Describing and expanding the clinical phenotype of anti-MDA5-associated rapidly progressive interstitial lung disease: case series of nine Canadian patients and literature review.

OBJECTIVE: To describe and expand the phenotype of anti-MDA5-associated rapidly progressive interstitial lung disease (MDA5-RPILD) in Canadian patients. METHOD: All proven cases of MDA5-RPILD hospitalized in the University of Montreal's affiliated centres from 2004 to 2015 were selected for inclusion. RESULTS: Of nine consecutive patients, RPILD was the presenting manifestation in seven, whereas two patients developed RPILD 2 years after the onset of arthritis and of chronic interstitial lung disease. In the case with arthritis, RPILD was probably triggered by initiation of tumour necrosis factor-α-inhibitor therapy. In most patients (89%), RPILD was accompanied by concomitant onset of palmar/lateral finger papules, skin ulcerations, and/or mechanic's hands. All patients experienced profound weight loss over 1-2 months (mean ± SD 10.2 ± 4.8 kg). All had arthralgias and/or arthritis. Six patients were clinically amyopathic; only one patient had creatine kinase (CK) levels > 500 U/L. Initial ferritin and transaminase levels were elevated in 86% and 67% of patients, respectively. The antinuclear antibody (ANA) test was negative for nuclear and cytoplasmic staining; antisynthetase autoantibodies were negative. Three patients died; time from initial symptoms to death ranged from 7 to 15 weeks. All six survivors received mycophenolate mofetil and/or tacrolimus as part of induction and/or maintenance therapy. CONCLUSION: In an inpatient setting, RPILD associated with characteristic skin rashes, profound weight loss, articular symptoms, normal or low CK with elevated ferritin, and absent fluorescence on ANA testing should alert the clinician to the possibility of MDA5-RPILD. T-cell-mediated therapies may play a role in this highly lethal condition.

Clinical and serological associations of autoantibodies to the Ku70/Ku80 heterodimer determined by a novel chemiluminescent immunoassay.

BACKGROUND: Autoantibodies targeting Ku, an abundant nuclear protein with DNA helicase activity, have been reported in patients with systemic autoimmune rheumatic diseases. Little is known about the clinical associations of anti-Ku antibodies, especially when novel diagnostic technologies are used. The objective of the present study was to analyse the prevalence of anti-Ku antibodies in different medical conditions using a novel chemiluminescent immunoassay. PATIENTS AND METHODS: Serum samples from adult patients with systemic lupus erythematosus (SLE, n=305), systemic sclerosis (SSc, n=70) and autoimmune myositis patients (AIM, n=109) were the primary focus of the study. Results were compared with disease controls (rheumatoid arthritis, RA, n=30; infectious diseases, n=17) and healthy individuals (n=167). In addition, samples submitted for routine autoantibody testing from patients referred to a rheumatology clinic (n=1078) were studied. All samples were tested for anti-Ku antibodies by QUANTA Flash Ku chemiluminescent immunoassay (research use only, Inova Diagnostics, San Diego, USA) using full length recombinant human Ku. SLE patient samples were also tested for other autoantibodies. Clinical data of anti-Ku antibody positive patients (high titres) were obtained by retrospective chart review. RESULTS AND FINDINGS: In the disease cohorts, 30/305 (9.8%) SLE, 3/70 (4.3%) systemic sclerosis and 4/109 (3.7%) autoimmune myositis (AIM) patients were positive, respectively. The four positive AIM patients had an overlap myositis syndrome that included two patients with SLE. The three systemic sclerosis (SSc) positive samples had diagnoses of SSc/SLE overlap, diffuse cutaneous SSc, and early edematous phase SSc. In the control cohorts, 2/170 (1.2%) healthy individuals (all low titre), 0/30 (0.0%) (RA) and 0/17 (0.0%) infectious disease patients were positive. The area under the curve values were: 0.75 for SLE vs. controls, 0.68 for SSc vs. controls and 0.37 for AIM vs. CONTROLS: In the rheumatology clinic referral cohort, 12/1078 (1.1%) were positive for anti-Ku antibodies, nine showing low and three high titres. The diagnoses of the three high positive anti-Ku positive patients were: probable SLE, mixed connective tissue disease (MCTD) and ANA positive RA. CONCLUSION: Anti-Ku antibodies detected by chemiluminescent immunoassay are most prevalent in SLE. When found in AIM and SSc, they were associated with overlap syndrome and early SSc.

Left heart disease: a frequent cause of early pulmonary hypertension in systemic sclerosis, unrelated to elevated NT-proBNP levels or overt cardiac fibrosis but associated with increased levels of MR-proANP and MR-proADM: retrospective analysis of a French Canadian cohort.

OBJECTIVES: Pulmonary hypertension (PH) causes mortality in systemic sclerosis (SSc). Pulmonary arterial hypertension (PAH) and left heart disease (LHD) are frequent causes of PH. Therefore, we studied PAH and LHD in early PH. METHOD: A total of 432 French Canadian SSc patients were studied retrospectively. All underwent screening for PH. We analysed clinical, serological, and radiographic data from 26 patients with early PH diagnosed by right heart catheterization (RHC). SSc patients with (n = 21) and without PH (n = 19) were prospectively re-evaluated by cardiac magnetic resonance imaging (MRI) and serial measurements of N-terminal pro-brain natriuretic peptide (NT-proBNP) and the haemodynamic biomarkers mid-regional pro-atrial natriuritic peptide (MR-proANP) and mid-regional pro-adrenomedullin (MR-proADM). RESULTS: The most frequent cause of early PH was LHD (58%). PAH was seen in 34% of patients. No association was found between the type of PH and autoantibodies. Early LHD-PH, but not early PAH, was associated with lower NT-proBNP (p = 0.024), but MR-proANP and MR-proADM levels were higher in early LHD-PH than in patients without PH (p = 0.014 and p = 0.012, respectively). Only one patient had abnormal cardiac MRI explaining LHD-PH. CONCLUSIONS: Early PH in SSc, like late PH, is heterogeneous and RHC is essential for determining its underlying cause. The most frequent cause of early PH was LHD. Levels of MR-proANP and MR-proADM, but not NT-proBNP, were increased in early LHD-PH, and may be more reliable than NT-proBNP as a biomarker of early PH in this subgroup of patients. Cardiac MRI did not explain LHD-PH. This study is the first to identify a high frequency of LHD in early PH correlating with normal NT-proBNP levels but increased MR-proANP and MR-proADM levels in SSc patients.

Breast cancer in systemic lupus erythematosus.

OBJECTIVE: Evidence points to a decreased breast cancer risk in systemic lupus erythematosus (SLE). We analyzed data from a large multisite SLE cohort, linked to cancer registries. METHODS: Information on age, SLE duration, cancer date, and histology was available. We analyzed information on histological type and performed multivariate logistic regression analyses of histological types according to age, SLE duration, and calendar year. RESULTS: We studied 180 breast cancers in the SLE cohort. Of the 155 cases with histology information, 11 were referred to simply as 'carcinoma not otherwise specified'. In the remaining 144 breast cancers, the most common histological type was ductal carcinoma (n = 95; 66%) followed by lobular adenocarcinoma (n = 11; 8%), 15 cancers were of mixed histology, and the remaining ones were special types. In our regression analyses, the independent risk factors for lobular versus ductal carcinoma was age [odds ratio (OR) 1.07, 95% confidence interval (CI) 1.01-1.14] and for the 'special' subtypes it was age (OR 1.06, 95% CI 1.01-1.10) and SLE duration (OR 1.05, 95% CI 1.00-1.11). CONCLUSIONS: Generally, up to 80% of breast cancers are ductal carcinomas. Though our results are not definitive, in the breast cancers that occur in SLE, there may be a slight decrease in the ductal histological type. In our analyses, age and SLE duration were independent predictors of histological status.

Expression of the gap junction protein connexin36 in small intensely fluorescent (SIF) cells in cardiac parasympathetic ganglia of rodents.

In mammals, several endocrine cell types are electrically coupled by connexin36 (Cx36)-containing gap junctions, which mediate intercellular communication and allow regulated and synchronized cellular activity through exchange of ions and small metabolites via formation of intercellular channels that link plasma membranes of apposing cells. One cell type thought to be endocrine-like in nature are small intensely fluorescent (SIF) cells that store catecholamines in their dense-core vesicles and reside in autonomic ganglia. Here, using immunofluorescence approaches, we examined whether SIF cells located specifically in cardiac parasympathetic ganglia of adult and neonatal mice and adult rats follow patterns of Cx36 expression seen in other endocrine cells. In these ganglia, SIF cells were identified by their distinct small soma size, autofluorescence at 475 nm, and immunolabelling for their markers tyrosine hydroxylase and vesicular monoamine transporter-1. SIF cells were often found in pairs or clusters among principal cholinergic neurons. Immunofluorescence labelling of Cx36 occurred exclusively as fine puncta that appeared at contacts between SIF cell processes and somata or at somato-somatic appositions of SIF cells. These puncta were absent in cardiac parasympathetic ganglia of Cx36 null mice. Transgenic mice expressing enhanced green fluorescent protein reporter for Cx36 expression displayed labelling for the reporter in SIF cells. The results suggest that Cx36-containing gap junctions electrically couple SIF cells, which is consistent with previous suggestions that these may be classified as endocrine-type cells that secrete catecholamines into the bloodstream in a regulated manner.

Characteristics of Electrical Synapses, C-terminals and Small-conductance Ca2+ activated Potassium Channels in the Sexually Dimorphic Cremaster Motor Nucleus in Spinal Cord of Mouse and Rat.

Sexually dimorphic motoneurons (MNs) located in lower lumbar spinal cord are involved in mating and reproductive behaviours and are known to be coupled by electrical synapses. The cremaster motor nucleus in upper lumbar spinal cord has also been suggested to support physiological processes associated with sexual behaviours in addition to its thermoregulatory and protective role in maintaining testes integrity. Using immunofluorescence approaches, we investigated whether cremaster MNs also exhibit features reflecting their potential for electrical synaptic communication and examined some of their other synaptic characteristics. Both mice and rats displayed punctate immunolabelling of Cx36 associated with cremaster MNs, indicative of gap junction formation. Transgenic mice with enhanced green fluorescent protein (eGFP) reporter for connexin36 expression showed that subpopulations of cremaster MNs in both male and female mice express eGFP, with greater proportions of those in male mice. The eGFP+ MNs within the cremaster nucleus vs. eGFP- MNs inside and outside this nucleus displayed a 5-fold greater density of serotonergic innervation and exhibited a paucity of innervation by C-terminals arising from cholinergic V0c interneurons. All MNs within the cremaster motor nucleus displayed prominent patches of immunolabelling for SK3 (K+) channels around their periphery, suggestive of their identity as slow MNs, many though not all of which were in apposition to C-terminals. The results provide evidence for electrical coupling of a large proportion of cremaster MNs and suggest the existence of two populations of these MNs with possibly differential innervation of their peripheral target muscles serving different functions.

Interrelationships Between Spinal Sympathetic Preganglionic Neurons, Autonomic Systems and Electrical Synapses Formed by Connexin36-containing Gap Junctions.

Spinal sympathetic preganglionic neurons (SPNs) are among the many neuronal populations in the mammalian central nervous system (CNS) where there is evidence for electrical coupling between cell pairs linked by gap junctions composed of connexin36 (Cx36). Understanding the organization of this coupling in relation to autonomic functions of spinal sympathetic systems requires knowledge of how these junctions are deployed among SPNs. Here, we document the distribution of immunofluorescence detection of Cx36 among SPNs identified by immunolabelling of their various markers, including choline acetyltransferase, nitric oxide and peripherin in adult and developing mouse and rat. In adult animals, labelling of Cx36 was exclusively punctate and dense concentrations of Cx36-puncta were distributed along the entire length of the spinal thoracic intermediolateral cell column (IML). These puncta were also seen in association with SPN dendritic processes in the lateral funiculus, the intercalated and central autonomic areas and those within and extending medially from the IML. All labelling for Cx36 was absent in spinal cords of Cx36 knockout mice. High densities of Cx36-puncta were already evident among clusters of SPNs in the IML of mouse and rat at postnatal days 10-12. In Cx36BAC::eGFP mice, eGFP reporter was absent in SPNs, thus representing false negative detection, but was localized to some glutamatergic and GABAergic synaptic terminals. Some eGFP+ terminals were found contacting SPN dendrites. These results indicate widespread Cx36 expression in SPNs, further supporting evidence of electrical coupling between these cells, and suggest that SPNs are innervated by neurons that themselves may be electrically coupled.

On the Organization of Connexin36 Expression in Electrically Coupled Cholinergic V0c Neurons (Partition Cells) in the Spinal Cord and Their C-terminal Innervation of Motoneurons.

Large cholinergic neurons (V0c neurons; aka, partition cells) in the spinal cord project profusely to motoneurons on which they form C-terminal contacts distinguished by their specialized postsynaptic subsurface cisterns (SSCs). The V0c neurons are known to be rhythmically active during locomotion and release of acetylcholine (ACh) from their terminals is known to modulate the excitability of motoneurons in what appears to be a task-dependent manner. Here, we present evidence that a subpopulation of V0c neurons express the gap junction forming protein connexin36 (Cx36), indicating that they are coupled by electrical synapses. Based on immunofluorescence imaging and the use of Cx36BAC-enhanced green fluorescent protein (eGFP) mice in which C-terminals immunolabelled for their marker vesicular acetylcholine transporter (vAChT) are also labelled for eGFP, we found a heterogeneous distribution of eGFP+ C-terminals on motoneurons at cervical, thoracic and lumber spinal levels. The density of C-terminals on motoneurons varied as did the proportion of those that were eGFP+ vs. eGFP-. We present evidence that fast vs. slow motoneurons have a greater abundance of these terminals and fast motoneurons also have the highest density that were eGFP+. Thus, our results indicate that a subpopulation of V0c neurons projects preferentially to fast motoneurons, suggesting that the capacity for synchronous activity conferred by electrical synapses among networks of coupled V0c neurons enhances their dynamic capabilities for synchronous regulation of motoneuron excitability during high muscle force generation. The eGFP+ vs. eGFP- V0c neurons were more richly innervated by serotonergic terminals, suggesting their greater propensity for regulation by descending serotonergic systems.

Nup180, a novel nuclear pore complex protein localizing to the cytoplasmic ring and associated fibrils.

Using an autoimmune serum from a patient with overlap connective tissue disease we have identified by biochemical and immunocytochemical approaches an evolutionarily conserved nuclear pore complex (NPC) protein with an estimated molecular mass of 180 kD and an isoelectric point of approximately 6.2 which we have designated as nup180. Extraction of isolated nuclear envelopes with 2 M urea and chromatography of the solubilized proteins on WGA-Sepharose demonstrated that nup180 is a peripheral membrane protein and does not react with WGA. Affinity-purified antibodies yielded a punctate immunofluorescent pattern of the nuclear surface of mammalian cells and stained brightly the nuclear envelope of cryosectioned Xenopus oocytes. Nuclei reconstituted in vitro in Xenopus egg extract were also stained in the characteristic punctate fashion. Immunogold EM localized nup180 exclusively to the cytoplasmic ring of NPCs and short fibers emanating therefrom into the cytoplasm. Antibodies to nup180 did not inhibit nuclear protein transport in vivo nor in vitro. Despite the apparent lack of involvement in NPC assembly or nucleocytoplasmic transport processes, the conservation of nup180 across species and its exclusive association with the NPC cytoplasmic ring suggests an important, though currently undefined function for this novel NPC protein.

Disseminated Histoplasma capsulatum infection presenting with panniculitis and focal myositis in rheumatoid arthritis treated with etanercept.

We report the case of a patient with rheumatoid arthritis (RA) on etanercept who presented with panniculitis and focal myositis as manifestations of disseminated histoplasmosis. Systematic search of the literature showed 11 additional case reports of disseminated histoplasmosis with tumour necrosis factor-alpha (TNFalpha) blockade therapy (infliximab, n = 8; etanercept, n = 3). Although disseminated histoplasmosis may manifest with classical symptoms of fever and respiratory complaints, it may also present atypically, such as with panniculitis and focal myositis. This review illustrates and emphasizes the importance of being highly suspicious for infection, including by opportunistic organisms, and to exclude such process in patients treated with a TNFalpha inhibitor when faced with unusual complications, even when an alternative aetiology appears plausible.

A survey of health care professionals and oncology patients at the McGill University Health Centre reveals enthusiasm for establishing a postmortem rapid tissue donation program.

Background: In the early developmental phase of a postmortem rapid tissue donation (rtd) program for patients with metastatic cancer, we surveyed health care professionals (hcps) and oncology patients at the McGill University Health Centre (muhc) to assess their knowledge and attitudes pertaining to rtd from metastatic cancer patients for research purposes. Methods: A 23-item survey was developed and distributed to hcps at tumour board meetings, and a related 26-item survey was developed and distributed to oncology patients at the muhc Cedars Cancer Centre. Results: The survey attracted participation from 73 hcps, including 37 attending physicians, and 102 oncology patients. Despite the fact that 88% of hcps rated their knowledge of rtd as none or limited, 42% indicated that they would feel comfortable discussing rtd with their cancer patients. Of the responding hcps, 67% indicated that their current knowledge of rtd would affect their decision to discuss such a program with patients, which implies the importance of education for hcps to facilitate enrolment of patients into a rtd program. Of responding patients, 78% indicated that they would not be uncomfortable if their doctor discussed rtd with them, and 61% indicated that they would like it if their doctor were to discuss rtd with them. The hcps and patients felt that the best time for patients to be approached about consenting to a rtd program would be at the transition to palliative care when no treatment options remain. Conclusions: At the muhc, hcps and patients are generally enthusiastic about adopting a rtd program for patients with metastatic cancer. Education of hcps and patients will be an important determinant of the program's success.

The relationship between cancer and medication exposures in systemic lupus erythaematosus: a case-cohort study.

OBJECTIVE: To examine if, in systemic lupus erythaematosus (SLE), exposure to immunosuppressive therapy (cyclophosphamide, azathioprine, methotrexate) increases cancer risk. METHODS: A case-cohort study was performed within a multi-site international SLE cohort; subjects were linked to regional tumour registries to determine cancer cases occurring after entry into the cohort. We calculated the hazard ratio (HR) for cancer after exposure to an immunosuppressive drug, in models that controlled for other medications (anti-malarial drugs, systemic glucocorticoids, non-steroidal anti-inflammatory drugs (NSAIDs), aspirin), smoking, age, sex, race/ethnicity, geographic location, calendar year, SLE duration, and lupus damage scores. In the primary analyses, exposures were treated categorically (ever/never) and as time-dependent. RESULTS: Results are presented from 246 cancer cases and 538 controls without cancer. The adjusted HR for overall cancer risk after any immunosuppressive drug was 0.82 (95% CI 0.50-1.36). Age > or = 65, and the presence of non-malignancy damage were associated with overall cancer risk. For lung cancer (n = 35 cases), smoking was also a prominent risk factor. When looking at haematological cancers specifically (n = 46 cases), there was a suggestion of an increased risk after immunosuppressive drug exposures, particularly when these were lagged by a period of 5 years (adjusted HR 2.29, 95% CI 1.02-5.15). CONCLUSIONS: In our SLE sample, age > or = 65, damage, and tobacco exposure were associated with cancer risk. Though immunosuppressive therapy may not be the principal driving factor for overall cancer risk, it may contribute to an increased risk of haematological malignancies. Future studies are in progress to evaluate independent influence of medication exposures and disease activity on risk of malignancy.

SLE patients with renal damage incur higher health care costs.

OBJECTIVES: To compare costs and quality of life (QoL) between SLE patients with and without renal damage. METHODS: Seven hundred and fifteen patients were surveyed semi-annually over 4 yrs on health care use and productivity loss and annually on QoL. Cumulative direct and indirect costs (2006 Canadian dollars) and QoL (average annual change in SF-36) were compared between patients with and without renal damage [Systemic Lupus International Collaborating Clinics/ACR Damage Index (SLICC/ACR DI)] using simultaneous regressions. RESULTS: At study conclusion, for patients with the renal subscale of the SLICC/ACR DI = 0 (n = 634), 1 (n = 54), 2 (n = 15) and 3 (n = 12), mean 4-yr cumulative direct costs per patient (95% CI) were $20,337 ($18,815, $21,858), $27,869 ($19,230, $36,509), $51,191 ($23,463, $78,919) and $99,544 ($57,102, $141,987), respectively. In a regression where the renal subscale of the SLICC/ACR DI was a single indicator variable, on average (95% CI), each unit increase in renal damage was associated with a 24% (15%, 33%) increase in direct costs. In a regression where each level in the renal subscale was an indicator variable, patients with end-stage renal disease incurred 103% (65%, 141%) higher direct costs than those without renal damage. Cumulative indirect costs and annual change in the SF-36 summary scores did not differ between patients. CONCLUSIONS: SLE patients with renal damage incurred higher direct costs, but did not experience a poorer QoL. QoL may be more influenced by concurrent renal activity than accumulated renal damage, which can occur at any time and patients may gradually habituate to their compromised health state.

Adaptive importance sampling to accelerate training of a neural probabilistic language model.

Previous work on statistical language modeling has shown that it is possible to train a feedforward neural network to approximate probabilities over sequences of words, resulting in significant error reduction when compared to standard baseline models based on n-grams. However, training the neural network model with the maximum-likelihood criterion requires computations proportional to the number of words in the vocabulary. In this paper, we introduce adaptive importance sampling as a way to accelerate training of the model. The idea is to use an adaptive n-gram model to track the conditional distributions produced by the neural network. We show that a very significant speedup can be obtained on standard problems.

Connexin36 Expression in Primary Afferent Neurons in Relation to the Axon Reflex and Modality Coding of Somatic Sensation.

Electrical coupling mediated by connexin36-containing gap junctions that form electrical synapses is known to be prevalent in the central nervous system, but such coupling was long ago reported also to occur between cutaneous sensory fibers. Here, we provide evidence supporting the capability of primary afferent fibers to engage in electrical coupling. In transgenic mice with enhanced green fluorescent protein (eGFP) serving as a reporter for connexin36 expression, immunofluorescence labeling of eGFP was found in subpopulations of neurons in lumbar dorsal root and trigeminal sensory ganglia, and in fibers within peripheral nerves and tissues. Immunolabeling of connexin36 was robust in the sciatic nerve, weaker in sensory ganglia than in peripheral nerve, and absent in these tissues from Cx36 null mice. Connexin36 mRNA was detected in ganglia from wild-type mice, but not in those from Cx36 null mice. Labeling of eGFP was localized within a subpopulation of ganglion cells containing substance P and calcitonin gene-releasing peptide, and in peripheral fibers containing these peptides. Expression of eGFP was also found in various proportions of sensory ganglion neurons containing transient receptor potential (TRP) channels, including TRPV1 and TRPM8. Ganglion cells labeled for isolectin B4 and tyrosine hydroxylase displayed very little co-localization with eGFP. Our results suggest that previously observed electrical coupling between peripheral sensory fibers occurs via electrical synapses formed by Cx36-containing gap junctions, and that some degree of selectivity in the extent of electrical coupling may occur between fibers belonging to subpopulations of sensory neurons identified according to their sensory modality responsiveness.

Immunoglobulin M autoantibody to vimentin intermediate filaments.

Serum from a patient with the CREST Syndrome and systemic lupus erythematosus contained an IgM antibody that reacted at dilutions up to 1:800 with a fibrous cytoplasmic network in several epithelioid and fibroblastic cell lines. The antibody was shown by immunofluorescence microscopy to label a specific subset of cytoskeletal polymers, the intermediate filaments. The reactive antigen from this biochemically heterogeneous group of filaments was established as the 58,000-mol wt protein, vimentin: (a) the patient's serum reacts with a range of cell lines that contain intermediate filaments composed of vimentin, but not with cells whose intermediate filaments are composed of different protein subunits; (b) in PTK2 epithelioid cells the serum reacts with the class of filaments that coils around the nucleus after colchicine treatment (vimentin) and not with the filaments that remain dispersed after colchicine (prekeratin); and (c) the component of reactive cells that combines with the serum is shown by immunoelectrophoresis to be a 58,000-mol wt protein antigen. A similar antibody that binds intermediate filaments of PTK2 cells was encountered at lower titer in some sera from other patients with connective tissue diseases and in control sera. Previous routine antinuclear antibody assays using mouse liver or commercially prepared HEp-2 cells have failed to reveal anticytoskeletal antibodies in patient sera, perhaps due to inadequate presentation or preservation of cytoplasmic antigens.

Anticytoskeletal autoantibody to microfilament anchorage sites recognizes novel focal contact proteins.

Actin microfilaments are anchored to the plasma membrane at focal contacts. Using an indirect immunofluorescence method, we detected an autoantibody reactive with focal contacts in PtK2, HEp-2, and BHK-21 cells in serum from two patients with early systemic sclerosis. With double immunofluorescence, using the actin-binding drug phalloidin, we localized the plaques decorated by these sera specifically at the termini of microfilament bundles. The reactive antigens were identified by immunoblotting as proteins of 80,000- and 75,300-mol wt in PtK2, and of 53,500-mol wt in HEp-2 and BHK-21 cells. The 53,500-mol wt protein was also identified in rat skeletal, myocardial, and smooth muscle tissues. The detergent solubility of these proteins suggested that they may be linked to the plasma membrane. The autoantigens were immunologically distinct from vinculin and alpha-actinin, two major proteins also known to be concentrated at the ends of microfilament bundles. Our observations suggest that this novel anticytoskeletal autoantibody may identify a novel family of vertebrate cell proteins involved in the linkage of microfilaments to the plasma membrane at focal contacts.

Catecholaminergic activation of G-protein coupling in rat spinal cord: further evidence for the existence of dopamine and noradrenaline receptors in spinal grey and white matter.

[35S]GTPgammaS autoradiography of slide-mounted tissue sections was used to examine G-protein coupling in the rat spinal cord, as stimulated by dopamine, the D1 receptor agonist SKF 38393, noradrenaline, and noradrenaline in the presence of the alpha adrenoceptor antagonist, phentolamine. Measurements were obtained from the different laminae of spinal grey and from the dorsal, lateral, and ventral columns of white matter, at cervical, thoracic, and lumbar levels. At every level, there was a relatively strong basal incorporation of GTPgammaS in laminae II-III>lamina IV-X of spinal grey, even in presence of DPCPX to block endogenous activation by adenosine A1 receptors. Dopamine, and to a lesser degree SKF 38393, but not the D2 receptor agonist quinpirole, stimulated G-protein coupling in laminae IV-X. Both dopamine and SKF 38393 also induced a weak but significant activation throughout the white matter. In both grey and white matter, the activation by dopamine was markedly reduced in presence of a selective D1 receptor antagonist. Noradrenaline strongly stimulated coupling throughout the spinal grey at all levels, an effect that was uniformly reduced in the presence of phentolamine. With or without phentolamine, there was also significant stimulation by noradrenaline in the white matter. Under the same experimental conditions, alpha 1, alpha 2, and beta adrenergic receptor agonists failed to activate GTPgammaS incorporation in either grey or white matter. However, in the presence of selective alpha 1 or alpha 2 receptor antagonist, significant reductions of noradrenaline-stimulated GTPgammaS incorporation were observed in both grey and white matter. The beta antagonist propanolol reduced GTPgammaS incorporation in grey matter only. Thus, the results confirmed the existence of D1 dopamine receptors and of alpha 1, alpha 2, and beta adrenergic receptors in the grey matter of rat spinal cord. In white matter, they strongly suggested the presence of dopamine D1, and of alpha 1 and alpha 2 adrenergic receptors on glia and/or microvessels, that might be activated by diffuse transmission in vivo.

Fate of pharmaceuticals and pesticides in fly larvae composting.

A novel and efficient organic waste management strategy currently gaining great attention is fly larvae composting. High resource recovery efficiency can be achieved in this closed-looped system, but pharmaceuticals and pesticides in waste could potentially accumulate in every loop of the treatment system and spread to the environment. This study evaluated the fate of three pharmaceuticals (carbamazepine, roxithromycin, trimethoprim) and two pesticides (azoxystrobin, propiconazole) in a fly larvae composting system and in a control treatment with no larvae. It was found that the half-life of all five substances was shorter in the fly larvae compost (<10% of control) and no bioaccumulation was detected in the larvae. Fly larvae composting could thus impede the spread of pharmaceuticals and pesticides into the environment.

Localization of the Ran-GTP binding protein RanBP2 at the cytoplasmic side of the nuclear pore complex.

A partial cDNA clone coding for the mouse homologue of the human Ran-GTP binding protein, RanBP2, has been isolated by screening of a murine expression library with antibodies to nup180, a previously identified nuclear pore complex protein (nucleoporin). Whether the antibodies cross-reacted with the polypeptide encoded by the cDNA clone or, alternatively, nup180 is proteolytically related to RanBP2, has not been determined. The 3795-bp open reading frame of the cDNA encodes a polypeptide consisting of 1265 amino acids with three Ran-GTP binding domains (RanBD) that are almost identical with published partial amino acid sequences of human RanBP2 as deduced from several partial cDNA clones of other authors. Sequence analysis further revealed that murine RanBP2 contains tandemly repeated zinc fingers of Cys2-Cys2 type and multiple copies of the FXFG nucleoporin "signature" motif clustered in regions preceding the RanBDs. Antibodies raised against a synthetic peptide of the derived amino acid sequence decorated the cytoplasmic rings of nuclear pore complexes (NPCs) as shown by immunogold electron microscopy. We suggest that the cytoplasmically disposed nucleoporin RanBP2 provides docking sites for import substrate-receptor complexes and, further, that the affinity of these sites to the transport substrate is modulated in a Ran-dependent fashion.