Parent-child communication and timing of interventions are challenges in the Duchenne muscular dystrophy care.

AIM: Duchenne muscular dystrophy (DMD) is an X-linked myopathy affecting one in 3600-6000 live male births. The aim of this study was to gain further insight into how parents experience caring for boys with DMD. METHODS: Using a qualitative design, parents in 12 families with boys with DMD, whereof two pairs of siblings, were interviewed in 2014 and 2015. Participants were parents of boys followed at Oslo University Hospital or other hospitals in the south-eastern health region of Norway. Mean age of the boys was 13 years, range 7-17. RESULTS: Parents expressed the importance of obtaining good information about the diagnosis, supplied with sensitivity for them as clients, and the parents must be involved in timing of interventions. Meeting with others in the same situation was highly appreciated. Many of the parents expressed their own need for support to cope with the difficult situation. Continuity of support in the boys' transition to adulthood was pointed out as important, as well as the need for professional help to talk to the boys about their diagnosis. CONCLUSION: Our study suggests that professional teams should expand the parental perspective and emphasise a holistic approach in their work with patients with DMD.

Analgesia and sedation of mechanically ventilated patients - a national survey of clinical practice.

BACKGROUND: The importance of balanced sedation and pain treatment in intensive care units (ICUs) is evident, but regimes and use of medication differ widely. Previous surveys have focused on the use of various medications and regimes. What has not been explored is the process by which nurses and physicians assess patients' needs and work together toward a defined level of sedation and pain for the ICU patient. The purpose of the study was to determine the use of protocols and medications for sedation and analgesia in Norwegian ICUs and the degree of cooperation between nurses and physicians in using them. METHODS: A national survey was conducted in autumn 2007, using postal self-administered questionnaires. RESULTS: Written pain treatment and sedation protocols were not routinely used in Norwegian ICUs; however, half of the departments titrated sedation according to a scoring system, most commonly the Motor Activity Assessment Score. The most commonly used sedatives were propofol and midazolam, while fentanyl and morphine were the most used analgesics. The majority of respondents were concerned about the side effects of sedation and analgesics, leading to circulatory instability and delayed awakening. Nurses and physicians agreed upon the main indications for sedation: patient tolerance for ventilation, tolerance for medical and nursing interventions, and patient symptoms. CONCLUSIONS: Potential factors which may improve sedation and pain management of mechanically ventilated patients in Norwegian ICUs are more systematic assessments of pain and sedation, and the use of written protocols. Strategies which reduce side effects should be addressed.

Targeting risk factors for impaired wound healing and wound complications after kidney transplantation.

BACKGROUND: Because of potent immunosuppression, impaired wound healing and complications are frequent features after kidney transplantation (KTx). OBJECTIVE: To investigate the incidence and nature of impaired wound healing and complications at a single transplantation center in Norway. PATIENTS: Of 226 patients who underwent KTx, 199 (87%) were followed up prospectively for 1 year (2005) via close and meticulous wound inspection. RESULTS: The study revealed a high rate of wound complications (200-250/y) in a high-volume center. Fifty-four patients (27%) experienced prolonged wound healing, defined as gaps, secretions, or wound complications, at 3 to 5 weeks posttransplantation, and 41 patients (21%) had impaired wound healing, defined as gaps, secretions, or wound complications after 5 weeks posttransplantation. In total, 50 patients (25%) required surgical or radiologic reintervention. Complications included lymphocele in 29 patients (14.6%), wound dehiscence in 16 (8.0%), bleeding or hematoma in 10 (5.0%), and infection in 9 (4.5%). Risk factors associated with wound complications included recipient older than 60 years, body mass index greater than 30, hemoglobin concentration less than 10 g/dL, albumin concentration less than 36 g/dL, duration of surgery more than 200 minutes, no subcutaneous sutures, and sirolimus or everolimus therapy. At nominal and logistic regression analysis, recipient older than 60 years, body mass index greater than 30, and no subcutaneous sutures were independent risk factors. CONCLUSION: Risk factor analysis and previous documentation suggest that wound complications might be counteracted using the following measures: subcutaneous sutures, predialysis transplantation, sealing or ligation of lymphatic trunks, prophylactic fenestration, reduction of corticosteroid load, and avoiding sirolimus/everolimus therapy.

T-cell receptor Vbeta repertoire after myeloablative and reduced intensity conditioning allogeneic haematopoietic stem cell transplantation.

T cells play an important role in the adaptive immune system. After haematopoietic stem cell transplantation (HSCT), T-cell function is impaired. This is reflected by the emergence of opportunistic infections, infections that are often difficult to treat because of the patient's insufficient immune function. T-cell receptor reconstitution was studied using CDR3 spectratyping to analyze the diversity of the T-cell repertoire at 3, 6 and 12 months after myeloablative and reduced intensity conditioning (RIC) HSCT in 23 patients. Immune function in vitro was tested by lymphocyte stimulation at 3, 6 and 12 months after HSCT. Lower diversity in the CDR3 repertoire was demonstrated in CD4+ cells after RIC HSCT at 3 and 6 months and in CD8+ cells at 3 months compared with healthy donors. After myeloablative HSCT, lower diversity was seen at 3, 6 and 12 months in CD4+ cells and at 6 and 12 months in CD8+ cells after HSCT. Acute and chronic graft-versus-host-disease (GVHD) did not affect diversity. Responses to phytohaemagglutinin (PHA), Concanavalin A (Con A) and Staphylococcus aureus protein A were significantly lower compared with healthy donors during the first 6 months after RIC HSCT. After myeloablative HSCT, lymphocyte response to Con A was significantly lower at 3 months compared with healthy donors. Decreased responses to cytomegalovirus and varicella zoster virus antigens were seen in patients suffering from acute GVHD grade II or chronic GVHD. The T-cell repertoire is skewed under the first year after HSCT, and immune reconstitution after HSCT with myeloablative and RIC conditioning seems to be comparable. GVHD, infections and age are more important for immune reconstitution than type of conditioning.

Reconstitution of the Ig heavy chain CDR3 repertoire after allogeneic haematopoietic stem cell transplantation with myeloablative or reduced-intensity conditioning regimens.

The objective of this study was to investigate B-lymphocyte reconstitution in patients undergoing allogeneic haematopoietic stem cell transplantation (HSCT) after myeloablative conditioning (MAC) or reduced-intensity conditioning (RIC) regimens. B-lymphocyte reconstitution was studied by monitoring the CDR3 repertoire with spectratyping. We demonstrate a delay in the recovery of the B-lymphocyte repertoire, measured by variation in size distribution of the immunoglobulin H CDR3 in patients conditioned with RIC compared to MAC. We found no general explanation for this finding, but when clinical data for each patient were studied in detail, we could identify a cause for the oligoclonality of the B-lymphocyte repertoire after HSCT with RIC for each of the patients. Older patients and donors, low cell dose at transplantation, relapse, graft-versus-host disease (GVHD) and its treatment as well as cytomegalovirus infection and its treatment are all possible causes for the restriction of the B-lymphocyte repertoire observed in this study. Taken together, reconstitution of the B-lymphocyte repertoire after HSCT is a process dependent on multiple factors and differs between patients. The conditioning regimen may be of importance, but data from this study suggest that individual factors and the various complications occurring after HSCT are more likely to determine the development of the B-lymphocyte repertoire.

Memory B lymphocytes determine repertoire oligoclonality early after haematopoietic stem cell transplantation.

The objective of this study was to investigate if oligoclonality of the Ig repertoire post-haematopoietic stem cell transplantation (HSCT) is restricted to memory B lymphocytes or if it is a general property among B lymphocytes. As a measure of B lymphocyte repertoire diversity, we have analysed size distribution of polymerase chain reaction (PCR) amplified Ig H complementarity determining region 3 (CDR3) in naive and memory B lymphocytes isolated from patients before HSCT and at 3, 6 and 12 months after HSCT as well as from healthy controls. We demonstrate a limited variation of the IgH CDR3 repertoire in the memory B lymphocyte population compared to the naive B cell population. This difference was significant at 3 and 6 months post-HSCT. Compared to healthy controls there is a significant restriction of the memory B lymphocyte repertoire at 3 months after HSCT, but not of the naive B lymphocyte repertoire. Twelve months after HSCT, the IgH CDR3 repertoire in both memory and naive B lymphocytes are as diverse as in healthy controls. Thus, our findings suggest a role for memory B cells in the restriction of the oligoclonal B cell repertoire observed early after HSCT, which may be of importance when considering reimmunization of transplanted patients.

Altered expression of receptors for thyroid hormone and insulin-like growth factor-I during reconstitution after allogeneic hematopoietic stem cell transplantation.

Treatment with neuroendocrine hormones has been suggested to promote reconstitution of the immune system after hematopoietic stem cell transplantation (HSCT). We investigated the expression of genes encoding receptors for growth hormone (GH), insulin-like growth factor-I (IGF-I) and triiodothyronine (T3), at various time points after HSCT in 16 patients and 15 healthy controls. Peripheral blood mononuclear cells were isolated and RNA for GH receptor (GHR), IGF-I receptor (IGF-IR) and thyroid hormone receptor (TRalpha1) was amplified by RT-PCR. The expression of the genes was compared with the expression of beta-actin. We demonstrate increased expression of TRalpha1 RNA in patients at 1.5 months post HSCT, compared to a group of healthy controls, and decreased expression of IGF-IR RNA at 2 and 3 months post HSCT, compared to the controls. Serum from three of the patients was also analyzed for levels of T3, T4, TSH and IGF-I at several time points after HSCT. Serum levels for T3, thyroxine (T4), thyroid stimulating hormone (TSH) and IGF-I were within the normal range in all samples. Our results on the molecular level indicate a role for thyroid hormones and IGF-I in immune reconstitution after HSCT, even though the serum levels of T3, T4, TSH and IGF-I are normal.

Lysine 114 of antithrombin is of crucial importance for the affinity and kinetics of heparin pentasaccharide binding.

Lys(114) of the plasma coagulation proteinase inhibitor, antithrombin, has been implicated in binding of the glycosaminoglycan activator, heparin, by previous mutagenesis studies and by the crystal structure of antithrombin in complex with the active pentasaccharide unit of heparin. In the present work, substitution of Lys(114) by Ala or Met was shown to decrease the affinity of antithrombin for heparin and the pentasaccharide by approximately 10(5)-fold at I 0.15, corresponding to a reduction in binding energy of approximately 50%. The decrease in affinity was due to the loss of two to three ionic interactions, consistent with Lys(114) and at least one other basic residue of the inhibitor binding cooperatively to heparin, as well as to substantial nonionic interactions. The mutation minimally affected the initial, weak binding of the two-step mechanism of pentasaccharide binding to antithrombin but appreciably (>40-fold) decreased the forward rate constant of the conformational change in the second step and greatly (>1000-fold) increased the reverse rate constant of this step. Lys(114) is thus of greater importance for the affinity of heparin binding than any of the other antithrombin residues investigated so far, viz. Arg(47), Lys(125), and Arg(129). It contributes more than Arg(47) and Arg(129) to increasing the rate of induction of the activating conformational change, a role presumably exerted by interactions with the nonreducing end trisaccharide unit of the heparin pentasaccharide. However, its major effect, also larger than that of these two residues, is in maintaining antithrombin in the activated state by interactions that most likely involve the reducing end disaccharide unit.

Role of the single cysteine residue, Cys 3, of human and bovine cystatin B (stefin B) in the inhibition of cysteine proteinases.

Cystatin B is unique among cysteine proteinase inhibitors of the cystatin superfamily in having a free Cys in the N-terminal segment of the proteinase binding region. The importance of this residue for inhibition of target proteinases was assessed by studies of the affinity and kinetics of interaction of human and bovine wild-type cystatin B and the Cys 3-to-Ser mutants of the inhibitors with papain and cathepsins L, H, and B. The wild-type forms from the two species had about the same affinity for each proteinase, binding tightly to papain and cathepsin L and more weakly to cathepsins H and B. In general, these affinities were appreciably higher than those reported earlier, perhaps because of irreversible oxidation of Cys 3 in previous work. The Cys-to-Ser mutation resulted in weaker binding of cystatin B to all four proteinases examined, the effect varying with both the proteinase and the species variant of the inhibitor. The affinities of the human inhibitor for papain and cathepsin H were decreased by threefold to fourfold and that for cathepsin B by approximately 20-fold, whereas the reductions in the affinities of the bovine inhibitor for papain and cathepsins H and B were approximately 14-fold, approximately 10-fold and approximately 300-fold, respectively. The decreases in affinity for cathepsin L could not be properly quantified but were greater than threefold. Increased dissociation rate constants were responsible for the weaker binding of both mutants to papain. By contrast, the reduced affinities for cathepsins H and B were due to decreased association rate constants. Cys 3 of both human and bovine cystatin B is thus of appreciable importance for inhibition of cysteine proteinases, in particular cathepsin B.

A novel anti-angiogenic form of antithrombin with retained proteinase binding ability and heparin affinity.

Latent antithrombin, an inactive antithrombin form with low heparin affinity, has previously been shown to efficiently inhibit angiogenesis and tumor growth. We now show that heat treatment similar to that used for preparation of latent antithrombin also transforms antithrombin to another form, which we denote prelatent, with potent anti-angiogenic and anti-tumor activity but with retained proteinase- and heparin-binding properties. The ability of prelatent antithrombin to inhibit angiogenesis is presumably due to a limited conformational change, which may partially resemble that in latent antithrombin. Such a change is evidenced by a different cleavage pattern of prelatent than of native antithrombin by nontarget proteinases. Prelatent antithrombin exerts its anti-angiogenic effect by a similar mechanism as latent antithrombin, i.e. by inhibiting focal adhesion formation and focal adhesion kinase activity, thereby leading to decreased proliferation of endothelial cells. The proteinase inhibitory fractions in commercial antithrombin preparations, which have been heat treated during production, also have anti-angiogenic activity, comparable with that of the prelatent antithrombin form.

Wild-type and met-65-->Leu variants of human cystatin A are functionally and structurally identical.

The solution structure of an N-terminally truncated and mutant form (M65L(2-98)) of the human cysteine protease inhibitor cystatin A has been reported that reveals extensive structural differences when compared to the previously published structure of full-length wild-type (WT) cystatin A. On the basis of the M65L(2-98) structure, a model of the inhibitory mechanism of cystatin A was proposed wherein specific interactions between the N- and C-terminal regions of cystatin A are invoked as critical determinants of protease binding. To test this model and to account for the reported differences between the two structures, we undertook additional structural and mechanistic analyses of WT and mutant forms of human cystatin A. These show that modification at the C-terminus of cystatin A by the addition of nine amino acids has no effect upon the affinity of papain inhibition (K(D) = 0.18+/-0.02 pM) and the consequences of such modification are not propagated to other parts of the structure. These findings indicate that perturbation of the C-terminus can be achieved without any measurable effect on the N-terminus or the proteinase binding loops. In addition, introduction of the methionine-65 --> leucine substitution into cystatin A that retains the N-terminal methionine (M65L(1-98)) has no significant effect upon papain binding (K(D) = 0.34+/-0.02 pM). Analyses of the structures of WT and M65L(1-98) using (1)H NMR chemical shifts and residual dipolar couplings in a partially aligning medium do not reveal any evidence of significant differences between the two inhibitors. Many of the differences between the published structures correspond to major violations by M65L(2-98) of the WT constraints list, notably in relation to the position of the N-terminal region of the inhibitor, one of three structural motifs indicated by crystallographic studies to be involved in protease binding by cystatins. In the WT structure, and consistent with the crystallographic data, this region is positioned adjacent to another inhibitory motif (the first binding loop), whereas in M65L(2-98) there is no proximity of these two motifs. As the NMR data for both WT9C and M65L(1-98) are wholly consistent with the published structure of WT cystatin A and incompatible with that of M65L(2-98), we conclude that the former represents the most reliable structural model of this protease inhibitor.

Antiangiogenic effects of latent antithrombin through perturbed cell-matrix interactions and apoptosis of endothelial cells.

Antithrombin is a plasma protein of the serpin superfamily that may occur as several conformational variants. The native form of antithrombin is a major regulator of blood clotting. In the present study, we have identified the mechanism underlying the antiangiogenic action of a heat-denatured form, denoted latent antithrombin. Fibroblast growth factor (FGF)-induced angiogenesis in the chick embryo and angiogenesis in mouse fibrosarcoma tumors were inhibited by treatment with latent antithrombin at 1 mg/kg/day. Thermolysin-cleaved and native antithrombin were less efficient in these respects. Treatment with latent antithrombin induced apoptosis of cultured endothelial cells and inhibited cell migration toward FGF-2. Under these conditions, FGF-2-stimulated FGF receptor kinase activity was unaffected. However, actin reorganization, activation of focal adhesion kinase, and focal adhesion formation were disturbed by latent antithrombin treatment of FGF-2-stimulated endothelial cells. These data indicate that latent antithrombin induces apoptosis of endothelial cells by disrupting cell-matrix interactions through uncoupling of focal adhesion kinase.

Tissue distribution of bovine cystatin C analysed by in situ hybridisation.

Cysteine proteinase inhibitors of the cystatin superfamily have been identified in many living organisms. However, knowledge of the tissue distribution of such inhibitors is limited. To elucidate this distribution in mammals, we have investigated the expression of the gene for cystatin C, belonging to cystatin family II, in several bovine tissues. In situ hybridisation with a digoxigenin-labelled cRNA probe demonstrated a high concentration of bovine cystatin C mRNA in the secretory epithelial cells of the choroid plexus, and also intense staining in cells of lymphoid tissue and in Sertoli cells. Cystatin C mRNA was also present in scattered neurons and glial cells throughout the cerebrum and the cerebellum. In the submandibular gland, specific mRNA was found mainly in striated intralobular ducts and interlobular ducts. The expression of cystatin C in brain tissue is of particular interest, as the inhibitor appears to be involved in certain neurological diseases. The main production of cystatin C within the brain is believed to be by astrocytes. However, this work shows that also neurons from young, normal individuals express cystatin C.

The region of antithrombin interacting with full-length heparin chains outside the high-affinity pentasaccharide sequence extends to Lys136 but not to Lys139.

The interaction of a well-defined pentasaccharide sequence of heparin with a specific binding site on antithrombin activates the inhibitor through a conformational change. This change increases the rate of antithrombin inhibition of factor Xa, whereas acceleration of thrombin inhibition requires binding of both inhibitor and proteinase to the same heparin chain. An extended heparin binding site of antithrombin outside the specific pentasaccharide site has been proposed to account for the higher affinity of the inhibitor for full-length heparin chains by interacting with saccharides adjacent to the pentasaccharide sequence. To resolve conflicting evidence regarding the roles of Lys136 and Lys139 in this extended site, we have mutated the two residues to Ala or Gln. Mutation of Lys136 decreased the antithrombin affinity for full-length heparin by at least 5-fold but minimally altered the affinity for the pentasaccharide. As a result, the full-length heparin and pentasaccharide affinities were comparable. The reduced affinity for full-length heparin was associated with the loss of one ionic interaction and was caused by both a lower overall association rate constant and a higher overall dissociation rate constant. In contrast, mutation of Lys139 affected neither full-length heparin nor pentasaccharide affinity. The rate constants for inhibition of thrombin and factor Xa by the complexes between antithrombin and full-length heparin or pentasaccharide were unaffected by both mutations, indicating that neither Lys136 nor Lys139 is involved in heparin activation of the inhibitor. Together, these results show that Lys136 forms part of the extended heparin binding site of antithrombin that participates in the binding of full-length heparin chains, whereas Lys139 is located outside this site.

Mixed chimerism in the B cell lineage is a rapid and sensitive indicator of minimal residual disease in bone marrow transplant recipients with pre-B cell acute lymphoblastic leukemia.

One of the major problems after allogeneic bone marrow transplantation (BMT) is a high frequency of leukemia relapse. We have prospectively studied the presence of donor- and recipient-derived chimeric cells in bone marrow recipients with pre-B cell acute lymphoblastic leukemia (pre-B-ALL). The chimeric status of BMT recipients was compared to minimal residual disease (MRD) detection by analysis of immunoglobulin heavy chain (IgH) and T cell receptor (TcR) genes. Post-transplant blood and bone marrow samples from 12 patients with pre-B-ALL were studied. Five patients showed mixed chimerism (MC) in the CD19-positive cell fraction. Four of them have relapsed to date. The remaining patient with MC in the B cell lineage was also MRD positive in the same samples. All seven patients with donor chimerism in the B cell fraction remain in clinical remission (P = 0.01). In samples from all five patients having MC in the B cell lineage, the patient-specific IgH or TcR rearrangement was also detected. In three of four patients who relapsed, MC in the B cell lineage was seen more than 2.5 months prior to morphologically verified relapse. The results of this comparison suggest that routinely performed MC analysis of the affected cell lineage may facilitate post-BMT monitoring and rapid therapeutic decisions in transplanted patients with pre-B-ALL.

Role of arginine 129 in heparin binding and activation of antithrombin.

The contribution of Arg(129) of the serpin, antithrombin, to the mechanism of allosteric activation of the protein by heparin was determined from the effect of mutating this residue to either His or Gln. R129H and R129Q antithrombins bound pentasaccharide and full-length heparins containing the antithrombin recognition sequence with similar large reductions in affinity ranging from 400- to 2500-fold relative to the control serpin, corresponding to a loss of 28-35% of the binding free energy. The salt dependence of pentasaccharide binding showed that the binding defect of the mutant serpin resulted from the loss of approximately 2 ionic interactions, suggesting that Arg(129) binds the pentasaccharide cooperatively with other residues. Rapid kinetic studies showed that the mutation minimally affected the initial low affinity binding of heparin to antithrombin, but greatly affected the subsequent conformational activation of the serpin leading to high affinity heparin binding, although not enough to disfavor activation. Consistent with these findings, the mutant antithrombin was normally activated by heparin for accelerated inhibition of factor Xa and thrombin. These results support an important role for Arg(129) in an induced-fit mechanism of heparin activation of antithrombin wherein conformational activation of the serpin positions Arg(129) and other residues for cooperative interactions with the heparin pentasaccharide so as to lock the serpin in the activated state.

The high stability of cruzipain against pH-induced inactivation is not dependent on its C-terminal domain.

Unlike mammalian lysosomal cysteine proteases, the trypanosomal cysteine protease cruzipain contains a 130-amino acid residue C-terminal domain, in addition to the catalytic domain, and it is stable at neutral pH. The endogenous (with C-terminal domain) and recombinant (without C-terminal domain) cruzipains exhibit similar stabilities at both acid (k(inac)=3.1x10(-3) s(-1) and 4.4x10(-3) s(-1) at pH 2.75 for endogenous and recombinant cruzipain, respectively) and alkaline pH (k(inac)=3.0x10(-3) s(-1) and 3. 7x10(-3) s(-1) at pH 9.15 for endogenous and recombinant cruzipain, respectively). The pH-induced inactivation, which is a highly pH dependent first order process, is irreversible and accompanied by significant changes of secondary and tertiary structure as revealed by circular dichroism measurements. The different stability of cruzipain as compared to related proteases, is therefore due mainly to the different number, nature and distribution of charged residues within the catalytic domain and not due to addition of the C-terminal domain.

Long-term persistence of oligoclonal serum IgM repertoires in patients treated with allogeneic bone marrow transplantation (BMT).

Immunoglobulin gene rearrangements in patients treated with BMT have restricted repertoire diversity. Clonal variability remains low for 3 months and reconstitution of the humoral immune system appears to follow a wave-like pattern. In the present study we analysed serum IgM and IgG repertoires in 44 patients from 1 week to 3 years after transplantation. We applied a quantitative immunoblot technique in combination with a newly developed method for estimation of repertoire diversity in complex mixtures of antibodies. Our results demonstrate that 60% of BMT patients have severely reduced diversity in the IgM repertoire during and after the first year post-BMT, compared with healthy controls. In contrast, the majority of patients have a polyclonal IgG repertoire, similar to that of healthy controls. Serum IgM repertoires remain oligoclonal even though the serum concentration of total IgM is within normal range around 6 months post-BMT. During the first years after transplantation IgM as well as IgG repertoires are less diverse in patients receiving a BM graft from a sibling donor compared with those receiving a graft from an HLA-matched unrelated donor. Patients in the latter group show a higher incidence of infections and minor antigen mismatches which may promote the development of a diverse immunoglobulin repertoire post-BMT.

Cystatin inhibition of cathepsin B requires dislocation of the proteinase occluding loop. Demonstration By release of loop anchoring through mutation of his110.

Cystatins A and C were both shown to inhibit cathepsin B by a two-step mechanism, involving an initial weak interaction followed by a conformational change. Disruption of the major salt bridge anchoring the occluding loop of cathepsin B to the main body of the enzyme by mutation of His110 to Ala converted the binding to an apparent one-step reaction. The second step of cystatin binding to cathepsin B must therefore be due to the inhibitor having to alter the conformation of the enzyme by displacing the occluding loop to allow a tight complex to be formed. Cystatin A was appreciably less effective in displacing the loop than cystatin C, resulting in a considerably lower overall inhibition rate constant.

From simplicity to complexity: developing a model of practical skill performance in nursing.

The purpose of this article is to present and discuss a new model of practical skill performance in nursing. The model is conceptualized as having five components: substance and sequence; accuracy; fluency; integration; and caring conduct. The model challenges the truism of 'simple' nursing procedures. It is argued that performance of practical skills in nursing is characterized by complexity on many levels. Complexity lies within and between the components of the performance model and in the interaction between the nurse and the clinical context where practical nursing actions are performed. These complexities are described. Examples that illustrate the complex and reciprocal nature of these components are drawn from an empirical study of graduate nurses' development of practical skill in surgical hospital units. Implications of the model for education, practice and research are discussed.