Localisation and expression of a myelin associated neurite inhibitor, Nogo-A and its receptor Nogo-receptor by mammalian CNS cells.

Axon regeneration failure in the adult mammalian central nervous system (CNS) is partly due to inhibitory molecules associated with myelin. The Nogo receptor (NgR) plays a role in this process through an extraordinary degree of cross reactivity with three structurally unrelated myelin-associated inhibitory ligands namely; Nogo-A, myelin associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp). The major aim of the study was to investigate and explore the cellular localisation and expression pattern of NgR and Nogo-A in the mammalian nervous system. We therefore generated a rabbit polyclonal anti-NgR antibody from the leucine rich repeat (LRR) No. 9 domain of the NgR polypeptide chain. Together with a commercially available polyclonal antibody specific for NgR, and in conjunction with double labeling immunofluorescence methods on cryosections and cell cultures, NgR immunoreactivity was observed in the CNS and dorsal root ganglia (DRG). In cellular populations, it was confined to neuronal cell bodies and their processes. NgR was also localised on the surface of extending DRG intact axons and growth cones in live staining experiments. Nogo-A, a member of the reticulon family protein, was widely distributed in the mammalian brain, spinal cord, and DRG. Intense Nogo-A immunoreactivity was also detected in oligodendrocyte cell bodies and their myelin sheaths in nerve fibre tracts of the CNS. Furthermore, numerous populations of neurons in the brain and spinal cord expressed Nogo-A to a variable extent in their cell bodies and neurites, suggesting additional, as-yet-unknown, functions of this protein. These results confirm results obtained by other researchers with different sets of antibodies. However, they also raise the question of the mechanism and circumstances under which NgR interacts with Nogo-A, as the latter appears to be confined to the cytoplasm and can therefore not be expected to bind NgR on the axon surface.

Glycosylphosphatidyl inositol-anchored proteins and fyn kinase assemble in noncaveolar plasma membrane microdomains defined by reggie-1 and -2.

Using confocal laser scanning and double immunogold electron microscopy, we demonstrate that reggie-1 and -2 are colocalized in < or =0.1-microm plasma membrane microdomains of neurons and astrocytes. In astrocytes, reggie-1 and -2 do not occur in caveolae but clearly outside these structures. Microscopy and coimmunoprecipitation show that reggie-1 and -2 are associated with fyn kinase and with the glycosylphosphatidyl inositol-anchored proteins Thy-1 and F3 that, when activated by antibody cross-linking, selectively copatch with reggie. Jurkat cells, after cross-linking of Thy-1 or GM1 (with the use of cholera toxin), exhibit substantial colocalization of reggie-1 and -2 with Thy-1, GM1, the T-cell receptor complex and fyn. This, and the accumulation of reggie proteins in detergent-resistant membrane fractions containing F3, Thy-1, and fyn imparts to reggie-1 and -2 properties of raft-associated proteins. It also suggests that reggie-1 and -2 participate in the formation of signal transduction centers. In addition, we find reggie-1 and -2 in endolysosomes. In Jurkat cells, reggie-1 and -2 together with fyn and Thy-1 increase in endolysosomes concurrent with a decrease at the plasma membrane. Thus, reggie-1 and -2 define raft-related microdomain signaling centers in neurons and T cells, and the protein complex involved in signaling becomes subject to degradation.

Osteoid osteoma of the lunate--a case report.

We report a rare case of an osteoid osteoma of the lunate bone in a young lady who presented to us with chronic wrist pain. She was treated by excision and cancellous bone grafting of the lesion with complete resolution of symptoms.

Identification of reggie-1 and reggie-2 as plasmamembrane-associated proteins which cocluster with activated GPI-anchored cell adhesion molecules in non-caveolar micropatches in neurons.

Neurons are believed to possess plasmalemmal microdomains and proteins analogous to the caveolae and caveolin of nonneuronal cells. Caveolae are plasmalemmal invaginations where activated glycosyl-phosphatidylinositol (GPI)-anchored proteins preferentially assemble and where transmembrane signaling may occur. Molecular cloning of rat reggie-1 and -2 (80% identical to goldfish reggie proteins) shows that reggie-2 is practically identical to mouse flotillin-1. Flotillin-1 and epidermal surface antigen (ESA) (flotillin-2) are suggested to represent possible membrane proteins in caveolae. Rat reggie-1 is 99% homologous to ESA in overlapping sequences but has a 49-amino-acid N-terminus not present in ESA. Antibodies (ABs) which recognize reggie-1 or -2 reveal that both proteins cluster at the plasmamembrane and occur in micropatches in neurons [dorsal root ganglia (DRGs), retinal ganglion, and PC-12 cells] and in nonneuronal cells. In neurons, reggie micropatches occur along the axon and in lamellipodia and filopodia of growth cones, but they do not occur in caveolae. By quantitative electronmicroscopic analysis we demonstrate the absence of caveolae in (anti-caveolin negative) neurons and show anti-reggie-1 immunogold-labeled clusters at the plasmamembrane of DRGs. When ABs against the GPI-anchored cell adhesion molecules (CAMs) F3 and Thy-1 are applied to live DRGs, the GPI-linked CAMs sequester into micropatches. Double immunofluorescence shows a colocalization of the CAMs with micropatches of anti-reggie antibodies. Thus, reggie-1 and reggie-2 identify sites where activated GPI-linked CAMs preferentially accumulate and which may represent noncaveolar micropatches (domains).

Exercise-induced bronchospasm in high school athletes via a free running test: incidence and epidemiology.

BACKGROUND: Exercise-induced bronchospasm (EIB) affects up to 35% of athletes and up to 90% of asthmatics. Asthma morbidity and mortality have increased over the past several decades among residents of Philadelphia, PA. It is possible that a simple free running test for EIB may serve as a tool to study the factors contributing to recent trends in asthma, and to screen for asthma in athletes in the urban setting. OBJECTIVES: The purposes of this study were to (1) assess a free running test to screen for EIB, and (2) examine prevalence of and epidemiologic factors associated with EIB in high school athletes. DESIGN: Cross-sectional observational study on the incidence and risk factors for EIB. To validate our method and criteria for the diagnosis of EIB, a repeat test was performed on a portion of the athletes. In a randomized single-blinded fashion, 15 athletes who had demonstrated EIB initially received albuterol or placebo prior to a repeat exercise test. SETTING: Community high school athletic facilities. PARTICIPANTS: We studied 238 male high school varsity football players. INTERVENTION: All athletes underwent an acquaintance session with a questionnaire, followed by a 1-mile outdoor run (6 to 8 mins). MEASUREMENTS: Peak expiratory flow (PEF) measurements were determined prior to and 5, 15, and 30 min after exercise. Heart rates (HRs) and dyspnea scores were measured. EIB was defined as a decrease of 15% in PEF at any time point after exercise. Associations of EIB with demographic factors were assessed by univariate and multivariate analyses. RESULTS: Two hundred thirty-eight athletes participated: 92 European-Americans (EA), 140 African-Americans (AA), 5 Hispanics, and 1 Native American. Mean age was 16+/-1 years. Average HR postexercise was 156+/-24 beats/min. Twenty-four (10%) reported a history of treated asthma. The prevalence of EIB among the remaining 214 athletes was 19 of 214 (9%). The rate of EIB among AA athletes was higher than among EA athletes: (17/126 [13%] AA vs 2/82 [2%] EA, p = 0.01). During the validation portion of the study, the placebo-treated group (n = 7) demonstrated a consistent drop in PEF after exercise on repeat testing, with a 16+/-5% fall in PEF on initial testing and a 14+/-13 drop with placebo. In contrast, the fall in airflow in the albuterol-treated athletes (n = 8) following exercise reversed with albuterol treatment, from a 15+/-6% fall in PEF at initial testing to an increase in PEF of 6+/-9% from baseline following albuterol administration. A history of wheezing (p < 0.001), residence in a poverty area (p < 0.0001), race (p = 0.01), remote history of asthma (p < 0.001), and absolute water content of the air on the day tested (p = 0.04) were significantly associated with EIB. By stepwise regression, EIB was most closely associated with a history of wheezing (p = 0.001) and poverty area residence (p = 0.003). CONCLUSIONS: Our findings indicate a substantial rate of unrecognized EIB exists among urban varsity athletes, and suggest that active screening for EIB, especially for students residing in poverty areas, may be indicated to identify individuals at risk for EIB and asthma.

Biologically active monomeric and heterodimeric recombinant human calpain I produced using the baculovirus expression system.

Calpain I is a heterodimeric protein that is part of a family of calcium-activated intracellular cysteine proteases presumed to play a role in mediating signals transduced by calcium. Expression of bioactive recombinant human calpain I has been achieved using the baculovirus expression system, by either co-infection with two viruses, each expressing one of the subunits, or infection with a single virus containing both subunits. The approximately 80 kDa catalytic subunit exhibited calcium-dependent proteolytic activity when expressed alone or with the approximately 30 kDa regulatory subunit. Baculoviral recombinant calpain I appeared fully active in that the catalytic subunit in unpurified cell extracts exhibited calcium-dependent autocatalytic cleavage at the correct locus. The amount of approximately 80 kDa subunit accumulated at steady state was greatly increased by co-expression of the approximately 30 kDa subunit, suggesting a possible role for enzyme stabilization by the latter subunit. The recombinant human calpain I was purified to near homogeneity and compared with purified native human erythrocyte calpain I. The recombinant and native enzymes had equivalent inhibition constants for structurally diverse calpain inhibitors, identical calcium activation profiles, and similar specific activities, demonstrating the suitability of using the recombinant protein for studies of the native enzyme.

Patellar resurfacing versus retention in total knee arthroplasty.

We studied 40 patients in whom the patella was not severely deformed and who were undergoing primary total knee arthroplasty (TKA) for osteoarthritis by one surgeon using one type of prosthesis. They were randomly allocated either to have the patella retained or resurfaced with a cemented, all-polyethylene component regardless of the state of the patellar articular cartilage. Apart from removal of osteophytes, no surgery was undertaken on the retained patellae. All 38 surviving patients were evaluated at three years using the HSS knee score and a new, specifically designed Patellar score (maximum score of 30). No TKA was revised, but two patients in the resurfacing group had a further unrelated procedure. The mean HSS and Patellar scores at follow-up were 89 and 28 in the patellar retention group and 83 and 26 in the patellar resurfacing group. Statistically significant lower scores for both were recorded in women and in heavier patients. Stair-climbing ability was significantly better in the retention group. Although there were no complications related to patellar resurfacing, in the medium term we did not find any significant benefit from resurfacing the patella during TKA for osteoarthritis if it was not severely deformed.

Safety and possible efficacy of fiberoptic bronchoscopy with lavage in the management of refractory asthma with mucous impaction.

Mucous impaction may be suspected in asthmatic exacerbation when, despite aggressive medical management, patients continue to produce sputum containing mucous plugs and exhibit prominent rhonchi and/or wheezes on chest auscultation. Spirometric measurements in this setting corroborate lack of improvement and reveal significant impairment in indices that may reflect small airways function (FEF25-75). We hypothesized that clearance of inspissated secretions by fiberoptic bronchoscopy with lavage (FOBwL) may promote or hasten the clinical improvement of such patients. Fifty-one therapeutic FOBwL were accomplished in 19 patients during 20 episodes of stabilized yet refractory asthma with mucous impaction. No significant complications were encountered. After FOBwL, spirometric measurements of FEV1, FEF25-75, and FVC increased significantly (P less than .01, paired t test), and correlated with relief of dyspnea and mobilization of secretions with cough. FOBwL can be safely performed in stabilized, refractory asthma, and with apparent efficacy. Further investigation is needed to document the therapeutic utility of FOBwL in refractory asthma.

High-molecular-weight kininogen is cleaved in active erythema multiforme.

Erythema multiforme (EM) is an inflammatory disorder of the skin, which may include mucous membrane involvement, that features target (iris) lesions. Mediators specifically involved in EM are not well characterized; its pathogenesis remains enigmatic. In this study, evidence for participation of kinins in the pathophysiology of inflammation in EM was investigated by assessing cleavage of high-molecular-weight kininogen (HMWK) in plasma. These data were compared with analyses of plasmas from patients with serum sickness, chronic idiopathic urticaria/angioedema, and from normal control subjects. Patients with EM demonstrated significant levels of circulating cleaved HMWK in plasma during active disease (p less than 0.01), which declined during remission/recovery. Plasmas from patients with EM obtained during active disease also demonstrated significant levels of 94 kd C1 inhibitor (p less than 0.01) and C1 inhibitor-kallikrein complexes. Patients with serum sickness and chronic idiopathic urticaria/angioedema did not demonstrate these findings and did not differ from normal control subjects (p = not significant). Although the kininogenase responsible for HMWK cleavage in EM has not been conclusively demonstrated, these findings suggest that HMWK cleavage resulted from activation of the contact system and that some of the manifestations of EM in selected patients may in part be accounted for by inflammatory and proinflammatory actions of kinins. Based on these preliminary findings, it will be important to establish whether or not HMWK cleavage in EM is a general finding in patients with this disorder. Further investigation is needed to characterize more clearly kininogenase activity and elucidate the possible role of kinin generation in EM.

Venous bypass for surgical resection of renal carcinoma invading the vena cava: a new approach.

A new technique is described which facilitates the surgical removal of renal carcinoma from the inferior vena cava. The use of cardiopulmonary bypass with or without cardiac arrest has been advocated but with this procedure only the inferior vena cava is bypassed, using femoral and right atrial cannulation, assisted by a closed system electromagnetic centrifugal pump. In appropriate cases this less complex technique allows prolonged access to the inferior vena cava whilst providing equal protection from pulmonary embolisation and tumour dissemination; it also reduces morbidity, operating time, difficulty and cost when compared with cardiopulmonary bypass.