Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 109
Filter
1.
Nurse Educ Today ; 131: 105975, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37729862

ABSTRACT

BACKGROUND: Internationally educated nurses attending a bridging program must demonstrate clinical competence and meet requirements to apply for a nursing license in Sweden. OBJECTIVES: To describe preceptors' experiences of supervising internationally educated nurses undergoing clinical practice education during a bridging program. DESIGN: A qualitative descriptive study. SETTINGS: Two universities offering the 1-year bridging program for nurses with a nursing degree from outside European Union/European Economic Area and Switzerland. PARTICIPANTS: Fifteen preceptors, all registered nurses, who supervised internationally educated nurses were included. METHODS: Semi-structured interviews were performed, and data were analyzed using qualitative content analysis. RESULTS: Supervising internationally educated nurses was not the same as supervising nursing students and raised feelings of both joy and frustration. Preceptors had to adapt supervision to the student's nursing knowledge and skills. They had to help students communicate in Swedish and form good relationships with other students, patients, and other professionals. Most preceptors requested more information about the student's nurse education, country of education/cultural background, and previous work experiences. Mixed experiences of support from the university, first-line managers, and colleagues were reported. CONCLUSIONS: Being a preceptor for internationally educated nurses is a challenge, and supervision training is important for managing preceptorship. To supervise students based on their level of knowledge and skills, more information must be shared with the preceptor. Encounters with others are of importance in the training, where teamwork and person-centered care must be in focus, both in prior theoretical education and in clinical practice education.


Subject(s)
Education, Nursing, Baccalaureate , Students, Nursing , Humans , Qualitative Research , Educational Status , Sweden , Preceptorship , Clinical Competence
2.
Nurse Educ Today ; 119: 105595, 2022 Dec.
Article in English | MEDLINE | ID: mdl-36265213

ABSTRACT

BACKGROUND: Bridging programs are offered to support migrated nurses, but in some countries, nurses can also choose to validate their nursing competence. Thus far, little is known about how migrated nurses estimate their competence when they are about to enter working life in a new country and how this differs from regular nursing students. OBJECTIVE: To compare two groups of internationally educated nurses' - those from bridging programs and those who chose validation - and one group of regular nursing students' self-rated professional competence when they are about to start working as registered nurses. The hypotheses were: 1) internationally educated nurses rate their competence higher than regular nursing students and 2) those from bridging programs rate their competence higher than those who chose validation. In addition, the aim was to compare the groups' self-efficacy and thriving. DESIGN: A cross-sectional, comparative design. SETTINGS: Five universities in Sweden. PARTICIPANTS: Nurses educated in non-European countries from a bridging program (n = 128, response rate 79.0 %) or validation process (n = 61, response rate 59.2 %) and students graduating from the regular nursing program (n = 213, response rate 68.3 %). METHODS: Data were collected with coded questionnaires (paper or online) between 2019 and 2021 and analyzed using non-parametric tests, e.g., Kruskal-Wallis. RESULTS: Both groups of internationally educated nurses had higher median scores on total nursing competence (both groups p < 0.001), general self-efficacy (bridging programs p < 0.001, validation p = 0.020), and total thriving (bridging programs p < 0.001, validation p = 0.012) than regular nursing students did. However, comparing the groups of internationally educated nurses showed no significant differences. CONCLUSION: Internationally educated nurses rated their competence high but with differences within the groups for different competence areas. More research is needed to investigate whether the different paths are important for nurses' competence later in working life, and some of the competence areas might need extra attention when nurses start working.


Subject(s)
Students, Nursing , Humans , Cross-Sectional Studies , Self Efficacy , Sweden , Professional Competence , Surveys and Questionnaires , Clinical Competence
3.
Nurse Educ Today ; 99: 104744, 2021 Apr.
Article in English | MEDLINE | ID: mdl-33549959

ABSTRACT

BACKGROUND: Bridging programs have been created to facilitate internationally educated nurses' integration process. Thus far, studies on bridging programs have been few and have only been conducted in English-speaking countries. Due to language barriers, it may be a greater challenge to attend a bridging program in a non-English-speaking country. OBJECTIVES: The aim was to examine internationally educated nurses' experience of attending a one-year bridging program to obtain a Swedish nursing license. DESIGN: A qualitative study with a descriptive design was applied. SETTINGS: The study setting was the five universities offering the one-year, full-time Swedish bridging program. PARTICIPANTS: Purposive sampling was used. Eighteen nurses participated in the study at the end of the program. METHODS: Semi-structured interviews were conducted and analyzed using qualitative content analysis. RESULTS: Studying in a new environment and language was challenging and intensive, as were adapting to a new healthcare system and relearning some nursing practices. However, attending the bridging program was also rewarding and gave feelings of happiness and pride; the nurses developed their nursing skills as well as their language and academic skills. Moreover, they became familiar with Sweden's nursing practices, healthcare system, and culture. Good support was important, but not always enough. CONCLUSIONS: By attending a bridging program, nurses can become familiar with the country's healthcare system and nursing practices. Moreover, develop their language skills and attain skills important to lifelong learning. Although the program may not eliminate all difficulties nurses often experience in a new country, it can offer the support nurses need to handle the challenges. However, for some nurses, due to different backgrounds and prerequisites, the support offered may need to be more individualized.


Subject(s)
Language , Nurses , Communication Barriers , Humans , Licensure , Sweden
4.
J Pediatr Urol ; 10(6): 1100-5, 2014 Dec.
Article in English | MEDLINE | ID: mdl-24881806

ABSTRACT

BACKGROUND: The aim of the present study was to investigate whether addition of transcutaneous electrical nerve stimulation (TENS) treatment improves the results of standard urotherapy in children with overactive bladder (OAB) symptoms. MATERIAL AND METHODS: Sixty-two children with symptoms of OAB and incontinence were included. The children were randomized either to standard urotherapy treatment alone or a combination of standard urotherapy and TENS. The effect variables were taken from a voiding-drinking diary: number of voiding, number of incontinence episodes, and maximum voided volume. RESULTS: Both treatment groups had good treatment results, with no significant difference between the groups. In the standard treatment group 13/28 (46%) were completely dry and 11/28 (40%) had a decrease in incontinence episodes, compared to 16/24 (67%) and 3/24 (13%), respectively, in the standard+TENS group (p=0.303). The number of voiding decreased in two-thirds of the patients in both groups. However, maximal voided volume only increased in the standard treatment group. Subjectively 72% and 80% considered themselves significantly improved or free of symptoms. Previous treatment was registered in 15/55 (27%). All efficacy variables decreased in those with previous treatment, but with no difference between the standard and standard+TENS treatment groups. The only difference noted was when comparing the children without previous treatment in the groups: a significantly higher proportion were completely dry in the TENS group (12/18 [71%] versus 10/22 [48%], p=0.05). CONCLUSION: Our results showed no significant difference overall in treatment response to OAB symptoms between urotherapy only and urotherapy+TENS, whereas a tendency to difference was found in children without previous treatment. Thus with good urotherapy support, TENS only seems to have marginal additional effects on OAB symptoms.


Subject(s)
Transcutaneous Electric Nerve Stimulation/methods , Urinary Bladder, Overactive/therapy , Urination/physiology , Child , Child, Preschool , Female , Follow-Up Studies , Humans , Male , Treatment Outcome , Urinary Bladder, Overactive/physiopathology
5.
Xenobiotica ; 39(8): 606-15, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19622003

ABSTRACT

The association of CYP1B1 gene alterations in primary congenital glaucoma individuals has been known for about a decade. Recent evidence has shown the involvement of CYP1B1 mutations in a number of forms of glaucoma and anterior segment disorders. This suggests a wide role for CYP1B1 in ocular physiology. Histochemical studies of eyes from individuals with primary congenital glaucoma revealed abnormalities in the anterior chamber angle, the region between the cornea and the iris, containing the trabecular meshwork. The cells of the trabecular meshwork serve as a filter to allow drainage of the aqueous humour, the fluid formed by the ciliary body that fills the anterior chamber. Mutations in CYP1B1 that affect its activity have frequently been shown to influence development of the trabecular meshwork, and it is thought that CYP1B1, a monooxygenase, acts to form or degrade some endobiotic compound that is necessary for proper development of the filtering structures. The rapidly developing area of stem cell research suggests a potential therapeutic approach for glaucomas resulting from deleterious mutations in CYP1B1, that is, the transfer of stem cells, differentiated to a specific lineage, containing wild-type CYP1B1 to specific regions of the eye, where they will develop into normal cells of that region and rectify the defect.


Subject(s)
Cytochrome P-450 Enzyme System/genetics , Genetic Diseases, Inborn/therapy , Glaucoma/therapy , Mutation , Stem Cell Transplantation , Animals , Aryl Hydrocarbon Hydroxylases , Cornea/embryology , Cornea/enzymology , Cytochrome P-450 CYP1B1 , Genetic Diseases, Inborn/enzymology , Genetic Diseases, Inborn/genetics , Glaucoma/enzymology , Glaucoma/genetics , Humans , Iris/embryology , Iris/enzymology , Stem Cells/enzymology , Trabecular Meshwork/embryology , Trabecular Meshwork/enzymology
7.
Drug Metab Rev ; 38(1-2): 337-52, 2006.
Article in English | MEDLINE | ID: mdl-16684663

ABSTRACT

Expression of 10 CYP orthologs (Families 1-3) in developing mouse conceptus is constitutive. These forms have specific temporal and spatial expression. Studies on CYP1B1 indicate its requirement for normal eye development, both in human and mouse. The distribution of the enzyme in the mouse eye is in three regions, which may reflect three different, perhaps equally important, functions in this organ. Its presence in the inner ciliary and lens epithelia appears to be necessary for normal development of the trabecular meshwork and its function in regulating intraocular pressure. Its expression in the retinal ganglion and inner nuclear layers may reflect a role in maintenance of the visual cycle. Its expression in the corneal epithelium may indicate a function in metabolism of environmental xenobiotics.


Subject(s)
Cytochrome P-450 Enzyme System/physiology , Eye/enzymology , Eye/growth & development , Animals , Aryl Hydrocarbon Hydroxylases/biosynthesis , Aryl Hydrocarbon Hydroxylases/genetics , Cytochrome P-450 CYP1B1 , Cytochrome P-450 Enzyme System/biosynthesis , Cytochrome P-450 Enzyme System/genetics , Humans
8.
Eur J Vasc Endovasc Surg ; 31(6): 651-60, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16427339

ABSTRACT

OBJECTIVES: To test if initial high-dose, pulse-spray thrombolysis improves the early and late outcome of lower limb ischaemia as compared with low-dose infusion alone. DESIGN: Prospective randomised multicentre study. MATERIAL AND METHODS: Patients with acute and sub-acute (<30 days) lower limb ischaemia were randomised following angiography. Group 1 (n=58) received pulse-spray infusion of recombinant plasminogen activator (rt-PA, 15 mg/h) for 2h followed by low-dose infusion if needed. Group 2 (n=63) were only treated with low-dose infusion (0.5mg/h) of rt-PA for 48 h. Underlying lesions were corrected if required. RESULTS: The study was stopped prematurely. Complications were equally frequent in both groups. More than 75% lysis was accomplished in 78 versus 67% of the patients (p=0.21). Primary endpoints (re-occlusion, incomplete lysis, life-threatening complication, amputation, or death) were reached in 24 versus 32% of the patients (p=0.35). Neither vascular patency nor clinical parameters differed during the first year, but re-interventions tended to be more frequent (p=0.040 at 1 month; p=0.090 at 1 year) and of a greater magnitude (p=0.028) in group 2. CONCLUSIONS: There was no obvious advantage with initial high-dose thrombolysis, which may be a type-2 error. A reduction of major re-interventions was recorded.


Subject(s)
Fibrinolytic Agents/therapeutic use , Ischemia/drug therapy , Lower Extremity/blood supply , Thrombolytic Therapy , Tissue Plasminogen Activator/therapeutic use , Aged , Aged, 80 and over , Drug Administration Schedule , Female , Fibrinolytic Agents/administration & dosage , Humans , Male , Middle Aged , Prospective Studies , Recombinant Proteins/therapeutic use , Thrombolytic Therapy/methods , Tissue Plasminogen Activator/administration & dosage , Tissue Plasminogen Activator/genetics , Treatment Outcome
9.
Tissue Antigens ; 63(6): 600-1, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15140042

ABSTRACT

We have identified a new human leukocyte antigen-C allele in a Caucasian potential stem-cell donor. The new allele is identical to the other Cw*05 alleles in exon 2 but differs from Cw*0501 and *0503 at nucleotide position 379 in exon 3, where a C is substituted with a G. This results in an amino acid substitution from leucine to valine at residue 103 in alpha2 helix.


Subject(s)
HLA-C Antigens/genetics , Base Sequence , Humans , Molecular Sequence Data
10.
Arch Biochem Biophys ; 414(1): 91-100, 2003 Jun 01.
Article in English | MEDLINE | ID: mdl-12745259

ABSTRACT

This study is the first systematic investigation of the gestational age-dependent and adult tissue-specific expression patterns of each known mouse CYP family (40 genes) using normalized cDNA panels and uniform reverse transcriptase polymerase chain reaction-based assays. Twenty-seven of the P450s were constitutively expressed during development. The number gradually increased through the phases of gastrulation E7 (n=14), neural patterning and somitogenesis E11 (n=17), organogenesis E15 (n=20), and fetal period E17 (n=21). Cyp2s1, Cyp8a1, Cyp20, Cyp21a1, Cyp26a1, Cyp46, and Cyp51 were detected in each of the four stages studied. Members of family CYP1 demonstrated complex, nonoverlapping embryonic patterns of expression, indicating that Cyp1a1 and Cyp1a2 may not compensate for Cyp1b1 deficiency associated with abnormal eye development. Multiple Cyp forms were found to be constitutively expressed in each of the adult tissues studied: liver (n=31), kidney (n=30), testis (n=26), lung (n=24), and heart (n=13). The tissue-specific P450-expression profiles reported in this study provide a reference for more focused analysis of the tissue-specific and developmental functions of the cytochrome P450 monooxygenases.


Subject(s)
Aging/genetics , Cytochrome P-450 Enzyme System/genetics , Gene Expression Profiling/methods , Gene Expression Regulation, Developmental/genetics , Gene Expression Regulation, Enzymologic/genetics , Aging/physiology , Animals , Base Sequence , Cytochrome P-450 Enzyme System/classification , Embryo, Mammalian/enzymology , Male , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis/methods , Organ Specificity , Reverse Transcriptase Polymerase Chain Reaction/methods , Sequence Alignment , Sequence Analysis, DNA/methods
11.
Tissue Antigens ; 59(4): 344-6, 2002 Apr.
Article in English | MEDLINE | ID: mdl-12135440

ABSTRACT

During routine typing of a potential bone marrow donor, a new HLA DPB1 allele was identified. The new allele, officially named HLA DPB1*9001, differs from HLA DPB1*01011 in the second hypervariable region, where a single nucleotide substitution in position 191 changes the codon 35 from TAC to TTC with a predicted amino acid change from Tyr to Phe.


Subject(s)
HLA-DP Antigens/genetics , Alleles , Base Sequence , HLA-DP beta-Chains , Humans , Molecular Sequence Data
12.
Tissue Antigens ; 59(3): 226-8, 2002 Mar.
Article in English | MEDLINE | ID: mdl-12074715

ABSTRACT

During recent years, the view of the relative importance of the HLA Cw locus has undergone substantial change. From being an HLA locus with both limited polymorphism and biological significance there are now more than a hundred different alleles known and the biological importance of HLA Cw, both as a transplantation antigen and as a receptor for NK cells, is well established. Sequence based typing has been shown to be a powerful tool, especially for HLA Cw typing. Here we describe two new HLA Cw* alleles found during routine typing of potential bone marrow donors and hematological patients. The HLA Cw*0105 differs from Cw*0102 at positions 361 and 368 in exon 3 leading to a Trp to Arg and Cys to Ser substitution, respectively. HLA Cw*1405 differs from Cw*14021 by a single nucleotide substitution at position 368. This mutation results in an amino acid substitution of Phe for Tyr.


Subject(s)
Alleles , HLA-C Antigens/genetics , Amino Acid Sequence , Exons/genetics , Histocompatibility Testing/methods , Humans , Molecular Sequence Data , Polymerase Chain Reaction
13.
Tissue Antigens ; 59(2): 159-61, 2002 Feb.
Article in English | MEDLINE | ID: mdl-12028550

ABSTRACT

Five different haplotypes in the human HLA D region are recognized based on their gene composition. The HLA DR52 associated DRB1 alleles include DRB1*03/08/11/12/13/14 and are characterized by the YST sequence motif in the codons 10-12 but vary in other polymorphic regions. The mechanisms generating the extensive variability are not entirely clear. Some alleles have probably arisen from point mutations, but most polymorphism has probably been caused by intralocus gene conversion, and the distinction between the ancient serologically defined groups is more and more difficult. This report describes the identification of a novel DRB1* allele - DRB1*1345 - found in a kidney transplant recipient from East Africa. The new allele shows the closest resemblance to DRB1*1114 and DRB1*1323. It differs from both those alleles at codons 57, 58 and 60, where the new allele carries the 'A-H' sequence motif common to DRB1*14 alleles such as 1401/04/07/10/16/25. This motif is only found in one other DRB1*13 allele namely DRB1*1343.


Subject(s)
Exons , HLA-DR Antigens/genetics , Africa, Eastern , Alleles , Amino Acid Sequence , Base Sequence , HLA-DR Antigens/immunology , HLA-DRB1 Chains , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Polymorphism, Genetic , Sequence Analysis, DNA
14.
Pharmacogenetics ; 11(9): 793-801, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11740343

ABSTRACT

CYP1B1 is linked to normal eye development by the disease phenotype, primary congenital glaucoma (PCG). CYP1B1 mRNA was expressed in a number of human fetal tissue cDNA libraries, supporting the suggestion of its involvement in tissue development. Highest expression levels were found in thymus and kidney, followed by spleen. A considerably lower level was observed in lung, cardiac and skeletal muscle. No expression was detected in liver or brain. CYP1B1 is able to metabolize steroid hormones. Testosterone was a poor substrate and activity with progesterone was 6-fold higher, but estradiol was the preferred substrate, exhibiting a greater metabolite profile with CYP1B1 than with CYP1A2. Major metabolites were A-ring hydroxylations (75-80%). Others were 15alpha-, 6alpha-, 16alpha- and 6beta-hydroxy metabolites. Two CYP1B1 mutations found in families with the PCG phenotype in which incomplete penetrance is seen were expressed in Escherichia coli. G61E, a hinge region mutation, and R469W, a heme region mutation, were shown to code for holoenzymes. G61E had greatly diminished stability, while the R469W holoenzyme, if anything, was stabilized. Both mutants showed compromised catalytic activity. The extents of isomeric site activity diminution were not proportional, resulting in alterations in the metabolite profiles. The results suggest that if a metabolite of CYP1B1 or elimination of a metabolite by CYP1B1 is necessary for normal embryonic or fetal tissue development, the appearance of these two mutations could result in developmental abnormalities. The altered activities of the mutants and ability of CYP1B1 to respond to external challenge may be the basis for the observed incomplete penetrance.


Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Glaucoma/genetics , Point Mutation , Steroids/metabolism , Cytochrome P-450 CYP1A2/metabolism , Cytochrome P-450 CYP1B1 , Enzyme Stability , Escherichia coli/genetics , Estradiol/metabolism , Fetus/metabolism , Glaucoma/enzymology , Humans , Hydroxylation , Isomerism , Penetrance , Polymorphism, Genetic , Progesterone/metabolism , RNA, Messenger/metabolism , Substrate Specificity , Testosterone/metabolism , Tissue Distribution
15.
Drug Metabol Drug Interact ; 18(1): 33-55, 2001.
Article in English | MEDLINE | ID: mdl-11522124

ABSTRACT

Cytochrome P450 (CYP) forms are ubiquitous in nature, appearing in almost all phyla, with many forms appearing in any organism. About 50 different forms have been identified in man, and some of these are found in the embryo, some showing temporal dependence. Many of the forms of cytochrome P450 present in one species have homologues in other species. For example, CYP1A2 is present in many species, including man, rabbits, rodents, fish and fowl. The amino acid sequence identity of these homologues is often in excess of 70%. CYP26, too, has more than 61% identity in amino acid sequence between fish, fowl and mammals. In view of the high degree of conservation of sequence as well as of enzymatic activities, it is only reasonable to assume that such strong conservation of sequence also reflects a conservation of function. Since the 'xenobiotic metabolizing' enzymes predate the production of the many xenobiotics they are known to metabolize, perhaps it is reasonable to consider endobiotics as natural substrates for their metabolism. Of the identified forms of cytochrome P450 that are present in embryonic tissue, we consider the possibility that they serve the organism in support of morphogenesis of the embryonic tissue. These forms may either function to generate morphogenic molecules or to keep regions free of them, thereby creating temporal and spatial regions of morphogen action and supporting region-specific changes in cells. One known morphogen, retinoic acid, has the enzymes retinal dehydrogenase (RALDH) and CYP26 maintaining its actions, the former responsible for its generation and the latter for its elimination. Another form of cytochrome P450, CYP1B1 appears also to be involved in differentiation of tissue, with its absence resulting in primary congenital glaucoma. However, the nature of the morphogen it may maintain still remains to be elucidated.


Subject(s)
Cytochrome P-450 Enzyme System/physiology , Embryonic and Fetal Development/physiology , Animals , Cytochrome P-450 Enzyme System/genetics , Glaucoma/congenital , Glaucoma/enzymology , Humans , Morphogenesis/physiology
16.
Arch Biochem Biophys ; 385(1): 78-87, 2001 Jan 01.
Article in English | MEDLINE | ID: mdl-11361029

ABSTRACT

Quartz crystal microbalance (QCM) resonance measurements were used to examine the surface charge characteristics of cytochrome P450 forms and the influence of charge on the docking of redox partners like cytochrome b5. The distal surface of cytochrome P450 (CYP)101 (pI = 4.5), relative to the heme, is fairly anionic, as is the proximal surface. The latter, however, also has two cationic clusters. A considerably greater extent of CYP101 binding was seen to the cationic, polyethylene-surfaced resonators. CYP2B4 (pI = 8.5) preferentially bound to the polyanionic, polystyrene sulfonate-surfaced resonators. Cytochrome b5 is an acidic protein that had a preferential binding to the poly(ethyleneimine (PEI)-surfaced resonators. When binding to CYP2B4-surfaced films, cytochrome b5 preferentially bound to those cytochrome P450 molecules that were adsorbed to cationic (PEI) films. It is suggested that adsorption of CYP2B4 to an anionic poly(styrenesulfonate) (PSS) surface is with cationic clusters that include the cytochrome b5 docking domain. This diminishes the extent of docking of the cytochrome b5. In contrast, when CYP2B4 is adsorbed to a cationic film the proximal surface with the cytochrome b5-docking site is available for cytochrome b5 binding. A film of the polycation PEI was adsorbed to the silver QCM surface. It formed polymer islands when viewed with atomic force microscopy. Polyanionic PSS was adsorbed intermittently with the PEI. By the third and fourth layer of polyions the polymer islands were essentially merged and protein adsorption as a fourth or fifth layer formed a nearly continuous film. CYP101 was seen to adsorb as globules with a molecular diameter of about 10 nm. CYP2B4 adsorbed to the polyionic films had a slightly elliptical globular shape, also with a molecular diameter of about 10 nm.


Subject(s)
Aryl Hydrocarbon Hydroxylases , Chemistry/instrumentation , Chemistry/methods , Cytochrome P-450 Enzyme System/chemistry , Adsorption , Animals , Anions , Binding Sites , Cations , Cytochrome P-450 Enzyme System/metabolism , Cytochromes b5/metabolism , Dose-Response Relationship, Drug , Ions , Kinetics , Microscopy, Atomic Force/methods , Models, Molecular , Polystyrenes/metabolism , Protein Binding , Protein Structure, Tertiary , Rabbits , Steroid Hydroxylases/metabolism
17.
Toxicology ; 144(1-3): 211-9, 2000 Apr 03.
Article in English | MEDLINE | ID: mdl-10781890

ABSTRACT

Conditions for the optimal expression of the human CYP1B1 hemoprotein in Escherichia coli have been investigated. CYP1B1 cDNA was prepared from a retinal cDNA template and used to generate cDNA fragments with modified 5'-sequences reported to allow enhanced expression in E. coli DH5alpha. Plasmids were constructed, using the pCWori+ expression vector and were used to examine necessity for thiamine, delta-aminolevulinic acid (ALA), and IPTG. The optimal shaking speed in an orbital incubator was 150 rpm at 30 degrees C. Higher speeds resulted in increased cell death and lower speeds resulted in lower expression of cytochrome P450. IPTG was necessary for this expression system, which makes use of the lac repressor, but levels above 0.5 mM were without additional benefit. We were able to show thiamine to be unnecessary in this expression system, although included by others expressing CYP1B1. ALA has been reported to enhance expression of several different forms of cytochrome P450. We examined the dependence of CYP1B1 expression on ALA. The expression proved to be highly dependent upon this heme precursor, with levels of CYP1B1 increasing approximately 20-fold, to 920 nmol/l in the presence of up to 2.5 mM ALA. The question of whether heme synthesis and apoprotein synthesis were coupled was then investigated. It could be shown that although heme synthesis was not limiting (CYP101 holoenzyme expression in the absence of ALA was four times higher than the ALA-supported CYP1B1 holoenzyme expression), it was necessary for optimal expression of CYP1B1. CYP1B1 protein synthesis appears to be coupled to heme precursor availability, as seen by SDS-PAGE, because in the absence of heme precursor apocytochrome P450 1B1 does not accumulate.


Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/biosynthesis , Escherichia coli/enzymology , Aminolevulinic Acid/metabolism , Cloning, Molecular , Cytochrome P-450 CYP1B1 , DNA Fragmentation , DNA, Complementary/analysis , DNA, Complementary/biosynthesis , Electrophoresis, Polyacrylamide Gel , Humans , Reverse Transcriptase Polymerase Chain Reaction
18.
J Colloid Interface Sci ; 224(1): 162-168, 2000 Apr 01.
Article in English | MEDLINE | ID: mdl-10708506

ABSTRACT

Layered, electrochemically active films of bacterial iron-sulfur protein putidaredoxin (Pdx) and poly(dimethyldiallyammonium) (PDDA) polycations were constructed on gold electrodes coated with mercaptopropane sulfonate (MPS) and on quartz slides. Second-derivative UV-vis spectra suggested similar structures of Pdx in films and solutions at pH 7. Direct electrochemistry was achieved between Pdx and gold electrodes in these films, with significantly better electrochemical reversibility than in cast Nafion-lipid-Pdx films. A formal potential dispersion model gave a good fit to square wave voltammograms by regression analysis and was used to estimate an average apparent rate constant of 4.5 s(-1). Reduced Pdx in the polyion films did not react with its natural redox partner cytochrome P450(cam) because of unfavorable thermodynamics in the film environment. Copyright 2000 Academic Press.

SELECTION OF CITATIONS
SEARCH DETAIL
...