Dystrophin and utrophin influence fiber type composition and post-synaptic membrane structure

The X-linked muscle wasting disease Duchenne muscular dystrophy is caused by the lack of dystrophin in muscle. Protein structure predictions, patient mutations, in vitro binding studies and transgenic and knockout mice suggest that dystrophin plays a mechanical role in skeletal muscle, linking the subsarcolemmal cytoskeleton with the extracellular matrix through its direct interaction with the dystrophin-associated protein complex (DAPC). Although a signaling role for dystrophin has been postulated, definitive data have been lacking. To identify potential non-mechanical roles of dystrophin, we tested the ability of various truncated dystrophin transgenes to prevent any of the skeletal muscle abnormalities associated with the double knockout mouse deficient for both dystrophin and the dystrophin-related protein utrophin. We show that restoration of the DAPC with Dp71 does not prevent the structural abnormalities of the post-synaptic membrane or the abnormal oxidative properties of utrophin/dystrophin-deficient muscle. In marked contrast, a dystrophin protein lacking the cysteine-rich domain, which is unable to prevent dystrophy in the mdx mouse, is able to ameliorate these abnormalities in utrophin/dystrophin-deficient mice. These experiments provide the first direct evidence that in addition to a mechanical role and relocalization of the DAPC, dystrophin and utrophin are able to alter both structural and biochemical properties of skeletal muscle. In addition, these mice provide unique insights into skeletal muscle fiber type composition.     

Potency of antileukoprotease and alpha 1-antitrypsin to inhibit degradation of fibrinogen by adherent polymorphonuclear leukocytes from normal subjects and patients with chronic granulomatous disease.

We have studied the relative efficacy of antileukoprotease (ALP) and alpha 1-antitrypsin (alpha 1AT) to inhibit the degradation of substrate by polymorphonuclear leukocytes (PMN) attached onto a fibrinogen matrix. PMN elastase activity was assayed by radioimmunoassay of a specific 21-residue cleavage product from the amino terminus of the A alpha chain, A alpha (1-21), of fibrinogen. The adherence of PMN (1.0 x 10(6)) to a fibrinogen matrix was facilitated by incubation with recombinant tumor necrosis factor-alpha (1 nM). Subsequently, the cells were exposed to inhibitors before stimulation with cytochalasin B and formylmethionyl-leucylphenylalanine. Under these conditions, ALP inhibited A alpha (1-21) formation with an IC50 of 85 +/- 30 nM and alpha 1AT gave an IC50 of 220 +/- 98 nM (mean +/- SD). The effect of oxidant production on A alpha (1-21) formation was evaluated by comparing the effect of PMN from normal subjects with PMN from subjects with X-linked NADPH oxidase deficiency. Stimulation of PMN from the latter subjects in a similar fashion as described above resulted in the formation of 40 +/- 4 pmol/ml A alpha (1-21), or approximately twice the amount seen with cells from normal subjects. Preincubation with ALP or alpha 1AT in a concentration range between 10 to 900 nM resulted in an IC50 of 50 +/- 13 nM for ALP compared with 150 +/- 21 nM for alpha 1AT. Both inhibitors are more effective to prevent fibrinogen degradation caused by chronic granulomatous disease (CGD) PMN than by normal PMN despite the fact that CGD PMN generated more A alpha (1-21) than did normal PMN.(ABSTRACT TRUNCATED AT 250 WORDS)     

ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to beta-dystroglycan

An intracellular protein, dystrophin, plays an important role in keeping muscle fibers intact by binding at its N-terminal end to the subsarcolemmal cytoskeletal actin network and via its C-terminal end to the transmembraneous protein beta-dystroglycan. Duchenne muscular dystrophy is caused by the loss of dystrophin, which can result from the loss of this binding. The N-terminal part of the latter binding site of dystrophin has been well documented using overlay assay and X-ray diffraction assays. However, the binding site at the C-terminal region of dystrophin has not been examined in detail. In the present work, we report a detailed analysis of the C-terminal binding domain as follows. (1). The full binding activity corresponding to the effective binding in vivo is expressed by the dystrophin fragment spanning amino acids 3026-3345 containing the ZZ domain at the C-terminus. Determination of this binding range is important not only for understanding of the mechanism of dystrophy, but also useful for the design of truncated dystrophin constructs for gene therapy. (2). The ZZ domain binds to EF1 domain in the dystrophin fragment to reinforce the binding activity. (3). The cysteine 3340 in the ZZ domain is essential for the binding of dystrophin to beta-dystroglycan. A reported case of DMD due to missense mutation C3340Y may be caused by inability to fix dystrophin beneath the cell membrane. (4). The binding mode of utrophin is different from that of dystrophin. The difference is conspicuous concerning the cysteine residues present in the ZZ domain.     

Studies on the dispersal of staphylococci

The dispersal of staphylococci during undressing in a cubicle has been investigated. The ability to disperse appears to be largely dependent on the degree of contamination of the skin with staphylococci. There are some major differences in the numbers of bacteria and skin scales disseminated by different groups of people, e.g., patients with skin disease disseminated more than did normal persons. Differences in dissemination between males and females appeared to be largely due to differences in clothing.     

Generation of thyroglobulin-specific T-cell clones derived from F1 Fisher rats.

We established T-cell clones from 'poor-responder' Fisher rats specific for thyroglobulin (Tg) with a view to examining Tg presentation by cloned Fisher rat thyroid (FRTL) cells. From the screening of 60 T-cell clones, three high-responding Tg-specific clones (B21.01, B21.04 and B21.05) were isolated from the lymph nodes of F1 generation (Fisher x 'high-responder' Buffalo) female rats immunized with murine Tg in complete Freund's adjuvant (CFA). Three T-cell clones expressed a W3/25+, OX-8- phenotype and responded specifically to murine and rat Tg in T-cell proliferation assays with Fisher rat antigen-presenting cells and secreted IL-2 as measured using a murine cytotoxic T-lymphocyte line (CTLL-2). Both B21.04 and B21.05 T-cell clones were capable of providing helper T-cell function for rat Tg antibody production in syngeneic reconstitution cultures in vitro. In contrast, clone B21.01 inhibited Tg antibody secretion. These data demonstrate that 'poor-responder' Fisher rats are capable of mounting significant T-cell responses to Tg in an F1 generation. Such Tg-specific T-cell clones will allow us to analyse their interaction with cloned Fisher rat thyroid cells and determine the role, if any, of thyroid cell antigen self-presentation to the immune system.     

Experimental staphylococcal endocarditis and aortitis

Summary The initial colonization, byStaphylococcus aureus, of the catheter damaged aortic valve and aorta of the rabbit, was examined by light and electron microscopy at 15 min, 3 h and 24 h post inoculation (PI). At 15 min PI, the majority of bacteria (80%) were located on the lateral surfaces of the thrombic vegetations while 20% were attached directly to the connective tissue of the aortic valve and aorta in areas where the endothelial lining was disrupted. By 3 h the bacteria on the thrombic vegetations were covered by fibrin. At this time, the bacteria both within the vegetations and on the surface of the vasculature were undergoing multiplication to form small groups. The precipitation of thrombus around the bacteria attached to the surface of the aorta to form microscopic infected vegetations had occurred by 24 h PI. The colonizing bacteria did not elicit any phagocytic response. The colonization of the cardiovasculature byStaph. aureus did not necessarily require pre-existing vegetations.     

Cytogenetic analysis of the Asian Plethodontid salamander, Karsenia koreana: Evidence for karyotypic conservation, chromosome repatterning, and genome size evolution

A cytogenetic analysis, including the karyotype, C-bands, silver-stained nucleolus organizer regions and genome size, was performed on the recently discovered species, Karsenia koreana, the first plethodontid salamander from Asia. The karyotype consists of 14 pairs of bi-armed chromosomes, with no evidence of heteromorphic sex chromosomes. C-banding reveals a concentration of heterochromatin at the centromeres as well as at interstitial locations. The smallest chromosome (pair number 14) has symmetrical interstitial C-bands in each arm, resembling chromosome no. 14 of North American species of its sister group taxon, supergenus Hydromantes. Acomparative analysis of C-band heterochromatin and silver-stained nucleolus organizer regions of Karsenia and other plethodontid genera reveals that chromosomal evolution may have featured chromosome 'repatterning' within the context of conserved chromosome number and shape in this clade. Genome size is correlated with geographic distribution in plethodontids and appears to have important phenotypic correlates as well. The genome size of Karsenia is relatively large, and resembles that of the geographically closest plethodontids from western North America, especially species of the genus Hydromantes. The biological significance of these cytogenetic characteristics of plethodontid salamanders is discussed within an evolutionary context.     

The morphology of immune reactions in normal, thymectomized and reconstituted mice: I. The response to sheep erythrocytes

The response to sheep red blood cells has been studied in the lymph nodes draining their site of injection in normal mice, and in thymectomized, irradiated, bone-marrow injected mice with and without a reconstituting thymus graft. By using a chromosome marker to differentiate between cells derived from the bone-marrow and thymus graft it has proved possible to show that the immune response should be thought of in terms of at least two cell populations. Cells of thymic origin are stimulated to mitotic activity in the interfollicular cortex, and their activity precedes both antibody production and morphological signs of activity in the follicular regions. Mitotic divisions of cells of bone-marrow origin reached a peak a day later than did the thymic cells and their activity was sustained. Follicular enlargement and germinal centre production were coincident in time both with antibody production and bone-marrow cell mitotic activity. Lymph nodes of animals lacking a thymic influence showed only minor changes after antigenic stimulation and these were restricted to the follicular regions. There appeared to be only a small quantitative difference between the responses of normal and of reconstituted animals.     

Preferences for self-care or professional advice for minor illness: a discrete choice experiment

AIM: To determine the relative importance of factors that influence decision making in the management of minor illness, and how people trade between these factors. DESIGN OF STUDY: Discrete choice experiment.Setting:Scottish electoral roll. METHOD: Six hundred and fifty-two responders of a previous national survey were invited to complete a discrete choice experiment questionnaire. This was used to measure relative preferences for managing symptoms of minor illness often associated with analgesic use. Three attributes were identified as important to participants: type of management, availability, and cost of managing symptoms. Trade-offs between these attributes were examined. RESULTS: A 57% response rate was achieved (51% valid response rate). People preferred to manage symptoms by self-care and were willing to pay almost pounds 23 to do so. Community pharmacy was the preferred source of advice. Responders preferred less waiting time and paying less money when managing symptoms, and were willing to trade between factors. A less preferred type of management became more attractive when waiting times and cost were reduced. CONCLUSION: Findings suggest that self-care is the preferred method of managing symptoms of minor illness. When developing services to support self-care, policy makers should invest in services that reduce waiting times and incur least cost to users.