Nurses' experiences providing palliative care to individuals living in rural communities: aspects of the physical residential setting.

INTRODUCTION: Efforts are needed to improve palliative care in rural communities, given the unique characteristics and inherent challenges with respect to working within the physical aspects of residential settings. Nurses who work in rural communities play a key role in the delivery of palliative care services. Hence, the purpose of this study was to explore nurses' experiences of providing palliative care in rural communities, with a particular focus on the impact of the physical residential setting. METHODS: This study was grounded in a qualitative approach utilizing an exploratory descriptive design. Individual telephone interviews were conducted with 21 community nurses. Data were analyzed by thematic content analysis. RESULTS: Nurses described the characteristics of working in a rural community and how it influences their perception of their role, highlighting the strong sense of community that exists but how system changes over the past decade have changed the way they provide care. They also described the key role that they play, which was often termed a 'jack of all trades', but focused on providing emotional, physical, and spiritual care while trying to manage many challenges related to transitioning and working with other healthcare providers. Finally, nurses described how the challenges of working within the physical constraints of a rural residential setting impeded their care provision to clients who are dying in the community, specifically related to the long distances that they travel while dealing with bad weather. CONCLUSIONS: These study findings contribute to our understanding of the experiences of nurses working in rural communities in terms of the provision of palliative care and the influence of the physical residential setting that surrounds them. These findings are important since nurses play a major role in caring for community-dwelling clients who are dying, but they are confronted with many obstacles. As such, these results may help inform future decisions about how to best improve access to important services and ways to support them while providing palliative care to rural individuals.

Cystosolic chaperonin subunits have a conserved ATPase domain but diverged polypeptide-binding domains.

CCT (also called the TCP-1 complex or TriC) is a chaperonin found in the eukaryotic cytosol, and has unique structural and functional features. Unlike homo-oligomeric chaperonins, CCT comprises at least eight different subunits, and appears to have a limited range of physiological substrates. We have analysed CCT sequences in light of the recent determination of the crystal structure and mutational identification of the functional domains of the bacterial chaperonin GroEL. A high level of identity among all chaperonin subunits is observed in those regions that correspond to the ATP-binding site of GroEL. By contrast, no significant identity is shared in the region corresponding to the polypeptide-binding region of GroEL, either between CCT subunits or between CCT subunits and GroEL. This suggests that the polypeptide-binding sites of CCT subunits have diverged both from each other and from GroEL, which may explain the apparently different range of substrates recognized by CCT.

Spatially selective sampling of single cells using optically trapped fusogenic emulsion droplets: a new single-cell proteomic tool.

We present a platform for the spatially selective sampling of the plasma membrane of single cells. Optically trapped lipid-coated oil droplets (smart droplet microtools, SDMs), typically 0.5-5 microm in size, composed of a hexadecane hydrocarbon core and fusogenic lipid outer coating (mixture of 1,2-dioleoyl-phosphatidylethanolamine and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) were brought into controlled contact with target colon cancer cells leading to the formation of connecting membrane tethers. Material transfer from the cell to the SDM across the membrane tether was monitored by tracking membrane-localized enhanced green fluorescent protein.

Identification of six Tcp-1-related genes encoding divergent subunits of the TCP-1-containing chaperonin.

BACKGROUND: TCP-1 is a 60 kD subunit of a cytosolic hetero-oligomeric chaperone that is known to be involved in the folding of actin and tubulin. This protein is a member of the chaperonin family, which includes Escherichia coli GroEL, the mitochondrial heat-shock protein Hsp60, the plastid Rubisco-subunit-binding protein and the archaebacterial protein TF55. These chaperonins assist the folding of proteins upon ATP hydrolysis. RESULTS: Using two-dimensional gel analysis, we have identified nine different subunits of TCP-1-containing chaperonin complexes from mammalian testis and seven different subunits of such complexes from mouse F9 cells. We have isolated full-length mouse cDNAs encoding six novel TCP-1-related polypeptides and show that these cDNAs encode subunits of the TCP-1-containing cytosolic chaperonin. These subunits are between 531 and 545 residues in length. Their sequences are 25-36% identical to one another, 27-35% identical to that of TCP-1 and 32-39% identical to that of the archaebacterial chaperonin, TF55. We have named these genes, Cctb, Cctg, Cctd, Ccte, Cctz and Ccth, which encode the CCT beta, CCT gamma, CCT delta, CCT epsilon, CCT zeta and CCT eta subunits, respectively, of the 'Chaperonin Containing TCP-1' (CCT). All the CCT subunits contain motifs that are also shared by all other known chaperonins of prokaryotes and eukaryotic organelles, and that probably relate to their common ATPase function. CONCLUSION: It is likely that each CCT subunit has a specific, independent function, as they are highly diverged from each other but conserved from mammals to yeast. We suggest that the expansion in the number of types of CCT subunit, compared with other chaperonins, has allowed CCT to carry out the more complex functions that are required for the folding and assembly of highly evolved eukaryotic proteins.

Electron microscopy and 3D reconstructions reveal that human ATM kinase uses an arm-like domain to clamp around double-stranded DNA.

The human tumor suppressor gene ataxia telangiectasia mutated (ATM) encodes a 3056 amino-acid protein kinase that regulates cell cycle checkpoints. ATM is defective in the neurodegenerative and cancer predisposition syndrome ataxia-telangiectasia. ATM protein kinase is activated by DNA damage and responds by phosphorylating downstream effectors involved in cell cycle arrest and DNA repair, such as p53, MDM2, CHEK2, BRCA1 and H2AX. ATM is probably a component of, or in close proximity to, the double-stranded DNA break-sensing machinery. We have observed purified human ATM protein, ATM-DNA and ATM-DNA-avidin bound complexes by single-particle electron microscopy and obtained three-dimensional reconstructions which show that ATM is composed of two main domains comprising a head and an arm. DNA binding to ATM induces a large conformational movement of the arm-like domain. Taken together, these three structures suggest that ATM is capable of interacting with DNA, using its arm to clamp around the double helix.

Defining the eukaryotic cytosolic chaperonin-binding sites in human tubulins.

The actins and tubulins are the obligate substrates in vivo of the chaperonin-containing TCP-1 (CCT). The precise elements of recognition between the chaperonin and its substrates remain largely unknown. We have used a solid phase peptide binding assay to screen the human alpha, beta and gamma-tubulin sequences for CCT recognition. Multiple regions seem to be implicated in interactions between tubulins and CCT. These potential CCT-binding sites are highly dispersed throughout the primary sequences of the human tubulins. In addition, using site-directed mutagenesis we assessed the contribution of the selected residues in the C-terminal domain of beta-tubulin to CCT binding. Various hot spots have been identified even though, in each case, their replacement by alanine does not reduce dramatically the total affinity of beta-tubulin for CCT. The CCT-binding information in the tubulins is probably confined to multiple specific regions each having weak or moderate affinity for CCT apical domains. The main binding region seems to be located between residues 263 and 384, but there are no single amino acid residues in this region, which make large contributions to the binding energy, although we have detected a minor contribution by F377. These biochemical results are understandable in the context of our recent structural analysis of CCT-tubulin complexes by cryo-electron microscopy and image reconstruction, which shows that, in one stage of an in vitro binding reaction between apo-CCT and tubulin diluted from guanidinium chloride, ten major, stable contacts between tubulin and CCT are involved. Therefore, specificity is achieved through the co-operation of many specific, albeit weak, interactions.

Nested allosteric interactions in the cytoplasmic chaperonin containing TCP-1.

Initial rates of ATP hydrolysis by the chaperonin containing TCP-1 (CCT) from bovine testis were measured as a function of ATP concentration. Two allosteric transitions are observed: one at relatively low concentrations of ATP (<100 microM) and the second at higher concentrations of ATP. The data suggest that CCT has positive intra-ring cooperativity and negative inter-ring cooperativity in ATP hydrolysis, with respect to ATP, as previously observed in the case of GroEL. It is shown that the relatively weak positive intra-ring cooperativity found in the case of CCT may be due to heterogeneity in its subunit composition. Our results suggest that nested allosteric behavior may be common to chaperone double-ring systems.

Brefeldin A and mannose 6-phosphate regulation of acrosomic related vesicular trafficking.

Acrosomal biogenesis represents a unique system for the molecular analysis of the various processes involved in vesicular membrane transport. To assess the basic membrane trafficking mechanisms used in spermatids, we have used two fluorescent lipid compounds that label: a) the Golgi and Golgi-derived vesicles (C5-DMB-Cer), and b) endocytic vesicles (FM4-64). Incubation of mouse testicular germ cells at 33 degrees C for 1.5 h with C5-DMB-Cer revealed that C5-DMB-Cer labeling is localized in the Golgi and acrosome of early-maid round spermatid stages, with no labeling of the acrosome in late round spermatid stages. Culturing germ cells with FM4-64 for 1.5 h at 33 degrees C, showed that FM4-64 labeling in spermatids was localized in endocytic vesicles and Golgi of early-mid round spermatid stages, whereas in a small population of late round spermatid stages, FM4-64 was also localized in the apex region of the acrosome. Incubation with brefeldin A (BFA) (5 micrograms/ml) inhibited the distribution of C5-DMB-Cer and FM4-64 to the acrosome, however, it did not affect the localization of acrosin-an acrosome-specific protein-indicating that there was no apparent acrosome disassembly in the presence of BFA. Localization of FM4-64 in late round spermatids in the presence of 2.5 mM mannose 6-phosphate was found in endocytic vesicles and the Golgi, but not the acrosome. These results show that there are at least two sources of vesicular transport to the acrosome derived from the Golgi and plasma membrane.

The eighth Cct gene, Cctq, encoding the theta subunit of the cytosolic chaperonin containing TCP-1.

The chaperonin containing t-complex polypeptide 1 (TCP-1), as one of its subunits, CCT, is a cytosolic heterooligomeric molecular chaperone assisting in the folding of proteins in eukaryotic cytosol. We have isolated a Tcp-1-related 119-bp cDNA fragment from a human cDNA library by polymerase chain reaction, and cloned full-length mouse cDNAs orthologous to the human cDNA by hybridization. The nucleotide (nt) sequence of the longest mouse clone (1844 bp) shows an open reading frame (ORF) encoding a TCP-1-related polypeptide of 548 amino acids (aa) (59,562 Da). This gene is different from Tcp-1 and the six Tcp-1-related genes reported previously, Tcp-1 (Ccta), Cctb, Cctg, Cctd, Ccte, Cctz and Ccth, which encode subunits of CCT. The product of the novel gene was analysed using an antibody raised against the C terminus of the polypeptide deduced from the nt sequence. We found that this gene encodes a subunit of CCT (polypeptide S1; 62 kDa and pI 6.25 by two-dimensional gel analysis). We have named it Cctq, encoding the theta subunit of CCT (CCT theta). The aa sequence of CCT theta shows 23-29% identity to the other CCT subunits, alpha, beta, gamma, delta, epsilon, zeta and eta, and 29% identity to the archaebacterial chaperonin TF55. CCT theta also contains the motifs common to all the other subunits of CCT which are postulated to be involved in ATPase activity.

Structure and expression of the gene encoding mouse t-complex polypeptide (Tcp-1).

The nucleotide (nt) sequence of the structural gene (Tcp-1) encoding mouse t-complex polypeptide 1 (TCP-1) has been determined. The nt sequence extending to 10,043 bp shows that the Tcp-1 gene is divided into 12 exons, 11 introns and 5'- and 3'-flanking regions. The Tcp-1 gene has a tight cluster of major transcription start points (tsp). Two GC boxes, one CCAAT box and some other possible regulatory elements are located in the region upstream from the tsp, but no TATA box was found. Extending from the 5'-flanking region to the first intron, a CpG dinucleotide-rich cluster is located. In addition, Tcp-1 gene transcripts in mouse organs, embryos and cultured cells were analyzed by Northern blotting. The Tcp-1 mRNA is enriched not only in testes, but also in early post-implantation embryos and some cultured cell lines, as compared with mouse organs other than the testis. The amount of Tcp-1 mRNA in embryos decreases during development. These results suggest that the expression of the Tcp-1 gene may be regulated spatially and temporally in embryonic and adult mice by transcriptional control or by mRNA stability.

Antibody characterisation of two distinct conformations of the chaperonin-containing TCP-1 from mouse testis.

We describe a panel of antibodies specific to individual subunits of the chaperonin-containing TCP-1 (CCT) and one antibody that reacts with all the subunits of CCT. Immunoblot analysis of CCT purified from mouse testis suggests that the testis-specific subunit, S6, may be related to CCT zeta and that a co-purifying 63 kDa protein may be a novel subunit of CCT. Using these antibodies in the analysis of CCT subjected to nondenaturing IEF we observed the resolution of two distinct conformations of CCT, which differ in their susceptibility to proteolysis and in the number of associated polypeptides.

Tissue-specific subunit of the mouse cytosolic chaperonin-containing TCP-1.

We have cloned a novel Tcp-1-related mouse testis cDNA encoding a polypeptide of 531 amino acids which shares 81.2% identity with the zeta subunit of the mouse cytosolic chaperonin-containing TCP-1 (CCT). Immunoblot analysis of mouse testis CCT subunits separated by 2-dimensional gel electrophoresis indicates that this novel gene, Cctz-2, encodes a CCT subunit of Mr 57 000 and pI 7.1. Cctz-2 mRNA is detected only in testis whereas the other Cctz gene, Cctz-1, is expressed in all tissues investigated. The CCTzeta-2 subunit may have specific functions in the folding of testicular proteins and for interactions with testicular molecular chaperones.

Perceived support needs of family caregivers and implications for a telephone support service.

The purposes of this study were: to identify the perceived support needs of family caregivers of persons living with chronic illness (physical or cognitive) and receiving home-care services, and to describe the types of telephone services that would meet the expressed needs of caregivers. The qualitative design used semi-structured interviews. A total of 34 caregivers (mean age 62 years) participated in the study. The care recipients (mean age 78 years) were primarily the husband/wife or parent of the caregiver. The most commonly expressed caregiver needs were: a social life, instrumental support (e.g., respite, assistance with physical care, financial compensation), informational support, and emotional support. Most caregivers said they would use a telephone support service provided by a professional (71%) or a fellow caregiver (59%) if available. The results of this study support a pilot study and evaluation of a telephone support service for family caregivers.

A single amino acid residue is responsible for species-specific incompatibility between CCT and alpha-actin.

Actin is dependent on the type-II chaperonin CCT (chaperonin containing TCP-1) to reach its native state. In vitro, yeast CCT folds yeast and also mammalian cytoplasmic (beta/gamma) actins but is now found to be incapable of folding mammalian skeletal muscle alpha-actin. Arrest of alpha-actin on yeast CCT at a folding cycle intermediate has been observed by electron microscopy. This discovery explains previous observations in vivo that yeast mutants expressing only the muscle actin gene are non-viable. Mutational analysis identified a single specific alpha-actin residue, Asn-297, that confers this species/isoform folding specificity. The implications of this incompatibility for chaperonin mechanism and actin-CCT co-evolution are discussed.

Sequence of the t complex Tcp-10at gene and examination of the Tcp-10t gene family.

Transmission ratio distortion (TRD) is a property of complete t haplotypes which results in the preferential transmission of the t haplotype chromosome from heterozygous t/+ males to the majority of the offspring. A candidate gene for one of the primary genetic elements in TRD, the t complex responder locus has recently been suggested to be Tcp-10bt. There are multiple, functional Tcp-10t genes, but genetic data suggest the presence of the Tcp-10at gene alone is compatible with normal transmission ratios. Here we present the complete sequence and genomic structure of the Tcp-10at gene which is compared with sequence data from a number of cDNAs and genomic subclones representing all active Tcp-10t family genes. A detailed table of all sequence variants discovered in the course of our investigation is presented, and we have clarified the extent of 5' untranslated alternative splicing patterns exhibited by this gene family. A 60 base pair (bp) in-frame deletion from the 5' end of exon 3 of the Tcp-10at gene is also presented and compared with the equivalent region of Tcp-10bt and Tcp-10ct. A search of the University of Edinburgh database has revealed a significant homology between the Tcp-10bt open reading frame and several cytosolic filament proteins. Interestingly, the region of homology is involved in the deletion from the Tcp-10at gene.