been indirect; using cell area and immunolocalization of focal adhesion components. In this study, we have developed a flow apparatus to assess the biophysical strength of cell attachment to biomaterials used in ITAP. We have demonstrated that dermal fibroblast attachment strength increases significantly up to 96 h and that data from direct and indirect methods of assessing cell attachment strength have a significant positive correlation. Additionally, we have used direct and indirect assessment methods to demonstrate that dermal fibroblast attachment strength is significantly greater on fibronectin-coated titanium alloy compared with uncoated controls at 1, 4, and 24 hours.


Assuntos
Ligas , Adesão Celular , Pele/citologia , Titânio , Linhagem Celular Transformada , Fibroblastos/citologia , Fibronectinas , Humanos , Teste de Materiais , Reprodutibilidade dos Testes
18.
Am J Intellect Dev Disabil ; 115(3): 250-62, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20441394

RESUMO

The pathways through services for offenders with intellectual disability were reviewed. Participants were 197 offenders with intellectual disability accepted into three types of community and three types of secure forensic intellectual disability services. They were first compared with 280 participants referred but not accepted into services and were then followed-up for 2 years to review pathways through services. Those accepted into services had a higher charge rate than did those who were referred (46% and 25%, respectively). The greatest diversity in pathway was seen in participants in community forensic intellectual disability and inpatient services. Individuals in secure settings showed the least diversity over time, and, similarly, a relatively high percentage of those accepted into generic community services remained in these services.


Assuntos
Acessibilidade aos Serviços de Saúde , Deficiência Intelectual/reabilitação , Transtornos Mentais/reabilitação , Serviços de Saúde Mental , Prisioneiros/psicologia , Encaminhamento e Consulta , Adulto , Transtorno da Personalidade Antissocial/psicologia , Transtorno da Personalidade Antissocial/reabilitação , Serviços Comunitários de Saúde Mental , Crime/psicologia , Feminino , Seguimentos , Hospitalização , Humanos , Deficiência Intelectual/psicologia , Masculino , Transtornos Mentais/psicologia , Equipe de Assistência ao Paciente , Medidas de Segurança , Medicina Estatal
19.
Acta Crystallogr D Biol Crystallogr ; 59(Pt 1): 171-3, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12499561

RESUMO

HolliDay junction endonuclease (Hje) from Sulfolobus solfataricus is a resolving enzyme involved in cleaving specific sites on either side of recombinant four-way HolliDay junctions. The HJE gene from S. solfataricus was cloned from genomic DNA into the pET19b Escherichia coli expression vector and recombinant protein was expressed to high levels. Hje was purified using heat treatment, cation exchange and gel filtration. Hanging-drop crystallization trials yielded primitive hexagonal crystals which diffract to 2.4 A on a laboratory source. Systematic absences (only 00l = 6n present) and poor scaling in P622 indicate that the space group is P6(1) or its enantiomer. Failed attempts at molecular replacement using models of a related archaeal resolving enzyme, Hjc, raise the possibility of a difference in quaternary structure between Hjc and Hje, which may be responsible for differences in their activities.


Assuntos
Endodesoxirribonucleases/química , Sulfolobus/enzimologia , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Primers do DNA/genética , DNA Arqueal/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/isolamento & purificação , Endodesoxirribonucleases/metabolismo , Escherichia coli/metabolismo , Resolvases de Junção Holliday , Modelos Moleculares , Conformação de Ácido Nucleico , Estrutura Quaternária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
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