Disruption of the talin gene arrests mouse development at the gastrulation stage.

Studies on cultured cells show that the cytoskeletal protein talin plays a key role in cell spreading and the assembly of cell-extracellular matrix junctions. To examine the role of talin in vivo, we have generated mice with a targeted disruption of the talin gene. Heterozygotes are normal, but no surviving homozygous mutant animals were obtained, proving that talin is required for embryogenesis. Mutant embryos develop normally to the blastocyst stage and implant, but there is a gross disorganization of the embryos at gastrulation (6.5-7.5 days post coitum), and they die around 8.5-9.5 days post coitum. The embryonic ectoderm is reduced in size, with fewer cells, and is incompletely organised compared with wild-type embryos. The mutant embryos show disorganised extraembryonic tissues, and the ectoplacental and excocoelomic cavities are not formed. This seems to be because embryonic mesoderm accumulates as a mass on the posterior side of the embryos and fails to migrate to extraembryonic regions, although mesodermal cells are evident in the embryo proper. Spreading of trophoblast cells derived from cultured mutant blastocysts on fibronectin and laminin is also considerably reduced. Therefore, the fundamental deficit in these embryos seems to be a failure of cell migration at gastrulation.

An evaluation of the nurse practitioner role in a major rural emergency department.

The purpose of this pilot study was to investigate whether nurse practitioners are able to provide a level of primary health service applicable to remote/isolated settings in wound management and treatment of blunt limb trauma. It was hypothesized that there would be no significant difference in the quality of care, or the level of client satisfaction, provided by the medical officers and the nurse practitioners in the study. Two groups participated in the study, nurse practitioners and medical officers. The study used a randomized trial design. Data were collected using quantitative and qualitative methods. Two hundred and thirty-two clients participated in the study. Of this number 63 were supervised cases in the pilot trial. In the randomized trial participants were distributed between nurse practitioners and medical officers (n = 169), of which 91 were randomized to medical officers and 78 to nurse practitioners. Telephone interviews were conducted to evaluate client satisfaction. The majority of study participants were surveyed for client satisfaction (n = 132). This represents approximately 78% of the randomized sample and multivariate analysis was carried out on the data. Study results indicate that there were no significant differences between the two groups in relation to client satisfaction. Very positive outcomes of treatment were consistent across groups in the study. The study also found that there was strong support for the role of the nurse practitioner in the rural emergency setting. Recommendations include further research to measure the efficacy of nurse practitioners utilizing the selected competencies in remote/isolated settings.

Integrin-mediated cell adhesion: the cytoskeletal connection.

Members of the integrin family of cell adhesion molecules play a pivotal role in the interaction between animal cells and the extracellular matrix. This article reviews the evidence (i) that the integrin beta-subunit cytoplasmic domain is important in the localization of integrins to focal adhesions, and for integrin-mediated cell adhesion/spreading; and (ii) that the integrin beta-subunit can be linked to F-actin via the actin-binding proteins talin, alpha-actinin and filamin. Talin has two or more actin-binding sites, and three binding sites for the cytoskeletal protein vinculin. Because vinculin can also bind F-actin, it may cross-link talin and actin, thereby stabilizing the interaction. In addition, vinculin contains a binding site for VASP (vasodilator-stimulated phospho-protein), a protein which may serve to recruit a profilin/G-actin complex to talin, which has actin-nucleating activity. Evidence that talin, vinculin and alpha-actinin are important in the assembly of focal adhesions, obtained using antisense technology and protein microinjection, is reviewed. To analyse the role of talin in focal adhesions, we have disrupted both copies of the talin gene in mouse embryonic stem (ES) cells. Undifferentiated talin (-/-) ES cell mutants are unable to assemble focal adhesions when plated on fibronectin, whereas vinculin (-/-) ES cells are able to do so. Finally, the role of small GTP-binding proteins in the assembly of focal adhesions is discussed, along with our recent studies using streptolysin-O-permeabilized Swiss 3T3 cells which suggest that the GTP-binding protein ADP-ribosylation factor-1 (ARF-1) is important in targeting the protein paxillin to focal adhesions.

Disruption of the talin gene compromises focal adhesion assembly in undifferentiated but not differentiated embryonic stem cells.

We have used gene disruption to isolate two talin (-/-) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of beta1 integrin, although levels of alpha5 and alphaV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (-/-) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (-/-) ES cells were able to assemble talin-containing focal adhesions. Both talin (-/-) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (-/-) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the beta1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for beta1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.

The support needs of pregnant and parenting adolescents in rural communities.

Pregnant adolescents and teenage mothers in rural communities face extraordinary difficulties accessing appropriate and adequate support services, despite having recognised specialist health needs and unique support requirements. The Australian Rural Health Research Institute at Charles Sturt University, Wagga Wagga, is currently engaged in a federally funded project aimed it significantly improving access to services for this disadvantaged and often neglected group, through the publication and wide dissemination of a resource booklet identifying successful aspects of service delivery across a range of rural support settings. Five existing health and welfare support services for pregnant or parenting adolescent: in rural Australia have been selected for inclusion in the booklet, which is being developed for community use. Assessment is being undertaken during visits to each of the services, and, following interviews with staff, referral agencies and consumers. This paper outlines the strengths, attributes and access difficulties associated with two contrasting service models involved in the project, and stresses the importance of improving access to services for rural adolescents. A framework for establishing innovative and effective new services is also presented.

The narrative skills of primary school children with a unilateral hearing impairment.

The discourse skills of four boys with a unilateral hearing impairment (UHI), aged 7·2-10·7 years, were appraised over a 2-year period by examining their oral narrative use. All subjects exhibited delayed narrative skills, including features typical of children with a language disorder. These findings of language difficulties within this population were at variance with previous findings showing that children with UHI do not experience language problems. The subjects' language skills were discussed in relation to their academic performance. The implications of the findings for the process of narrative assessment were also discussed.

Talin contains three actin-binding sites each of which is adjacent to a vinculin-binding site.

We have determined the sequence of chicken talin (2,541 amino acids, M(r) 271,881) which is very similar (89% identity) to that of the mouse protein. Alignments with the Caenorhabditis elegans and Dictyostelium discoideum talin sequences show that the N- and C-terminal regions of the protein are conserved whereas the central part of the molecule is more divergent. By expressing overlapping talin polypeptides as fusion proteins, we have identified at least three regions of the protein which can bind F-actin: residues 102-497, 951-1,327 and 2,269-2,541. The N-terminal binding site contains a region with homology to the ERM family of actin-binding proteins, and the C-terminal site is homologous to the yeast actin-binding protein Sla2p. Each of the actin-binding sites is close to, but distinct from a binding site for vinculin, a protein which also binds actin. The Pro1176 to Thr substitution found in talin from Wistar-Furth rats does not destroy the capacity of this region of the protein to bind actin or vinculin. Microinjection studies showed that a fusion protein containing the N-terminal actin-binding site localised weakly to stress fibres, whereas one containing the C-terminal site initially localised predominantly to focal adhesions. The former was readily solubilised, and the latter was resistant to Triton extraction. The N-terminal talin polypeptide eventually disrupted actin stress fibres whereas the C-terminal polypeptide was without effect. However, a larger C-terminal fusion protein also containing a vinculin-binding site did disrupt stress fibres and focal adhesions. The results suggest that, although both the N- and C-terminal regions of talin bind actin, the properties of these two regions of the protein are distinct.

Vietnam memories: Australian army nurses, the Vietnam war, and oral history.

This paper is about women nurse veterans from the Royal Australian Army Nursing Corps (RAANC) who served in Vietnam. I aim to develop an understanding about these nurses that might place their experiences into a wider context. My conclusions provide starting points for future studies on myth, remembering and oral history.

Utrophin actin binding domain: analysis of actin binding and cellular targeting.

Utrophin, or dystrophin-related protein, is an autosomal homologue of dystrophin. The protein is apparently ubiquitously expressed and in muscle tissues the expression is developmentally regulated. Since utrophin has a similar domain structure to dystrophin it has been suggested that it could substitute for dystrophin in dystrophic muscle. Like dystrophin, utrophin has been shown to be associated with a membrane-bound glycoprotein complex. Here we demonstrate that expressed regions of the predicted actin binding domain in the NH2 terminus of utrophin are able to bind to F-actin in vitro, but do not interact with G-actin. The utrophin actin binding domain was also able to associate with actin-containing structures, stress fibres and focal contacts, when microinjected into chick embryo fibroblasts. The expressed NH2-terminal 261 amino acid domain of utrophin has an affinity for skeletal F-action (Kd 19 +/- 2.8 microM), midway between that of the corresponding domains of alpha-actinin (Kd 4 microM) and dystrophin (Kd 44 microM). Moreover, this utrophin domain binds to non-muscle actin with a approximately 4-fold higher affinity than to skeletal muscle actin. These data (together with those of Matsumura et al. (1992) Nature, 360, 588-591) demonstrate for the first time that utrophin is capable of performing a functionally equivalent role to that of dystrophin. The NH2 terminus of utrophin binds to actin and the COOH terminus binds to the membrane associated glycoprotein complex, thus in non-muscle and developing muscle utrophin performs the same predicted 'spacer' or 'shock absorber' role as dystrophin in mature muscle tissues. These data suggest that utrophin could replace dystrophin functionally in dystrophic muscle.

The identification and characterisation of an actin-binding site in alpha-actinin by mutagenesis.

We have shown previously that the N-terminal actin-binding domain of alpha-actinin retains activity when expressed in E. coli as a fusion protein with glutathione-S-transferase. In the present study we have made a series of N- and C-terminal deletions within this domain and show that an actin-binding site is contained within residues 120-134. Amino acid substitutions within this region indicate that several highly conserved hydrophobic residues are involved in binding to F-actin. The hypothesis that the interaction between alpha-actinin and F-actin is predominantly hydrophobic in nature is supported by the observation that binding is relatively independent of salt concentration.

Analysis of the actin-binding domain of alpha-actinin by mutagenesis and demonstration that dystrophin contains a functionally homologous domain.

To define the actin-binding site within the NH2-terminal domain (residues 1-245) of chick smooth muscle alpha-actinin, we expressed a series of alpha-actinin deletion mutants in monkey Cos cells. Mutant alpha-actinins in which residues 2-19, 217-242, and 196-242 were deleted still retained the ability to target to actin filaments and filament ends, suggesting that the actin-binding site is located within residues 20-195. When a truncated alpha-actinin (residues 1-290) was expressed in Cos cells, the protein localized exclusively to filament ends. This activity was retained by a deletion mutant lacking residues 196-242, confirming that these are not essential for actin binding. The actin-binding site in alpha-actinin was further defined by expressing both wild-type and mutant actin-binding domains as fusion proteins in E. coli. Analysis of the ability of such proteins to bind to F-actin in vitro showed that the binding site was located between residues 108 and 189. Using both in vivo and in vitro assays, we have also shown that the sequence KTFT, which is conserved in several members of the alpha-actinin family of actin-binding proteins (residues 36-39 in the chick smooth muscle protein) is not essential for actin binding. Finally, we have established that the NH2-terminal domain of dystrophin is functionally as well as structurally homologous to that in alpha-actinin. Thus, a chimeric protein containing the NH2-terminal region of dystrophin (residues 1-233) fused to alpha-actinin residues 244-888 localized to actin-containing structures when expressed in Cos cells. Furthermore, an E. coli-expressed fusion protein containing dystrophin residues 1-233 was able to bind to F-actin in vitro.

Clinical performance of university nursing graduates.

This study investigated the clinical performance of new nurse graduates during their first year of nursing and the relationship between this clinical performance and the length of clinical experience in their pre-service nursing education program. The study was carried out over the period of one calendar year beginning in early 1990. From an initial sample of 402 graduates, a representative sub-sample of 115 was chosen for closer study. The sub-sample was comprised of a high and low group of graduates, with the low group having received fewer hours of clinical experience during their pre-service program. On various occasions during the research the sample and sub-sample were surveyed using a modified version of the Scale of Nursing Performance (SNP). The Nursing Unit Managers (NUMs) of the sub-sample were also regularly asked to assess the nursing performance of these graduates. The findings question whether unreserved support can be given to the argument that more hours of clinical experience for student nurses will always result in more clinically competent registered nurses.

The diagnostic value and molecular characterisation of an Echinococcus multilocularis antigen gene clone.

A diagnostic Echinococcus multilocularis antigen gene (EM4) has been expressed using the Escherichia coli expression vector pGEX-1 resulting in the high level synthesis of a readily purifiable, soluble, non-denatured peptide fused to the 26-kDa glutathione-S-transferase of Schistosoma japonicum. This recombinant antigen, on testing by enzyme-linked immunosorbent assay (ELISA) with heterologous human antisera, demonstrated 100% E. multilocularis specificity. Specific anti-EM4 antibody immunoprecipitated a single 66-kDa protein from protoscolex total RNA directed in vitro translation products indicating the probable involvement of post-translational modification in the production of the native EM4 antigens. Southern blotting analysis suggests that the EM4 native antigens are coded for by a single-copy gene and that the genomic organisation of the EM4 related genes in other parasites is not conserved. The nucleotide sequence of the cloned EM4 cDNA molecule has been obtained and the derived amino acid sequence shows no significant homology with other existing protein sequences.

The isolation, by differential antibody screening, of Echinococcus multilocularis antigen gene clones with potential for immunodiagnosis.

A lambda gt11 cDNA expression library consisting of a million recombinant clones has been constructed from Echinococcus multilocularis protoscolex mRNA. Differential screening of the library with pools of E. multilocularis and Echinococcus granulosus human infection sera has revealed 13 potentially immunodiagnostic clones. On the basis of plaque immunoassays and lysogen characteristics, two of these clones, designated EM2 and EM4, have been further characterised. The recombinant fusion-peptides have been purified and their potential as immunodiagnostic reagents has been assessed by immunoblotting and, in the case of one fusion-peptide (EM4), by enzyme-linked immunosorbent assay (ELISA). Furthermore, the native parasite antigens coded for by these clones have been identified. EM2 corresponds to a 70 kDa protein and epitopes coded for by EM4 have been found on three antigens of 62, 49 and 44 kDa. The native antigens of both clones are present in the protoscolex and those corresponding to EM4 appear to be excreted/secreted products. They are not recognised in ELISA by a variety of human parasitic infection sera other than sera taken from patients infected with E. multilocularis. Nevertheless, the native antigens for both clones are present in E. granulosus protoscoleces and Taenia solium cysticerci. These antigens are not detectable in E. granulosus cyst fluid, and this may, in part, explain the lack of immune response to them in human E. granulosus and T. solium infections.