A specialist seating assessment clinic: changing pressure relief practice.

STUDY DESIGN: Description of a clinical service, evaluation of pressure relief practices. OBJECTIVES: To describe a specialist seating assessment clinic and a change in clinical practice arising from its work. SETTING: National Spinal Injuries Centre, Stoke Mandeville Hospital, UK. METHODS: Retrospective review of the ischial transcutaneous oxygen measurements of 50 newly injured and chronic spinal cord-injured (SCI) individuals seen in a specialist seating assessment clinic. Tissue oxygenation was measured in the sitting position (loaded) and during pressure relief (unloaded). RESULTS: Mean duration of pressure relief required to raise tissue oxygen to unloaded levels was 1 min 51 s (range 42 s-3 min 30 s). CONCLUSION: These results confirmed the clinical perception that brief pressure lifts of 15-30 s are ineffective in raising transcutaneous oxygen tension (TcPO(2)) to the unloaded level for most individuals. Sustaining the traditional pressure relief by lifting up from the seat for the necessary extended duration is neither practical nor desirable for the majority of clients. It was found that alternative methods of pressure relief were more easily sustainable and very efficient.

The Caenorhabditis elegans polarity gene ooc-5 encodes a Torsin-related protein of the AAA ATPase superfamily.

The PAR proteins are required for polarity and asymmetric localization of cell fate determinants in C. elegans embryos. In addition, several of the PAR proteins are conserved and localized asymmetrically in polarized cells in Drosophila, Xenopus and mammals. We have previously shown that ooc-5 and ooc-3 mutations result in defects in spindle orientation and polarity in early C. elegans embryos. In particular, mutations in these genes affect the re-establishment of PAR protein asymmetry in the P(1) cell of two-cell embryos. We now report that ooc-5 encodes a putative ATPase of the Clp/Hsp100 and AAA superfamilies of proteins, with highest sequence similarity to Torsin proteins; the gene for human Torsin A is mutated in individuals with early-onset torsion dystonia, a neuromuscular disease. Although Clp/Hsp100 and AAA family proteins have roles in diverse cellular activities, many are involved in the assembly or disassembly of proteins or protein complexes; thus, OOC-5 may function as a chaperone. OOC-5 protein co-localizes with a marker of the endoplasmic reticulum in all blastomeres of the early C. elegans embryo, in a pattern indistinguishable from that of OOC-3 protein. Furthermore, OOC-5 localization depends on the normal function of the ooc-3 gene. These results suggest that OOC-3 and OOC-5 function in the secretion of proteins required for the localization of PAR proteins in the P(1) cell, and may have implications for the study of torsion dystonia.

Analysis of the roles of kinesin and dynein motors in microtubule-based transport in the Caenorhabditis elegans nervous system.

The heteromeric kinesins constitute a subfamily of kinesin-related motor complexes that function in several distinct intracellular transport events. The founding member of this subfamily, heterotrimeric kinesin II, has been purified and characterized from early sea urchin embryos, where it was shown using antibody perturbation to be required for the synthesis of motile cilia, presumably by driving the anterograde transport of raft complexes. To further characterize heteromeric kinesin transport pathways, and to attempt to identify cargo molecules, we are using the model organism Caenorhabditis elegans to exploit its well-characterized nervous system and simple genetics. Here we describe methods for large-scale nematode growth and partial purification of kinesin-related holoenzymes from C. elegans, and an in vivo transport assay that allows the direct labeling and visualization of motor complexes and putative cargo molecules moving in living C. elegans neurons. This transport assay is being used to characterize the in vivo transport properties of motor enzymes in living cells, and to exploit a number of existing mutations in C. elegans that may represent constituents of heteromeric kinesin-driven transport pathways, for example, the retrograde intraflagellar transport motor CHE-3 dynein, as well as cargo molecules and/or regulatory molecules.

Mutations in ooc-5 and ooc-3 disrupt oocyte formation and the reestablishment of asymmetric PAR protein localization in two-cell Caenorhabditis elegans embryos.

The early development of Caenorhabditis elegans embryos is characterized by a series of asymmetric divisions in which the mitotic spindle is repeatedly oriented on the same axis due to a rotation of the nuclear-centrosome complex. To identify genes involved in the control of spindle orientation, we have screened maternal-effect lethal mutants for alterations in cleavage pattern. Here we describe mutations in ooc-5 and ooc-3, which were isolated on the basis of a nuclear rotation defect in the P(1) cell of two-cell embryos. These mutations are novel in that they affect the asymmetric localization of PAR proteins at the two-cell stage, but not at the one-cell stage. In wild-type two-cell embryos, PAR-3 protein is present around the entire periphery of the AB cell and prevents nuclear rotation in this cell. In contrast, PAR-2 functions to allow nuclear rotation in the P(1) cell by restricting PAR-3 localization to the anterior periphery of P(1). In ooc-5 and ooc-3 mutant embryos, PAR-3 was mislocalized around the periphery of P(1), while PAR-2 was reduced or absent. The germ-line-specific P granules were also mislocalized at the two-cell stage. Mutations in ooc-5 and ooc-3 also result in reduced-size oocytes and embryos. However, par-3 ooc double-mutant embryos can exhibit nuclear rotation, indicating that small size per se does not prevent rotation and that PAR-3 mislocalization contributes to the failure of rotation in ooc mutants. We therefore postulate that wild-type ooc-5 and ooc-3 function in oogenesis and in the reestablishment of asymmetric domains of PAR proteins at the two-cell stage.

Role of a class DHC1b dynein in retrograde transport of IFT motors and IFT raft particles along cilia, but not dendrites, in chemosensory neurons of living Caenorhabditis elegans.

The heterotrimeric motor protein, kinesin-II, and its presumptive cargo, can be observed moving anterogradely at 0.7 microm/s by intraflagellar transport (IFT) within sensory cilia of chemosensory neurons of living Caenorhabditis elegans, using a fluorescence microscope-based transport assay (Orozco, J.T., K.P. Wedaman, D. Signor, H. Brown, L. Rose, and J.M. Scholey. 1999. Nature. 398:674). Here, we report that kinesin-II, and two of its presumptive cargo molecules, OSM-1 and OSM-6, all move at approximately 1.1 microm/s in the retrograde direction along cilia and dendrites, which is consistent with the hypothesis that these proteins are retrieved from the distal endings of the cilia by a retrograde transport pathway that moves them along cilia and then dendrites, back to the neuronal cell body. To test the hypothesis that the minus end-directed microtubule motor protein, cytoplasmic dynein, drives this retrograde transport pathway, we visualized movement of kinesin-II and its cargo along dendrites and cilia in a che-3 cytoplasmic dynein mutant background, and observed an inhibition of retrograde transport in cilia but not in dendrites. In contrast, anterograde IFT proceeds normally in che-3 mutants. Thus, we propose that the class DHC1b cytoplasmic dynein, CHE-3, is specifically responsible for the retrograde transport of the anterograde motor, kinesin-II, and its cargo within sensory cilia, but not within dendrites.

Two heteromeric kinesin complexes in chemosensory neurons and sensory cilia of Caenorhabditis elegans.

Chemosensation in the nervous system of the nematode Caenorhabditis elegans depends on sensory cilia, whose assembly and maintenance requires the transport of components such as axonemal proteins and signal transduction machinery to their site of incorporation into ciliary structures. Members of the heteromeric kinesin family of microtubule motors are prime candidates for playing key roles in these transport events. Here we describe the molecular characterization and partial purification of two heteromeric kinesin complexes from C. elegans, heterotrimeric CeKinesin-II and dimeric CeOsm-3. Transgenic worms expressing green fluorescent protein driven by endogenous heteromeric kinesin promoters reveal that both CeKinesin-II and CeOsm-3 are expressed in amphid, inner labial, and phasmid chemosensory neurons. Additionally, immunolocalization experiments on fixed worms show an intense concentration of CeKinesin-II and CeOsm-3 polypeptides in the ciliated endings of these chemosensory neurons and a punctate localization pattern in the corresponding cell bodies and dendrites. These results, together with the phenotypes of known mutants in the pathway of sensory ciliary assembly, suggest that CeKinesin-II and CeOsm-3 drive the transport of ciliary components required for sequential steps in the assembly of chemosensory cilia.

The let-99 gene is required for proper spindle orientation during cleavage of the C. elegans embryo.

The orientation of cell division is a critical aspect of development. In 2-cell C. elegans embryos, the spindle in the posterior cell is aligned along the long axis of the embryo and contributes to the unequal partitioning of cytoplasm, while the spindle in the anterior cell is oriented transverse to the long axis. Differing spindle alignments arise from blastomere-specific rotations of the nuclear-centrosome complex at prophase. We have found that mutations in the maternally expressed gene let-99 affect spindle orientation in all cells during the first three cleavages. During these divisions, the nuclear-centrosome complex appears unstable in position. In addition, in almost half of the mutant embryos, there are reversals of the normal pattern of spindle orientations at second cleavage: the spindle of the anterior cell is aligned with the long axis of the embryo and nuclear rotation fails in the posterior cell causing the spindle to form transverse to the long axis. In most of the remaining embryos, spindles in both cells are transverse at second cleavage. The distributions of several asymmetrically localized proteins, including P granules and PAR-3, are normal in early let-99 embryos, but are perturbed by the abnormal cell division orientations at second cleavage. The accumulation of actin and actin capping protein, which marks the site involved in nuclear rotation in 2-cell wild-type embryos, is abnormal but is not reversed in let-99 mutant embryos. Based on these data, we conclude that let-99(+) is required for the proper orientation of spindles after the establishment of polarity, and we postulate that let-99(+) plays a role in interactions between the astral microtubules and the cortical cytoskeleton.

Early patterning of the C. elegans embryo.

Studies of about 20 maternally expressed genes are providing an understanding of mechanisms of patterning and cell-fate determination in the early Caenorhabditis elegans embryo. The analyses have revealed that fates of the early blastomeres are specified by a combination of intrinsically asymmetric cell divisions and two types of cell-cell interactions: inductions and polarizing interactions. In this review we summarize the current level of understanding of the molecular mechanisms underlying these processes in the specification of cell fates in the pregastrulation embryo.

Pseudocleavage is dispensable for polarity and development in C. elegans embryos.

The first cleavage of the Caenorhabditis elegans embryo is asymmetrical, producing daughters with different cell fates. During the first cell cycle, P granules, cytoplasmic components that are segregated to the germ-line, are localized to the posterior of the embryo. It has been hypothesized that the asymmetrical behavior of the daughters of the first division results from a similar localization of developmental determinants. A process called pseudocleavage also occurs during the first cell cycle: Anterior cortical contractions culminate in a single partial constriction of the embryo called the pseudocleavage furrow. Coincident with pseudocleavage, there is an anteriorly directed flow of cortical cytoplasm and a posteriorly directed flow of internal cytoplasm. Foci of filamentous cortical actin become asymmetrically distributed into an anterior cap. Roles for these various first cell cycle events in cytoplasmic localization and development have been suggested but remain unclear. We have isolated a maternal effect mutation, nop-1(it142), which abolishes the anterior cortical contractions and the pseudocleavage furrow. In addition, cortical actin foci remain uniformly distributed in most embryos. Despite these defects, cytoplasmic and cortical streaming is present and P granules are localized to the posterior of early embryos. In most embryos from mutant mothers, development proceeds normally and the embryos hatch and grow into fertile adults. We conclude that the pseudocleavage contractions and furrow are dispensable for the development of C. elegans.

The Drosophila cellularization gene nullo produces a blastoderm-specific transcript whose levels respond to the nucleocytoplasmic ratio.

The initial development of the Drosophila embryo is characterized by rapid nuclear mitosis without cytokinesis. After 13 such mitoses, a coordinated cell division process called cellularization occurs, during which membranes simultaneously enclose each nucleus in a cell. Cellularization requires the establishment of a hexagonal network of actin and myosin filaments in the cortex of the embryo; the filaments are located on the cytoplasmic face of the invaginating membrane furrows. Zygotic expression of the nullo gene is essential for the maintenance of an intact actin-myosin network. We have cloned the nullo gene and present its sequence as well as a characterization of nullo transcript levels in wild-type and mutant embryos. The nullo gene encodes a predicted protein of 213 amino acids, a large proportion of which is basic. nullo transcripts are first detectable at nuclear cell cycle 11, peak in accumulation at the end of cycle 13, and disappear rapidly as cellularization begins. The gene does not appear to be expressed at any other time in the life of the organism. The normal accumulation of nullo transcripts does not require gene activity of other zygotic cellularization genes. The regulation of nullo RNA levels during cycle 14, however, is coupled to the nucleocytoplasmic ratio, which also controls the cessation of rapid, synchronous mitosis just before cellularization.

Effect of abdominal binders on breathing in tetraplegic patients.

We studied the effect on breathing of a conventional and a newly designed abdominal binder in seven patients with complete tetraplegia. The indices of respiratory ability used were the transdiaphragmatic pressure on maximal sniff (sniff Pdi), the maximum static inspiratory mouth pressure (PImax), and the vital capacity (VC). These were measured in patients with and without binders, in the supine position, raised up to 70 degrees on a tilt table, and seated upright. When patients were raised from the supine to the 70 degrees tilt and to the seated posture, sniff Pdi and VC decreased. Both binders improved VC in the seated position and at 70 degrees tilt, and sniff Pdi at 70 degrees tilt. The new binder was as effective as but no better than the conventional binder. PImax was too variable to be a valuable index of inspiratory power. These findings support the view that abdominal binders assist breathing in tetraplegic patients who are seated or raised to near vertical positions.

Measurement of abdominal wall compliance in normal subjects and tetraplegic patients.

On inspiration descent of the diaphragm is opposed by the passive properties of the abdominal wall, the tone of its muscles, and the inertia of the abdominal contents. As a result, intra-abdominal pressure rises and promotes rib cage expansion. In patients with high spinal injury the diaphragm is the most important muscle of inspiration and abdominal wall displacement is more evident than in normal subjects. Abdominal wall compliance has been measured by relating gastric pressure to abdominal wall displacement, which was determined by means of an optical contour mapping system. Six normal subjects and six tetraplegic patients were studied in the supine posture, during passive expiration from total lung capacity to functional residual capacity. Over this lung volume range the normal subjects partitioned an average of 31% of expired volume to the abdominal compartment, while the corresponding average figure in the patients was 77% of expired volume. Since the range of gastric pressure was similar in the two groups, it is concluded that abdominal wall compliance is greater in tetraplegic patients. This high compliance could have a detrimental effect on lower rib cage expansion.

Physical ability in relation to anthropometric measurements in persons with complete spinal cord lesion below the sixth cervical segment.

A study was undertaken to determine the ability of patients with complete tetraplegia below cervical sixth segment to transfer in relation to their anthropometric characteristics. Thirty-six chronic patients were assessed and spasticity was measured. A discriminant function analysis was carried out to assess the extent to which a number of anthropometric and anatomical variables could predict the patients' final ability to effect a transfer. Using nine of the original 23 predictor variables it is possible to correctly classify a patient's eventual ability to transfer in 92% of cases.