Characterization of the in vitro expressed autoimmune rippling muscle disease immunogenic domain of human titin encoded by TTN exons 248-249.

Autoimmune rippling muscle disease (ARMD) is an autoimmune neuromuscular disease associated with myasthenia gravis (MG). Past studies in our laboratory recognized a very high molecular weight skeletal muscle protein antigen identified by ARMD patient antisera as the titin isoform. These past studies used antisera from ARMD and MG patients as probes to screen a human skeletal muscle cDNA library and several pBluescript clones revealed supporting expression of immunoreactive peptides. This study characterizes the products of subcloning the titin immunoreactive domain into pGEX-3X and the subsequent fusion protein. Sequence analysis of the fusion gene indicates the cloned titin domain (GenBank ID: EU428784) is in frame and is derived from a sequence of N2-A spanning the exons 248-250 an area that encodes the fibronectin III domain. PCR and EcoR1 restriction mapping studies have demonstrated that the inserted cDNA is of a size that is predicted by bioinformatics analysis of the subclone. Expression of the fusion protein result in the isolation of a polypeptide of 52 kDa consistent with the predicted inferred amino acid sequence. Immunoblot experiments of the fusion protein, using rippling muscle/myasthenia gravis antisera, demonstrate that only the titin domain is immunoreactive.

Response of Eucalyptus largiflorens to floodplain salinisation.

An analysis of the stable isotopes from the tree rings of Eucalyptus largiflorens on the Chowilla Floodplain was undertaken. This chronology of tree water use responses was compared to the known hydrological changes that have occurred due to the effects of river regulation. The isotope chronology indicates that E. largiflorens has always oscillated between groundwater and rain-derived soil water and river regulation has apparently had little effect on tree water sources over time. This result is surprising, but no doubt reflects the complexity of floodplain processes and interactions between trees, water and the landscape.

A novel applicator for low-dose-rate brachytherapy of gynecological cancers.

The standard low-dose-rate (LDR) delivery system utilized in the definitive management of patients with cervical carcinoma involves an intrauterine tandem and a pair of vaginal colpostats (ovoids). This well-known application system may deliver inadequate dosage if the tumor extends to the lower vaginal mucosa. During the gauze packing of the ovoids, either operator error or narrowing of the vaginal apex can result in mal-alignment of the colpostats and subsequent inadequate dosing to the ecto-cervix. A novel vaginal cylinder has been designed to address these concerns. Beginning January 1, 2001, patients with cancer of the cervix, endometrium, or vagina requiring LDR brachytherapy have been enrolled into an institutionally sanctioned clinical trial. As of May 31, 2001, a total of 11 patients have been entered but only 10 were successfully implanted with the test device. Patient follow-up has ranged from 0.81 years to 1.2 years (median: 0.96 years). Using our study applicator, all patients received within 10% of the preimplant prescribed dose to tumor. Also, no one had cumulative dosage that exceeded 10% of the maximum allowed dose to the critical normal tissues. Thus far, all study patients have had no clinical evidence of persistence/recurrence of disease or complications from treatment. The preliminary results presented herein clearly demonstrate the feasibility of this novel LDR vaginal cylinder in the treatment of a variety of clinical situations involving gynecological cancers. Our institutional trial is continuing.

Salinization of a fresh palaeo-ground water resource by enhanced recharge.

Deterioration of fresh ground water resources caused by salinization is a growing issue in many arid and semi-arid parts of the world. We discuss here the incipient salinization of a 10(4) km2 area of fresh ground water (<3,000 mg/L) in the semiarid Murray Basin of Australia caused by widespread changes in land use. Ground water 14C concentrations and unsaturated zone Cl soil water inventories indicate that the low salinity ground water originated mainly from palaeo-recharge during wet climatic periods more than 20,000 years ago. However, much of the soil water in the 20 to 60 m thick unsaturated zone throughout the area is generally saline (>15,000 mg/L) because of relatively high evapotranspiration during the predominantly semiarid climate of the last 20,000 years. Widespread clearing of native vegetation over the last 100 years and replacement with crops and pastures leads to enhancement of recharge rates that progressively displace the saline soil-water from the unsaturated zone into the ground water. To quantify the impact of this new hydrologic regime, a one-dimensional model that simulates projected ground water salinities as a function of depth to ground water, recharge rates, and soil water salt inventory was developed. Results from the model suggest that, in some areas, the ground water salinity within the top 10 m of the water table is likely to increase by a factor of 2 to 6 during the next 100 years. Ground water quality will therefore potentially degrade beyond the point of usefulness well before extraction of the ground water exhausts the resource.

Regional planning for the siting of local evaporation basins for the disposal of saline irrigation drainage: development and testing of a GIS-based suitability approach.

In order to prevent salinisation of the streams of the Riverine Plain of the Murray-Darling Basin in southern Australia, evaporation basins are used to dispose of saline irrigation drainage water. Local on-farm (individual landholder) and community (shared between multiple landholders) basins are increasingly being used to prevent export of salt outside irrigation districts. There are questions regarding the availability of land suitable for these basins and their impact on the surrounding environment. We describe the use of currently available spatial data to assist in regional planning for the environmentally safe use of these basins. A GIS-based approach was developed using suitability criteria expected to minimise the risk of off-site effects of basin leakage. The criteria were proximity to surface water features, urban areas and infrastructure, water table depth and salinity, and soil hydraulic conductivity. The approach was applied to all of the major irrigation districts at 1:250,000, the scale at which data are available over the entire Riverine Plain. Confidence in well-defined parameters such as proximity to infrastructure, urban areas and surface water features was higher than for those involving interpolated point data such as water table depth, salinity, and hydraulic conductivity. Most critically, hydraulic conductivity, the most important factor for basin leakage, was found to be unreliable at this scale. Use of higher resolution data (up to 1:100,000) available for two of the irrigation districts improved confidence in both water table depth and salinity but not in hydraulic conductivity. Despite these limitations, it was found that: (i) on-farm basins can only be used on an opportunistic basis in the eastern irrigation districts, but can be widely used in the western districts; (ii) community basins can be used anywhere there is suitable land; and (iii) the results raise serious questions as to whether there is enough suitable land in the eastern districts to dispose of all of the drainage water that is produced.

Resident continuity of care experience in a Canadian general surgery training program.

OBJECTIVES: To provide baseline data on resident continuity of care experience, to describe the effect of ambulatory centre surgery on continuity of care, to analyse continuity of care by level of resident training and to assess a resident-run preadmission clinic's effect on continuity of care. DESIGN: Data were prospectively collected for 4 weeks. All patients who underwent a general surgical procedure were included if a resident was present at operation. SETTING: The Division of General Surgery, Queen's University, Kingston, Ont. OUTCOME MEASURES: Preoperative, operative and inhospital postoperative involvement of each resident with each case was recorded. RESULTS: Residents assessed preoperatively (before entering the operating room) 52% of patients overall, 20% of patients at the ambulatory centre and 83% of patients who required emergency surgery. Of patients assessed by the chief resident, 94% were assessed preoperatively compared with 32% of patients assessed by other residents (p < 0.001). Of the admitted patients, 40% had complete resident continuity of care (preoperative, operative and postoperative). There was no statistical difference between this rate and that for emergency, chief-resident and non-chief-resident subgroups. Of the eligible patients, 58% were seen preoperatively by the resident on the preadmission clinic service compared with 54% on other services (p < 0.1). CONCLUSIONS: This study serves as a reference for the continuity of care experience in Canadian surgical programs. Residents assessed only 52% of patients preoperatively, and only 40% of patients had complete continuity of care. Factors such as ambulatory surgery and junior level of training negatively affected continuity experience. Such factors must be taken into account in planning surgical education.

Identification of autoantibodies associated with rippling muscles and myasthenia gravis that recognize skeletal muscle proteins: possible relationship of antigens and stretch-activated ion channels.

The role of mechanosensitive calcium channels in skeletal muscle physiology is not understood. This study takes advantage of an autoimmune neuromuscular disorder (myasthenia gravis associated with rippling muscles) to identify components in the skeletal muscle myocyte that may play a role in mechanosensitive calcium channel activity. Rippling muscles are characterized by stretch or percussion activated wave-like muscle contractions that do not require motor unit action potentials for propagation. Autoantibodies from the sera of patients with autoimmune rippling muscles (associated with myasthenia gravis) are directed against high molecular weight muscle proteins. Some of these proteins are uniquely recognized by antisera from patients with autoimmune rippling muscles. This suggests these autoantigens are distinct from those normally associated with myasthenia gravis, and may play a role in the mechanosensitive activation of muscle contraction.

Microtubule-entrained kinase activities associated with the cortical cytoskeleton during cytokinesis.

Research over the past few years has demonstrated the central role of protein phosphorylation in regulating mitosis and the cell cycle. However, little is known about how the mechanisms regulating the entry into mitosis contribute to the positional and temporal regulation of the actomyosin-based contractile ring formed during cytokinesis. Recent studies implicate p34cdc2 as a negative regulator of myosin II activity, suggesting a link between the mitotic cycle and cytokinesis. In an effort to study the relationship between protein phosphorylation and cytokinesis, we examined the in vivo and in vitro phosphorylation of actin-associated cortical cytoskeletal (CSK) proteins in an isolated model of the sea urchin egg cortex. Examination of cortices derived from eggs or zygotes labeled with 32P-orthophosphate reveals a number of cortex-associated phosphorylated proteins, including polypeptides of 20, 43 and 66 kDa. These three major phosphoproteins are also detected when isolated cortices are incubated with [32P]ATP in vitro, suggesting that the kinases that phosphorylate these substrates are also specifically associated with the cortex. The kinase activities in vivo and in vitro are stimulated by fertilization and display cell cycle-dependent activities. Gel autophosphorylation assays, kinase assays and immunoblot analysis reveal the presence of p34cdc2 as well as members of the mitogen-activated protein kinase family, whose activities in the CSK peak at cell division. Nocodazole, which inhibits microtubule formation and thus blocks cytokinesis, significantly delays the time of peak cortical protein phosphorylation as well as the peak in whole-cell histone H1 kinase activity. These results suggest that a key element regulating cortical contraction during cytokinesis is the timing of protein kinase activities associated with the cortical cytoskeleton that is in turn regulated by the mitotic apparatus.

Isolation and characterization of a sea urchin zygote cortex that supports in vitro contraction and reactivation of furrowing.

The isolation of the cortex of the sea urchin blastomere by detergent lysis was explored with the aim of analyzing components important in the structure and function of the cortical cytoskeleton, and their relationship to such phenomena as contraction. Buffered EGTA medium supplemented with isotonic glycerol and with magnesium, at a level close to the reported internal cellular concentration, yields stable cytoskeletal cortices that retain their spherical shape. Cortices prepared this way contain actin, myosin, fascin and spectrin, components normally associated with the cortical cytoskeleton in a similar distribution to that in intact zygotes. They retain the organized cortical filamentous structure, including the actin-fascin bundles that form cores of microvilli. ATP and NaCl caused changes in cortical shape, described as either contraction or expansion, respectively. Spectrin, but not myosin, was partially extracted by NaCl, resulting in expansion of the cortex that suggests a role for spectrin in maintenance of cortical structure. ATP (but not ADP nor ATP gamma S), which caused the partial removal of myosin and spectrin, led to the contraction of the cortex, consistent with a role for myosin in cortical tension. In cortices isolated from dividing eggs, the zygotes retained their cleavage furrows and ATP induced continuation of furrow progression. This preparation appears to be a useful in vitro model for cytokinesis.

Noncompliant behavior of people with mental retardation.

Literature pertaining to noncompliant behavior of people with mental retardation was reviewed. Noncompliant behavior was considered in terms of antecedents (characteristics of instructions), behaviors (characteristics of tasks being refused), and consequences (environmental results of noncompliance). Literature pertaining to nondisabled "oppositional" children was incorporated along with literature pertaining to persons with mental retardation. Research involving antecedent conditions suggested that noncompliance is more likely when instructions are vague and interrupted with other instructions. It also suggested that a series of high probability requests could enhance compliance to a subsequent low probability request. Research into behavior variables suggested that noncompliance is more likely when task demands are too difficult. Research into consequent conditions indicates that either positive or negative reinforcement may maintain noncompliant behavior. Treatment studies suggested that oppositional children respond poorly to verbal reward alone and somewhat better to tangible rewards and to programs utilizing timeout. By contrast, noncompliance by people with mental retardation appears to respond well to programs utilizing combinations of social and tangible rewards, but results also suggest that results of timeout programs are mixed. Implications for treatment were discussed along with suggestions for future research.

Immunofluorescence evidence for cytoskeletal rearrangement accompanying pigment redistribution in goldfish xanthophores.

Immunofluorescence and phase-contrast microscopic studies of goldfish xanthophores with aggregated or dispersed pigment show two unusual features. First, immunofluorescence studies with anti-actin show punctate structures instead of filaments. These punctate structures are unique for the xanthophores and are absent from both goldfish dermal non-pigment cells and a dedifferentiated cell line (GEM-81) derived from a goldfish xanthophore tumor. Comparison of immunofluorescence and phase-contrast microscopic images with electron microscopic images of thin sections and of Triton-insoluble cytoskeletons show that these punctate structures represent pterinosomes with radiating F-actin. The high local concentration of actin around the pterinosomes results in strong localized fluorescence such that, when the images have proper brightness for these structures, individual actin filaments elsewhere in the cell are too weak in their fluorescence to be visible in the micrographs. Second, whereas immunofluorescence images with anti-tubulin show typical patterns in xanthophores with either aggregated or dispersed pigment, namely, filaments radiating out from the microtubule organizing center, immunofluorescence images with anti-actin or with anti-intermediate filament proteins show different patterns in xanthophores with aggregated versus dispersed pigment. In cells with dispersed pigment, the punctate structures seen with anti-actin are relatively evenly distributed in the cytoplasm, and intermediate filaments appear usually as a dense perinuclear band and long filaments elsewhere in the cytoplasm. In cells with aggregated pigment, both intermediate filaments and pterinosomes with associated actin are largely excluded from the space occupied by the pigment aggregate, and the band of intermediate filaments surrounds not only the nucleus but also the pigment aggregate. The patterns of distribution of the different cytoskeleton components, together with previous results from this laboratory, indicate that formation of the pigment aggregate depends at least in part on the interaction between pigment organelles and microtubules. The possibility that intermediate filaments may play a role in the formation/stabilization of the pigment aggregate is discussed.

Regulation of the distribution of carotenoid droplets in goldfish xanthophores and possible implication to secretory processes.

In goldfish xanthophores, the formation of pigment aggregate requires: 1) that a pigment organelle (carotenoid droplet) protein p57 be in the unphosphorylated state; 2) that self-association of pigment organelles occur in a microtubule-independent manner; and 3) that pigment organelles via p57 associate with microtubules. In the fully aggregated state, the pigment organelles are completely stationary. Pigment dispersion is initiated by activation of a cAMP-dependent protein kinase, which phosphorylates p57 and allows pigment dispersion via an active process dependent on F-actin and a cytosolic factor. This factor is not an ATPase, and its function is unknown. However, its abundance in different tissues parallels secretory activity of the tissues, suggesting a similarity between secretion and pigment dispersion in xanthophores. The identity of the motor for pigment dispersion is unclear. Experimental results show that pigment organelles isolated from cells with dispersed pigment have associated actin and ATPase activity comparable to myosin ATPase. This ATPase is probably an organelle protein of relative molecular mass approximately 72,000, and unlikely to be an ion pump. Isolated pigment organelles without associated actin have 5x lower ATPase activity. Whether this organelle ATPase is the motor for pigment dispersion is under investigation. The process of pigment aggregation is poorly understood, with conflicting results for and against the involvement of intermediate filaments.

Intermediate filaments with novel protein composition from certain goldfish cells.

Using the conditions for vimentin filament recycling, intermediate filaments (approximately 10 nm) were prepared from the cytoskeleton of a goldfish tumor cell line (erythrophoroma or xanthophoroma). 2-D analysis showed unusual protein composition, with four proteins of molecular weights of 60, 45, 56 and 51 kilodaltons in ratios of approximately 4:4:1:1. These correspond to four of the major cytoskeletal proteins of both the tumor cells and normal xanthophores.

Positive practice overcorrection combined with additional procedures to teach signed words to an autistic child.

The present study evaluated the effectiveness of using positive practice overcorrection in combination with other techniques to teach two manual signs ("milk" and "cookie") to an autistic boy. This boy had a great deal of difficulty in forming any type of discrimination and often became confused in learning the most simple simultaneous discrimination. Intervention primarily consisted of positive practice overcorrection in which the subject was physically guided to form a required hand sign 10 times when he responded incorrectly and was positively reinforced when he signed correctly. The study used a changing criterion within a multiple-baseline design across responses. The results indicated that overcorrection plus positive reinforcement was effective in teaching one sign (milk), however, and added contingent exercise (having to stand up and sit down 10 times for an incorrect response) was required to teach the second sign (cookie). Once the two signs were learned to a criterion level, it was a relatively easy task for the subject to respond correctly with the signs in a matching-to-sample task.

Detection, localization, and treatment of occult bronchogenic carcinoma in nickel workers.

Asymptomatic men exposed to nickel sulfide prior to 1963 were studied prospectively to investigate cytologic abnormalities in the sputum. We discovered a considerably greater incidence of squamous cell tumors that would have been expected from subjects exposed only to cigarette smoke.