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  1. Light chain (AL) amyloidosis: update on diagnosis and management

    PubMed Central

    2011-01-01

    Light chain (AL) amyloidosis is a plasma cell dyscrasia characterized by the pathologic production of fibrillar proteins comprised of monoclonal light chains which deposit in tissues and cause organ dysfunction. The diagnosis can be challenging, requiring a biopsy and often specialized testing to confirm the subtype of systemic disease. The goal of treatment is eradication of the monoclonal plasma cell population and suppression of the pathologic light chains which can result in organ improvement and extend patient survival. Standard treatment approaches include high dose melphalan (HDM) followed by autologous hematopoietic stem cell transplantation (SCT) or oral melphalan with dexamethasone (MDex). The use of novel agents (thalidomide, lenalidomide and bortezomib) alone and in combination with steroids and alkylating agents has shown efficacy and continues to be explored. A risk adapted approach to SCT followed by novel agents as consolidation reduces treatment related mortality with promising outcomes. Immunotherapeutic approaches targeting pathologic plasma cells and amyloid precursor proteins or fibrils are being developed. Referral of patients to specialized centers focusing on AL amyloidosis and conducting clinical trials is essential to improving patient outcomes. PMID:22100031

  2. Atypical immunoglobulin light chain amyloidosis

    PubMed Central

    Wu, Xia; Feng, Jun; Cao, Xinxin; Zhang, Lu; Zhou, Daobin; Li, Jian

    2016-01-01

    Abstract Background: Primary immunoglobulin light chain amyloidosis (AL amyloidosis) is a plasma cell disorder which mainly affects heart, kidneys, liver, and peripheral nervous system. Cases of atypical AL amyloidosis presented as spontaneous vertebral compression fractures have been rarely reported, and data about the management and clinical outcomes of the patients are scarce. Methods: Herein, we present 3 new cases of AL amyloidosis with spontaneous vertebral compression fracture and review 13 cases retrieved from the literature. Results: Moreover, we observed overrepresentations of liver involvement and bone marrow involvement in AL amyloidosis with spontaneous vertebral compression fracture. Conclusion: We believe that better awareness of the rare clinical presentation as spontaneous vertebral compression fracture of AL amyloidosis can facilitate earlier diagnosis and earlier treatment. PMID:27603350

  3. Simultaneous presentation of kappa-restricted chronic lymphocytic leukemia and lambda light chain AL amyloidosis.

    PubMed

    von Keudell, Gottfried; Sanchorawala, Vaishali; O'Hara, Carl; C Seldin, David; Sloan, J Mark

    2014-06-01

    We report on a 58-year-old man who presented with simultaneous kappa-restricted chronic lymphocytic leukemia (CLL) and a lambda-restricted plasma cell dyscrasia causing AL amyloidosis involving the kidney and GI tract. While monoclonal immunoglobulins occasionally produced by CLL has previously been implicated in AL amyloidosis, this is the first case of AL amyloidosis resulting from a distinct plasma cell dyscrasia that is not clonally related to the concurrent CLL. Appropriate treatment depended on detailed pathologic diagnosis of both disease processes.

  4. Disease burden of systemic light-chain (AL) amyloidosis: a systematic literature review.

    PubMed

    Lin, Huamao Mark; Gao, Xin; Cooke, Catherine E; Berg, Deborah; Labotka, Richard; Faller, Douglas V; Seal, Brian; Hari, Parameswaran

    2017-02-21

    A systematic literature review on AL amyloidosis was conducted in order to understand the disease burden, and identify unmet medical needs and knowledge gaps. MEDLINE, EMBASE and COCHRANE databases were searched for English language studies published in the last 10 years using search terms that focused on the clinical, economic, and patient-reported outcome (PRO) aspects of AL amyloidosis. There was a low yield of articles in the economic and PRO categories and additional searches were conducted in clinical conference proceedings, and using Google and Google Scholar. After review, there were 65 articles included for data extraction. AL amyloidosis is a rare disorder without any FDA or EMA approved indications for drug therapy. Using off-label therapies, there is a high rate, 42-64%, of non-response or progression, and an associated high mortality. Toxicities during therapy are common with estimates of up to 30-40% of patients experiencing severity of grade 3 or higher. Patients with AL amyloidosis report severe psychological distress, anxiety and clinical depression. There is a deficiency in the literature on the economic costs associated with AL amyloidosis, and information on costs has been derived from studies that examined multiple myeloma or other disease or treatment components common to AL amyloidosis.

  5. Myocardial infarction with "clean coronaries" caused by amyloid light-chain AL amyloidosis: a case report and literature review.

    PubMed

    Tsai, Stephanie B; Seldin, David C; Wu, Hao; O'Hara, Carl; Ruberg, Frederick L; Sanchorawala, Vaishali

    2011-09-01

    In AL (amyloid light-chain) amyloidosis, the greatest risk of death occurs in patients with cardiac involvement, who typically develop diastolic dysfunction and then systolic heart failure, with predisposition to arrhythmias and sudden death. Here, we present an alternate variation of cardiac amyloidosis. This patient had recent non-obstructive coronary angiography, yet suffered a fatal myocardial infarction shortly after stem cell collection and mobilization in preparation for treatment with high-dose melphalan and autologous stem cell transplantation (HDM/SCT). On autopsy, widespread deposition of amyloid was found in the small vessels of the heart with evidence of associated acute infarction. While the typical presentation of cardiac amyloidosis is an infiltrating restrictive cardiomyopathy, this case report and literature review illustrate that ischemic small vessel amyloidosis may also occur. Small vessel coronary disease and associated myocardial ischemia should be considered in patients with AL amyloidosis with angina, as its presence may increase treatment-related complications. Contemporary testing should aim to detect both forms of cardiac amyloidosis, which may impact management and prognosis.

  6. Aggregation of Full-length Immunoglobulin Light Chains from Systemic Light Chain Amyloidosis (AL) Patients Is Remodeled by Epigallocatechin-3-gallate.

    PubMed

    Andrich, Kathrin; Hegenbart, Ute; Kimmich, Christoph; Kedia, Niraja; Bergen, H Robert; Schönland, Stefan; Wanker, Erich; Bieschke, Jan

    2017-02-10

    Intervention into amyloid deposition with anti-amyloid agents like the polyphenol epigallocatechin-3-gallate (EGCG) is emerging as an experimental secondary treatment strategy in systemic light chain amyloidosis (AL). In both AL and multiple myeloma (MM), soluble immunoglobulin light chains (LC) are produced by clonal plasma cells, but only in AL do they form amyloid deposits in vivo We investigated the amyloid formation of patient-derived LC and their susceptibility to EGCG in vitro to probe commonalities and systematic differences in their assembly mechanisms. We isolated nine LC from the urine of AL and MM patients. We quantified their thermodynamic stabilities and monitored their aggregation under physiological conditions by thioflavin T fluorescence, light scattering, SDS stability, and atomic force microscopy. LC from all patients formed amyloid-like aggregates, albeit with individually different kinetics. LC existed as dimers, ∼50% of which were linked by disulfide bridges. Our results suggest that cleavage into LC monomers is required for efficient amyloid formation. The kinetics of AL LC displayed a transition point in concentration dependence, which MM LC lacked. The lack of concentration dependence of MM LC aggregation kinetics suggests that conformational change of the light chain is rate-limiting for these proteins. Aggregation kinetics displayed two distinct phases, which corresponded to the formation of oligomers and amyloid fibrils, respectively. EGCG specifically inhibited the second aggregation phase and induced the formation of SDS-stable, non-amyloid LC aggregates. Our data suggest that EGCG intervention does not depend on the individual LC sequence and is similar to the mechanism observed for amyloid-β and α-synuclein.

  7. Light chain amyloidosis: Where are the light chains from and how they play their pathogenic role?

    PubMed

    Zhang, Chunlan; Huang, Xufei; Li, Jian

    2017-03-08

    Amyloid light-chain (AL) amyloidosis is a plasma-cell dyscrasia, as well as the most common type of systematic amyloidosis. Pathogenic plasma cells that have distinct cytogenetic and molecular properties secrete an excess amount of amyloidogenic light chains. Assisted by post-translational modifications, matrix components, and other environmental factors, these light chains undergo a conformational change that triggers the formation of amyloid fibrils that overrides the extracellular protein quality control system. Moreover, the amyloidogenic light-chain itself is cytotoxic. As a consequence, organ dysfunction is caused by both organ architecture disruption and the direct cytotoxic effect of amyloidogenic light chains. Here, we reviewed the molecular mechanisms underlying this sequence of events that ultimately leads to AL amyloidosis and also discuss current in vitro and in vivo models, as well as relevant novel therapeutic approaches.

  8. Systemic Light Chain Amyloidosis Mimicking Rheumatic Disorders

    PubMed Central

    2016-01-01

    Secondary amyloidosis can complicate chronic inflammatory autoimmune diseases. However, the clinical findings of primary amyloidosis may mimic those of primary rheumatologic disorders. We present the case of a 53-year-old woman who presented with dystrophic nail changes, dry eyes, bilateral carpal tunnel syndrome, Raynaud's phenomenon, and high titer positive nucleolar pattern antinuclear antibody. She was initially misdiagnosed as having Undifferentiated Connective Tissue Disease (UCTD). On further workup, she was eventually diagnosed with lambda light chain systemic amyloidosis by abdominal fat pad biopsy. Her symptoms completely resolved after autologous stem cell transplantation. With this case, we would like to highlight the similarities in the clinical features between light chain amyloidosis and rheumatological disorders. We would also like to emphasize the importance of the prompt recognition of the clinical features of amyloidosis which are crucial to triggering appropriate diagnostic procedures, since early diagnosis is a key to improving outcomes in this disease with an otherwise poor prognosis. PMID:28042297

  9. Exceptional oral manifestations of amyloid light chain protein (AL) systemic amyloidosis.

    PubMed

    Elad, Sharon; Czerninski, Rakefet; Fischman, Stuart; Keshet, Na'ama; Drucker, Scott; Davidovich, Tzipporah; Goldschmidt, Neta

    2010-03-01

    Oral amyloidosis is usually presented in the tongue and is often associated with multiple myeloma. We present three patients with unusual oral manifestations of primary amyloidosis, which to the best of our knowledge have not been previously published. In two cases the oral manifestation was overt at the time of diagnosis and all cases ended in patient mortality. Since these oral manifestations can contribute to the diagnosis of systemic amyloidosis, clinicians should be made aware of them. Future research should assess the significance of oral manifestation as a prognostic indicator.

  10. Neuropathy and efficacy of once weekly subcutaneous bortezomib in multiple myeloma and light chain (AL) amyloidosis

    PubMed Central

    Sidana, Surbhi; Narkhede, Mayur; Elson, Paul; Hastings, Debbie; Faiman, Beth; Valent, Jason; Samaras, Christy; Hamilton, Kimberly; Liu, Hien K.; Smith, Mitchell R.; Reu, Frederic J.

    2017-01-01

    Introduction Randomized studies have shown that bortezomib (BTZ) can be given weekly via intravenous (IV) route or twice weekly via subcutaneous (SC) route with lower neuropathy risk and no loss of anti-myeloma efficacy compared to original standard IV twice weekly schedule. Weekly SC should therefore yield the best therapeutic index and is widely used but has not been compared to established administration schedules in the context of a clinical trial. Methods Comprehensive electronic medical record review was done for disease control and neuropathy symptoms of 344 consecutive patients who received their first BTZ-containing regimen for myeloma or AL amyloidosis before or after we changed to SC weekly in December 2010. Univariate and multivariable analyses were carried out that adjusted for age, underlying disease, concurrently used anticancer agents, underlying conditions predisposing to neuropathy, and number of prior regimens compared SC weekly to other schedules. Results Fifty-three patients received BTZ SC weekly, 17 SC twice weekly, 127 IV weekly and 147 IV twice weekly. Risk for neuropathy of any grade was higher with other schedules compared to SC weekly (44.3% vs. 26.9%, p = 0.001) while response rate was similar (72.1% vs. 76.6%, respectively, p = 0.15). Multivariable analyses upheld higher neuropathy risk (Odds ratio 2.45, 95% CI 1.26–4.76, p = 0.008) while the likelihood of not achieving a response (= partial response or better) was comparable (Odds ratio 1.25, 95% CI 0.58–2.71, p = 0.56) for other schedules compared to SC weekly, respectively. Lower neuropathy risk translated into longer treatment duration when BTZ was started SC weekly (p = 0.001). Conclusions Weekly SC BTZ has activity comparable to other schedules and causes low rates of neuropathy. PMID:28278302

  11. 2A4 binds soluble and insoluble light chain aggregates from AL amyloidosis patients and promotes clearance of amyloid deposits by phagocytosis †

    PubMed Central

    Renz, Mark; Torres, Ronald; Dolan, Philip J.; Tam, Stephen J.; Tapia, Jose R.; Li, Lauri; Salmans, Joshua R.; Barbour, Robin M.; Shughrue, Paul J.; Nijjar, Tarlochan; Schenk, Dale; Kinney, Gene G.; Zago, Wagner

    2016-01-01

    Abstract Amyloid light chain (AL) amyloidosis is characterized by misfolded light chain (LC) (amyloid) deposition in various peripheral organs, leading to progressive dysfunction and death. There are no regulatory agency–approved treatments for AL amyloidosis, and none of the available standard of care approaches directly targets the LC protein that constitutes the amyloid. NEOD001, currently in late-stage clinical trials, is a conformation-specific, anti-LC antibody designed to specifically target misfolded LC aggregates and promote phagocytic clearance of AL amyloid deposits. The present study demonstrated that the monoclonal antibody 2A4, the murine form of NEOD001, binds to patient-derived soluble and insoluble LC aggregates and induces phagocytic clearance of AL amyloid in vitro. 2A4 specifically labeled all 21 fresh-frozen organ samples studied, which were derived from 10 patients representing both κ and λ LC amyloidosis subtypes. 2A4 immunoreactivity largely overlapped with thioflavin T–positive labeling, and 2A4 bound both soluble and insoluble LC aggregates extracted from patient tissue. Finally, 2A4 induced macrophage engagement and phagocytic clearance of AL amyloid deposits in vitro. These findings provide further evidence that 2A4/NEOD001 can effectively clear and remove human AL-amyloid from tissue and further support the rationale for the evaluation of NEOD001 in patients with AL amyloidosis. PMID:27494229

  12. Nutritional status independently affects quality of life of patients with systemic immunoglobulin light-chain (AL) amyloidosis.

    PubMed

    Caccialanza, Riccardo; Palladini, Giovanni; Klersy, Catherine; Cereda, Emanuele; Bonardi, Chiara; Cameletti, Barbara; Montagna, Elisabetta; Russo, Paola; Foli, Andrea; Milani, Paolo; Lavatelli, Francesca; Merlini, Giampaolo

    2012-03-01

    Nutritional status is an independent prognostic factor in immunoglobulin light-chain amyloidosis (AL), but its influence on quality of life (QoL) is unknown. The aim of this cross-sectional study was to investigate the association between nutritional status and QoL in AL patients at diagnosis. One hundred and fifty consecutive patients with biopsy-proven AL were assessed for nutritional status by anthropometry [body mass index, unintentional weight loss (WL) in the previous 6 months and mid-arm muscle circumference (MAMC)], biochemistry (serum prealbumin), and semiquantitative food intake at referral. QoL was assessed by the Medical Outcomes Study 36-item Short Form General Health Survey. The composite physical component summary (PCS) and the mental component summary (MCS) for AL outpatients were 36.2 ± 10.1 and 44.9 ± 11.3, respectively (p < 0.001 for both vs the population norms of 50). In multivariate linear regression models adjusted for gender, age, Eastern Cooperative Oncology Group performance status, the number of organs involved, the severity of cardiac damage, C-reactive protein, energy intake, and WL, PCS was significantly lower for serum prealbumin <200 mg/L and MAMC <10th percentile (adjusted difference 3.8, 95% CI 0.18-7.5, p = 0.040 and 5.3, 95% CI 2.0-8.7, p = 0.002, respectively). MCS was decreased by 0.47 (95% CI 0.18-0.75, p = 0.002) for each kilogram of body weight lost in the previous 6 months. Nutritional status independently affects QoL in AL patients since diagnosis. Nutritional evaluation should be integral part of the clinical assessment of AL patients. Nutritional support intervention trials are warranted in such patients' population.

  13. Thermal Stability Threshold for Amyloid Formation in Light Chain Amyloidosis

    PubMed Central

    Poshusta, Tanya L.; Katoh, Nagaaki; Gertz, Morie A.; Dispenzieri, Angela; Ramirez-Alvarado, Marina

    2013-01-01

    Light chain (AL) amyloidosis is a devastating disease characterized by amyloid deposits formed by immunoglobulin light chains. Current available treatments involve conventional chemotherapy and autologous stem cell transplant. We have recently concluded a phase III trial comparing these two treatments. AL amyloidosis patients who achieve hematological complete response (CR) do not necessarily achieve organ response regardless of the treatment they received. In order to investigate the possible correlation between amyloid formation kinetics and organ response, we selected AL amyloidosis patients from the trial with kidney involvement and CR after treatment. Six patients were selected and their monoclonal immunoglobulin light chains were characterized. The proteins showed differences in their stability and their kinetics of amyloid formation. A correlation was detected at pH 7.4, showing that less stable proteins are more likely to form amyloid fibrils. AL-T03 is too unstable to form amyloid fibrils at pH 7.4. This protein was found in the only patient in the study that had organ response, suggesting that partially folded species are required for amyloid formation to occur in AL amyloidosis. PMID:24248061

  14. Update on treatment of light chain amyloidosis

    PubMed Central

    Mahmood, Shameem; Palladini, Giovanni; Sanchorawala, Vaishali; Wechalekar, Ashutosh

    2014-01-01

    Light chain amyloidosis is the most common type of amyloidosis as a consequence of protein misfolding of aggregates composed of amyloid fibrils. The clinical features are dependent on the organs involved, typically cardiac, renal, hepatic, peripheral and autonomic neuropathy and soft tissue. A tissue biopsy or fat aspirate is needed to confirm the presence/type of amyloid and prognostic tools are important in a risk stratified approach to treatment. Autologous stem cell transplant eligibility should be assessed at baseline, weighing the reversible or non-reversible contraindications, toxicity of treatment and chemotherapy alternatives available. Chemotherapy options include melphalan, thalidomide, bortezomib, lenalidomide, bendamustine in combination with dexamethasone. Many studies have explored these treatment modalities, with ongoing debate about the optimal first line and sequential treatment thereafter. Attaining a very good partial response or better is the treatment goal coupled with early assessment central to optimizing treatment. One major challenge remains increasing the awareness of this disease, frequently diagnosed late as the presenting symptoms mimic many other medical conditions. This review focuses on the treatments for light chain amyloidosis, how these treatments have evolved over the years, improved patient risk stratification, toxicities encountered and future directions. PMID:24497558

  15. AL Amyloidosis

    PubMed Central

    2012-01-01

    Definition of the disease AL amyloidosis results from extra-cellular deposition of fibril-forming monoclonal immunoglobulin (Ig) light chains (LC) (most commonly of lambda isotype) usually secreted by a small plasma cell clone. Most patients have evidence of isolated monoclonal gammopathy or smoldering myeloma, and the occurrence of AL amyloidosis in patients with symptomatic multiple myeloma or other B-cell lymphoproliferative disorders is unusual. The key event in the development of AL amyloidosis is the change in the secondary or tertiary structure of an abnormal monoclonal LC, which results in instable conformation. This conformational change is responsible for abnormal folding of the LC, rich in β leaves, which assemble into monomers that stack together to form amyloid fibrils. Epidemiology AL amyloidosis is the most common type of systemic amyloidois in developed countries with an estimated incidence of 9 cases/million inhabitant/year. The average age of diagnosed patients is 65 years and less than 10% of patients are under 50. Clinical description The clinical presentation is protean, because of the wide number of tissues or organs that may be affected. The most common presenting symptoms are asthenia and dyspnoea, which are poorly specific and may account for delayed diagnosis. Renal manifestations are the most frequent, affecting two thirds of patients at presentation. They are characterized by heavy proteinuria, with nephrotic syndrome and impaired renal function in half of the patients. Heart involvement, which is present at diagnosis in more than 50% of patients, leading to restrictive cardiopathy, is the most serious complication and engages prognosis. Diagnostic methods The diagnosis relies on pathological examination of an involved site showing Congo red-positive amyloid deposits, with typical apple-green birefringence under polarized light, that stain positive with an anti-LC antibody by immunohistochemistry and/or immunofluorescence. Due to the

  16. Clinical and imaging predictors of 1-year and long-term mortality in light chain (AL) amyloidosis: a 5-year follow-up study.

    PubMed

    Migrino, Raymond Q; Harmann, Leanne; Christenson, Richard; Hari, Parameswaran

    2014-11-01

    Light chain amyloidosis (AL) involves multiorgan failure induced by amyloidogenic light chain proteins, and is associated with high mortality. We aimed to identify clinical, laboratory, and imaging parameters that would predict 1-year and long-term AL mortality. Forty-four biopsy-proven AL patients (61.5 ± 12 years, 20 females) underwent clinical evaluation including laboratory assays, echocardiography, and contrast cardiac magnetic resonance imaging (CMR, n = 31) prior to chemotherapy. Patients were prospectively followed for median duration of 62.7 months (interquartile range 35.5 months). Clinical and laboratory parameters were compared between 1-year survivors and nonsurvivors. Univariate Kaplan-Meier survival plots were calculated followed by stepwise logistic regression analysis to assess independent predictors of long-term survival. Eighteen (40.9 %) patients died within 1 year and an additional 10 subjects died during long-term follow-up. Patients who expired within 1 year presented with more advanced class of heart failure, higher alkaline phosphatase and uric acid, lower limb lead voltage on electrocardiography, shorter left ventricular ejection time (ET) on echocardiography, and a higher proportion of late gadolinium enhancement on CMR. On multivariable analysis, only ET ≤240 ms on echocardiography (hazard ratio (HR) 5.07, 95 % confidence interval (CI) 1.83-14.1, P = 0.002) and New York Heart Association functional class II-IV presentation (HR 1.0058, 95 % CI 1.0014-1.0103, P = 0.01) were independent predictors of AL mortality. In conclusion, AL amyloidosis is associated with high 1-year and long-term mortality. Among clinical, laboratory, and imaging parameters tested, an echocardiographic finding of ET ≤240 ms has independent and additive prognostic value to clinical heart failure evaluation in determining long-term survival of AL patients. This result may be important in the early identification of patients at risk.

  17. Apolipoprotein C-II Deposition Amyloidosis: A Potential Misdiagnosis as Light Chain Amyloidosis

    PubMed Central

    Schuiteman, Emily; Zarouk, Sami

    2016-01-01

    Hereditary amyloidoses are rare and pose a diagnostic challenge. We report a case of hereditary amyloidosis associated with apolipoprotein C-II deposition in a 61-year-old female presenting with renal failure and nephrotic syndrome misdiagnosed as light chain amyloidosis. Renal biopsy was consistent with amyloidosis on microscopy; however, immunofluorescence was inconclusive for the type of amyloid protein. Monoclonal gammopathy evaluation revealed kappa light chain. Bone marrow biopsy revealed minimal involvement with amyloidosis with kappa monotypic plasma cells on flow cytometry. She was started on chemotherapy for light chain amyloidosis. She was referred to the Mayo clinic where laser microdissection and liquid chromatography mass spectrometry detected high levels of apolipoprotein C-II, making a definitive diagnosis. Apolipoprotein C-II is a component of very low-density lipoprotein and aggregates in lipid-free conditions to form amyloid fibrils. The identification of apolipoprotein C-II as the cause of amyloidosis cannot be solely made with routine microscopy or immunofluorescence. Further evaluation of biopsy specimens with laser microdissection and mass spectrometry and DNA sequencing of exons should be done routinely in patients with amyloidoses for definitive diagnosis. Our case highlights the importance of determining the subtype of amyloidosis that is critical for avoiding unnecessary therapy such as chemotherapy. PMID:27840752

  18. Long-term outcomes of primary systemic light chain (AL) amyloidosis in patients treated upfront with bortezomib or lenalidomide and the importance of risk adapted strategies.

    PubMed

    Kastritis, Efstathios; Roussou, Maria; Gavriatopoulou, Maria; Migkou, Magdalini; Kalapanida, Despina; Pamboucas, Constantinos; Kaldara, Elisavet; Ntalianis, Argyrios; Psimenou, Erasmia; Toumanidis, Savvas T; Tasidou, Anna; Terpos, Evangelos; Dimopoulos, Meletios A

    2015-04-01

    Bortezomib and lenalidomide are increasingly used in patients with AL amyloidosis, but long term data on their use as primary therapy in AL amyloidosis are lacking while early mortality remains significant. Thus, we analyzed the long term outcomes of 85 consecutive unselected patients, which received primary therapy with bortezomib or lenalidomide and we prospectively evaluated a risk adapted strategy based on bortezomib/dexamethasone to reduce early mortality. Twenty-six patients received full-dose bortezomib/dexamethasone, 36 patients lenalidomide with oral cyclophosphamide and low-dose dexamethasone and 23 patients received bortezomib/dexamethasone at a dose and schedule adjusted to the risk of early death. On intent to treat, 67% of patients achieved a hematologic response (24% hemCRs) and 34% an organ response; both were more frequent with bortezomib. An early death occurred in 20%: in 36% of those treated with full-dose bortezomib/dexamethasone, in 22% of lenalidomide-treated patients but only in 4.5% of patients treated with risk-adapted bortezomib/dexamethasone. Activity of full vs. adjusted dose bortezomib/dexamethasone was similar; twice weekly vs. weekly administration of bortezomib also had similar activity. After a median follow up of 57 months, median survival is 47 months and is similar for patients treated with bortezomib vs. lenalidomide-based regimens. However, risk adjusted-bortezomib/dexamethasone was associated with improved 1-year survival vs. full-dose bortezomib/dexamethasone or lenalidomide-based therapy (81% vs. 56% vs. 53%, respectively). In conclusion, risk-adapted bortezomib/dexamethasone may reduce early mortality and preserve activity while long term follow up indicates that remissions obtained with lenalidomide or bortezomib may be durable, even without consolidation with alkylators.

  19. Phase 2 trial of daily, oral epigallocatechin gallate in patients with light-chain amyloidosis.

    PubMed

    Meshitsuka, Sohsuke; Shingaki, Sumito; Hotta, Masatoshi; Goto, Miku; Kobayashi, Makoto; Ukawa, Yuuichi; Sagesaka, Yuko M; Wada, Yasuyo; Nojima, Masanori; Suzuki, Kenshi

    2017-03-01

    Previous studies have suggested that an increase in mitochondrial reactive oxygen species may cause organ damage in patients with light-chain (AL) amyloidosis; however, this damage can be decreased by antioxidant-agent treatment. Epigallocatechin gallate (EGCG), the major natural catechin in green tea, has potent antioxidant activity. Because EGCG has recently been reported to have a favorable toxicity profile for treating amyloidosis, we sought to examine the clinical efficacy and toxicity of EGCG in patients with AL amyloidosis. Fifty-seven patients were randomly assigned to the EGCG and observation groups and observed for six months. There were no increases in grade 3-5 adverse events and EGCG therapy was well tolerated. Although a decrease in the urinary albumin level was found in the EGCG group in patients with obvious albuminuria after treatment initiation, its antioxidant activity may not be sufficient to clarify the potential effect of EGCG in patients with AL amyloidosis. Because some of the biological markers responsible for organ damage were well correlated to the level of antioxidant potential in patients' plasma, the status of oxidative stress in the blood may indicate the extent of organ damage in clinical situations.

  20. Chronic myopathy due to immunoglobulin light chain amyloidosis

    PubMed Central

    Manoli, Irini; Kwan, Justin Y.; Wang, Qian; Rushing, Elisabeth J.; Tsokos, Maria; Arai, Andrew E.; Burch, Warner M.; Dispenzieri, Angela; McPherron, Alexandra C.; Gahl, William A.

    2013-01-01

    Amyloid myopathy associated with a plasma cell dyscrasia is a rare cause of muscle hypertrophy. It can be a challenging diagnosis, since pathological findings are often elusive. In addition, the mechanism by which immunoglobulin light-chain deposition stimulates muscle overgrowth remains poorly understood. We present a 53–year old female with a 10-year history of progressive generalized muscle overgrowth. Congo-red staining and immunohistochemistry revealed perivascular lambda light chain amyloid deposits, apparent only in a second muscle biopsy. The numbers of central nuclei and satellite cells were increased, suggesting enhanced muscle progenitor cell formation. Despite the chronicity of the light chain disease, the patient showed complete resolution of hematologic findings and significant improvement of her muscle symptoms following autologous bone marrow transplantation. This case highlights the importance of early diagnosis and therapy for this treatable cause of a chronic myopathy with muscle hypertrophy. PMID:23465863

  1. AL Amyloidosis and Agent Orange

    MedlinePlus

    ... for survivors' benefits . Research on AL amyloidosis and herbicides The Health and Medicine Division (formally known as ... to the compounds of interest found in the herbicide Agent Orange and AL amyloidosis." VA made a ...

  2. The prognostic value of diagnosing concurrent multiple myeloma in immunoglobulin light chain amyloidosis.

    PubMed

    Dinner, Shira; Witteles, Wesley; Witteles, Ronald; Lam, Anthony; Arai, Sally; Lafayette, Richard; George, Tracy I; Schrier, Stanley L; Liedtke, Michaela

    2013-05-01

    The prevalence and prognostic value of a concomitant diagnosis of symptomatic or asymptomatic multiple myeloma (MM), as defined by the current International Myeloma Working Group (IMWG) criteria, in patients with immunoglobulin light chain amyloidosis (AL), are unknown. We studied 46 consecutive patients with AL who underwent quantification of serum M-protein and clonal bone marrow plasma cells, as well as a comprehensive evaluation for end organ damage by MM. Using standard morphology and CD138 immunohistochemical staining, 57% and 80% of patients were found to have concomitant MM, respectively. Nine patients exhibited end organ damage consistent with a diagnosis of symptomatic MM. While overall survival was similar between AL patients with or without concurrent myeloma (1-year overall survival 68% vs. 87%; P = 0.27), a diagnosis of symptomatic myeloma was associated with inferior outcome (1-year overall survival 39% vs. 81%; P = 0.005). Quantification of bone marrow plasma cells by both standard morphology and CD138 immunohistochemistry identified a much higher prevalence of concurrent MM in patients with AL than previously reported. Evaluation of bone marrow plasma cell infiltration and presence of myeloma associated end organ damage could be clinically useful for prognostication of patients with AL.

  3. T1 mapping and survival in systemic light-chain amyloidosis

    PubMed Central

    Banypersad, Sanjay M.; Fontana, Marianna; Maestrini, Viviana; Sado, Daniel M.; Captur, Gabriella; Petrie, Aviva; Piechnik, Stefan K.; Whelan, Carol J.; Herrey, Anna S.; Gillmore, Julian D.; Lachmann, Helen J.; Wechalekar, Ashutosh D.; Hawkins, Philip N.; Moon, James C.

    2015-01-01

    Aims To assess the prognostic value of myocardial pre-contrast T1 and extracellular volume (ECV) in systemic amyloid light-chain (AL) amyloidosis using cardiovascular magnetic resonance (CMR) T1 mapping. Methods and results One hundred patients underwent CMR and T1 mapping pre- and post-contrast. Myocardial ECV was calculated at contrast equilibrium (ECVi) and 15 min post-bolus (ECVb). Fifty-four healthy volunteers served as controls. Patients were followed up for a median duration of 23 months and survival analyses were performed. Mean ECVi was raised in amyloid (0.44 ± 0.12) as was ECVb (mean 0.44 ± 0.12) compared with healthy volunteers (0.25 ± 0.02), P < 0.001. Native pre-contrast T1 was raised in amyloid (mean 1080 ± 87 ms vs. 954 ± 34 ms, P < 0.001). All three correlated with pre-test probability of cardiac involvement, cardiac biomarkers, and systolic and diastolic dysfunction. During follow-up, 25 deaths occurred. An ECVi of >0.45 carried a hazard ratio (HR) for death of 3.84 [95% confidence interval (CI): 1.53–9.61], P = 0.004 and pre-contrast T1 of >1044 ms = HR 5.39 (95% CI: 1.24–23.4), P = 0.02. Extracellular volume after primed infusion and ECVb performed similarly. Isolated post-contrast T1 was non-predictive. In Cox regression models, ECVi was independently predictive of mortality (HR = 4.41, 95% CI: 1.35–14.4) after adjusting for E:E′, ejection fraction, diastolic dysfunction grade, and NT-proBNP. Conclusion Myocardial ECV (bolus or infusion technique) and pre-contrast T1 are biomarkers for cardiac AL amyloid and they predict mortality in systemic amyloidosis. PMID:25411195

  4. In situ characterization of protein aggregates in human tissues affected by light chain amyloidosis: a FTIR microspectroscopy study

    PubMed Central

    Ami, Diletta; Lavatelli, Francesca; Rognoni, Paola; Palladini, Giovanni; Raimondi, Sara; Giorgetti, Sofia; Monti, Luca; Doglia, Silvia Maria; Natalello, Antonino; Merlini, Giampaolo

    2016-01-01

    Light chain (AL) amyloidosis, caused by deposition of amyloidogenic immunoglobulin light chains (LCs), is the most common systemic form in industrialized countries. Still open questions, and premises for developing targeted therapies, concern the mechanisms of amyloid formation in vivo and the bases of organ targeting and dysfunction. Investigating amyloid material in its natural environment is crucial to obtain new insights on the molecular features of fibrillar deposits at individual level. To this aim, we used Fourier transform infrared (FTIR) microspectroscopy for studying in situ unfixed tissues (heart and subcutaneous abdominal fat) from patients affected by AL amyloidosis. We compared the infrared response of affected tissues with that of ex vivo and in vitro fibrils obtained from the pathogenic LC derived from one patient, as well as with that of non amyloid-affected tissues. We demonstrated that the IR marker band of intermolecular β-sheets, typical of protein aggregates, can be detected in situ in LC amyloid-affected tissues, and that FTIR microspectroscopy allows exploring the inter- and intra-sample heterogeneity. We extended the infrared analysis to the characterization of other biomolecules embedded within the amyloid deposits, finding an IR pattern that discloses a possible role of lipids, collagen and glycosaminoglycans in amyloid deposition in vivo. PMID:27373200

  5. Deposition of kappa and lambda light chains in amyloid filaments of dialysis-related amyloidosis.

    PubMed

    Brancaccio, D; Ghiggeri, G M; Braidotti, P; Garberi, A; Gallieni, M; Bellotti, V; Zoni, U; Gusmano, R; Coggi, G

    1995-10-01

    beta 2-Microglobulin (beta 2m) is considered to be the amyloidogenic precursor in dialysis-related amyloidosis, although the implication of other relevant cofactors in the pathogenesis of this disease has also been hypothesized. It is conceivable that substances found in amyloid deposits might represent something more than simple codeposition, possibly playing a pathogenic role in amyloidogenesis. Along these lines, a detailed analysis of the protein composition of amyloid fibrils purified from synovial material surgically obtained from nine patients on long-term dialysis was carried out. By the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, several other protein components, in addition to beta 2m, were found. These were characterized by NH2 amino-terminal sequencing and immunoblotting. In fibrils obtained by water extraction, which fulfill the electron microscopy criteria of highly pure amyloid material, polyclonal kappa and lambda light chains were detected with a concentration of 15 micrograms/mL in the water extraction material; the beta 2m concentration was 200 micrograms/mL. Light microscopy immunohistochemistry was performed on samples from five patients. Amyloid deposits reacted with anti-beta 2m, and anti-light (kappa, lambda), chain antibodies. The immunoreaction of amyloid filaments to anti-beta 2m, anti-lambda, and anti-kappa light chain antibodies was also tested by electron microscopy by use of the immunogold staining procedure. Amyloid filaments were labeled by the three antibodies and showed a different intensity of immunostaining apparently related to their different aggregation pattern. These observations demonstrate that polyclonal immunoglobulin light chains (kappa and lambda) are not contaminants but, together with beta 2m, represent a major constituent of amyloid deposits in dialysis-related osteoarticular amyloidosis, thus indicating their possible role in amyloidogenesis.

  6. Sequential cyclophosphamide-bortezomib-dexamethasone unmasks the harmful cardiac effect of dexamethasone in primary light-chain cardiac amyloidosis.

    PubMed

    Le Bras, Fabien; Molinier-Frenkel, Valerie; Guellich, Aziz; Dupuis, Jehan; Belhadj, Karim; Guendouz, Soulef; Ayad, Karima; Colombat, Magali; Benhaiem, Nicole; Tissot, Claire Marie; Hulin, Anne; Jaccard, Arnaud; Damy, Thibaud

    2017-03-20

    Chemotherapy combining cyclophosphamide, bortezomib and dexamethasone is widely used in light-chain amyloidosis. The benefit is limited in patients with cardiac amyloidosis mainly because of adverse cardiac events. Retrospective analysis of our cohort showed that 39 patients died with 42% during the first month. A new escalation-sequential regimen was set to improve the outcomes. Nine newly-diagnosed patients were prospectively treated with close monitoring of serum N-terminal pro-brain natriuretic peptide, troponin-T and free light chains. The results show that corticoids may destabilise the heart through fluid retention. Thus, a sequential protocol may be a promising approach to treat these patients.

  7. Recurrent mutations of MAPK pathway genes in multiple myeloma but not in amyloid light-chain amyloidosis

    PubMed Central

    Kim, Nayoung K.D.; Yun, Jae Won; Hwang, Jee Hyang; Kim, Kihyun; Park, Woong-Yang

    2016-01-01

    Clinically applicable platforms revealing actionable genomic alterations may improve the treatment efficacy of myeloma patients. In this pilot study, we used a high depth targeted sequencing panel containing 83 anti-cancer drug target genes to sequence genomic DNAs extracted from bone marrow aspirates of 23 patients with myeloma and 12 patients with amyloid light-chain (AL) amyloidosis. Mutation analysis revealed NRAS as the most commonly mutated gene (30%, 7/23) in myeloma patients followed by KRAS (26%, 6/23) and BRAF (22%, 5/23). However, no significant mutations were found in the 12 patients with AL amyloidosis. Notably, 6 of the 23 myeloma patients showed multi-site and/or multi-gene mutations in NRAS, KRAS, or BRAF, indicating compound aberrations in the Mitogen activated protein kinase (MAPK) pathway. Gene panel sequencing also revealed cytogenetic abnormalities associated with prognosis in myeloma patients. In conclusion, our pilot study suggests that targeted gene sequencing may have an important prognostic value for myeloma patients for the identification of actionable genomic alterations and cytogenetic aberrations. PMID:27634910

  8. Solid-state NMR chemical shift assignments for AL-09 VL immunoglobulin light chain fibrils.

    PubMed

    Piehl, Dennis W; Blancas-Mejía, Luis M; Ramirez-Alvarado, Marina; Rienstra, Chad M

    2017-04-01

    Light chain (AL) amyloidosis is a systemic disease characterized by the formation of immunoglobulin light-chain fibrils in critical organs of the body. The light-chain protein AL-09 presents one severe case of cardiac AL amyloidosis, which contains seven mutations in the variable domain (VL) relative to its germline counterpart, κI O18/O8 VL. Three of these mutations are non-conservative-Y87H, N34I, and K42Q-and previous work has shown that they are responsible for significantly reducing the protein's thermodynamic stability, allowing fibril formation to occur with fast kinetics and across a wide-range of pH conditions. Currently, however, there is extremely limited structural information available which explicitly describes the residues that are involved in supporting the misfolded fibril structure. Here, we assign the site-specific (15)N and (13)C chemical shifts of the rigid residues of AL-09 VL fibrils by solid-state NMR, reporting on the regions of the protein involved in the fibril as well as the extent of secondary structure.

  9. Extracorporeal Membrane Oxygenation as Bridge-to-Decision in Acute Heart Failure due to Systemic Light-Chain Amyloidosis

    PubMed Central

    Silva, Jennifer Mancio; Fontes-Carvalho, Ricardo; Valente, Dília; Almeida, Cristiana; Cruz, António José; Tente, David; Coelho, Henrique; Oliveira, Marco; Albuquerque, Aníbal; Ribeiro, Vasco Gama

    2015-01-01

    Patient: Female, 58 Final Diagnosis: Acute hear failure Symptoms: Dispnoea • edema • fatigue Medication: — Clinical Procedure: Bone marrow biopsy • endomyocardial biopsy • abdominal subcutaneous fat biopsy under ECMO support Specialty: Cardiology Objective: Rare disease Background: Cardiac amyloidosis results from the amyloid deposition in heart tissue, either in the context of a systemic disease or as a localized form. Several pro-amyloid proteins can produce amyloid deposits in the heart. Each of these amyloidoses has characteristic clinical (cardiac and extracardiac) features, and a specific diagnosis and treatment. Case Report: A 58-year-old woman who presented with acute heart failure and echocardiographic findings strongly suggestive of infiltrative cardiomyopathy needed percutaneous veno-arterial extracorporeal membrane oxygenation (ECMO) as bridge-to-decision. Amyloid deposition was found on endomyocardial and bone marrow biopsies. Bone marrow plasma cell infiltrate with acute renal lesion and hypercalcemia confirmed the diagnosis of multiple myeloma-associated systemic light-chain amyloidosis (AL). Refractory shock with multi-organic failure syndrome persisted and no improvements in left ventricular function and structure were seen. After extensive discussion by a multidisciplinary team, and with the patients’ family, she was not considered eligible for high-dose chemotherapy and/or autologous stem cell transplantation, heart transplantation, or sequential heart with autologous stem cell transplantation. The patient died a few hours after ECMO withdrawal. During the 14 days of ECMO support no major bleeding or thrombotic complications occurred. Conclusions: The clinician must consider a diagnosis of cardiac amyloidosis in patients with heart failure, a restrictive type of cardiomyopathy with ventricular hypertrophy in the absence of valve abnormalities, or uncontrolled arterial hypertension. Although developments in chemotherapy have greatly

  10. Clinical Presentation of Tubulointerstitial Nephritis Caused by Amyloid Light-chain Amyloidosis in a Patient with Sjögren's Syndrome

    PubMed Central

    Inoue, Reiko; Fujigaki, Yoshihide; Kobayashi, Kana; Tamura, Yoshifuru; Ota, Tatsuru; Shibata, Shigeru; Ishida, Tsuyoshi; Kondo, Fukuo; Yamaguchi, Yutaka; Uchida, Shunya

    2017-01-01

    We report a 70-year-old woman with Sjögren's syndrome who had severe renal dysfunction with mild proteinuria and elevated urinary low-molecular-weight proteins. Based on these clinical presentations, interstitial nephritis due to Sjögren's syndrome was strongly suspected. Unexpectedly, renal pathology revealed amyloid light-chain (AL) lambda-type depositions predominantly in the vasculatures with severe tubulointerstitial damage. Concentrated urine immunofixation was positive for Bence Jones lambda-type monoclonal proteins. Given the involvement in other organs, systemic AL amyloidosis was diagnosed. The patient underwent chemotherapy, but hemodialysis was ultimately instituted. It should be remembered that renal amyloidosis occurs as a clinical presentation of interstitial nephritis. PMID:28202864

  11. Stabilisation of Laryngeal AL Amyloidosis with Long Term Curcumin Therapy

    PubMed Central

    Golombick, Terry; Diamond, Terrence H.; Manoharan, Arumugam; Ramakrishna, Rajeev

    2015-01-01

    Multiple myeloma (MM), smoldering myeloma (SMM), and monoclonal gammopathy of undetermined significance (MGUS) represent a spectrum of plasma cell dyscrasias (PCDs). Immunoglobulin light chain amyloidosis (AL) falls within the spectrum of these diseases and has a mortality rate of more than 80% within 2 years of diagnosis. Curcumin, derived from turmeric, has been shown to have a clinical benefit in some patients with PCDs. In addition to a clinical benefit in these patients, curcumin has been found to have a strong affinity for fibrillar amyloid proteins. We thus administered curcumin to a patient with laryngeal amyloidosis and smoldering myeloma and found that the patient has shown a lack of progression of his disease for a period of five years. This is in keeping with our previous findings of clinical benefits of curcumin in patients with plasma cell dyscrasias. We recommend further evaluation of curcumin in patients with primary AL amyloidosis. PMID:26199769

  12. Treatment Patterns and Outcome Following Initial Relapse or Refractory Disease in Patients with Systemic Light Chain Amyloidosis.

    PubMed

    Tandon, Nidhi; Sidana, Surbhi; Gertz, Morie A; Dispenzieri, Angela; Lacy, Martha Q; Buadi, Francis K; Dingli, David; Fonder, Amie L; Hobbs, Miriam A; Hayman, Suzanne R; Gonsalves, Wilson I; Hwa, Yi Lisa; Kapoor, Prashant; Kyle, Robert A; Leung, Nelson; Go, Ronald S; Lust, John A; Russell, Stephen J; Zeldenrust, Steven R; Rajkumar, S Vincent; Kumar, Shaji K

    2017-03-17

    We analyzed the outcomes following initial relapse or refractory disease in systemic light chain amyloidosis (AL) and the impact of type of therapy employed.A total of 1327 patients with AL seen at Mayo Clinic within 90 days of diagnosis, between 2006 and 2015, were reviewed. The study included 366 patients experiencing a documented hematological or organ relapse or refractory disease requiring start of second line therapy. Overall survival (OS) and time to next treatment (TTNT) were calculated from start of second line treatment.The median time to require second line treatment was 16.2 months (1-93) from the start of first line therapy. At relapse, patients received proteasome inhibitors (PI; 45.1%), immunomodulators (IMiD; 22.7%), alkylators (9%), PI and IMiD combination (4.1%), autologous transplant (3.8%), steroids and other therapies (4.9%). Among these, 124 (33.9%) required change or reinstitution of therapy. The median time to require third line treatment was 31 months (95% CI; 24, 40.5) and the median overall survival (OS) was 38.8 months (95% CI; 29.6, 52.6) from the start of second line treatment. Retreatment with same therapy at relapse significantly reduced TTNT (22m vs 32.3m; p= 0.01) as compared to different therapy; but did not have any impact OS (30.8m vs 51.1m; p = 0.5). In conclusion, this study provides important information about outcomes of patients with AL who require second line treatment for relapsed/refractory disease . Treatment with a different therapy at relapse improves time to next therapy but does not impact OS. This article is protected by copyright. All rights reserved.

  13. The Coexistence of Multiple Myeloma-associated Amyloid Light-chain Amyloidosis and Fabry Disease in a Hemodialysis Patient.

    PubMed

    Taguchi, Kensei; Moriyama, Atsuo; Kodama, Goh; Nakayama, Yosuke; Fukami, Kei

    2017-01-01

    Fabry disease (FD) is an inherited lysosomal disorder caused by an X-linked α-galactosidase A deficiency. We report the case of a 50-year-old male FD patient on hemodialysis who presented with macroglossia-related speaking difficulty and gastrointestinal symptoms. An endoscopic analysis revealed multiple gastric ulcers, and a histological examination led to a diagnosis of amyloid light-chain amyloidosis. Serum free light-chain and bone marrow analyses detected multiple myeloma (MM). Treatment with bortezomib and dexamethasone significantly improved the patient's symptoms. This is the first case to demonstrate a potential pathogenic relationship between FD and MM. The similar gastrointestinal manifestations might have contributed to the diagnostic difficulty.

  14. Long-term event-free and overall survival after risk-adapted melphalan and SCT for systemic light chain amyloidosis

    PubMed Central

    Landau, H; Smith, M; Landry, C; Chou, J F; Devlin, S M; Hassoun, H; Bello, C; Giralt, S; Comenzo, R L

    2017-01-01

    Stem cell transplantation (SCT), an effective therapy for amyloid light chain (AL) amyloidosis patients, is associated with low treatment-related mortality (TRM) with appropriate patient selection and risk-adapted dosing of melphalan (RA-SCT). Consolidation after SCT increases hematologic complete response (CR) rates and may improve overall survival (OS) for patients with AL amyloidosis. PMID:27560108

  15. Autoimmune disease leading to pulmonary AL amyloidosis and pulmonary hypertension

    PubMed Central

    Ellender, Claire M; McLean, Catriona; Williams, Trevor J; Snell, Gregory I; Whitford, Helen M

    2015-01-01

    A 33-year-old woman with past history of Sjögren's syndrome and systemic lupus erythematosus presented with dyspnea and syncope secondary to pulmonary hypertension. After progressive symptoms over 4 years, she received bilateral lung transplantation. Histopathology of the explanted lungs showed isolated pulmonary amyloid light-chain amyloidosis and pulmonary cysts. No evidence of systemic amyloidosis was found at the time of transplantation. Seven years post lung transplantation, she remains well with no evidence of systemic amyloidosis recurrence. PMID:26090118

  16. Life-saving implantable cardioverter defibrillator therapy in cardiac AL amyloidosis

    PubMed Central

    Patel, Ketna S; Hawkins, Philip N; Whelan, Carol J; Gillmore, Julian D

    2014-01-01

    Cardiac involvement is the main determinant of prognosis in systemic monoclonal immunoglobulin light chain (AL) amyloidosis. Ventricular arrhythmias and sudden cardiac death are not uncommon. The electrical events that precede sudden death, and their potential to be treated effectively, remain undefined. There are no European guidelines for the use of implantable cardioverter defibrillator (ICD) in amyloidosis. ICDs in general are not usually offered to patients with a life expectancy of less than 1 year. We describe a patient who presented with cardiac AL amyloidosis who underwent prophylactic ICD implantation for the prevention of sudden cardiac death during treatment with chemotherapy, in whom life-threatening ventricular arrhythmia was successfully terminated over a 3-year period. PMID:25535224

  17. Improved Outcomes After Autologous Hematopoietic Cell Transplantation for Light Chain Amyloidosis: A Center for International Blood and Marrow Transplant Research Study

    PubMed Central

    D'Souza, Anita; Dispenzieri, Angela; Wirk, Baldeep; Zhang, Mei-Jie; Huang, Jiaxing; Gertz, Morie A.; Kyle, Robert A.; Kumar, Shaji; Comenzo, Raymond L.; Peter Gale, Robert; Lazarus, Hillard M.; Savani, Bipin N.; Cornell, Robert F.; Weiss, Brendan M.; Vogl, Dan T.; Freytes, César O.; Scott, Emma C.; Landau, Heather J.; Moreb, Jan S.; Costa, Luciano J.; Ramanathan, Muthalagu; Callander, Natalie S.; Kamble, Rammurti T.; Olsson, Richard F.; Ganguly, Siddhartha; Nishihori, Taiga; Kindwall-Keller, Tamila L.; Wood, William A.; Mark, Tomer M.; Hari, Parameswaran

    2015-01-01

    Purpose Autologous hematopoietic cell transplantation, or autotransplantation, is effective in light-chain amyloidosis (AL), but it is associated with a high risk of early mortality (EM). In a multicenter randomized comparison against oral chemotherapy, autotransplantation was associated with 24% EM. We analyzed trends in outcomes after autologous hematopoietic cell transplantation for AL in North America. Patients and Methods Between 1995 and 2012, 1,536 patients with AL who underwent autotransplantation at 134 centers were identified in the Center for International Blood and Marrow Transplant Research database. EM and overall survival (OS) were analyzed in three time cohorts: 1995 to 2000 (n = 140), 2001 to 2006 (n = 596), and 2007 to 2012 (n = 800). Hematologic and renal responses and factors associated with EM, relapse and/or progression, progression-free survival and OS were analyzed in more recent subgroups from 2001 to 2006 (n = 197) and from 2007 to 2012 (n = 157). Results Mortality at 30 and 100 days progressively declined over successive time periods from 11% and 20%, respectively, in 1995 to 2000 to 5% and 11%, respectively, in 2001 to 2006, and to 3% and 5%, respectively, in 2007 to 2012. Correspondingly, 5-year OS improved from 55% in 1995 to 2000 to 61% in 2001 to 2006 and to 77% in 2007 to 2012. Hematologic response to transplantation improved in the latest cohort. Renal response rate was 32%. Centers performing more than four AL transplantations per year had superior survival outcomes. In the multivariable analysis, cardiac AL was associated with high EM and inferior progression-free survival and OS. Autotransplantation in 2007 to 2012 and use of higher dosages of melphalan were associated with a lowered relapse risk. A Karnofsky score less than 80 and creatinine levels 2 mg/m2 or greater were associated with worsened OS. Conclusion Post-transplantation survival in AL has improved, with a dramatic reduction in early post-transplantation mortality

  18. Amyloidosis

    MedlinePlus

    ... your tissues as amyloid, interfering with normal function. AA amyloidosis mostly affects your kidneys but occasionally your ... infectious or inflammatory disease increases your risk of AA amyloidosis. Family history. Some types of amyloidosis are ...

  19. Development of Renal Failure without Proteinuria in a Patient with Monoclonal Gammopathy of Undetermined Significance: An Unusual Presentation of AL Kappa Amyloidosis.

    PubMed

    Sun, Yijuan; Sandhu, Amarpreet; Gabaldon, Darlene; Danaraj, Jonathan; Servilla, Karen S; Tzamaloukas, Antonios H

    2012-01-01

    AL amyloidosis complicating monoclonal gammopathy of undetermined significance (MGUS) has usually a predominant glomerular deposition of lambda light chain. Heavy proteinuria is one of its cardinal manifestations. A 78-year-old man with a 9-year history of IgG kappa light-chain-MGUS and normal urine protein excretion developed severe renal failure. Serum levels of kappa light chain and serum IgG had been stable while proteinuria was absent throughout the nine-year period. For the first eight years, he had stable stage III chronic kidney disease attributed to bladder outlet obstruction secondary to prostatic malignancy. In the last year, he developed progressive serum creatinine elevation, without any increase in the serum or urine levels of paraproteins or any sign of malignancy. Renal ultrasound and furosemide renogram showed no evidence of urinary obstruction. Renal biopsy revealed AL amyloidosis, with reactivity exclusive for kappa light chains, affecting predominantly the vessels and the interstitium. Glomerular involvement was minimal. Melphalan and prednisone were initiated. However, renal function continues deteriorating. Deposition of AL kappa amyloidosis developing during the course of MGUS predominantly in the wall of the renal vessels and the renal interstitium, while the involvement of the glomeruli is minimal, leads to progressive renal failure and absence of proteinuria. Renal biopsy is required to detect both the presence and the sites of deposition of renal AL kappa light chain amyloidosis.

  20. Development of Renal Failure without Proteinuria in a Patient with Monoclonal Gammopathy of Undetermined Significance: An Unusual Presentation of AL Kappa Amyloidosis

    PubMed Central

    Sun, Yijuan; Sandhu, Amarpreet; Gabaldon, Darlene; Danaraj, Jonathan; Servilla, Karen S.; Tzamaloukas, Antonios H.

    2012-01-01

    AL amyloidosis complicating monoclonal gammopathy of undetermined significance (MGUS) has usually a predominant glomerular deposition of lambda light chain. Heavy proteinuria is one of its cardinal manifestations. A 78-year-old man with a 9-year history of IgG kappa light-chain-MGUS and normal urine protein excretion developed severe renal failure. Serum levels of kappa light chain and serum IgG had been stable while proteinuria was absent throughout the nine-year period. For the first eight years, he had stable stage III chronic kidney disease attributed to bladder outlet obstruction secondary to prostatic malignancy. In the last year, he developed progressive serum creatinine elevation, without any increase in the serum or urine levels of paraproteins or any sign of malignancy. Renal ultrasound and furosemide renogram showed no evidence of urinary obstruction. Renal biopsy revealed AL amyloidosis, with reactivity exclusive for kappa light chains, affecting predominantly the vessels and the interstitium. Glomerular involvement was minimal. Melphalan and prednisone were initiated. However, renal function continues deteriorating. Deposition of AL kappa amyloidosis developing during the course of MGUS predominantly in the wall of the renal vessels and the renal interstitium, while the involvement of the glomeruli is minimal, leads to progressive renal failure and absence of proteinuria. Renal biopsy is required to detect both the presence and the sites of deposition of renal AL kappa light chain amyloidosis. PMID:24555136

  1. Assessment of renal response with urinary exosomes in patients with AL amyloidosis: A proof of concept.

    PubMed

    Ramirez-Alvarado, Marina; Barnidge, David R; Murray, David L; Dispenzieri, Angela; Marin-Argany, Marta; Dick, Christopher J; Cooper, Shawna A; Nasr, Samih H; Ward, Christopher J; Dasari, Surendra; Jiménez-Zepeda, Víctor H; Leung, Nelson

    2017-03-10

    Immunoglobulin light chain (AL) amyloidosis is a fatal complication of B-cell proliferation secondary to deposition of amyloid fibrils in various organs. Urinary exosomes (UEX) are the smallest of the microvesicles excreted in the urine. Previously, we found UEX of patients with AL amyloidosis contained immunoglobulin light chain (LC) oligomers that patients with multiple myeloma did not have. To further explore the role of the LC oligomers, UEX was isolated from an AL amyloidosis patient with progressive renal disease despite achieving a complete response. LC oligomers were identified. Mass spectrometry (MS) of the UEX and serum identified two monoclonal lambda LCs. Proteomics of the trypsin digested amyloid fragments in the kidney by laser microdissection and MS analysis identified a λ6 LC. The cDNA from plasma cell clone was from the IGLV- 6-57 family and it matched the amino acid sequences of the amyloid peptides. The predicted mass of the peptide product of the cDNA matched the mass of one of the two LCs identified in the UEX and serum. UEX combined with MS were able to identify 2 monoclonal lambda LCs that current clinical methods could not. It also identified the amyloidogenic LC which holds potential for response assessment in the future. This article is protected by copyright. All rights reserved.

  2. 75 FR 65279 - Schedule for Rating Disabilities; AL Amyloidosis (Primary Amyloidosis)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-22

    ... presumptive service connection based on herbicide exposure for this disease. The intended effects are to... adding AL amyloidosis to the list of diseases associated with exposure to certain herbicide...

  3. AL-Base: a visual platform analysis tool for the study of amyloidogenic immunoglobulin light chain sequences

    PubMed Central

    Bodi, Kip; Prokaeva, Tatiana; Spencer, Brian; Eberhard, Maurya; Connors, Lawreen H.; Seldin, David C.

    2014-01-01

    AL-Base, a curated database of human immunoglobulin (Ig) light chain (LC) sequences derived from patients with AL amyloidosis and controls, is described, along with a collection of analytical and graphic tools designed to facilitate their analysis. AL-Base is designed to compile and analyse amyloidogenic Ig LC sequences and to compare their predicted protein sequence and structure to non-amyloidogenic LC sequences. Currently, the database contains over 3000 de-identified LC nucleotide and amino acid sequences, of which 433 encode monoclonal proteins that were reported to form fibrillar deposits in AL patients. Each sequence is categorised according to germline gene usage, clinical status and sample source. Currently, tools are available to search for sequences by various criteria, to analyse the biochemical properties of the predicted amino acids at each position and to display the results in a graphical fashion. The likelihood that each sequence has evolved through somatic hypermutation can be predicted using an automated binomial or multinomial distribution model. AL-Base is available to the scientific community for research purposes. PMID:19291508

  4. Lenalidomide, melphalan and dexamethasone in a population of patients with immunoglobulin light chain amyloidosis with high rates of advanced cardiac involvement.

    PubMed

    Dinner, Shira; Witteles, Wesley; Afghahi, Anosheh; Witteles, Ronald; Arai, Sally; Lafayette, Richard; Schrier, Stanley L; Liedtke, Michaela

    2013-10-01

    Immunoglobulin light chain amyloidosis remains incurable despite recent therapeutic advances, and is particularly difficult to treat in patients with amyloid cardiomyopathy. Based on evidence of activity in multiple myeloma, we designed a pilot study of an oral regimen of lenalidomide in combination with dexamethasone and low-dose melphalan in order to evaluate its safety and efficacy in patients with amyloidosis, including those with advanced cardiac involvement. Twenty-five patients were enrolled. Ninety-two percent of patients had cardiac involvement by amyloidosis, and 36% of patients met the criteria for Mayo Clinic cardiac stage III disease. Patients received up to nine cycles of treatment, consisting of lenalidomide 10 mg/day orally on days 1 - 21 (28-day cycle); melphalan 0.18 mg/kg orally on days 1-4; and dexamethasone 40 mg orally on days 1, 8, 15, and 22. High rates (33%) of cardiac arrhythmias and low rates of treatment completion (12.5%) were observed. Ten patients died during the study, all within the first several months of treatment due to acute cardiac events. The overall hematologic response rate was 58%, however organ responses were seen in only 8% of patients. The overall survival rate at 1 year was 58%. While we confirmed the hematologic response rates observed with similar regimens, front-line treatment with melphalan, lenalidomide and dexamethasone was toxic, ineffective, and did not alter survival outcomes for patients with high-risk cardiac disease. Our data highlight the importance of developing novel treatment approaches for amyloid cardiomyopathy. This trial was registered at www.clinicaltrials.gov (NCT00890552).

  5. Doxycycline reduces fibril formation in a transgenic mouse model of AL amyloidosis

    PubMed Central

    Ward, Jennifer Ellis; Ren, Ruiyi; Toraldo, Gianluca; SooHoo, Pam; Guan, Jian; O'Hara, Carl; Jasuja, Ravi; Trinkaus-Randall, Vickery; Liao, Ronglih; Connors, Lawreen H.

    2011-01-01

    Systemic AL amyloidosis results from the aggregation of an amyloidogenic immunoglobulin (Ig) light chain (LC) usually produced by a plasma cell clone in the bone marrow. AL is the most rapidly fatal of the systemic amyloidoses, as amyloid fibrils can rapidly accumulate in tissues including the heart, kidneys, autonomic or peripheral nervous systems, gastrointestinal tract, and liver. Chemotherapy is used to eradicate the cellular source of the amyloidogenic precursor. Currently, there are no therapies that target the process of LC aggregation, fibril formation, or organ damage. We developed transgenic mice expressing an amyloidogenic λ6 LC using the cytomegalovirus (CMV) promoter to circumvent the disruption of B cell development by premature expression of recombined LC. The CMV-λ6 transgenic mice develop neurologic dysfunction and Congophilic amyloid deposits in the stomach. Amyloid deposition was inhibited in vivo by the antibiotic doxycycline. In vitro studies demonstrated that doxycycline directly disrupted the formation of recombinant LC fibrils. Furthermore, treatment of ex vivo LC amyloid fibrils with doxycycline reduced the number of intact fibrils and led to the formation of large disordered aggregates. The CMV-λ6 transgenic model replicates the process of AL amyloidosis and is useful for testing the antifibril potential of orally available agents. PMID:21998211

  6. Systemic AL amyloidosis in a Beech Marten (Martes foina).

    PubMed

    Scaglione, F E; Mignone, W; Ferrero, E; Poggi, M; Biolatti, B; Bollo, E

    2013-10-01

    A wild Beech Marten (Martes foina), was referred for necropsy to the Department of Animal Pathology of the University of Turin (Italy). At gross examination, whitish and firm masses, 10-mm in diameter, were found on the heart and in the kidney. Spleen showed lighter color and greater consistency, and the cut surface of the liver appeared scattered with whitish-yellow coalescing foci homogeneously distributed. Amyloid deposits were present in the perivascular and intercellular spaces of the visceral organs, such as the heart, liver, and kidneys. Amyloid stained positively with Congo red with and without 5% potassium permanganate pretreatment and showed green birefringence observable under polarized light. A diagnosis of systemic AL amyloidosis was made. This is the first description of systemic AL amyloidosis in a wild Stone Marten.

  7. Uninvolved immunoglobulins predicting hematological response in newly diagnosed AL amyloidosis.

    PubMed

    Muchtar, Eli; Magen, Hila; Itchaki, Gilad; Cohen, Amos; Rosenfeld, Ra'ama; Shochat, Tzippy; Kornowski, Ran; Iakobishvili, Zaza; Raanani, Pia

    2016-02-01

    Immunoparesis serves as a marker for elevated risk for progression in plasma cell proliferative disorders. However, the impact of immunoparesis in AL amyloidosis has not been addressed. Immunoparesis was defined qualitatively as any decrease below the low reference levels of the uninvolved immunoglobulins and quantitatively, as the relative difference between the uninvolved immunoglobulins and the lower reference values. Forty-one newly diagnosed AL amyloidosis patients were included. Sixty-six percent of patients had a suppression of the uninvolved immunoglobulins. The median relative difference of the uninvolved immunoglobulins was 18% above the low reference levels [range (-71%)-210%]. Ninety percent of the patients were treated with novel agents-based regimens, mostly bortezomib-containing regimens. Nineteen percent of the patients did not attain response to first line treatment. Patients with relative difference of uninvolved immunoglobulins below -25% of the low reference levels were less likely to respond to first line treatment compared to patients with a relative difference of -25% and above [odds ratio for no response vs. partial response and better 30 [(95% CI 4.1-222.2), P=0.0004]. Patients who failed first line treatment were successfully salvaged with lenalidomide-based treatment. Immunoparesis, if assessed quantitatively, may serve as a predictor of response in AL amyloidosis patients treated with bortezomib-containing regimens.

  8. A reappraisal of immunoglobulin variable gene primers and its impact on assessing clonal relationships between PB B cells and BM plasma cells in AL amyloidosis.

    PubMed

    Katoh, Nagaaki; Poshusta, Tanya L; Manske, Michelle K; Dispenzieri, Angela; Gertz, Morie A; Abraham, Roshini S; Ramirez-Alvarado, Marina

    2011-12-01

    Monoclonal tumor plasma cells as well as non-terminally differentiated B cells having a clonal relationship to the tumor cells have been detected in the peripheral blood (PB) of some multiple myeloma (MM) patients but rarely in light chain (primary systemic) amyloidosis (AL) patients. Previously, our group found these peripheral clonotypic B cells in three AL patients. Here, we report detailed analysis of a larger cohort of AL patients to validate the prior findings and to investigate the effect of this cell population on clinical outcome. Fourteen AL patients were selected from a clinical prospective trial, and the relationship between immunoglobulin light chain variable gene (V(L)) representation in PB B cells and the clonal population in the bone marrow (BM) was investigated. A clonal relationship was not detected, and the present study provides important insights into the disparity with the earlier data, including clinical history of the patients and methodological analysis.

  9. Pulmonary arterial hypertension in primary amyloidosis

    PubMed Central

    Emerson, Lyska L.; Bull, David A.; Hatton, Nathan; Nativi-Nicolai, Jose; Hildebrandt, Gerhard C.; Ryan, John J.

    2016-01-01

    Abstract Amyloidosis involves extravascular deposition of fibrillar proteins within tissues and organs. Primary light chain amyloidosis represents the most common form of systemic amyloidosis involving deposition of monoclonal immunoglobulin light chains. Although pulmonary amyloid deposition is common in primary amyloidosis, clinically significant pulmonary amyloidosis is uncommon, and elevated pulmonary artery pressures are rarely observed in the absence of other underlying etiologies for pulmonary hypertension, such as elevated filling pressures secondary to cardiac amyloid. In this case report, we present a patient with primary light chain amyloidosis and pulmonary arterial hypertension in the setting of pulmonary vascular and right ventricular myocardial amyloid deposition. PMID:27252852

  10. 77 FR 6466 - Schedule for Rating Disabilities; AL Amyloidosis (Primary Amyloidosis)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-08

    ... herbicide exposure for this disease. The intended effects are to provide consistency in disability ratings... presumptive service connection based on herbicide exposure for this disease. A final rule regarding that... amyloidosis to the list of diseases associated with exposure to certain herbicide agents. For these...

  11. Immunoglobulin light chains, glycosaminoglycans and amyloid.

    SciTech Connect

    Stevens, F. J.; Kisilevsky, R.; Biosciences Division; Queen's Univ.

    2000-03-01

    Immunoglobulin light chains are the precursor proteins for fibrils that are formed during primary amyloidosis and in amyloidosis associated with multiple myeloma. As found for the approximately 20 currently described forms of focal, localized, or systemic amyloidoses, light chain-related fibrils extracted from physiological deposits are invariably associated with glycosaminoglycans, predominantly heparan sulfate. Other amyloid-related proteins are either structurally normal, such as g2-microglobulin and islet amyloid polypeptide, fragments of normal proteins such as serum amyloid A protein or the precursor protein of the g peptide involved in Alzheimer's disease, or are inherited forms of single amino acid variants of a normal protein such as found in the familial forms of amyloid associated with transthyretin. In contrast, the primary structures of light chains involved in fibril formation exhibit extensive mutational diversity rendering some proteins highly amyloidogenic and others non-pathological. The interactions between light chains and glycosaminoglycans are also affected by amino acid variation and may influence the clinical course of disease by enhancing fibril stability and contributing to resistance to protease degradation. Relatively little is currently known about the mechanisms by which glycosaminoglycans interact with light chains and light-chain fibrils. It is probable that future studies of this uniquely diverse family of proteins will continue o shed light on the processes of amyloidosis, and contribute as well to a greater understanding of the normal physiological roles of glycosaminoglycans.

  12. Tertiary structure of human {Lambda}6 light chains.

    SciTech Connect

    Pokkuluri, P. R.; Solomon, A.; Weiss, D. T.; Stevens, F. J.; Schiffer, M.; Center for Mechanistic Biology and Biotechnology; Univ. of Tennessee Medical Center /Graduate School of Medicine

    1999-01-01

    AL amyloidosis is a disease process characterized by the pathologic deposition of monoclonal light chains in tissue. To date, only limited information has been obtained on the molecular features that render such light chains amyloidogenic. Although protein products of the major human V kappa and V lambda gene families have been identified in AL deposits, one particular subgroup--lambda 6--has been found to be preferentially associated with this disease. Notably, the variable region of lambda 6 proteins (V lambda 6) has distinctive primary structural features including the presence in the third framework region (FR3) of two additional amino acid residues that distinguish members of this subgroup from other types of light chains. However, the structural consequences of these alterations have not been elucidated. To determine if lambda 6 proteins possess unique tertiary structural features, as compared to light chains of other V lambda subgroups, we have obtained x-ray diffraction data on crystals prepared from two recombinant V lambda 6 molecules. These components, isolated from a bacterial expression system, were generated from lambda 6-related cDNAs cloned from bone marrow-derived plasma cells from a patient (Wil) who had documented AL amyloidosis and another (Jto) with multiple myeloma and tubular cast nephropathy, but no evident fibrillar deposits. The x-ray crystallographic analyses revealed that the two-residue insertion located between positions 68 and 69 (not between 66 and 67 as previously surmised) extended an existing loop region that effectively increased the surface area adjacent to the first complementarity determining region (CDR1). Further, an unusual interaction between the Arg 25 and Phe 2 residues commonly found in lambda 6 molecules was noted. However, the structures of V lambda 6 Wil and Jto also differed from each other, as evidenced by the presence in the latter of certain ionic and hydrophobic interactions that we posit increased protein

  13. Bendamustine-Induced Nephrogenic Diabetes Insipidus in a Patient With AL Amyloidosis.

    PubMed

    Uwumugambi, Nsabimana A; Sanchorawala, Vaishali; Shelton, Anthony C; Stern, Lauren; Gordon, Craig E

    2017-02-01

    Nephrogenic diabetes insipidus is a condition characterized by polyuria with dilute urine due to the inability of the principal cells of the renal collecting ducts to respond to antidiuretic hormone and concentrate urine. Nephrogenic diabetes insipidus can be drug induced, and several chemotherapeutic agents have been reported to cause it. Bendamustine is a traditional chemotherapeutic agent being studied for treatment for relapsed systemic AL amyloidosis. We report a case of a 59-year-old man with AL amyloidosis who developed partial nephrogenic diabetes insipidus after receiving bendamustine for treatment of AL amyloidosis. The nephrogenic diabetes insipidus responded well to sodium restriction, hydrochlorothiazide, and desmopressin treatment, allowing the patient to receive subsequent bendamustine cycles without polyuria. Nephrogenic diabetes insipidus resolved shortly after completion of bendamustine therapy.

  14. Systemic AA amyloidosis: epidemiology, diagnosis, and management

    PubMed Central

    Real de Asúa, Diego; Costa, Ramón; Galván, Jose María; Filigheddu, María Teresa; Trujillo, Davinia; Cadiñanos, Julen

    2014-01-01

    The term “amyloidosis” encompasses the heterogeneous group of diseases caused by the extracellular deposition of autologous fibrillar proteins. The global incidence of amyloidosis is estimated at five to nine cases per million patient-years. While amyloid light-chain (AL) amyloidosis is more frequent in developed countries, amyloid A (AA) amyloidosis is more common in some European regions and in developing countries. The spectrum of AA amyloidosis has changed in recent decades owing to: an increase in the median age at diagnosis; a percent increase in the frequency of primary AL amyloidosis with respect to the AA type; and a substantial change in the epidemiology of the underlying diseases. Diagnosis of amyloidosis is based on clinical organ involvement and histological evidence of amyloid deposits. Among the many tinctorial characteristics of amyloid deposits, avidity for Congo red and metachromatic birefringence under unidirectional polarized light remain the gold standard. Once the initial diagnosis has been made, the amyloid subtype must be identified and systemic organ involvement evaluated. In this sense, the 123I-labeled serum amyloid P component scintigraphy is a safe and noninvasive technique that has revolutionized the diagnosis and monitoring of treatment in systemic amyloidosis. It can successfully identify anatomical patterns of amyloid deposition throughout the body and enables not only an initial estimation of prognosis, but also the monitoring of the course of the disease and the response to treatment. Given the etiologic diversity of AA amyloidosis, common therapeutic strategies are scarce. All treatment options should be based upon a greater control of the underlying disease, adequate organ support, and treatment of symptoms. Nevertheless, novel therapeutic strategies targeting the formation of amyloid fibrils and amyloid deposition may generate new expectations for patients with AA amyloidosis. PMID:25378951

  15. Primary Hepatic Amyloidosis Presenting as Acute-on-Chronic Liver Failure

    PubMed Central

    Rangegowda, Devaraja; Vyas, Tanmay; Kulkarni, Anand; Grover, Shrruti; Mahiwall, Rakhi; Sarin, Shiv Kumar

    2017-01-01

    Systemic amyloidosis of amyloid light chain associated protein (AL), also called primary amyloidosis, frequently involves the liver, but rarely causes clinically apparent liver disease. The more common presentation is with acute renal failure. Hepatomegaly and mild elevation of alkaline phosphatase are the most common clinical and biochemical findings, respectively. We report a case of systemic amyloidosis of AL that clinically presented as acute-on-chronic liver failure and resulted in a fatal clinical course in a 56-year-old man. PMID:28286788

  16. Short- and long-term outcomes of AL amyloidosis patients admitted into intensive care units.

    PubMed

    Guinault, Damien; Canet, Emmanuel; Huart, Antoine; Jaccard, Arnaud; Ribes, David; Lavayssiere, Laurence; Venot, Marion; Cointault, Olivier; Roussel, Murielle; Nogier, Marie-Béatrice; Pichereau, Claire; Lemiale, Virginie; Arnulf, Bertrand; Attal, Michel; Chauveau, Dominique; Azoulay, Elie; Faguer, Stanislas

    2016-09-01

    Amyloidosis is a rare and threatening condition that may require intensive care because of amyloid deposit-related organ dysfunction or therapy-related adverse events. Although new multiple myeloma drugs have dramatically improved outcomes in AL amyloidosis, the outcomes of AL patients admitted into intensive care units (ICUs) remain largely unknown. Admission has been often restricted to patients with low Mayo Clinic staging and/or with a complete or very good immunological response at admission. In a retrospective multicentre cohort of 66 adult AL (n = 52) or AA (n = 14) amyloidosis patients, with similar causes of admission to an ICU, the 28-d and 6-month survival rates of AA patients were significantly higher compared to AL patients (93% vs. 60%, P = 0·03; 71% vs. 45%, P = 0·02, respectively). In AL patients, the simplified Index of Gravity Score (IGS2) was the only independent predictive factor for death by day 28, whereas the Mayo-Clinic classification stage had no influence. In Cox's multivariate regression model, only cardiac arrest and on-going chemotherapy at ICU admission significantly predicted death at 6 months. Short-term outcomes of AL patients admitted into an ICU were mainly related to the severity of the acute medical condition, whereas on-going chemotherapy for active amyloidosis impacted on long-term outcomes.

  17. Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

    PubMed Central

    McWilliams-Koeppen, Helen P.; Foster, James S.; Hackenbrack, Nicole; Ramirez-Alvarado, Marina; Donohoe, Dallas; Williams, Angela; Macy, Sallie; Wooliver, Craig; Wortham, Dale; Morrell-Falvey, Jennifer; Foster, Carmen M.; Kennel, Stephen J.; Wall, Jonathan S.

    2015-01-01

    Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. These data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils. PMID:26393799

  18. Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

    SciTech Connect

    McWilliams-Koeppen, Helen P.; Foster, James S.; Hackenbrack, Nicole; Ramirez-Alvarado, Marina; Donohoe, Dallas; Williams, Angela; Macy, Sallie; Wooliver, Craig; Wortham, Dale; Morrell-Falvey, Jennifer; Foster, Carmen M.; Kennel, Stephen J.; Wall, Jonathan S.

    2015-09-22

    Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. Ultimately, these data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.

  19. Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

    DOE PAGES

    McWilliams-Koeppen, Helen P.; Foster, James S.; Hackenbrack, Nicole; ...

    2015-09-22

    Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presencemore » of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. Ultimately, these data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.« less

  20. New Light Chain Amyloid Response Criteria Help Risk Stratification of Patients by Day 100 after Autologous Hematopoietic Cell Transplantation.

    PubMed

    D'Souza, Anita; Huang, Jiaxing; Hari, Parameswaran

    2016-04-01

    Hematologic response criteria in light chain (AL) amyloidosis were updated in 2012 to incorporate free light chain responses. These criteria have been validated in autologous hematopoietic cell transplantation in AL at 6 and 12 months after transplantation. Using a transplantation registry, we assessed day 100 responses in AL amyloidosis. We validate the prognostic significance of the new criteria at this time point. Further, we show that patients who do not achieve at least a very good partial response by this time point have equally worse outcomes, regardless of depth of response (partial versus no response). Thus, we conclude that the new criteria help identify the poor responders by day 100 after transplantation and that this subset of patients should be studied for early evaluation in consolidation trials.

  1. Clinical profile and treatment outcome of older (>75 years) patients with systemic AL amyloidosis

    PubMed Central

    Sachchithanantham, Sajitha; Offer, Mark; Venner, Christopher; Mahmood, Shameem A.; Foard, Darren; Rannigan, Lisa; Lane, Thirusha; Gillmore, Julian D.; Lachmann, Helen J.; Hawkins, Philip N.; Wechalekar, Ashutosh D.

    2015-01-01

    Systemic AL amyloidosis, a disease with improving outcomes using novel therapies, is increasingly recognized in the elderly but treatment and outcomes have not been systematically studied in this group of patients in whom comorbidities and frailty may compound morbidity and mortality. We report the outcomes of 295 patients with systemic AL amyloidosis ≥75 years seen at the UK National Amyloidosis Centre from 2005–2012. The median age was 78.5 years. The median overall survival was 20 months. Two hundred and thirty-eight patients received chemotherapy and 57 elected for supportive care only (overall survival – 24 and 8.4 months, respectively). On intention-to-treat analysis, 44% achieved a hematologic response including a very good partial response or better in 23%. The median overall survival was 6.2 years in patients achieving very good partial response or better at the 6-month landmark analysis and 1.5 years in non-responders. Factors independently indicating a poor prognosis were: cardiac involvement, performance status ≥2; systolic blood pressure <100 mmHg and, on landmark analysis, achieving less than a very good partial response. Treatment of systemic AL amyloidosis in the elderly is challenging. Deep clonal responses are associated with excellent survival and organ responses. Achieving a response to the first-line regimen appears particularly important as outcomes of non-responders are similar to those of untreated patients. Prospective trials with lower toxicity, outpatient treatment regimens are needed. PMID:26294730

  2. Etiology of amyloidosis determines myocardial 99mTc-DPD uptake in amyloidotic cardiomyopathy.

    PubMed

    Longhi, Simone; Bonfiglioli, Rachele; Obici, Laura; Gagliardi, Christian; Milandri, Agnese; Lorenzini, Massimiliano; Guidalotti, Pier Luigi; Merlini, Giampaolo; Rapezzi, Claudio

    2015-05-01

    Tc-DPD (Tc-3,3-diphosphono-1,2-propanodicarboxylic acid) has a high affinity for transthyretin (TTR)-infiltrated myocardium, allowing a differential diagnosis with light chain cardiac amyloidosis and other nonamyloidotic cardiomyopathies with a hypertrophic phenotype, in which myocardial tracer uptake is low or absent. Myocardial bone tracer uptake in the rarer forms of amyloidosis (eg, apolipoprotein-related) has been rarely studied. We present 4 cases of cardiac amyloidosis that underwent Tc-DPD scintigraphy; myocardial DPD uptake was present in patients with ATTR, wtTTR and apolipoprotein AI and negative in cases with AL and apolipoprotein AII-related disease.

  3. Effect of lysine modification on the stability and cellular binding of human amyloidogenic light chains.

    PubMed

    Davern, S; Murphy, C L; O'Neill, H; Wall, J S; Weiss, D T; Solomon, A

    2011-01-01

    AL amyloidosis is characterized by the pathologic deposition as fibrils of monoclonal light chains (i.e., Bence Jones proteins [BJPs]) in particular organs and tissues. This phenomenon has been attributed to the presence in amyloidogenic proteins of particular amino acids that cause these molecules to become unstable, as well as post-translational modifications and, in regard to the latter, we have investigated the effect of biotinylation of lysyl residues on cell binding. We utilized an experimental system designed to test if BJPs obtained from patients with AL amyloidosis or, as a control, multiple myeloma (MM), bound human fibroblasts and renal epithelial cells. As documented by fluorescence microscopy and ELISA, the amyloidogenic BJPs, as compared with MM components, bound preferentially and this reactivity increased significantly after chemical modification of their lysyl residues with sulfo-NHS-biotin. Further, based on tryptophan fluorescence and circular dichroism data, it was apparent that their conformation was altered, which we hypothesize exposed a binding site not accessible on the native protein. The results of our studies indicate that post-translational structural modifications of pathologic light chains can enhance their capacity for cellular interaction and thus may contribute to the pathogenesis of AL amyloidosis and multiple myeloma.

  4. Effect of Lysine Modification on the Stability and Cellular Binding of Human Amyloidogenic Light Chains

    SciTech Connect

    O'Neill, Hugh Michael; Davern, Sandra M.; Murphy, Charles L.; Wall, Jonathan; Deborah, Weiss T.; Solomon, Alan

    2011-01-01

    AL amyloidosis is characterized by the pathologic deposition as fibrils of monoclonal light chains (i.e., Bence Jones proteins [BJPs]) in particular organs and tissues. This phenomenon has been attributed to the presence in amyloidogenic proteins of particular amino acids that cause these molecules to become unstable, as well as post-translational modifications and, in regard to the latter, we have investigated the effect of biotinylation of lysyl residues on cell binding. We utilized an experimental system designed to test if BJPs obtained from patients with AL amyloidosis or, as a control, multiple myeloma (MM), bound human fibroblasts and renal epithelial cells. As documented by fluorescent microscopy and ELISA, the amyloidogenic BJPs, as compared with MM components, bound preferentially and this reactivity increased significantly after chemical modification of their lysyl residues with sulfo-NHS-biotin. Further, based on tryptophan fluorescence and circular dichorism data, it was apparent that their conformation was altered, which we hypothesize exposed a binding site not accessible on the native protein. The results of our studies indicate that post-translational structural modifications of pathologic light chains can enhance their capacity for cellular interaction and thus may contribute to the pathogenesis of AL amyloidosis and multiple myeloma.

  5. Differential recruitment efficacy of patient-derived amyloidogenic and myeloma light chain proteins by synthetic fibrils—A metric for predicting amyloid propensity

    PubMed Central

    Wooliver, Craig; Heidel, R. Eric; Adams, Sarah; Dunlap, John; Lands, Ronald H.

    2017-01-01

    Background Monoclonal free light chain (LC) proteins are present in the circulation of patients with immunoproliferative disorders such as light chain (AL) amyloidosis and multiple myeloma (MM). Light chain-associated amyloid is a complex pathology composed of proteinaceous fibrils and extracellular matrix proteins found in all patients with AL and in ~10–30% of patients who presented with MM. Amyloid deposits systemically in multiple organs and tissues leading to dysfunction and ultimately death. The overall survival of patients with amyloidosis is worse than for those with early stage MM. Methods and findings We have developed a sensitive binding assay quantifying the recruitment of full length, patient-derived LC proteins by synthetic amyloid fibrils, as a method for studying their amyloidogenic potential. In a survey of eight urinary LC, both AL and MM-associated proteins were recruited by synthetic amyloid fibrils; however, AL-associated LC bound significantly more efficiently (p < 0.05) than did MM LCs. The LC proteins used in this study were isolated from urine and presumed to represent a surrogate of serum free light chains. Conclusion The binding of LC to synthetic fibrils in this assay accurately differentiated LC with amyloidogenic propensity from MM LC that were not associated with clinical amyloid disease. Notably, the LC from a MM patient who subsequently developed amyloid behaved as an AL-associated protein in the assay, indicating the possibility for identifying MM patients at risk for developing amyloidosis based on the light chain recruitment efficacy. With this information, at risk patients can be monitored more closely for the development of amyloidosis, allowing timely administration of novel, amyloid-directed immunotherapies—this approach may improve the prognosis for these patients. PMID:28350808

  6. Confocal Cornea Microscopy Detects Involvement of Corneal Nerve Fibers in a Patient with Light-Chain Amyloid Neuropathy Caused by Multiple Myeloma: A Case Report

    PubMed Central

    Sturm, Dietrich; Schmidt-Wilcke, Tobias; Greiner, Tineke; Maier, Christoph; Schargus, Marc; Tegenthoff, Martin; Vorgerd, Matthias

    2016-01-01

    Changes in the subbasal corneal plexus detected by confocal cornea microscopy (CCM) have been described for various types of neuropathy. An involvement of these nerves within light-chain (AL) amyloid neuropathy (a rare cause of polyneuropathy) has never been shown. Here, we report on a case of a patient suffering from neuropathy caused by AL amyloidosis and underlying multiple myeloma. Small-fiber damage was detected by CCM. PMID:27482195

  7. The evolving use of serum free light chain assays in haematology.

    PubMed

    Pratt, Guy

    2008-05-01

    Over the last few years new immunoassays have emerged that allow the measurement of free immunoglobulin light chains (FLCs) in serum to a level of 2-4 mg/l and provide a much greater sensitivity than older methods, such as immunofixation, which is able to detect FLCs at a minimum concentration of 100-150 mg/l. The new FLC assay has enabled the detection of monoclonal protein in some patients with non-secretory myeloma and amyloidosis that were previously undetectable. FLC measurements are quantitative, correlating with disease activity, and are an advance in monitoring light chain only multiple myeloma, AL amyloidosis, non-secretory and oligo-secretory multiple myeloma. Serum FLC concentrations also reflect the disease course in the majority of myeloma patients producing intact monoclonal immunoglobulin proteins and have been incorporated into the new response criteria. The rapid half life of lambda and kappa free light chains means that FLC assays may provide a more rapid indication of the response to treatment but their clinical utility in this setting needs further study. An abnormal FLC ratio has been shown to be a risk factor for progression of monoclonal gammopathy of undetermined significance, smouldering myeloma and solitary plasmacytoma of bone and is prognostic in multiple myeloma.

  8. Formation of Amyloid Fibers by Monomeric Light Chain Variable Domains*

    PubMed Central

    Brumshtein, Boris; Esswein, Shannon R.; Landau, Meytal; Ryan, Christopher M.; Whitelegge, Julian P.; Phillips, Martin L.; Cascio, Duilio; Sawaya, Michael R.; Eisenberg, David S.

    2014-01-01

    Systemic light chain amyloidosis is a lethal disease characterized by excess immunoglobulin light chains and light chain fragments composed of variable domains, which aggregate into amyloid fibers. These fibers accumulate and damage organs. Some light chains induce formation of amyloid fibers, whereas others do not, making it unclear what distinguishes amyloid formers from non-formers. One mechanism by which sequence variation may reduce propensity to form amyloid fibers is by shifting the equilibrium toward an amyloid-resistant quaternary structure. Here we identify the monomeric form of the Mcg immunoglobulin light chain variable domain as the quaternary unit required for amyloid fiber assembly. Dimers of Mcg variable domains remain stable and soluble, yet become prone to assemble into amyloid fibers upon disassociation into monomers. PMID:25138218

  9. Sinonasal Globular Amyloidosis Simulating Malignancy: A Rare Presentation.

    PubMed

    Kumar, Binay; Pant, Bhawna; Kumar, Vikrant; Negi, Meghna

    2016-09-01

    Primary localized amyloidosis in the head and neck region is a rare entity. The most commonly involved organ is larynx. Primary amyloidosis localized to the sinonasal tract is extremely rare. We report one such case along with a brief review of the associated literature. The aim of reporting this case is to emphasize the fact that sometimes nasal amyloidosis can also present with signs and symptoms of nasal and nasopharyngeal malignancy. The definitive diagnosis in such cases depends upon histopathology and further confirmed by immunohistochemistry. A 55-year old male presented with recurrent episodes of nasal bleed, bilateral nasal obstruction, and bilateral hearing loss from last 7 years. On clinical examination a mass was found in the nasal cavity on both sides reaching up to the nasopharynx. Contrast enhanced CT scan revealed that the mass was extending up to the skull base and destroying bony landmarks of the nasal cavity and paranasal sinuses. Mass was proved to be amyloidosis after histopathological examination. It showed multiple blotches of globular submucosal deposit of amyloid, on staining with Congo red. Immunohistochemistry confirmed AL amyloidosis with expression of mixed kappa and lambda light chain immunoglobulin (κ > λ). No evidence of systemic amyloidosis was found after proper work up. It was managed by conservative surgery.

  10. Serum free light chains for monitoring multiple myeloma.

    PubMed

    Mead, G P; Carr-Smith, H D; Drayson, M T; Morgan, G J; Child, J A; Bradwell, A R

    2004-08-01

    Monoclonal immunoglobulin free light chains (FLC) are found in the serum and urine of patients with a number of B-cell proliferative disorders, including multiple myeloma. Automated immunoassays, which can measure FLC in serum, are useful for the diagnosis and monitoring of light chain (AL) amyloidosis, Bence Jones myeloma and non-secretory myeloma patients. We report the results of a study investigating the utility of serum FLC measurements in myeloma patients producing monoclonal intact immunoglobulin proteins. FLC concentrations were measured in presentation sera from 493 multiple myeloma patients with monoclonal, intact immunoglobulin proteins. Serial samples were assayed from 17 of these patients and the FLC measurements were compared with other disease markers. Serum FLC concentrations were abnormal in 96% of patients at presentation. FLC concentrations fell more rapidly in response to treatment than intact immunoglobulin G (IgG) and showed greater concordance with serum beta2 microglobulin concentrations and bone marrow plasma cell assessments. It was concluded that serum FLC assays could be used to follow the disease course in nearly all multiple myeloma patients. In addition, because of their short serum half-life, changes in serum FLC concentrations provide a rapid indication of the response to treatment.

  11. Serum free light chains in clinical laboratory diagnostics.

    PubMed

    Jenner, Ellen

    2014-01-01

    Monoclonal free light chains (FLCs) are important disease biomarkers in patients with plasma cell-proliferative disorders. The increasing evidence for clonal diversity and evolution in multiple myeloma highlights the importance of laboratory algorithms that measure both intact immunoglobulins and monoclonal FLCs, at diagnosis and when monitoring response to treatment. A particular focus in the field has been on the utility of serum FLC (sFLC) assays to replace urine electrophoresis for monoclonal FLC measurement. Due to the limited sensitivity and practical constraints of urine analysis, a serum-based algorithm of SPE and sFLC has been adopted by many laboratories as a first line screen in patients with suspected monoclonal gammopathies. This review will discuss the data supporting the use of this simple serum-based algorithm at initial diagnosis, including its utility for the rapid identification of monoclonal FLC in the setting of unexplained acute kidney injury, and provide a comprehensive review of the diagnostic sensitivity of sFLC in patients with multiple myeloma, AL amyloidosis and light chain deposition disease.

  12. In vitro aggregation behavior of a non-amyloidogenic λ light chain dimer deriving from U266 multiple myeloma cells.

    PubMed

    Arosio, Paolo; Owczarz, Marta; Müller-Späth, Thomas; Rognoni, Paola; Beeg, Marten; Wu, Hua; Salmona, Mario; Morbidelli, Massimo

    2012-01-01

    Excessive production of monoclonal light chains due to multiple myeloma can induce aggregation-related disorders, such as light chain amyloidosis (AL) and light chain deposition diseases (LCDD). In this work, we produce a non-amyloidogenic IgE λ light chain dimer from human mammalian cells U266, which originated from a patient suffering from multiple myeloma, and we investigate the effect of several physicochemical parameters on the in vitro stability of this protein. The dimer is stable in physiological conditions and aggregation is observed only when strong denaturating conditions are applied (acidic pH with salt at large concentration or heating at melting temperature T(m) at pH 7.4). The produced aggregates are spherical, amorphous oligomers. Despite the larger β-sheet content of such oligomers with respect to the native state, they do not bind Congo Red or ThT. The impossibility to obtain fibrils from the light chain dimer suggests that the occurrence of amyloidosis in patients requires the presence of the light chain fragment in the monomer form, while dimer can form only amorphous oligomers or amorphous deposits. No aggregation is observed after denaturant addition at pH 7.4 or at pH 2.0 with low salt concentration, indicating that not a generic unfolding but specific conformational changes are necessary to trigger aggregation. A specific anion effect in increasing the aggregation rate at pH 2.0 is observed according to the following order: SO(4)(-)≫Cl(-)>H(2)PO(4)(-), confirming the peculiar role of sulfate in promoting protein aggregation. It is found that, at least for the investigated case, the mechanism of the sulfate effect is related to protein secondary structure changes induced by anion binding.

  13. In Vitro Aggregation Behavior of a Non-Amyloidogenic λ Light Chain Dimer Deriving from U266 Multiple Myeloma Cells

    PubMed Central

    Arosio, Paolo; Owczarz, Marta; Müller-Späth, Thomas; Rognoni, Paola; Beeg, Marten; Wu, Hua; Salmona, Mario; Morbidelli, Massimo

    2012-01-01

    Excessive production of monoclonal light chains due to multiple myeloma can induce aggregation-related disorders, such as light chain amyloidosis (AL) and light chain deposition diseases (LCDD). In this work, we produce a non-amyloidogenic IgE λ light chain dimer from human mammalian cells U266, which originated from a patient suffering from multiple myeloma, and we investigate the effect of several physicochemical parameters on the in vitro stability of this protein. The dimer is stable in physiological conditions and aggregation is observed only when strong denaturating conditions are applied (acidic pH with salt at large concentration or heating at melting temperature Tm at pH 7.4). The produced aggregates are spherical, amorphous oligomers. Despite the larger β-sheet content of such oligomers with respect to the native state, they do not bind Congo Red or ThT. The impossibility to obtain fibrils from the light chain dimer suggests that the occurrence of amyloidosis in patients requires the presence of the light chain fragment in the monomer form, while dimer can form only amorphous oligomers or amorphous deposits. No aggregation is observed after denaturant addition at pH 7.4 or at pH 2.0 with low salt concentration, indicating that not a generic unfolding but specific conformational changes are necessary to trigger aggregation. A specific anion effect in increasing the aggregation rate at pH 2.0 is observed according to the following order: SO4−≫Cl−>H2PO4−, confirming the peculiar role of sulfate in promoting protein aggregation. It is found that, at least for the investigated case, the mechanism of the sulfate effect is related to protein secondary structure changes induced by anion binding. PMID:22432016

  14. Renal Outcomes in Patients with AL Amyloidosis: Prognostic Factors, Renal response and the Impact of Therapy.

    PubMed

    Kastritis, Efstathios; Gavriatopoulou, Maria; Roussou, Maria; Migkou, Magdalini; Fotiou, Despina; Ziogas, Dimitrios C; Kanellias, Nikos; Eleutherakis-Papaiakovou, Evangelos; Panagiotidis, Ioannis; Giannouli, Stavroula; Psimenou, Erasmia; Marinaki, Smaragdi; Apostolou, Theofanis; Gakiopoulou, Hariklia; Tasidou, Anna; Papassotiriou, Ioannis; Terpos, Evangelos; Dimopoulos, Meletios A

    2017-03-28

    A staging system for patients with renal AL amyloidosis, based on eGFR (<50 ml/min/1.73 m(2) ) and proteinuria (≥5gr/day) at diagnosis, as well as criteria for renal progression (≥25% eGFR reduction) and response (≥30% reduction of proteinuria without renal progression) were recently proposed. We validated these criteria in a cohort of 125 patients with renal AL amyloidosis, mostly treated with bortezomib or lenalidomide. We confirmed the prognostic value of the renal staging system but also identified the limitations of renal progression criteria which are based only on eGFR reduction. We identified the ratio of 24h proteinuria to eGFR as a sensitive marker of renal risk which also accounts for changes in both proteinuria and eGFR: 24h proteinuria/eGFR ratio <30 (in mg/ml/min/1.73 m(2) ) was associated with a 2-year progression to dialysis rate of 0% compared to 9% for a ratio of 31-99 and 35% for a ratio ≥100 (p<0.001). In landmark analysis, patients who achieved a reduction of this ratio by at least 25% or ≤100 (if initially >100) at 3 months had a 2-year progression to dialysis of 0% vs 24% for patients who either did not reduce to or still had a ratio >100 (p=0.001); similar results were obtained by applying the same criteria at 6 months; thus, the evaluation of treatment effect on renal function may be identified early. Furthermore, primary bortezomib-based therapy was more effective than lenalidomide-based therapy, in terms of renal outcomes, especially in patients at intermediate renal risk, but without affecting overall survival. This article is protected by copyright. All rights reserved.

  15. Serum-free light-chain analysis in diagnosis and management of multiple myeloma and related conditions.

    PubMed

    Milani, Paolo; Palladini, Giovanni; Merlini, Giampaolo

    2016-01-01

    The introduction of the serum-free light-chain (S-FLC) assay has been a breakthrough in the diagnosis and management of plasma cell dyscrasias, particularly monoclonal light-chain diseases. The first method, proposed in 2001, quantifies serum-free light-chains using polyclonal antibodies. More recently, assays based on monoclonal antibodies have entered into clinical practice. S-FLC measurement plays a central role in the screening for multiple myeloma and related conditions, in association with electrophoretic techniques. Analysis of S-FLC is essential in assessing the risk of progression of precursor diseases to overt plasma cell dyscrasias. It is also useful for risk stratification in solitary plasmacytoma and AL amyloidosis. The S-FLC measurement is part of the new diagnostic criteria for multiple myeloma, and provides a marker to follow changes in clonal substructure over time. Finally, the evaluation of S-FLC is fundamental for assessing the response to treatment in monoclonal light chain diseases.

  16. Hereditary amyloidosis

    MedlinePlus

    ... MA. Amyloidosis. In: Goldman L, Schafer AI, eds. Goldman's Cecil Medicine . 25th ed. Philadelphia, PA: Elsevier Saunders; 2016:chap 188. Seldin DC, Skinner M. Amyloidosis. In: Firestein GS, Budd RC, Gabriel SE, McInnes IB, O'Dell ...

  17. Immunoglobulin Light Chains Form an Extensive and Highly Ordered Fibril Involving the N- and C-Termini

    PubMed Central

    2017-01-01

    Light-chain (AL)-associated amyloidosis is a systemic disorder involving the formation and deposition of immunoglobulin AL fibrils in various bodily organs. One severe instance of AL disease is exhibited by the patient-derived variable domain (VL) of the light chain AL-09, a 108 amino acid residue protein containing seven mutations relative to the corresponding germline protein, κI O18/O8 VL. Previous work has demonstrated that the thermodynamic stability of native AL-09 VL is greatly lowered by two of these mutations, Y87H and N34I, whereas a third mutation, K42Q, further increases the kinetics of fibril formation. However, detailed knowledge regarding the residues that are responsible for stabilizing the misfolded fibril structure is lacking. In this study, using solid-state NMR spectroscopy, we show that the majority of the AL-09 VL sequence is immobilized in the fibrils and that the N- and C-terminal portions of the sequence are particularly well-structured. Thus, AL-09 VL forms an extensively ordered and β-strand-rich fibril structure. Furthermore, we demonstrate that the predominant β-sheet secondary structure and rigidity observed for in vitro prepared AL-09 VL fibrils are qualitatively similar to those observed for AL fibrils extracted from postmortem human spleen tissue, suggesting that this conformation may be representative of a common feature of AL fibrils. PMID:28261692

  18. Concomitant diagnosis of primary Sjögren's syndrome and systemic AL amyloidosis.

    PubMed

    Delèvaux, I; André, M; Amoura, Z; Kémény, J L; Piette, J C; Aumaître, O

    2001-07-01

    A 48 year old woman was referred to hospital for buccal discomfort. Physical examination showed a macroglossia and features of xerostomia. She was diagnosed as having primary Sjögren's syndrome according to the criteria proposed by the European Community study group in 1993. Furthermore, a lower lip salivary gland biopsy showed amyloid deposits that were also seen in the stomach and in the bone marrow. Echocardiography was consistent with cardiac amyloidosis. Serum immunofixation identified a monoclonal IgGlambda. As far as is known, this is the first report of systemic primary amyloidosis associated with Sjögren's syndrome. The relation between these two disorders is discussed.

  19. Inhibition of pathologic immunoglobulin free light chain production by small interfering RNA molecules

    PubMed Central

    Phipps, Jonathan E.; Kestler, Daniel P.; Foster, James S.; Kennel, Stephen J.; Donnell, Robert; Weiss, Deborah T.; Solomon, Alan; Wall, Jonathan S.

    2010-01-01

    Objectives Morbidity and mortality occurring in patients with multiple myeloma, AL amyloidosis, and light chain deposition disease can result from the pathologic deposition of monoclonal Ig light chains (LCs) in kidneys and other organs. To reduce synthesis of such components, therapy for these disorders typically has involved anti-plasma cell agents; however, this approach is not always effective and can have adverse consequences. We have investigated another means to achieve this objective; namely, RNA interference (RNAi). Materials and Methods SP2/O mouse myeloma cells were stably transfected with a construct encoding a λ6 LC (Wil) under control of the CMV promoter, while λ2-producing myeloma cell line RPMI 8226 was purchased from the ATCC. Both were treated with small interfering RNA (siRNA) directed specifically to the V, J, or C portions of the molecules and then analyzed by ELISA, flow cytometry and real time PCR. Results Transfected cells were found to constitutively express detectable quantities of mRNA and protein Wil and, after exposure to siRNAs, an ~40% reduction in mRNA and LC production was evidenced at 48 hours. An even greater effect was seen with the 8226 cells. Conclusion Our results have shown that RNAi can markedly reduce LC synthesis and provide the basis for testing the therapeutic potential of this strategy using in vivo experimental models of multiple myeloma. PMID:20637260

  20. Site-directed mutagenesis reveals regions implicated in the stability and fiber formation of human λ3r light chains.

    PubMed

    Villalba, Miryam I; Canul-Tec, Juan C; Luna-Martínez, Oscar D; Sánchez-Alcalá, Rosalba; Olamendi-Portugal, Timoteo; Rudiño-Piñera, Enrique; Rojas, Sonia; Sánchez-López, Rosana; Fernández-Velasco, Daniel A; Becerril, Baltazar

    2015-01-30

    Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this work, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, the second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40-60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. This mutagenic approach helped to identify key regions implicated in λ3 AL.

  1. Site-directed Mutagenesis Reveals Regions Implicated in the Stability and Fiber Formation of Human λ3r Light Chains

    SciTech Connect

    Villalba, Miryam I.; Canul-Tec, Juan C.; Luna-Martínez, Oscar D.; Sánchez-Alcalá, Rosalba; Olamendi-Portugal, Timoteo; Rudiño-Piñera, Enrique; Rojas, Sonia; Sánchez-López, Rosana; Fernández-Velasco, Daniel A.; Becerril, Baltazar

    2014-12-11

    Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this paper, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, the second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40–60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. Finally, this mutagenic approach helped to identify key regions implicated in λ3 AL.

  2. Site-directed Mutagenesis Reveals Regions Implicated in the Stability and Fiber Formation of Human λ3r Light Chains

    DOE PAGES

    Villalba, Miryam I.; Canul-Tec, Juan C.; Luna-Martínez, Oscar D.; ...

    2014-12-11

    Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this paper, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, themore » second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40–60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. Finally, this mutagenic approach helped to identify key regions implicated in λ3 AL.« less

  3. Site-directed Mutagenesis Reveals Regions Implicated in the Stability and Fiber Formation of Human λ3r Light Chains*

    PubMed Central

    Villalba, Miryam I.; Canul-Tec, Juan C.; Luna-Martínez, Oscar D.; Sánchez-Alcalá, Rosalba; Olamendi-Portugal, Timoteo; Rudiño-Piñera, Enrique; Rojas, Sonia; Sánchez-López, Rosana; Fernández-Velasco, Daniel A.; Becerril, Baltazar

    2015-01-01

    Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this work, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, the second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40–60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. This mutagenic approach helped to identify key regions implicated in λ3 AL. PMID:25505244

  4. Waldenström's macroglobulinemia associated with AA amyloidosis.

    PubMed

    Gardyn, J; Schwartz, A; Gal, R; Lewinski, U; Kristt, D; Cohen, A M

    2001-07-01

    It is widely accepted that amyloidosis in Waldenström's macroglobulinemia (WM) is exclusively due to amyloid light-chain deposition. However, only a small number of previous reports have actually characterized the type of amyloid in WM. We now report the third patient with WM and amyloid A protein (AA) amyloidosis. This patient developed malabsorption, nephrotic syndrome, and orthostatic hypotension. AA was immunohistochemically demonstrated in the rectal biopsy. In conjunction with previous examples of AA amyloidosis, the present report raises the possibility that AA amyloidosis may also occur in WM patients.

  5. [Amyloidosis: Up-to-date].

    PubMed

    Magy-Bertrand, N

    2016-08-01

    Amyloidosis is mainly a systemic disease belonging to protein-folding diseases. The past 10 years have shown significant progress in typing and the clinical management of amyloidosis, in the identification of novel prognostic markers for risk-stratification, and also in the development of new therapeutic agents. Biological molecular techniques are now able to type amyloidosis which were unidentified. Cardiac MRI and biomarkers allow a precise risk-stratification, especially in AL amyloidosis. If necessary, this prognostic evaluation may lead to rapid changes in the chemotherapy treatment. Emerging treatments rely on biotherapies, gene therapy, immunotherapy and blocking analogous agents. They give hope about an increase of survival of patients with systemic amyloidosis.

  6. Heart transplantation and cardiac amyloidosis: approach to screening and novel management strategies.

    PubMed

    Varr, Brandon C; Liedtke, Michaela; Arai, Sally; Lafayette, Richard A; Schrier, Stanley L; Witteles, Ronald M

    2012-03-01

    Limited data exist regarding screening methods and outcomes for orthotopic heart transplantation (OHT) in cardiac amyloidosis. As a result, uncertainty exists over the best approach to OHT for cardiac amyloidosis and for the timing of critical post-transplant therapies. This article reviews 6 patients who underwent OHT for cardiac amyloidosis at the Stanford University Amyloid Center from 2008 to present. All patients with light-chain amyloidosis received chemotherapy in the interval between OHT and autologous hematopoietic stem cell transplant. Five patients remain alive up to 25 months after OHT, without evidence of recurrent cardiac amyloid deposition. A novel strategy of OHT, followed by light-chain suppressive chemotherapy before autologous hematopoietic stem cell transplant, is feasible for patients with light-chain amyloidosis.

  7. Localized amyloidosis of the stomach mimicking a superficial gastric cancer.

    PubMed

    Kagawa, Miwako; Fujino, Yasuteru; Muguruma, Naoki; Murayama, Noriaki; Okamoto, Koichi; Kitamura, Shinji; Kimura, Tetsuo; Kishi, Kazuhiro; Miyamoto, Hiroshi; Uehara, Hisanori; Takayama, Tetsuji

    2016-06-01

    A 73-year-old man was referred to our hospital for further examination of a depressed lesion in the stomach found by cancer screening gastroscopy. A barium upper gastrointestinal series showed an area of irregular mucosa measuring 15 mm on the anterior wall of the gastric body. Esophagogastroduodenoscopy revealed a 15 mm depressed lesion on the anterior wall of the lower gastric body. We suspected an undifferentiated adenocarcinoma from the appearance and took some biopsies. However, histology of the specimens revealed amyloidal deposits in the submucosal layer without malignant findings. Congo red staining was positive for amyloidal protein and green birefringence was observed under polarized light microscopy. Congo red staining with prior potassium permanganate incubation confirmed the light chain (AL) amyloid type. There were no amyloid deposits in the colon or duodenum. Computed tomography of the chest, abdomen, and pelvis showed no remarkable findings. Thus, this case was diagnosed as a localized gastric amyloidosis characterized by AL type amyloid deposition in the mucosal or submucosal layer. As the clinical outcome of gastric AL amyloidosis seems favorable, this case is scheduled for periodic examination to recognize potential disease progression and has been stable for 2 years.

  8. AL amyloidosis with IgD-lambda monoclonal gammopathy and lambda-type Bence-Jones protein: successful treatment by autologous stem cell transplantation.

    PubMed

    Sakurai-Chin, Chanhyok; Ubara, Yoshifumi; Suwabe, Tatsuya; Hoshino, Junichi; Yonaha, Tomoki; Hasegawa, Eiko; Sumida, Keiichi; Hiramatsu, Rikako; Yamanouchi, Masayuki; Hayami, Noriko; Yamauchi, Junji; Tominaga, Naoyuki; Sawa, Naoki; Takemoto, Fumi; Masuoka, Kazuhiro; Takaichi, Kenmei; Oohashi, Kenichi

    2010-10-01

    A 45-year-old Japanese woman had been diagnosed with monoclonal gammopathy of undetermined significance (MGUS) featuring urinary Bence-Jones protein of the lambda type (BJP-lambda) for 11 years. She then developed eyelid purpura, dyspnea, and flank pain. Abdominal CT scans revealed renal infarction. Biopsy of the kidney, heart, jejunum, and skin demonstrated amyloid deposits in the vessel walls, but not in the glomeruli. She was diagnosed as having AL amyloidosis with IgD-lambda monoclonal gammopathy and BJP-lambda. Autologous stem cell transplantation (SCT) was done after chemotherapy with vincristine, daunorubicin, dexamethasone (VAD), and high-dose melphalan (HDM). This reduced the IgD level from 156 to 0.1 mg/dL, along with the disappearance of BJP, despite cerebral infarction during chemotherapy. We recommend SCT for patients with IgD-associated AL amyloidosis.

  9. Nuclear imaging of amyloidosis.

    PubMed

    Cytawa, Wojciech; Teodorczyk, Jacek; Lass, Piotr

    2014-01-01

    Summary Amyloidosis is a clinical condition caused by deposition of various protein fibrills in extracellular space. The presented symptoms depend on the type of deposits and the organ or organs involved. The correct diagnosis is often difficult, due to lack of nonivasive imaging techniques and insufficiency of morphological imaging procedures delievered by radiology. We presented a list of potential radiopharmaceuticals that can be used in detecting various types of amyloidoses. (123)I-SAP proved to have high sensitivity in imaging of AA and AL amyloidosis in visceral organs. (99m)Tc-Aprotinin was found to be useful in detecting cardiac amyloidosis. A couple of classical radiotracers, such as (201)Tl, (123)I-mIBG, together with (111)In-antimyosin were also tested for accuracy in cardiac imaging, however the main problem was low specificity. Potential applicability was also found in case of some bone-seeking agents and other radiotracers, e.g. (67)Ga-citrate and (99m)Tc-penta-DMSA. High sensitivity and specificity was achieved with β2-microglobulin labeled with (131)I or (111)In. Among PET tracers, (11)C-PIB deserves more attention, because it may have an important role in diagnosing of AD in the near future. Further clinical studies are expected to take place, because noninvasive diagnosing and monitoring of amyloidosis is still a challenge.

  10. Amyloidosis: an unusual cause of portal hypertension

    PubMed Central

    Laborda, Lorena Silva; Bernardelli, Raquel; Pinesi, Henrique Trombini; Silva, Marilia Polo Minguete e; Chiavelli, Viviane; Simões, Angélica Braz; Felipe-Silva, Aloisio

    2016-01-01

    Amyloidosis comprises a group of diseases that occurs in five to nine cases per million patients per year worldwide irrespective of its classification. Although the hepatic involvement in primary amyloidosis is frequent, the clinical manifestations of liver amyloidosis are mild or even absent. The authors report the case of an aged man who complained of diffuse abdominal pain and marked weight loss and presented clinical signs of hepatopathy. Clinical workup revealed portal hypertension with ascites, hemorrhoids, and esophageal varices. The laboratory tests showed the cholestatic pattern of liver enzymes, hyperbilirubinemia, renal insufficiency and massive proteinuria accompanied by the presence of serum pike of monoclonal lambda light chain protein. The outcome was unfavorable, and the patient died. The autopsy findings revealed the diagnosis of amyloidosis predominantly involving the liver and kidneys. The bone marrow examination demonstrated the deposition of amyloid material associated with clonal plasma cells infiltration. The authors call attention to portal hypertension as a rare manifestation of primary amyloidosis. Meanwhile, this diagnosis should be taken into account whenever the hepatopathy is accompanied by laboratory abnormalities consistent with hepatic space-occupying lesions concomitantly with other organs involvement. In the case reported herein, kidney involvement was also present with renal failure, massive proteinuria with monoclonal serum gammopathy, what reinforced the diagnostic possibility of primary amyloidosis. PMID:27547738

  11. IgD multiple myeloma: Clinical, biological features and prognostic value of the serum free light chain assay.

    PubMed

    Djidjik, R; Lounici, Y; Chergeulaïne, K; Berkouk, Y; Mouhoub, S; Chaib, S; Belhani, M; Ghaffor, M

    2015-09-01

    IgD multiple myeloma (MM) is a rare subtype of myeloma, it affects less than 2% of patients with MM. To evaluate the clinical and prognostic attributes of serum free light chains (sFLCs) analysis, we examined 17 cases of IgD MM. From 1998 to 2012, we obtained 1250 monoclonal gammapathies including 590 multiple myeloma and 17 patients had IgD MM. With preponderance of men patients with a mean age at diagnosis of: 59±12years. Patients with IgD MM have a short survival (Median survival=9months). The presenting features included: bone pain (75%), lymphadenopathy (16%), hepatomegaly (25%), splenomegaly (8%), associated AL amyloidosis (6%), renal impairment function (82%), infections (47%), hypercalcemia (37%) and anemia (93%). Serum electrophoresis showed a subtle M-spike (Mean=13.22±10g/L) in all patients associated to a hypogammaglobulinemia. There was an over-representation of Lambda light chain (65%); high serum β2-microglobulin in 91% and Bence Jones proteinuria was identified in 71%. The median rate of sFLCs κ was 19.05mg/L and 296.75mg/L for sFLCs λ. sFLCR was abnormal in 93% of patients and it showed concordance between baseline sFLCR and the survival (P=0.034). The contribution of FLC assay is crucial for the prognosis of patients with IgD MM.

  12. Acquired factor X deficiency developed four years after autologous transplantation in a patient with multiple myeloma associated with systemic AL amyloidosis.

    PubMed

    Takemura, Tomonari; Fukatsu, Yusuke; Nagata, Yasuyuki; Asahina, Aya; Yokota, Daisuke; Hirano, Isao; Yagyu, Tomohiro; Ono, Takaaki; Katsumi, Akira; Ohnishi, Kazunori

    2014-05-01

    We describe a case of acquired factor X deficiency after high-dose melphalan with autologous stem cell transplantation (HDM/ASCT) for multiple myeloma (MM) with systemic AL amyloidosis. A 68-year-old woman with renal amyloidosis was diagnosed as having MM in 2007. She achieved a partial response after VAD (vincristine, adriamycin, dexamethasone) therapy and HDM/ASCT. In December 2011, coagulation tests revealed a prolonged prothrombin time (PT) of 17.6 sec and she was administered vitamin K. In January 2012, she received low anterior resection with colostomy for rectal cancer. She received fresh frozen plasma (FFP) infusion but the perioperative bleeding tendency persisted. In February 2012, she was referred from surgery for colostomy closure. She showed no progression of MM and had prolonged PT, corrected by mixing with normal plasma. Factor X activity was markedly decreased. She was diagnosed as having an acquired factor X deficiency and was given FFP infusion for colostomy closure. Although acquired factor X deficiency after HDM/ASCT for MM with systemic AL amyloidosis is rare, we should be aware of the possibility of this disease in MM patients with a bleeding tendency.

  13. 18F-Florbetapir Binds Specifically to Myocardial Light Chain and Transthyretin Amyloid Deposits: An Autoradiography Study

    PubMed Central

    Park, Mi-Ae; Padera, Robert F.; Belanger, Anthony; Dubey, Shipra; Hwang, David H.; Veeranna, Vikas; Falk, Rodney H.; Di Carli, Marcelo F.; Dorbala, Sharmila

    2015-01-01

    Background 18F-florbetapir is a promising imaging biomarker for light chain (AL) and transthyretin (ATTR) cardiac amyloidosis. Our aim, using human autopsy myocardial specimens, was to test the hypothesis that 18F-florbetapir binds specifically to myocardial AL and ATTR amyloid deposits. Methods and Results We studied myocardial sections from 30 subjects with autopsy documented AL (N = 10), ATTR (N = 10) and non-amyloid controls (N = 10), using 18F-florbetapir and cold florbetapir compound and digital autoradiography. Total and non-specific binding of 18F-florbetapir was determined using the maximum signal intensity values. Specific binding of 18F-florbetapir was calculated by subtracting non-specific from total binding measurements (in decays per minute/mm2, DPM mm2), and was compared to cardiac structure and function on echocardiography and the histological extent of amyloid deposits. Diffuse or focally increased 18F-florbetapir uptake was noted in all AL and ATTR samples and in none of the control samples. Compared to control samples, mean 18F-florbetapir specific uptake was significantly higher in the amyloid samples (0.94 ± 0.43 vs. 2.00 ± 0.58 DPM/mm2, p < 0.001), and in the AL compared to the ATTR samples (2.48 ± 0.40 vs. 1.52 ± 0.22 DPM/mm2, p < 0.001). The samples from subjects with atypical echocardiographic features of amyloidosis showed quantitatively more intense 18F-florbetapir specific uptake compared to control samples (1.50 ± 0.17 vs. 0.94 ± 0.43 DPM/mm2, p = 0.004), despite smaller amyloid extent than in subjects with typical echocardiograms. Conclusions 18F-florbetapir specifically binds to myocardial AL and ATTR deposits in humans and offers the potential to screen for the two most common types of myocardial amyloid. PMID:26259579

  14. A molecular model for self-assembly of amyloid fibrils: Immunoglobulin light chains

    SciTech Connect

    Stevens, F.J.; Myatt, E.A.; Westholm, F.A.

    1995-08-29

    The formation and pathological deposition of amyloid fibrils are defining features of many acquired and inherited disorders, including primary or light-chain-associated amyloidosis, Alzheimer`s disease, and adult-onset diabetes. No pharmacological methods exist to block this process or to effect the removal of fibrils from tissue, and thus, little can be done to prevent organ failure and ultimate death that result from deposition of amyloid. Knowledge of the pathogenesis, treatment, or prevention of these presently incurable diseases is limited due to the relative paucity of information regarding the biophysical basis of amyloid formation. Antibody light chains of different amino acid sequence show differential amyloid-forming tendencies and, as such, can provide insight into the structural organization of amyloid fibrils as well as into basic mechanisms of protein self-assembly. We have compared primary structures of 180 human monoclonal light chains and have identified particular residues and positions within the variable domain that differentiate amyloid-from nonamyloid-associated proteins. We propose a molecular model that accounts for amyloid formation by antibody light chains and might also have implications for other forms of amyloidosis. 24 refs., 2 figs., 1 tab.

  15. Extrusion of amyloid fibrils to the extracellular space in experimental mesangial AL-amyloidosis: transmission and scanning electron microscopy studies and correlation with renal biopsy observations.

    PubMed

    Teng, Jiamin; Turbat-Herrera, Elba A; Herrera, Guillermo A

    2014-04-01

    In vitro studies have provided much information regarding the process of glomerular AL-amyloidogenesis. Research efforts have been successful in deciphering how glomerulopathic light chains interact with mesangial cells. The sequential steps involved in the genesis of amyloid fibrils include interactions with surface caveolae in mesangial cells and internalization of the monoclonal light chains through a clathrin-mediated process followed by trafficking in the mesangial cells to the mature lysosomal compartment where fibrils are formed. This manuscript focuses on how mesangial cells, once amyloid has been formed, deliver the fibrils to the extracellular matrix. The delivery of amyloid fibrils to the outside of the cells is carried out by lysosomes, which abut the mesangial cell membranes and extrude their contents into the extracellular space. This final step responsible for the fibrils to be present predominantly in the extracellular space is well demonstrated with scanning electron microscopy.

  16. Clathrin heavy chain, light chain interactions.

    PubMed Central

    Winkler, F K; Stanley, K K

    1983-01-01

    Purified pig brain clathrin can be reversibly dissociated and separated into heavy chain trimers and light chains in the presence of non-denaturing concentrations of the chaotrope thiocyanate. The isolated heavy chain trimers reassemble into regular polygonal cage structures in the absence of light chains. The light chain fraction can be further resolved into its two components L alpha and L beta which give different one-dimensional peptide maps. Radiolabelled light chains bind with high affinity (KD < 10(-10) M) to heavy chain trimers, to heavy chain cages and to a 110,000 mol. wt. tryptic fragment of the heavy chain. Both light chains compete with each other and with light chains from other sources for the same binding sites on heavy chains and c.d. spectroscopy shows that the two pig brain light chains possess very similar structures. We conclude that light chains from different sources, despite some heterogeneity, have a highly conserved, high affinity binding site on the heavy chain but are not essential for the formation of regular cage structures. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 8. PMID:10872336

  17. Physicochemical consequences of amino acid variations that contribute to fibril formation by immunoglobulin light chains.

    PubMed Central

    Raffen, R.; Dieckman, L. J.; Szpunar, M.; Wunschl, C.; Pokkuluri, P. R.; Dave, P.; Wilkins Stevens, P.; Cai, X.; Schiffer, M.; Stevens, F. J.

    1999-01-01

    The most common form of systemic amyloidosis originates from antibody light chains. The large number of amino acid variations that distinguish amyloidogenic from nonamyloidogenic light chain proteins has impeded our understanding of the structural basis of light-chain fibril formation. Moreover, even among the subset of human light chains that are amyloidogenic, many primary structure differences are found. We compared the thermodynamic stabilities of two recombinant kappa4 light-chain variable domains (V(L)s) derived from amyloidogenic light chains with a V(L) from a benign light chain. The amyloidogenic V(L)s were significantly less stable than the benign V(L). Furthermore, only the amyloidogenic V(L)s formed fibrils under native conditions in an in vitro fibril formation assay. We used site-directed mutagenesis to examine the consequences of individual amino acid substitutions found in the amyloidogenic V(L)s on stability and fibril formation capability. Both stabilizing and destabilizing mutations were found; however, only destabilizing mutations induced fibril formation in vitro. We found that fibril formation by the benign V(L) could be induced by low concentrations of a denaturant. This indicates that there are no structural or sequence-specific features of the benign V(L) that are incompatible with fibril formation, other than its greater stability. These studies demonstrate that the V(L) beta-domain structure is vulnerable to destabilizing mutations at a number of sites, including complementarity determining regions (CDRs), and that loss of variable domain stability is a major driving force in fibril formation. PMID:10091653

  18. Nasal and ocular amyloidosis in a 15-year-old horse.

    PubMed

    Østevik, Liv; Gunnes, Gjermund; de Souza, Gustavo A; Wien, Tale N; Sørby, Randi

    2014-08-27

    Localized nasal, conjunctival and corneal amyloidosis was diagnosed in a 15-year-old pony with nasal and conjunctival masses and severe dyspnoea. Multiple swellings had been evident in the nostrils for at least two years and had gradually increased in size before presentation due to dyspnoea and exercise intolerance. Surgical debulking of the masses was performed and histological examination revealed large amounts of extracellular, hyaline, eosinophilic, Congo red positive material in the lamina propria of the nasal mucosa. A tentative diagnosis of localized nasal amyloidosis was made. The treatment relieved the clinical signs, however, the nasal masses recurred and bilateral conjunctival, papillary masses developed. The horse was euthanized. Nodular nasal and papillary conjunctival masses consisting of rubbery, grey to yellow tissue were found at necropsy. At the limbus this tissue infiltrated and expanded the cornea. The masses consisted of amyloid and moderate infiltrates of T lymphocytes and B lymphocytes were present in the tissue. No predominance of either cell type was observed and no distinct neoplastic mass could be identified. Ultrastructural examination of the nasal mucosa and cornea confirmed the presence of abundant extracellular deposits of non-branching fibrils ranging from 9-11 nm in diameter consistent with amyloid. Immunohistochemistry of amyloid revealed no labelling for AA amyloid, and no peptides representing serum amyloid A (SAA) were detected by microscopic laser dissection and subsequent mass spectrometry. Peptides from immunoglobulin kappa-like light chains were detected and are suggestive of AL amyloidosis, however the results were inconclusive and a final identification of the amyloid protein could not be made.Nasal amyloidosis is a clinical entity of localized amyloid deposits in the horse. Localized amyloidosis involving the conjunctiva of the horse is previously described in only seven cases and the present case is the first case of

  19. A Case of Conjunctival Amyloidosis with Repeated Subconjunctival Hemorrhage

    PubMed Central

    Ando, Takaaki; Saito, Mamiko; Tawada, Ayako; Yotsukura, Jiro; Yamamoto, Shuichi

    2017-01-01

    Conjunctival amyloidosis is a very rare disease, and its presence may be a sign of systemic amyloidosis. We present our ocular and systemic findings in a patient with conjunctival amyloidosis. A 43-year-old man had repeated subconjunctival hemorrhages (SCHs) for two years and was referred to the Chiba University Hospital. He had comprehensive ophthalmological and systemic examinations to determine the cause of the SCHs. His visual acuities were 1.2 OU, and the intraocular pressures were 13-14 mmHg OU. Magnetic resonance imaging was normal. Initially, the SCH was the only abnormality. After 3 months, the SCH had partially cleared, and a pink mass was detected in the superior area of the subconjunctiva. Partial biopsy and histopathological examinations showed a greenish birefringence and dichroism under polarized light illumination. The birefringence was located in amyloid fibers. Immunofixation electrophoresis detected λ-light chain abnormality in the ocular biopsy specimen but systemic examinations did not find any lesions. Multiple myeloma was ruled out, and the patient is being followed closely to detect any early signs of systemic amyloidosis. Because repeated SCHs might be initial signs of systemic amyloidosis, patients with conjunctival amyloidosis should be comprehensively examined for systemic amyloidosis because of its poor life prognosis. PMID:28326212

  20. [White blood cell lysis syndrome after autologous peripheral blood stem cell transplantation in the treatment of renal AL amyloidosis. Case report].

    PubMed

    Gatica, Antonio; Bertin, Pablo; Tagle, Rodrigo

    2006-06-01

    The treatment of AL amyloidosis was not successful until the advent of myeloablative chemotherapy consisting of high-dose intravenous melphalan followed by autologous peripheral blood stem cell transplantation. This new treatment has achieved better survival rates and, remarkably, it has obtained complete remission. Among patients with renal involvement, achievement of a complete hematological response was associated with a 50% reduction in proteinuria and stable creatinine clearance in more than 2/3 of patients. Despite of these excellent results, this new therapy is associated with significant toxicity, including the development of acute renal failure due to white blood cell lysis syndrome. We report a 59 year-old female with a nephrotic syndrome due to primary amyloidosis successfully treated autologous stem cell transplantation who developed acute renal failure caused by white blood cell lysis syndrome. The patient required treatment with granulocytic colony stimulating factor and intermittent hemofiltration and was discharged 23 days after melphalan administration with a satisfactory renal function and white blood cell count. After one year of follow up, she maintains a good glomerular filtration rate, a proteinuria of less than, 1 g/day and normal hematological values.

  1. Epigallocatechin-3-gallate preferentially induces aggregation of amyloidogenic immunoglobulin light chains

    PubMed Central

    Hora, Manuel; Carballo-Pacheco, Martin; Weber, Benedikt; Morris, Vanessa K.; Wittkopf, Antje; Buchner, Johannes; Strodel, Birgit; Reif, Bernd

    2017-01-01

    Antibody light chain amyloidosis is a rare disease caused by fibril formation of secreted immunoglobulin light chains (LCs). The huge variety of antibody sequences puts a serious challenge to drug discovery. The green tea polyphenol epigallocatechin-3-gallate (EGCG) is known to interfere with fibril formation in general. Here we present solution- and solid-state NMR studies as well as MD simulations to characterise the interaction of EGCG with LC variable domains. We identified two distinct EGCG binding sites, both of which include a proline as an important recognition element. The binding sites were confirmed by site-directed mutagenesis and solid-state NMR analysis. The EGCG-induced protein complexes are unstructured. We propose a general mechanistic model for EGCG binding to a conserved site in LCs. We find that EGCG reacts selectively with amyloidogenic mutants. This makes this compound a promising lead structure, that can handle the immense sequence variability of antibody LCs. PMID:28128355

  2. Unusual Case of Cardiac Amyloidosis Preceded by a Twenty-year History of Dilated Cardiomyopathy and Heart Failure.

    PubMed

    Shimada, Seijiro; Maekura, Shunji; Ino, Hikaru; Matsuura, Masayosi; Masunaga, Nobutaka; Matsumoto, Takahiro; Hama, Junkichi

    2016-01-01

    Amyloidosis is a well-known but uncommon disease, and the physician must maintain a high index of suspicion in order to make a timely diagnosis. The expected survival of patients with cardiac amyloidosis is generally poor. In particular, survival has been reported to be 4-12 months for patients with amyloid light-chain amyloidosis with congestive heart failure. We herein report a rare case of cardiac amyloidosis in which the patient presented with cardiac hypertrophy after a 20-year history of dilated cardiomyopathy and heart failure.

  3. Structural basis of light chain amyloidogenicity: comparison of the thermodynamic properties, fibrillogenic potential and tertiary structural features of four vλ6 proteins

    SciTech Connect

    Wall, J.S.; Gupta, V.; Wilkerson, M.; Schell, M.; Loris, R.; Adams, P.; Solomon, A.; Stevens, F.; Dealwis, C.

    2004-04-01

    Primary (AL) amyloidosis results from the pathologic deposition of monoclonal light chains as amyloid fibrils. Studies of recombinant-derived variable region (V{sub L}) fragments of these proteins have shown an inverse relationship between thermodynamic stability and fibrillogenic potential. Further, ionic interactions within the V{sub L} domain were predicted to influence the kinetics of light chain fibrillogenicity, as evidenced from our analyses of a relatively stable V{sub {lambda}}6 protein (Jto) with a long range electrostatic interaction between Asp and Arg side chains at position 29 and 68, respectively, and an unstable, highly fibrillogenic V{sub {lambda}}6 protein (Wil) that had neutral amino acids at these locations. To test this hypothesis, we have generated two Jto-related mutants designed to disrupt the interaction between Asp 29 and Arg 68 (JtoD29A and JtoR68S). Although the thermodynamic stabilities of unfolding for these two molecules were identical, they exhibited very different kinetics of fibril formation: the rate of JtoD29A fibrillogenesis was slow and comparable to the parent molecule, whereas that of JtoR68S was significantly faster. High-resolution X-ray diffraction analyses of crystals prepared from the two mutants having the same space group and unit cell dimensions revealed no significant main-chain conformational changes. However, several notable side-chain alterations were observed in JtoR68S, as compared with JtoD29A, that resulted in the solvent exposure of a greater hydrophobic surface and modifications in the electrostatic potential surface. We posit that these differences contributed to the enhanced fibrillogenic potential of the Arg 68 mutant, since both Jto mutants lacked the intrachain ionic interaction and were equivalently unstable. The information gleaned from our studies has provided insight into structural parameters that in addition to overall thermodynamic stability, contribute to the fibril forming propensity of

  4. The amino acid sequence of a carbohydrate-containing immunoglobulin-light-chain-type amyloid-fibril protein.

    PubMed Central

    Tveteraas, T; Sletten, K; Westermark, P

    1985-01-01

    The amino acid sequence of an amyloid-fibril protein Es492 of immunoglobulin-lambda-light-chain origin (AL) was elucidated. The amyloid fibrils were obtained from the spleen of a patient who died from systemic amyloidosis. The amino acid sequence was elucidated from structural studies of peptides derived from digestion of the protein with trypsin, thermolysin, chymotrypsin and Staphylococcus aureus V8 proteinase and from cleavage of the protein with CNBr and BNPS-skatole. A heterogeneity in the length of the polypeptide was seen in the C-terminal region. The protein was by sequence homology to other lambda-chains shown to be of the V lambda II subgroup. Although an extensive homology was seen, some amino acid residues in positions 26, 31, 32, 40, 44, 93, 97, 98 and 99 have not previously been reported in these positions of V lambda II proteins. The significance of these residues in the fibril formation is unclear. The protein was found to contain carbohydrate, with glycosylation sites in two of the hypervariable regions. PMID:3936482

  5. Amyloid Goiter Associated with Amyloidosis Secondary to Rheumatoid Arthritis

    PubMed Central

    Uzum, Gungor; Kaya, Fatih Oner; Uzum, Ayse Kubat; Kucukyilmaz, Meltem; Duzkoylu, Yigit; Leblebici, Cem; Koc, Oguz

    2013-01-01

    Amyloidosis refers to a variety of conditions in which amyloid proteins are abnormally deposited in organs and/or tissues. The most common forms of systemic amyloidosis are primary amyloidosis (PA) of light chains and secondary amyloidosis (SA) caused by chronic inflammatory diseases such as rheumatoid arthritis (RA). Although involvement of the thyroid gland by amyloid is a relatively common phenomenon, clinically significant enlargement of the thyroid owing to amyloid deposition is a rare occurrence. In SA, the deposition of amyloid associated (AA) protein is associated with atrophy of thyroid follicles. The clinical picture of these patients is characterized by rapid, painless thyroid gland enlargement which may be associated with dysphagia, dyspnea, or hoarseness. Thyroid function is not impaired in most cases. Although amyloid goitre secondary to systemic amyloidosis due to chronic inflammatory diseases is relatively common, specifically related to RA is much more uncommon one and it is reported less in the literature. In this report, A 52-old-year female patient with amyloid goiter associated with amyloidosis secondary to rheumatoid arthritis is presented. PMID:24368922

  6. Hereditary apolipoprotein AI-associated renal amyloidosis: A diagnostic challenge.

    PubMed

    Samillán-Sosa, Kelly Del Rocío; Sención-Martínez, Gloria; Lopes-Martín, Vanessa; Martínez-González, Miguel Angel; Solé, Manel; Arostegui, Jose Luis; Mesa, Jose; García-Díaz, Juan de Dios; Rodríguez-Puyol, Diego; Martínez-Miguel, Patricia

    2015-01-01

    Hereditary renal amyloidosis is an autosomal dominant condition with considerable overlap with other amyloidosis types. Differential diagnosis is complicated, but is relevant for prognosis and treatment. We describe a patient with nephrotic syndrome and progressive renal failure, who had a mother with renal amiloidosis. Renal biopsy revealed amyloid deposits in glomerular space, with absence of light chains and protein AA. We suspected amyloidosis with fibrinogen A alpha chain deposits, which is the most frequent cause of hereditary amyloidosis in Europe, with a glomerular preferential affectation. However, the genetic study showed a novel mutation in apolipoprotein AI. On reviewing the biopsy of the patient's mother similar glomerular deposits were found, but there were significant deposits in the renal medulla as well, which is typical in APO AI amyloidosis. The diagnosis was confirmed by immunohistochemistry. Apo AI amyloidosis is characterized by slowly progressive renal disease and end-stage renal disease occurs aproximately 3 to 15 years from initial diagnosis. Renal transplantation offers an acceptable graft survival and in these patients with hepatorenal involvement simultaneous liver and kidney transplantation could be considered.

  7. Leukocyte chemotactic factor 2 amyloidosis (ALECT2) is a common form of renal amyloidosis among Egyptians.

    PubMed

    Larsen, Christopher P; Ismail, Wesam; Kurtin, Paul J; Vrana, Julie A; Dasari, Surendra; Nasr, Samih H

    2016-04-01

    Large case series of renal amyloidosis subtypes have recently been published in the United States and Europe showing AL amyloidosis to be the predominant subtype in this part of the world. However, the most common subtypes of renal amyloidosis throughout the rest of the world are unknown. We present here the first large case series detailing the subtypes of renal amyloidosis among Egyptians. In this population, AA amyloidosis was the most common type of amyloidosis on renal biopsy at 48%. The newly described leukocyte chemotactic factor 2 amyloidosis (ALECT2) was the second most common type and represented nearly one-third of renal amyloid cases at 31%. AL accounted for only 20% of cases. The pathologic findings in ALECT2 cases were similar to those previously described in other case series. Thus ALECT2, which was initially thought to affect mainly Hispanics in the United States, appears to represent an important and likely underrecognized etiology of chronic kidney disease among Egyptians and probably in other ethnic groups around the world.

  8. Leukocyte Cell-Derived Chemotaxin 2-Associated Amyloidosis: A Recently Recognized Disease with Distinct Clinicopathologic Characteristics.

    PubMed

    Nasr, Samih H; Dogan, Ahmet; Larsen, Christopher P

    2015-11-06

    derived from leukocyte cell-derived chemotaxin 2 amyloidosis as Ig light chain-derived amyloidosis to avoid harmful chemotherapy.

  9. Myosin light chains: Teaching old dogs new tricks

    PubMed Central

    Heissler, Sarah M; Sellers, James R

    2014-01-01

    The myosin holoenzyme is a multimeric protein complex consisting of heavy chains and light chains. Myosin light chains are calmodulin family members which are crucially involved in the mechanoenzymatic function of the myosin holoenzyme. This review examines the diversity of light chains within the myosin superfamily, discusses interactions between the light chain and the myosin heavy chain as well as regulatory and structural functions of the light chain as a subunit of the myosin holoenzyme. It covers aspects of the myosin light chain in the localization of the myosin holoenzyme, protein-protein interactions and light chain binding to non-myosin binding partners. Finally, this review challenges the dogma that myosin regulatory and essential light chain exclusively associate with conventional myosin heavy chains while unconventional myosin heavy chains usually associate with calmodulin. PMID:26155737

  10. Primary cerebral low-grade B-cell lymphoma, monoclonal immunoglobulin deposition disease, cerebral light chain deposition disease and “aggregoma”: an update on classification and diagnosis

    PubMed Central

    2013-01-01

    Background This work aims to add evidence and provide an update on the classification and diagnosis of monoclonal immunoglobulin deposition disease (MIDD) and primary central nervous system low-grade lymphomas. MIDD is characterized by the deposition of light and heavy chain proteins. Depending on the spatial arrangement of the secreted proteins, light chain-derived amyloidosis (AL) can be distinguished from non-amyloid light chain deposition disease (LCDD). We present a case of an extremely rare tumoral presentation of LCDD (aggregoma) and review the 3 previously published LCDD cases and discuss their presentation with respect to AL. Case presentation A 61-year-old woman presented with a 3½-year history of neurologic symptoms due to a progressive white matter lesion of the left subcortical parieto-insular lobe and basal ganglia. 2 former stereotactic biopsies conducted at different hospitals revealed no evidence of malignancy or inflammation; thus, no therapy had been initiated. After performing physiological and functional magnetic resonance imaging (MRI), the tumor was removed under intraoperative monitoring at our department. Histological analysis revealed large amorphous deposits and small islands of lymphoid cells. Conclusion LCCD is a very rare and obscure manifestation of primary central nervous system low-grade lymphomas that can be easily misdiagnosed by stereotactic biopsy sampling. If stereotactic biopsy does not reveal a definite result, a “wait-and-see” strategy can delay possible therapy for this disease. The impact of surgical removal, radiotherapy and chemotherapy in LCDD obviously remains controversial because of the low number of relevant cases. PMID:23947787

  11. Myeloma light chains are ligands for cubilin (gp280).

    PubMed

    Batuman, V; Verroust, P J; Navar, G L; Kaysen, J H; Goda, F O; Campbell, W C; Simon, E; Pontillon, F; Lyles, M; Bruno, J; Hammond, T G

    1998-08-01

    Although myeloma light chains are known to undergo receptor-mediated endocytosis in the kidney, the molecular identity of the receptor has not been characterized. We examined the interaction between cubilin (gp280) and four species of light chains isolated from the urine of patients with multiple myeloma. Four lines of evidence identify cubilin, a giant glycoprotein receptor, which is restricted in distribution to endocytic scavenger pathways and which has potent effects on endosomal trafficking, as a potentially physiologically relevant binding site for light chains: 1) light chains coeluted during immunoaffinity purification of cubilin; 2) polyclonal antisera to cubilin but not control sera, displaced human light chain binding from rat renal brush-border membranes; 3) cubilin bound to multiple species of light chains during surface plasmon resonance; 4) anti-cubilin antiserum interfered with light chain endocytosis by visceral yolk sac epithelial cells. However, both binding of light chains to brush-border membranes and endocytosis of light chains by yolk sac epithelial cells were only partially inhibited by anticubilin antibodies, suggesting presence of additional or alternate binding sites for light chains. Excess light chain had a potent inhibitory effect on endosomal fusion in vitro. Binding showed dose and time-dependent saturability with low-affinity, high-capacity equilibrium binding parameters. These data demonstrate that cubilin plays a role in the endocytosis and trafficking of light chains in renal proximal tubule cells.

  12. Peptide p5 binds both heparinase-sensitive glycosaminoglycans and fibrils in patient-derived AL amyloid extracts.

    PubMed

    Martin, Emily B; Williams, Angela; Heidel, Eric; Macy, Sallie; Kennel, Stephen J; Wall, Jonathan S

    2013-06-21

    In previously published work, we have described heparin-binding synthetic peptides that preferentially recognize amyloid deposits in a mouse model of reactive systemic (AA) amyloidosis and can be imaged by using positron and single photon emission tomographic imaging. We wanted to extend these findings to the most common form of visceral amyloidosis, namely light chain (AL); however, there are no robust experimental animal models of AL amyloidosis. To further define the binding of the lead peptide, p5, to AL amyloid, we characterized the reactivity in vitro of p5 with in situ and patient-derived AL amyloid extracts which contain both hypersulfated heparan sulfate proteoglycans as well as amyloid fibrils. Histochemical staining demonstrated that the peptide specifically localized with tissue-associated AL amyloid deposits. Although we anticipated that p5 would undergo electrostatic interactions with the amyloid-associated glycosaminoglycans expressing heparin-like side chains, no significant correlation between peptide binding and glycosaminoglycan content within amyloid extracts was observed. In contrast, following heparinase I treatment, although overall binding was reduced, a positive correlation between peptide binding and amyloid fibril content became evident. This interaction was further confirmed using synthetic light chain fibrils that contain no carbohydrates. These data suggest that p5 can bind to both the sulfated glycosaminoglycans and protein fibril components of AL amyloid. Understanding these complex electrostatic interactions will aid in the optimization of synthetic peptides for use as amyloid imaging agents and potentially as therapeutics for the treatment of amyloid diseases.

  13. Isolation of cardiac myosin light-chain isotypes by chromatofocusing. Comparison of human cardiac atrial light-chain 1 and foetal ventricular light-chain 1.

    PubMed

    Vincent, N D; Cummins, P

    1985-04-01

    Cardiac myosin light chain isotypes have been resolved using chromatofocusing, a new preparative column chromatographic technique. The method relies on production of narrow-range, shallow and stable pH gradients using ion-exchange resins and buffers with even buffering capacity over the required pH range. Light chains were resolved in order of decreasing isoelectric point in the pH range 5.2-4.5. Gradients of delta pH = 0.004-0.006/ml elution volume were achieved which were capable of resolving light chains with isoelectric point differences of only 0.03. Analytical isoelectric focusing of light chains in polyacrylamide gels could be used to predict the results of preparative chromatofocusing for method development. Chromatofocusing was capable of resolving human and bovine cardiac light chain 1 and 2 subunits, atrial (ALC) and ventricular (VLC) light chain isotypes and homologous VLC-2 and VLC-2* light chains. The technique was used to purify and resolve the human foetal ventricular light chain 1 (FLC-1) from adult ventricular light chain 1 (VLC-1) present in foetal ventricles and the atrial light chain 1 (ALC-1) in adult atria. Comparative peptide mapping studies and amino acid analyses were carried out on FLC-1 and ALC-1. No differences were detected between FLC-1 and ALC-1 using three different proteases and amino acid compositions were similar with the exception of glycine content. The studies indicate that FLC-1 and ALC-1 are homologous, and possibly identical, light chains. Comparison of human FLC-1/ALC-1 with VLC-1 suggested marked structural and chemical differences in these light chain isotypes, in particular in the contents of methionine, proline, lysine and alanine residues. Differences in the contents of these residues were also apparent in the corresponding bovine atrial and ventricular light chains [Wikman-Coffelt, J. & Srivastava, S. (1979) FEBS Lett. 106, 207-212]. The latter three residues are known to be rich in the N-termini of cardiac and

  14. Influence of the germline sequence on the thermodynamic stability and fibrillogenicity of human lambda 6 light chains.

    PubMed

    del Pozo Yauner, Luis; Ortiz, Ernesto; Sánchez, Rosalba; Sánchez-López, Rosana; Güereca, Leopoldo; Murphy, Charles L; Allen, Amy; Wall, Jonathan S; Fernández-Velasco, D Alejandro; Solomon, Alan; Becerril, Baltazar

    2008-08-01

    Light chain-associated amyloidosis is a fatal disease characterized by the aggregation and pathologic deposition of monoclonal light chain-related fragments as amyloid fibrils in organs or tissues throughout the body. Notably, it has been observed that proteins encoded by the lambda variable light chain (V(L)) gene segment 6a are invariably associated with amyloid deposition; however, the contribution of the gene to this phenomenon has not been established. In this regard, we have determined the thermodynamic stability and kinetics of in vitro fibrillogenesis of a recombinant (r) V(L) protein, designated 6aJL2, which contains the predicted sequences encoded by the 6a and JL2 germline genes. Additionally, we studied a 6a mutant (6aJL2-Arg25Gly), that is present in approximately 25% of all amyloid-associated lambda6 light chains. Remarkably, the wild-type 6aJL2 protein was more stable than were all known amyloidogenic kappa and lambda light chains for which stability parameters are available; more importantly, it was even more so (and less fibrillogenic) than the only clinically proven nonamyloidogenic lambda6 protein, Jto. Conversely, the mutated 6aJL2-R25G molecule was considerably less stable and more fibrillogenic than was the native 6aJL2. Our data indicate that the propensity of lambda6 light chains to form amyloid can not be attributed to thermodynamic instability of the germline-encoded Vlambda6 domain, but rather, is dependent on sequence alterations that render such proteins amyloidogenic.

  15. Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

    SciTech Connect

    Josephson, Matthew P.; Sikkink, Laura A.; Penheiter, Alan R.; Burghardt, Thomas P.; Ajtai, Katalin

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V{sub max} and K{sub M} for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant

  16. Amyloidosis: A cancer-derived paraproteinemia and kidney involvement.

    PubMed

    Małyszko, Jolanta; Kozłowska, Klaudia; Małyszko, Jacek Stanisław

    2017-01-30

    Amyloidosis is the general term describing the extracellular tissue deposition of fibrils composed of low molecular weight subunits of a variety of proteins. There are multiple different human protein precursors of amyloid fibrils. Amyloid deposits are stained using Congo Red and show typical apple-green birefringence in polarized microscopy. Nowadays, a novel technique LMD/MS technique or laser microdissection combined with mass spectrometry help to diagnose amyloidosis. Amyloidosis of the kidney is typically classified as being either one of two types: AL or AA. Less common is the hereditary amyloidosis. Clinical manifestations are usually determined by the type of precursor protein, the tissue distribution, and the amount of amyloid deposition. Renal manifestation is usually present as asymptomatic proteinuria or clinically apparent nephrotic syndrome. In some patients clinical presentation include impaired kidney function with no or mild proteinuria. Patients with renal amyloidosis who progress to end-stage renal disease (ESRD) can be treated with either dialysis or renal transplantation. Diagnosis of amyloidosis is prerequisite to consider treatment options to avoid unnecessary chemotherapy. Treatment of amyloidosis is aimed at decreasing the precursors of fibrillary proteins and/or decrease in synthesis/deposition of amyloid fibrils. It depends upon the type of amyloidosis and cause of excess fibril production.

  17. Intestinal pseudo-obstruction associated with amyloidosis.

    PubMed

    Liapis, Konstantinos; Michelis, Fotios V; Delimpasi, Sosanna; Karmiris, Themistoklis

    2011-06-01

    Intestinal pseudo-obstruction is a condition characterised by clinical manifestations of mechanical obstruction of the intestine in the absence of any organic occlusion of the lumen. This syndrome has rarely been reported to complicate the course of systemic amyloidosis. We describe the case of a 64-year-old man who presented with the syndrome of small bowel pseudo-obstruction secondary to AL amyloid infiltration of the gastrointestinal tract. We comment on the pathophysiology and on the clinical importance of amyloidosis-associated intestinal pseudo-obstruction.

  18. Genetic factors in amyloidosis.

    PubMed Central

    Thomas, P K

    1975-01-01

    In the absence of biochemical distinctions, the nosography of the inherited amyloidoses must at present depend largely upon clinical subdivisions. In the broad classification adopted here, the disorders have for convenience been grouped according to the anatomical system that is predominantly affected. It is evident that the amyloid syndromes display considerable heterogeneity. However, they overlap. Thus in the Iowa type classified with the hereditary amyloid neuropathies (van Allen et al, 1969; Gimeno et al, 1974), renal involvement was frequent and was the usual cause of death. In the English (Zalin et al, 1974) and Scandinavian (Andersson, 1970) families with neuropathy as the predominant feature, cardiac involvement was a common finding. In certain of the conditions discussed, such as medullary carcinoma of the thyroid and Down's syndrome, amyloid deposition is merely an incidental aspect of the disorder. In those conditions in which generalized or localized amyloid deposition occupies a more central position in the clinical syndrome, an autosomal dominant inheritance has been established or suggested in the majority. An autosomal recessive inheritance has so far only been recognized in familial Mediterranean fever. In the family with hereditary amyloid heart diseases reported by Fredricksen et al (1962), the disorder was confined to a single sibship, raising the possibility of recessive inheritance. This could also be true in sporadic examples of primary amyloidosis. The dominantly inherited amyloidoses comprise a number of geographically widely scattered families with clinical pictures that do not show consistent differences between some families. The families that do not show consistent differences are not necessarily harbouring nutations at the same locus, or the same mutation at any particular locus. However, many of these dominantly inherited clinical syndromes are sufficiently different from each other and the clinical manifestations of each

  19. Structural repertoire of immunoglobulin λ light chains.

    PubMed

    Chailyan, Anna; Marcatili, Paolo; Cirillo, Davide; Tramontano, Anna

    2011-05-01

    The immunoglobulin λ isotype is present in nearly all vertebrates and plays an important role in the human immune system. Despite its importance, few systematic studies have been performed to analyze the structural conformation of its variable regions, contrary to what is the case for κ and heavy chains. We show here that an analysis of the structures of λ chains allows the definition of a discrete set of recurring conformations (canonical structures) of their hypervariable loops and, most importantly, the identification of sequence constraints that can be used to predict their structure. We also show that the structural repertoire of λ chains is different and more varied than that of the κ chains, consistently with the current view of the involvement of the two major light-chain families in complementary strategies of the immune system to ensure a fine tuning between diversity and stability in antigen recognition.

  20. Method for altering antibody light chain interactions

    DOEpatents

    Stevens, Fred J.; Stevens, Priscilla Wilkins; Raffen, Rosemarie; Schiffer, Marianne

    2002-01-01

    A method for recombinant antibody subunit dimerization including modifying at least one codon of a nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in the interface segment of the light polypeptide variable region, the charged amino acid having a first polarity; and modifying at least one codon of the nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in an interface segment of the heavy polypeptide variable region corresponding to a position in the light polypeptide variable region, the charged amino acid having a second polarity opposite the first polarity. Nucleic acid sequences which code for novel light chain proteins, the latter of which are used in conjunction with the inventive method, are also provided.

  1. Comparison of serum free light chain and urine electrophoresis for the detection of the light chain component of monoclonal immunoglobulins in light chain and intact immunoglobulin multiple myeloma.

    PubMed

    Dejoie, Thomas; Attal, Michel; Moreau, Philippe; Harousseau, Jean-Luc; Avet-Loiseau, Herve

    2016-03-01

    Response criteria for multiple myeloma are based upon changes in monoclonal protein levels quantified using serum and/or urine protein electrophoresis. The latter lacks sensitivity at low monoclonal protein levels and since 2001, the serum free light chain test has been available and its clinical utility proven, yet guidelines have not recommended it as a replacement for urine assessment. Herein we evaluated responses using serum free light chain measurements and serum and urine electrophoresis after 2 and 4 cycles of therapy and after stem cell transplantation in 25 light chain and 157 intact immunoglobulin myeloma patients enrolled in the IFM 2007-02 MM trial. All 25 light chain patients had measurable disease by serum free light chain and urine methods at presentation. By contrast 98 out of 157 intact immunoglobulin patients had measurable disease by serum free light chain compared to 55 out of 157 by urine electrophoresis. In all patients there was substantial agreement between predicate (serum/urine protein electrophoresis) and test (serum protein electrophoresis and serum free light chain) methods for response assessment (Weighted Kappa=0.83). Urine immunofixation became negative in 47% light chain and 43% intact immunoglobulin patients after 2 cycles of therapy. At this time the serum free light chain ratio normalised in only 11% and 27% patients, respectively. In summary we found good agreement between methods for response assessment, but the serum free light chain test provided greater sensitivity than urine electrophoresis for monitoring. To our knowledge this is the first report comparing both methods for response assignment based on the International Myeloma Working Group guidelines. (Clinical Trials Register.eu identifier: 2007-005204-40).

  2. Globular hepatic amyloid is highly sensitive and specific for LECT2 amyloidosis.

    PubMed

    Chandan, Vishal S; Shah, Sejal S; Lam-Himlin, Dora M; Petris, Giovanni De; Mereuta, Oana M; Dogan, Ahmet; Torbenson, Michael S; Wu, Tsung-Teh

    2015-04-01

    Globular hepatic amyloid (GHA) is rare, and its clinical significance remains unclear. Recently, leukocyte chemotactic factor-associated amyloidosis (ALECT2) has been reported to involve the liver, showing a globular pattern. We reviewed 70 consecutive cases of hepatic amyloidosis to determine the prevalence and morphology of hepatic amyloid subtypes, especially ALECT2 and its association with GHA. Each case was reviewed for amyloid subtype (immunohistochemistry and/or mass spectrometry), its pattern (linear or globular), and distribution (vascular, perisinusoidal, or stromal). In addition, 24 cases of confirmed hepatic ALECT2 on mass spectrometry from our consultation files were also reviewed. LECT2 immunostaining was performed in 49 cases. Of the 70 cases, immunoglobulin light chain (AL) type was most common with 41 cases (59%), followed by transthyretin (ATTR) 15 cases (22%), 3 cases each of fibrinogen A (AFib) (4%), serum amyloid A (AA) (4%), and ALECT2 (4%), 2 cases of apolipoproteins (AApoA1) (3%), and 3 cases (4%) were unclassified. Three of our 70 cases (4%), with ALECT2, and all 24 cases (100%) of mass spectrometry-confirmed hepatic ALECT2 showed only GHA deposits in the hepatic sinusoids and portal tracts. Three (4%) other cases of AL type showed a focal globular pattern admixed with prominent linear amyloid. None of the other amyloid subtypes showed GHA. LECT2 immunostain was positive in all 27 cases (100%) of ALECT2 and negative in the other 22 non-ALECT2 cases (100%) (14 AL, 5 ATTR, 1 AA, 1 AFib, 1 AApoA1). Pure GHA is uncommon (4%) but is highly specific for ALECT2, and LECT2 immunostain is helpful in confirming this amyloid type.

  3. Biochemical characterization of human enteropeptidase light chain.

    PubMed

    Gasparian, M E; Ostapchenko, V G; Dolgikh, D A; Kirpichnikov, M P

    2006-02-01

    The synthetic gene encoding human enteropeptidase light chain (L-HEP) was cloned into plasmid pET-32a downstream from the gene of fusion partner thioredoxin immediately after the DNA sequence encoding the enteropeptidase recognition site. The fusion protein thioredoxin (Trx)/L-HEP was expressed in Escherichia coli BL21(DE3). Autocatalytic cleavage of the fusion protein and activation of recombinant L-HEP were achieved by solubilization of inclusion bodies and refolding of Trx/L-HEP fusion protein. The kinetic parameters of human and bovine enteropeptidases in the presence of different concentrations of Ca2+ and Na+ for cleavage of the specific substrate GD4K-na and nonspecific substrates such as small ester Z-Lys-SBzl and chromogenic substrates Z-Ala-X-Arg-pNA have been comparatively analyzed. It is demonstrated that positively charged ions increased the Michaelis constant (Km) for cleavage of specific substrate GD4K-na, while the catalytic constant (k(cat)) remained practically unchanged. L-HEP demonstrated secondary specificity to the chromogenic substrate Z-Ala-Phe-Arg-pNA with k(cat)/Km 260 mM(-1) x sec(-1). Enzymatic activity of L-HEP was suppressed by inhibitors of trypsin-like and cysteine (E-64), but not metallo-, amino-, or chymotrypsin-like proteinases. L-HEP was active over a broad range of pH (6-9) with optimum activity at pH 7.5, and it demonstrated high stability to different denaturing agents.

  4. Myosin, Transgelin, and Myosin Light Chain Kinase

    PubMed Central

    Léguillette, Renaud; Laviolette, Michel; Bergeron, Celine; Zitouni, Nedjma; Kogut, Paul; Solway, Julian; Kachmar, Linda; Hamid, Qutayba; Lauzon, Anne-Marie

    2009-01-01

    Rationale: Airway smooth muscle (SM) of patients with asthma exhibits a greater velocity of shortening (Vmax) than that of normal subjects, and this is thought to contribute to airway hyperresponsiveness. A greater Vmax can result from increased myosin activation. This has been reported in sensitized human airway SM and in models of asthma. A faster Vmax can also result from the expression of specific contractile proteins that promote faster cross-bridge cycling. This possibility has never been addressed in asthma. Objectives: We tested the hypothesis that the expression of genes coding for SM contractile proteins is altered in asthmatic airways and contributes to their increased Vmax. Methods: We quantified the expression of several genes that code for SM contractile proteins in mild allergic asthmatic and control human airway endobronchial biopsies. The function of these contractile proteins was tested using the in vitro motility assay. Measurements and Main Results: We observed an increased expression of the fast myosin heavy chain isoform, transgelin, and myosin light chain kinase in patients with asthma. Immunohistochemistry demonstrated the expression of these genes at the protein level. To address the functional significance of this overexpression, we purified tracheal myosin from the hyperresponsive Fisher rats, which also overexpress the fast myosin heavy chain isoform as compared with the normoresponsive Lewis rats, and found a faster rate of actin filament propulsion. Conversely, transgelin did not alter the rate of actin filament propulsion. Conclusions: Selective overexpression of airway smooth muscle genes in asthmatic airways leads to increased Vmax, thus contributing to the airway hyperresponsiveness observed in asthma. PMID:19011151

  5. Prevalence, Severity, and Prognostic Value of Sleep Apnea Syndromes in Cardiac Amyloidosis

    PubMed Central

    Bodez, Diane; Guellich, Aziz; Kharoubi, Mounira; Covali-Noroc, Ala; Tissot, Claire-Marie; Guendouz, Soulef; Hittinger, Luc; Dubois-Randé, Jean-Luc; Lefaucheur, Jean-Pascal; Planté-Bordeneuve, Violaine; Adnot, Serge; Boyer, Laurent; Damy, Thibaud

    2016-01-01

    Study Objectives: To assess prevalence, severity, and prognostic value of sleep-disordered breathing (SDB), in the three main cardiac amyloidosis (CA) types, i.e., light-chain (AL), transthyretin-related familial (m-TTR), or senile (WT-TTR). Methods: Patients consecutively referred for CA diagnosis work-up underwent cardiac assessment and nocturnal polygraphy. SDB was defined as apnea-hypopnea index (AHI) ≥ 5/h. Multivariate analysis was used to identify predictors of a major adverse cardiac event (MACE) defined as death, heart transplantation and acute heart failure. Results: Seventy CA patients were included (31 AL, 22 m-TTR, 17 WT-TTR). The mean ± standard deviation age and left ventricular ejection fraction were 71 ± 12 years and 49% ± 13% and median (interquartile range) N terminal pro brain natriuretic peptide (NT-proBNP) was 3,932 (1,607; 7,028) pg/mL. The prevalence of SDB was 90% without difference between amyloidosis types. SDB was central in 27% and obstructive in 73%. AL had less frequent severe SDB compared to m-TTR and WT-TTR (P = 0.015) but longer time with peripheral capillary oxygen saturation (SpO2) < 90% (P = 0.037). After a median follow-up of 7.5 (2.8; 14.9) months, 49% patients experienced MACE. Time with nocturnal SpO2 < 90% was the only independent predictor of MACE. The best-identified threshold was 30 min. Values > 30 min were associated with bad prognosis (Log-rank χ2: 8.01, P value = 0.005). Using binomial logistic regression, determinants of time with nocturnal SpO2 < 90% were New York Heart Association class (P = 0.011), and log-NT-proBNP (P = 0.04) but not AHI. Conclusions: In CA population, prevalence of SDB is high (90%) and dominated by the obstructive pattern. Bad prognosis in this population was driven by nocturnal desaturation, reflecting heart failure severity and respiratory involvement. Citation: Bodez D, Guellich A, Kharoubi M, Covali-Noroc A, Tissot CM, Guendouz S, Hittinger L, Dubois-Randé JL, Lefaucheur JP, Plant

  6. Amyloidosis and Anesthesia

    PubMed Central

    Wani, Zara; Harkawat, Dev Kumar; Sharma, Meenaxi

    2017-01-01

    Aim: The aim of this article is to provide a view of amyloidosis and discuss implications for the anesthetic management of patients with this condition. Material and Method: Urine samples from patients with plasma cell dyscrasias were obtained from a urine bank that gathers urine samples from patients who gave research use consent for specimens that would otherwise be considered waste. Results: Patients with amyloidosis may present to the anesthesiologist for procedures relating to diagnosis, surgery relating to the underlying condition (e.g., bronchial laser and organ transplant), or for incidental surgery. The condition carries a significant risk of perioperative morbidity and mortality. Conclusion: The term amyloid was coined by Virchow in the mid 19th century, meaning “starch like.” Amyloidosis is a disease complex, in which there is an abnormal deposition of extracellular hyaline material with particular staining characteristics and which contains protein fibrils embedded in a relatively amorphous ground substance. There are numerous clinical manifestations, the onset is insidious, and the diagnosis may not be made in a patient undergoing anesthesia and surgery for an apparently straightforward problem. Unexpected complications such as heart or kidney failure may arise, either before operation or in the postoperative period. Bullous lesions of the skin or oral mucosa and extensive areas of purpura are but two of the ways, in which amyloidosis may first present. The disease spectrum may be inherited or acquired, localized or systemic, and life threatening or an incidental finding. PMID:28298791

  7. Higher stem cell dose infusion after intensive chemotherapy does not improve symptom burden in older patients with multiple myeloma and amyloidosis

    PubMed Central

    Shah, Nina; Shi, Qiuling; Williams, Loretta A.; Mendoza, Tito R.; Wang, Xin Shelley; Reuben, James M.; Dougherty, Patrick M.; Bashir, Qaiser; Qazilbash, Muzaffar H.; Champlin, Richard E.; Cleeland, Charles S.; Giralt, Sergio A.

    2015-01-01

    Autologous hematopoietic stem cell transplantation (ASCT) for multiple myeloma (MM) is associated with high symptom burden, particularly for older patients and those with amyloid light-chain (AL) amyloidosis. Symptom burden peaks during leukopenia. We hypothesized that higher doses of CD34+ stem cells would be associated with an improved symptom outcome. Patients undergoing ASCT for MM who were ≥60 years old or had AL amyloidosis were randomized to receive either a standard (4–6×106 cells/kg) or high dose (10–15×106 cells/kg) of CD34+ cells after melphalan 200 mg/m2. Symptom burden was assessed via the MD Anderson Symptom Inventory MM module (MDASI-MM). Eighty patients were enrolled. Median CD34+ cell doses were 5.1×106 cells/kg (standard dose) and 10.5×106 cells/kg (high dose). The most severe symptoms during the first week were fatigue, lack of appetite, drowsiness, disturbed sleep, and pain. The AUC for the mean composite severity score of these symptoms was similar between treatment arms (P = .819). Median times to neutrophil, lymphocyte, and platelet engraftment were also similar between groups. IL-6 increased similarly for both groups throughout the ASCT course. Infusion of higher autologous stem cell dose after high-dose chemotherapy does not yield a difference in symptom burden or engraftment time in the first few weeks post-ASCT. PMID:26253006

  8. AL Amyloidosis and Agent Orange

    MedlinePlus

    ... Z) Hepatitis HIV Mental Health Mental Health Home Suicide Prevention Substance Abuse Military Sexual Trauma PTSD Research ( ... eligible for a free Agent Orange registry health exam . Surviving spouses, dependent children and dependent parents of ...

  9. Light chain replacement: a new model for antibody gene rearrangement

    PubMed Central

    1995-01-01

    A functional B cell antigen receptor is thought to regulate antibody gene rearrangement either by stopping further rearrangement (exclusion) or by promoting additional rearrangement (editing). We have developed a new model to study the regulation of antibody gene rearrangement. In this model, we used gene targeting to replace the J kappa region with a functional V kappa-J kappa light chain gene. Two different strains of mice were created; one, V kappa 4R, has a V kappa 4-J kappa 4 rearrangement followed by a downstream J kappa 5 segment, while the other, V kappa 8R, has a V kappa 8-J kappa 5 light chain. Here, we analyze the influence of these functional light chains on light chain rearrangement. We show that some V kappa 4R and V kappa 8R B cells only have the V kappa R light chain rearrangement, whereas others undergo additional rearrangements. Additional rearrangement can occur not only at the other kappa allele or isotype (lambda), but also at the targeted locus in both V kappa 4R and V kappa 8R. Rearrangement to the downstream J kappa 5 segment is observed in V kappa 4R, as is deletion of the targeted locus in both V kappa 4R and V kappa 8R. The V kappa R models illustrate that a productively rearranged light chain can either terminate further rearrangement or allow further rearrangement. We attribute the latter to editing of autoantibodies and to corrections of dysfunctional receptors. PMID:7629511

  10. Amyloidosis and POEMS Syndrome

    PubMed Central

    Chee, Cheng E. M.D.; Dispenzieri, Angela M.D.; Gertz, Morie A. M.D

    2010-01-01

    Importance of the field Treatment options for amyloidosis and POEMS have rapidly increased in the past years, but many patients are diagnosed late in the disease course and do not receive state of the art therapy Areas covered in this review Stem cell transplantation and novel agents have widened the chemotherapy alternatives available in these disorders and combinations of novel agents with high dose therapy further improve treatment opotions. This review covers the main areas of debate in the optimal treatment amyloidosis and POEMS patients, focusing on the implications for everyday clinical practice and management strategies published in the past 36 months. What the reader will gain Insights into treatment strategies are provided in the review. Keys to early recognition of the syndromes are reviewed Take home message With early diagnosis most patients are therapy candidates. New agents and new application of stem cell transplantation have dramatically improved outcomes for these previously uniformly poor prognosis disorders. PMID:20426710

  11. A novel approach for the purification and proteomic analysis of pathogenic immunoglobulin free light chains from serum.

    PubMed

    Lavatelli, Francesca; Brambilla, Francesca; Valentini, Veronica; Rognoni, Paola; Casarini, Simona; Di Silvestre, Dario; Perfetti, Vittorio; Palladini, Giovanni; Sarais, Gabriele; Mauri, Pierluigi; Merlini, Giampaolo

    2011-03-01

    An excess of circulating monoclonal free immunoglobulin light chains (FLC) is common in plasma cell disorders. A subset of FLC, as amyloidogenic ones, possess intrinsic pathogenicity. Because of their complex purification, little is known on the biochemical features of serum FLC, possibly related to their pathogenic spectrum. We developed an immunopurification approach to isolate serum FLC from patients with monoclonal gammopathies, followed by proteomic characterization. Serum monoclonal FLC were detected and quantified by immunofixation and immunonephelometry. Immunoprecipitation was performed by serum incubation with agarose beads covalently linked to polyclonal anti-κ or λ FLC antibodies. Isolated FLC were analyzed by SDS-PAGE, 2D-PAGE, immunoblotting, mass spectrometry (MS). Serum FLC were immunoprecipitated from 15 patients with ALλ amyloidosis (serum λ FLC range: 98-2350mg/L), 5 with ALκ amyloidosis and 1 with κ light chain (LC) myeloma (κ FLC range: 266-2660mg/L), and 3 controls. Monoclonal FLC were the prevalent eluted species in patients. On 2D-PAGE, both λ and κ FLC originated discrete spots with multiple pI isoforms. The nature of eluted FLC and coincidence with the LC sequence from the bone marrow clone was confirmed by MS, which also detected post-translational modifications, including truncation, tryptophan oxidation, cysteinylation, peptide dimerization. Serum FLC were purified in soluble form and adequate amounts for proteomics, which allowed studying primary sequence and detecting post-translational modifications. This method is a novel instrument for studying the molecular bases of FLC pathogenicity, allowing for the first time the punctual biochemical description of the circulating forms.

  12. Hsp70 and antifibrillogenic peptides promote degradation and inhibit intracellular aggregation of amyloidogenic light chains.

    SciTech Connect

    Dul, J. L.; Davis, D. P.; Williamson, E. K.; Stevens, F. J.; Argon, Y.; Univ. of Chicago

    2001-02-19

    In light chain (LC) amyloidosis an immunoglobulin LC assembles into fibrils that are deposited in various tissues. Little is known about how these fibrils form in vivo. We previously showed that a known amyloidogenic LC, SMA, can give rise to amyloid fibrils in vitro when a segment of one of its {beta} sheets undergoes a conformational change, exposing an Hsp70 binding site. To examine SMA aggregation in vivo, we expressed it and its wild-type counterpart, LEN, in COS cells. While LEN is rapidly oxidized and subsequently secreted, newly synthesized SMA remains in the reduced state. Most SMA molecules are dislocated out of the ER into the cytosol, where they are ubiquitinylated and degraded by proteasomes. A parallel pathway for molecules that are not degraded is condensation into perinuclear aggresomes that are surrounded by vimentin-containing intermediate filaments and are dependent upon intact microtubules. Inhibition of proteasome activity shifts the balance toward aggresome formation. Intracellular aggregation is decreased and targeting to proteasomes improved by overexpression of the cytosolic chaperone Hsp70. Importantly, transduction into the cell of an Hsp70 target peptide, derived from the LC sequence, also reduces aggresome formation and increases SMA degradation. These results demonstrate that an amyloidogenic LC can aggregate intracellularly despite the common presentation of extracellular aggregates, and that a similar molecular surface mediates both in vitro fibril formation and in vivo aggregation. Furthermore, rationally designed peptides can be used to suppress this aggregation and may provide a feasible therapeutic approach.

  13. Antibody elbow angles are influenced by their light chain class

    SciTech Connect

    Stanfield, R; Zemla, A; Wilson, I; Rupp, B

    2006-01-12

    We have examined the elbow angles for 365 different Fab fragments, and observe that Fabs with lambda light chains have adopted a wider range of elbow angles than their kappa-chain counterparts, and that the lambda light chain Fabs are frequently found with very large (>195{sup o}) elbow angles. This apparent hyperflexibility of lambda-chain Fabs may be due to an insertion in their switch region, which is one residue longer than in kappa chains, with glycine occurring most frequently at the insertion position. A new, web-based computer program that was used to calculate the Fab elbow angles is also described.

  14. Coexistence of chronic neutrophilic leukemia with light chain myeloma.

    PubMed

    Cehreli, C; Undar, B; Akkoc, N; Onvural, B; Altungoz, O

    1994-01-01

    A 60-year-old woman who presented with weakness, night sweats, bone pain, easy bruising and weight loss was found to have ecchymoses and hepatosplenomegaly. Blood counts showed persistent neutrophilia of mature cell type with Döhle bodies and toxic granulation. Coexistence of chronic neutrophilic leukemia and multiple myeloma of kappa light chain type was documented by bone marrow examination and immunofixation.

  15. [Pulmonary Amyloidosis: A Diagnostic Challenge].

    PubMed

    Alves, Ana; Alfaro, Tiago M; Madama, Daniela; Freitas, Sara; Robalo-Cordeiro, Carlos; Gamboa, Fernanda

    2015-01-01

    Amyloidosis is characterized by amyloid extracellular deposition in organs and tissues. Pulmonary involvement is a rare manifestation of the disease and it can be focal or as part of systemic amyloidosis. We report two cases. Case 1: 71 year-old female with bronchiectasis and Sjogrenâ syndrome, who complained of anorexia, weight loss and a productive cough. The diagnostic study included a surgical lung biopsy and histological examination demonstrated pulmonary amyloidosis. Case 2: 83 year-old male patient, ex-smoker, asymptomatic, whose routine chest x-ray showed a nodular opacity in the right lung field. A transthoracic biopsy revealed an amyloid lung tumor. These cases illustrate a rare disease which in Case 1 also coexisted with Sjögrenâs syndrome and bronchiectasis. The most important differential diagnosis is cancer and so a definitive diagnosis is essential, as amyloidosis is usually benign and indolent.

  16. Evaluation of myosin light chain phosphorylation in isolated pancreatic acini

    SciTech Connect

    Burnham, D.B.; Soeling, H.D.; Williams, J.A. Universitaet Goettingen )

    1988-01-01

    The role of contractile proteins in secretory granule exocytosis was evaluated by determining whether myosin light chain phosphorylation was altered during stimulation of secretion in mouse pancreatic acini. Acinar myosin was purified by extraction into isosmotic sucrose solution containing 40 mM pyrophosphate followed by ammonium sulfate precipitation and Sepharose 4B-CL chromatography. Myosin was eluted as a single peak of K{sup +}-EDTA ATPase activity and was purified over 2,000-fold to a final ATPase specific activity of 0.96 {mu}mol{center dot}min{sup {minus}1}{center dot}mg protein {sup {minus}1}. Three major myosin subunits of apparent M{sub r} of 200,000, 20,000, and 17,000 were present in the purified myosin preparation. A fourth protein of M{sub r} 21,000 was also present. Purification of myosin from {sup 32}P-labeled acini revealed that M{sub r} 200,000, 21,000, and 20,000 proteins to be heavily labeled. The effect of cholecystokinin octapeptide (CCK-8) on myosin phosphorylation was studied after isolation of myosin from {sup 32}P-labeled acinar lysates by immunoprecipitation. Treatment of acini for 1-10 min with a concentration of CCK-8 that gives a maximal secretory response caused a 25-40% increase in light chain labeling. Treatment with a supramaximal CCK-8 concentration produced a 50-80% increase in light chain labeling. Phosphorylation of myosin heavy chain was not significantly affected by secretagogue treatment. These results indicate that stimulation of pancreatic acinar secretion is accompanied by an increase in myosin light chain phosphorylation.

  17. Indolent systemic mastocytosis associated with light chain deposition disease

    PubMed Central

    Sasaki, Kotaro; Chang, Alice; Najafian, Behzad

    2012-01-01

    Systemic mastocytosis (SM) is characterized by infiltration of neoplastic mast cells in one or more organ systems. SM in association with plasma cell dyscrasia is very rare. We report a first case of indolent SM (ISM) associated with light chain deposition disease (LCDD) in a kidney biopsy from a 59-year-old female presenting with skin rash, elevated serum creatinine, hematuria and mild proteinuria. Subsequent workup demonstrated IgG kappa monoclonal protein in serum and urine. A bone marrow biopsy revealed neoplastic mast cells involving bone marrow without evidence of clonal myeloid or lymphoid proliferation. Kidney biopsy demonstrated modest mesangial expansion detected by light microscopy and unequivocal evidence of monoclonal kappa light chain deposition within glomerular capillaries, tubular basement membranes and vascular walls detected by immunofluorescence and/or electron microscopy, along with equivocal evidence of light chain cast nephropathy. Despite treatment with bortezomib and dexamethasone, her renal function was progressively declined over the next 6 months. This case is a reminder that SM can coincide with LCDD, which requires clinical suspicion and multimodality workup on a kidney biopsy including immunofluorescence and electron microscopy to reach the correct diagnosis. PMID:26019820

  18. Kinesin light chains are essential for axonal transport in Drosophila.

    PubMed

    Gindhart, J G; Desai, C J; Beushausen, S; Zinn, K; Goldstein, L S

    1998-04-20

    Kinesin is a heterotetramer composed of two 115-kD heavy chains and two 58-kD light chains. The microtubule motor activity of kinesin is performed by the heavy chains, but the functions of the light chains are poorly understood. Mutations were generated in the Drosophila gene Kinesin light chain (Klc), and the phenotypic consequences of loss of Klc function were analyzed at the behavioral and cellular levels. Loss of Klc function results in progressive lethargy, crawling defects, and paralysis followed by death at the end of the second larval instar. Klc mutant axons contain large aggregates of membranous organelles in segmental nerve axons. These aggregates, or organelle jams (Hurd, D.D., and W.M. Saxton. 1996. Genetics. 144: 1075-1085), contain synaptic vesicle precursors as well as organelles that may be transported by kinesin, kinesin-like protein 68D, and cytoplasmic dynein, thus providing evidence that the loss of Klc function blocks multiple pathways of axonal transport. The similarity of the Klc and Khc (. Cell 64:1093-1102; Hurd, D.D., and W.M. Saxton. 1996. Genetics 144: 1075-1085) mutant phenotypes indicates that KLC is essential for kinesin function, perhaps by tethering KHC to intracellular cargos or by activating the kinesin motor.

  19. Yeast clathrin has a distinctive light chain that is important for cell growth

    PubMed Central

    1990-01-01

    The structure and physiologic role of clathrin light chain has been explored by purification of the protein from Saccharomyces cerevisiae, molecular cloning of the gene, and disruption of the chromosomal locus. The single light chain protein from yeast shares many physical properties with the mammalian light chains, in spite of considerable sequence divergence. Within the limited amino acid sequence identity between yeast and mammalian light chains (18% overall), three regions are notable. The carboxy termini of yeast light chain and mammalian light chain LCb are 39% homologous. Yeast light chain contains an amino- terminal region 45% homologous to a domain that is completely conserved among mammalian light chains. Lastly, a possible homolog of the tissue- specific insert of LCb is detected in the yeast gene. Disruption of the yeast gene (CLC1) leads to a slow-growth phenotype similar to that seen in strains that lack clathrin heavy chain. However, light chain gene deletion is not lethal to a strain that cannot sustain a heavy chain gene disruption. Light chain-deficient strains frequently give rise to variants that grow more rapidly but do not express an immunologically related light chain species. These properties suggest that clathrin light chain serves an important role in cell growth that can be compensated in light chain deficient cells. PMID:2211819

  20. [Amyloidosis of the tongue as a possible diagnostic manifestation of plasmacytoma].

    PubMed

    Hoefert, S; Schilling, E; Philippou, S; Eufinger, H

    1999-01-01

    Plasmocytic non-Hodgkin's lymphoma is the most common tumor of bone and bone marrow, typically diagnosed by symptoms such as monoclonal paraproteinemia, proteinuria, anemia and hypercalcemia. In its progress, deposits of amyloids in almost all organs can be observed. However, plasmacytomas which are diagnosed by macroglossia of primarily unknown etiology are rare. This case report presents a 61-year-old woman who suffered from a persistent swelling of the tongue with painful ulcerations. A biopsy led to the diagnosis of primary systemic amyloidosis of the light-chain type, which subsequently proved to be a plasmacytoma with lambda light-chains stage II after Durie and Salmon. In the course of the disease the patient developed further deposits of amyloids in the whole gastro-enteric system. Macroglossia as a primary manifestation of plasmacytoma is rarely described in medical literature. However, reports on deposits of amyloid in the tongue in advanced stages of disease are well known.

  1. Constitutive phosphorylation of cardiac myosin regulatory light chain in vivo.

    PubMed

    Chang, Audrey N; Battiprolu, Pavan K; Cowley, Patrick M; Chen, Guohua; Gerard, Robert D; Pinto, Jose R; Hill, Joseph A; Baker, Anthony J; Kamm, Kristine E; Stull, James T

    2015-04-24

    In beating hearts, phosphorylation of myosin regulatory light chain (RLC) at a single site to 0.45 mol of phosphate/mol by cardiac myosin light chain kinase (cMLCK) increases Ca(2+) sensitivity of myofilament contraction necessary for normal cardiac performance. Reduction of RLC phosphorylation in conditional cMLCK knock-out mice caused cardiac dilation and loss of cardiac performance by 1 week, as shown by increased left ventricular internal diameter at end-diastole and decreased fractional shortening. Decreased RLC phosphorylation by conventional or conditional cMLCK gene ablation did not affect troponin-I or myosin-binding protein-C phosphorylation in vivo. The extent of RLC phosphorylation was not changed by prolonged infusion of dobutamine or treatment with a β-adrenergic antagonist, suggesting that RLC is constitutively phosphorylated to maintain cardiac performance. Biochemical studies with myofilaments showed that RLC phosphorylation up to 90% was a random process. RLC is slowly dephosphorylated in both noncontracting hearts and isolated cardiac myocytes from adult mice. Electrically paced ventricular trabeculae restored RLC phosphorylation, which was increased to 0.91 mol of phosphate/mol of RLC with inhibition of myosin light chain phosphatase (MLCP). The two RLCs in each myosin appear to be readily available for phosphorylation by a soluble cMLCK, but MLCP activity limits the amount of constitutive RLC phosphorylation. MLCP with its regulatory subunit MYPT2 bound tightly to myofilaments was constitutively phosphorylated in beating hearts at a site that inhibits MLCP activity. Thus, the constitutive RLC phosphorylation is limited physiologically by low cMLCK activity in balance with low MLCP activity.

  2. Russell body duodenitis with immunoglobulin kappa light chain restriction.

    PubMed

    Munday, William R; Kapur, Lucy Harn; Xu, Mina; Zhang, Xuchen

    2015-01-16

    Russell bodies are eosinophilic intracytoplasmic globules which are likely the result of disturbed secretion of immunoglobulins that accumulate within the plasma cell. Russell body collections have been identified within the stomach, known as Russell body gastritis. Similar lesions within the duodenum are referred to as Russell body duodenitis, which is rare. Several Russell body gastritis case reports are associated with Helicobacter pylori. However, the etiology of Russell body duodenitis remains unclear. Here we report the first case of Russell body duodenitis with immunoglobulin light chain restriction in a background of peptic duodenitis.

  3. [Light chain deposition disease as a cause of renal failure].

    PubMed

    Wohl, P; Chadimová, M; Englis, M; Táborský, P; Rossmann, P; Matl, I

    1998-11-30

    The objective of the paper is to draw attention to a rare cause of rapidly progressing renal failure which developed in the course of four months as a result of light chain deposition disease. The authors submit two case-histories of the disease assessed by renal biopsy after previous clinical and laboratory suspicion of monoclonal gammapathy. In one patient in the sternal punctate plasmacytoma was diagnosed and in the second case it was not possible to detect any type of monoclonal gammapathy or another possible cause of disease. Renal failure was in both cases irreversible and both patients were enlisted in regular haemodialyzation treatment.

  4. A Single Mutation at the Sheet Switch Region Results in Conformational Changes Favoring 6 Light-Chain Fibrillogenesis

    SciTech Connect

    Hernández-Santoyo, A.; Del Pozo Yauner, L; Fuentes-Silva, D; Ortiz, E; Rudiño-Piñera, E; Sánchez-López, R; Horjales, E; Becerril, B; Rodríguez-Romero, A

    2010-01-01

    Systemic amyloid light-chain (LC) amyloidosis is a disease process characterized by the pathological deposition of monoclonal LCs in tissue. All LC subtypes are capable of fibril formation although {lambda} chains, particularly those belonging to the {lambda}6 type, are overrepresented. Here, we report the thermodynamic and in vitro fibrillogenic properties of several mutants of the {lambda}6 protein 6aJL2 in which Pro7 and/or His8 was substituted by Ser or Pro. The H8P and H8S mutants were almost as stable as the wild-type protein and were poorly fibrillogenic. In contrast, the P7S mutation decreased the thermodynamic stability of 6aJL2 and greatly enhanced its capacity to form amyloid-like fibrils in vitro. The crystal structure of the P7S mutant showed that the substitution induced both local and long-distance effects, such as the rearrangement of the VL (variable region of the light chain)-VL interface. This mutant crystallized in two orthorhombic polymorphs, P2{sub 1}2{sub 1}2{sub 1} and C222{sub 1}. In the latter, a monomer that was not arranged in the typical Bence-Jones dimer was observed for the first time. Crystal-packing analysis of the C222{sub 1} lattice showed the establishment of intermolecular {beta}-{beta} interactions that involved the N-terminus and {beta}-strand B and that these could be relevant in the mechanism of LC fibril formation. Our results strongly suggest that Pro7 is a key residue in the conformation of the N-terminal sheet switch motif and, through long-distance interactions, is also critically involved in the contacts that stabilized the VL interface in {lambda}6 LCs.

  5. Hereditary Amyloidosis with Recurrent Lung Infiltrates

    PubMed Central

    Revelo, Alberto E.; Magaspi, Crischelle; Maguire, George; Aronow, Wilbert S.

    2016-01-01

    Patient: Male, 51 Final Diagnosis: Familial amyloidotic polyneuropathy with lung involvement Symptoms: Cough • dyspnea • lethargy Medication: Diflunisal Clinical Procedure: Fiberoptic bronchoscopy with trans-bronchial biopsy Specialty: Pulmonary Medicine Objective: Rare co-existance of disease or patholog Background: Amyloidosis is a protein conformational disorder characterized by extracellular deposition of amyloid fibrils in extracellular tissue. Lung involvement is most commonly caused by secondary AL amyloidosis. The familial autosomal-dominant senile transthyretin (ATTR) disease manifests mainly as polyneuropathy and restrictive cardiomyopathy denoting the name familial amyloidotic polyneuropathy (FAP). Rarely, this form manifests with clinical and radiologically relevant respiratory tract symptoms and lung involvement. Case Report: A 51-year-old male former smoker presented with progressive lower-extremity weakness of several months’ duration. He was ultimately diagnosed with chronic demyelinating polyneuropathy and treated with intravenous immunoglobulin therapy. Subsequently, he was admitted with heart failure symptoms and pulmonary infiltrates and his echocardiogram showed a ‘myocardial speckled pattern’, prompting an endomyocardial biopsy, which showed transthyretin amyloid deposition. He was started on diflunisal. Additionally, serial radiographic imaging of his chest over 3 different admissions for cough, dyspnea, hypoxemia, and lethargy demonstrated recurrent pulmonary infiltrates. A fiberoptic bronchoscopy with trans-bronchial biopsies revealed amyloid deposition in the lung tissue. Conclusions: The clinical presentation of recurrent or persistent pulmonary symptoms and fleeting infiltrates on imaging in a patient with familial amyloidotic polyneuropathy is not common; when present, it should raise the suspicion of respiratory tract involvement. PMID:27872470

  6. Serum free light chains, not urine specimens, should be used to evaluate response in light-chain multiple myeloma

    PubMed Central

    Dejoie, Thomas; Corre, Jill; Caillon, Helene; Hulin, Cyrille; Perrot, Aurore; Caillot, Denis; Boyle, Eileen; Chretien, Marie-Lorraine; Fontan, Jean; Belhadj, Karim; Brechignac, Sabine; Decaux, Olivier; Voillat, Laurent; Rodon, Philippe; Fitoussi, Olivier; Araujo, Carla; Benboubker, Lotfi; Fontan, Charlotte; Tiab, Mourad; Godmer, Pascal; Luycx, Odile; Allangba, Olivier; Pignon, Jean-Michel; Fuzibet, Jean-Gabriel; Legros, Laurence; Stoppa, Anne Marie; Dib, Mamoun; Pegourie, Brigitte; Orsini-Piocelle, Frederique; Karlin, Lionel; Arnulf, Bertrand; Roussel, Murielle; Garderet, Laurent; Mohty, Mohamad; Meuleman, Nathalie; Doyen, Chantal; Lenain, Pascal; Macro, Margaret; Leleu, Xavier; Facon, Thierry; Moreau, Philippe; Attal, Michel

    2016-01-01

    Guidelines for monitoring multiple myeloma (MM) patients expressing light chains only (light-chain MM [LCMM]) rely on measurements of monoclonal protein in urine. Alternatively, serum free light chain (sFLC) measurements have better sensitivity over urine methods, however, demonstration that improved sensitivity provides any clinical benefit is lacking. Here, we compared performance of serum and urine measurements in 113 (72κ, 41λ) newly diagnosed LCMM patients enrolled in the Intergroupe Francophone du Myélome (IFM) 2009 trial. All diagnostic samples (100%) had an abnormal κ:λ sFLC ratio, and involved (monoclonal) FLC (iFLC) expressed at levels deemed measurable for monitoring (≥100 mg/L). By contrast, only 64% patients had measurable levels of monoclonal protein (≥200 mg per 24 hours) in urine protein electrophoresis (UPEP). After 1 and 3 treatment cycles, iFLC remained elevated in 71% and 46% of patients, respectively, whereas UPEP reported a positive result in 37% and 18%; all of the patients with positive UPEP at cycle 3 also had elevated iFLC levels. Importantly, elevated iFLC or an abnormal κ:λ sFLC ratio after 3 treatment cycles associated with poorer progression-free survival (P = .006 and P < .0001, respectively), whereas positive UPEP or urine immunofixation electrophoresis (uIFE) did not. In addition, patients with an abnormal κ:λ sFLC ratio had poorer overall survival (P = .022). Finally, early normalization of κ:λ sFLC ratio but not negative uIFE predicted achieving negative minimal residual disease, as determined by flow cytometry, after consolidation therapy (100% positive predictive value). We conclude that improved sensitivity and prognostic value of serum over urine measurements provide a strong basis for recommending the former for monitoring LCMM patients. PMID:27729323

  7. Immunoglobulin light chain isotypes in the teleost Trematomus bernacchii.

    PubMed

    Coscia, Maria Rosaria; Giacomelli, Stefano; De Santi, Concetta; Varriale, Sonia; Oreste, Umberto

    2008-06-01

    Three immunoglobulin light chain (IgL) isotypes TrbeL1, TrbeL2, and TrbeL3 were identified in the Antarctic teleost Trematomus bernacchii by immunoscreening a cDNA expression library, and using RT-PCR, and 5' RACE. One of them was distinguished in two subisotypes TrbeL1A and TrbeL1B. Real-time PCR experiments showed that the different isotypes were expressed in similar ratios in the various tissues analyzed. Interestingly, the expression level of TrbeL1A isotype was very high in all tissues. Molecular models of the CH1-CL domain pairings were built and minimized for the different isotypes. Several differences were identified in the superimposable structures mainly in the loops. In addition, the isotype-specific residues determined a different distribution of the charges on the external CL domain surface. Phylogenetic trees of 43 isotype representative sequences of CL domain from teleost species, built by different methods, indicated that all teleost light chain isotypes are distributed into three groups. Furthermore, the split of the group IgL1 into two subgroups, one of them carrying a micro-satellite DNA insertion, may have occurred in the Acanthopterygean ancestor.

  8. Regulatory myosin light-chain genes of Caenorhabditis elegans.

    PubMed Central

    Cummins, C; Anderson, P

    1988-01-01

    We have cloned and analyzed the Caenorhabditis elegans regulatory myosin light-chain genes. C. elegans contains two such genes, which we have designated mlc-1 and mlc-2. The two genes are separated by 2.6 kilobases and are divergently transcribed. We determined the complete nucleotide sequences of both mlc-1 and mlc-2. A single, conservative amino acid substitution distinguishes the sequences of the two proteins. The C. elegans proteins are strongly homologous to regulatory myosin light chains of Drosophila melanogaster and vertebrates and weakly homologous to a superfamily of eucaryotic calcium-binding proteins. Both mlc-1 and mlc-2 encode abundant mRNAs. We mapped the 5' termini of these transcripts by using primer extension sequencing of mRNA templates. mlc-1 mRNAs initiate within conserved hexanucleotides at two different positions, located at -28 and -38 relative to the start of translation. The 5' terminus of mlc-2 mRNA is not encoded in the 4.8-kilobase genomic region upstream of mlc-2. Rather, mlc-2 mRNA contains at its 5' end a short, untranslated leader sequence that is identical to the trans-spliced leader sequence of three C. elegans actin genes. Images PMID:3244358

  9. The N-terminal strand modulates immunoglobulin light chain fibrillogenesis.

    PubMed

    del Pozo-Yauner, Luis; Wall, Jonathan S; González Andrade, Martín; Sánchez-López, Rosana; Rodríguez-Ambriz, Sandra L; Pérez Carreón, Julio I; Ochoa-Leyva, Adrián; Fernández-Velasco, D Alejandro

    2014-01-10

    It has been suggested that the N-terminal strand of the light chain variable domain (V(L)) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V(L) protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.

  10. [Amyloidosis and familial Mediterranean fever].

    PubMed

    Pras, M

    1986-01-01

    Familial Mediterranean Fever (F. M. F.) is an autosomal recessive disorder occurring most commonly in Sepharadi Jews and Armenians. Two phenotypic features characterize the disease: brief episodic febrile attacks of peritonitis, pleuritis or synovitis recurring from childhood or adolescence and the development of systemic amyloidosis. Attacks are accompanied by striking elevations of acute phase proteins, including serum amyloid A protein. The amyloidosis of Familial Mediterranean Fever is of the AA type, and manifest clinically as a nephropathy that passes through proteinuria, nephrotic and uremic stages to renal death. Although there is ethnic variation in the incidence of amyloidosis of F. M. F. in our patient population--predominantly Sepharadi Jews of North African extraction--an amyloidotic death at an early age is their genetic destiny. Since the introduction in 1972 of colchicine to prevent the febrile attacks, the drug has been proven and become the main stay of therapy. Today, colchicine has been shown to be effective in preventing amyloidosis as well as the febrile attacks in Familial Mediterranean Fever. End stage renal disease is not the end of the road for patients with F.M.F. because of improving outlook for dialysis and renal transplantation in these patients.

  11. Amyloidosis in Retinal Neurodegenerative Diseases

    PubMed Central

    Masuzzo, Ambra; Dinet, Virginie; Cavanagh, Chelsea; Mascarelli, Frederic; Krantic, Slavica

    2016-01-01

    As a part of the central nervous system, the retina may reflect both physiological processes and abnormalities related to pathologies that affect the brain. Amyloidosis due to the accumulation of amyloid-beta (Aβ) was initially regarded as a specific and exclusive characteristic of neurodegenerative alterations seen in the brain of Alzheimer’s disease (AD) patients. More recently, it was discovered that amyloidosis-related alterations, similar to those seen in the brain of Alzheimer’s patients, also occur in the retina. Remarkably, these alterations were identified not only in primary retinal pathologies, such as age-related macular degeneration (AMD) and glaucoma, but also in the retinas of Alzheimer’s patients. In this review, we first briefly discuss the biogenesis of Aβ, a peptide involved in amyloidosis. We then discuss some pathological aspects (synaptic dysfunction, mitochondrial failure, glial activation, and vascular abnormalities) related to the neurotoxic effects of Aβ. We finally highlight common features shared by AD, AMD, and glaucoma in the context of Aβ amyloidosis and further discuss why the retina, due to the transparency of the eye, can be considered as a “window” to the brain. PMID:27551275

  12. Primary diffuse alveolar septal amyloidosis.

    PubMed Central

    Poh, S C; Tjia, T S; Seah, H C

    1975-01-01

    The case is reported of a 61-year-old man with primary diffuse alveolar septal pulmonary amyloidosis. Amyloid infiltration of the heart and other organs was also observed. The clinical findings and laboratory investigations reveal features characteristic of defective gas transfer with pulmonary oedema due to left ventricular failure from myocardial involvement. Images PMID:1179316

  13. Neurofilament Light Chain in Blood and CSF as Marker of Disease Progression in Mouse Models and in Neurodegenerative Diseases.

    PubMed

    Bacioglu, Mehtap; Maia, Luis F; Preische, Oliver; Schelle, Juliane; Apel, Anja; Kaeser, Stephan A; Schweighauser, Manuel; Eninger, Timo; Lambert, Marius; Pilotto, Andrea; Shimshek, Derya R; Neumann, Ulf; Kahle, Philipp J; Staufenbiel, Matthias; Neumann, Manuela; Maetzler, Walter; Kuhle, Jens; Jucker, Mathias

    2016-07-06

    A majority of current disease-modifying therapeutic approaches for age-related neurodegenerative diseases target their characteristic proteopathic lesions (α-synuclein, Tau, Aβ). To monitor such treatments, fluid biomarkers reflecting the underlying disease process are crucial. We found robust increases of neurofilament light chain (NfL) in CSF and blood in murine models of α-synucleinopathies, tauopathy, and β-amyloidosis. Blood and CSF NfL levels were strongly correlated, and NfL increases coincided with the onset and progression of the corresponding proteopathic lesions in brain. Experimental induction of α-synuclein lesions increased CSF and blood NfL levels, while blocking Aβ lesions attenuated the NfL increase. Consistently, we also found NfL increases in CSF and blood of human α-synucleinopathies, tauopathies, and Alzheimer's disease. Our results suggest that CSF and particularly blood NfL can serve as a reliable and easily accessible biomarker to monitor disease progression and treatment response in mouse models and potentially in human proteopathic neurodegenerative diseases.

  14. The N-terminal strand modulates immunoglobulin light chain fibrillogenesis

    SciTech Connect

    Pozo-Yauner, Luis del; Wall, Jonathan S.; González Andrade, Martín; Sánchez-López, Rosana; Rodríguez-Ambriz, Sandra L.; Pérez Carreón, Julio I.; and others

    2014-01-10

    Highlights: •We evaluated the impact of mutations in the N-terminal strand of 6aJL2 protein. •Mutations destabilized the protein in a position-dependent manner. •Destabilizing mutations accelerated the fibrillogenesis by shortening the lag time. •The effect on the kinetic of fibril elongation by seeding was of different nature. •The N-terminal strand is buried in the fibrillar state of 6aJL2 protein. -- Abstract: It has been suggested that the N-terminal strand of the light chain variable domain (V{sub L}) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V{sub L} protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.

  15. THE AUTOIMMUNE CONSTELLATION IN LICHEN AMYLOIDOSIS.

    PubMed

    Andrese, Elena; Vâţă, D; Ciobanu, Delia; Stătescu, Laura; Solovăstru, Laura Gheucă

    2015-01-01

    Localized cutaneous amyloidosis is a rare disease among white people, being more common in South-Asia, China and South America. The disease is characterized by deposition of amyloid material in the papillary dermis without visceral involvement. Nevertheless, there is a growing list of immune-mediated disorders that have been linked to cutaneous amyloidosis. We present two cases of concomitant occurrence of lichen amyloidosis and autoimmune thyroiditis/atopic dermatitis in two Caucasian women.

  16. Malabsorption Secondary to Gout-Induced Amyloidosis

    PubMed Central

    Balasubramaniam, Renuka; Safa, Shahram; McIvor, Carolyn; Mollee, Peter

    2017-01-01

    Many chronic inflammatory conditions can lead to systemic amyloidosis. However, secondary amyloidosis has rarely been associated with gout, and the literature reports only a handful of cases, all presenting with renal disease. We report a patient with a history of poorly controlled gout who presented with malabsorption. Endoscopic biopsies confirmed a diagnosis of small intestinal amyloidosis. This was believed to be a consequence of gout. Interestingly, renal involvement was subclinical. Our case raises awareness of this rare association and highlights the importance of considering a diagnosis of amyloidosis in patients who present with the combination of gout and gastrointestinal symptoms. PMID:28286797

  17. Clinical and laboratory findings in primary generalized and multiple-myeloma-related amyloidosis.

    PubMed Central

    Pruzanski, W.; Katz, A.

    1976-01-01

    Thirty-four patients with primary generalized amyloidosis (PGA) and 14 with multiple-myeloma-related amyloidosis (MRA) were studied. The commonest clinical manifestations in PGA were nephrotic syndrome, hepatomegaly and congestive heart failure, and in MRA, low back pain, plasmacytoma and rheumatoid-arthritis-like syndrome. Eight patients with PGA had limited clinical expression of the disease, such as involvement of only kidneys, joints, parotid glands or gastrointestinal tract; in one patient amyloidosis was limited to lymph nodes. Low serum concentrations of total protein and albumin were common. M components were detected in the serum of 91% of patients with PGA and 92% of patients with MRA: 70% of the M components in PGA and 25% of those in MRA had lambda light chains. Bence Jones proteinemia was detected in 56% of the patients with PGA and in 77% of those with MRA. The serum concentration of immunoglobulins was decreased substantially in more than two thirds of the patients with PGA. Proteinuria (greater than 250 mg/24 h) was observed in 78% of patients with PGA and in 93% of patients with MRA. Bence Jones proteinuria was noted in 75 and 77% of patients, respectively. Plasmacytic infiltration of the bone marrow was found in 90% of the patients with PGA. The mean survival time of the patients with PGA was 28 months and of those with MRA, 29 months from the time of diagnosis. PMID:1268776

  18. Polyclonal free light chain of Ig may interfere with interpretation of monoclonal free light chain κ/λ ratio.

    PubMed

    Levinson, Stanley S

    2010-01-01

    There is controversy about whether a sensitive assay for the serum Ig free light chain (FLC) κ/λ ratio can replace urine immunofixation electrophoresis (UIFE). This report describes two untreated patients in whom monoclonal FLCs were identified in urine despite normal serum FLC κ/λ ratios. Unlike the classical serum electrophoretic patterns in multiple myeloma, both serum samples showed adequate amounts of polyclonal Ig. The most likely explanation is a masking effect by polyclonal FLC on the serum κ/λ ratio when sufficient concentrations of polyclonal FLC exist. These cases illustrate this likely effect and attest to the continued importance of UIFE for initial screening of patients for Bence-Jones protein.

  19. Verification of serum reference intervals for free light chains in a local South African population.

    PubMed

    Zemlin, Annalise E; Rensburg, Megan A; Ipp, Hayley; Germishuys, Jurie J; Erasmus, Rajiv T

    2013-11-01

    Monoclonal serum free light chain measurements are used to follow up and manage patients with monoclonal gammopathies, and abnormal serum free light chain ratios are associated with risk of progression in certain diseases. We aimed to validate the reference intervals in our population. Reference intervals for κ and λ free light chains were established on 120 healthy adults. Creatinine levels were measured to exclude renal dysfunction and serum protein electrophoresis was performed. All creatinine values were within normal limits. After exclusion of subjects with abnormal serum protein electrophoreses, 113 subjects were available for analysis. The 95% reference interval was 6.3-20.6 mg/L for κ free light chains, 8.7-25.9 mg/L for λ free light chains and 0.46-1.23 for free light chain ratio. Most of the values fell within the manufacturer's recommended limits and therefore could be used for our population.

  20. CVID Associated with Systemic Amyloidosis

    PubMed Central

    Esenboga, Saliha; Çagdas Ayvaz, Deniz; Saglam Ayhan, Arzu; Peynircioglu, Banu; Sanal, Ozden; Tezcan, Ilhan

    2015-01-01

    Common variable immunodeficiency (CVID) is a frequent primary immune deficiency (PID), which consists of a heterogeneous group of disorders and can present with recurrent infections, chronic diarrhea, autoimmunity, chronic pulmonary and gastrointestinal diseases, and malignancy. Secondary amyloidosis is an uncommon complication of CVID. We report an unusual case of a 27-year-old male patient who presented with recurrent sinopulmonary infections, chronic diarrhea, and hypogammaglobulinemia and was diagnosed with CVID. The patient was treated with intravenous immunoglobulin (IVIg) therapy once every 21 days and daily trimethoprim-sulfamethoxazole for prophylaxis. Two years after initial diagnosis, the patient was found to have progressive decline in IgG levels (as low as 200–300 mg/dL) despite regular Ig infusions. The laboratory tests revealed massive proteinuria and his kidney biopsy showed accumulation of AA type amyloid. We believe that the delay in the diagnosis of CVID and initiation of Ig replacement therapy caused chronic inflammation due to recurrent infections in our patient and this led to an uncommon and life-threatening complication, amyloidosis. Patients with CVID require regular follow-up for the control of infections and assessment of adequacy of Ig replacement therapy. Amyloidosis should be kept in the differential diagnosis when managing patients with CVID. PMID:26346511

  1. Amyloidosis in six dairy cows.

    PubMed

    Johnson, R; Jamison, K

    1984-12-15

    Amyloidosis was diagnosed in 6 Holstein cows that were examined because of chronic intractable diarrhea. Besides diarrhea, the chief finding was a nephrotic-like syndrome, in that there was edema, hypoproteinemia, and proteinuria. Other consistent clinicopathologic abnormalities were hyperfibrinogenemia, low-normal serum calcium content or hypocalcemia, hypomagnesemia, prolonged bromosulphalein half time, high serum urea nitrogen concentration, high serum creatinine concentration, and low urine specific gravity. Foci of inflammation including traumatic reticuloperitonitis, traumatic pericarditis, salpingitis, mastitis, and metritis were found. There was histologic evidence of amyloid in the kidneys, liver, adrenal glands, and spleen. The iodine-sulfuric acid test for amyloid was positive in 2 cows. The Congo red dye test for amyloid was positive in 2 other cows. In spite of supportive care, all the cows either died naturally or were euthanatized. Because foci of inflammation were found in each cow, it was concluded that the most likely classification of amyloidosis in these cases would be reactive systemic amyloidosis and that the major amyloid fibril protein would be type AA.

  2. Duodenal Amyloidosis Masquerading as Iron Deficiency Anemia

    PubMed Central

    Hurairah, Abu

    2016-01-01

    The present study is a unique illustration of duodenal amyloidosis initially manifesting with iron deficiency anemia. It underscores the importance of clinical suspicion of amyloidosis while performing upper gastrointestinal endoscopy with a biopsy to establish the definite diagnosis in patients with unexplained iron deficiency anemia. PMID:27625911

  3. Amyloidosis: diffuse involvement of the retroperitoneum.

    PubMed

    Glynn, T P; Kreipke, D L; Irons, J M

    1989-03-01

    Systemic amyloidosis diffusely involving the retroperitoneum has not, to the authors' knowledge, been previously reported. The computed tomographic scans of a 68-year-old man showed evidence of diffuse, nonenhancing thickening of the entire retroperitoneum. This appearance mimicked retroperitoneal fibrosis, but the diagnosis of amyloidosis was confirmed at autopsy.

  4. Tracheobronchial Amyloidosis-A Series of Two Cases

    PubMed Central

    Raiyani, Palak D; Vyas, Sunil S

    2014-01-01

    Amyloidosis is a rare disorder characterised by accumulation of insoluble fibrillar proteins in extracellular space. Respiratory amyloidosis presents as two tracheobronchial forms (local and diffuse) and two parenchymal forms (nodular and diffuse), of which diffuse tracheobronchial amyloidosis is the least common. We herein present two cases of tracheobronchial amyloidosis. PMID:25386446

  5. 21 CFR 866.5550 - Immunoglobulin (light chain specific) immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Immunoglobulin (light chain specific) immunological test system. 866.5550 Section 866.5550 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Test Systems § 866.5550 Immunoglobulin (light chain specific) immunological test system....

  6. 21 CFR 866.5550 - Immunoglobulin (light chain specific) immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Immunoglobulin (light chain specific) immunological test system. 866.5550 Section 866.5550 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Test Systems § 866.5550 Immunoglobulin (light chain specific) immunological test system....

  7. 21 CFR 866.5550 - Immunoglobulin (light chain specific) immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Immunoglobulin (light chain specific) immunological test system. 866.5550 Section 866.5550 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Test Systems § 866.5550 Immunoglobulin (light chain specific) immunological test system....

  8. 21 CFR 866.5550 - Immunoglobulin (light chain specific) immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Immunoglobulin (light chain specific) immunological test system. 866.5550 Section 866.5550 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Test Systems § 866.5550 Immunoglobulin (light chain specific) immunological test system....

  9. The use of immunoglobulin light chain assays in the diagnosis of paraprotein-related kidney disease

    PubMed Central

    Yadav, Punit; Leung, Nelson; Sanders, Paul W.; Cockwell, Paul

    2016-01-01

    Kidney involvement is common in paraprotein-related diseases. A diversity of clinical presentations and histopathological features can occur secondary to tissue injury caused by precipitation or deposition of a clonal immunoglobulin, usually an immunoglobulin light chain. The paraprotein is either produced by multiple myeloma or by a clone of B-cell lineage that does not fulfill diagnostic criteria for multiple myeloma. The recent introduction of serum immunoglobulin free light chain assays, which accurately quantify both light chain isotypes to produce a ratio that indicates the presence or absence of a light chain paraprotein, is a major clinical development. However, as the interpretation of the assay can be challenging, the aim of this review is to clarify the role of serum and urinary light chain assays in the screening and diagnosis of paraprotein-related kidney disease. PMID:25296094

  10. [Non-amyloidotic glomerular disease caused by light-chain deposits: a case report].

    PubMed

    Cantillo, Jorge de Jesús; López, Rocío del Pilar; Andrade, Rafael Enrique

    2009-12-01

    The nephropathy of monoclonal gammopathies is principally caused by light chain deposits of fragmented immunoglobins. Paraprotein-related renal diseases are associated with such deposits of intact (heavy chain) or fragmentary (light chain) immunoglobins. A condition of pathological light chain deposits is rare and characterized by deposits of fragments of monoclonal immunoglobulins in many organs. Renal deposits occur primarily in glomeruli and tubular basement membranes. This disease is frequently associated with lymphoproliferative disorders. The majority of cases are caused by deposits of kappa light chains. Whereas this disease is most frequently associated with hematologic malignancies, occasionally a case occurs without detectable hematological pathologies; these cases are called idiopathic or primary. They usually manifest themselves as severe renal insufficiencies with nephrotic-range proteinuria. No treatment regime has been clearly established and the prognosis is poor. Herein, the clinical and histological characteristics are described regarding the first case in Colombia of light chain deposit disease without symptoms of malignancy.

  11. Mutations in Myosin Light Chain Kinase Cause Familial Aortic Dissections

    PubMed Central

    Wang, Li; Guo, Dong-chuan; Cao, Jiumei; Gong, Limin; Kamm, Kristine E.; Regalado, Ellen; Li, Li; Shete, Sanjay; He, Wei-Qi; Zhu, Min-Sheng; Offermanns, Stephan; Gilchrist, Dawna; Elefteriades, John; Stull, James T.; Milewicz, Dianna M.

    2010-01-01

    Mutations in smooth muscle cell (SMC)-specific isoforms of α-actin and β-myosin heavy chain, two major components of the SMC contractile unit, cause familial thoracic aortic aneurysms leading to acute aortic dissections (FTAAD). To investigate whether mutations in the kinase that controls SMC contractile function (myosin light chain kinase [MYLK]) cause FTAAD, we sequenced MYLK by using DNA from 193 affected probands from unrelated FTAAD families. One nonsense and four missense variants were identified in MYLK and were not present in matched controls. Two variants, p.R1480X (c.4438C>T) and p.S1759P (c.5275T>C), segregated with aortic dissections in two families with a maximum LOD score of 2.1, providing evidence of linkage of these rare variants to the disease (p = 0.0009). Both families demonstrated a similar phenotype characterized by presentation with an acute aortic dissection with little to no enlargement of the aorta. The p.R1480X mutation leads to a truncated protein lacking the kinase and calmodulin binding domains, and p.S1759P alters amino acids in the α-helix of the calmodulin binding sequence, which disrupts kinase binding to calmodulin and reduces kinase activity in vitro. Furthermore, mice with SMC-specific knockdown of Mylk demonstrate altered gene expression and pathology consistent with medial degeneration of the aorta. Thus, genetic and functional studies support the conclusion that heterozygous loss-of-function mutations in MYLK are associated with aortic dissections. PMID:21055718

  12. Assessment of Intrathecal Free Light Chain Synthesis: Comparison of Different Quantitative Methods with the Detection of Oligoclonal Free Light Chains by Isoelectric Focusing and Affinity-Mediated Immunoblotting

    PubMed Central

    Kušnierová, Pavlína; Švagera, Zdeněk; Všianský, František; Byrtusová, Monika; Hradílek, Pavel; Kurková, Barbora; Zapletalová, Olga; Bartoš, Vladimír

    2016-01-01

    Objectives We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups. Methods We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing. Results Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF. Conclusions Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance. PMID:27846293

  13. [New trends in the treatment of amyloidosis].

    PubMed

    Martínez-Valle, Fernando; Gironella-Mesa, Mercedes; Solans-Laqué, Roser

    2012-05-26

    Amyloidosis is a clinical disorder caused by extracellular deposition of proteins that are normally soluble as insoluble fibrils that damage different organs. More than 20 proteins can form amyloid deposits. All types of amyloid fibrils have a secondary structure with a β folded shape that is characteristic and makes them to adopt a green birefringence after stained with Congo red and viewed under cross-polarized light. Amyloidosis can be acquired or hereditary, systemic or localized, and are classified by the fibril precursor protein. Advances in the knowledge of the pathogenesis of amyloidosis allows the development of new diagnostic and therapeutical schemes that are currently under investigation.

  14. Primary systemic amyloidosis presenting as constrictive pericarditis.

    PubMed

    Singh, Vikas; Fishman, Joel E; Alfonso, Carlos E

    2011-01-01

    The most frequent presentation of cardiac amyloidosis is with endomyocardial deposition, and resultant restrictive cardiomyopathy. We present a case of primary systemic amyloidosis causing constrictive pericarditis (CP) and congestive heart failure without clinical evidence of endomyocardial deposition. A comprehensive evaluation by noninvasive and invasive studies facilitated the differentiation of CP from restrictive cardiomyopathy and the patient was effectively treated with pericardectomy. To our knowledge, this is the first documented case of primary systemic amyloidosis causing selective CP with successful antemortem diagnosis and treatment in a young man.

  15. Magnetic resonance imaging in cardiac amyloidosis

    SciTech Connect

    O'Donnell, J.K.; Go, R.T.; Bott-Silverman, C.; Feiglin, D.H.; Salcedo, E.; MacIntyre, W.J.

    1984-01-01

    Primary amyloidosis (AL) involves the myocardium in 90% of cases and may present as apparent ischemia, vascular disease, or congestive heart failure. Two-dimensional echocardiography (echo) has proven useful in the diagnosis, particularly in differentiating AL from constrictive pericarditis. The findings of thickened RV and LV myocardium, normal LV cavity dimension, and a diffuse hyperrefractile ''granular sparkling'' appearance are virtually diagnostic. Magnetic resonance (MR) imaging may improve the resolution of anatomic changes seen in cardiac AL and has the potential to provide more specific information based on biochemical tissue alterations. In this preliminary study, the authors obtained both MR and echo images in six patients with AL and biopsy-proven myocardial involvement. 5/6 patients also had Tc-99 PYP myocardial studies including emission tomography (SPECT). MR studies utilized a 0.6 Tesla superconductive magnet. End diastolic gated images were obtained with TE=30msec and TR=R-R interval on the ECG. 6/6 pts. showed LV wall thickening which was concentric and included the septum. Papillary muscles were identified in all and were enlarged in 3/6. 4/6 pts. showed RV wall thickening but to a lesser degree than LV. Pericardial effusions were present in 4 cases. These findings correlated well with the results of echo although MR gave better RV free wall resolution. PYP scans were positive in 3 pts. but there was no correlation with degree of LV thickening. The authors conclude that there are no identifiable MR findings in patients with cardiac AL which encourage further attempts to characterize myocardial involvement by measurement of MR relaxation times in vivo.

  16. Antigen nature and complexity influence human antibody light chain usage and specificity.

    PubMed

    Smith, Kenneth; Shah, Hemangi; Muther, Jennifer J; Duke, Angie L; Haley, Kathleen; James, Judith A

    2016-05-27

    Human antibodies consist of a heavy chain and one of two possible light chains, kappa (κ) or lambda (λ). Here we tested how these two possible light chains influence the overall antibody response to polysaccharide and protein antigens by measuring light chain usage in human monoclonal antibodies from antibody secreting cells obtained following vaccination with Pneumovax23. Remarkably, we found that individuals displayed restricted light chain usage to certain serotypes and that lambda antibodies have different specificities and modes of cross-reactivity than kappa antibodies. Thus, at both the monoclonal (7 kappa, no lambda) and serum levels (145μg/mL kappa, 2.82μg/mL lambda), antibodies to cell wall polysaccharide were nearly always kappa. The pneumococcal reference serum 007sp was analyzed for light chain usage to 12 pneumococcal serotypes for which it is well characterized. Similar to results at the monoclonal level, certain serotypes tended to favor one of the light chains (14 and 19A, lambda; 6A and 23F, kappa). We also explored differences in light chain usage at the serum level to a variety of antigens. We examined serum antibodies to diphtheria toxin mutant CRM197 and Epstein-Barr virus protein EBNA-1. These responses tended to be kappa dominant (average kappa-to-lambda ratios of 4.52 and 9.72 respectively). Responses to the influenza vaccine were more balanced with kappa-to-lambda ratio averages having slight strain variations: seasonal H1N1, 1.1; H3N2, 0.96; B, 0.91. We conclude that antigens with limited epitopes tend to produce antibodies with restricted light chain usage and that in most individuals, antibodies with lambda light chains have specificities different and complementary to kappa-containing antibodies.

  17. Judging disease activity in rheumatoid arthritis by serum free kappa and lambda light chain levels.

    PubMed

    Ye, Yun; Li, Su-Liang; Xie, Ming; Jiang, Ping; Liu, Kai-Ge; Li, Ya-Jun

    2013-10-01

    The study aimed to evaluate the levels of serum free kappa (κ) and lambda (λ) light chains in patients with rheumatoid arthritis (RA) as well as exploring the association between serum free κ and λ light chains and activity of RA. For this purpose, healthy individuals and patients with active RA and RA in remission were enrolled, and their serum levels of free κ and λ light chains were measured using rate nephelometry. The diagnostic accuracy of serum free κ and λ light chains was evaluated by receiver operating characteristic curves and 95% confidence intervals for areas under the curve (AUC). The results obtained indicated that the levels of serum free κ and λ light chains in patients with active RA were significantly higher than those of patients in remission and of healthy controls (p < 0.05). Further, the AUC values in patients with active RA were 0.871 for free κ light chain and 0.781 for free λ light chain. When the optimal cut-off point for serum κ light chain was 8.02 g/L, the maximum sensitivity and specificity were 82.5% and 82.5%, respectively, and when the optimal cut-off point for serum λ light chain was 3.57 g/L, the maximum sensitivity and specificity were 80% and 82.5%, respectively. It was thus found that serum levels of free κ and λ light chains were positively correlated with disease activity in RA, the Disease Activity Score 28 (DAS28), and values for C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), platelet count (PLT), rheumatoid factor (RF), and anticitrullinated protein antibody (ACPA) (p < 0.05). In conclusion, high serum levels of free κ and λ light chains in patients with active RA are closely correlated with disease activity parameters including DAS28, CRP, ESR, PLT, RF, and ACPA. Thus, the above-mentioned levels of serum free κ and λ light chains may be used as important indicators of activity of RA.

  18. Renal amyloidosis. Evaluation by gallium imaging

    SciTech Connect

    Lee, V.W.; Skinner, M.; Cohen, A.S.; Ngai, S.; Peng, T.T.

    1986-09-01

    A study has been performed to evaluate the efficacy of gallium imaging in the detection of renal amyloidosis. Ten of the 11 patients who had biopsy-proven renal amyloidosis demonstrated marked uptake in both kidneys. One patient revealed moderate gallium uptake in his kidneys. None of the patients had underlying renal or extrarenal pathology other than amyloidosis, which could account for renal gallium uptake (renal infection, neoplasm, hepatic failure or frequent blood transfusions). Four patients also had extrarenal foci of abnormal gallium uptake, suggesting other sites of amyloid deposits. Our data strongly suggest that gallium imaging has a high sensitivity for detection of renal amyloidosis. Its specificity is enhanced significantly by careful review of the clinical history to exclude other known causes of renal gallium uptake. Potentially, gallium imaging may be used to monitor the progress of patients under experimental therapy.

  19. Renal amyloidosis in a drug abuser.

    PubMed

    Tan, A U; Cohen, A H; Levine, B S

    1995-03-01

    Drug abusers, particularly those who inject drugs s.c. ("skin popping"), may develop amyloidosis. Chronic infections are thought to play a pathogenetic role in this setting. A patient is presented who had a history of "skin popping" cocaine and heroin and developed nephrotic syndrome, with an elevated serum creatinine and a creatinine clearance of 61 mL/min. Renal biopsy demonstrated amyloidosis. Treatment with colchicine was initiated, and proteinuria decreased to near normal levels after 12 months. Concomitant with the decrease in proteinuria, creatinine clearance improved, although a repeat renal biopsy failed to show any significant improvement in amyloid burden. These observations suggest that colchicine may be a useful treatment in reversing the proteinuria of renal amyloidosis associated with drug abuse. Furthermore, clinical improvement may occur before any demonstrable regression in the amyloidosis.

  20. Genetics Home Reference: primary localized cutaneous amyloidosis

    MedlinePlus

    ... AP, Hans-Filho G, Sakuma TH, Lai-Cheong J, Clements S, Odashiro M, Odashiro DN, Hans-Neto G, ... mutations underlie familial primary localized cutaneous amyloidosis. Am J Hum Genet. 2008 Jan;82(1):73-80. ...

  1. Primary amyloidosis presenting as renal infarction.

    PubMed

    Arias, M; Abreu, J A; Iglesias, A; Longo, J; Lecumberri, F; Vega, F

    1996-01-01

    We report a case of primary amyloidosis affecting the kidney and presenting as a renal infarction on computed tomography and ultrasound examination. To our knowledge, it is the first case in the radio-logical literature with these imaging characteristics.

  2. Amyloidosis and auto-inflammatory syndromes.

    PubMed

    Grateau, Gilles; Jéru, Isabelle; Rouaghe, Saad; Cazeneuve, Cécile; Ravet, Nathalie; Duquesnoy, Philippe; Cuisset, Laurence; Dodé, Catherine; Delpech, Marc; Amselem, Serge

    2005-02-01

    Amyloidosis remains currently a severe potential complication of many chronic inflammatory disorders. It is not exactly know why some patients develop a progressive amyloidosis, whereas others do not although latent deposits may be present. A permanent acute phase response, ideally evaluated with serial measurement of serum protein SAA, the precursor of the AA protein deposited in tissues, seems to be a prerequisite to the development of inflammatory (AA) amyloidosis. Genetic factors have however been recently emphasized. Among persistent or emerging causes of AA amyloidosis, hereditary periodic fever syndromes also known as auto-inflammatory syndromes are a group of diseases characterised by intermittent bouts of clinical inflammation with focal organ involvement mainly: abdomen, musculoskeletal system and skin. The most frequent is familial Mediterranean fever which affects patients of Mediterranean descent all over the world. Three other types have been recently clinically as well as genetically characterised. A thorough diagnosis is warranted, as clinical and therapeutic management is specific for each of these diseases.

  3. Amyloidosis

    MedlinePlus

    ... Content ASCO Conquer Cancer Foundation Journal of Clinical Oncology Journal of Oncology Practice ASCO University Donate eNEWS SIGNUP f Cancer. ... of medical, surgical, radiation, gynecologic, and pediatric oncologists, oncology nurses, physician assistants, social workers, and patient advocates. ...

  4. Immunoglobulin K light chain deficiency: A rare, but probably underestimated, humoral immune defect.

    PubMed

    Sala, Pierguido; Colatutto, Antonio; Fabbro, Dora; Mariuzzi, Laura; Marzinotto, Stefania; Toffoletto, Barbara; Perosa, Anna R; Damante, Giuseppe

    2016-04-01

    Human immunoglobulin molecules are generated by a pair of identical heavy chains, which identify the immunoglobulin class, and a pair of identical light chains, Kappa or Lambda alternatively, which characterize the immunoglobulin type. In normal conditions, Kappa light chains represent approximately 2/3 of the light chains of total immunoglobulins, both circulating and lymphocyte surface bound. Very few cases of immunoglobulin Kappa or Lambda light chain defects have been reported. Furthermore, the genetic basis of this defect has been extensively explored only in a single case. We report a case of a patient suffering of serious recurrent bacterial infections, which was caused by a very rare form of immunoglobulin disorder, consisting of a pure defect of Kappa light chain. We evaluated major serum immunoglobulin concentrations, as well as total and free Kappa and Lambda light chain concentrations. Lymphocyte phenotyping was also performed and finally we tested the Kappa chain VJ rearrangement as well as the constant Kappa region sequence. Studies performed on VJ rearrangement showed a polyclonal genetic arrangement, whereas the gene sequencing for the constant region of Kappa chain showed a homozygous T to G substitution at the position 1288 (rs200765148). This mutation causes a substitution from Cys to Gly in the protein sequence and, therefore, determines the abnormal folding of the constant region of Kappa chain. We suggest that this defect could lead to an effective reduction of the variability of total antibody repertoire and a consequent defect of an apparently normal immunoglobulin response to common antigens.

  5. IMMUNOGLOBULIN LIGHT CHAIN IMMUNOHISTOCHEMISTRY REVISITED, WITH EMPHASIS ON REACTIVE FOLLICULAR HYPERPLASIA VS. FOLLICULAR LYMPHOMA

    PubMed Central

    Weiss, Lawrence M.; Loera, Sofia; Bacchi, Carlos E.

    2009-01-01

    The identification of monotypic light chains is an important adjunct to the diagnosis of B-cell lymphoma, yet is often difficult to reliably perform on formalin-fixed paraffin sections. We have evaluated a new set of monoclonal antibodies to kappa and lambda light chain that are reactive in paraffin sections. In reactive lymphoid tissues, polytypic staining was noted in greater than 95% of cases, with strong staining of plasma cells, moderate staining of the follicular dendritic cell network, and weak staining of mantle zone cells. Strong staining for the appropriate light chain was seen in each of 7 cases of multiple myeloma. In a series of 58 cases of B-cell lymphoma, correlation between the results of immunohistochemistry and flow cytometry was obtained in 36 cases (62%), including 32 cases (21 kappa and 11 lambda) in which a single light chain was expressed. Monotypic staining also seen in 6 additional cases (10%) in which the flow cytometry had been negative. Thirty of 46 cases (65%) of follicular lymphoma showed monotypic light chain expression, in contrast to 64 of 67 cases (95%) of reactive lymphoid hyperplasia, which showed polytypic light chain expression. These antibodies may provide an effective adjunct to the diagnosis of B-cell lymphoma in routine diagnostic work. PMID:20042853

  6. Macular Amyloidosis and Epstein-Barr Virus

    PubMed Central

    Nahidi, Yalda; Tayyebi Meibodi, Naser; Meshkat, Zahra; Nazeri, Narges

    2016-01-01

    Background. Amyloidosis is extracellular precipitation of eosinophilic hyaline material of self-origin with special staining features and fibrillar ultrastructure. Macular amyloidosis is limited to the skin, and several factors have been proposed for its pathogenesis. Detection of Epstein-Barr virus (EBV) DNA in this lesion suggests that this virus can play a role in pathogenesis of this disease. Objective. EBV DNA detection was done on 30 skin samples with a diagnosis of macular amyloidosis and 31 healthy skin samples in the margin of removed melanocytic nevi by using PCR. Results. In patients positive for beta-globin gene in PCR, BLLF1 gene of EBV virus was positive in 23 patients (8 patients in case and 15 patients in the control group). There was no significant difference in presence of EBV DNA between macular amyloidosis (3.8%) and control (23.8%) groups (P = 0.08). Conclusion. The findings of this study showed that EBV is not involved in pathogenesis of macular amyloidosis. PMID:26981113

  7. Tracheobronchial Amyloidosis Mimicking Tracheal Tumor.

    PubMed

    Tanrıverdi, Elif; Özgül, Mehmet Akif; Uzun, Oğuz; Gül, Şule; Çörtük, Mustafa; Yaşar, Zehra; Acat, Murat; Arda, Naciye; Çetinkaya, Erdoğan

    2016-01-01

    Tracheobronchial amyloidosis is a rare presentation and accounts for about 1% of benign tumors in this area. The diagnosis of disease is delayed due to nonspecific pulmonary symptoms. Therapeutic approaches are required to control progressive pulmonary symptoms for most of the patients. Herein, we report a case of a 68-year-old man admitted with progressive dyspnea to our institution for further evaluation and management. He was initially diagnosed with and underwent management for bronchial asthma for two years but had persistent symptoms despite optimal medical therapy. Pulmonary computed tomography scan revealed severe endotracheal stenosis. Bronchoscopy was performed and showed endotracheal mass obstructing 70% of the distal trachea and mimicking a neoplastic lesion. The mass was successfully resected by mechanical resection, argon plasma coagulation (APC), and Nd-YAG laser during rigid bronchoscopy. Biopsy materials showed deposits of amorphous material by hematoxylin and eosin staining and these deposits were selectively stained with Congo Red. Although this is a rare clinical condition, this case indicated that carrying out a bronchoscopy in any patient complaining of atypical bronchial symptoms or with uncontrolled asthma is very important.

  8. Tracheobronchial Amyloidosis Mimicking Tracheal Tumor

    PubMed Central

    Özgül, Mehmet Akif; Uzun, Oğuz; Yaşar, Zehra; Acat, Murat; Arda, Naciye; Çetinkaya, Erdoğan

    2016-01-01

    Tracheobronchial amyloidosis is a rare presentation and accounts for about 1% of benign tumors in this area. The diagnosis of disease is delayed due to nonspecific pulmonary symptoms. Therapeutic approaches are required to control progressive pulmonary symptoms for most of the patients. Herein, we report a case of a 68-year-old man admitted with progressive dyspnea to our institution for further evaluation and management. He was initially diagnosed with and underwent management for bronchial asthma for two years but had persistent symptoms despite optimal medical therapy. Pulmonary computed tomography scan revealed severe endotracheal stenosis. Bronchoscopy was performed and showed endotracheal mass obstructing 70% of the distal trachea and mimicking a neoplastic lesion. The mass was successfully resected by mechanical resection, argon plasma coagulation (APC), and Nd-YAG laser during rigid bronchoscopy. Biopsy materials showed deposits of amorphous material by hematoxylin and eosin staining and these deposits were selectively stained with Congo Red. Although this is a rare clinical condition, this case indicated that carrying out a bronchoscopy in any patient complaining of atypical bronchial symptoms or with uncontrolled asthma is very important. PMID:27594885

  9. Phosphorylation/dephosphorylation of the beta light chain of clathrin from rat liver coated vesicles.

    PubMed

    Loeb, J E; Cantournet, B; Vartanian, J P; Goris, J; Merlevede, W

    1989-06-01

    The phosphorylation in vitro, on serine residues by endogenous casein kinase 2, of the clathrin beta light chain (33 kDa) of rat liver coated vesicles requires the presence of poly(L-lysine) which acts through binding to the beta light chain. The phosphorylation of other proteins is also increased in the presence of poly(L-lysine) and casein kinase 2. In contrast, the phosphorylation of the upper band of the 50-kDa protein doublet from rat liver coated vesicles is inhibited. Rat liver coated vesicles display a protein phosphatase activity which preferentially dephosphorylates clathrin beta light chain. This activity is different from the protein phosphatase which dephosphorylates the 50-kDa protein. This enzyme seems to be unrelated to the ATP/Mg-dependent protein phosphatase, or the polycation-stimulated protein phosphatases, which dephosphorylate the 50-kDa protein and beta light chain very efficiently, but with a different specificity. After dissociation of coated vesicles the beta-light-chain phosphatase activity is recovered in the membrane fraction. This phosphatase activity is inhibited by 50 microM orthovanadate and 5 mM p-nitrophenyl phosphate but not by 10 mM EDTA.

  10. Myosin subunit interactions. Properties of the 19,000-dalton light chain-deficient myosin.

    PubMed

    Pastra-Landis, S C; Lowey, S

    1986-11-05

    The 19,000-dalton light chain (LC2) can be completely and reversibly removed from chicken pectoralis myosin in 1 mM EDTA and 5 mM ATP using immunoaffinity chromatography at 37 degrees C. Earlier methods have led to only partial removal of LC2 or have caused limited degradation of the heavy chain. Electron microscopy of LC2-deficient myosin showed it to have a marked tendency to aggregate into oligomers through the "neck" region of the myosin head. Myosin reverted to the monomeric form when it was reconstituted with light chains. LC2-deficient myosin retained full K+ (EDTA) or Ca2+-ATPase activity, and the actin-activated Mg2+-ATPase was similar to that of the native molecule. Alkali light chain exchange at 37 degrees C, which has been demonstrated in subfragment 1 prepared with chymotrypsin, does not occur with intact myosin molecules or with papain subfragment 1, both of which contain LC2. However, a temperature-dependent exchange of alkali light chains was observed in myosin lacking LC2. The interaction of the alkali light chain with the heavy chain thus appears to be influenced by the presence of LC2, which may have an important stabilizing effect on the myosin molecule.

  11. Demonstration of structural polymorphism among MB3 light chains by two-dimensional gel electrophoresis.

    PubMed

    Ishikawa, N; Kasahara, M; Ikeda, H; Ogasawara, K; Hawkin, S; Takenouchi, T; Wakisaka, A; Kikuchi, Y; Aizawa, M

    1985-01-01

    The heavy and light chain subunits of MB3 molecules were isolated from KT2 (DKT2, DR4, MB3 homozygous), ER (Dw4, DR4, MB3 homozygous), JMe (Dw5, DR5, MB3 homozygous), EBV-Sh (DSh, DRw6.2, MB3 homozygous), and EBV-Ky (DKy, DRw9, MB3 homozygous) cells and were compared with one another by two-dimensional gel electrophoresis. The MB3 light chains from KT2, ER, and EBV-Ky cells were clearly different in terms of their isoelectric points, whereas those from ER, JMe, and EBV-Sh cells were indistinguishable. No differences in charge or m.w. were noted for the MB3 heavy chains from the five cell lines. Thus, three out of the five MB3-positive, D/DR-disparate cell lines were found to express structurally distinct MB3 molecules, demonstrating that MB3 is a public serologic specificity shared by at least three structurally distinct MB (human I-A-like) molecules. Because the DR light chain subunits isolated from EBV-Wa, KT2, ER, JMe, EBV-Sh, and EBV-Ky cells differed from one another in their isoelectric points, the DR light chains were apparently more polymorphic than the MB3 light chains.

  12. Characteristics of light chains of Chara myosin revealed by immunological investigation

    PubMed Central

    KAKEI, Toshihito; SUMIYOSHI, Hiroki; HIGASHI-FUJIME, Sugie

    2012-01-01

    Chara myosin is plant myosin responsible for cytoplasmic streaming and moves actin filaments at 60 µm/s, which is the fastest of all myosins examined. The neck of the myosin molecule has usually mechanical and regulatory roles. The neck of Chara myosin is supposed to bind six light chains, but, at present, we have no knowledge about them. We found Ca++-calmodulin activated Chara myosin motility and its actin-activated ATPase, and actually bound with the Chara myosin heavy chain, indicating calmodulin might be one of candidates for Chara myosin light chains. Antibody against essential light chain from Physarum myosin, and antibodies against Chara calmodulin and chicken myosin light chain from lens membranes reacted with 20 kDa and 18 kDa polypeptides of Chara myosin preparation, respectively. Correspondingly, column purified Chara myosin had light chains of 20 kDa, and 18 kDa with the molar ratio of 0.7 and 2.5 to the heavy chain, respectively. PMID:22687741

  13. Minimum requirements for inhibition of smooth-muscle myosin light-chain kinase by synthetic peptides.

    PubMed Central

    Hunt, J T; Floyd, D M; Lee, V G; Little, D K; Moreland, S

    1989-01-01

    Although the amino acid residues that are important for peptide substrates of myosin light-chain kinase have been reported, those that are important for peptide inhibitors of this enzyme have not previously been investigated. Synthetic peptides based on the sequence Lys11-Lys12-Arg13-Ala-Ala-Arg16-Ala-Thr-Ser19 -Asn-Val21-Phe22-Ala of the chicken gizzard myosin light chain were tested as inhibitors of pig carotid-artery myosin light-chain kinase. The basic amino acid residues of the known myosin light-chain kinase inhibitor Lys-Lys-Arg-Ala-Ala-Arg-Ala-Thr-Ser-NH2 (IC50 = 14 microM) [Pearson, Misconi & Kemp (1986) J. Biol. Chem. 261, 25-27] were shown to be the important residues that contribute to inhibitor potency, as evidence by the finding that the hexapeptide Lys-Lys-Arg-Ala-Ala-Arg-NH2 had an IC50 value of 22 microM. This indicates that binding of the phosphorylatable serine residue to myosin light-chain kinase, which is of obvious importance for a substrate, does not enhance the potency of an inhibitor. With the aim of preparing more potent inhibitors, peptides Lys-Lys-Arg-Ala-Ala-Arg-Ala-Ala-Xaa-NH2 were prepared with a variety of amino acids substituted for the phosphorylatable serine residue. None of these peptides was a more potent inhibitor than the serine peptide. PMID:2920029

  14. Primarily isolated hepatic involvement of amyloidosis

    PubMed Central

    Ye, Lei; Shi, Hui; Wu, Hui-Min; Wang, Fang-Yu

    2016-01-01

    Abstract Background: Amyloidosis is particularly difficult to diagnose because the signs and symptoms are subtle. Additionally, there are no specific imaging or laboratory tests, except histopathology. Although it is considered to be a systemic disorder, a small portion of cases may be localized. Introduction of the case: A 54-year-old man presented with nonspecific symptoms (jaundice and back pruritus). Biochemical tests showed a high level of bilirubin and elevated serum tumor markers (CA19–9 and CA125). Routine imaging showed hepatomegaly without heterogeneous enhancement. Liver biopsy confirmed the diagnosis of hepatic amyloidosis. No cardiac or renal involvement was found. The patient accepted treatment involving oral chemotherapy. Conclusion: A rare and unique presentation of hepatic amyloidosis was highlighted in this case. PMID:28033255

  15. Colchicine use in isolated renal AA amyloidosis.

    PubMed

    Meneses, Carlos F; Egües, César A; Uriarte, Miren; Belzunegui, Joaquín; Rezola, Marta

    2015-01-01

    We present the case of a 45-year-old woman, with two-year history of chronic renal insufficiency and proteinuria. A kidney biopsy showed the presence of AA amyloidosis (positive Congo red staining and immunohistochemistry). There was no evidence of amyloid deposits in other organs and there was no underlying disease. AA amyloidosis normally is secondary to chronic inflammatory or infectious diseases. High levels of IL-1, IL-6 and TNF-α play a role in the pathogenesis of amyloidosis and induce the synthesis of serum amyloid A protein (SAA), a precursor of tissue amyloid deposits. We empirically treated the patient with a low dose colchicine. The patient responded well. Colchicine has been used for the treatment of Familiar Mediterranean Fever and related auto-inflammatory diseases. To monitor treatment responses, we measured SAA finding low titers. Soon after treatment onset there were signs of improvement pertaining to proteinuria and stabilization of renal function.

  16. The interdomain disulfide bond of a homogeneous rabbit pneumococcal antibody light chain.

    PubMed

    Strosberg, A D; Margolies, M N; Haber, E

    1975-11-01

    Rabbit light chain 3315, prepared from a homogeneous antipneumococcal antibody, was subjected to hydrolysis by pepsin without prior reduction and alkylation of the intrachain disulfide bonds. Gel filtration of the hydrolysate on Sephadex G-10, G-15, and G-25 and ion exchange chromatography on SP-Sephadex yielded several disulfide bridge peptides. These were fully reduced and alkulated and sequenced by Edman degradation. The peptides were located in the light chain sequence determined in independent studies from our laboratory. The half-cystine residues in this KB rabbit chain are located at positions 23, 80, 88, 134, 171, 194, and 214. The extra disulfide bridge extends between residues 80 and 171, thus joining the variable and constant domains. This is consistent with x-ray diffraction crystallographic studies showing that the corresponding residues in human light chains are separated by a distance compatible with disulfide bond formation.

  17. K-252a, a novel microbial product, inhibits smooth muscle myosin light chain kinase

    SciTech Connect

    Nakanishi, S.; Yamada, K.; Kase, H.; Nakamura, S.; Nonomura, Y.

    1988-05-05

    Effects of K-252a, purified from the culture broth of Nocardiopsis sp., on the activity of myosin (light chain kinase were investigated. 1) K-252a affected three characteristic properties of chicken gizzard myosin-B, natural actomyosin, to a similar degree: the Ca/sup 2 +/-dependent activity of ATPase, superprecipitation, and the phosphorylation of the myosin light chain. 2) K-252a inhibited the activities of the purified myosin light chain kinase and a Ca/sup 2 +/-independent form of the enzyme which was constructed by cross-linking of myosin light chain kinase and calmodulin using glutaraldehyde. The degrees of inhibition by 3 x 10/sup -6/ M K-252a were 69 and 48% of the control activities with the purified enzyme and the cross-linked complex, respectively. Chlorpromazine (3 x 10/sup -4/ M), a calmodulin antagonist, inhibited the native enzyme, but not the cross-linked one. These results suggested that K-252a inhibited myosin light chain kinase by direct interaction with the enzyme, whereas chlorpromazine suppressed the enzyme activation by interacting with calmodulin. 3) The inhibition by K-252a of the cross-linked kinase was affected by the concentration of ATP, a phosphate donor. The concentration causing 50% inhibition was two orders magnitude lowere in the presence of 100 ..mu..M ATP than in the presence of 2 mM ATP. 4) Kinetic analyses using (..gamma..-/sup 32/P)ATP indicated that the inhibitory mode of K-252a was competitive with respect to ATP. These results suggest that K-252a interacts at the ATP-binding domain of myosin light chain kinase.

  18. Molecular tweezers targeting transthyretin amyloidosis.

    PubMed

    Ferreira, Nelson; Pereira-Henriques, Alda; Attar, Aida; Klärner, Frank-Gerrit; Schrader, Thomas; Bitan, Gal; Gales, Luís; Saraiva, Maria João; Almeida, Maria Rosário

    2014-04-01

    Transthyretin (TTR) amyloidoses comprise a wide spectrum of acquired and hereditary diseases triggered by extracellular deposition of toxic TTR aggregates in various organs. Despite recent advances regarding the elucidation of the molecular mechanisms underlying TTR misfolding and pathogenic self-assembly, there is still no effective therapy for treatment of these fatal disorders. Recently, the "molecular tweezers", CLR01, has been reported to inhibit self-assembly and toxicity of different amyloidogenic proteins in vitro, including TTR, by interfering with hydrophobic and electrostatic interactions known to play an important role in the aggregation process. In addition, CLR01 showed therapeutic effects in animal models of Alzheimer's disease and Parkinson's disease. Here, we assessed the ability of CLR01 to modulate TTR misfolding and aggregation in cell culture and in an animal model. In cell culture assays we found that CLR01 inhibited TTR oligomerization in the conditioned medium and alleviated TTR-induced neurotoxicity by redirecting TTR aggregation into the formation of innocuous assemblies. To determine whether CLR01 was effective in vivo, we tested the compound in mice expressing TTR V30M, a model of familial amyloidotic polyneuropathy, which recapitulates the main pathological features of the human disease. Immunohistochemical and Western blot analyses showed a significant decrease in TTR burden in the gastrointestinal tract and the peripheral nervous system in mice treated with CLR01, with a concomitant reduction in aggregate-induced endoplasmic reticulum stress response, protein oxidation, and apoptosis. Taken together, our preclinical data suggest that CLR01 is a promising lead compound for development of innovative, disease-modifying therapy for TTR amyloidosis.

  19. Lambda light chain myeloma presenting as nodular hepatic lesion: a clinical rarity.

    PubMed

    Pal, Santanu; Chattopadhyay, Bitoti; Chatterjee, Atri; Bhattacharya, Biswamit

    2014-01-01

    We report a case of a 63-year-old lady presenting with pain in the right hypochondrium, jaundice, anorexia, and firm tender hepatomegaly with remarkably high serum alkaline phosphatase. Abdominal ultrasonography revealed a hypoechoic solid space-occupying lesion in right lobe of liver which was cytologically diagnosed as hepatic plasmacytoma. Serum and urine immunofixation electrophoresis, serum free light chain ratio, and bone marrow examination further confirmed the presence of lambda light chain multiple myeloma in the background. The patient achieved complete remission after four cycles of induction therapy with thalidomide and dexamethasone protocol and consolidated with further four cycles of the same regimen.

  20. Systemic lambda light-chain deposition presenting with predominant cardiac involvement.

    PubMed Central

    Garton, M. J.; Walton, S.; Ewen, S. W.

    1993-01-01

    An 82 year old woman with suspected Bence Jones myeloma developed intractable fluid retention presumed secondary to cardiac failure. In addition she experienced angina pectoris, and required permanent cardiac pacing for symptomatic sinus bradycardia. Postmortem studies revealed prominent myocardial and renal deposits of lambda light-chains which were Congo Red negative, and had a non-fibrillar ultrastructure. Non-amyloidotic light-chain deposition is uncommon, and a rare cause of cardiac disease. Previous work regarding possible pathogenetic mechanisms, clinical and laboratory features and treatment is reviewed. Images Figure 1 Figure 2 Figure 3 PMID:8415352

  1. Study on epidemiology of cutaneous amyloidosis in northern India and effectiveness of dimethylsulphoxide in cutaneous amyloidosis

    PubMed Central

    Krishna, Arvind; Nath, Bhola; Dhir, G. G.; Kumari, Ranjeeta; Budhiraja, Virendra; Singh, Kalpana

    2012-01-01

    Context: Amyloidosis, which is characterized by the extracellular deposition of a proteinaceous substance, is usually associated with considerable tissue dysfunction. However, the etiology of the disease remains uncertain and the treatment disappointing. Aim: 1. To know the epidemiology of cutaneous amyloidosis 2. To evaluate the effect of dimethylsulphoxide on cutaneous amyloidosis. Settings and Design: Data was collected from patients attending the Outpatient Department (OPD) over a period of one year. Material and Methods: Patients were screened on the basis of signs and symptoms and then confirmed histologically. A total of 62 patients who were suspected to be suffering from amyloidosis on the basis of clinical signs and symptoms and 38 patients who were further confirmed histopathologically underwent the treatment. Statistical Analysis Used: Chi-square test was used for testing the significance of proportions. Results: 63.15 percent of the patients had macular amyloidosis and the interscapular area was the most common area involved (52.63%). Pruritus, pigmentation, and papules responded excellently to dimethylsulphoxide after one month of treatment. Conclusions: Cutaneous amyloidosis is a disease found in middle-aged persons, with a female preponderance, and dimethylsulphoxide seems to be an effective therapy. PMID:23189250

  2. Evaluation of systemic amyloidosis by scintigraphy with sup 123 I-labeled serum amyloid P component

    SciTech Connect

    Hawkins, P.N.; Lavender, J.P.; Pepys, M.B. )

    1990-08-23

    In systemic amyloidosis the distribution and progression of disease have been difficult to monitor, because they can be demonstrated only by biopsy. Serum amyloid P component (SAP) is a normal circulating plasma protein that is deposited on amyloid fibrils because of its specific binding affinity for them. We investigated whether labeled SAP could be used to locate amyloid deposits. Purified human SAP labeled with iodine-123 was given intravenously to 50 patients with biopsy-proved systemic amyloidosis--25 with the AL (primary) type and 25 with the AA (secondary) type--and to 26 control patients with disease and 10 healthy subjects. Whole-body images and regional views were obtained after 24 hours and read in a blinded fashion. In the patients with amyloidosis the 123I-SAP was localized rapidly and specifically in amyloid deposits. The scintigraphic images obtained were characteristic and appeared to identify the extent of amyloid deposition in all 50 patients. There was no uptake of the 123I-SAP by the control patients and the healthy subjects. In all patients with AA amyloidosis the spleen was affected, whereas the scans showed uptake in the heart, skin, carpal region, and bone marrow only in patients with the AL type. Positive images were seen in six patients in whom biopsies had been negative or unsuccessful; in all six, amyloid was subsequently found on biopsy or at autopsy. Progressive amyloid deposition was observed in 9 of 11 patients studied serially. Scintigraphy after the injection of 123I-SAP can be used for diagnosing, locating, and monitoring the extent of systemic amyloidosis.

  3. Primary amyloidosis presenting as "dropped head syndrome".

    PubMed

    Chuquilin, Miguel; Al-Lozi, Muhammad

    2011-06-01

    A 77-year-old man, with a history of lymphoma, presented with isolated neck extensor weakness and a 2-year history of bilateral carpal tunnel syndrome (CTS). Needle electromyography showed myopathic changes, and biopsy of cervical paraspinal muscles showed amyloid deposition in blood vessels. Amyloidosis should be considered in the differential diagnosis of dropped head syndrome.

  4. Early Prognostic Value of Monitoring Serum Free Light Chain in Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplantation.

    PubMed

    Özkurt, Zübeyde Nur; Sucak, Gülsan Türköz; Akı, Şahika Zeynep; Yağcı, Münci; Haznedar, Rauf

    2017-03-16

    We hypothesized the levels of free light chains obtained before and after autologous stem cell transplantation can be useful in predicting transplantation outcome. We analyzed 70 multiple myeloma patients. Abnormal free light chain ratios before stem cell transplantation were found to be associated early progression, although without any impact on overall survival. At day +30, the normalization of levels of involved free light chain related with early progression. According to these results almost one-third reduction of free light chain levels can predict favorable prognosis after autologous stem cell transplantation.

  5. Silencing megalin and cubilin genes inhibits myeloma light chain endocytosis and ameliorates toxicity in human renal proximal tubule epithelial cells.

    PubMed

    Li, Min; Balamuthusamy, Saravanan; Simon, Eric E; Batuman, Vecihi

    2008-07-01

    Using target-specific short interfering (si) RNAs, we silenced the tandem endocytic receptors megalin and cubilin genes in cultured human renal proximal tubule epithelial cells. Transfection by siRNA resulted in up to 90% suppression of both megalin and cubilin protein and mRNA expression. In HK-2 cells exposed to kappa-light chain for up to 24 h, light chain endocytosis was reduced in either megalin- or cubilin-silenced cells markedly but incompletely. Simultaneous silencing of both the cubilin and megalin genes, however, resulted in near-complete inhibition of light chain endocytosis, as determined by measuring kappa-light chain protein concentration in cell cytoplasm and by flow cytometry using FITC-labeled kappa-light chain. In these cells, light chain-induced cytokine responses (interleukin-6 and monocyte chemoattractant protein-1) and epithelial-to-mesenchymal transition as well as the associated cellular and morphological alterations were also markedly suppressed. The results demonstrate that light chain endocytosis is predominantly mediated by the megalin-cubilin tandem endocytic receptor and identify endocytosis as a key step in light chain cytotoxicity. Blocking light chain endocytosis prevents its nephrotoxic effects on human kidney proximal tubule cells.

  6. ATYPICAL MACULOPATHY IN A PATIENT WITH LIGHT CHAIN DEPOSITION DISEASE MIMICKING ADVANCED GEOGRAPHIC ATROPHY

    PubMed Central

    Oshry, Lauren J.; Reichel, Elias

    2017-01-01

    Purpose: To report a previously unreported presentation of advanced geographic atrophy of the macula mimicking nonneovascular (dry) age-related macular degeneration in a patient with light chain deposition disease. Methods: Ocular examination included dilated fundus examination, fundus autofluorescence, full-field electroretinography, and spectral domain optical coherence tomography. Patients: Single-patient case report. Results: Dilated fundus examination demonstrated diffuse loss of the retinal pigment epithelium in a geographic atrophy pattern in the macula and drusenlike deposits localized to the outer retina and retinal pigment epithelium. There were no signs of choroidal neovascularization or retinal pigment epithelium detachments. Fundus autofluorescence demonstrated wide areas of retinal pigment epithelium loss. Full-field electroretinography was normal. Spectral domain optical coherence tomography displayed atrophy of the outer retinal layers. Discussion: This is the first documented case of drusenlike deposits and maculopathy in a patient with light chain deposition disease that mimics advanced geographic atrophy that is typically observed in nonneovascular age-related macular degeneration. Physicians should be aware of the macular changes that can be associated with light chain deposition disease, and patients with light chain deposition disease should be regularly evaluated for associated macular disease. PMID:26934302

  7. A novel approach for the chromatographic purification and peptide mass fingerprinting of urinary free light chains.

    PubMed

    Mali, Bhupesh C; Badgujar, Shamkant B; Shukla, Kunal K; Bhanushali, Paresh B

    2017-02-01

    We describe a chromatographic approach for the purification of urinary free light chains (FLCs) viz., lambda free light chains (λ-FLCs) and kappa free light chains (κ-FLCs). Isolated urinary FLCs were analyzed by SDS-PAGE, immunoblotting and mass spectrometry (MS). The relative molecular masses of λ-FLC and κ-FLC are 22,933.397 and 23,544.336Da respectively. Moreover, dimer forms of each FLC were also detected in mass spectrum which corresponds to 45,737.747 and 47,348.028Da respectively for λ-FLCs and κ-FLCs. Peptide mass fingerprint analysis of the purified λ-FLCs and κ-FLCs has yielded peptides that partially match with known light chain sequences viz., gi|218783338 and gi|48475432 respectively. The tryptic digestion profile of isolated FLCs infers the exclusive nature of them and they may be additive molecules in the dictionary of urinary proteins. This is the first report of characterization and validation of FLCs from large volume samples by peptide sequencing. This simple and cost-effective approach to purification of FLCs, together with the easy availability of urine samples make the large-scale production of FLCs possible, allowing exploration of various bioclinical as well as biodiagnostic applications.

  8. Purification, Characterization and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkia.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in close species. In this study, MLC with the molecular mass of 18kDa was purified from crayfish (Procambarus clarkii) muscle fibrils. Its physicochemical characterization showed that the...

  9. Bone marrow lambda-type light chain crystalline structures associated with multiple myeloma.

    PubMed

    Schvartz, H; Bonhomme, P; Caulet, S; Beorchia, A; Patey, M; Caulet, T

    1985-01-01

    A 58-year-old man showed bone marrow crystalline structures associated with a lambda light chain producing multiple myeloma. Analysis and processing of electron images clearly displayed the periodic structure of the crystals. Immunochemistry suggested that they contained the whole or a fragmented constant portion of immunoglobulin.

  10. Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity

    PubMed Central

    Vanam, Ram P; Schneider, Michael A; Marlow, Michael S

    2015-01-01

    An alternative method to traditional 2-dimensional gel electrophoresis (2D-PAGE) and its application in characterizing the inherent charge heterogeneity of chromatographically isolated monoclonal antibody heavy and light chains is described. This method, referred to as ChromiCE, utilizes analytical size-exclusion chromatography (SEC), performed under reducing and denaturing conditions, followed by imaged capillary isoelectric focusing (icIEF) of the chromatographically separated heavy and light chains. Under conditions suitable for the subsequent icIEF analysis, the absolute and relative SEC elution volumes of the heavy and light chains were found to be highly pH dependent, a phenomenon that can be exploited in optimizing chromatographic separation. Compared to 2D-PAGE, the ChromiCE method substantially decreases the time and labor needed to complete the analysis, improves reproducibility, and provides fully quantitative assessment of charge heterogeneity. The ChromiCE methodology was applied to a set of diverse monoclonal antibodies to demonstrate suitability for quantitative charge variant analysis of heavy and light chains. A typical application of ChromiCE in extended characterization and stability studies of a purified antibody is shown. PMID:26305772

  11. Phenotyping polyclonal kappa and lambda light chain molecular mass distributions in patient serum using mass spectrometry.

    PubMed

    Barnidge, David R; Dasari, Surendra; Ramirez-Alvarado, Marina; Fontan, Adrian; Willrich, Maria A V; Tschumper, Renee C; Jelinek, Diane F; Snyder, Melissa R; Dispenzieri, Angela; Katzmann, Jerry A; Murray, David L

    2014-11-07

    We previously described a microLC-ESI-Q-TOF MS method for identifying monoclonal immunoglobulins in serum and then tracking them over time using their accurate molecular mass. Here we demonstrate how the same methodology can be used to identify and characterize polyclonal immunoglobulins in serum. We establish that two molecular mass distributions observed by microLC-ESI-Q-TOF MS are from polyclonal kappa and lambda light chains using a combination of theoretical molecular masses from gene sequence data and the analysis of commercially available purified polyclonal IgG kappa and IgG lambda from normal human serum. A linear regression comparison of kappa/lambda ratios for 74 serum samples (25 hypergammaglobulinemia, 24 hypogammaglobulinemia, 25 normal) determined by microflowLC-ESI-Q-TOF MS and immunonephelometry had a slope of 1.37 and a correlation coefficient of 0.639. In addition to providing kappa/lambda ratios, the same microLC-ESI-Q-TOF MS analysis can determine the molecular mass for oligoclonal light chains observed above the polyclonal background in patient samples. In 2 patients with immune disorders and hypergammaglobulinemia, we observed a skewed polyclonal molecular mass distribution which translated into biased kappa/lambda ratios. Mass spectrometry provides a rapid and simple way to combine the polyclonal kappa/lambda light chain abundance ratios with the identification of dominant monoclonal as well as oligoclonal light chain immunoglobulins. We anticipate that this approach to evaluating immunoglobulin light chains will lead to improved understanding of immune deficiencies, autoimmune diseases, and antibody responses.

  12. Amyloidosis of the Tongue-Report of A Rare Case

    PubMed Central

    Babburi, Suresh; B, Ramya; RV, Subramanyam; V, Aparna; Srivastava, Gautam

    2013-01-01

    Amyloid involvement of the tongue is almost always secondary to systemic amyloidosis. Isolated amyloidosis of the tongue is relatively rare and it accounts for less than 9% of all types of amyloidosis. We are presenting a case of a 54–year–old male patient who complained of an enlarged tongue and bilateral multiple swellings on the lateral borders of the tongue, which had been there since one year. Bilaterally symmetrical, violaceous, purpuric patches interspersed with nodules were seen surrounding the eyes. Histopathologically, the lesion exhibited homogenous eosinophilic amyloid-like material. Special staining with Congo red showed amyloid material as peach red colour under light microscopy and as apple green birefringence under polarized light. Based on these observations, a definitive diagnosis of amyloidosis of tongue was made. Amyloidosis of tongue is uncommon and its features resemble those of a benign tumour. A battery of tests is necessary to differentiate localized amyloidosis from its systemic forms PMID:24551740

  13. λ Light Chain Bias Associated With Enhanced Binding and Function of Anti-HIV Env Glycoprotein Antibodies.

    PubMed

    Sajadi, Mohammad M; Farshidpour, Maham; Brown, Eric P; Ouyang, Xin; Seaman, Michael S; Pazgier, Marzena; Ackerman, Margaret E; Robinson, Harriet; Tomaras, Georgia; Parsons, Matthew S; Charurat, Manhattan; DeVico, Anthony L; Redfield, Robert R; Lewis, George K

    2016-01-01

    The humoral response to human immunodeficiency virus (HIV) remains incompletely understood. In this report, we describe biased λ light chain use during the HIV Env glycoprotein (Env) response in HIV infection and vaccination. We examined HIV Env binding (and neutralization) in the context of light chain use in subjects with acute HIV infection, chronic HIV infection, and among HIV vaccinees. In all populations tested, there was a λ chain bias for HIV Env binding antibodies, compared with other HIV antigens (such as p24) or tetanus toxoid. In subjects with chronic HIV infection, a λ bias was noted for neutralization, with λ antibodies accounting for up to 90% of all neutralization activity observed. This is the first report of antibody function in a human infection being tied to light chain use. In HIV infection, antibodies expressing λ light chains tended to have longer CDRL3s, increased light chain contact with HIV Env, and less hypermutation in the heavy chain, compared with antibodies using the κ light chain. These data also support an evolutionary model for the understanding the various κ to λ light chain ratios observed across species and suggest that the λ light chain bias against HIV provides the host an advantage in developing a more efficient humoral response.

  14. Poor Outcomes in Hepatic Amyloidosis: A Report of 2 Cases

    PubMed Central

    Kertowidjojo, Elizabeth; Zhang, Yue; Patel, Pruthvi

    2016-01-01

    Hepatic amyloidosis is a rare disease entity that results from insoluble amyloid protein deposition in the liver. The disease often presents with vague, nonspecific clinical features. Currently, there is little literature describing treatment outcomes for biopsy-proven hepatic amyloidosis and current treatment guidelines recommend that patients enroll in a clinical trial due to insufficient evidence to suggest an optimal treatment regimen. Here, we present two cases of hepatic amyloidosis at an academic medical center and describe their presentation, treatment, and outcomes. These cases highlight the poor outcomes and difficult management of hepatic amyloidosis. Further understanding and investigation of this rare disease are warranted. PMID:27774327

  15. Gastrointestinal Amyloidosis Presenting with Multiple Episodes of Gastrointestinal Bleeding

    SciTech Connect

    Kim, Sang Hyeon Kang, Eun Ju; Park, Jee Won; Jo, Jung Hyun; Kim, Soo Jin; Cho, Jin Han; Kang, Myong Jin; Park, Byeong Ho

    2009-05-15

    Amyloidosis is characterized by the extracellular deposition of amyloid protein in various organs. Gastrointestinal involvement in amyloidosis is common, but a diagnosis of amyloidosis is often delayed. Severe gastrointestinal hemorrhage in amyloidosis is rare but can be fatal in some cases. We experienced a case of a 49-year-old man who presented with recurrent massive hematochezia. Although embolization was performed eight times for bleeding from different sites of the small intestine, hematochezia did not cease. We report the case, with a review of the literature.

  16. A Rare Case of Ascites due to Peritoneal Amyloidosis

    PubMed Central

    Stofer, Fernanda; Barretto, Maria Fernanda; Gouvea, Ana Luisa; Ribeiro, Mario; Neves, Marcio; Gismondi, Ronaldo Altenburg; Mocarzel, Luís Otavio

    2016-01-01

    Patient: Male, 65 Final Diagnosis: Peritoneal amyloidosis Symptoms: Anasarca • Dyspnea • Orthopnea Medication: — Clinical Procedure: Paracentesis and peritoneal biopsy Specialty: Gastroenterology and Hepatology Objective: Unusual clinical course Background: The clinical manifestations of amyloidosis depend on the type of insoluble protein as well as the location of amyloid deposits in tissues or organs. In the gastrointestinal tract, the small intestine is the most common site of amyloid deposits, whereas peritoneal involvement and ascites are rare. Case Report: We report on a case of ascites due to peritoneal amyloidosis. A 65-year-old patient was admitted to our institution due to anasarca and pulmonary congestion, mimicking heart failure. We started the patient on diuretics and vasodilators. Despite improvement in pulmonary congestion and peripheral edema, his ascites was not reduced. Echocardiogram revealed restrictive cardiomyopathy and a speckle-tracking pattern suggestive of cardiac amyloidosis. Subcutaneous and peritoneal biopsies revealed amyloidosis. Conclusions: Amyloidosis is rare in the peritoneum and is usually asymptomatic. Ascites occurs in only 20% of patients with peritoneal amyloidosis. We searched PubMed using “ascites” and “amyloidosis” and identified only eight case reports of amyloidosis with ascites. Physicians should be particularly careful in heart failure and anasarca cases when ascites is disproportional or not responsive to diuretic treatment. To date, there is no specific treatment for peritoneal amyloidosis. PMID:27353538

  17. Interaction of protein-bound polysaccharide (PSK) with smooth muscle myosin regulatory light chain.

    PubMed

    Fujii, Toshihiro; Kunimatsu, Mitoshi

    2003-06-01

    The interaction of a protein-bound polysaccharide (PSK) isolated from Basidiomycetes with smooth muscle myosin components was evaluated by limited digestion, urea/glycerol gel electrophoresis, affinity chromatography and overlay assay using a peptide array. PSK was bound to the regulatory light chain (RLC) of myosin, but not to the essential light chain. The binding to PSK was definitely observed for unphosphorylated RLC, compared to phosphorylated one. From the amino acid sequence of the RLC, 490 peptides were synthesized on a cellulose membrane. Overlay assays showed that the PSK-binding on the molecule of RLC were localized in the N- and C-terminal basic regions and these sites were conserved in RLC from the human smooth muscle and nonmuscle cells.

  18. Light-chain deposition disease of the kidney: a case report.

    PubMed

    Darouich, Sihem; Goucha, Rym; Jaafoura, Mohamed Habib; Zekri, Semy; Kheder, Adel; Maiz, Hedi Ben

    2012-04-01

    A 41-year-old man was admitted for evaluation of nephrotic syndrome associated with microhematuria, hypertension, and moderate renal failure. In serum and urine samples, monoclonal IgG-lambda was detected. Bone marrow examination showed normal representation of all cell lines with normal range of plasma cells. Renal biopsy demonstrated diabetes-like nodular glomerulosclerosis. Immunofluorescence failed to demonstrate the presence of kappa or lambda light chains in the kidney. Electron microcopy showed granular electron-dense deposits along the glomerular basement membranes and in the mesangial nodules. The patient was diagnosed as having light-chain deposition disease (LCDD) without evidence of plasma cell dyscrasia. This report was designed to stress the significant challenges that remain in the diagnosis of LCDD-related glomerulopathy. The salient morphological features that help in making an accurate diagnosis are discussed.

  19. Surface supercharged human enteropeptidase light chain shows improved solubility and refolding yield.

    PubMed

    Simeonov, Peter; Berger-Hoffmann, Renate; Hoffmann, Ralf; Sträter, Norbert; Zuchner, Thole

    2011-03-01

    Enteropeptidase is a serine protease used in different biotechnological applications. For many applications the smaller light chain can be used to avoid the expression of the rather large holoenzyme. Recombinant human enteropeptidase light chain (hEPL) shows high activity but low solubility and refolding yields, currently limiting its use in biotechnological applications. Here we describe several protein modifications that lead to improved solubility and refolding yield of human hEPL whilst retaining the enzyme activity. Specifically, protein surface supercharging (N6D, G21D, G22D, N141D, K209E) of the protein increased the solubility more than 100-fold. Replacement of a free cysteine residue with serine (C112S) improved the refolding yield by 50%. The heat stability of this C112S variant was also significantly improved by supercharging. This study shows that even mild protein surface supercharging can have pronounced effects on protein solubility and stability.

  20. Bacterial kinesin light chain (Bklc) links the Btub cytoskeleton to membranes.

    PubMed

    Akendengue, Lurlène; Trépout, Sylvain; Graña, Martín; Voegele, Alexis; Janke, Carsten; Raynal, Bertrand; Chenal, Alexandre; Marco, Sergio; Wehenkel, Anne Marie

    2017-03-30

    Bacterial kinesin light chain is a TPR domain-containing protein encoded by the bklc gene, which co-localizes with the bacterial tubulin (btub) genes in a conserved operon in Prosthecobacter. Btub heterodimers show high structural homology with eukaryotic tubulin and assemble into head-to-tail protofilaments. Intriguingly, Bklc is homologous to the light chain of the microtubule motor kinesin and could thus represent an additional eukaryotic-like cytoskeletal element in bacteria. Using biochemical characterization as well as cryo-electron tomography we show here that Bklc interacts specifically with Btub protofilaments, as well as lipid vesicles and could thus play a role in anchoring the Btub filaments to the membrane protrusions in Prosthecobacter where they specifically localize in vivo. This work sheds new light into possible ways in which the microtubule cytoskeleton may have evolved linking precursors of microtubules to the membrane via the kinesin moiety that in today's eukaryotic cytoskeleton links vesicle-packaged cargo to microtubules.

  1. Five Sequential Evaluations of Renal Histology in a Patient with Light Chain Deposition Disease

    PubMed Central

    Ueno, Toshiharu; Kikuchi, Koichi; Hazue, Ryo; Mise, Koki; Sumida, Keiichi; Hayami, Noriko; Suwabe, Tatsuya; Hoshino, Junichi; Sawa, Naoki; Arizono, Kenji; Hara, Shigeko; Takaichi, Kenmei; Fujii, Takeshi; Ohashi, Kenichi; Ubara, Yoshifumi

    2016-01-01

    A 58-year-old man was referred to our institution for an evaluation of nephrotic range proteinuria. Renal biopsy showed a marked expansion of the mesangial matrix and thickening of glomerular basement membrane (GBM) in periodic acid-silver methenamine (PAM). Immunofluorescence (IF) revealed strong staining for the monoclonal kappa light chain. EM demonstrated massive subendothelial and mesangial dense deposits. As a result, light chain deposition disease (LCDD) was diagnosed. Melphalan and prednisolone (MP) therapy was started, which was continued for 10 years with minimal complications. Serial evaluations of renal histology revealed the resolution of nodular lesions and the glomeruli became nearly normal. MP therapy can therefore be an effective therapeutic option for LCDD if it is continued over the long term. PMID:27746438

  2. Crystal Structure of a Phosphorylated Light Chain Domain of Scallop Smooth-Muscle Myosin

    SciTech Connect

    Kumar, V.S.; Robinson, H.; O-Neall-Hennessey, E.; Reshetnikova, L.; Brown, J. H.; Szent-Gyorgyi, A. G.; Cohen, C.

    2011-11-02

    We have determined the crystal structure of a phosphorylated smooth-muscle myosin light chain domain (LCD). This reconstituted LCD is of a sea scallop catch muscle myosin with its phosphorylatable regulatory light chain (RLC SmoA). In the crystal structure, Arg{sup 16}, an arginine residue that is present in this isoform but not in vertebrate smooth-muscle RLC, stabilizes the phosphorylation site. This arginine interacts with the carbonyl group of the phosphorylation-site serine in the unphosphorylated LCD (determined previously), and with the phosphate group when the serine is phosphorylated. However, the overall conformation of the LCD is essentially unchanged upon phosphorylation. This result provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.

  3. Expression, purification, and characterization of Clostridium botulinum type B light chain.

    PubMed

    Gilsdorf, Janice; Gul, Nizamettin; Smith, Leonard A

    2006-04-01

    A full-length synthetic gene encoding the light chain of botulinum neurotoxin serotype B, approximately 50 kDa (BoNT/B LC), has been cloned into a bacterial expression vector pET24a+. BoNT/B LC was expressed in Escherichia coli BL21.DE3.pLysS and isolated from the soluble fraction. The resultant protein was purified to homogeneity by cation chromatography and was determined to be >98% pure as assessed by SDS-polyacrylamide gel stained with SilverXpress and analyzed by densitometry. Mass spectroscopic analysis indicated the protein to be 50.8 kDa, which equaled the theoretically expected mass. N-terminal sequencing of the purified protein showed the sequence corresponded to the known reported sequence. The recombinant BoNT/B light chain was found to be highly stable, catalytically active, and has been used to prepare antisera that neutralizes against BoNT/B challenge. Characterization of the protein including pH, temperature, and the stability of the protein in the presence or absence of zinc is described within. The influence of pH differences, buffer, and added zinc on secondary and tertiary structure of BoNT/B light chain was analyzed by circular dichroism and tryptophan fluorescence measurements. Optimal conditions for obtaining maximum metalloprotease activity and stabilizing the protein for long term storage were determined. We further analyzed the thermal denaturation of BoNT/B LC as a function of temperature to probe the pH and added zinc effects on light chain stability. The synthetic BoNT/B LC has been found to be highly active on its substrate (vesicle associated membrane protein-2) and, therefore, can serve as a useful reagent for BoNT/B research.

  4. Cardiac amyloidosis in a heart transplant patient - A case report and retrospective analysis of amyloidosis evolution

    PubMed Central

    Kintsler, Svetlana; Jäkel, Jörg; Brandenburg, Vincent; Kersten, Katrin; Knuechel, Ruth; Röcken, Christoph

    2015-01-01

    Summary Cardiac amyloidosis is a very rare cause of heart failure in heart transplant recipients but an important differential diagnosis in cases of progressive cardiac failure. We report a 72-year-old male patient with the diagnosis of senile systemic amyloidosis (SSA) in a transplanted heart 15 years after transplantation by the initial diagnosis of the dilated cardiomyopathy. Additionally performed immunohistochemical analysis with anti-transthyretin antibody of the cardiac biopsies of the last 15 years enabled the possibility to show the evolution of this disease with characteristic biphasic pattern. PMID:25674390

  5. Familial mutations in fibrinogen Aα (FGA) chain identified in renal amyloidosis increase in vitro amyloidogenicity of FGA fragment.

    PubMed

    Sivalingam, Vishwanath; Patel, Basant K

    2016-08-01

    Amyloidoses are clinical disorders where deposition of β-sheet rich, misfolded protein aggregates called amyloid occurs in vital organs like brain, kidney, liver or heart etc. Aggregation of several proteins such as immunoglobulin light chain, fibrinogen Aα chain (FGA) and lysozyme have been found to be associated with renal amyloidosis. Fibrinogen amyloidosis (AFib) is predominantly familial and is associated with the deposition of mutant FGA amyloid, primarily in kidneys. Over ten substitution and frame-shift mutations in FGA have been identified from AFib patients. Whether wild-type FGA is also involved in AFib is yet unknown. The affected tissues from AFib patients usually show ∼10 kDA peptide from C-terminal 80 amino acid residues of mutant FGA. Notably, this region also encompasses all known disease-related mutations. Whether these point mutations increase the amyloidogenicity of FGA leading to disease progression, have not been studied yet. Here, we have investigated the role of two disease-related mutations in affecting amyloidogenic propensity of an FGA(496-581) fragment. We found that at physiological pH, the wild-type FGA(496-581) fragment remains monomeric, whereas its E540V mutant forms amyloid-like fibrils as observed by AFM. Also, FGA(496-581) harbouring another familial mutation, R554L, converts in vitro into globular, β-sheet rich aggregates, showing amyloid-like properties. These findings suggest that familial mutations in FGA may have role in renal amyloidosis via enhanced amyloid formation.

  6. High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire.

    PubMed

    DeKosky, Brandon J; Ippolito, Gregory C; Deschner, Ryan P; Lavinder, Jason J; Wine, Yariv; Rawlings, Brandon M; Varadarajan, Navin; Giesecke, Claudia; Dörner, Thomas; Andrews, Sarah F; Wilson, Patrick C; Hunicke-Smith, Scott P; Willson, C Grant; Ellington, Andrew D; Georgiou, George

    2013-02-01

    Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (V(H) and V(L)) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 10(4) capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion V(H):V(L) linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of V(H):V(L) pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets-peripheral blood IgG(+) B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination.

  7. A novel monoclonal antibody against the constant region of goose immunoglobulin light chain.

    PubMed

    Guo, Yongli; Gao, Mingchun; Ma, Bo; Sheng, Qiaoling; Wang, Qian; Liu, Dandan; Wang, Junwei

    2014-04-01

    A monoclonal antibody (MAb) against the antigenic determinant of the constant region of goose immunoglobulin light chain (GoIgCL) was produced and characterized for the first time here. Goose immunoglobulin (Ig) in serum was purified by immunoaffinity chromatography and the resulting protein was used as immunogen to immunize BALB/c mice. At the same time, the GoIgCL gene was expressed and purified as the screening antigen for selecting MAb against GoIgCL. One hybridoma that produces antibodies against GoIgCL was selected by indirect ELISA. Then the characterization of the MAb was analyzed by ELISA, Western blot, and flow cytometry. It was found to be IgG1 with κ light chain; the MAB has high specificity to Ig in goose serum, bile, and B lymphocytes from peripheral blood, reacts only with the light chain of goose Ig, and can distinguish Ig from other birds. Therefore, the MAb generated in this study can be used as a specific reagent for detection of goose disease-specific antibodies and as a powerful tool for basic immunology research on geese.

  8. Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis.

    PubMed

    Valencia-Expósito, Andrea; Grosheva, Inna; Míguez, David G; González-Reyes, Acaimo; Martín-Bermudo, María D

    2016-02-18

    Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies.

  9. Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis

    PubMed Central

    Valencia-Expósito, Andrea; Grosheva, Inna; Míguez, David G.; González-Reyes, Acaimo; Martín-Bermudo, María D.

    2016-01-01

    Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies. PMID:26888436

  10. A novel method of preparing the monoform structure of catalytic antibody light chain.

    PubMed

    Hifumi, Emi; Matsumoto, Shingo; Nakashima, Hiroki; Itonaga, Shogo; Arakawa, Mitsue; Katayama, Yoshiki; Kato, Ryuichi; Uda, Taizo

    2016-02-01

    Along with the development of antibody drugs and catalytic antibodies, the structural diversity (heterogeneity) of antibodies has been given attention. For >20 yr, detailed studies on the subject have not been conducted, because the phenomenon presents many difficult and complex problems. Structural diversity provides some (or many) isoforms of an antibody distinguished by different charges, different molecular sizes, and modifications of amino acid residues. For practical use, the antibody and the subunits must have a defined structure. In recent work, we have found that the copper (Cu) ion plays a substantial role in solving the diversity problem. In the current study, we used several catalytic antibody light chains to examine the effect of the Cu ion. In all cases, the different electrical charges of the molecule converged to a single charge, giving 1 peak in cation-exchange chromatography, as well as a single spot in 2-dimensional gel electrophoresis. The Cu-binding site was investigated by using mutagenesis, ultraviolet-visible spectroscopy, atomic force microscope analysis, and molecular modeling, which suggested that histidine and cysteine residues close to the C-terminus are involved with the binding site. The constant region domain of the antibody light chain played an important role in the heterogeneity of the light chain. Our findings may be a significant tool for preparing a single defined, not multiple, isoform structure.

  11. Two kappa immunoglobulin light chains are secreted by an anti-DNA hybridoma: implications for isotypic exclusion.

    PubMed

    Zack, D J; Wong, A L; Stempniak, M; Weisbart, R H

    1995-12-01

    An anti-DNA hybridoma derived from an MRL/lpr mouse secretes two different kappa light chains in combination with a single heavy chain. Multiple single cell clones express and secrete immunoglobulin containing both kappa light chains. The N-terminal protein sequences of the light chains correspond to sequences predicted from functionally rearranged mRNAs subjected to reverse transcription and amplified by polymerase chain reaction (PCR). Karyotype analysis of the hybridoma indicates a clonal line derived from the fusion of two cells. By amino acid sequence comparison and PCR analysis, both functional kappa light chains are derived from the MRL/lpr spleen. The two functional light chain cDNAs were cloned and co-transfected into COS-7 cells with the heavy chain cDNA. Only one of the light chains in combination with mAb 3E10 heavy chain confers anti-DNA reactivity. The presence of two separate kappa light chains and, therefore, two separate antigen receptors on a single B cell may have ramifications for both polyclonal activation and toleration of lupus B cells.

  12. Idiopathic systemic AA-amyloidosis in a skunk (Mephitis mephitis).

    PubMed

    Elhensheri, Mohamed; Linke, Reinhold P; Blankenburg, Anja; Beineke, Andreas

    2012-03-01

    This report describes a case of systemic amyloidosis in a captive striped skunk. At necropsy, bilateral alopecia, as well as reno-, hepato-, and splenomegaly were present. Congo red staining and immunohistochemistry revealed depositions of AA-amyloid in different organs. The lack of a predisposing disease is suggestive of idiopathic systemic AA-amyloidosis.

  13. Nephrotic Syndrome Associated with Lung Cancer: A Rare Case of Malignancy Associated with AA Amyloidosis.

    PubMed

    Gueutin, Victor; Langlois, Anne-Lyse; Shehwaro, Nathalie; Elharraqui, Ryme; Rouvier, Philippe; Izzedine, Hassane

    2013-01-01

    Nonhematologic malignancies are rarely reported to be associated with AA amyloidosis. Although the association between renal cell carcinoma and systemic AA amyloidosis has been established, the evidence linking pulmonary cancer to AA amyloidosis is scarce. Here, a case of biopsy-proven renal AA amyloidosis complicated with nephrotic syndrome associated with lung carcinoma is reported.

  14. Nephrotic Syndrome Associated with Lung Cancer: A Rare Case of Malignancy Associated with AA Amyloidosis

    PubMed Central

    Gueutin, Victor; Langlois, Anne-Lyse; Shehwaro, Nathalie; Elharraqui, Ryme; Rouvier, Philippe; Izzedine, Hassane

    2013-01-01

    Nonhematologic malignancies are rarely reported to be associated with AA amyloidosis. Although the association between renal cell carcinoma and systemic AA amyloidosis has been established, the evidence linking pulmonary cancer to AA amyloidosis is scarce. Here, a case of biopsy-proven renal AA amyloidosis complicated with nephrotic syndrome associated with lung carcinoma is reported. PMID:24558629

  15. Cardiac amyloidosis: the importance of a multidisciplinary approach.

    PubMed

    Gómez-Bueno, Manuel; Segovia, Javier; García-Pavía, Pablo; Barceló, Juan M; Krsnik, Isabel; Sánchez-Turrión, Víctor; Salas, Clara; Alonso-Pulpón, Luis

    2009-06-01

    Cardiac amyloidosis is associated with the interstitial deposition of abnormal protein in the myocardium, which can lead to a form of restrictive cardiomyopathy with a poor prognosis. This protein can have a number of different origins, which give rise to various subtypes of amyloidosis that have different prognoses and that require different therapeutic approaches. Drugs commonly used in heart failure have little effect in amyloidosis and the use of heart transplantation is controversial because amyloidosis is a multi-organ disease and because there is a possibility of disease recurrence in the graft. The use of new techniques to identify the specific amyloidosis subtype, the emergence of novel ways of preventing or decreasing amyloid production, the ability to monitor responses to therapy and, above all, the introduction of multidisciplinary teams that can implement a combination of therapies, including multiple organ transplantation, have contributed to a substantial improvement in the prognosis of this disease.

  16. Seminal Tract Amyloidosis: Synchronous Amyloidosis of the Seminal Vesicles, Deferent Ducts and Ejaculatory Ducts.

    PubMed

    Rath-Wolfson, Lea; Bubis, Golan; Shtrasburg, Shmuel; Shvero, Asaf; Koren, Rumelia

    2017-01-17

    Senile Seminal Vesicle Amyloidosis (SSVA) increases with age. Involvement of the whole seminal tract, i.e. the seminal vesicles, ejaculatory and deferent ducts was first reported by us in the International Symposium on Amyloidosis 1998. Since then we encountered four more cases of SSVA. In all these cases the ejaculatory and deferent ducts were also involved by amyloid. The amyloid was located mostly sub-epithelially, stained positively with Congo red, gave green birefringence under polarized light and was permanganate sensitive, slightly positive for lactoferrin immunostaining and negative for all known amyloid types. In recent years the amyloid was found to be derived from Semenogelin I, a major constituent of the seminal fluid which is present in the epithelial cells of the seminal vesicle and vas deference. This would explain the deposition of amyloid not only in the seminal vesicles but also in the deferent an ejaculatory ducts which transport the seminal fluid. In a review of the literature we found three more articles on SSVA in which the amyloid was not limited to the seminal vesicles alone. We propose to designate this type of amyloid as "Senile seminal Tract Amyloidosis" (SSTA) instead of "Senile Seminal Vesicle Amyloidosis (SSVA)".

  17. A Case of Amyloidosis Presenting as Chronic Cholecystitis, Misdiagnosed as Polymyalgia Rheumatica.

    PubMed

    Um, Yoo Jin; Kim, Hyoun Ah; Jung, Jin Hee; Cho, Hundo; Kang, Joon Koo

    2016-07-25

    Amyloidosis is a rare disease defined by extracellular deposits of amorphous fibrillar proteins, derived from aggregations of misfolded proteins. Localization of amyloidosis in the gallbladder is uncommon; only eight cases have been reported. We describe a case of amyloidosis diagnosed by cholecystectomy, which possibly also affected the liver and kidney. The patient was misdiagnosed with polymyalgia rheumatica, but after a cholecystectomy to treat chronic cholecystitis, we ultimately diagnosed him with amyloidosis. We review amyloidosis with gallbladder involvement in the literature.

  18. Urinary monoclonal free light chains in primary Sjögren's syndrome: an aid to the diagnosis of malignant lymphoma.

    PubMed Central

    Walters, M T; Stevenson, F K; Herbert, A; Cawley, M I; Smith, J L

    1986-01-01

    Three patients, two with typical primary Sjögren's syndrome (SS) and the third with several features of SS, including abnormal sialography and reduced tear secretion, developed B cell non-Hodgkin's lymphoma (NHL) of parotid or lung, or both. Isoelectric focusing of concentrated urine specimens in agarose, followed by immunofixation, demonstrated the presence in each patient's urine of monoclonal free light chains of the same class as that shown on the tumour cells. In one patient the level of urinary free light chains was monitored and found to correlate with disease activity. Similar techniques showed no monoclonal light chains in the urine from a further 26 cases of SS with no clinical evidence of lymphoma. The detection of monoclonal urinary free light chains may provide an early diagnostic clue to the development of lymphoma in patients with SS and be a means of tumour monitoring. Images PMID:3082300

  19. Dialysis-related amyloidosis: challenges and solutions

    PubMed Central

    Scarpioni, R; Ricardi, M; Albertazzi, V; De Amicis, S; Rastelli, F; Zerbini, L

    2016-01-01

    Amyloidosis refers to the extracellular tissue deposition of fibrils composed of low-molecular-weight subunits of a variety of proteins. These deposits may result in a wide range of clinical manifestations depending upon their type, location, and the amount of deposition. Dialysis-related amyloidosis is a serious complication of long-term dialysis therapy and is characterized by the deposition of amyloid fibrils, principally composed of β2 microglobulins (β2M), in the osteoarticular structures and viscera. Most of the β2M is eliminated through glomerular filtration and subsequent reabsorption and catabolism by the proximal tubules. As a consequence, the serum levels of β2M are inversely related to the glomerular filtration rate; therefore, in end-stage renal disease patients, β2M levels increase up to 60-fold. Serum levels of β2M are also elevated in several pathological conditions such as chronic inflammation, liver disease, and above all, in renal dysfunction. Retention of amyloidogenic protein has been attributed to several factors including type of dialysis membrane, prolonged uremic state and/or decreased diuresis, advanced glycation end products, elevated levels of cytokines and dialysate. Dialysis treatment per se has been considered to be an inflammatory stimulus, inducing cytokine production (such as interleukin-1, tumor necrosis factor-α, interleukin-6) and complement activation. The released cytokines are thought to stimulate the synthesis and release of β2M by the macrophages and/or augment the expression of human leukocyte antigens (class I), increasing β2M expression. Residual renal function is probably the best determinant of β2M levels. Therefore, it has to be maintained as long as possible. In this article, we will focus our attention on the etiology of dialysis-related amyloidosis, its prevention, therapy, and future solutions. PMID:27994478

  20. Amyloidosis: The Newer Discovered ALECT2 Associated with der7q add(7)

    PubMed Central

    Samal, Priyanka; Chakrabartty, Joydeep

    2016-01-01

    Amyloidosis is characterized by pathological deposition of abnormal protein aggregates in various tissues, AL protein being the commonest. ALECT 2 is the newest protein described, having a predisposition to affect the kidneys, sometimes the liver and rarely other organs. We present a case of renal amyloid ALECT 2 due to leucocyte cell derived chemotaxin 2, a novel amyloidogenic protein. The patient presented with mild proteinuria, scattered plasma cells on bone marrow examination and altered kappa/lambda ratio with associated cytogenetic abnormality of der7q add(7). It is essential to correctly type this protein and differentiate it from AL during diagnosis for appropriate and effective clinical management. PMID:27790444

  1. Clathrin light chain B: gene structure and neuron-specific splicing.

    PubMed Central

    Stamm, S; Casper, D; Dinsmore, J; Kaufmann, C A; Brosius, J; Helfman, D M

    1992-01-01

    The clathrin light chains are components of clathrin coated vesicles, structural constituents involved in endocytosis and membrane recycling. The clathrin light chain B (LCB) gene encodes two isoforms, termed LCB2 and LCB3, via an alternative RNA splicing mechanism. We have determined the structure of the rat clathrin light chain B gene. The gene consists of six exons that extend over 11.9 kb. The first four exons and the last exon are common to the LCB2 and LCB3 isoforms. The fifth exon, termed EN, is included in the mRNA in brain, giving rise to the brain specific form LCB2 but is excluded in other tissues, generating the LCB3 isoform. Primary rat neuronal cell cultures express predominantly the brain specific LCB2 isoform, whereas primary rat cultures of glia express only the LCB3 isoform, suggesting that expression of the brain-specific LCB2 form is limited to neurons. Further evidence for neuronal localization of the LCB2 form is provided using a teratocarcinoma cell line, P19, which can be induced by retinoic acid to express a neuronal phenotype, concomitant with the induction of the LCB2 form. In order to determine the sequences involved in alternative splice site selection, we constructed a minigene containing the alternative spliced exon EN and its flanking intron and exon sequences. This minigene reflects the splicing pattern of the endogenous gene upon transfection in HeLa cell and primary neuronal cell cultures, indicating that this region of the LCB gene contains all the necessary information for neuron-specific splicing. Images PMID:1408826

  2. Molecular characterization of the immunoglobulin light chain variable region repertoire of human autoantibodies

    SciTech Connect

    Victor, K.D.

    1992-01-01

    The molecular structures of the light chain variable regions encoding human autoantibodies have been studied in detail. The variable region repertoire among this group of antibodies is diverse. There is no evidence for preferential utilization of specific V[sub L] gene families or over-representation of certain V[sub L] gene segments in autoantibodies. Many autoreactive antibodies utilize direct copies of known germline gene segments with little evidence of somatic mutation, supporting the conclusion that at least some germline gene segments encode autoreactivity. Additionally, the structures of several autoantibodies are clearly the product of somatic mutation. Lastly, affinity maturation has been demonstrated in two clonally related IgM rheumatoid factors suggestive of an antigen driven response. The heterogeneity of the V[sub L] region repertoire in human autoantibodies challenges evidence in the literature suggesting that the majority of human autoantibodies utilize the same or closely related germline gene segments with no evidence of somatic mutation. In addition, this study has documented that variation in the length of the light chain is a common feature in human antibodies. Length variation is confined to the V[sub k]-J[sub k] joint of CDR3 and occurs in all V[sub k] gene families. Analysis of the structures of the V[sub k]-J[sub k] joints suggests that both germline derived and non-germline encoded nucleotides (N-segments), probably the result of terminal deoxynucleotidyl transferase activity, contribute to the junctional diversity of the immunoglobulin light chain variable region. Thus, length variation at the V[sub L]-J[sub L] joint is a frequent event having the potential to expand the diversity of the antibody molecule.

  3. Systemic lupus erythematosus: molecular cloning and analysis of recombinant DNase monoclonal κ light chain NGK-1.

    PubMed

    Kostrikina, Irina A; Odintsova, Elena S; Buneva, Valentina N; Nevinsky, Georgy A

    2014-08-01

    Because DNase antibodies are cytotoxic, enter the nucleus and cause DNA fragmentation inducing cell death by apoptosis, they can play an important role in the pathogenesis of different autoimmune pathologies and especially systemic lupus erythematosus (SLE). The interesting goal of catalytic antibodies research is not only to study a possible biological role of such antibodies, but also to develop in future new human and animal therapies that use the advantages offered by abzymes. An immunoglobulin κ light chain library from SLE patients was cloned into a phagemid vector. Phage particles displaying recombinant monoclonal antibody light chains (MLChs) capable of binding DNA were isolated by affinity chromatography on DNA-cellulose. Sixteen of the 46 MLChs efficiently hydrolyzed DNA; one MLCh (approximately 27-28kDa) was expressed in Escherichia coli and purified by metal chelating and gel filtration. MLCh NGK-1 was electrophoretically homogeneous and demonstrated a positive answer with mouse IgGs against light chains of human antibodies after western blotting. SDS-PAGE in a gel containing DNA demonstrated that the MLCh hydrolyzes DNA and is not contaminated by canonical DNases. The DNase MLCh was activated by several metal ions. The protein sequence of the DNase MLCh has homology with mammalian DNases I and shares with them several identical or similar (with the same side chain functionality) important amino acid residues, which are necessary for DNA hydrolysis and binding of Mg(2+) and Ca(2+) ions. The affinity of DNA for this first example of a MLCh (K(M) = 0.3 microM) was 150- to 200-fold higher than for human DNase I.

  4. Effect of specimen type on free immunoglobulin light chains analysis on the Roche Diagnostics cobas 8000 analyzer.

    PubMed

    Nelson, Louis S; Steussy, Bryan; Morris, Cory S; Krasowski, Matthew D

    2015-01-01

    The measurement of free immunoglobulin light chains is typically performed on serum; however, the use of alternative specimen types has potential benefits. Using the Freelite™ kappa and lambda free light chains assay on a Roche Diagnostics cobas 8000 c502 analyzer, we compared three specimen types (serum, EDTA-plasma and lithium heparin plasma separator gel-plasma) on 100 patients. Using Deming regression and eliminating outliers (limiting data to light chain concentrations below 400 mg/L), the three specimen types showed comparable results for kappa light chain concentration, lambda light chain concentration, and kappa/lambda ratio with slopes close to 1.0 and y-intercepts close to zero. EDTA-plasma showed slightly more positive bias relative to serum than lithium heparin. Analysis using EDTA-plasma and lithium heparin plasma showed comparable linearity, precision, and temperature stability. A single sample showing hook effect (not in the comparison set) gave comparable results using either plasma specimen type. For the Freelite™ kappa and lambda free light chains assay, both EDTA-plasma or lithium heparin-plasma can serve as acceptable substitutes for serum, at least for the Roche cobas 8000 analyzer.

  5. Clathrin self-assembly is regulated by three light-chain residues controlling the formation of critical salt bridges.

    PubMed

    Ybe, J A; Greene, B; Liu, S H; Pley, U; Parham, P; Brodsky, F M

    1998-08-10

    Clathrin self-assembly into a polyhedral lattice mediates membrane protein sorting during endocytosis and organelle biogenesis. Lattice formation occurs spontaneously in vitro at low pH and, intracellularly, is triggered by adaptors at physiological pH. To begin to understand the cellular regulation of clathrin polymerization, we analyzed molecular interactions during the spontaneous assembly of recombinant hub fragments of the clathrin heavy chain, which bind clathrin light-chain subunits and mimic the self-assembly of intact clathrin. Reconstitution of hubs using deletion and substitution mutants of the light-chain subunits revealed that the pH dependence of clathrin self-assembly is controlled by only three acidic residues in the clathrin light-chain subunits. Salt inhibition of hub assembly identified two classes of salt bridges which are involved and deletion analysis mapped the clathrin heavy-chain regions participating in their formation. These combined observations indicated that the negatively charged regulatory residues, identified in the light-chain subunits, inhibit the formation of high-affinity salt bridges which would otherwise induce clathrin heavy chains to assemble at physiological pH. In the presence of light chains, clathrin self-assembly depends on salt bridges that form only at low pH, but is exquisitely sensitive to regulation. We propose that cellular clathrin assembly is controlled via the simple biochemical mechanism of reversing the inhibitory effect of the light-chain regulatory sequence, thereby promoting high-affinity salt bridge formation.

  6. Structural Characterization of the Partially Folded Intermediates of An Immunoglobulin Light Chain Leading to Amyloid Fibrillation And Amorphous Aggregation

    SciTech Connect

    Qin, Z.; Hu, D.; Zhu, M.; Fink, A.L.; /UC, Santa Cruz

    2007-07-12

    Immunoglobulin light chain deposition diseases involve various types of extracellular deposition of light chain variable domains, including amyloid fibrils and amorphous deposits. The decreased thermodynamic stability of the light chain is believed to be the major factor leading to fibrillation. However, the differences in the nature of the deposits among the light chain deposition diseases raise the question of whether the mechanisms leading to fibrillar or amorphous aggregation is different. In this study, we generated two partially folded intermediates of the light chain variable domain SMA in the presence of guanidine hydrochloride (GuHCl) and characterized their conformations. The more unfolded intermediate formed fibrils most rapidly, while the more native-like intermediate predominantly led to amorphous deposits. The results also show that the monomeric, rather than the dimeric state, was critical for fibrillation. The data also indicate that fibril elongation involves addition of a partially unfolded intermediate, rather than the native state. We postulate that a more highly unfolded intermediate is more suited to undergo the topological rearrangements necessary to form amyloid fibrils than a more structured one and that this also correlates with increased destabilization. In the case of light chain aggregation, it appears that more native-like intermediate conformations are more prone to form amorphous deposits.

  7. Prevalence of amyloid deposition in mature healthy chickens in the flock that previously had outbreaks of vaccine-associated amyloidosis

    PubMed Central

    IBI, Kanata; MURAKAMI, Tomoaki; GODA, Wael Mohamed; KOBAYASHI, Naoki; ISHIGURO, Naotaka; YANAI, Tokuma

    2015-01-01

    Avian amyloid A (AA) amyloidosis is commonly observed in adult birds with chronic inflammation, such as that caused by bacterial infection. We previously described vaccine-associated AA amyloidosis in juvenile chickens. In this study, the prevalence of amyloid deposition was measured in mature healthy chickens that survived a previous outbreak of avian AA amyloidosis while they were juveniles. Herein, we analyzed the amyloid deposition in mature chickens and compared the prevalence of amyloid deposition with juvenile chickens obtained in our previous study (Murakami et al., 2013). We found that: 1) amyloid deposition in the liver was absent in mature chickens, while juvenile chickens had a rate of 24%; 2) amyloid deposition in the spleen was observed in 36% of juvenile chickens and in 40% of mature chickens; 3) amyloid deposition in the pectoral muscle of mature chickens (43.75%) was approximately half that of juvenile chickens (88%). These results suggest that additional amyloid deposition in chickens previously exposed to AA amyloidosis may not worsen with age. Further, amyloid deposition in chickens may tend to regress when causative factors, such as vaccinations and/or chronic inflammation, are absent. PMID:25985816

  8. Prevalence of amyloid deposition in mature healthy chickens in the flock that previously had outbreaks of vaccine-associated amyloidosis.

    PubMed

    Ibi, Kanata; Murakami, Tomoaki; Goda, Wael Mohamed; Kobayashi, Naoki; Ishiguro, Naotaka; Yanai, Tokuma

    2015-10-01

    Avian amyloid A (AA) amyloidosis is commonly observed in adult birds with chronic inflammation, such as that caused by bacterial infection. We previously described vaccine-associated AA amyloidosis in juvenile chickens. In this study, the prevalence of amyloid deposition was measured in mature healthy chickens that survived a previous outbreak of avian AA amyloidosis while they were juveniles. Herein, we analyzed the amyloid deposition in mature chickens and compared the prevalence of amyloid deposition with juvenile chickens obtained in our previous study (Murakami et al., 2013). We found that: 1) amyloid deposition in the liver was absent in mature chickens, while juvenile chickens had a rate of 24%; 2) amyloid deposition in the spleen was observed in 36% of juvenile chickens and in 40% of mature chickens; 3) amyloid deposition in the pectoral muscle of mature chickens (43.75%) was approximately half that of juvenile chickens (88%). These results suggest that additional amyloid deposition in chickens previously exposed to AA amyloidosis may not worsen with age. Further, amyloid deposition in chickens may tend to regress when causative factors, such as vaccinations and/or chronic inflammation, are absent.

  9. Transthyretin amyloidosis: an under-recognized neuropathy and cardiomyopathy.

    PubMed

    Galant, Natalie J; Westermark, Per; Higaki, Jeffrey N; Chakrabartty, Avijit

    2017-03-01

    Transthyretin (TTR) amyloidosis (ATTR amyloidosis) is an underdiagnosed and important type of cardiomyopathy and/or polyneuropathy that requires increased awareness within the medical community. Raising awareness among clinicians about this type of neuropathy and lethal form of heart disease is critical for improving earlier diagnosis and the identification of patients for treatment. The following review summarizes current criteria used to diagnose both hereditary and wild-type ATTR (ATTRwt) amyloidosis, tools available to clinicians to improve diagnostic accuracy, available and newly developing therapeutics, as well as a brief biochemical and biophysical background of TTR amyloidogenesis.

  10. Bacterial kinesin light chain (Bklc) links the Btub cytoskeleton to membranes

    PubMed Central

    Akendengue, Lurlène; Trépout, Sylvain; Graña, Martín; Voegele, Alexis; Janke, Carsten; Raynal, Bertrand; Chenal, Alexandre; Marco, Sergio; Wehenkel, Anne Marie

    2017-01-01

    Bacterial kinesin light chain is a TPR domain-containing protein encoded by the bklc gene, which co-localizes with the bacterial tubulin (btub) genes in a conserved operon in Prosthecobacter. Btub heterodimers show high structural homology with eukaryotic tubulin and assemble into head-to-tail protofilaments. Intriguingly, Bklc is homologous to the light chain of the microtubule motor kinesin and could thus represent an additional eukaryotic-like cytoskeletal element in bacteria. Using biochemical characterization as well as cryo-electron tomography we show here that Bklc interacts specifically with Btub protofilaments, as well as lipid vesicles and could thus play a role in anchoring the Btub filaments to the membrane protrusions in Prosthecobacter where they specifically localize in vivo. This work sheds new light into possible ways in which the microtubule cytoskeleton may have evolved linking precursors of microtubules to the membrane via the kinesin moiety that in today’s eukaryotic cytoskeleton links vesicle-packaged cargo to microtubules. PMID:28358387

  11. High level expression of human enteropeptidase light chain in Pichia pastoris.

    PubMed

    Pepeliaev, Stanislav; Krahulec, Ján; Černý, Zbyněk; Jílková, Jana; Tlustá, Marcela; Dostálová, Jana

    2011-10-20

    Human enterokinase (enteropeptidase, rhEP), a serine protease expressed in the proximal part of the small intestine, converts the inactive form of trypsinogen to active trypsin by endoproteolytic cleavage. The high specificity of the target site makes enterokinase an ideal tool for cleaving fusion proteins at defined cleavage sites. The mature active enzyme is comprised of two disulfide-linked polypeptide chains. The heavy chain anchors the enzyme in the intestinal brush border membrane, whereas the light chain represents the catalytic enzyme subunit. The synthetic gene encoding human enteropeptidase light chain with His-tag added at the C-terminus to facilitate protein purification was cloned into Pichia pastoris expression plasmids under the control of an inducible AOX1 or constitutive promoters GAP and AAC. Cultivation media and conditions were optimized as well as isolation and purification of the target protein. Up to 4 mg/L of rhEP was obtained in shake-flask experiments and the expression level of about 60-70 mg/L was achieved when cultivating in lab-scale fermentors. The constitutively expressing strains proved more efficient and less labor-demanding than the inducible ones. The rhEP was immobilized on AV 100 sorbent (Iontosorb) to allow repeated use of enterokinase, showing specific activity of 4U/mL of wet matrix.

  12. Nonmuscle Myosin IIA Regulates Platelet Contractile Forces Through Rho Kinase and Myosin Light-Chain Kinase.

    PubMed

    Feghhi, Shirin; Tooley, Wes W; Sniadecki, Nathan J

    2016-10-01

    Platelet contractile forces play a major role in clot retraction and help to hold hemostatic clots against the vessel wall. Platelet forces are produced by its cytoskeleton, which is composed of actin and nonmuscle myosin filaments. In this work, we studied the role of Rho kinase, myosin light-chain kinase, and myosin in the generation of contractile forces by using pharmacological inhibitors and arrays of flexible microposts to measure platelet forces. When platelets were seeded onto microposts, they formed aggregates on the tips of the microposts. Forces produced by the platelets in the aggregates were measured by quantifying the deflection of the microposts, which bent in proportion to the force of the platelets. Platelets were treated with small molecule inhibitors of myosin activity: Y-27632 to inhibit the Rho kinase (ROCK), ML-7 to inhibit myosin light-chain kinase (MLCK), and blebbistatin to inhibit myosin ATPase activity. ROCK inhibition reduced platelet forces, demonstrating the importance of the assembly of actin and myosin phosphorylation in generating contractile forces. Similarly, MLCK inhibition caused weaker platelet forces, which verifies that myosin phosphorylation is needed for force generation in platelets. Platelets treated with blebbistatin also had weaker forces, which indicates that myosin's ATPase activity is necessary for platelet forces. Our studies demonstrate that myosin ATPase activity and the regulation of actin-myosin assembly by ROCK and MLCK are needed for the generation of platelet forces. Our findings illustrate and explain the importance of myosin for clot compaction in hemostasis and thrombosis.

  13. Calcyon, a novel partner of clathrin light chain, stimulates clathrin-mediated endocytosis.

    PubMed

    Xiao, Jiping; Dai, Rujuan; Negyessy, Laszlo; Bergson, Clare

    2006-06-02

    In the central nervous system, clathrin-mediated endocytosis is crucial for efficient synaptic transmission. Clathrin-coated vesicle assembly and disassembly is regulated by some 30 adaptor and accessory proteins, most of which interact with clathrin heavy chain. Using the calcyon cytosolic domain as bait, we isolated clathrin light chain in a yeast two-hybrid screen. The interaction domain was mapped to the heavy chain binding domain and C-terminal regions of light chain. Further, the addition of the calcyon C terminus stimulated clathrin self-assembly in a dose-dependent fashion. Calcyon, which is a single transmembrane protein predominantly expressed in brain, localized to vesicular compartments within pre- and postsynaptic structures. There was a high degree of overlap in the distribution of LC and calcyon in neuronal dendrites, spines, and cell bodies. Co-immunoprecipitation studies further suggested an association of calcyon with the clathrin-mediated endocytic machinery. Compared with controls, HEK293 cells overexpressing calcyon exhibited significantly enhanced transferrin uptake but equivalent levels of recycling. Conversely, transferrin uptake was largely abolished in neocortical neurons obtained from mice homozygous for a calcyon null allele, whereas recycling proceeded at wild type levels. Collectively, these data indicate a role for calcyon in clathrin-mediated endocytosis in brain.

  14. Unique Substrate Recognition Mechanism of the Botulinum Neurotoxin D Light Chain*

    PubMed Central

    Guo, Jiubiao; Chen, Sheng

    2013-01-01

    Botulinum neurotoxins are the most potent protein toxins in nature. Despite the potential to block neurotransmitter release at the neuromuscular junction and cause human botulism, they are widely used in protein therapies. Among the seven botulinum neurotoxin serotypes, mechanisms of substrate recognition and specificity are known to a certain extent in the A, B, E, and F light chains, but not in the D light chain (LC/D). In this study, we addressed the unique substrate recognition mechanism of LC/D and showed that this serotype underwent hydrophobic interactions with VAMP-2 at its V1 motif. The LC/D B3, B4, and B5 binding sites specifically recognize the hydrophobic residues in the V1 motif of VAMP-2. Interestingly, we identified a novel dual recognition mechanism employed by LC/D in recognition of VAMP-2 sites at both the active site and distal binding sites, in which one site of VAMP-2 was recognized by two independent, but functionally similar LC/D sites that were complementary to each other. The dual recognition strategy increases the tolerance of LC/D to mutations and renders it a good candidate for engineering to improve its therapeutic properties. In conclusion, in this study, we identified a unique multistep substrate recognition mechanism by LC/D and provide insights for LC/D engineering and antitoxin development. PMID:23963459

  15. Planarian myosin essential light chain is involved in the formation of brain lateral branches during regeneration.

    PubMed

    Yu, Shuying; Chen, Xuhui; Yuan, Zuoqing; Zhou, Luming; Pang, Qiuxiang; Mao, Bingyu; Zhao, Bosheng

    2015-08-01

    The myosin essential light chain (ELC) is a structure component of the actomyosin cross-bridge, however, the functions in the central nervous system (CNS) development and regeneration remain poorly understood. Planarian Dugesia japonica has revealed fundamental mechanisms and unique aspects of neuroscience and neuroregeneration. In this study, the cDNA DjElc, encoding a planarian essential light chain of myosin, was identified from the planarian Dugesia japonica cDNA library. It encodes a deduced protein with highly conserved functionally domains EF-Hand and Ca(2+) binding sites that shares significant similarity with other members of ELC. Whole mount in situ hybridization studies show that DjElc expressed in CNS during embryonic development and regeneration of adult planarians. Loss of function of DjElc by RNA interference during planarian regeneration inhibits brain lateral branches regeneration completely. In conclusion, these results demonstrated that DjElc is required for maintenance of neurons and neurite outgrowth, particularly for involving the brain later branch regeneration.

  16. Regulatory light chain phosphorylation increases eccentric contraction-induced injury in skinned fast-twitch fibers.

    PubMed

    Childers, Martin K; McDonald, Kerry S

    2004-02-01

    During contraction, activation of Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) results in phosphorylation of myosin's regulatory light chain (RLC), which potentiates force and increases speed of force development over a wide range of [Ca(2+)]. We tested the hypothesis that RLC phosphorylation by MLCK mediates the extent of eccentric contraction-induced injury as measured by force deficit in skinned fast-twitch skeletal muscle fibers. Results indicated that RLC phosphorylation in single skinned rat psoas fibers significantly increased Ca(2+) sensitivity of isometric force; isometric force from 50 +/- 16 to 59 +/- 18 kN/m(2) during maximal Ca(2+) activation; peak absolute power output from 38 +/- 15 to 48 +/- 14 nW during maximal Ca(2+) activation; and the magnitude of contraction-induced force deficit during maximal (pCa 4.5) activation from 26 +/- 9.8 to 35 +/- 9.6%. We conclude that RLC phosphorylation increases force deficits following eccentric contractions, perhaps by increasing the number of force-generating cross-bridges.

  17. Myosin light chain kinase and Src control membrane dynamics in volume recovery from cell swelling

    PubMed Central

    Barfod, Elisabeth T.; Moore, Ann L.; Van de Graaf, Benjamin G.; Lidofsky, Steven D.

    2011-01-01

     The expansion of the plasma membrane, which occurs during osmotic swelling of epithelia, must be retrieved for volume recovery, but the mechanisms are unknown. Here we have identified myosin light chain kinase (MLCK) as a regulator of membrane internalization in response to osmotic swelling in a model liver cell line. On hypotonic exposure, we found that there was time-dependent phosphorylation of the MLCK substrate myosin II regulatory light chain. At the sides of the cell, MLCK and myosin II localized to swelling-induced membrane blebs with actin just before retraction, and MLCK inhibition led to persistent blebbing and attenuated cell volume recovery. At the base of the cell, MLCK also localized to dynamic actin-coated rings and patches upon swelling, which were associated with uptake of the membrane marker FM4-64X, consistent with sites of membrane internalization. Hypotonic exposure evoked increased biochemical association of the cell volume regulator Src with MLCK and with the endocytosis regulators cortactin and dynamin, which colocalized within these structures. Inhibition of either Src or MLCK led to altered patch and ring lifetimes, consistent with the concept that Src and MLCK form a swelling-induced protein complex that regulates volume recovery through membrane turnover and compensatory endocytosis under osmotic stress. PMID:21209319

  18. [Ontogenetic and phylogenetic analysis of myosin light chain proteins from skeletal muscles of loach Misgurnus fossilis].

    PubMed

    Miuge, N S; Tikhonov, A V; Ozerniuk, N D

    2005-01-01

    mRNAs of all three types of myosin light chain proteins are expressed in skeletal muscles of both larval and adult stages of loach Misgurnus fossilis (Cobitidae) and these proteins are encoded by different genes (mlc1, mlc2, and mlc3). No difference was revealed between transcripts from larval stage and adult fish for all three mlc proteins. Our approach (RT-PCR with fish-specific mlc1, mlc2, and mlc3 primers) failed to reveal the larval form of myosin light chain protein found previously by protein electrophoresis of loach fry muscle extract. Comparative analysis of the protein structure shows high homology of MLC1 and MLC3 proteins sharing a large EF-hand calcium-binding domain. Phylogenetic analysis of MLC1 from skeletal muscles of fish and other vertebrate species is concordant with the traditional phylogeny of the group. Within the Teleostei, loach MLC1 had the highest homology with other Cyprinidae, and least with Salmonidae fishes.

  19. Immunohistochemical and morphometric analysis of immunoglobulin light-chain immunoreactive amyloid in psammoma bodies of the human choroid plexus.

    PubMed

    Jovanović, Ivan; Ugrenović, Sladjana; Vasović, Ljiljana; Stojanović, Ivan

    2014-03-01

    The aim of this research was to establish the presence of amyloid and to quantify immunohistochemical reactions of kappa and lambda light chains of psammoma bodies of the choroid plexus. Choroid plexus tissue obtained from 14 right lateral ventricles postmortem was processed histologically and stained with Congo red, thioflavin T, and monoclonal antibodies for kappa and lambda light chains. Morphological analysis was performed with a light microscope at lens magnifications of 4×, 10×, 20×, 25×, and 40×. The morphometric characteristics of psammoma bodies that were kappa and lambda positive and negative were analyzed with ImageJ. Histological analysis showed that the psammoma bodies, stromal blood vessel walls, and some epithelial cells reacted positively with Congo red and thioflavin T. Psammoma bodies were predominantly positive for lambda light chains. Lambda positivity was detected inside some stromal blood vessels, which pointed to a probable systemic origin for these light chains. Morphometric analysis showed that the mean optical densities of lambda- and kappa-positive psammoma bodies were significantly higher than those that gave a negative reaction. The percentage of lambda-positive psammoma bodies was significantly higher than the percentage of lambda-negative psammoma bodies in 80% of the cases, while the reaction with kappa light chains was negative in the majority of the cases. Linear regression analysis showed a significant increase in the percentage of lambda-positive psammoma bodies and their mean optical density with age. Finally, it can be concluded that the positive reaction of psammoma bodies in the choroid plexus with respect to amyloid and lambda light chains may point to the presence of light-chain amyloid in their structures.

  20. Antibodies to myelin-associated glycoprotein (anti-Mag) in IgM amyloidosis may influence expression of neuropathy in rare patients.

    PubMed

    Garces-Sanchez, Mercedes; Dyck, Peter J; Kyle, Robert A; Zeldenrust, Steven; Wu, Yanhong; Ladha, Shafeeq S; Klein, Christopher J

    2008-04-01

    We have examined whether antibodies to myelin-associated glycoprotein (anti-MAG) influence neuropathy occurrence and phenotype in primary (AL IgM) amyloidosis. Anti-MAG and the cross-reacted sulfoglucuronyl paragloboside antibodies (SGPG) were studied in 46 patients with IgM amyloidosis (21 with polyneuropathy), and 21 matched IgM MGUS (monoclonal gammopathies of undetermined significance) controls without neuropathy. We assessed the occurrence, phenotype of neuropathy, and attributes of nerve conduction and their relation to antibody activity. Twenty of 46 patients with IgM amyloidosis (7 with and 13 without polyneuropathy) had elevation of anti-MAG or SGPG by enzyme-linked immunosorbent assay (ELISA). Two of the polyneuropathy patients with IgM amyloidosis had antibodies to MAG based on Western blot (WB) positivity. One of these patients, with the highest anti-MAG titer, had a painful sensory ataxia, with prominent demyelination, and amyloid deposition in sural nerve. The other anti-MAG WB-positive amyloid patient had an axonal neuropathy and dysautonomia. Low levels of anti-MAG antibodies were found in 12 of 21 IgM MGUS controls without neuropathy (mean follow-up, 11 years). We conclude that finding serum anti-MAG antibodies does not exclude the diagnosis of primary amyloidosis. They do not appear to affect the occurrence or expression of polyneuropathy, except possibly in occasional cases with WB positivity.

  1. [Otolaryngological complaints in tongue amyloidosis: a case report].

    PubMed

    Pons Rocher, F; Guallart Doménech, F; Mompó Romero, L; Artazkozl del Toro, J J; Serrano Badía, E; Dalmau Galofré, J; Faubel Serra, M

    1994-01-01

    We present a case of Amyloidosis of the oral cavity associated to multiple mieloma, with otolaryngological symptom. Review of structural characterization of the disease, its pathogenesis and clinical disorders when displayed in thyroid, oral cavity and upper respiratory tract.

  2. AA amyloidosis associated with systemic-onset juvenile idiopathic arthritis.

    PubMed

    Saha, Abhijeet; Chopra, Yogiraj; Theis, Jason D; Vrana, Julie A; Sethi, Sanjeev

    2013-10-01

    We report a 12-year-old boy with nephrotic syndrome due to renal AA amyloidosis. The AA amyloidosis was associated with a 3-year history of systemic-onset juvenile idiopathic arthritis. The presence of serum amyloid A protein was confirmed by laser microdissection of Congo Red-positive glomeruli and vessels followed by liquid chromatography and tandem mass spectrometry; this analysis excluded hereditary and familial amyloidosis. Aggressive management of the systemic-onset juvenile idiopathic arthritis resulted in improvement in clinical and laboratory parameters. The case represents an unusual cause of nephrotic syndrome in children. Early diagnosis of renal amyloidosis and management of systemic-onset juvenile idiopathic arthritis is paramount to preventing progression of kidney disease.

  3. Severe cranial nerve involvement in a patient with monoclonal anti-MAG/SGPG IgM antibody and localized hard palate amyloidosis.

    PubMed

    Yoshida, Takuhiro; Yazaki, Masahide; Gono, Takahisa; Tazawa, Ko-ichi; Morita, Hiroshi; Matsuda, Masayuki; Funakoshi, Kei; Yuki, Nobuhiro; Ikeda, Shu-ichi

    2006-05-15

    We report a patient with severe cranial polyneuropathy as well as sensory limb neuropathy. Biclonal serum IgM-kappa/IgM-lambda gammopathy was found and serum anti-myelin-associated glycoprotein (MAG)/sulfoglucuronyl paragloboside (SGPG) IgM antibody was also detected. Immunofluorescence analysis of a sural nerve biopsy specimen revealed binding of IgM and lambda-light chain on myelin sheaths. No amyloid deposition was detected in biopsied tissues except for the hard palate, suggesting that the amyloidosis was of the localized type and had no relation to the pathogenesis of cranial neuropathy. Our observations indicate that the anti-MAG/SGPG IgM antibody may be responsible for this patient's cranial polyneuropathy, which is a rare manifestation in anti-MAG/SGPG-associated neuropathy.

  4. A Rare Case of Amyloidosis of the Eyelid and Conjunctiva

    PubMed Central

    Fernando, Bertie

    2016-01-01

    Amyloidosis of the eyelid is uncommon and is typically associated with systemic associations. In contrast, amyloidosis of the conjunctiva is often localised with no other associations. We present a rare case of a 92-year-old gentleman with both cutaneous lid lesions and conjunctival amyloid with no systemic involvement. Biopsy demonstrated the hallmarks of amyloid and treatment has remained conservative. He remains at the department to be monitored for secondary glaucoma. PMID:27752377

  5. Extracellular volume quantification by dynamic equilibrium cardiac computed tomography in cardiac amyloidosis

    PubMed Central

    Treibel, Thomas A.; Bandula, Steve; Fontana, Marianna; White, Steven K.; Gilbertson, Janet A.; Herrey, Anna S.; Gillmore, Julian D.; Punwani, Shonit; Hawkins, Philip N.; Taylor, Stuart A.; Moon, James C.

    2015-01-01

    Background Cardiac involvement determines outcome in patients with systemic amyloidosis. There is major unmet need for quantification of cardiac amyloid burden, which is currently only met in part through semi-quantitative bone scintigraphy or Cardiovascular Magnetic Resonance (CMR), which measures ECVCMR. Other accessible tests are needed. Objectives To develop cardiac computed tomography to diagnose and quantify cardiac amyloidosis by measuring the myocardial Extracellular Volume, ECVCT. Methods Twenty-six patients (21 male, 64 ± 14 years) with a biopsy-proven systemic amyloidosis (ATTR n = 18; AL n = 8) were compared with twenty-seven patients (19 male, 68 ± 8 years) with severe aortic stenosis (AS). All patients had undergone echocardiography, bone scintigraphy, NT-pro-BNP measurement and EQ-CMR. Dynamic Equilibrium CT (DynEQ-CT) was performed using a prospectively gated cardiac scan prior to and after (5 and 15 minutes) a standard Iodixanol (1 ml/kg) bolus to measure ECVCT. ECVCT was compared to the reference ECVCMR and conventional amyloid measures: bone scintigraphy and clinical markers of cardiac amyloid severity (NT-pro-BNP, Troponin, LVEF, LV mass, LA and RA area). Results ECVCT and ECVCMR results were well correlated (r2 = 0.85 vs r2 = 0.74 for 5 and 15 minutes post bolus respectively). ECVCT was higher in amyloidosis than AS (0.54 ± 0.11 vs 0.28 ± 0.04, p<0.001) with no overlap. ECVCT tracked clinical markers of cardiac amyloid severity (NT-pro-BNP, Troponin, LVEF, LV mass, LA and RA area), and bone scintigraphy amyloid burden (p<0.001). Conclusion Dynamic Equilibrium CT, a 5 minute contrast-enhanced gated cardiac CT, has potential for non-invasive diagnosis and quantification of cardiac amyloidosis. PMID:26209459

  6. Insulin-induced myosin light-chain phosphorylation during receptor capping in IM-9 human B-lymphoblasts.

    PubMed Central

    Majercik, M H; Bourguignon, L Y

    1988-01-01

    We have examined further the interaction between insulin surface receptors and the cytoskeleton of IM-9 human lymphoblasts. Using immunocytochemical techniques, we determined that actin, myosin, calmodulin and myosin light-chain kinase (MLCK) are all accumulated directly underneath insulin-receptor caps. In addition, we have now established that the concentration of intracellular Ca2+ (as measured by fura-2 fluorescence) increases just before insulin-induced receptor capping. Most importantly, we found that the binding of insulin to its receptor induces phosphorylation of myosin light chain in vivo. Furthermore, a number of drugs known to abolish the activation properties of calmodulin, such as trifluoperazine (TFP) or W-7, strongly inhibit insulin-receptor capping and myosin light-chain phosphorylation. These data imply that an actomyosin cytoskeletal contraction, regulated by Ca2+/calmodulin and MLCK, is involved in insulin-receptor capping. Biochemical analysis in vitro has revealed that IM-9 insulin receptors are physically associated with actin and myosin; and most interestingly, the binding of insulin-receptor/cytoskeletal complex significantly enhances the phosphorylation of the 20 kDa myosin light chain. This insulin-induced phosphorylation is inhibited by calmodulin antagonists (e.g. TFP and W-7), suggesting that the phosphorylation is catalysed by MLCK. Together, these results strongly suggest that MLCK-mediated myosin light-chain phosphorylation plays an important role in regulating the membrane-associated actomyosin contraction required for the collection of insulin receptors into caps. Images Fig. 2. Fig. 4. PMID:3048249

  7. Two distinct myosin light chain structures are induced by specific variations within the bound IQ motifs—functional implications

    PubMed Central

    Terrak, Mohammed; Wu, Guanming; Stafford, Walter F.; Lu, Renne C.; Dominguez, Roberto

    2003-01-01

    IQ motifs are widespread in nature. Mlc1p is a calmodulin-like myosin light chain that binds to IQ motifs of a class V myosin, Myo2p, and an IQGAP-related protein, Iqg1p, playing a role in polarized growth and cytokinesis in Saccharomyces cerevisiae. The crystal structures of Mlc1p bound to IQ2 and IQ4 of Myo2p differ dramatically. When bound to IQ2, Mlc1p adopts a compact conformation in which both the N- and C-lobes interact with the IQ motif. However, in the complex with IQ4, the N-lobe no longer interacts with the IQ motif, resulting in an extended conformation of Mlc1p. The two light chain structures relate to two distinct subfamilies of IQ motifs, one of which does not interact with the N-lobes of calmodulin-like light chains. The correlation between light chain structure and IQ sequence is demonstrated further by sedimentation velocity analysis of complexes of Mlc1p with IQ motifs from Myo2p and Iqg1p. The resulting ‘free’ N-lobes of myosin light chains in the extended conformation could mediate the formation of ternary complexes during protein localization and/or partner recruitment. PMID:12554638

  8. Hereditary renal amyloidosis with a novel variant fibrinogen.

    PubMed Central

    Uemichi, T; Liepnieks, J J; Benson, M D

    1994-01-01

    Two families with hereditary renal amyloidosis were found to have a novel mutation in the fibrinogen A alpha chain gene. This form of amyloidosis is an autosomal dominant condition characterized by proteinuria, hypertension, and subsequent azotemia. DNAs of patients with amyloidosis were screened for a polymorphism in fibrinogen A alpha chain gene by single-strand conformation polymorphism analysis, and affected individuals from two kindreds were found to have a mutation. Both of these kindreds are American of Irish descent presenting with non-neuropathic, nephropathic amyloidosis in the fifth to the seventh decade of life. DNA sequencing showed a point mutation in the fibrinogen A alpha chain gene that is responsible for substitution of valine for glutamic acid at position 526. By restriction fragment length polymorphism analysis, 7 affected individuals and 14 asymptomatic individuals in these two kindreds were positive for the fibrinogen A alpha chain Val 526 gene. Fibrinogen was isolated from plasma of a heterozygous gene carrier and shown to contain approximately 50% variant fibrinogen. Discovery of this new mutation confirms the association between fibrinogen A alpha chain variant and hereditary renal amyloidosis and establishes a new biochemical subtype of amyloidosis. Images PMID:8113408

  9. Amyloidosis in a Captive Zebra Finch (Taeniopygia guttata) Research Colony

    PubMed Central

    Shientag, Lisa J; Garlick, David S; Galati, Erin

    2016-01-01

    Five birds in a captive zebra finch research colony were diagnosed with systemic amyloidosis within a 7-mo period by means of postmortem Congo red staining and green birefringence under polarized light. The liver was the most frequently and usually the most seriously affected organ, followed by the spleen and then the kidney. All 5 birds had been clinically affected with various inflammatory, infectious, and neoplastic conditions associated with amyloid A (AA) amyloidosis in humans and animals. Immunohistochemistry using antisera against duck AA protein revealed that tissues from 2 of the 5 birds were positive for the presence of AA protein and systemic inflammation-associated amyloidosis. Although the development of AA amyloidosis has been associated with chronic inflammation, trauma, and various infectious and neoplastic diseases as well as possible genetic predispositions and stresses linked to overcrowding, the root causes for individual cases of AA amyloidosis are incompletely understood. As far as we know, this report is the first description of AA amyloidosis in captive, research zebra finches. PMID:27298248

  10. Primary amyloidosis and severe intrahepatic cholestatic jaundice.

    PubMed Central

    Peters, R A; Koukoulis, G; Gimson, A; Portmann, B; Westaby, D; Williams, R

    1994-01-01

    Liver involvement in systemic amyloidosis is frequent but is rarely of clinical importance. Five patients with severe cholestatic jaundice are described and an additional 20 from published reports are reviewed. The most frequent presenting symptoms were lethargy and abdominal pain, which were present for a median of 11 months before the onset of jaundice. Hepatomegaly, usually marked, was present in 92%, with ascites in 56% of the cases. The serum bilirubin concentration was noticeably high and the serum globulin low. Histology of the liver showed considerable perisinusoidal deposition with a slight predilection for the periportal area. Two patients presented with predominant centrilobular deposition. Congo red staining was not uniformly positive. A variety of treatment regimens was tried but median survival was only three months from the onset of jaundice. PMID:7959246

  11. Design and efficient production of bovine enterokinase light chain with higher specificity in E. coli.

    PubMed

    Chun, Haarin; Joo, Keehyoung; Lee, Jooyoung; Shin, Hang-Cheol

    2011-06-01

    Enterokinase light chain (EKL) is a serine protease that recognizes Asp-Asp-Asp-Asp-Lys (D(4)K) sequence and cleaves the C-terminal peptide bond of the lysine residue. The utility of EKL as a site-specific cleavage enzyme is hampered by sporadic cleavage at other sites than the canonical D(4)K recognition sequence. In order to produce more site-specific EKL, we have generated several EKL mutants in E. coli with substitutions at Tyr174 and Lys99 using PDI (protein disulfide isomerase) fusion system. Substitution of Tyr174 by basic residues confers higher specificity on EKL. The production of EKL with higher specificity could widen the utility of EKL as a site-specific cleavage enzyme to produce various recombinant proteins with therapeutic or industrial values.

  12. Crystal structure of a supercharged variant of the human enteropeptidase light chain.

    PubMed

    Simeonov, Peter; Zahn, Michael; Sträter, Norbert; Zuchner, Thole

    2012-07-01

    The highly specific serine protease human enteropeptidase light chain cleaves the Asp4Lys recognition sequence and represents an interesting enzyme for biotechnological applications. The human enzyme shows 10 times faster kinetics compared to other animal sources but low solubility under low salt conditions, which hampers protein production and crystallization. Therefore, a supercharged variant (N6D/G21D/G22D/N142D/K210E/C112S) with increased solubility was used for crystallization. The structure (resolution, 1.9 Å) displays a typical α/β trypsin-like serine protease-fold. The mutations introduced for protein supercharging generate larger clusters of negative potential on both sites of the active cleft but do not affect the structural integrity of the protein.

  13. RNA metabolism in isolated nuclei: processing and transport of immunoglobulin light chain sequences.

    PubMed Central

    Otegui, C; Patterson, R J

    1981-01-01

    Transport of prelabeled RNA from isolated myeloma nuclei is studied using conditions that permit RNA synthesis. Cytosol and spermidine are not required to maintain nuclear stability and inhibited RNA release. Omission of ATP or GTP decreased release 25 to 40%. The stimulatory effect of ATP or GTP is not due to hydrolysis of the triphosphates by the nuclear envelope NTPase, since addition of quercetin (an inhibitor of this NTPase) has no effect on the quantity of RNA released. The size distribution and percentage of poly A-containing species released from nuclei incubated with or without ATP or the other rNTPs are identical. Hybridization analysis of nuclear RNA before the transport assay revealed mature and precursor k light chain mRNA sequences. Following the transport assay, a significant fraction of k mRNA precursors is chased into mature k mRNA which is found both in nuclear-retained and released RNA. PMID:6795596

  14. Cryo-EM reveals the steric zipper structure of a light chain-derived amyloid fibril

    PubMed Central

    Schmidt, Andreas; Annamalai, Karthikeyan; Schmidt, Matthias; Grigorieff, Nikolaus; Fändrich, Marcus

    2016-01-01

    Amyloid fibrils are proteinaceous aggregates associated with diseases in humans and animals. The fibrils are defined by intermolecular interactions between the fibril-forming polypeptide chains, but it has so far remained difficult to reveal the assembly of the peptide subunits in a full-scale fibril. Using electron cryomicroscopy (cryo-EM), we present a reconstruction of a fibril formed from the pathogenic core of an amyloidogenic immunoglobulin (Ig) light chain. The fibril density shows a lattice-like assembly of face-to-face packed peptide dimers that corresponds to the structure of steric zippers in peptide crystals. Interpretation of the density map with a molecular model enabled us to identify the intermolecular interactions between the peptides and rationalize the hierarchical structure of the fibril based on simple chemical principles. PMID:27185936

  15. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

    NASA Technical Reports Server (NTRS)

    Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

    2002-01-01

    Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

  16. Purification, Characterization, and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkii.

    PubMed

    Zhang, Yong-Xia; Chen, Heng-Li; Maleki, Soheila J; Cao, Min-Jie; Zhang, Ling-Jing; Su, Wen-Jin; Liu, Guang-Ming

    2015-07-15

    Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in shrimp. In this study, MLC with a molecular mass of 18 kDa was purified from crayfish (Procambarus clarkii) muscle. Its physicochemical characterization showed that the purified MLC is a glycoprotein with 4.3% carbohydrate, highly stable to heat, acid-alkali, and digestion, and weakly retains IgE-binding activity when its secondary structure was altered. Serological assays suggested that conformational epitopes predominate over linear epitopes in the purified MLC. Two isoforms of the MLC gene (MLC1 and MLC2) were cloned, and the purified MLC was identified as MLC1. Analysis of the secondary and tertiary structures of the MLCs indicated that MLC1 has four conformational epitopes and three linear epitopes, whereas MLC2 had a major conformational epitope and three linear epitopes. These results are significant for understanding hypersensitization of humans to crayfish.

  17. Light chain crystalline kidney disease: diagnostic urine microscopy as the "liquid kidney biopsy".

    PubMed

    Luciano, Randy L; Castano, Ekaterina; Fogazzi, Giovanni B; Perazella, Mark A

    2014-12-01

    Multiple myeloma (MM) is a plasma cell disorder, which often causes parenchymal kidney disease. Light chain (LC) cast nephropathy represents the most common renal lesion. In some instances, LC crystals precipitate within renal tubular lumens and deposit within proximal tubular cell cytoplasms. Importantly, urine microscopy in such patients can provide insight into the underlying LC-related lesion. Here we present two patients with MM complicated by acute kidney injury (AKI) where LC crystalline casts were observed on urinary sediment analysis. Kidney biopsy revealed acute tubular injury with LC crystal casts within both tubular lumens and renal tubular epithelial cell cytoplasms. These findings suggest that the urinary sediment may be a non-invasive way to diagnose LC crystalline-induced AKI in patients with MM.

  18. [Light chain escape followed by leukemic transformation in a patient with IgD myeloma].

    PubMed

    Hatsuse, Mayumi; Fuchida, Shin-Ichi; Okano, Akira; Murakami, Satoshi; Shimazaki, Chihiro

    2015-01-01

    A 63-year-old male with multiple myeloma (IgD-λ) received autologous peripheral blood stem cell transplantation (PBSCT) after induction of VAD in March 2008, and obtained a very good partial response. However, he required BOR/DEX and a second PBSCT for relapse, and in August 2012, treatment with LEN/DEX was started. After 4 cycles of LEN/DEX, IgD decreased but FLC-λ increased paradoxically, indicating a clonal change. In January 2013, an LCD regimen was started and after 4 cycles, IgD showed normalization, but his condition worsened as FLC-λ increased. This case showed a fulminant clinical course with light chain escape in this era of treating multiple myeloma with novel agents.

  19. Prevalence and progression of monoclonal gammopathy of undetermined significance and light-chain MGUS in Germany.

    PubMed

    Eisele, Lewin; Dürig, Jan; Hüttmann, Andreas; Dührsen, Ulrich; Assert, Roland; Bokhof, Beate; Erbel, Raimund; Mann, Klaus; Jöckel, Karl-Heinz; Moebus, Susanne

    2012-02-01

    We determined the prevalence and progression rate of monoclonal gammopathy of undetermined significance (MGUS) and light-chain MGUS (LCMGUS) in Germany utilizing the biobank of the population-based Heinz Nixdorf Recall Study. The Heinz Nixdorf Recall Study comprises 4,814 men and women aged 45-75 years. To detect monoclonal proteins, standard serum electrophoresis was combined with parallel screening immunofixation using pentavalent antisera. Additionally, free light chains (FLC) were measured in all samples. Definition of MGUS included M-protein concentration, laboratory results, and disease history. LCMGUS was defined as abnormal FLC ratio, increase in FLC causing the abnormal ratio, and lack of intact immunoglobulin. One hundred sixty-five MGUS cases were identified among 4,702 screened samples (prevalence 3.5%, 95% confidence interval (CI) 3.0-4.1; median age 63 years, range 47-75 years; 103 (62%) male; IgG 59%, IgA 17%, IgM 17%, biclonal 4.8%, kappa 56%, and lambda 44%). Five cases progressed (0.6%/year, 95% CI 0.2-1.4). An abnormal FLC ratio was detected in 220 samples. Thirty-nine of these showed intact immunoglobulin. Thirty-four of the remaining met LCMGUS criteria (prevalence 0.7%, 95% CI 0.5-1.0). None of the LCMGUS cases progressed. We demonstrate a MGUS prevalence of 3.5% and a LCMGUS prevalence of 0.7% in the general population aged 45-75 years in Germany using a sensitive screening approach.

  20. Serum levels of immunoglobulin free light chains in patients with chronic hepatitis C presenting cryoglobulinemia.

    PubMed

    Oliveira, Isabela S; Cabral, Milena S; Jesus, Larissa S; Paraná, Raymundo; Atta, Ajax M; Sousa Atta, Maria Luiza B

    2014-01-01

    Hepatitis C virus (HCV) infects B-lymphocytes, provokes cellular dysfunction and causes lymphoproliferative diseases such as cryoglobulinemia and non-Hodgkin's B-cell lymphoma. In the present study, we investigated the serum levels of kappa and lambda free light chains (FLC) of immunoglobulins and the kappa/lambda FLC ratio in Brazilian patients with chronic HCV infection and cryoglobulinemia. We also analyzed the immunochemical composition of the cryoglobulins in these patients. Twenty-eight cryoglobulinemic HCV patients composed the target group, while 37 HCV patients without cryoglobulinemia were included as controls. The median levels of kappa and lambda FLC were higher in patients with cryoglobulinemia compared to controls (p=0.001 and p=0.003, respectively), but the kappa/lambda FLC ratio was similar in patients with and without cryoglobulinemia (p>0.05). The median FLC ratio was higher in HCV patients presenting with advanced fibrosis of the liver compared to HCV patients without fibrosis (p=0.004). Kappa and lambda FLC levels were strongly correlated with the IgA, IgG and IgM levels in the patients with cryoglobulinemia. In patients without cryoglobulinemia, the kappa FLC level was only correlated with the IgG level, whereas the lambda FLC were weakly correlated with the IgA, IgG and IgM levels. An immunochemical pattern of mixed cryoglobulins (MC), predominantly IgM, IgG, IgA and kappa light chain, was verified in these immune complexes. We concluded that HCV-infected patients presenting cryoglobulinemia have vigorous polyclonal B-lymphocyte activation due to chronic HCV infection and persistent immune stimulation.

  1. Novel Polymorphisms in the Myosin Light Chain Kinase Gene Confer Risk for Acute Lung Injury

    PubMed Central

    Gao, Li; Grant, Audrey; Halder, Indrani; Brower, Roy; Sevransky, Jonathan; Maloney, James P.; Moss, Marc; Shanholtz, Carl; Yates, Charles R.; Meduri, Gianfranco Umberto; Shriver, Mark D.; Ingersoll, Roxann; Scott, Alan F.; Beaty, Terri H.; Moitra, Jaideep; Ma, Shwu Fan; Ye, Shui Q.; Barnes, Kathleen C.; Garcia, Joe G. N.

    2006-01-01

    The genetic basis of acute lung injury (ALI) is poorly understood. The myosin light chain kinase (MYLK) gene encodes the nonmuscle myosin light chain kinase isoform, a multifunctional protein involved in the inflammatory response (apoptosis, vascular permeability, leukocyte diapedesis). To examine MYLK as a novel candidate gene in sepsis-associated ALI, we sequenced exons, exon–intron boundaries, and 2 kb of 5′ UTR of the MYLK, which revealed 51 single-nucleotide polymorphisms (SNPs). Potential association of 28 MYLK SNPs with sepsis-associated ALI were evaluated in a case-control sample of 288 European American subjects (EAs) with sepsis alone, subjects with sepsis-associated ALI, or healthy control subjects, and a sample population of 158 African American subjects (AAs) with sepsis and ALI. Significant single locus associations in EAs were observed between four MYLK SNPs and the sepsis phenotype (P < 0.001), with an additional SNP associated with the ALI phenotype (P = 0.03). A significant association of a single SNP (identical to the SNP identified in EAs) was observed in AAs with sepsis (P = 0.002) and with ALI (P = 0.01). Three sepsis risk-conferring haplotypes in EAs were defined downstream of start codon of smooth muscle MYLK isoform, a region containing putative regulatory elements (P < 0.001). In contrast, multiple haplotypic analyses revealed an ALI-specific, risk-conferring haplotype at 5′ of the MYLK gene in both European and African Americans and an additional 3′ region haplotype only in African Americans. These data strongly implicate MYLK genetic variants to confer increased risk of sepsis and sepsis-associated ALI. PMID:16399953

  2. Light chain types of IgD in human bone marrow and serum.

    PubMed Central

    van Nieuwkoop, J A; Radl, J

    1985-01-01

    One of the unexplained features of human IgD is its preferential expression with either kappa or lambda light chains in different situations. While the membrane IgD on B lymphocytes shows a predominance of the kappa type, about 90% of all known IgD myeloma proteins and 87% of normal IgD producing plasma cells in spleens of healthy individuals were shown to belong to the lambda type. Very little is known of the kappa/lambda light chain distribution of normal polyclonal IgD in the serum and in the bone marrow plasma cells. In this study, the kappa/lambda representation of IgD in bone marrow plasma cells and in the serum of 25 adult persons (two healthy and 23 suffering from various nonmalignant diseases) was investigated. The kappa/lambda ratio of IgD+ bone marrow plasma cells showed a large variation among the individuals of this group, in 84% of the cases being below 1.0. While about 1/3 of the investigated subjects had 80% or more of IgD of the lambda type (kappa/lambda ratio below 0.2), most showed a kappa/lambda ratio of IgD higher than that, with four persons exhibiting a clear cut predominance of IgD of the kappa type. A positive correlation (Spearman's correlation co-efficient, P = 0.005) between the percentages of IgD+ plasma cells and their kappa/lambda ratio was found. Semiquantitative evaluation of the kappa/lambda composition within the serum IgD by immunoselection was in agreement with the kappa/lambda ratio of IgD+ plasma cells in all individual cases. Images Fig. 1 PMID:3926359

  3. Crystal Structures of the Tetratricopeptide Repeat Domains of Kinesin Light Chains: Insight into Cargo Recognition Mechanisms

    SciTech Connect

    Zhu, Haizhong; Lee, Han Youl; Tong, Yufeng; Hong, Bum-Soo; Kim, Kyung-Phil; Shen, Yang; Lim, Kyung Jik; Mackenzie, Farrell; Tempel, Wolfram; Park, Hee-Won

    2012-10-23

    Kinesin-1 transports various cargos along the axon by interacting with the cargos through its light chain subunit. Kinesin light chains (KLC) utilize its tetratricopeptide repeat (TPR) domain to interact with over 10 different cargos. Despite a high sequence identity between their TPR domains (87%), KLC1 and KLC2 isoforms exhibit differential binding properties towards some cargos. We determined the structures of human KLC1 and KLC2 tetratricopeptide repeat (TPR) domains using X-ray crystallography and investigated the different mechanisms by which KLCs interact with their cargos. Using isothermal titration calorimetry, we attributed the specific interaction between KLC1 and JNK-interacting protein 1 (JIP1) cargo to residue N343 in the fourth TRP repeat. Structurally, the N343 residue is adjacent to other asparagines and lysines, creating a positively charged polar patch within the groove of the TPR domain. Whereas, KLC2 with the corresponding residue S328 did not interact with JIP1. Based on these finding, we propose that N343 of KLC1 can form 'a carboxylate clamp' with its neighboring asparagine to interact with JIP1, similar to that of HSP70/HSP90 organizing protein-1's (HOP1) interaction with heat shock proteins. For the binding of cargos shared by KLC1 and KLC2, we propose a different site located within the groove but not involving N343. We further propose a third binding site on KLC1 which involves a stretch of polar residues along the inter-TPR loops that may form a network of hydrogen bonds to JIP3 and JIP4. Together, these results provide structural insights into possible mechanisms of interaction between KLC TPR domains and various cargo proteins.

  4. Cytoplasmic free calcium, myosin light chain phosphorylation, and force in phasic and tonic smooth muscle

    PubMed Central

    1988-01-01

    The time course of [Ca2+]i, tension, and myosin light chain phosphorylation were determined during prolonged depolarization with high K+ in intact tonic (rabbit pulmonary artery) and phasic (longitudinal layer of guinea pig ileum) smooth muscles. [Ca2+]i was monitored with the 340 nm/380 nm signal ratio of the fluorescent indicator fura-2. The fluorescence ratio had a similar time course in both muscle types during depolarization with 109 mM [K+]o; after a transient peak, there was a decline to 70% of its peak value in tonic smooth muscle, and to 60% in phasic smooth muscle. Tension, however, continued to increase in the pulmonary artery, while in the ileum it declined in parallel with the [Ca2+]i. On changing [K+]o from 109 to 20 mM, tension and [Ca2+]i either remained unchanged or declined in parallel in the pulmonary artery. Phosphorylation of the 20-kD myosin light chain, measured during stimulation of muscle strips with 109 mM [K+]o in another set of experiments, increased from 3% to a peak of 50% in the intact pulmonary artery, and then declined to a steady state value of 23%. In the intact ileum, a very rapid, early transient phosphorylation (up to 50%) at 2-3 s was seen. This transient declined by 30 s to a value that was close to the resting level (7%), while tension remained at 55% of its peak force. A quick release during maintained stimulation induced no detectable change in the [Ca2+]i in either type of smooth muscle. We discuss the possibility that the slowly rising tonic tension in pulmonary artery could be due to cooperativity between phosphorylated and nonphosphorylated crossbridges. PMID:3216188

  5. Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities

    PubMed Central

    Mahajan, Pravin; Knapp, Stefan; Barton, Hannah; Sweeney, H. Lee; Kamm, Kristine E.; Stull, James T.

    2016-01-01

    The well-known, muscle-specific smooth muscle myosin light chain kinase (MLCK) (smMLCK) and skeletal muscle MLCK (skMLCK) are dedicated protein kinases regulated by an autoregulatory segment C terminus of the catalytic core that blocks myosin regulatory light chain (RLC) binding and phosphorylation in the absence of Ca2+/calmodulin (CaM). Although it is known that a more recently discovered cardiac MLCK (cMLCK) is necessary for normal RLC phosphorylation in vivo and physiological cardiac performance, information on cMLCK biochemical properties are limited. We find that a fourth uncharacterized MLCK, MLCK4, is also expressed in cardiac muscle with high catalytic domain sequence similarity with other MLCKs but lacking an autoinhibitory segment. Its crystal structure shows the catalytic domain in its active conformation with a short C-terminal “pseudoregulatory helix” that cannot inhibit catalysis as a result of missing linker regions. MLCK4 has only Ca2+/CaM-independent activity with comparable Vmax and Km values for different RLCs. In contrast, the Vmax value of cMLCK is orders of magnitude lower than those of the other three MLCK family members, whereas its Km (RLC and ATP) and KCaM values are similar. In contrast to smMLCK and skMLCK, which lack activity in the absence of Ca2+/CaM, cMLCK has constitutive activity that is stimulated by Ca2+/CaM. Potential contributions of autoregulatory segment to cMLCK activity were analyzed with chimeras of skMLCK and cMLCK. The constitutive, low activity of cMLCK appears to be intrinsic to its catalytic core structure rather than an autoinhibitory segment. Thus, RLC phosphorylation in cardiac muscle may be regulated by two different protein kinases with distinct biochemical regulatory properties. PMID:27325775

  6. Monoclonal gammopathy of renal significance with light-chain deposition disease diagnosed postrenal transplant: a diagnostic and therapeutic challenge.

    PubMed

    Nambirajan, Aruna; Bhowmik, Dipankar; Singh, Geetika; Agarwal, Sanjay Kumar; Dinda, Amit Kumar

    2015-03-01

    Patients with light-chain deposition disease (LCDD) frequently do not meet criteria for myeloma. In such cases, despite low tumor burden, the circulating monoclonal immunoglobulins cause renal damage, are responsible for post-transplant recurrence, and are rightly categorized as monoclonal gammopathy of renal significance (MGRS) requiring chemotherapy. A 65-year male with uncharacterized nodular glomerulopathy presented with proteinuria 3 years postrenal transplant. His allograft biopsies were diagnostic of light-chain deposition disease (likely recurrent), and in the absence of myeloma, he was labeled as MGRS. Based on the limited literature available, he was treated with bortezomib which resulted in normalization of serum-free light-chain ratios and resolution of proteinuria. He, however, later succumbed to complications of chemotherapy. This case highlights the diagnostic difficulties in LCDD, the importance of an accurate pretransplant diagnosis, and treatment of the malignant clone, in the absence of which post-transplant management of recurrence is challenging with poor outcomes.

  7. Rearrangement of immunoglobulin light chain genes in the chicken occurs prior to colonization of the embryonic bursa of Fabricius.

    PubMed Central

    Mansikka, A; Sandberg, M; Lassila, O; Toivanen, P

    1990-01-01

    We have applied polymerase-chain-reaction-directed immunoglobulin gene analysis to study the embryonic differentiation of chicken B cells. Immunoglobulin light chain DNA segments in the rearranged configuration were amplified from cells of the intraembryonic mesenchyme as early as day 7 of incubation. We showed by sequencing that the rearranged variable region genes in these early B-cell progenitors were not different from the germ-line V lambda 1 gene (the single functional light chain variable region gene in chickens). In the bursal B lymphocytes, on the other hand, clear gene conversion events were first observed at day 15 of embryonic development. The present data indicate that rearrangement of light chain genes in the chicken occurs independently of the bursa of Fabricius and that diversification of the variable region begins only later, when the surface immunoglobulin-positive B cells are proliferating in the bursal follicles. Images PMID:2123557

  8. Chromosomal orientation of the lambda light chain locus: V lambda is proximal to C lambda in 22q11.

    PubMed Central

    Emanuel, B S; Cannizzaro, L A; Magrath, I; Tsujimoto, Y; Nowell, P C; Croce, C M

    1985-01-01

    We have demonstrated that the chromosomal breakpoint at 22q11 of a Burkitt lymphoma cell line (PA682) with an 8;22 translocation interrupts the variable region of the lambda light chain locus. In these cells, all of the C lambda and some V lambda sequences translocate to the 8q+ chromosome whereas some V lambda sequences remain on the 22q-. These results indicate that the lambda light chain locus on the long arm of chromosome 22 is oriented such that V lambda is proximal to C lambda. Images PMID:3923432

  9. Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice

    SciTech Connect

    Yuan, Chen-Ching; Muthu, Priya; Kazmierczak, Katarzyna; Liang, Jingsheng; Huang, Wenrui; Irving, Thomas C.; Kanashiro-Takeuchi, Rosemeire M.; Hare, Joshua M.; Szczesna-Cordary, Danuta

    2015-06-29

    Myosin light chain kinase (MLCK)-dependent phosphorylation of the regulatory light chain (RLC) of cardiac myosin is known to play a beneficial role in heart disease, but the idea of a phosphorylation-mediated reversal of a hypertrophic cardiomyopathy (HCM) phenotype is novel. Our previous studies on transgenic (Tg) HCM-RLC mice revealed that the D166V (Aspartate166 →Valine) mutation-induced changes in heart morphology and function coincided with largely reduced RLC phosphorylation in situ. In this paper, we hypothesized that the introduction of a constitutively phosphorylated Serine15 (S15D) into the hearts of D166V mice would prevent the development of a deleterious HCM phenotype. In support of this notion, MLCK-induced phosphorylation of D166V-mutated hearts was found to rescue some of their abnormal contractile properties. Tg-S15D-D166V mice were generated with the human cardiac RLC-S15D-D166V construct substituted for mouse cardiac RLC and were subjected to functional, structural, and morphological assessments. The results were compared with Tg-WT and Tg-D166V mice expressing the human ventricular RLC-WT or its D166V mutant, respectively. Echocardiography and invasive hemodynamic studies demonstrated significant improvements of intact heart function in S15D-D166V mice compared with D166V, with the systolic and diastolic indices reaching those monitored in WT mice. A largely reduced maximal tension and abnormally high myofilament Ca2+ sensitivity observed in D166V-mutated hearts were reversed in S15D-D166V mice. Low-angle X-ray diffraction study revealed that altered myofilament structures present in HCM-D166V mice were mitigated in S15D-D166V rescue mice. Finally, our collective results suggest that expression of pseudophosphorylated RLC in the hearts of HCM mice is sufficient to prevent the development of the pathological HCM phenotype.

  10. Myosin Light Chain Kinase (MLCK) Gene Influences Exercise Induced Muscle Damage during a Competitive Marathon

    PubMed Central

    Valero, Marjorie; Lara, Beatriz; Salinero, Juan José; Gallo-Salazar, César; Areces, Francisco

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin producing increases in force development during skeletal muscle contraction. It has been suggested that MLCK gene polymorphisms might alter RLC phosphorylation thereby decreasing the ability to produce force and to resist strain during voluntary muscle contractions. Thus, the genetic variations in the MLCK gene might predispose some individuals to higher values of muscle damage during exercise, especially during endurance competitions. The aim of this investigation was to determine the influence of MLCK genetic variants on exercise-induced muscle damage produced during a marathon. Sixty-seven experienced runners competed in a marathon race. The MLCK genotype (C37885A) of these marathoners was determined. Before and after the race, a sample of venous blood was obtained to assess changes in serum myoglobin concentrations and leg muscle power changes were measured during a countermovement jump. Self-reported leg muscle pain and fatigue were determined by questionnaires. A total of 59 marathoners (88.1%) were CC homozygotes and 8 marathoners (11.9%) were CA heterozygotes. The two groups of participants completed the race with a similar time (228 ± 33 vs 234 ± 39 min; P = 0.30) and similar self-reported values for fatigue (15 ± 2 vs 16 ± 2 A.U.; P = 0.21) and lower-limb muscle pain (6.2 ± 1.7 vs 6.6 ± 1.8 cm; P = 0.29). However, CC marathoners presented higher serum myoglobin concentrations (739 ± 792 vs 348 ± 144 μg·mL-1; P = 0.03) and greater pre-to-post- race leg muscle power reduction (-32.7 ± 15.7 vs -21.2 ± 21.6%; P = 0.05) than CA marathoners. CA heterozygotes for MLCK C37885A might present higher exercise-induced muscle damage after a marathon competition than CC counterparts. PMID:27483374

  11. Clinical responses with T lymphocytes targeting malignancy-associated κ light chains

    PubMed Central

    Ramos, Carlos A.; Savoldo, Barbara; Torrano, Vicky; Ballard, Brandon; Zhang, Huimin; Dakhova, Olga; Liu, Enli; Carrum, George; Kamble, Rammurti T.; Gee, Adrian P.; Mei, Zhuyong; Wu, Meng-Fen; Liu, Hao; Grilley, Bambi; Rooney, Cliona M.; Brenner, Malcolm K.; Heslop, Helen E.; Dotti, Gianpietro

    2016-01-01

    BACKGROUND. Treatment of B cell malignancies with adoptive transfer of T cells with a CD19-specific chimeric antigen receptor (CAR) shows remarkable clinical efficacy. However, long-term persistence of T cells targeting CD19, a pan–B cell marker, also depletes normal B cells and causes severe hypogammaglobulinemia. Here, we developed a strategy to target B cell malignancies more selectively by taking advantage of B cell light Ig chain restriction. We generated a CAR that is specific for the κ light chain (κ.CAR) and therefore recognizes κ-restricted cells and spares the normal B cells expressing the nontargeted λ light chain, thus potentially minimizing humoral immunity impairment. METHODS. We conducted a phase 1 clinical trial and treated 16 patients with relapsed or refractory κ+ non-Hodgkin lymphoma/chronic lymphocytic leukemia (NHL/CLL) or multiple myeloma (MM) with autologous T cells genetically modified to express κ.CAR (κ.CARTs). Other treatments were discontinued in 11 of the 16 patients at least 4 weeks prior to T cell infusion. Six patients without lymphopenia received 12.5 mg/kg cyclophosphamide 4 days before κ.CART infusion (0.2 × 108 to 2 × 108 κ.CARTs/m2). No other lymphodepletion was used. RESULTS. κ.CART expansion peaked 1–2 weeks after infusion, and cells remained detectable for more than 6 weeks. Of 9 patients with relapsed NHL or CLL, 2 entered complete remission after 2 and 3 infusions of κ.CARTs, and 1 had a partial response. Of 7 patients with MM, 4 had stable disease lasting 2–17 months. No toxicities attributable to κ.CARTs were observed. CONCLUSION. κ.CART infusion is feasible and safe and can lead to complete clinical responses. TRIAL REGISTRATION. ClinicalTrials.gov NCT00881920. FUNDING. National Cancer Institute (NCI) grants 3P50CA126752 and 5P30CA125123 and Leukemia and Lymphoma Society (LLS) Specialized Centers of Research (SCOR) grant 7018. PMID:27270177

  12. Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice

    DOE PAGES

    Yuan, Chen-Ching; Muthu, Priya; Kazmierczak, Katarzyna; ...

    2015-06-29

    Myosin light chain kinase (MLCK)-dependent phosphorylation of the regulatory light chain (RLC) of cardiac myosin is known to play a beneficial role in heart disease, but the idea of a phosphorylation-mediated reversal of a hypertrophic cardiomyopathy (HCM) phenotype is novel. Our previous studies on transgenic (Tg) HCM-RLC mice revealed that the D166V (Aspartate166 →Valine) mutation-induced changes in heart morphology and function coincided with largely reduced RLC phosphorylation in situ. In this paper, we hypothesized that the introduction of a constitutively phosphorylated Serine15 (S15D) into the hearts of D166V mice would prevent the development of a deleterious HCM phenotype. In supportmore » of this notion, MLCK-induced phosphorylation of D166V-mutated hearts was found to rescue some of their abnormal contractile properties. Tg-S15D-D166V mice were generated with the human cardiac RLC-S15D-D166V construct substituted for mouse cardiac RLC and were subjected to functional, structural, and morphological assessments. The results were compared with Tg-WT and Tg-D166V mice expressing the human ventricular RLC-WT or its D166V mutant, respectively. Echocardiography and invasive hemodynamic studies demonstrated significant improvements of intact heart function in S15D-D166V mice compared with D166V, with the systolic and diastolic indices reaching those monitored in WT mice. A largely reduced maximal tension and abnormally high myofilament Ca2+ sensitivity observed in D166V-mutated hearts were reversed in S15D-D166V mice. Low-angle X-ray diffraction study revealed that altered myofilament structures present in HCM-D166V mice were mitigated in S15D-D166V rescue mice. Finally, our collective results suggest that expression of pseudophosphorylated RLC in the hearts of HCM mice is sufficient to prevent the development of the pathological HCM phenotype.« less

  13. Lambda Immunoglobulin Light Chain Restricted B Cells in the Ascitic Fluid in Association with Terminal Ileal Florid Follicular Hyperplasia.

    PubMed

    Aqil, Barina; Xie, Wei; Szigeti, Reka

    2016-01-01

    Distinguishing reactive changes from neoplastic processes during lymphoid tissue evaluation is oftentimes difficult. Ancillary studies, such as flow cytometry, may aid the diagnosis by demonstrating monotypic or polytypic light chain expression on the B cells. The detection of immunoglobulin light chain restricted B cell population is considered a surrogate marker of clonality, which can be confirmed by molecular assays. In general, the presence of a monotypic B cell population in the ascitic fluid is considered lymphomatous involvement rather than a reactive condition. We describe a young, previously healthy male patient who developed ascites with a lambda light chain restricted B cell population. Further investigation revealed florid follicular hyperplasia, histologically mimicking diffuse large B cell lymphoma, in the terminal ileum. Follicular hyperplasia in the gastrointestinal tract with lambda light chain restricted B cells has been recently described in the pediatric population. Importantly, our case demonstrates that such entity can occur in older age groups. This recognition could prevent misdiagnosis and unnecessary treatment in similar cases.

  14. Hematogones With Lambda Light Chain Restriction in a 4-Year-Old Boy With Burkitt Lymphoma: A Potential Diagnostic Pitfall.

    PubMed

    Guillory, Tesha; Li, Shiyong; Bergsagel, Daniel J; Weinzierl, Elizabeth; Bunting, Silvia T

    2016-05-01

    Hematogones are immature normal B cell precursors with a characteristic immunophenotype profile on flow cytometry that typically do not express surface immunoglobulin light chains. In this report, we describe a case in which the hematogones exhibit light chain restriction. Our patient was a 4-year-old boy with a complicated medical history involving treatment for a presumed bilateral Wilms tumor of the kidney that on later resection was diagnosed as Burkitt lymphoma. Flow cytometry analysis of his bone marrow revealed a small distinct population of cells expressing dim cluster of differentiation (CD)10, CD19, CD22, CD38, dim CD58, human leukocyte antigen-D related (HLA-DR), and dim CD45, which are characteristic of hematogones. These cells, however, demonstrated dim surface immunoglobulin lambda light-chain restriction. Molecular study results for immunoglobulin heavy and kappa light-chain gene rearrangements were negative. We present this case to raise awareness of the potential pitfalls of working up bone marrow for involvement by B cell lymphoproliferative disorder.

  15. Catalytic antibody light chain capable of cleaving a chemokine receptor CCR-5 peptide with a high reaction rate constant.

    PubMed

    Mitsuda, Yukie; Hifumi, Emi; Tsuruhata, Kumi; Fujinami, Hiroko; Yamamoto, Naoki; Uda, Taizo

    2004-04-20

    A monoclonal antibody (MAb), ECL2B-2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR-5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B-2 MAb, molecular modeling was conducted to calculate the steric conformation of the antibody. Modeling suggested that the structure of ECL2B-2 could possess one or two catalytic triad(s), composed of Asp(1), Ser(27a) (or Ser(27e)), and His(93) (or His(27d)), in the light chain of ECL2B-2. The three amino acid residues, Asp(1), Ser(27a), and His(93), are identical to those of catalytic antibody light chains such as VIPase and i41SL1-2. The light chain of ECL2B-2 MAb degraded the antigenic peptide CCR-5 within about 100 h. Surprisingly, the light chain had a very high catalytic reaction rate constant (k(cat)) of 2.23 min(-1), which is greater by factors of tens to hundreds than those of natural catalytic antibodies obtained previously. The heavy chain of ECL2B-2 MAb, which has no catalytic triad because of a lack of His residue, did not degrade the CCR-5 peptide.

  16. Identification and partial characterization of a low affinity metal-binding site in the light chain of tetanus toxin.

    PubMed

    Wright, J F; Pernollet, M; Reboul, A; Aude, C; Colomb, M G

    1992-05-05

    Tetanus toxin was shown to contain a metal-binding site for zinc and copper. Equilibrium dialysis binding experiments using 65Zn indicated an association constant of 9-15 microM, with one zinc-binding site/toxin molecule. The zinc-binding site was localized to the toxin light chain as determined by binding of 65Zn to the light chain but not to the heavy chain after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to Immobilon membranes. Copper was an efficient inhibitor of 65Zn binding to tetanus toxin and caused two peptide bond cleavages in the toxin light chain in the presence of ascorbate. These metal-catalyzed oxidative cleavages were inhibited by the presence of zinc. Partial characterization of metal-catalyzed oxidative modifications of a peptide based on a putative metal-binding site (HELIH) in the toxin light chain was used to map the metal-binding site in the protein.

  17. Characterization and ontogenetic expression analysis of the myosin light chains from the fast white muscle of mandarin fish Siniperca chuatsi.

    PubMed

    Chu, W Y; Chen, J; Zhou, R X; Zhao, F L; Meng, T; Chen, D X; Nong, X X; Liu, Z; Lu, S Q; Zhang, J S

    2011-04-01

    Three full-length complementary DNA (cDNA) clones were isolated encoding the skeletal myosin light chain 1 (MLC1; 1237 bp), myosin light chain 2 (MLC2; 1206 bp) and myosin light chain 3 (MLC3; 1079 bp) from the fast white muscle cDNA library of mandarin fish Siniperca chuatsi. The sequence analysis indicated that MLC1 and MLC3 were not produced from differentially spliced messenger RNAs (mRNA) as reported in birds and rodents but were encoded by different genes. The MLC2 encodes 170 amino acids, which include four EF-hand (helix-loop-helix) structures. The primary structures of the Ca(2+)-binding domain were well conserved among the MLC2s of seven other fish species. The ontogenetic expression analysis by real-time PCR showed that the three light-chain mRNAs were first detected in the gastrula stage, and their expression increased from the tail bud stage to the larval stage. All three MLC mRNAs showed longitudinal expression variation in the fast white muscle of S. chuatsi, especially MLC1 which was highly expressed at the posterior area. Taken together, the study provides a better understanding about the MLC gene structure and their expression pattern in muscle development of S. chuatsi.

  18. Case report: isolated cardiac amyloidosis: an enigma unravelled.

    PubMed

    Khalid, Umair; Awar, Omar; Verstovsek, Gordana; Cheong, Benjamin; Yellapragada, Sarvari Venkata; Jneid, Hani; Deswal, Anita; Virani, Salim S

    2015-01-01

    Amyloidosis is a rare, multisystem disease characterized by deposition of fibrils in extracellular tissue involving kidney, liver, heart, autonomic nervous system, and several other organs. This report discusses a 75-year-old male who presented with worsening dyspnea on exertion, orthopnea, and lower-extremity edema. On physical exam, he had elevated jugular venous pressure and lower-extremity edema. Electrocardiogram depicted low voltage in limb leads and a prolonged PR interval. Echocardiogram revealed left ventricular hypertrophy, severe biatrial dilatation, and restrictive filling physiology. Coronary angiography showed absence of significant epicardial coronary artery disease. On right heart catheterization, a "dip-and-plateau sign" was noted on right ventricular pressure tracings. A diagnosis of cardiac amyloidosis was considered, but a complete hematology work-up for systemic amyloidosis was negative. Cardiac magnetic resonance imaging was pursued, showing delayed gadolinium enhancement, and this ultimately led to the myocardial biopsy confirming the diagnosis of isolated cardiac amyloidosis. Further genetic analyses confirmed isolated cardiac amyloid caused by mutant transthyretin protein (Val-122-Ile). Isolated cardiac amyloidosis is an extremely rare entity, and diagnosis may be difficult despite the use of multimodality imaging. If the index of suspicion is high, then myocardial biopsy should be considered.

  19. Case Report: Isolated Cardiac Amyloidosis: An Enigma Unravelled

    PubMed Central

    Khalid, Umair; Awar, Omar; Verstovsek, Gordana; Cheong, Benjamin; Yellapragada, Sarvari Venkata; Jneid, Hani; Deswal, Anita; Virani, Salim S.

    2015-01-01

    Amyloidosis is a rare, multisystem disease characterized by deposition of fibrils in extracellular tissue involving kidney, liver, heart, autonomic nervous system, and several other organs. This report discusses a 75-year-old male who presented with worsening dyspnea on exertion, orthopnea, and lower-extremity edema. On physical exam, he had elevated jugular venous pressure and lowerextremity edema. Electrocardiogram depicted low voltage in limb leads and a prolonged PR interval. Echocardiogram revealed left ventricular hypertrophy, severe biatrial dilatation, and restrictive filling physiology. Coronary angiography showed absence of significant epicardial coronary artery disease. On right heart catheterization, a “dip-and-plateau sign” was noted on right ventricular pressure tracings. A diagnosis of cardiac amyloidosis was considered, but a complete hematology work-up for systemic amyloidosis was negative. Cardiac magnetic resonance imaging was pursued, showing delayed gadolinium enhancement, and this ultimately led to the myocardial biopsy confirming the diagnosis of isolated cardiac amyloidosis. Further genetic analyses confirmed isolated cardiac amyloid caused by mutant transthyretin protein (Val-122-Ile). Isolated cardiac amyloidosis is an extremely rare entity, and diagnosis may be difficult despite the use of multimodality imaging. If the index of suspicion is high, then myocardial biopsy should be considered. PMID:25793032

  20. [Systemic amyloidosis associated with IgD-λ multiple myeloma].

    PubMed

    Nagamachi, Yasuhiro; Yamauchi, Naofumi; Muramatsu, Hirohito; Inomata, Hidetoshi; Nozawa, Eri; Koyama, Ryuzo; Ihara, Koji; Nishisato, Takuji; Yamada, Hideyuki; Yano, Tomohiro; Kikuchi, Shohei; Hirako, Tasuku; Kitaoka, Keisuke; Ono, Kaoru; Ihara, Hideyuki; Kato, Junji

    2011-12-01

    We describe here a case of systemic amyloidosis associated with IgD multiple myeloma. A 59-year-old man was admitted to our hospital in April 2009, because of macroglossia and swelling in both wrists and fingers. He had difficulty moving his limbs and was aware of peripheral neuropathy. Skin biopsy revealed extensive deposition of amyloidosis, which was positive by Congo red staining. Laboratory findings were as follows: serum electrophoresis revealed IgD λ monoclonal protein, and Bence-Jones protein was detected. Monoclonal IgD protein had a concentration of 727 mg/dl, and a bone marrow aspiration revealed 49.6% of plasma cells. These findings led to a diagnosis of IgD multiple myeloma with systemic amyloidosis. The patient was treated with MP (melphalan and methylprednisolone), high-dose dexamethasone and VAD therapy (vincristine, adriamycin and dexamethasone), but systemic amyloidosis progressed, and his general condition deteriorated. Coexistence of IgD multiple myeloma and systemic amyloidosis is rare, and accumulation of case reports is needed to gain a better understanding of this condition.

  1. Evolving landscape in the management of transthyretin amyloidosis

    PubMed Central

    Hawkins, Philip N.; Ando, Yukio; Dispenzeri, Angela; Gonzalez-Duarte, Alejandra; Adams, David; Suhr, Ole B.

    2015-01-01

    Transthyretin (TTR) amyloidosis (ATTR amyloidosis) is a multisystemic, multigenotypic disease resulting from deposition of insoluble ATTR amyloid fibrils in various organs and tissues. Although considered rare, the prevalence of this serious disease is likely underestimated because symptoms can be non-specific and diagnosis largely relies on amyloid detection in tissue biopsies. Treatment is guided by which tissues/organs are involved, although therapeutic options are limited for patients with late-stage disease. Indeed, enthusiasm for liver transplantation for familial ATTR amyloidosis with polyneuropathy was dampened by poor outcomes among patients with significant neurological deficits or cardiac involvement. Hence, there remains an unmet medical need for new therapies. The TTR stabilizers tafamidis and diflunisal slow disease progression in some patients with ATTR amyloidosis with polyneuropathy, and the postulated synergistic effect of doxycycline and tauroursodeoxycholic acid on dissolution of amyloid is under investigation. Another therapeutic approach is to reduce production of the amyloidogenic protein, TTR. Plasma TTR concentration can be significantly reduced with ISIS-TTRRx, an investigational antisense oligonucleotide-based drug, or with patisiran and revusiran, which are investigational RNA interference-based therapeutics that target the liver. The evolving treatment landscape for ATTR amyloidosis brings hope for further improvements in clinical outcomes for patients with this debilitating disease. PMID:26611723

  2. Analysis of heavy and light chain sequences of conventional camelid antibodies from Camelus dromedarius and Camelus bactrianus species.

    PubMed

    Griffin, Laura M; Snowden, James R; Lawson, Alastair D G; Wernery, Ulrich; Kinne, Jorg; Baker, Terry S

    2014-03-01

    Camel antibodies have been widely investigated, but work has focused upon the unique heavy chain antibodies found across camelid species. These are homodimers, devoid of light chains and the first constant heavy chain domain. Camelid species also display conventional hetero-tetrameric antibodies with identical pairs of heavy and light chains; in Camelus dromedarius these constitute 25% of circulating antibodies. Few investigations have been made on this subset of antibodies and complete conventional camel IgG sequences have not been reported. Here we study the sequence diversity of functional variable and constant regions observed in 57 conventional heavy, 18 kappa and 35 lambda light chains of C. dromedarius and Camelus bactrianus. We detail sequences of the full kappa and lambda light chain, variable and CH1 region for IgG1a and IgG1b and the CH2 and CH3 region for IgG1a. The majority (60%) of IgG1 variable region sequences aligned with the human IgHV3 family (clan III) and had leader sequences beginning with MELG whereas the remaining sequences aligned with the IgHV4 (clan II) and had leader sequences beginning with MRLL. Distinct differences in CDR length were observed between the two; where CDR1 was typically 5 and 7 residues and CDR2 at 17 and 16 residues, respectively. CDR3 length of IgHV4 (range 11 to 20) was closer to that typical of VHH antibodies than that of IgHV3 (range 3 to 18 residues). Designed oligonucleotide primers have enabled identification of paired heavy and light chains of conventional camel antibodies from individual B cell clones.

  3. Subset of Kappa and Lambda Germline Sequences Result in Light Chains with a Higher Molecular Mass Phenotype.

    PubMed

    Barnidge, David R; Lundström, Susanna L; Zhang, Bo; Dasari, Surendra; Murray, David L; Zubarev, Roman A

    2015-12-04

    In our previous work, we showed that electrospray ionization of intact polyclonal kappa and lambda light chains isolated from normal serum generates two distinct, Gaussian-shaped, molecular mass distributions representing the light-chain repertoire. During the analysis of a large (>100) patient sample set, we noticed a low-intensity molecular mass distribution with a mean of approximately 24 250 Da, roughly 800 Da higher than the mean of the typical kappa molecular-mass distribution mean of 23 450 Da. We also observed distinct clones in this region that did not appear to contain any typical post-translational modifications that would account for such a large mass shift. To determine the origin of the high molecular mass clones, we performed de novo bottom-up mass spectrometry on a purified IgM monoclonal light chain that had a calculated molecular mass of 24 275.03 Da. The entire sequence of the monoclonal light chain was determined using multienzyme digestion and de novo sequence-alignment software and was found to belong to the germline allele IGKV2-30. The alignment of kappa germline sequences revealed ten IGKV2 and one IGKV4 sequences that contained additional amino acids in their CDR1 region, creating the high-molecular-mass phenotype. We also performed an alignment of lambda germline sequences, which showed additional amino acids in the CDR2 region, and the FR3 region of functional germline sequences that result in a high-molecular-mass phenotype. The work presented here illustrates the ability of mass spectrometry to provide information on the diversity of light-chain molecular mass phenotypes in circulation, which reflects the germline sequences selected by the immunoglobulin-secreting B-cell population.

  4. The long myosin light chain kinase is differentially phosphorylated during interphase and mitosis.

    PubMed

    Dulyaninova, Natalya G; Bresnick, Anne R

    2004-10-01

    We have shown previously that the activity of the long myosin light chain kinase (MLCK) is cell cycle regulated with a decrease in specific activity during mitosis that can be restored following treatment with alkaline phosphatase. To better understand the role and significance of phosphorylation in regulating MLCK function during mitosis, we examined the phosphorylation state of in vivo derived MLCK. Phosphoamino acid analysis and phosphopeptide mapping demonstrate that the long MLCK is differentially phosphorylated on serine residues during interphase and mitosis with the majority of the phosphorylation sites located within the N-terminal IgG domain. Biochemical assays show that Aurora B binds and phosphorylates the IgG domain of the long MLCK. In addition, phosphopeptide maps of the endogenous full-length MLCK from mitotic cells and in vitro phosphorylated IgG domain demonstrate that Aurora B phosphorylates the same sites as those observed in vivo. Altogether, these studies suggest that the long MLCK may be a cellular target for Aurora B during mitosis.

  5. Myosin light chain kinase accelerates vesicle endocytosis at the calyx of Held synapse.

    PubMed

    Yue, Hai-Yuan; Xu, Jianhua

    2014-01-01

    Neuronal activity triggers endocytosis at synaptic terminals to retrieve efficiently the exocytosed vesicle membrane, ensuring the membrane homeostasis of active zones and the continuous supply of releasable vesicles. The kinetics of endocytosis depends on Ca(2+) and calmodulin which, as a versatile signal pathway, can activate a broad spectrum of downstream targets, including myosin light chain kinase (MLCK). MLCK is known to regulate vesicle trafficking and synaptic transmission, but whether this kinase regulates vesicle endocytosis at synapses remains elusive. We investigated this issue at the rat calyx of Held synapse, where previous studies using whole-cell membrane capacitance measurement have characterized two common forms of Ca(2+)/calmodulin-dependent endocytosis, i.e., slow clathrin-dependent endocytosis and rapid endocytosis. Acute inhibition of MLCK with pharmacological agents was found to slow down the kinetics of both slow and rapid forms of endocytosis at calyces. Similar impairment of endocytosis occurred when blocking myosin II, a motor protein that can be phosphorylated upon MLCK activation. The inhibition of endocytosis was not accompanied by a change in Ca(2+) channel current. Combined inhibition of MLCK and calmodulin did not induce synergistic inhibition of endocytosis. Together, our results suggest that activation of MLCK accelerates both slow and rapid forms of vesicle endocytosis at nerve terminals, likely by functioning downstream of Ca(2+)/calmodulin.

  6. [Refolding of the fusion protein of recombinant enterokinase light chain rEKL].

    PubMed

    Yi, Jin-Hua; Zhang, Yuan-Xing

    2006-09-01

    The fusion protein of enterokinase light chain, DsbA-rEKL, was expressed mainly in inclusion body in E. coli. The recombinant bacteria was fermented to high density, with high expression of the fusion protein. After being washed with 0.5% Triton X-100 and 4mol/L urea, the inclusion body was dissolved in 6mol/L guanidine and 100mmol/L DTP, derivatized by cystine and refolded by pulse refolding. The strategy of pulse refolding involved the addition of 0.03mg/mL of fusion protein until its final concentration reached 0.3mg/mL. The refolded protein was autocleaved and the active EKL molecule was released after adding 2mmol/L CaCl2. Using the two-step purification processes of IDA-Sepharose chromatography and Q-Sepharose chromatography, the purity of rEKL was found to be above 95%, with a high activity to cleave the recombinant reteplase fusion protein Trx-rPA. The yield of purified rEKL was more than 60mg/L of cultures. As a result, the therapeutic proteins like rPA could be produced on a large-scale in a way such as expressed in the form of fusion proteins.

  7. Functional expression and purification of bovine enterokinase light chain in recombinant Escherichia coli.

    PubMed

    Huang, Lei; Ruan, Hong; Gu, Weiyan; Xu, Zhinan; Cen, Peilin; Fan, Limei

    2007-01-01

    Enterokinase (EC 3.4.21.9) is a serine proteinase of the intestinal brush border that exhibits specificity for the sequence (Asp)(4)-Lys and converts trypsinogen into its active form, trypsin. A codon optimized sequence coding light chain (catalytic subunit) of bovine enterokinase gene (sBEKLC) was synthesized, and it was fused with DsbA to construct the expression vector (pET39-sBEKLC). Then, the plasmid was transformed into E. coli BL21 (DE3) for expression. Under optimal conditions, the volumetric productivity of fusion protein reached 151.2 mg L(-1), i.e., 80.6 mg sBEKLC L(-1). The cold osmotic shock technique was successfully used to extract sBEKLC from periplasmic space, and nickel affinity chromatography was employed to obtain mature sBEKLC. Finally, about 6.8 mg of bioactive sBEKLC was purified from 1 liter fermentation broth and could be used to cleave one tested fusion protein with an inter-domain enteropeptidase recognition site. This work will be helpful for large-scale production of this increasingly demanded enterokinase.

  8. Purification and refolding optimization of recombinant bovine enterokinase light chain overexpressed in Escherichia coli.

    PubMed

    Tan, Haidong; Wang, Jinxia; Zhao, Zongbao Kent

    2007-11-01

    The nucleotide sequence encoding bovine enterokinase light chain (EK) from Chinese northern yellow bovine was isolated. Two single-nucleotide mutations, namely, C245G and A528T were identified. The gene encoding the Pro82Arg/Glu176Asp variant of known bovine EK was fused with glutathione S-transferase and overexpressed mainly as an inclusion body in Escherichia coli BL21 (DE3), upon induction with IPTG and glucose. Effective fusion protein purification, refolding, auto-catalytic cleavage and mature EK recovery were described. The specific activity of the purified EK was determined as 110+/- 10 U/mg, which was comparable to a specific activity of > or =20 U/mg of the E. coli expressed EK sample provided by Sigma (Cat. No. E4906). This procedure produced approximately 53 mg of EK per 500 mL of cell culture, which was much higher than previous reports, thus providing a basis for large-scale production of EK and for further applications in biotechnology.

  9. High-level secretory production of recombinant bovine enterokinase light chain by Pichia pastoris.

    PubMed

    Peng, Lisheng; Zhong, Xiaofen; Ou, Jingxing; Zheng, Suilan; Liao, Jian; Wang, Lei; Xu, Anlong

    2004-03-04

    Enterokinase (EC 3.4.21.9) is a serine proteinase with a specific digest sequence (Asp)4-Lys in the duodenum. Its high specificity for the recognition site makes enterokinase (EK) a useful tool for an in vitro cleavage of fusion proteins. In this work, an active bovine enterokinase light chain (EK(L)) was produced in secretory form by a recombinant strain of the methylotrophic yeast Pichia pastoris. The influences of methanol utilization phenotype of the host strain, induction pH, and carbon source on the recombinant production were studied. The production of recombinant EK(L) by Mut(s) strain was much higher than that by Mut+ strain. When inducted at pH 6.0, on a glycerol/methanol medium, the concentration of recombinant EK(L) (rEK(L)) reached 350 mg l(-1), which was 20-fold higher than that reported previously. The recombinant EK(L) was purified in a simple procedure on the anion exchange chromatography and 15 mg pure active EK(L) were obtained from 100 ml culture broth supernatant. The specific activity of purified rEK(L) was approximately 9000 u mg(-1). To facilitate purification and removal of rEKL after cleavage of fusion protein, the C-terminal His-tagged EK(L) (EK(L)/His) was also expressed in P. pastoris, and this His-tagged EK(L) exhibited a similar enzymatic activity to the untagged EK(L).

  10. Olanzapine May Inhibit Colonic Motility Associated with the 5-HT Receptor and Myosin Light Chain Kinase

    PubMed Central

    Zhang, Jiarui; Qiao, Ying; Le, Jingjing

    2016-01-01

    Objective To study whether the effects of olanzapine on gastrointestinal motility is related to the serotonin antagonism and myosin light chain kinase. Methods Male Sprague-Dawley rats were randomly divided into four groups. Olanzapine gavage was performed for each treatment group during the course of 30 continuous days, while the same volume of saline was given to the rats in the control group. Defecation of the rats was observed on days 7 and 30 after olanzapine gavage. The effects of olanzapine on contraction of colonic smooth muscles were observed in ex vivo experiments. A Western blot was used to evaluate expression levels of the serotonin transporter (SERT) and MLCK in colon segments of the rats. Results ResultsaaCompared to the control group, 5-160 µ M of olanzapine could inhibit dose-dependently the contraction of colonic smooth muscle ex vivo experiments. The maximum smooth muscle contraction effects of 5-HT and acetylcholine significantly decreased after treatment with 40-160 µ M of olanzapine. Constipation was found in the olanzapine-treated rats on day 7 and have sustained day 30 after gavage. Expression of MLCK in olanzapine-treated rats was significantly decreased, whereas the expression of SERT significantly increased on the day 7, then significantly decreased on the day 30 after olanzapine gavage. Conclusion SERT and MLCK may involve in the inhibition of colonic contraction induced by olanzapine. PMID:27081386

  11. Phosphorylation of myosin light chain from adrenomedullary chromaffin cells in culture.

    PubMed Central

    Gutierrez, L M; Hidalgo, M J; Palmero, M; Ballesta, J J; Reig, J A; Garcia, A G; Viniegra, S

    1989-01-01

    The myosin-light-chain (MLC) phosphorylation accompanying catecholamine release in chromaffin cells was investigated with the objective of assessing the possible role of this contractile protein in catecholamine secretion. The electrophoretic characteristics of adrenomedullary MLC were determined by immunochemical techniques using two different specific antibodies. The identified 22 kDa phosphoprotein was mainly present in the cytosol, as demonstrated by ultracentrifugation and immunocytochemical analysis. A part of this protein was located on, or close to, the plasma membrane. Cell stimulation by secretagogues resulted in a Ca2(+)-dependent 32P incorporation into MLC, the time course of this process being related to catecholamine release. These findings were supported by a two-dimensional gel-electrophoretic analysis by which means this protein was resolved into two acidic forms. A role for Ca2(+)-calmodulin and Ca2(+)-phospholipid kinases in adrenomedullary MLC phosphorylation is reported. The results obtained suggest a regulatory role for such a protein in the underlying exocytotic event. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 6. PMID:2481449

  12. Circulating antibody free light chains and risk of posttransplant lymphoproliferative disorder.

    PubMed

    Engels, E A; Preiksaitis, J; Zingone, A; Landgren, O

    2012-05-01

    Posttransplant lymphoproliferative disorder (PTLD) is a major complication of solid-organ transplantation. With human immunodeficiency virus infection (an analogous immunosuppressive state), elevated kappa and lambda immunoglobulin free light chains (FLCs) in peripheral blood are associated with increased risk of lymphoma. To assess the role of B-cell dysfunction in PTLD, we measured circulating FLCs among Canadian transplant recipients, including 29 individuals with PTLD and 57 matched transplant recipients who were PTLD-free. Compared with controls, PTLD cases had higher kappa FLCs (median 1.53 vs. 1.07 times upper limit of normal) and lambda FLCs (1.03 vs. 0.68). Using samples obtained on average 3.5 months before PTLD diagnosis, cases were more likely to have polyclonal FLC elevations (i.e. elevated kappa and/or lambda with normal kappa/lambda ratio: odds ratio [OR] 4.2, 95%CI 1.1-15) or monoclonal elevations (elevated kappa and/or lambda with abnormal ratio: OR 3.0, 95%CI 0.5-18). Strong FLC-PTLD associations were also observed at diagnosis/selection. Among recipients with Epstein-Barr virus (EBV) DNA measured in blood, EBV DNAemia was associated with FLC abnormalities (ORs 6.2 and 3.2 for monoclonal and polyclonal elevations). FLC elevations are common in transplant recipients and associated with heightened PTLD risk. FLCs likely reflect B-cell dysfunction, perhaps related to EBV-driven lymphoproliferation.

  13. Free light chain monomer-dimer patterns in the diagnosis of multiple sclerosis.

    PubMed

    Kaplan, Batia; Golderman, Sizilia; Yahalom, Gilad; Yeskaraev, Regina; Ziv, Tamar; Aizenbud, Boris M; Sela, Ben-Ami; Livneh, Avi

    2013-04-30

    In our search of new biomarkers for multiple sclerosis (MS), we aimed to characterize the immunoglobulin (Ig) free light chains (FLC) in patients' cerebrospinal fluid (CSF) and serum, and to evaluate the diagnostic utility of FLC monomer-dimer patterns for MS. FLC were analyzed by Western blotting and mass spectroscopy. CSF and serum samples were examined for the presence of oligoclonal Ig bands by a conventional laboratory test for MS. Three distinct pathological FLC monomer-dimer patterns, typical of MS but not of other neurological diseases, were revealed. In 31 out 56 MS patients the highly increased CSF levels of κ monomers and dimers were demonstrated. In 18 MS patients, the increased κ-FLC levels were accompanied by highly elevated λ dimers. Five MS cases showed no significant elevation in κ-FLC, but they displayed abnormally high λ dimer levels. The intensity of the immunoreactive FLC bands was measured to account for κ and λ monomer and dimer levels and their ratios in the CSF and serum. Combined usage of different FLC parameters allowed the determination of the appropriate FLC threshold values to diagnose MS. The developed method showed higher sensitivity and specificity (96% and 90%, respectively), as compared to those of the conventional OCB test (82% and 70%, respectively). Our study highlights the role of the differential analysis of monomeric and dimeric κ- and λ-FLC for the precise diagnosis of MS.

  14. Serum free light chains and post-transplant lymphoproliferative disorder in patients with renal transplant.

    PubMed

    Fernando, Rodrigo C; Rizzatti, Edgar G; Braga, Walter M T; Santos, Melina G; de Oliveira, Mariana B; Pestana, José O M; Baiocchi, Otavio C G; Colleoni, Gisele W B

    2013-10-01

    The aim of the present study was to determine whether there is an association between serum free light chains (sFLC) quantification and the development of post-transplant lymphoproliferative disorder (PTLD), using serum samples from a nested case-control cohort of patients with renal transplant. Ten new cases of PTLD and 46 controls were enrolled. Additional comparison groups consisted of five human immunodeficiency virus (HIV)-infected individuals, five with untreated Hodgkin lymphoma and six normal individuals. Serum κ and λ FLC concentrations were measured by nephelometry and compared with reference ranges (normal and renal ranges). κ and/or λ were above the normal range in 90% of cases and in 65% of matched controls. There was no statistically significant difference between all groups, except for λ FLC concentrations between cases of PTLD and normal individuals (p = 0.016). The κ/λ sFLC ratios of cases and controls were within the renal range and normal range. Our results suggest that sFLC are not useful to predict PTLD development in renal transplant recipients.

  15. Interaction of the Rabies Virus P Protein with the LC8 Dynein Light Chain

    PubMed Central

    Raux, Hélène; Flamand, Anne; Blondel, Danielle

    2000-01-01

    The rabies virus P protein is involved in viral transcription and replication but its precise function is not clear. We investigated the role of P (CVS strain) by searching for cellular partners by using a two-hybrid screening of a PC12 cDNA library. We isolated a cDNA encoding a 10-kDa dynein light chain (LC8). LC8 is a component of cytoplasmic dynein involved in the minus end-directed movement of organelles along microtubules. We confirmed that this molecule interacts with P by coimmunoprecipitation in infected cells and in cells transfected with a plasmid encoding P protein. LC8 was also detected in virus particles. Series of deletions from the N- and C-terminal ends of P protein were used to map the LC8-binding domain to the central part of P (residues 138 to 172). These results are relevant to speculate that dynein may be involved in the axonal transport of rabies virus along microtubules through neuron cells. PMID:11024151

  16. Myosin light chain kinase regulates cell polarization independently of membrane tension or Rho kinase

    PubMed Central

    Lou, Sunny S.; Diz-Muñoz, Alba; Weiner, Orion D.; Fletcher, Daniel A.

    2015-01-01

    Cells polarize to a single front and rear to achieve rapid actin-based motility, but the mechanisms preventing the formation of multiple fronts are unclear. We developed embryonic zebrafish keratocytes as a model system for investigating establishment of a single axis. We observed that, although keratocytes from 2 d postfertilization (dpf) embryos resembled canonical fan-shaped keratocytes, keratocytes from 4 dpf embryos often formed multiple protrusions despite unchanged membrane tension. Using genomic, genetic, and pharmacological approaches, we determined that the multiple-protrusion phenotype was primarily due to increased myosin light chain kinase (MLCK) expression. MLCK activity influences cell polarity by increasing myosin accumulation in lamellipodia, which locally decreases protrusion lifetime, limiting lamellipodial size and allowing for multiple protrusions to coexist within the context of membrane tension limiting protrusion globally. In contrast, Rho kinase (ROCK) regulates myosin accumulation at the cell rear and does not determine protrusion size. These results suggest a novel MLCK-specific mechanism for controlling cell polarity via regulation of myosin activity in protrusions. PMID:25918227

  17. New insights into the regulation of myosin light chain phosphorylation in retinal pigment epithelial cells.

    PubMed

    Ruiz-Loredo, Ariadna Yolanda; López-Colomé, Ana María

    2012-01-01

    The retinal pigment epithelium (RPE) plays an essential role in the function of the neural retina and the maintenance of vision. Most of the functions displayed by RPE require a dynamic organization of the acto-myosin cytoskeleton. Myosin II, a main cytoskeletal component in muscle and non-muscle cells, is directly involved in force generation required for organelle movement, selective molecule transport within cell compartments, exocytosis, endocytosis, phagocytosis, and cell division, among others. Contractile processes are triggered by the phosphorylation of myosin II light chains (MLCs), which promotes actin-myosin interaction and the assembly of contractile fibers. Considerable evidence indicates that non-muscle myosin II activation is critically involved in various pathological states, increasing the interest in studying the signaling pathways controlling MLC phosphorylation. Particularly, recent findings suggest a role for non-muscle myosin II-induced contraction in RPE cell transformation involved in the establishment of numerous retinal diseases. This review summarizes the current knowledge regarding myosin function in RPE cells, as well as the signaling networks leading to MLC phosphorylation under pathological conditions. Understanding the molecular mechanisms underlying RPE dysfunction would improve the development of new therapies for the treatment or prevention of different ocular disorders leading to blindness.

  18. Crystal Structure of Botulinum Neurotoxin Type G Light Chain - Serotype Divergence in Substrate Recognition†,‡

    PubMed Central

    Arndt, Joseph W.; Yu, Wayne; Bi, Fay; Stevens, Raymond C.

    2008-01-01

    The seven serotypes (A–G) of botulinum neurotoxins (BoNTs) block neurotransmitter release through their specific proteolysis of one of the three proteins of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex. BoNTs have stringent substrate specificities that are unique for metalloprotease in that they require exceptionally long substrates (1). In order to understand the molecular reasons for the unique specificities of the BoNTs, we determined the crystal structure of the catalytic light chain (LC) of Clostridium botulinum neurotoxin type G (BoNT/G-LC) at 2.35 Å resolution. The structure of BoNT/G-LC reveals a C-terminal β-sheet that is critical for LC oligomerization and is unlike that seen in the other LC structures. Its structural comparison with thermolysin and the available pool of LC structures reveals important serotype differences that are likely to be involved in substrate recognition of the P1’ residue. In addition, structural and sequence analysis have identified a potential exosite of BoNT/G-LC that recognizes a SNARE recognition motif of VAMP. PMID:16008342

  19. Crystal structure of botulinum neurotoxin type G light chain: serotype divergence in substrate recognition.

    PubMed

    Arndt, Joseph W; Yu, Wayne; Bi, Fay; Stevens, Raymond C

    2005-07-19

    The seven serotypes (A-G) of botulinum neurotoxins (BoNTs) block neurotransmitter release through their specific proteolysis of one of the three proteins of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex. BoNTs have stringent substrate specificities that are unique for metalloprotease in that they require exceptionally long substrates (1). To understand the molecular reasons for the unique specificities of the BoNTs, we determined the crystal structure of the catalytic light chain (LC) of Clostridium botulinum neurotoxin type G (BoNT/G-LC) at 2.35 A resolution. The structure of BoNT/G-LC reveals a C-terminal beta-sheet that is critical for LC oligomerization and is unlike that seen in the other LC structures. Its structural comparison with thermolysin and the available pool of LC structures reveals important serotype differences that are likely to be involved in substrate recognition of the P1' residue. In addition, structural and sequence analyses have identified a potential exosite of BoNT/G-LC that recognizes a SNARE recognition motif of VAMP.

  20. A comparison between mammalian and avian fast skeletal muscle alkali myosin light chain genes: regulatory implications.

    PubMed Central

    Daubas, P; Robert, B; Garner, I; Buckingham, M

    1985-01-01

    A single locus in the mouse, rat and chicken encodes both alkali myosin light chains, MLC1F and MLC3F. This gene has two distinct promoters and gives rise to two different primary transcripts, which are processed by alternative and different modes of splicing to form MLC1F and MLC3F mRNAs. The MLC1F/MLC3F gene is very similar between mouse, rat and chicken, in terms of its overall structure, the length and location of the introns, and the splice site consensus sequences. Nucleotide sequences of coding regions are very conserved but 3' and 5' non coding regions of the mRNAs have diverged. In the MLC1F promoter regions, several blocks of nucleotides are highly conserved (more than 70% homology), especially a sequence of about 70 nucleotides, located between positions -80 and -150 relative to the Cap site. Conserved blocks of homology are also found in the MLC3F promoter regions, although the common sequences are shorter. The presence of such highly conserved nucleotide sequences in the 5' flanking regions suggests that these sequences are functionally important in initiation of transcription and regulation of expression of this complex gene. Primer extension experiments indicate multiple cap sites for MLC3F mRNA. Images PMID:4022770

  1. Evolutionary redefinition of immunoglobulin light chain isotypes in tetrapods using molecular markers.

    PubMed

    Das, Sabyasachi; Nikolaidis, Nikolas; Klein, Jan; Nei, Masatoshi

    2008-10-28

    The phylogenetic relationships of Ig light chain (IGL) genes are difficult to resolve, because these genes are short and evolve relatively fast. Here, we classify the IGL sequences from 12 tetrapod species into three distinct groups (kappa, lambda, and sigma isotypes) using conserved amino acid residues, recombination signal sequences, and genomic organization of IGL genes as cladistic markers. From the distribution of the markers we conclude that the earliest extant tetrapods, the amphibians, possess three IGL isotypes: kappa, lambda, and sigma. Of these, two (kappa and lambda) are also found in reptiles and some mammals. The lambda isotype is found in all tetrapods tested to date, whereas the kappa isotype seems to have been lost at least in some birds and in the microbat. Conservation of the cladistic molecular markers suggests that they are associated with functional specialization of the three IGL isotypes. The genomic maps of IGL loci reveal multiple gene rearrangements that occurred in the evolution of tetrapod species. These rearrangements have resulted in interspecific variation of the genomic lengths of the IGL loci and the number and order of IGL constituent genes, but the overall organization of the IGL loci has not changed.

  2. An outer arm dynein light chain acts in a conformational switch for flagellar motility

    PubMed Central

    Patel-King, Ramila S.

    2009-01-01

    A system distinct from the central pair–radial spoke complex was proposed to control outer arm dynein function in response to alterations in the mechanical state of the flagellum. In this study, we examine the role of a Chlamydomonas reinhardtii outer arm dynein light chain that associates with the motor domain of the γ heavy chain (HC). We demonstrate that expression of mutant forms of LC1 yield dominant-negative effects on swimming velocity, as the flagella continually beat out of phase and stall near or at the power/recovery stroke switchpoint. Furthermore, we observed that LC1 interacts directly with tubulin in a nucleotide-independent manner and tethers this motor unit to the A-tubule of the outer doublet microtubules within the axoneme. Therefore, this dynein HC is attached to the same microtubule by two sites: via both the N-terminal region and the motor domain. We propose that this γ HC–LC1–microtubule ternary complex functions as a conformational switch to control outer arm activity. PMID:19620633

  3. Clinical and prognostic differences among patients with light chain deposition disease, myeloma cast nephropathy and both.

    PubMed

    Zand, Ladan; Nasr, Samih H; Gertz, Morie A; Dispenzieri, Angela; Lacy, Martha Q; Buadi, Francis K; Kumar, Shaji; Kyle, Robert A; Fervenza, Fernando C; Sethi, Sanjeev; Dingli, David; Rajkumar, S Vincent; Kapoor, Prashant; McCurdy, Arleigh; Leung, Nelson

    2015-01-01

    In some patients with light chain deposition disease (LCDD) there is also evidence of myeloma cast nephropathy (MCN) on renal biopsy. The purpose of this study was to evaluate the renal and survival outcome of patients with concomitant diagnosis of MCN and LCDD to LCDD and MCN alone. Eighty seven patients were identified and divided into LCDD (n=45), MCN (n=29), and LCDD+ MCN (n=13). Patients with LCDD+ MCN had a worse overall survival (OS) compared to patients with LCDD (p=0.03), but similar to patients with MCN (p=0.4). Death-censored renal survival was no different amongst the groups. Presenting with acute renal failure at time of renal biopsy (HR 7.2, p=0.0002) was an independent poor renal prognostic factor while older age (HR 1.06, p=0.0002), presence of osteolytic lesions (HR 4.4, p<0.0001), and requirement for dialysis or creatinine≥5 mg/dL (HR 3.2, p=0.0006) at time of renal biopsy were independent poor prognostic factors for OS.

  4. Dynein light chain family genes in 15 plant species: Identification, evolution and expression profiles.

    PubMed

    Cao, Jun; Li, Xiangyang; Lv, Yueqing

    2017-01-01

    Dynein light chain (DLC) is one important component of the dynein complexes, which have been proved involving in a variety of cellular functions. However, higher plants lack all other components of the complexes except DLCs, suggesting that in plants, the DLC protein does not carry out the same function as it in animals. Therefore, the function of this family in plants is mysterious. In this study, we investigated the DLC gene family in 15 plant species and analyzed their expression profiles. In total, 128 DLC genes were identified from the 15 studied plant species and were divided into eight groups by their phylogenetic relation. Highly conserved gene structure and motif arrangement was discovered within each group, indicating their functional correlation. Genetic variation and recombination events were also detected in DLC genes. Through selection analyses, we also identified some significant site-specific constraints in most of the DLC paralogs. In addition, DLC genes presented various expression profiles in different development stages, or under different abiotic stresses or phytohormone treatments. This may be associated with a variety of cis-elements responding to stress and phytohormone in the upstream sequences of the DLC genes. Functional network analysis exhibited 123 physical or functional interactions. The results provide a foundation for exploring the characterization of the DLC genes in plants and offer insights for additional functional studies.

  5. Structure and function of outer dynein arm intermediate and light chain complex

    PubMed Central

    Oda, Toshiyuki; Abe, Tatsuki; Yanagisawa, Haruaki; Kikkawa, Masahide

    2016-01-01

    The outer dynein arm (ODA) is a molecular complex that drives the beating motion of cilia/flagella. Chlamydomonas ODA is composed of three heavy chains (HCs), two ICs, and 11 light chains (LCs). Although the three-dimensional (3D) structure of the whole ODA complex has been investigated, the 3D configurations of the ICs and LCs are largely unknown. Here we identified the 3D positions of the two ICs and three LCs using cryo–electron tomography and structural labeling. We found that these ICs and LCs were all localized at the root of the outer-inner dynein (OID) linker, designated the ODA-Beak complex. Of interest, the coiled-coil domain of IC2 extended from the ODA-Beak to the outer surface of ODA. Furthermore, we investigated the molecular mechanisms of how the OID linker transmits signals to the ODA-Beak, by manipulating the interaction within the OID linker using a chemically induced dimerization system. We showed that the cross-linking of the OID linker strongly suppresses flagellar motility in vivo. These results suggest that the ICs and LCs of the ODA form the ODA-Beak, which may be involved in mechanosignaling from the OID linker to the HCs. PMID:26864626

  6. Probing BoNT/A Protease Exosites: Implications for Inhibitor Design and Light Chain Longevity

    PubMed Central

    2015-01-01

    Botulinum neurotoxin serotype A (BoNT/A) is one of the most lethal toxins known. Its extreme toxicity is due to its light chain (LC), a zinc protease that cleaves SNAP-25, a synaptosome-associated protein, leading to the inhibition of neuronal activity. Studies on BoNT/A LC have revealed that two regions, termed exosites, can play an important role in BoNT catalytic activity. A clear understanding of how these exosites influence neurotoxin catalytic activity would provide a critical framework for deciphering the mechanism of SNAP-25 cleavage and the design of inhibitors. Herein, based on the crystallographic structure of BoNT/A LC complexed with its substrate, we designed an α-exosite binding probe. Experiments with this unique probe demonstrated that α-exosite binding enhanced both catalytic activity and stability of the LC. These data help delineate why α-exosite binding is needed for SNAP-25 cleavage and also provide new insights into the extended lifetime observed for BoNT/A LC in vivo. PMID:25295706

  7. Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains.

    PubMed

    Muyldermans, S; Atarhouch, T; Saldanha, J; Barbosa, J A; Hamers, R

    1994-09-01

    We cloned 17 different PCR fragments encoding VH genes of camel (Camelus dromedarius). These clones were derived from the camel heavy chain immunoglobulins lacking the light chain counterpart of normal immunoglobulins. Insight into the camel VH sequences and structure may help the development of single domain antibodies. The most remarkable difference in the camel VH, consistent with the absence of the VL interaction, is the substitution of the conserved Leu45 by an Arg or Cys. Another noteworthy substitution is the Leu11 to Ser. This amino acid normally interacts with the CH1 domain, a domain missing in the camel heavy chain immunoglobulins. The nature of these substitutions agrees with the increased solubility behavior of an isolated camel VH domain. The VH domains of the camels are also characterized by a long CDR3, possibly compensating for the absence of the VL contacts with the antigen. The CDR3 lacks the salt bridge between Arg94 and Asp101. However, the frequent occurrence of additional Cys residues in both the CDR1 and CDR3 might lead to the formation of a second internal disulfide bridge, thereby stabilizing the CDR structure as in the DAW antibody. Within CDRs of the camel VH domains we observe a broad size distribution and a different amino acid pattern compared with the mouse or human VH. Therefore the camel hypervariable regions might adopt structures which differ substantially from the known canonical structures, thereby increasing the repertoire of the camel antigen binding sites within a VH.

  8. Notes on individual sequence variation in humans: Immunoglobulin kappa light chain

    SciTech Connect

    Kurth, J.H. ); Cavalli-Sforza, L.L. )

    1994-06-01

    Little is known concerning the magnitude of variability in the nucleic acid sequence of DNA at the individual level. The authors have collected a large set of sequence data from the human immunoglobulin kappa light-chain-locus constant region (10,444 bp) and subgroup IV variable region (18,580 bp). For the constant region, absolute conservation of sequence was observed, even in intron and coding-region silent sites, with the exception of one previously defined polymorphic site. For the variable region, 12 heterozygous positions were identified, giving a heterozygosity of 6 x 10[sup [minus]4] per nucleotide site. The amount of nucleic acid sequence variation differs significantly ([chi][sup 2] = 4.88) between these two regions, and the observed variation is two orders of magnitude lower than that reported for two Drosophila melanogaster loci. These data suggest that, for at least some regions of the human genome, nucleic acid sequence may be less variable than previously estimated. 13 refs., 2 figs.

  9. Effects of a Fluorescent Myosin Light Chain Phosphatase Inhibitor on Prostate Cancer Cells

    PubMed Central

    Grindrod, Scott; Suy, Simeng; Fallen, Shannon; Eto, Masumi; Toretsky, Jeffrey; Brown, Milton L.

    2011-01-01

    Myosin light chain phosphatase (MLCP) is an enzyme important to regulation of cell cycle and motility that is shown to be upregulated in aggressive prostate cancer cells and tissue. We developed a fluorescent small molecule inhibitor of MLCP using structure based design in recombinant protein phosphatase 1C. Several best fit compounds were synthesized and evaluated by their inhibition of MLCP/32P-MLC dephosphorylation, which resulted in the identification of novel MLCP inhibitors. Androgen dependent (AD) and castration resistant prostate cancer cell (CRPC) lines were treated with the lead inhibitor resulting in decreased growth rate, reduced DNA synthesis, and G2/M cell cycle arrest. Moreover, CRPC cell lines showed an increased sensitivity to drug treatment having GI50 values four times lower than the AD prostate cancer cell line. This was reinforced by reduced BrdU DNA incorporation into CRPC cells compared to AD cells. β-actin disruption was also seen at much lower drug concentrations in CR cells which caused a dose dependent reduction in cellular chemotaxis of PC-3 cells. Since there are currently few clinical therapeutics targeting CR prostate cancer, MLCP represents a new target for preclinical and clinical development of new potential therapeutics which inhibit this disease phenotype. PMID:22655237

  10. Amyloid A amyloidosis secondary to rheumatoid arthritis: pathophysiology and treatments.

    PubMed

    Nakamura, Tadashi

    2011-01-01

    The introduction of biological therapies targeting specific inflammatory mediators revolutionised the treatment of rheumatoid arthritis (RA). Targeting key components of the immune system allows efficient suppression of the pathological inflammatory cascade that leads to RA symptoms and subsequent joint destruction. Reactive amyloid A (AA) amyloidosis, one of the most severe complications of RA, is a serious, potentially life-threatening disorder caused by deposition of AA amyloid fibrils in multiple organs. These AA amyloid fibrils derive from the circulatory acute-phase reactant serum amyloid A protein (SAA), and may be controlled by treatment. New biologics may permit AA amyloidosis secondary to RA to become a treatable, manageable disease. Rheumatologists, when diagnosing and treating patients with AA amyloidosis secondary to RA, must understand the pathophysiology and clinical factors related to development and progression of the disease, including genetic predisposition and biological versatility of SAA.

  11. 99mTc DPD is the preferential bone tracer for diagnosis of cardiac transthyretin amyloidosis.

    PubMed

    Rossi, Pascal; Tessonnier, Laurent; Frances, Yves; Mundler, Olivier; Granel, Brigitte

    2012-08-01

    We emphasize the role of Tc-99m-3,3-diphosphono-1,2-propanodicarboxylicacid (DPD) scintigraphy as a noninvasive tool to distinguish transthyretin (TTR)-related cardiac amyloidosis from other forms of cardiac amyloidosis. We report the case of a 76-year-old male patient suffering from congestive heart failure in whom imaging investigation by DPD scintigraphy showed a strong cardiac uptake highly suggestive of TTR amyloidosis variant. TTR-related cardiac amyloidosis was confirmed on myocardial biopsies by immunohistochemistry analysis. This case supports the growing interest in DPD scintigraphy for typing cardiac amyloidosis and for its contribution in the place of invasive myocardial biopsy.

  12. Diagnostic approach to cardiac amyloidosis: A case report.

    PubMed

    Fernandes, Andreia; Caetano, Francisca; Almeida, Inês; Paiva, Luís; Gomes, Pedro; Mota, Paula; Trigo, Joana; Botelho, Ana; Cachulo, Maria do Carmo; Alves, Joana; Francisco, Luís; Leitão Marques, António

    2016-05-01

    The authors present a case of systemic amyloidosis with cardiac involvement. We discuss the need for a high level of suspicion to establish a diagnosis, diagnostic techniques and treatment options. Our patient was a 78-year-old man with chronic renal disease and atrial fibrillation admitted with acute decompensated heart failure of unknown cause. The transthoracic echocardiogram revealed severely impaired left ventricular function with phenotypic overlap between hypertrophic and restrictive cardiomyopathy. After an extensive diagnostic workup, which included an abdominal fat pad biopsy, the final diagnosis was amyloidosis.

  13. Mice with Alopecia, Osteoporosis, and Systemic Amyloidosis Due to Mutation in Zdhhc13, a Gene Coding for Palmitoyl Acyltransferase

    PubMed Central

    Hsiao, Ya-Wen; Huang, Hong-Wen; Kao, Hsiao-Jung; Liu, Kai-Ming; Shen, Li-Fen; Song, I-wen; Tu, Chen-Pei D.; Wu, Jer-Yuarn; Kikuchi, Tateki; Justice, Monica J.; Yen, Jeffrey J. Y.; Chen, Yuan-Tsong

    2010-01-01

    Protein palmitoylation has emerged as an important mechanism for regulating protein trafficking, stability, and protein–protein interactions; however, its relevance to disease processes is not clear. Using a genome-wide, phenotype driven N-ethyl-N-nitrosourea–mediated mutagenesis screen, we identified mice with failure to thrive, shortened life span, skin and hair abnormalities including alopecia, severe osteoporosis, and systemic amyloidosis (both AA and AL amyloids depositions). Whole-genome homozygosity mapping with 295 SNP markers and fine mapping with an additional 50 SNPs localized the disease gene to chromosome 7 between 53.9 and 56.3 Mb. A nonsense mutation (c.1273A>T) was located in exon 12 of the Zdhhc13 gene (Zinc finger, DHHC domain containing 13), a gene coding for palmitoyl transferase. The mutation predicted a truncated protein (R425X), and real-time PCR showed markedly reduced Zdhhc13 mRNA. A second gene trap allele of Zdhhc13 has the same phenotypes, suggesting that this is a loss of function allele. This is the first report that palmitoyl transferase deficiency causes a severe phenotype, and it establishes a direct link between protein palmitoylation and regulation of diverse physiologic functions where its absence can result in profound disease pathology. This mouse model can be used to investigate mechanisms where improper palmitoylation leads to disease processes and to understand molecular mechanisms underlying human alopecia, osteoporosis, and amyloidosis and many other neurodegenerative diseases caused by protein misfolding and amyloidosis. PMID:20548961

  14. Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation

    PubMed Central

    1995-01-01

    The phosphorylation of regulatory myosin light chains by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK) has been shown to be essential and sufficient for initiation of endothelial cell retraction in saponin permeabilized monolayers (Wysolmerski, R. B. and D. Lagunoff. 1990. Proc. Natl. Acad. Sci. USA. 87:16-20). We now report the effects of thrombin stimulation on human umbilical vein endothelial cell (HUVE) actin, myosin II and the functional correlate of the activated actomyosin based contractile system, isometric tension development. Using a newly designed isometric tension apparatus, we recorded quantitative changes in isometric tension from paired monolayers. Thrombin stimulation results in a rapid sustained isometric contraction that increases 2- to 2.5-fold within 5 min and remains elevated for at least 60 min. The phosphorylatable myosin light chains from HUVE were found to exist as two isoforms, differing in their molecular weights and isoelectric points. Resting isometric tension is associated with a basal phosphorylation of 0.54 mol PO4/mol myosin light chain. After thrombin treatment, phosphorylation rapidly increases to 1.61 mol PO4/mol myosin light chain within 60 s and remains elevated for the duration of the experiment. Myosin light chain phosphorylation precedes the development of isometric tension and maximal phosphorylation is maintained during the sustained phase of isometric contraction. Tryptic phosphopeptide maps from both control and thrombin-stimulated cultures resolve both monophosphorylated Ser-19 and diphosphorylated Ser-19/Thr-18 peptides indicative of MLCK activation. Changes in the polymerization of actin and association of myosin II correlate temporally with the phosphorylation of myosin II and development of isometric tension. Activation results in a 57% increase in F-actin content within 90 s and 90% of the soluble myosin II associates with the reorganizing F-actin. Furthermore, the disposition of actin and

  15. Use of Nonclonal Serum Immunoglobulin Free Light Chains to Predict Overall Survival in the General Population

    PubMed Central

    Dispenzieri, Angela; Katzmann, Jerry A.; Kyle, Robert A.; Larson, Dirk R.; Therneau, Terry M.; Colby, Colin L.; Clark, Raynell J.; Mead, Graham P.; Kumar, Shaji; Melton, L. Joseph; Rajkumar, S. Vincent

    2012-01-01

    Objective To determine whether the free light chain (FLC) assay provides prognostic information relevant to the general population. Methods After excluding persons with a known plasma cell disorder, we studied 15,859 Olmsted County, Minnesota, residents 50 years or older in whom unmasked data and samples for FLC testing were available. Baseline information was obtained between March 13, 1995, and November 21, 2003, and follow-up status and cause of death were identified through June 30, 2009. The κ and λ FLC sum (Σ FLC) was evaluated for its ability to predict overall survival. Specific causes of death were also investigated. Results In 158,003 person-years of follow-up, 4348 individuals died. A high Σ FLC was significantly predictive of worse overall survival; the risk ratio for death for those with the highest decile of Σ FLC (ie, ≥4.72 mg/dL) was 4.4 (95% confidence interval, 4.1-4.7) relative to the remaining study participants. Multivariate analyses demonstrated that this excess risk of death was independent of age, sex, and renal insufficiency, with a corrected risk ratio of 2.1 (95% confidence interval, 1.9-2.2). The increased mortality was not restricted to any particular cause of death because the observed-to-expected risk of death from most causes was significantly higher among those individuals with an antecedent Σ FLC of 4.72 mg/dL or higher, which is near the upper limit of normal for the test. Conclusion A nonclonal elevation of Σ FLC is a significant predictor of worse overall survival in the general population of persons without plasma cell disorders. PMID:22677072

  16. Serum free-light chain removal by high cutoff hemodialysis: optimizing removal and supportive care.

    PubMed

    Hutchison, Colin A; Harding, Stephen; Mead, Graham; Goehl, Hermann; Storr, Markus; Bradwell, Arthur; Cockwell, Paul

    2008-12-01

    In multiple myeloma the predominant cause of irreversible renal failure is cast nephropathy, secondary to excess kappa or lambda serum free light chains (FLCs). These molecules are efficiently cleared by hemodialysis (HD) using the Gambro HCO 1100 dialyzer. To optimize the removal of FLCs by this dialyzer we have studied the effect of dialyzers in series, dialyzer change, and hemodiafiltration in 14 patients with multiple myeloma and renal failure. The clearance rates of both kappa FLCs and lambda FLCs were significantly increased on two dialyzers from 19 (7.3-34)-15.3 (9-28) mL/min to 47 (17-79)-35.5 (20-57) mL/min, respectively. Clearance rates of both FLCs decreased over the course of the dialysis sessions (both P < 0.001). Changing the dialyzer during a HD session increased lambda FLC clearance rates (22.5 [6-41] to 37.6 [9-52] mL/min; P < 0.001) and decreased kappa FLC clearance rates (39.6 [9-72] to 19 [8-59] mL/min; P < 0.003). Ultrafiltration during HD increased the clearance rates of kappa FLCs (R 0.52, P < 0.01) but not lambda FLCs (R -0.25; P < 0.076). Hemodiafiltration increased the clearance rates of both kappa (19 [SD 6.8] to 32 [SD 9.8] mL/min) and lambda FLCs (15 [SD 7.8] to 20 [SD 7.7] mL/min). Albumin replacement requirements for 8 h of HD increased from 12 g for a single dialyzer to 45 g for two dialyzers in series (P < 0.001). Different protocols are required to optimize the removal of kappa and lambda FLCs in patients with myeloma and renal failure.

  17. Disruption of the murine dynein light chain gene Tcte3-3 results in asthenozoospermia.

    PubMed

    Rashid, Sajid; Grzmil, Pawel; Drenckhahn, Joerg-Detlef; Meinhardt, Andreas; Adham, Ibrahim; Engel, Wolfgang; Neesen, Juergen

    2010-01-01

    To elucidate the role of the mouse gene Tcte3 (Tctex2), which encodes a putative light chain of the outer dynein arm of cilia and sperm flagella, we have inactivated this gene in mice using targeted disruption. Breeding of heterozygous males and females resulted in normal litter size; however, we were not able to detect homozygous Tcte3-deficent mice using standard genotype techniques. In fact, our results indicate the presence of at least three highly similar copies of the Tcte3 gene (Tcte3-1, Tcte3-2, and Tcte3-3) in the murine genome. Therefore, quantitative real-time PCR was established to differentiate between mice having one or two targeted Tcte3-3 alleles. By this approach, Tcte3-3(-/-) animals were identified, which were viable and revealed no obvious malformation. Interestingly, some homozygous Tcte3-3-deficient male mice bred with wild-type female produced no offspring while other Tcte3-3-deficient males revealed decreased sperm motility but were fertile. In infertile Tcte3-3(-/-) males, spermatogenesis was affected and sperm motility was reduced, too, resulting in decreased ability of Tcte3-3-deficient spermatozoa to move from the uterus into the oviduct. Impaired flagellar motility is not correlated with any gross defects in the axonemal structure, since outer dynein arms are detectable in sperm of Tcte3-3(-/-) males. However, in infertile males, deficient Tcte3-3 function is correlated with increased apoptosis during male germ cell development, resulting in a reduction of sperm number. Moreover, multiple malformations in developing haploid germ cells are present. Our results support a role of Tcte3-3 in generation of sperm motility as well as in male germ cell differentiation.

  18. Immunoglobulin Free Light Chains Are Increased in Hypersensitivity Pneumonitis and Idiopathic Pulmonary Fibrosis

    PubMed Central

    Groot Kormelink, Tom; Pardo, Annie; Knipping, Karen; Buendía-Roldán, Ivette; García-de-Alba, Carolina; Blokhuis, Bart R.; Selman, Moises; Redegeld, Frank A.

    2011-01-01

    Background Idiopathic pulmonary fibrosis (IPF), a devastating lung disorder of unknown aetiology, and chronic hypersensitivity pneumonitis (HP), a disease provoked by an immunopathologic reaction to inhaled antigens, are two common interstitial lung diseases with uncertain pathogenic mechanisms. Previously, we have shown in other upper and lower airway diseases that immunoglobulin free light chains (FLCs) are increased and may be involved in initiating a local inflammation. In this study we explored if such a mechanism may also apply to HP and IPF. Methods In this study we examined the presence of FLC in serum and BAL fluid from 21 IPF and 22 HP patients and controls. IgG, IgE and tryptase concentrations were measured in BAL fluid only. The presence of FLCs, plasma cells, B cells and mast cells in lung tissue of 3 HP and 3 IPF patients and 1 control was analyzed using immunohistochemistry. Results FLC concentrations in serum and BAL fluid were increased in IPF and HP patients as compared to control subjects. IgG concentrations were only increased in HP patients, whereas IgE concentrations were comparable to controls in both patient groups. FLC-positive cells, B cells, plasma cells, and large numbers of activated mast cells were all detected in the lungs of HP and IPF patients, not in control lung. Conclusion These results show that FLC concentrations are increased in serum and BAL fluid of IPF and HP patients and that FLCs are present within affected lung tissue. This suggests that FLCs may be involved in mediating pathology in both diseases. PMID:21980441

  19. Comparing domain interactions within antibody Fabs with kappa and lambda light chains

    PubMed Central

    Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W.; Dickey, Mark; Froning, Karen; Conner, Elaine M.; Cujec, Thomas P.; Demarest, Stephen J.

    2016-01-01

    ABSTRACT IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates. PMID:27454112

  20. Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction

    SciTech Connect

    Muthu, Priya; Wang, Li; Yuan, Chen-Ching; Kazmierczak, Katarzyna; Huang, Wenrui; Hernandez, Olga M.; Kawai, Masataka; Irving, Thomas C.; Szczesna-Cordary, Danuta

    2012-04-02

    The myosin essential light chain (ELC) is a structural component of the actomyosin cross-bridge, but its function is poorly understood, especially the role of the cardiac specific N-terminal extension in modulating actomyosin interaction. Here, we generated transgenic (Tg) mice expressing the A57G (alanine to glycine) mutation in the cardiac ELC known to cause familial hypertrophic cardiomyopathy (FHC). The function of the ELC N-terminal extension was investigated with the Tg-{Delta}43 mouse model, whose myocardium expresses a truncated ELC. Low-angle X-ray diffraction studies on papillary muscle fibers in rigor revealed a decreased interfilament spacing ({approx} 1.5 nm) and no alterations in cross-bridge mass distribution in Tg-A57G mice compared to Tg-WT, expressing the full-length nonmutated ELC. The truncation mutation showed a 1.3-fold increase in I{sub 1,1}/I{sub 1,0}, indicating a shift of cross-bridge mass from the thick filament backbone toward the thin filaments. Mechanical studies demonstrated increased stiffness in Tg-A57G muscle fibers compared to Tg-WT or Tg-{Delta}43. The equilibrium constant for the cross-bridge force generation step was smallest in Tg-{Delta}43. These results support an important role for the N-terminal ELC extension in prepositioning the cross-bridge for optimal force production. Subtle changes in the ELC sequence were sufficient to alter cross-bridge properties and lead to pathological phenotypes.

  1. Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

    PubMed

    Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W; Dickey, Mark; Froning, Karen; Conner, Elaine M; Cujec, Thomas P; Demarest, Stephen J

    2016-10-01

    IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

  2. Myosin light chain kinase controls voltage-dependent calcium channels in vascular smooth muscle.

    PubMed

    Martinsen, A; Schakman, O; Yerna, X; Dessy, C; Morel, N

    2014-07-01

    The Ca(2+)-dependent kinase myosin light chain kinase (MLCK) is the activator of smooth muscle contraction. In addition, it has been reported to be involved in Ca(2+) channel regulation in cultured cells, and we previously showed that the MLCK inhibitor ML-7 decreases arginine vasopressin (AVP)-induced Ca(2+) influx in rat aorta. This study was designed to investigate whether MLCK is involved in Ca(2+) regulation in resistance artery smooth muscle cell, which plays a major role in the control of blood pressure. As ML compounds were shown to have off-target effects, MLCK was downregulated by transfection with a small interfering RNA targeting MLCK (MLCK-siRNA) in rat small resistance mesenteric artery (RMA) and in the rat embryonic aortic cell line A7r5. Noradrenaline-induced contraction and Ca(2+) signal were significantly depressed in MLCK-siRNA compared to scramble-siRNA-transfected RMA. Contraction and Ca(2+) signal induced by high KCl and voltage-activated Ca(2+) current were also significantly decreased in MLCK-siRNA-transfected RMA, suggesting that MLCK depletion modifies voltage-operated Ca(2+) channels. KCl- and AVP-induced Ca(2+) signals and voltage-activated Ca(2+) current were decreased in MLCK-depleted A7r5 cells. Eventually, real-time quantitative PCR analysis indicated that in A7r5, MLCK controlled mRNA expression of CaV1.2 (L-type) and CaV3.1 (T-type) voltage-dependent Ca(2+) channels. Our results suggest that MLCK controls the transcription of voltage-dependent Ca(2+) channels in vascular smooth muscle cells.

  3. Regulatory light chain mutants linked to heart disease modify the cardiac myosin lever arm.

    PubMed

    Burghardt, Thomas P; Sikkink, Laura A

    2013-02-19

    Myosin is the chemomechanical energy transducer in striated heart muscle. The myosin cross-bridge applies impulsive force to actin while consuming ATP chemical energy to propel myosin thick filaments relative to actin thin filaments in the fiber. Transduction begins with ATP hydrolysis in the cross-bridge driving rotary movement of a lever arm converting torque into linear displacement. Myosin regulatory light chain (RLC) binds to the lever arm and modifies its ability to translate actin. Gene sequencing implicated several RLC mutations in heart disease, and three of them are investigated here using photoactivatable GFP-tagged RLC (RLC-PAGFP) exchanged into permeabilized papillary muscle fibers. A single-lever arm probe orientation is detected in the crowded environment of the muscle fiber by using RLC-PAGFP with dipole orientation deduced from the three-spatial dimension fluorescence emission pattern of the single molecule. Symmetry and selection rules locate dipoles in their half-sarcomere, identify those at the minimal free energy, and specify active dipole contraction intermediates. Experiments were performed in a microfluidic chamber designed for isometric contraction, total internal reflection fluorescence detection, and two-photon excitation second harmonic generation to evaluate sarcomere length. The RLC-PAGFP reports apparently discretized lever arm orientation intermediates in active isometric fibers that on average produce the stall force. Disease-linked mutants introduced into RLC move intermediate occupancy further down the free energy gradient, implying lever arms rotate more to reach stall force because mutant RLC increases lever arm shear strain. A lower free energy intermediate occupancy involves a lower energy conversion efficiency in the fiber relating a specific myosin function modification to the disease-implicated mutant.

  4. N-terminal or signal peptide sequence engineering prevents truncation of human monoclonal antibody light chains.

    PubMed

    Gibson, S J; Bond, N J; Milne, S; Lewis, A; Sheriff, A; Pettman, G; Pradhan, R; Higazi, D R; Hatton, D

    2017-03-28

    Monoclonal antibodies (mAbs) contain short N-terminal signal peptides on each individual polypeptide that comprises the mature antibody, targeting them for export from the cell in which they are produced. The signal peptide is cleaved from each heavy chain (Hc) and light chain (Lc) polypeptide after translocation to the ER and prior to secretion. This process is generally highly efficient, producing a high proportion of correctly cleaved Hc and Lc polypeptides. However, mis-cleavage of the signal peptide can occur, resulting in truncation or elongation at the N-terminus of the Hc or Lc. This is undesirable for antibody manufacturing as it can impact efficacy and can result in product heterogeneity. Here, we describe a truncated variant of the Lc that was detected during a routine developability assessment of the recombinant human IgG1 MEDI8490 in Chinese hamster ovary cells. We found that the truncation of the Lc was caused due to the use of the murine Hc signal peptide together with a lambda Lc containing an SYE amino acid motif at the N-terminus. This truncation was not caused by mis-processing of the mRNA encoding the Lc and was not dependent on expression platform (transient or stable), the scale of the fed-batch culture or clonal lineage. We further show that using alternative signal peptides or engineering the Lc SYE N-terminal motif prevented the truncation and that this strategy will improve Lc homogeneity of other SYE lambda Lc-containing mAbs. This article is protected by copyright. All rights reserved.

  5. Constraints on intron evolution in the gene encoding the myosin alkali light chain in Drosophila

    SciTech Connect

    Leicht, B.G.; Muse, S.V.; Hanczyc, M.

    1995-01-01

    Interspecific comparisons of intron sequences reveal conserved blocks of invariant nucleotides and several other departures from the strictly neutral model of molecular evolution. To distinguish the past action of evolutionary forces in introns known to have regulatory information, we examined nucleotide sequence variation at 991 sites in a random sample of 16 Drosophila melanogaster alleles of the gene encoding the myosin alkali light chain (Mlc1). The Mlc1 gene of D. melanogaster encodes two Mlc1 isoforms via developmentally regulated alternative pre-mRNA splicing. Analyses of these data reveal that introns 4 and 5, which flank the alternatively spliced exon 5, have reduced levels of both intraspecific polymorphism and interspecific divergence relative to intron 3. No polymorphism was observed in any of the exons examined in D. melanogaster. A genealogical analysis clearly demonstrates the occurrence of intragenic recombination in the ancestral history of Mlc1. Recombination events are estimated to be 13 times more likely than mutation events over the span of the sequenced region. Although there is little evidence for pairwise linkage disequilibrium in the Mlc1 region, higher order disequilibrium. does seem to be present in the 5{prime} half of the portion of the gene that was examined. Predictions of the folding free energy of the pre-mRNA reveal that sampled alleles have a significantly higher (less stable) free energy than do randomly permuted sequences. These results are consistent with the hypothesis that introns surrounding an alternatively spliced exon are subjected to additional constraints, perhaps due to specific aspects of secondary structure required for appropriate splicing of the pre-mRNA molecule. 48 refs., 5 figs., 3 tabs.

  6. Antibody light chain variable domains and their biophysically improved versions for human immunotherapy

    PubMed Central

    Kim, Dae Young; To, Rebecca; Kandalaft, Hiba; Ding, Wen; van Faassen, Henk; Luo, Yan; Schrag, Joseph D; St-Amant, Nadereh; Hefford, Mary; Hirama, Tomoko; Kelly, John F; MacKenzie, Roger; Tanha, Jamshid

    2014-01-01

    We set out to gain deeper insight into the potential of antibody light chain variable domains (VLs) as immunotherapeutics. To this end, we generated a naïve human VL phage display library and, by using a method previously shown to select for non-aggregating antibody heavy chain variable domains (VHs), we isolated a diversity of VL domains by panning the library against B cell super-antigen protein L. Eight domains representing different germline origins were shown to be non-aggregating at concentrations as high as 450 µM, indicating VL repertoires are a rich source of non-aggregating domains. In addition, the VLs demonstrated high expression yields in E. coli, protein L binding and high reversibility of thermal unfolding. A side-by-side comparison with a set of non-aggregating human VHs revealed that the VLs had similar overall profiles with respect to melting temperature (Tm), reversibility of thermal unfolding and resistance to gastrointestinal proteases. Successful engineering of a non-canonical disulfide linkage in the core of VLs did not compromise the non-aggregation state or protein L binding properties. Furthermore, the introduced disulfide bond significantly increased their Tms, by 5.5–17.5 °C, and pepsin resistance, although it somewhat reduced expression yields and subtly changed the structure of VLs. Human VLs and engineered versions may make suitable therapeutics due to their desirable biophysical features. The disulfide linkage-engineered VLs may be the preferred therapeutic format because of their higher stability, especially for oral therapy applications that necessitate high resistance to the stomach’s acidic pH and pepsin. PMID:24423624

  7. Residual structure and dynamics in DMSO-d6 denatured dynein light chain protein.

    PubMed

    Chakraborty, Swagata; Mohan, P M Krishna; Hosur, Ramakrishna V

    2012-01-01

    Structural and motional features in the denatured state of a protein dictate the early folding events starting from that state and these features vary depending upon the nature of the denaturant used. Here, we have attempted to decipher the early events in the folding of Dynein Light Chain protein (DLC8), starting from DMSO-d6 denatured state. Multinuclear NMR experiments were used to obtain the full spectral assignment. The HSQC spectrum shows the presence of two sets of peaks for the residues Met 1, Ser 2, Arg 4, Ala 11, Met 17, Thr 26, Lys 44, Tyr 50, Asn 51, Trp 54, His 55, Val 58, Gly 59, Ser 64, Tyr 65, His 68, Phe 86, Lys 87 indicating the presence of slow conformational transition in the heterogeneous ensemble. Analysis of residual structural propensities with secondary (13)C chemical shifts, (3)J(H(N)(-)H(α)) coupling constants and (1)H-(1)H NOE revealed the presence of local preferences which encompass both native and non-native like structures. The spectral density calculations, as obtained from measured R(1), R(2) and (1)H-(15)N steady state NOE values provide insights into the backbone dynamics on the milli to picosecond timescale. The segment Ser 14 - His 55 exhibits slow motions on the milli- to microsecond timescale arising from conformational exchange. The presence of native like structural preference, as well as conformational exchange classifies the above segment as the nucleation site of folding. Based on the observations, we propose here, the probable hierarchy of folding of DLC8 on dilution of denaturant: the two helices are formed first followed by the formation of β2 and β5.

  8. Localization of dynein light chains 1 and 2 and their pro-apoptotic ligands.

    PubMed Central

    Day, Catherine L; Puthalakath, Hamsa; Skea, Gretchen; Strasser, Andreas; Barsukov, Igor; Lian, Lu-Yun; Huang, David C S; Hinds, Mark G

    2004-01-01

    The dynein and myosin V motor complexes are multi-protein structures that function to transport molecules and organelles within the cell. DLC (dynein light-chain) proteins, found as components of both dynein and myosin V motor complexes, connect the complexes to their cargoes. One of the roles of these motor complexes is to selectively sequester the pro-apoptotic 'BH3-only' (Bcl-2 homology 3-only) proteins, Bim (Bcl-2-interacting mediator of cell death) and Bmf (Bcl-2-modifying factor), and so regulate their cell death-inducing function. In vivo DLC2 is found exclusively as a component of the myosin V motor complex and Bmf binds DLC2 selectively. On the other hand, Bim interacts with DLC1 (LC8), an integral component of the dynein motor complex. The two DLCs share 93% sequence identity yet show unambiguous in vivo specificity for their respective BH3-only ligands. To investigate this specificity the three-dimensional solution structure of DLC2 was elucidated using NMR spectroscopy. In vitro structural and mutagenesis studies show that Bmf and Bim have identical binding characteristics to recombinant DLC2 or DLC1. Thus the selectivity shown by Bmf and Bim for binding DLC1 or DLC2, respectively, does not reside in their DLC-binding domains. Remarkably, mutational analysis of DLC1 and DLC2 indicates that a single surface residue (residue 41) determines the specific localization of DLCs with their respective motor complexes. These results suggest a molecular mechanism for the specific compartmentalization of DLCs and their pro-apoptotic cargoes and implicate other protein(s) in defining the specificity between the cargoes and the DLC proteins. PMID:14561217

  9. Myosin regulatory light chain phosphorylation enhances cardiac β-myosin in vitro motility under load.

    PubMed

    Karabina, Anastasia; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R

    2015-08-15

    Familial hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and myofibrillar disarray, and often results in sudden cardiac death. Two HCM mutations, N47K and R58Q, are located in the myosin regulatory light chain (RLC). The RLC mechanically stabilizes the myosin lever arm, which is crucial to myosin's ability to transmit contractile force. The N47K and R58Q mutations have previously been shown to reduce actin filament velocity under load, stemming from a more compliant lever arm (Greenberg, 2010). In contrast, RLC phosphorylation was shown to impart stiffness to the myosin lever arm (Greenberg, 2009). We hypothesized that phosphorylation of the mutant HCM-RLC may mitigate distinct mutation-induced structural and functional abnormalities. In vitro motility assays were utilized to investigate the effects of RLC phosphorylation on the HCM-RLC mutant phenotype in the presence of an α-actinin frictional load. Porcine cardiac β-myosin was depleted of its native RLC and reconstituted with mutant or wild-type human RLC in phosphorylated or non-phosphorylated form. Consistent with previous findings, in the presence of load, myosin bearing the HCM mutations reduced actin sliding velocity compared to WT resulting in 31-41% reductions in force production. Myosin containing phosphorylated RLC (WT or mutant) increased sliding velocity and also restored mutant myosin force production to near WT unphosphorylated values. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the HCM-induced phenotype at the molecular level.

  10. Expression of Calmodulin and Myosin Light Chain Kinase during Larval Settlement of the Barnacle Balanus amphitrite

    PubMed Central

    Chen, Zhang-Fan; Wang, Hao; Matsumura, Kiyotaka; Qian, Pei-Yuan

    2012-01-01

    Barnacles are one of the most common organisms in intertidal areas. Their life cycle includes seven free-swimming larval stages and sessile juvenile and adult stages. The transition from the swimming to the sessile stages, referred to as larval settlement, is crucial for their survivor success and subsequent population distribution. In this study, we focused on the involvement of calmodulin (CaM) and its binding proteins in the larval settlement of the barnacle, Balanus ( = Amphibalanus) amphitrite. The full length of CaM gene was cloned from stage II nauplii of B. amphitrite (referred to as Ba-CaM), encoding 149 amino acid residues that share a high similarity with published CaMs in other organisms. Quantitative real-time PCR showed that Ba-CaM was highly expressed in cyprids, the stage at which swimming larvae are competent to attach and undergo metamorphosis. In situ hybridization revealed that the expressed Ba-CaM gene was localized in compound eyes, posterior ganglion and cement glands, all of which may have essential functions during larval settlement. Larval settlement assays showed that both the CaM inhibitor compound 48/80 and the CaM-dependent myosin light chain kinase (MLCK) inhibitor ML-7 effectively blocked barnacle larval settlement, whereas Ca2+/CaM-dependent kinase II (CaMKII) inhibitors did not show any clear effects. The subsequent real-time PCR assay showed a higher expression level of Ba-MLCK gene in larval stages than in adults, suggesting an important role of Ba-MLCK gene in larval development and competency. Overall, the results suggest that CaM and CaM-dependent MLCK function during larval settlement of B. amphitrite. PMID:22348072

  11. Structure of the Single-lobe Myosin Light Chain C in Complex with the Light Chain-binding Domains of Myosin-1C Provides Insights into Divergent IQ Motif Recognition.

    PubMed

    Langelaan, David N; Liburd, Janine; Yang, Yidai; Miller, Emily; Chitayat, Seth; Crawley, Scott W; Côté, Graham P; Smith, Steven P

    2016-09-09

    Myosin light chains are key regulators of class 1 myosins and typically comprise two domains, with calmodulin being the archetypal example. They bind IQ motifs within the myosin neck region and amplify conformational changes in the motor domain. A single lobe light chain, myosin light chain C (MlcC), was recently identified and shown to specifically bind to two sequentially divergent IQ motifs of the Dictyostelium myosin-1C. To provide a molecular basis of this interaction, the structures of apo-MlcC and a 2:1 MlcC·myosin-1C neck complex were determined. The two non-functional EF-hand motifs of MlcC pack together to form a globular four-helix bundle that opens up to expose a central hydrophobic groove, which interacts with the N-terminal portion of the divergent IQ1 and IQ2 motifs. The N- and C-terminal regions of MlcC make critical contacts that contribute to its specific interactions with the myosin-1C divergent IQ motifs, which are contacts that deviate from the traditional mode of calmodulin-IQ recognition.

  12. Light Chain Deposition Disease Diagnosed with Laser Micro-dissection, Liquid Chromatography, and Tandem Mass Spectrometry of Nodular Glomerular Lesions

    PubMed Central

    Kasagi, Tomomichi; Nobata, Hironobu; Suzuki, Keisuke; Miura, Naoto; Banno, Shogo; Takami, Akiyoshi; Yamashita, Taro; Ando, Yukio; Imai, Hirokazu

    2017-01-01

    A 42-year-old man developed nephrotic syndrome and rapidly progressive renal failure. Kidney biopsy demonstrated nodular glomerulosclerosis, negative Congo red staining, and no deposition of light or heavy chains. Laser micro-dissection and liquid chromatography with tandem mass spectrometry of nodular lesions revealed the presence of a kappa chain constant region and kappa III variable region, which signified light chain deposition disease. Dexamethasone and thalidomide were effective in decreasing the serum levels of free kappa light chain from 147.0 to 38.0 mg/L, eliminating proteinuria, and halting the worsening of the kidney dysfunction, with serum creatinine levels stable around 4.0 mg/dL for 3 years. PMID:28050001

  13. Phylogeny, genomic organization and expression of lambda and kappa immunoglobulin light chain genes in a reptile, Anolis carolinensis.

    PubMed

    Wu, Qian; Wei, Zhiguo; Yang, Zhi; Wang, Tao; Ren, Liming; Hu, Xiaoxiang; Meng, Qingyong; Guo, Ying; Zhu, Qinghong; Robert, Jacques; Hammarström, Lennart; Li, Ning; Zhao, Yaofeng

    2010-05-01

    The reptiles are the last major taxon of jawed vertebrates in which immunoglobulin light chain isotypes have not been well characterized. Using the recently released genome sequencing data, we show in this study that the reptile Anolis carolinensis expresses both lambda and kappa light chain genes. The genomic organization of both gene loci is structurally similar to their respective counterparts in mammals. The identified lambda locus contains three constant region genes each preceded by a joining gene segment, and a total of 37 variable gene segments. In contrast, the kappa locus contains only a single constant region gene, and two joining gene segments with a single family of 14 variable gene segments located upstream. Analysis of junctions of the recombined VJ transcripts reveals a paucity of N and P nucleotides in both expressed lambda and kappa sequences. These results help us to understand the generation of the immunoglobulin repertoire in reptiles and immunoglobulin evolution in vertebrates.

  14. Differences in potential for amino acid change after mutation reveals distinct strategies for kappa and lambda light-chain variation.

    PubMed

    Hershberg, Uri; Shlomchik, Mark J

    2006-10-24

    B cells generate varied yet functional clones under high rates of mutation of their V genes. It has been proposed that as a result of the opposing demands of diversification and preservation of integrity, the V genes of heavy and light chains have evolved to overexpress codons prone to amino acid change in their complementarity determining regions (CDR) compared with the framework (FW) regions. We have analyzed the germ-line V genes of heavy and light chains (both kappa and lambda), comparing codons of CDR and FW of the germ-line V regions both to each other and to control regions. We found that in both germ-line heavy chains and lambda chains, CDR codons are prone to replacement mutations, whereas in the FW, the opposite is true. Furthermore, the difference between CDR and FW in heavy chains and lambda chains is based on codons that are prone to nonconservative changes of amino acid. In contrast, in germ-line kappa chains, the codons in both CDR and FW are more prone to replacement mutations. We also demonstrated that negative selection during immune responses is more sensitive to nonconservative amino acid substitutions than overall amino acid change, demonstrating the applicability of our analysis to real-time process of selection in the immune system. The differences in germ-line kappa and lambda light chains' potential reaction to mutation suggests that via these two differently evolved light-chain types, the B cell repertoire encompasses two different strategies to balance diversity and stability in an immune response.

  15. Papaverine Prevents Vasospasm by Regulation of Myosin Light Chain Phosphorylation and Actin Polymerization in Human Saphenous Vein

    PubMed Central

    Hocking, Kyle M.; Putumbaka, Gowthami; Wise, Eric S.; Cheung-Flynn, Joyce; Brophy, Colleen M.; Komalavilas, Padmini

    2016-01-01

    Objective Papaverine is used to prevent vasospasm in human saphenous veins (HSV) during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i) and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation. Approach and Results HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin. Conclusions These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm. PMID:27136356

  16. Systemic lupus erythematosus: molecular cloning of several recombinant DNase monoclonal kappa light chains with different catalytic properties.

    PubMed

    Botvinovskaya, Alina V; Kostrikina, Irina A; Buneva, Valentina N; Nevinsky, Georgy A

    2013-10-01

    An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of three patients with systemic lupus erythematosus was used. Phage particles displaying DNA binding light chains were isolated by affinity chromatography on DNA-cellulose, and the fraction eluted by an acidic buffer (pH 2.6) was used for preparation of individual monoclonal light chains (MLChs, 28 kDa). Thirty three of 687 individual colonies obtained were randomly chosen for study of MLCh DNase activity. Nineteen of 33 clones contained MLChs with DNase activity. Four preparations of MLChs were expressed in Escherichia coli in soluble form, purified by metal chelating chromatography followed by gel filtration, and studied in detail. Detection of DNase activity after SDS-PAGE in a gel containing DNA demonstrated that the four MLChs are not contaminated by canonical DNases. The MLChs demonstrated one or two pH optima. They were inactive after the dialysis against ethylenediaminetetraacetic acid but could be activated by several externally added metal ions; the ratio of relative activity in the presence of Mg(2+) , Mn(2+) , Ni(2+) , Ca(2+) , Zn(2+) , and Co(2+) was individual for each MLCh preparation. K(+) and Na(+) inhibited the DNase activity of various MLChs at different concentrations. Hydrolysis of DNA by all four MLCh was saturable and consistent with Michaelis-Menten kinetics. These clones are the first examples of recombinant MLChs possessing high affinity for DNA (Km  = 3-9 nM) and demonstrating high kcat values (3.4-6.9 min(-1) ). These observations suggest that the systemic lupus erythematosus light chain repertoire can serve as a source of new types of DNases.

  17. Primary hepatic amyloidosis: A case report and review of literature

    PubMed Central

    Sonthalia, Nikhil; Jain, Samit; Pawar, Sunil; Zanwar, Vinay; Surude, Ravindra; Rathi, Praveen M

    2016-01-01

    We describe a case of 42-year-old female presenting with abdominal pain associated with loss of weight and fever for 8 mo. On evaluation she had gross hepatomegaly with raised alkaline phosphatase and raised GGT levels with normal transaminases and bilirubin. On imaging she had diffuse enlargement of liver with heterogeneous contrast uptake in liver. Her viral marker and autoimmune markers were negative. Liver biopsy depicted massive deposition of amyloid in peri-sinusoidal spaces which revealed apple green birefringence on polarizing microscopy after Congo red staining. Cardiac and renal evaluation was unremarkable. Abdominal fat pad and rectum biopsy was negative for amyloid deposit. There was no evidence of primary amyloidosis as bone marrow examination was normal. Serum and urine immunofixation electrophoresis were normal. Immunoperoxidase staining for serum amyloid associated protein for secondary amyloidosis was negative from liver biopsy. We present this rare case of primary hepatic amyloidosis and review the literature regarding varied presentations of hepatic involvement in amyloidosis. PMID:26962400

  18. [Osteoarticular amyloidosis and dysglobulinemia: apropos of 2 cases].

    PubMed

    Morillon-Vié, M A; Petit, E; Galezowski, N; Rebischung, J L; Baviéra, E; Herreman, G

    1997-01-01

    We report two cases of bone and joint amyloidosis involvement related to plasma cell dyscrasia. The radiographic appearances mimic numerous benign or malignant diseases. MR imaging shows a diffuse low signal in T1 and an heterogeneous low or mild low signal in T2 weighted spin-echo sequence.

  19. A Case of Cardiac Amyloidosis Initially Misdiagnosed as Syndrome X

    PubMed Central

    Sohn, Hyung Rae; Song, Bong Gun; Jeong, Seong Yeon; Hong, Su-Min; Jung, Hyun Gul; Jung, Hye-Jin; Cho, Wook-Hyun; Choi, Suk-Koo

    2011-01-01

    Cardiac infiltration of amyloid fibril results in progressive cardiomyopathy with a grave prognosis and results in cardiac diseases such as congestive heart disease, cardiomyopathy, valvular heart disease, and arrhythmias. We present a rare case of cardiac amyloidosis initially misdiagnosed as syndrome X in which recurrent chest pain and progressive heart failure could be managed finally by heart transplantation.

  20. [New advances in the subtyping of systemic amyloidosis].

    PubMed

    Sun, Wei-Yi; Li, Jian

    2014-02-01

    Amyloidosis is a heterogeneous group of diseases caused by deposition of misfolded proteins, which usually leads to organ dysfunction. Accurate typing of amyloid deposits is of paramount importance because organ involvements and disease prognosis differ widely among different subtypes, and its treatments are type specific. Correct identification of amyloidogenic protein is crucial to proper treatment. Traditional antibody-based diagnostic methods such as immunohistochemistry and immunofluorescence are helpful in amyloid typing, but limitations of those approaches including antibody availability and serum protein contamination impair sensitivity and specificity of diagnosis. Sometimes misdiagnosis can lead to catastrophic therapeutic outcome. Genetic testing is important to confirm the diagnosis of hereditary amyloidosis. Nowadays proteomic analysis has been used as an advanced strategy for amyloid typing and the gold-standard today is laser microdissection followed by mass spectrometry (LMD/MS), which can identify causal protein without additional clinical information. Furthermore, LMD/MS is performed on formalin-fixed paraffin-embedded (FFPE) specimens, thus large scale retrospective studies based on archival material can be conducted. In recent studies, LMD/MS has been proven superior to traditional methods without the drawbacks mentioned above. This proteomic approach provides guarantee of appropriate clinical management and probability of new insights into the mechanism of amyloidosis.In this article the new advances of studies on subtyping of systemic amyloidosis are reviewed.

  1. Subunit Heterogeneity of Cytoplasmic Dynein: Differential Expression of 14 kDa Dynein Light Chains in Rat Hippocampus

    PubMed Central

    Chuang, Jen-Zen; Milner, Teresa A.; Sung, Ching-Hwa

    2013-01-01

    Cytoplasmic dynein is a multi-subunit protein complex in which each subunit is encoded by a few genes. How these subunit isoforms are assembled and regulated to mediate the diverse functions of cytoplasmic dynein is unknown. We previously have shown that two highly conserved 14 kDa dynein light chains, Tctex-1 and RP3, have different cargo-binding abilities. In this report, coimmunoprecipitation revealed that Tctex-1 and RP3 were present in mutually exclusive dynein complexes of brain. Two specific antibodies were used to examine the localization of these two dynein light chains in adult rat hippocampal formation and cerebral cortex. By light microscopy, Tctex-1 and RP3 immunoreactivities exhibited distinct and almost complementary distribution patterns in both brain regions. In hippocampal formation, Tctex-1 immunoreactivity was most enriched in somata of newly generated granule cells and scant in the mature granule and pyramidal cell somata. In contrast, RP3 immunoreactivity was abundant in pyramidal and granule cell somata. Ultrastructural analysis of the dentate gyrus revealed both dynein light chains were associated with various membranous organelles that often were affiliated with microtubules. In addition, Tctex-1 and RP3 immunoreactivities were preferentially and highly enriched on membranous organelles and/or vesicles of axon terminals and dendritic spines, respectively. These results suggest that dynein complexes with different subunit composition, and possibly function, are expressed differentially in a spatially and temporally regulated manner. Furthermore, Tctex-1 and RP3 may play important roles in synaptic functions. PMID:11466421

  2. Human lambda light-chain constant region gene CMor lambda: the primary structure of lambda VI Bence Jones protein Mor.

    PubMed Central

    Frangione, B; Moloshok, T; Prelli, F; Solomon, A

    1985-01-01

    Serologic, structural, and genetic analyses have shown that the constant (C) region of human kappa light chains is encoded by a single gene, whereas that of lambda chains is encoded by multiple genes. We have determined the complete C region amino acid sequence of two monoclonal lambda VI light chains, Bence Jones proteins Sut and Mor. The C region of lambda chains Sut and Mor consists of 105 residues, as is characteristic for human lambda light chains, of which 102 are identical in sequence. Protein Sut has the C region sequence associated with the C lambda isotype Mcg-, Kern-, Oz+ and represents a product of the C lambda 3 (Kern-, Oz+) gene. Protein Mor has a C region sequence associated with Mcg-, Kern-, and Oz- proteins but differs from protein Sut by the presence of three amino acid interchanges at positions 168, 176, and 194. These substitutions distinguish protein Mor from lambda chains encoded by the C lambda 1 (Mcg+), C lambda 2 (Kern-, Oz-), and C lambda 3 (Kern-, Oz+) genes and provide further evidence for polymorphism of the human C lambda genome. The gene encoding the C region sequence of lambda chain Mor is designated CMor lambda. PMID:3923477

  3. Detection of kappa and lambda light chain monoclonal proteins in human serum: automated immunoassay versus immunofixation electrophoresis.

    PubMed

    Jaskowski, Troy D; Litwin, Christine M; Hill, Harry R

    2006-02-01

    Recently, turbidimetric immunoassays for detecting and quantifying kappa and lambda free light chains (FLC) have become available and are promoted as being more sensitive than immunofixation electrophoresis (IFE) in detecting FLC monoclonal proteins. In this study, we assessed the ability of these turbidimetric assays to detect serum monoclonal proteins involving both free and heavy-chain-bound kappa and lambda light chains compared to standard immunofixation electrophoresis. Sera demonstrating a restricted band of protein migration (other than a definite M spike) by serum protein electrophoresis (SPE), which may represent early monoclonal proteins, were also examined. When compared to IFE, percent agreement, sensitivity, and specificity for the kappa-FLC and lambda-FLC were 94.6, 72.9, and 99.5% and 98.5, 91.4, and 99.7%, respectively, in detecting monoclonal proteins involving free and heavy-chain-bound light chains. The majority of sera (73.7%) demonstrating a restricted band of protein migration on SPE demonstrated abnormal IFE patterns suggestive of multiple myeloma or monoclonal gammopathy of unknown significance, but gave normal kappa/lambda FLC ratios using the turbidimetric immunoassays. In conclusion, the kappa and lambda FLC assays are significantly less sensitive (72.9 to 91.4%) than IFE, but specific in detecting serum monoclonal proteins. Moreover, the kappa/lambda ratio has little value in routine screening since the majority of sera with abnormal IFE patterns had normal kappa/lambda FLC ratios.

  4. [Ssp DnaB intein-mediated ligation of heavy and light chains of coagulation factor VIII in Escherichia coli].

    PubMed

    Zhu, Fuxiang; Liu, Zelong; Qu, Huige; Xin, Xiaolin; Dong, Hongxin; Liu, Xiangqin

    2009-07-01

    We studied the ligation of coagulation factor VIII heavy and light chains in Escherichia coli by utilizing the intein-mediated protein trans-splicing. A B-domain deleted factor VIII (BDD-FVIII) gene was broken into two halves of heavy and light chains before Ser1657 which meets the splicing required conserved residue and then fused to 106 and 48 amino acid-containing N-part termed Int-N and C-part termed Int-C coding sequences of split mini Ssp DnaB intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pBV220. Through induction for expression of recombinant protein it displayed an obvious protein band as predicted size of BDD-FVIII protein on SDS-PAGE gel. Western blotting using factor VIII specific antibodies confirmed that this protein band is BDD-FVIII produced by protein trans-splicing. It demonstrated that the heavy and light chains of BDD-FVIII can be efficiently ligated with the Ssp DnaB intein-mediated protein trans-splicing. These results provided evidence for encouraging our ongoing investigation with intein as a means in dual AAV vectors carrying the factor VIII gene to overcome the packaging size limitation of a single AAV vector in hemophilia A gene therapy.

  5. Cloning, high level expression, purification, and crystallization of the full length Clostridium botulinum neurotoxin type E light chain.

    PubMed

    Agarwal, Rakhi; Eswaramoorthy, Subramaniam; Kumaran, Desigan; Dunn, John J; Swaminathan, Subramanyam

    2004-03-01

    The catalytic activity of the highly potent botulinum neurotoxins are confined to their N-terminal light chains ( approximately 50kDa). A full-length light chain for the type E neurotoxin with a C-terminal 6x His-tag, BoNT/E-LC, has been cloned in a pET-9c vector and over-expressed in BL21 (DE3) cells. BoNT/E-LC was purified to homogeneity by affinity chromatography on Ni-NTA agarose followed by exclusion chromatography using a Superdex-75 sizing column. The purified protein has very good solubility and can be stored stably at -20 degrees C; however, it seems to undergo auto-proteolysis when stored at temperature #10878;4-10 degrees C. BoNT/E-LC is active on its natural substrate, the synaptosomal associated 25kDa protein, SNAP-25, indicating that it retains a native-like conformation and therefore can be considered as a useful tool in studying the structure/function of the catalytic light chain. Recombinant BoNT/E-LC has been crystallized under five different conditions and at various pHs. Crystals diffract to better than 2.1A.

  6. Age- and Activity-Related Differences in the Abundance of Myosin Essential and Regulatory Light Chains in Human Muscle

    PubMed Central

    Cobley, James N.; Ab. Malik, Zulezwan; Morton, James P.; Close, Graeme L.; Edwards, Ben J.; Burniston, Jatin G.

    2016-01-01

    Traditional methods for phenotyping skeletal muscle (e.g., immunohistochemistry) are labor-intensive and ill-suited to multixplex analysis, i.e., assays must be performed in a series. Addressing these concerns represents a largely unmet research need but more comprehensive parallel analysis of myofibrillar proteins could advance knowledge regarding age- and activity-dependent changes in human muscle. We report a label-free, semi-automated and time efficient LC-MS proteomic workflow for phenotyping the myofibrillar proteome. Application of this workflow in old and young as well as trained and untrained human skeletal muscle yielded several novel observations that were subsequently verified by multiple reaction monitoring (MRM). We report novel data demonstrating that human ageing is associated with lesser myosin light chain 1 content and greater myosin light chain 3 content, consistent with an age-related reduction in type II muscle fibers. We also disambiguate conflicting data regarding myosin regulatory light chain, revealing that age-related changes in this protein more closely reflect physical activity status than ageing per se. This finding reinforces the need to control for physical activity levels when investigating the natural process of ageing. Taken together, our data confirm and extend knowledge regarding age- and activity-related phenotypes. In addition, the MRM transitions described here provide a methodological platform that can be fine-tuned to suite multiple research needs and thus advance myofibrillar phenotyping. PMID:28248225

  7. Expression of heavy chain‐only antibodies can support B‐cell development in light chain knockout chickens

    PubMed Central

    Schusser, Benjamin; Collarini, Ellen J.; Pedersen, Darlene; Yi, Henry; Ching, Kathryn; Izquierdo, Shelley; Thoma, Theresa; Lettmann, Sarah; Kaspers, Bernd; Etches, Robert J.; van de Lavoir, Marie‐Cecile; Harriman, William

    2016-01-01

    Since the discovery of antibody‐producing B cells in chickens six decades ago, chickens have been a model for B‐cell development in gut‐associated lymphoid tissue species. Here we describe targeting of the immunoglobulin light chain locus by homologous recombination in chicken primordial germ cells (PGCs) and generation of VJCL knockout chickens. In contrast to immunoglobulin heavy chain knockout chickens, which completely lack mature B cells, homozygous light chain knockout (IgL−/−) chickens have a small population of B lineage cells that develop in the bursa and migrate to the periphery. This population of B cells expresses the immunoglobulin heavy chain molecule on the cell surface. Soluble heavy‐chain‐only IgM and IgY proteins of reduced molecular weight were detectable in plasma in 4‐week‐old IgL−/− chickens, and antigen‐specific IgM and IgY heavy chain proteins were produced in response to immunization. Circulating heavy‐chain‐only IgM showed a deletion of the CH1 domain of the constant region enabling the immunoglobulin heavy chain to be secreted in the absence of the light chain. Our data suggest that the heavy chain by itself is enough to support all the important steps in B‐cell development in a gut‐associated lymphoid tissue species. PMID:27392810

  8. Abnormal heavy/light chain ratio after treatment is associated with shorter survival in patients with IgA myeloma.

    PubMed

    Suehara, Yasuhito; Takamatsu, Hiroyuki; Fukumoto, Kota; Fujisawa, Manabu; Narita, Kentaro; Usui, Yoshiaki; Takeuchi, Masami; Endean, Kelly; Matsue, Kosei

    2017-02-01

    Immunoglobulin (Ig) heavy/light chain (HLC) assays enable the separate quantification of the different light chain types of each Ig class. We retrospectively analyzed the correlation of heavy/light chain ratio (HLCR) with clinical status and its impact on outcome in 120 patients with multiple myeloma (MM). Abnormal HLCR was seen more frequently in patients with poorer myeloma response, and it appeared to be more sensitive for detecting clonality in IgA myeloma compared to IgG myeloma after treatment. Among the 85 patients who achieved ≥VGPR, the patients remained HLCR abnormal were showed significantly shorter overall survival (OS) compared to those achieving a normal HLCR (not reached vs 55.5 months, P = 0.032). This correlation was seen in IgA myeloma patients (not reached vs 30.1 months, P = 0.014), but not in IgG myeloma patients when patients were analyzed separately. Univariate and multivariate analysis of factors that may affect survival identified abnormal HLCR at the best response as the only independent risk factor (hazard ratio, 4.7; 95% confidence interval, 1.4 - 15.26; P = 0.012) for shorter OS in this subset of patients. This study highlighted the HLC assay as a prognostic predictor in patients with IgA myeloma.

  9. Lambda light chain myeloma with co-migrating paraprotein at beta region on agarose gel electrophoresis: a case report.

    PubMed

    Siti Sarah, M; Nor Aini, U; Nurismah, M I; Hafiza, A; Khalidah, M; Mokhtar, A B; Das, S

    2014-01-01

    Paraproteinemia is one of the diagnostic features of multiple myeloma. A commonly used method is the detection of paraprotein by agarose gel electrophoresis (AGE) followed by by immunofixation electrophoresis (IFE) to confirm monoclonality. Due to their smaller size, immunoglobulin A (IgA) and light chain only paraproteins may appear at the beta or even alpha 2 protein fractions. Here, we discuss a case report of a 47-year-old man who presented with pathological fracture of third thoracic (T3) vertebra. Serum protein electrophoresis (SPE) was initially reported as no paraprotein detected. However, a bone biopsy was reported to show plasma cell proliferation with light chain restriction. A repeat sample for protein electrophoresis together with IFE revealed lambda light chain paraprotein co-migrating at the beta region. The beta band plus paraprotein was quantitated as 4.3 g/L (7.0%), which was within normal limits of the beta protein fraction. Hence, it has to be remembered that if the SPE is negative, it does not necessarily mean that the paraprotein is absent in cases which are highly suspicious.

  10. Serum immunoglobulin free light chain assessment in rheumatoid arthritis and primary Sjögren's syndrome

    PubMed Central

    Gottenberg, J‐E; Aucouturier, F; Goetz, J; Sordet, C; Jahn, I; Busson, M; Cayuela, J‐M; Sibilia, J; Mariette, X

    2007-01-01

    Background B cell activation may result in an increased secretion of immunoglobulin free light chains (FLCs) in autoimmune diseases. Objective To analyse serum FLC levels in patients with rheumatoid arthritis and in those with primary Sjögren's syndrome (pSS). Patients and methods Blood samples were collected from 80 healthy blood donors, 50 patients with rheumatoid arthritis and 139 patients with pSS. Serum FLC level was measured using a new quantitative immunoassay. Results Mean (standard error (SE)) serum κ and λ FLC levels were significantly higher in patients with rheumatoid arthritis and in those with pSS than in controls (κ : 18.9 (1.1) and 16.3 (1.4) v 10.5 (0.4) mg/l, p<0.001 and p = 0.001, respectively; λ: 16.7 (1.2) and 19.3 (1.5) v 11.6 (0.6) mg/l, p<0.001 for both). 18 (36%) patients with rheumatoid arthritis and 31 (22.3%) patients with pSS had abnormal serum FLC levels (increased κ or λ levels and abnormal ratio of κ:λ). Serum κ and λ levels were correlated with other B cell activation markers in both diseases. FLC levels increased with disease activity, because, unlike total gammaglobulin and immunoglobulin G levels, they were significantly correlated with Disease Activity Score 28 in patients with rheumatoid arthritis (p = 0.004 for κ, p = 0.05 for λ) and with extraglandular involvement in pSS (p = 0.01 for κ, p = 0.04 for λ). Conclusion FLC levels are increased and correlate with disease activity in patients with rheumatoid arthritis and in those with pSS, two diseases in which increased risk of lymphoma could result from persistent B cell activation and disease activity. Further studies are required to determine whether FLC assessment could represent a relevant biomarker for response to treatment (especially B cell depletion) and for the risk of lymphoma in autoimmune diseases. PMID:16569685

  11. Heart Failure Induced by Perinatal Ablation of Cardiac Myosin Light Chain Kinase.

    PubMed

    Islam, Yasmin F K; Joseph, Ryan; Chowdhury, Rajib R; Anderson, Robert H; Kasahara, Hideko

    2016-01-01

    Background: Germline knockout mice are invaluable in understanding the function of the targeted genes. Sometimes, however, unexpected phenotypes are encountered, due in part to the activation of compensatory mechanisms. Germline ablation of cardiac myosin light chain kinase (cMLCK) causes mild cardiac dysfunction with cardiomyocyte hypertrophy, whereas ablation in adult hearts results in acute heart failure with cardiomyocyte atrophy. We hypothesized that compensation after ablation of cMLCK is dependent on developmental staging and perinatal-onset of cMLCK ablation will result in more evident heart failure than germline ablation, but less profound when compared to adult-onset ablation. Methods and Results: The floxed-Mylk3 gene was ablated at the beginning of the perinatal stage using a single intra-peritoneal tamoxifen injection of 50 mg/kg into pregnant mice on the 19th day of gestation, this being the final day of gestation. The level of cMLCK protein level could no longer be detected 3 days after the injection, with these mice hereafter denoted as the perinatal Mylk3-KO. At postnatal day 19, shortly before weaning age, these mice showed reduced cardiac contractility with a fractional shortening 22.8 ± 1.0% (n = 7) as opposed to 31.4 ± 1.0% (n = 11) in controls. The ratio of the heart weight relative to body weight was significantly increased at 6.68 ± 0.28 mg/g (n = 12) relative to the two control groups, 5.90 ± 0.16 (flox/flox, n = 11) and 5.81 ± 0.33 (wild/wild/Cre, n = 5), accompanied by reduced body weight. Furthermore, their cardiomyocytes were elongated without thickening, with a long-axis of 101.8 ± 2.4 μm (n = 320) as opposed to 87.1 ± 1.6 μm (n = 360) in the controls. Conclusion: Perinatal ablation of cMLCK produces an increase of heart weight/body weight ratio, a reduction of contractility, and an increase in the expression of fetal genes. The perinatal Mylk3-KO cardiomyocytes were elongated in the absence of thickening, differing from the

  12. Serum neurofilament light chain protein is a measure of disease intensity in frontotemporal dementia

    PubMed Central

    Woollacott, Ione O.C.; Dick, Katrina M.; Brotherhood, Emilie; Gordon, Elizabeth; Fellows, Alexander; Toombs, Jamie; Druyeh, Ronald; Cardoso, M. Jorge; Ourselin, Sebastien; Nicholas, Jennifer M.; Norgren, Niklas; Mead, Simon; Andreasson, Ulf; Blennow, Kaj; Schott, Jonathan M.; Fox, Nick C.; Warren, Jason D.; Zetterberg, Henrik

    2016-01-01

    Objective: To investigate serum neurofilament light chain (NfL) concentrations in frontotemporal dementia (FTD) and to see whether they are associated with the severity of disease. Methods: Serum samples were collected from 74 participants (34 with behavioral variant FTD [bvFTD], 3 with FTD and motor neuron disease and 37 with primary progressive aphasia [PPA]) and 28 healthy controls. Twenty-four of the FTD participants carried a pathogenic mutation in C9orf72 (9), microtubule-associated protein tau (MAPT; 11), or progranulin (GRN; 4). Serum NfL concentrations were determined with the NF-Light kit transferred onto the single-molecule array platform and compared between FTD and healthy controls and between the FTD clinical and genetic subtypes. We also assessed the relationship between NfL concentrations and measures of cognition and brain volume. Results: Serum NfL concentrations were higher in patients with FTD overall (mean 77.9 pg/mL [SD 51.3 pg/mL]) than controls (19.6 pg/mL [SD 8.2 pg/mL]; p < 0.001). Concentrations were also significantly higher in bvFTD (57.8 pg/mL [SD 33.1 pg/mL]) and both the semantic and nonfluent variants of PPA (95.9 and 82.5 pg/mL [SD 33.0 and 33.8 pg/mL], respectively) compared with controls and in semantic variant PPA compared with logopenic variant PPA. Concentrations were significantly higher than controls in both the C9orf72 and MAPT subgroups (79.2 and 40.5 pg/mL [SD 48.2 and 20.9 pg/mL], respectively) with a trend to a higher level in the GRN subgroup (138.5 pg/mL [SD 103.3 pg/mL). However, there was variability within all groups. Serum concentrations correlated particularly with frontal lobe atrophy rate (r = 0.53, p = 0.003). Conclusions: Increased serum NfL concentrations are seen in FTD but show wide variability within each clinical and genetic group. Higher concentrations may reflect the intensity of the disease in FTD and are associated with more rapid atrophy of the frontal lobes. PMID:27581216

  13. [Seasonal changes in phosphorylation of myosin regulatory light chains and C-protein in myocardium of hibernating ground squirrel Citellus undulatus].

    PubMed

    Malyshev, S L; Osipova, D A; Vikhliantsev, I M; Podlubnaia, Z A

    2006-01-01

    A comparative study concerning the extent of phosphorylation of myosin regulatory light chains and C-protein from the left ventricle of hibernating ground squirrel Citellus undulatus during the periods of hibernation and activity was carried out. During hibernation, regulatory light chains of ground squirrel were found to be completely dephosphorylated. In active animals, the share of phosphorylated light chains averages 40-45% of their total amount. The extent of phosphorylation of the cardiac C-protein during hibernation is about two times higher than that in the active state. Seasonal differences in phosphorylation of the two proteins of ground squirrel myocardium are discussed in the context of adaptation to hibernation.

  14. [Effective dimethyl sulfoxide (DMSO) occlusive dressing technique for amyloidosis of the urinary bladder].

    PubMed

    Hasegawa, Yoshihiro; Kanda, Hideki; Miki, Manabu; Masui, Satoru; Yoshio, Yuko; Yamada, Yasushi; Soga, Norihito; Arima, Kiminobu; Sugimura, Yoshiki

    2013-10-01

    A 48-year-old married woman complaining of macroscopic hematuria and cystitis symptom was admitted to our institute. Flexible cystoscopy revealed many yellowish, nodular masses at the paries posterior of the urinary bladder, and cold-punch biopsy proved it to be amyloidosis. Serum amyloid protein A (SAA) was high, and suggested systemic amyloidosis. Renal biopsy and colon fiberscopy did not reveal any abnormalities. We therefore diagnosed a primary localized amyloidosis of the urinary bladder. Transurethral resection and dimethyl sulfoxide (DMSO) infusion therapy are used to treat amyloidosis of the urinary bladder. However there is no definite cure for amyloidosis of the urinary bladder. Therefore we selected DMSO occlusive dressing technique therapy. After 5 years of therapy, there was no evidence of a recurrence of amyloidosis.

  15. Fecal transmission of AA amyloidosis in the cheetah contributes to high incidence of disease.

    PubMed

    Zhang, Beiru; Une, Yumi; Fu, Xiaoying; Yan, Jingmin; Ge, FengXia; Yao, Junjie; Sawashita, Jinko; Mori, Masayuki; Tomozawa, Hiroshi; Kametani, Fuyuki; Higuchi, Keiichi

    2008-05-20

    AA amyloidosis is one of the principal causes of morbidity and mortality in captive cheetahs (Acinonyx jubatus), which are in danger of extinction, but little is known about the underlying mechanisms. Given the transmissible characteristics of AA amyloidosis, transmission between captive cheetahs may be a possible mechanism involved in the high incidence of AA amyloidosis. In this study of animals with AA amyloidosis, we found that cheetah feces contained AA amyloid fibrils that were different from those of the liver with regard to molecular weight and shape and had greater transmissibility. The infectious activity of fecal AA amyloid fibrils was reduced or abolished by the protein denaturants 6 M guanidine.HCl and formic acid or by AA immunodepletion. Thus, we propose that feces are a vehicle of transmission that may accelerate AA amyloidosis in captive cheetah populations. These results provide a pathogenesis for AA amyloidosis and suggest possible measures for rescuing cheetahs from extinction.

  16. Prognostic Value of Serum Free Light Chains Measurements in Multiple Myeloma Patients

    PubMed Central

    Bermudo Guitarte, Carmen; Menéndez Valladares, Paloma; Rojas Noboa, Johanna Carolina; Kestler, Krysta; Duro Millán, Rafael

    2016-01-01

    Background The outcome for patients with Multiple Myeloma (MM) is highly variable, therefore, the existence of robust and easy to determine prognostic markers is extremely important for an efficient management of these patients. Presently, there is a debate about the role of the serum free light chains (sFLC) in the prognosis of MM patients both at diagnosis and after treatment. The aim of this study is to evaluate in a cohort of newly diagnosed MM patients from the Southern area of Spain, the prognostic value of sFLC both at baseline and after treatment. Materials and Methods 180 patients with a median age of 69 years were followed-up for a median time of 35 (18–61) months. The sFLC ratio (sFLCR) was calculated using the monoclonal sFLC as numerator. Patients were divided in two groups according to a sFLCR cut-off based on ROC analysis. The primary endpoints were the Overall Survival (OS) and the Progression-free Survival (PFS). Additionally, thirty-six MM patients treated with novel agents (Bortezomib/Dexamethasone) that achieved Complete Response (CR) or stringent CR (sCR) before autologous stem cell transplantation were studied to assess the impact of sCR in Disease Free Survival (DFS) and OS. Results During follow-up there were 72 disease-related deaths. The 5-years OS for the whole group was 51%. However, separate analysis of patients with sFLCR above (group “high”) or below (groups “low”) the cut-off value of 47 shows an OS of 23% and 73%, respectively (HR = 5.03, 95%CI 2.99–8.50, p<0.001). In addition, analysis by ISS stage, showed that the presence of high sFLCR was always significantly associated with a worse OS. Multivariate analysis identified sFLCR (HR = 4.42, 95%CI 2.57–7.60, p<0.001) and beta-2-microglobulin (B2M) (HR = 3.04, 95%IC 1.75–5.31, p<0.001) as independent risk factors for adverse outcome. A new risk stratification model based on sFLCR≥47 and B2M>3.5 mg/L provided a statistically more significant result for this cohort

  17. [A case of lambda-expressing pulmonary MALT lymphoma with dual clonal rearrangements of kappa and lambda immunoglobulin light chain gene].

    PubMed

    Oh, Hye Ryong; Lee, Mi Ja; Park, Geon; Moon, Dae Soo; Park, Young Jin; Jang, Sook Jin

    2009-06-01

    A 70-yr-old woman was hospitalized with a history of dry cough. Bronchial endoscopy and transbronchial lung biopsy were performed. However, the findings of histopathology and immunohistochemistry were not sufficient to decide whether the lesion was benign or malignant, because of the presence of crush artifacts in the biopsy specimens. We performed B-cell clonality studies using BIOMED-2 multiplex PCR (InVivoScribe Technologies, USA) to detect clonal rearrangements in the immunoglobulin gene. The results of multiplex PCR showed clonal rearrangements of both kappa and lambda immunoglobulin light chain genes. The findings of immunochemistry revealed that the lesion expressed lambda light chain, but not kappa light chain. Based on the clinical, pathologic, and molecular findings, this case was diagnosed as pulmonary MALT lymphoma. We report the first case in Korea of lambda-expressing MALT lymphoma that is shown to have dual clonal rearrangements of kappa and lambda immunoglobulin light chain gene by multiplex PCR.

  18. Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab.

    PubMed

    Spooner, Jennifer; Keen, Jenny; Nayyar, Kalpana; Birkett, Neil; Bond, Nicholas; Bannister, David; Tigue, Natalie; Higazi, Daniel; Kemp, Benjamin; Vaughan, Tristan; Kippen, Alistair; Buchanan, Andrew

    2015-07-01

    Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab.

  19. Macular posterior pigmentary incontinence: its relation to macular amyloidosis and notalgia paresthetica.

    PubMed

    Westermark, P; Ridderström, E; Vahlquist, A

    1996-07-01

    Patients with clinical features of dorsal macular amyloidosis but without subepidermal amyloid deposits were followed for 2-11 years. The clinical appearance was fairly stable during this period of time, with little tendency of healing. Only 2 of the patients developed typical macular amyloidosis during the follow-up. It is concluded that a condition strongly resembling macular amyloidosis but without amyloid is an entity, and the designation "macular posterior pigmentary incontinence" is proposed. The relationship between macular posterior pigmentary incontinence and the two conditions macular amyloidosis and notalgia paresthetica is discussed.

  20. Anakinra induces complete remission of nephrotic syndrome in a patient with familial mediterranean fever and amyloidosis.

    PubMed

    Sevillano, Ángel M; Hernandez, Eduardo; Gonzalez, Esther; Mateo, Isabel; Gutierrez, Eduardo; Morales, Enrique; Praga, Manuel

    2016-01-01

    Renal amyloidosis is one of the most severe complications of familial Mediterranean fever (FMF). Colchicine has reduced the incidence of this complication, which now only appears in untreated, under-treated and resistant patients, but it is usually ineffective in patients with advanced amyloidosis. Here we report a patient with FMF and biopsy-proven amyloidosis who presented with nephrotic syndrome despite colchicine treatment. Anakinra (an interleukin-1β inhibitor) was started and a dramatic complete remission of nephrotic syndrome was observed in the following months. Anakinra can be an effective treatment for FMF patients with severe secondary amyloidosis.

  1. AA amyloidosis in the renal allograft: a report of two cases and review of the literature

    PubMed Central

    Rojas, Rebecca; Josephson, Michelle A.; Chang, Anthony; Meehan, Shane M.

    2012-01-01

    AA amyloidosis is a disorder characterized by the abnormal formation, accumulation and systemic deposition of fibrillary material that frequently involves the kidney. Recurrent AA amyloidosis in the renal allograft has been documented in patients with tuberculosis, familial Mediterranean fever, ankylosing spondylitis, chronic pyelonephritis and rheumatoid arthritis. De novo AA amyloidosis is rarely described. We report two cases of AA amyloidosis in the renal allograft. Our first case is a 47-year-old male with a history of ankylosing spondylitis who developed end-stage renal disease reportedly from tubulointerstitial nephritis from non-steroidal anti-inflammatory agent use. A biopsy was never performed. One year after transplantation, AA amyloidosis was identified in the femoral head and 8 years post-transplantation, AA amyloidosis was identified in the renal allograft. He was treated with colchicine and adalimumab and has stable renal function at 1 year-follow-up. Our second case is a 57-year-old male with a long history of intravenous drug use and hepatitis C infection who developed end-stage kidney disease due to AA amyloidosis. Our second patient's course was complicated by renal adenovirus, pulmonary aspergillosis and hepatitis C with AA amyloidosis subsequently being identified in the allograft 2.5 years post-transplantation. Renal allograft function remains stable 4-years post-transplantation. These reports describe clinical and pathologic features of two cases of AA amyloidosis presenting with proteinuria and focal involvement of the renal allograft. PMID:22833808

  2. Pregnancy outcome of five patients with renal amyloidosis regarding familial Mediterranean fever.

    PubMed

    Turgal, Mert; Selcuk, Ilker; Ozyuncu, Ozgur

    2014-03-01

    Familial Mediterranean fever (FMF) is an autosomal recessive disease affecting mainly patients of the Mediterranean basin and its major complication is the development of renal AA amyloidosis. On the other hand pregnancy with amyloidosis is not common; nevertheless, amyloidosis will complicate pregnancies also with the underlying disease and may cause terrible perinatal morbidities and mortalities. We report here the cases of five pregnant women and their pregnancy outcomes, who have been diagnosed with FMF complicated by renal amyloidosis. In the five cases, we observed that increased pregnancy complication such as small for gestational age, intrauterine growth restriction, preeclampsia and preterm birth.

  3. [Ulcerative colitis and amyloidosis. Presentation of a case and review of the literature].

    PubMed

    Triviño, A; Sánchez Lombraña, J L; Linares, A; Pérez, R; Herrero Zapatero, A; Rodrigo, L

    1992-08-01

    A case of ulcerative colitis associated with secondary amyloidosis in a 62-year-old man who died from septic shock and pneumonia complicating head injury is reported. Amyloid deposition was incidentally found at autopsy. Proteinuria and hepatomegaly discovered a few days before his death were the only signs of amyloidosis. The postmortem examination showed chronic ulcerative colitis (remitting form) with pseudo-polyps and amyloid deposition in the liver, spleen, pancreas, rectum, adrenals and kidneys. Although secondary amyloidosis complicating with Crohn's disease has been frequently reported, amyloidosis associated with ulcerative colitis has been exceptionally described and only 10 cases have been collected from the literature.

  4. Homology of the NH2-terminal amino acid sequences of the heavy and light chains of human monoclonal lupus autoantibodies containing the dominant 16/6 idiotype.

    PubMed Central

    Atkinson, P M; Lampman, G W; Furie, B C; Naparstek, Y; Schwartz, R S; Stollar, B D; Furie, B

    1985-01-01

    The NH2-terminal amino acid sequences have been determined by automated Edman degradation for the heavy and light chains of five monoclonal IgM anti-DNA autoantibodies that were produced by human-human hybridomas derived from lymphocytes of two patients with systemic lupus erythematosus. Four of the antibodies were closely related to the idiotype system 16/6, whereas the fifth antibody was unrelated idiotypically. The light chains of the 16/6 idiotype-positive autoantibodies (HF2-1/13b, HF2-1/17, HF2-18/2, and HF3-16/6) had identical amino acid sequences from residues 1 to 40. Their framework structures were characteristic of VKI light chains. The light chain of the 16/6 idiotype-negative autoantibody HF6-21/28 was characteristic of the VKII subgroup. The heavy chains of the 16/6 idiotype-positive autoantibodies had nearly identical amino acid sequences from residues 1 to 40. The framework structures were characteristic of the VHIII subgroup. In contrast, the GM4672 fusion partner of the hybridoma produced small quantities of an IgG with a VHI heavy chain and a VKI light chain. The heavy chains of the lupus autoantibodies and the light chains of those autoantibodies that were idiotypically related to the 16/6 system had marked sequence homology with WEA, a Waldenstrom IgM that binds to Klebsiella polysaccharides and expresses the 16/6 idiotype. These results indicate a striking homology in the amino termini of the heavy and light chains of the lupus autoantibodies studied and suggest that the V regions of the heavy and light chains of the 16/6 idiotype-positive DNA-binding lupus auto-antibodies are each encoded by a single germ line gene. PMID:3921567

  5. Circulating advanced glycation peptides in streptozotocin-induced diabetic rats: evidence for preferential modification of IgG light chains.

    PubMed

    Gugliucci, A; Menini, T

    1998-01-01

    As the glycation/glycoxidation hypothesis for the genesis of diabetic complications is achieving widespread acceptance, much attention is being paid to the role of low molecular weight advanced glycation (AGE) adducts, as second generation glycating agents. We set out a study with the objective of attesting the presence of increased amounts of AGE-peptides in the circulation of streptozotocin-induced diabetic rats and to determine the nature of the plasma proteins which are main targets for advanced glycation. AGE (Ex 370/Em 440 nm) and pentosidine fluorescence (Ex 335/Em 385 nm) were significantly higher in plasma from diabetic rats after only one month of hyperglycemia as compared to controls (35 +/- 7 vs 25 +/- 2 AU, p< 0.05 and 54 +/- 14 vs 27 +/- 3 AU, p< 0.01 respectively). AGE-peptides (<10 kDa) were more than two-fold higher in diabetic animals. Immunoblots after SDS-PAGE of plasma proteins showed that AGE-IgG displayed a selective predominant increment in the same animals. When native rat IgG was incubated in the presence of AGE-peptides isolated from diabetic animals, AGE modification was already apparent after only 24 h of incubation, and was particularly important for light chains. AGE-immunoreactive light chains displayed an apparent increase in molecular weight. Aminoguanidine prevented, while copper enhanced AGE binding to IgG light chains. Our data validate the streptozotocin-induced diabetic rat as a model reproducing the presence of circulating AGE-peptides, give evidence that IgG are preferential targets for advanced glycation in plasma and suggest that this modification, mediated by AGE-peptides, can be prevented by aminoguanidine.

  6. FhCaBP3: a Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains.

    PubMed

    Banford, Samantha; Drysdale, Orla; Hoey, Elizabeth M; Trudgett, Alan; Timson, David J

    2013-04-01

    A DNA sequence encoding a protein with predicted EF-hand and dynein light chain binding domains was identified in a Fasciola hepatica EST library. Sequence analysis of the encoded protein revealed that the most similar known protein was the Fasciola gigantica protein FgCaBP3 and so this newly identified protein was named FhCaBP3. Molecular modelling of FhCaBP3 predicted a highly flexible N-terminal region, followed by a domain containing two EF-hand motifs the second of which is likely to be a functioning divalent ion binding site. The C-terminal domain of the protein contains a dynein light chain like region. Interestingly, molecular modelling predicts that calcium ion binding to the N-terminal domain destabilises the β-sheet structure of the C-terminal domain. FhCaBP3 can be expressed in, and purified from, Escherichia coli. The recombinant protein dimerises and the absence of calcium ions appeared to promote dimerisation. Native gel shift assays demonstrated that the protein bound to calcium and manganese ions, but not to magnesium, barium, zinc, strontium, nickel, copper or cadmium ions. FhCaBP3 interacted with the calmodulin antagonists trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine as well as the myosin regulatory light chain-binding drug praziquantel. Despite sequence and structural similarities to other members of the same protein family from F. hepatica, FhCaBP3 has different biochemical properties to the other well characterised family members, FH22 and FhCaBP4. This suggests that each member of this trematode calcium-binding family has discrete functional roles within the organism.

  7. Guideline of transthyretin-related hereditary amyloidosis for clinicians

    PubMed Central

    2013-01-01

    Transthyretin amyloidosis is a progressive and eventually fatal disease primarily characterized by sensory, motor, and autonomic neuropathy and/or cardiomyopathy. Given its phenotypic unpredictability and variability, transthyretin amyloidosis can be difficult to recognize and manage. Misdiagnosis is common, and patients may wait several years before accurate diagnosis, risking additional significant irreversible deterioration. This article aims to help physicians better understand transthyretin amyloidosis—and, specifically, familial amyloidotic polyneuropathy—so they can recognize and manage the disease more easily and discuss it with their patients. We provide guidance on making a definitive diagnosis, explain methods for disease staging and evaluation of disease progression, and discuss symptom mitigation and treatment strategies, including liver transplant and several pharmacotherapies that have shown promise in clinical trials. PMID:23425518

  8. Senile cardiac amyloidosis: an underappreciated cause of heart failure

    PubMed Central

    Shah, Shreena; Dungu, Jason; Dubrey, Simon William

    2013-01-01

    This case presents a patient with biopsy-proven, wild-type transthyretin (TTR) senile amyloidosis. The case was that of a man in his early 70s who presented with gradually progressive symptoms and signs of heart failure. The recent history included an episode of severe pancreatitis secondary to cholelithiasis and subsequently (and incidentally) noted hepatomegaly and marked ascites. Further evaluation of the aetiology of the heart failure, through echocardiography, coronary angiography and endomyocardial biopsy, led to an exact diagnosis of SSA. The patient is being treated with conventional heart failure medications while consideration is given to the use of diflusinal as an antiamyloidogenic small molecular stabiliser of TTR. Monitoring and further management advice are being coordinated by the National Amyloidosis Centre. PMID:23391947

  9. [Hereditary cerebral hemorrhage. Dementia with cystatin C amyloidosis].

    PubMed

    Blöndal, H; Guomundsson, G; Benedikz, E; Jóhannesson, G

    1990-01-01

    Nineteen cases of hereditary cystatin C amyloidosis with cerebral haemorrhage are described. The first haemorrhage occurred between the ages of 20 and 41 years and the period of survival varied from 10 days to 23 years after the first insult. Progressive dementia was a striking clinical symptom in 17 of the patients and in two cases dementia was the first sign. At the last examination severe dementia and pronounced pathological EEG were established in the majority of the patients. Infiltration of amyloid substance positive for anti-cystatin C was found in the proximity of the blood vessels and in their walls. Lesions in the cerebral microvascular system together with haemorrhages and infarcts caused thereby were considered to be an adequate explanation of the dementia in these patients. In view of the discovery of amyloid discharges in tissues outside the CNS it is adjudged more correct to use the name Hereditary Cystatin C Amyloidosis (HCCA).

  10. Cardiac amyloidosis imaged by dual-source computed tomography.

    PubMed

    Marwan, Mohamed; Pflederer, Tobias; Ropers, Dieter; Schmid, Michael; Wasmeier, Gerald; Söder, Stephan; Daniel, Werner G; Achenbach, Stephan

    2008-11-01

    The ability of contrast-enhanced CT to detect "late enhancement" in a fashion similar to magnetic resonance imaging has been reported previously. Typical myocardial distribution patterns of "late enhancement" have been described for MRI. The same patterns can be observed in CT imaging, albeit at a lower signal to noise ratio. We report a case of cardiac amyloidosis with a typical pattern of subendocardial, circumferential late enhancement in all four cardiac chambers.

  11. Amyloid fibril proteins and amyloidosis: chemical identification and clinical classification International Society of Amyloidosis 2016 Nomenclature Guidelines.

    PubMed

    Sipe, Jean D; Benson, Merrill D; Buxbaum, Joel N; Ikeda, Shu-Ichi; Merlini, Giampaolo; Saraiva, Maria J M; Westermark, Per

    2016-12-01

    The Nomenclature Committee of the International Society of Amyloidosis (ISA) met during the XVth Symposium of the Society, 3 July-7 July 2016, Uppsala, Sweden, to assess and formulate recommendations for nomenclature for amyloid fibril proteins and the clinical classification of the amyloidoses. An amyloid fibril must exhibit affinity for Congo red and with green, yellow or orange birefringence when the Congo red-stained deposits are viewed with polarized light. While congophilia and birefringence remain the gold standard for demonstration of amyloid deposits, new staining and imaging techniques are proving useful. To be included in the nomenclature list, in addition to congophilia and birefringence, the chemical identity of the protein must be unambiguously characterized by protein sequence analysis when possible. In general, it is insufficient to identify a mutation in the gene of a candidate amyloid protein without confirming the variant changes in the amyloid fibril protein. Each distinct form of amyloidosis is uniquely characterized by the chemical identity of the amyloid fibril protein that deposits in the extracellular spaces of tissues and organs and gives rise to the disease syndrome. The fibril proteins are designated as protein A followed by a suffix that is an abbreviation of the parent or precursor protein name. To date, there are 36 known extracellular fibril proteins in humans, 2 of which are iatrogenic in nature and 9 of which have also been identified in animals. Two newly recognized fibril proteins, AApoCII derived from apolipoprotein CII and AApoCIII derived from apolipoprotein CIII, have been added. AApoCII amyloidosis and AApoCIII amyloidosis are hereditary systemic amyloidoses. Intracellular protein inclusions displaying some of the properties of amyloid, "intracellular amyloid" have been reported. Two proteins which were previously characterized as intracellular inclusions, tau and α-synuclein, are now recognized to form extracellular

  12. Selection of RNA Aptamers Against Botulinum Neurotoxin Type A Light Chain Through a Non-Radioactive Approach.

    PubMed

    Chang, Tzuu-Wang; Janardhanan, Pavithra; Mello, Charlene M; Singh, Bal Ram; Cai, Shuowei

    2016-09-01

    Botulinum neurotoxin (BoNT), a category A agent, is the most toxic molecule known to mankind. The endopeptidase activity of light chain domain of BoNT is the cause for the inhibition of the neurotransmitter release and the flaccid paralysis that leads to lethality in botulism. Currently, antidotes are not available to reverse the flaccid paralysis caused by BoNT. In the present study, a non-radioactive-based systematic evolution of ligands by exponential enrichment (SELEX) process is developed by utilizing surface plasmon resonance to monitor the binding enrichment. Two RNA aptamers have been identified as strong binders against light chain of botulinum neurotoxin type A. These two aptamers showed strong inhibition activity on LCA, with IC50 in nanomolar range. Inhibition kinetic studies reveal mid nanomolar KI and non-competitive nature of their inhibition, suggesting that they have strong potential as antidotes that can reverse the symptom caused by BoNT/A. More importantly, we observed that the 2'-fluorine-pyrimidine-modified RNA aptamers identified here do not change their binding and biological activities. This observation could lead to a cost-effective way for SELEX, by using regular nucleotide during SELEX, and 2'-fluorine-pyrimidine-modified nucleotide for final application to enhance their RNase-resistance.

  13. Histopathological analysis of B-cell non-Hodgkin lymphomas without light chain restriction by using flow cytometry.

    PubMed

    Ohmoto, Akihiro; Maeshima, Akiko Miyagi; Taniguchi, Hirokazu; Tanioka, Kensaku; Makita, Shinichi; Kitahara, Hideaki; Fukuhara, Suguru; Munakata, Wataru; Suzuki, Tatsuya; Maruyama, Dai; Kobayashi, Yukio; Tobinai, Kensei

    2015-01-01

    Detection of immunoglobulin light chain restriction (LCR) by flow cytometry (FCM) is a useful tool for B-cell non-Hodgkin lymphoma (B-NHL) diagnosis. Here, we identified B-NHLs without LCR by FCM and investigated the pathological causes for lack of LCR. A total of 89/471 cases (19%) of B-NHL were LCR-negative. The incidence of lack of LCR was 30% both in diffuse large B-cell lymphoma (DLBCL) and marginal zone lymphoma (MZL), and was 6% in follicular lymphoma (FL). In DLBCL cases, low expression of surface membrane light chain (33%), low proportion of lymphoma cells (11%), CD45 negativity (9%), and destruction or sampling error were suggested as reasons for lack of LCR. In MZL cases, the low proportion of lymphoma cells owing to admixture of many reactive germinal centres, and non-detection of plasmacytoid lymphoma cells by CD45 gating might be the reasons. Based on pathological subtypes, the frequency and reasons for lack of LCR by FCM varied.

  14. Surrogate light chain expression beyond the pre-B cell stage promotes tolerance in a dose-dependent fashion.

    PubMed

    Kil, Laurens P; Corneth, Odilia B J; de Bruijn, Marjolein J W; Asmawidjaja, Patrick S; Krause, Arndt; Lubberts, Erik; van Loo, Pieter Fokko; Hendriks, Rudi W

    2015-02-01

    While surrogate light chain (SLC) expression is normally terminated in differentiating pre-B cells, co-expression of SLC and conventional light chains has been reported in a small population of autoreactive peripheral human B cells that accumulate in arthritic joints. Despite this association with autoimmunity the contribution of SLC expressing mature B cells to disease development is still unknown. We studied the pathogenicity of SLC(+) B cells in a panel of mice that transgenically express the SLC components VpreB and λ5 throughout B cell development. Here we report that although VpreB or λ5 expression mildly activated mature B cells, only moderate VpreB expression levels - in the absence of λ5 - enhanced IgG plasma cell formation. However, no autoantibody production was detectable in VpreB or λ5 transgenic mice and VpreB expression could not accelerate autoimmunity. Instead, moderate VpreB expression partially protected mice from induced autoimmune arthritis. In support of a tolerogenic role of SLC-transgenic B cells, we observed that in a dose-dependent manner SLC expression beyond the pre-B cell stage enhanced clonal deletion among immature and transitional B cells and rendered mature B cells anergic. These findings suggest that SLC expression does not propagate autoimmunity, but instead may impose tolerance.

  15. Immunogenicity and antigenicity of immunoglobulins. XII. Intact light chain and heavy chain isotype-restricted Vk-associated epitopes.

    PubMed

    Walker, M; Hardie, D; Lowe, J; Ling, N R; De Lange, G; Jefferis, R

    1985-06-01

    Immunization with intact IgG has allowed the isolation of four hybridomas producing antibodies recognizing epitopes expressed within subpopulations of human kappa light chains unrelated to known polymorphisms (Km) and previously defined V-region subgroups. The V-region-associated epitopes recognized are conformation-dependent, being expressed on intact light chain but not on isolated VK or CK fragments. The frequency of expression within paraprotein panels of different heavy chain isotypes varied between individual antibodies. An epitope recognized by B2A6, expressed by greater than 85% IgGK paraproteins, was not represented in 16 IgM paraproteins tested, suggesting that association of VK with mu chains does not result in display of the epitope recognized, or alternatively, that selective association between VK and CH gene products occurs. These data contrast with the reactivity of other McAb for CK epitopes which were reactive with isolated CK fragments, and for all kappa-bearing paraproteins, regardless of heavy chain isotypes.

  16. The identification of a nonclassical cadherin expressed during B cell development and its interaction with surrogate light chain.

    PubMed

    Ohnishi, K; Shimizu, T; Karasuyama, H; Melchers, F

    2000-10-06

    A 130-kDa glycoprotein (p130) has been found to be associated with surrogate light chain on pro- and pre-B I cells. Using peptide sequences obtained from purified p130 we have cloned its gene. The gene encodes a typical cadherin type 1 membrane protein with six extracellular cadherin domains (one pseudo domain) but lacking the catenin-binding site in its cytoplasmic part. Even without this catenin-binding site, p130 mediates Ca(2+)-dependent homotypic adhesion of cells. The interaction of p130 with surrogate light chain is confirmed by co-transfection and co-immunoprecipitation experiments. The expression of p130 is biphasic during the B cell development. Reverse transcriptase-polymerase chain reaction and flow cytometric analyses revealed that it is expressed on B220(+)c-Kit(+) pro-B and pre-B-I cells as well as on B220(+)CD25(-)IgM(+) immature and mature B cells but not on B220(+)CD25(+) pre-B-II cells. It is also expressed in fetal liver, at low levels in myeloid cells, and strongly in intestinal epithelial cells. In the spleen, p130-expressing cells are mainly localized in the marginal zone. We call this B lineage-, intestine-, liver- and leukocyte-expressed gene BILL-cadherin. The possible functions of BILL-cadherin in B cell development are discussed.

  17. Surrogate or conventional light chains are required for membrane immunoglobulin mu to activate the precursor B cell transition [published erratum appears in J Exp Med 1997 Jan 6;185(1):183

    PubMed Central

    1996-01-01

    To examine the role of light chains in early B cell development we combined RAG-1 and lambda 5 mutations to produce mice that expressed neither conventional nor surrogate light chains (RAG-1-/-, lambda 5-/- ). Unique heavy and light chain genes were then introduced into the double and single mutant backgrounds. Membrane immunoglobulin (Ig)mu (mIg mu) associated with Ig alpha-Ig beta but was unable to activate the pre-B cell transition in RAG-1-/-lambda 5-/- mice. Either lambda 5 or kappa light chains were sufficient to complement this deficiency. Therefore light chains are absolutely required for a functional Ig signaling module in early B cell development. Our data provide direct evidence for the existence of two pathways for induction of early B cell development: one which is activated through surrogate light chains and mIg mu, and an alternative pathway which uses conventional light chains and mIg mu. PMID:8920890

  18. Diagnosing Multiple Myeloma from Test Results

    MedlinePlus

    ... does not need to be treated right away. Light chain amyloidosis A diagnosis of light chain amyloidosis is made when the patient has ... A biopsy that shows amyloid made up of light chains, PLUS any of the following: Elevated free ...

  19. BiP and immunoglobulin light chain cooperate to control the folding of heavy chain and ensure the fidelity of immunoglobulin assembly.

    PubMed

    Lee, Y K; Brewer, J W; Hellman, R; Hendershot, L M

    1999-07-01

    The immunoglobulin (Ig) molecule is composed of two identical heavy chains and two identical light chains (H2L2). Transport of this heteromeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. Although other heavy chain-constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (CH1) of the heavy chain, causing it to be retained in the ER. Using a simplified two-domain Ig heavy chain (VH-CH1), we have determined why BiP remains bound to free heavy chains and how light chains facilitate their transport. We found that in the absence of light chain expression, the CH1 domain neither folds nor forms its intradomain disulfide bond and therefore remains a substrate for BiP. In vivo, light chains are required to facilitate both the folding of the CH1 domain and the release of BiP. In contrast, the addition of ATP to isolated BiP-heavy chain complexes in vitro causes the release of BiP and allows the CH1 domain to fold in the absence of light chains. Therefore, light chains are not intrinsically essential for CH1 domain folding, but play a critical role in removing BiP from the CH1 domain, thereby allowing it to fold and Ig assembly to proceed. These data suggest that the assembly of multimeric protein complexes in the ER is not strictly dependent on the proper folding of individual subunits; rather, assembly can drive the complete folding of protein subunits.

  20. Diagnostic and prognostic significance of neurofilament light chain NF-L, but not progranulin and S100B, in the course of amyotrophic lateral sclerosis: Data from the German MND-net.

    PubMed

    Steinacker, Petra; Huss, André; Mayer, Benjamin; Grehl, Torsten; Grosskreutz, Julian; Borck, Guntram; Kuhle, Jens; Lulé, Dorothée; Meyer, Thomas; Oeckl, Patrick; Petri, Susanne; Weishaupt, Jochen; Ludolph, Albert C; Otto, Markus

    2017-02-01

    There is a need for diagnostic, prognostic, and monitoring blood biomarkers for ALS. We aimed to analyse and compare proposed candidate markers for disease progression in the course of ALS. Blood samples were taken from 125 ALS patients, including nine patients with C9orf72 or SOD1 mutation, at regular intervals of six months. ALS patients were characterized by the ALS functional rating scale (ALSFRS-R) and the Edinburgh Cognitive and Behavioural ALS Screen (ECAS). We quantified neurofilament light chain (NF-L), S100B, and progranulin (PGRN) and analysed it in relation to disease progression. Results showed that, at baseline, serum concentrations of NF-L but not PGRN or S100B discriminated significantly between ALS and controls. Within 24 months follow-up the marker concentrations remained stable. Baseline serum NF-L levels correlated with survival time, which was confirmed in subgroups with fast, intermediate, and slow disease progression and there was a weak association with disease duration. For S100B and PGRN we found an association with ALSFRS-R score changes and a trend for decreased levels in the fast progressor subgroup. In conclusion, serum NF-L in any ALS disease stage is a promising marker to support diagnosis and predict outcome, while serum PGRN and S100B are only of minor prognostic value.

  1. Transthyretin Amyloidosis: Chaperone Concentration Changes and Increased Proteolysis in the Pathway to Disease

    PubMed Central

    Ribeiro, Raquel; Gilberto, Samuel; Gomes, Ricardo A.; Ferreira, António; Mateus, Élia; Barroso, Eduardo; Coelho, Ana V.; Freire, Ana Ponces; Cordeiro, Carlos

    2015-01-01

    Transthyretin amyloidosis is a conformational pathology characterized by the extracellular formation of amyloid deposits and the progressive impairment of the peripheral nervous system. Point mutations in this tetrameric plasma protein decrease its stability and are linked to disease onset and progression. Since non-mutated transthyretin also forms amyloid in systemic senile amyloidosis and some mutation bearers are asymptomatic throughout their lives, non-genetic factors must also be involved in transthyretin amyloidosis. We discovered, using a differential proteomics approach, that extracellular chaperones such as fibrinogen, clusterin, haptoglobin, alpha-1-anti-trypsin and 2-macroglobulin are overrepresented in transthyretin amyloidosis. Our data shows that a complex network of extracellular chaperones are over represented in human plasma and we speculate that they act synergistically to cope with amyloid prone proteins. Proteostasis may thus be as important as point mutations in transthyretin amyloidosis. PMID:26147092

  2. Behçet's disease associated with amyloidosis in Turkey and in the world.

    PubMed Central

    Dilşen, N; Koniçe, M; Aral, O; Erbengi, T; Uysal, V; Koçak, N; Ozdogan, E

    1988-01-01

    The association of amyloidosis with Behçet's disease has infrequently been reported in published works. Twenty four such cases have been observed in the world, of which 12 are from Turkey, including eight of ours. In all our eight cases renal biopsy showed amyloidosis of type AA. Behçet's disease of male preponderance, long duration, complete type, multiple organ involvement, and positive skin pathergy test were the main characteristics of all 24 cases of Behçet's disease with amyloidosis. We conclude that amyloidosis associated with Behçet's disease is a secondary AA amyloidosis occurring as an intrinsic manifestation of Behçet's disease. Images PMID:3281606

  3. Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain.

    PubMed

    Kaneko, Mika K; Abe, Shinji; Ogasawara, Satoshi; Fujii, Yuki; Yamada, Shinji; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Nishioka, Yasuhiko; Kato, Yukinari

    2017-02-01

    Podoplanin (PDPN), a type I transmembrane 36-kDa glycoprotein, is expressed not only in normal cells, such as renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, but also in cancer cells, including brain tumors and lung squamous cell carcinomas. Podoplanin activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and the podoplanin/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced neutralizing anti-human podoplanin monoclonal antibody (mAb), clone NZ-1 (rat IgG2a, lambda), which neutralizes the podoplanin/CLEC-2 interaction and inhibits platelet aggregation and cancer metastasis. Human-rat chimeric antibody, NZ-8, was previously developed using variable regions of NZ-1 and human constant regions of heavy chain (IgG1) and light chain (kappa chain). Although NZ-8 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cells, the binding affinity of NZ-8 was lower than that of NZ-1. Herein, we produced a novel human-rat chimeric antibody, NZ-12, the constant regions of which consist of IgG1 heavy chain and lambda light chain. Using flow cytometry, we demonstrated that the binding affinity of NZ-12 was much higher than that of NZ-8. Furthermore, ADCC and CDC activities of NZ-12 were significantly increased against glioblastoma cell lines (LN319 and D397) and lung cancer cell line (PC-10). These results suggested that NZ-12 could become a promising therapeutic antibody against podoplanin-expressing brain tumors and lung cancers.

  4. Agonist-induced changes in the phosphorylation of the myosin- binding subunit of myosin light chain phosphatase and CPI17, two regulatory factors of myosin light chain phosphatase, in smooth muscle.

    PubMed Central

    Niiro, Naohisa; Koga, Yasuhiko; Ikebe, Mitsuo

    2003-01-01

    The inhibition of myosin light chain phosphatase (MLCP) enhances smooth muscle contraction at a constant [Ca2+]. There are two components, myosin-binding subunit of MLCP (MBS) and CPI17, thought to be responsible for the inhibition of MLCP by external stimuli. The phosphorylation of MBS at Thr-641 and of CPI17 at Thr-38 inhibits the MLCP activity in vitro. Here we determined the changes in the phosphorylation of MBS and CPI17 after agonist stimulation in intact as well as permeabilized smooth muscle strips using phosphorylation-site-specific antibodies as probes. The CPI17 phosphorylation transiently increased after agonist stimulation in both alpha-toxin skinned and intact fibres. The time course of the increase in CPI17 phosphorylation after stimulation correlated with the increase in myosin regulatory light chain (MLC) phosphorylation. The increase in CPI17 phosphorylation was significantly diminished by Y27632, a Rho kinase inhibitor, and GF109203x, a protein kinase C inhibitor, suggesting that both the protein kinase C and Rho kinase pathways influence the change in CPI17 phosphorylation. On the other hand, a significant level of MBS phosphorylation at Thr-641, an inhibitory site, was observed in the resting state for both skinned and intact fibres and the agonist stimulation did not significantly alter the MBS phosphorylation level at Thr-641. While the removal of the agonist markedly decreased MLC phosphorylation and induced relaxation, the phosphorylation of MBS was unchanged, while CPI17 phosphorylation markedly diminished. These results strongly suggest that the phosphorylation of CPI17 plays a more significant role in the agonist-induced increase in myosin phosphorylation and contraction of smooth muscle than MBS phosphorylation in the Ca2+-independent activation mechanism of smooth muscle contraction. PMID:12296769

  5. Catalytic Features of the Botulinum Neurotoxin A Light Chain Revealed by High Resolution Structure of an Inhibitory Peptide Complex

    SciTech Connect

    Silvaggi,N.; Wilson, D.; Tzipori, S.; Allen, K.

    2008-01-01

    The Clostridium botulinum neurotoxin serotype A light chain (BoNT/A-LC) is a Zn(II)-dependent metalloprotease that blocks the release of acetylcholine at the neuromuscular junction by cleaving SNAP-25, one of the SNARE proteins required for exocytosis. Because of the potential for use of the toxin in bioterrorism and the increasingly widespread application of the toxin in the medical field, there is significant interest in the development of small-molecule inhibitors of the metalloprotease. Efforts to design such inhibitors have not benefited from knowledge of how peptides bind to the active site since the enzyme-peptide structures available previously either were not occupied in the vicinity of the catalytic Zn(II) ion or did not represent the product of SNAP-25 substrate cleavage. Herein we report the 1.4 Angstroms-resolution X-ray crystal structure of a complex between the BoNT/A-LC and the inhibitory peptide N-Ac-CRATKML, the first structure of the light chain with an inhibitory peptide bound at the catalytic Zn(II) ion. The peptide is bound with the Cys S? atom coordinating the metal ion. Surprisingly, the cysteine sulfur is oxidized to the sulfenic acid form. Given the unstable nature of this species in solution, is it likely that oxidation occurs on the enzyme. In addition to the peptide-bound structure, we report two structures of the unliganded light chain with and without the Zn(II) cofactor bound at 1.25 and 1.20 Angstroms resolution, respectively. The two structures are nearly identical, confirming that the Zn(II) ion plays a purely catalytic role. Additionally, the structure of the Zn(II)-bound uncomplexed enzyme allows identification of the catalytic water molecule and a second water molecule that occupies the same position as the peptidic oxygen in the tetrahedral intermediate. This observation suggests that the enzyme active site is prearranged to stabilize the tetrahedral intermediate of the protease reaction.

  6. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

    PubMed Central

    Lima, V.V.; Lobato, N.S.; Filgueira, F.P.; Webb, R.C.; Tostes, R.C.; Giachini, F.R.

    2014-01-01

    O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction. PMID:25140811

  7. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain.

    PubMed

    Lima, V V; Lobato, N S; Filgueira, F P; Webb, R C; Tostes, R C; Giachini, F R

    2014-10-01

    O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2 ± 2 vs 7.9 ± 1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4 ± 2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3 ± 2 vs 7.5 ± 2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1 ± 2 vs 7.4 ± 2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca(2+)/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

  8. A mutation in the atrial-specific myosin light chain gene (MYL4) causes familial atrial fibrillation.

    PubMed

    Orr, Nathan; Arnaout, Rima; Gula, Lorne J; Spears, Danna A; Leong-Sit, Peter; Li, Qiuju; Tarhuni, Wadea; Reischauer, Sven; Chauhan, Vijay S; Borkovich, Matthew; Uppal, Shaheen; Adler, Arnon; Coughlin, Shaun R; Stainier, Didier Y R; Gollob, Michael H

    2016-04-12

    Atrial fibrillation (AF), the most common arrhythmia, is a growing epidemic with substantial morbidity and economic burden. Mechanisms underlying vulnerability to AF remain poorly understood, which contributes to the current lack of highly effective therapies. Recognizing mechanistic subtypes of AF may guide an individualized approach to patient management. Here, we describe a family with a previously unreported syndrome characterized by early-onset AF (age <35 years), conduction disease and signs of a primary atrial myopathy. Phenotypic penetrance was complete in all mutation carriers, although complete disease expressivity appears to be age-dependent. We show that this syndrome is caused by a novel, heterozygous p.Glu11Lys mutation in the atrial-specific myosin light chain gene MYL4. In zebrafish, mutant MYL4 leads to disruption of sarcomeric structure, atrial enlargement and electrical abnormalities associated with human AF. These findings describe the cause of a rare subtype of AF due to a primary, atrial-specific sarcomeric defect.

  9. Novel Familial Dilated Cardiomyopathy Mutation in MYL2 Affects the Structure and Function of Myosin Regulatory Light Chain

    PubMed Central

    Huang, Wenrui; Liang, Jingsheng; Yuan, Chen-Ching; Kazmierczak, Katarzyna; Zhou, Zhiqun; Morales, Ana; McBride, Kim L.; Fitzgerald-Butt, Sara M.; Hershberger, Ray E.; Szczesna-Cordary, Danuta

    2015-01-01

    Dilated Cardiomyopathy (DCM) is a disease of the myocardium characterized by left ventricular dilatation and diminished contractile function. In this report we describe a novel DCM mutation identified for the first time in the myosin regulatory light chain (RLC), replacing Aspartic Acid at position 94 with Alanine (D94A). The mutation was identified by exome sequencing of three adult first-degree relatives who met formal criteria for idiopathic DCM. To gain insight into the functional significance of this pathogenic MYL2 variant, we have cloned and purified the human ventricular RLC wild-type (WT) and D94A-mutant proteins and performed in vitro experiments using RLC-exchanged porcine cardiac preparations. The mutation was observed to induce a reduction in the α-helical content of the RLC and imposed intra-molecular rearrangements. The Ca2+-calmodulin-activated myosin light chain kinase phosphorylation of RLC was not affected by D94A. The mutation was seen to impair the binding of RLC to the MHC (myosin heavy chain), and its incorporation into the RLC-depleted porcine myosin. The actin-activated ATPase activity of mutant-reconstituted porcine cardiac myosin was significantly higher compared to ATPase of WT. No changes in myofibrillar ATPase-pCa relationship were observed in WT- or D94A-reconstituted preparations. Measurements of contractile force showed a slightly reduced maximal tension per cross-section of muscle with no change in calcium sensitivity of force in D94A-reconstituted skinned porcine papillary muscle strips compared with WT. Our data indicate that subtle structural rearrangements in the RLC molecule followed by its impaired interaction with the MHC may trigger functional abnormalities contributing to the DCM phenotype. PMID:25825243

  10. [Inhibition by IFN-(Asp)4-Lys-HIV chimeric protein of hydrolysis of the low molecular substrate by the enteropeptidase light chain].

    PubMed

    Shibanova, E D; Grishina, Iu B; Rumsh, L D

    2002-01-01

    The full length enteropeptidase or it's light chain have often used for the limited proteolysis of recombinant chimeric proteins incorporating the linker-(Asp)4Lys- to obtain the target protein. Any chimeric proteins were not cleaved by the full length enteropeptidase efficiently. The resistant to the hydrolysis chimeric protein IFN-(Asp)4Lys-HIV earlier was shown to be the competitive inhibitor (Ki = 3,4 x 10(-6) M) in relation to the low molecular substrate. In present study we were determined this chimeric protein competitive inhibited the same substrate hydrolysis by enteropeptidase light chain (Ki = 2,7 x 10(-5) M). Comparison the Ki values for the substrate hydrolysis by full length enzyme and its light chain suggests that the enteropeptidase heavy chain may participate in chimeric protein binding.

  11. Transthyretin-related hereditary amyloidosis with recurrent vomiting and renal insufficiency as the initial presentation

    PubMed Central

    Xu, Jing; Yang, Meng; Pan, Xiaoxia; Yu, Xialian; Xie, Jingyuan; Ren, Hong; Li, Xiao; Chen, Nan

    2017-01-01

    Abstract Rationale: Hereditary amyloidosis is diagnosed worldwidely with an increasing incidence. As the most common form, transthyretin-related hereditary amyloidosis (ATTR amyloidosis) is an autosomal dominant inherited disease due to mutations of TTR. Over the past several decades, more than 130 mutations have been reported. Previous studies suggested that ATTR amyloidosis initially showed polyneuropathy and autonomic dysfunction but later involving many visceral organs, such as kidney. Patient concerns: A young proband carrying TTR p.Leu75Pro mutation, a reported aggressive variant, initially presenting repeat vomiting and impaired renal function was described in a Chinese family. Diagnoses: ATTR amyloidosis patient was diagnosed by renal biopsy and gene sequencing. Interventions: Allograft liver transplantation (LT). Outcomes: Symptom relief but serum creatinine increased. Lessons subsections: This case illustrated the clinical and pathologic phenotype of an ATTR amyloidosis patient who initially presented impaired renal function and p.Leu75Pro variant was found by sequencing the coding region of TTR gene. Kidney is one of the most common and vulnerable organs of amyloidosis, and renal function should be closely monitored. PMID:28272196

  12. Amyloidosis involving the respiratory system: 5-year's experience of a multi-disciplinary group's activity.

    PubMed

    Scala, Raffaele; Maccari, Uberto; Madioni, Chiara; Venezia, Duccio; La Magra, Lidia Calogera

    2015-01-01

    Amyloidosis may involve the respiratory system with different clinical-radiological-functional patterns which are not always easy to be recognized. A good level of knowledge of the disease, an active integration of the pulmonologist within a multidisciplinary setting and a high level of clinical suspicion are necessary for an early diagnosis of respiratory amyloidosis. The aim of this retrospective study was to evaluate the number and the patterns of amyloidosis involving the respiratory system. We searched the cases of amyloidosis among patients attending the multidisciplinary rare and diffuse lung disease outpatients' clinic of Pulmonology Unit of the Hospital of Arezzo from 2007 to 2012. Among the 298 patients evaluated during the study period, we identified three cases of amyloidosis with involvement of the respiratory system, associated or not with other extra-thoracic localizations, whose diagnosis was histo-pathologically confirmed after the pulmonologist, the radiologist, and the pathologist evaluation. Our experience of a multidisciplinary team confirms that intra-thoracic amyloidosis is an uncommon disorder, representing 1.0% of the cases of rare and diffuse lung diseases referred to our center. The diagnosis of the disease is not always easy and quick as the amyloidosis may involve different parts of the respiratory system (airways, pleura, parenchyma). It is therefore recommended to remind this orphan disease in the differential diagnosis of the wide clinical scenarios the pulmonologist may intercept in clinical practice.

  13. Caloric restriction reduces the systemic progression of mouse AApoAII amyloidosis

    PubMed Central

    Ding, Xin; Yang, Mu; Xu, Zhe; Miyahara, Hiroki; Mori, Masayuki; Higuchi, Keiichi

    2017-01-01

    In mouse senile amyloidosis, apolipoprotein (Apo) A-II is deposited extracellularly in many organs in the form of amyloid fibrils (AApoAII). Reduction of caloric intake, known as caloric restriction (CR), slows the progress of senescence and age-related disorders in mice. In this study, we intravenously injected 1 μg of isolated AApoAII fibrils into R1.P1-Apoa2c mice to induce experimental amyloidosis and investigated the effects of CR for the next 16 weeks. In the CR group, AApoAII amyloid deposits in the liver, tongue, small intestine and skin were significantly reduced compared to those of the ad libitum feeding group. CR treatment led to obvious reduction in body weight, improvement in glucose metabolism and reduction in the plasma concentration of ApoA-II. Our molecular biological analyses of the liver suggested that CR treatment might improve the symptoms of inflammation, the unfolded protein response induced by amyloid deposits and oxidative stress. Furthermore, we suggest that CR treatment might improve mitochondrial functions via the sirtuin 1-peroxisome proliferator-activated receptor γ coactivator 1α (SIRT1-PGC-1α) pathway. We suggest that CR is a promising approach for treating the onset and/or progression of amyloidosis, especially for systemic amyloidosis such as senile AApoAII amyloidosis. Our analysis of CR treatment for amyloidosis should provide useful information for determining the cause of amyloidosis and developing effective preventive treatments. PMID:28225824

  14. Experimental transmission of systemic AA amyloidosis in autoimmune disease and type 2 diabetes mellitus model mice

    PubMed Central

    Maeda, Mayuko; Murakami, Tomoaki; Muhammad, Naeem; Inoshima, Yasuo; Ishiguro, Naotaka

    2016-01-01

    AA amyloidosis is a protein misfolding disease characterized by extracellular deposition of amyloid A (AA) fibrils. AA amyloidosis has been identified in food animals, and it has been postulated that AA amyloidosis may be transmissible to different animal species. Since the precursor protein of AA fibrils is serum amyloid A (SAA), which is an inflammatory acute phase protein, AA amyloidosis is considered to be associated with inflammatory diseases such as rheumatoid arthritis. Chronic diseases such as autoimmune disease and type 2 diabetes mellitus could be potential factors for AA amyloidosis. In this study, to examine the relationship between the induction of AA amyloidosis and chromic abnormalities such as autoimmune disease or type 2 diabetes mellitus, amyloid fibrils from mice, cattle, or chickens were experimentally injected into disease model mice. Wild-type mice were used as controls. The concentrations of SAA, IL-6, and IL-10 in autoimmune disease model mice were higher than those of control mice. However, induction of AA amyloidosis in autoimmune disease and type 2 diabetes mellitus model mice was lower than that in control mice, and the amount of amyloid deposits in the spleens of both mouse models was lower than that of control mice according to Congo red staining and immunohistochemistry. These results suggest that factors other than SAA levels, such as an inflammatory or anti-inflammatory environment in the immune response, may be involved in amyloid deposition. PMID:27321428

  15. Renal AA Amyloidosis in Patients with Type 2 Diabetes Mellitus

    PubMed Central

    Díez, Ramón; Madero, Magdalena; Gamba, Gerardo; Soriano, Juan; Soto, Virgilia

    2014-01-01

    Background Type 2 diabetes mellitus (T2DM) is the leading cause of chronic kidney disease and a major cause of cardiovascular disease (CVD) mortality. Inflammation is closely involved in the pathogenesis of T2DM, and reactive amyloidosis occurs in the presence of chronic inflammation. We hypothesized that patients with T2DM may have a higher prevalence of renal AA amyloidosis (RAAA) and that this could contribute to worse atherosclerosis and CVD. Materials and Methods We analyzed 330 autopsy kidneys from patients with a previous T2DM diagnosis. The kidney tissue was evaluated in order to determine the presence of diabetic nephropathy and RAAA, and systemic vessels were evaluated for the presence of atherosclerosis. Results RAAA was detected in 9% of our study population and was associated with an increased risk for nodular sclerosis [OR (95% CI)] [11 (2.04-59.16)], for chronic ischemic cardiomyopathy [4.59 (2.02-10.42)], for myocardial infarction [3.41 (1.52-7.64)] as well as for aortic [4.75 (1.09-20.69)], coronary [3.22 (1.47-7.04)], and intrarenal atherosclerosis [3.84 (1.46-10.09)]. Conclusions RAAA is prevalent in T2DM and is associated with worse CVD and renal disease, likely because RAAA is a marker of severe chronic inflammation. PMID:25337080

  16. Secondary Cutaneous Amyloidosis in a Patient with Mycosis Fungoides

    PubMed Central

    Nam, Chan Hee; Park, Min Kee; Choi, Mi Soo; Hong, Seung Phil; Park, Byung Cheol

    2017-01-01

    Secondary cutaneous amyloidosis refers to clinically unapparent amyloid deposits within the skin in association with a pre-existing skin condition or skin tumors, such as basal cell carcinoma, porokeratosis, solar elastosis, Bowen's disease, and mycosis fungoides. A 70-year-old woman presented with a 6-month history of asymptomatic multiple yellowish plaques on both legs. She had been diagnosed with mycosis fungoides 7 years ago and was treated with psoralen and ultraviolet A radiation (PUVA) therapy, narrow-band ultraviolet B (UVB) therapy, and acitretin for 5 years. Finally, she reached complete remission of mycosis fungoides. However, new yellowish lesions started to appear 1 year after discontinuing the phototherapy. A physical examination revealed multiple yellowish plaques on both extremities. The plaques were well circumscribed and slightly elevated. All laboratory tests were normal. A biopsy specimen showed multiple nodular deposits of eosinophilic amorphous material in papillary dermis and upper reticular dermis. The deposits represented apple green birefringence on Congo red stain viewed under polarized light. Acellular small nodules in the upper dermis consisted of randomly oriented, non-branching, 6.67~12.7 nm thick amyloid fibrils on electron microscopy. We report an interesting and rare case of secondary cutaneous amyloidosis after narrow-band UVB therapy and PUVA therapy in a patient with mycosis fungoides. PMID:28223751

  17. Secondary Cutaneous Amyloidosis in a Patient with Mycosis Fungoides.

    PubMed

    Nam, Chan Hee; Park, Min Kee; Choi, Mi Soo; Hong, Seung Phil; Park, Byung Cheol; Kim, Myung Hwa

    2017-02-01

    Secondary cutaneous amyloidosis refers to clinically unapparent amyloid deposits within the skin in association with a pre-existing skin condition or skin tumors, such as basal cell carcinoma, porokeratosis, solar elastosis, Bowen's disease, and mycosis fungoides. A 70-year-old woman presented with a 6-month history of asymptomatic multiple yellowish plaques on both legs. She had been diagnosed with mycosis fungoides 7 years ago and was treated with psoralen and ultraviolet A radiation (PUVA) therapy, narrow-band ultraviolet B (UVB) therapy, and acitretin for 5 years. Finally, she reached complete remission of mycosis fungoides. However, new yellowish lesions started to appear 1 year after discontinuing the phototherapy. A physical examination revealed multiple yellowish plaques on both extremities. The plaques were well circumscribed and slightly elevated. All laboratory tests were normal. A biopsy specimen showed multiple nodular deposits of eosinophilic amorphous material in papillary dermis and upper reticular dermis. The deposits represented apple green birefringence on Congo red stain viewed under polarized light. Acellular small nodules in the upper dermis consisted of randomly oriented, non-branching, 6.67~12.7 nm thick amyloid fibrils on electron microscopy. We report an interesting and rare case of secondary cutaneous amyloidosis after narrow-band UVB therapy and PUVA therapy in a patient with mycosis fungoides.

  18. Primary Systemic Amyloidosis and High Levels of Angiotensin-Converting Enzyme: Two Case Reports

    PubMed Central

    Praena-Segovia, J.; Sanchez-Gastaldo, A.; Bernabeu-Wittel, M.; Ocete-Pérez, R.; Ávila-Polo, R.; Martino, M. L.

    2013-01-01

    Infiltrative heart diseases are caused by a heterogeneous group of disorders; amyloidosis and sarcoidosis are two frequent causes of myocardial infiltration, which differ in clinical and biological outcome and treatment issues. The presence of high levels of angiotensin-converting enzyme (ACE) in a patient with infiltrative heart disease may increase suspicion of sarcoidosis. Nevertheless, no mention about increased ACE levels in extracerebral primary systemic amyloidosis is available. We present two cases of primary systemic amyloidosis, which are cardiac involvement and elevated ACE levels. PMID:24826302

  19. Human myeloma light chains with increased molecular weight: high frequency among lambda chains.

    PubMed

    Bouvet, J P; Pillot, J; Liacopoulos, P

    1983-04-01

    The discovery of a human myeloma protein comprising a kappa L-chain with an increased mol. wt of 30,000) (Bouvet et. al., 1980) prompted investigations on the incidence of such heavier L-chains among other human myeloma proteins. In 105 samples examined, 34 were found to have L-chains heavier than normal (23,000-24,000), ranging from 25,000 up to 31,000, and five of lighter mol. wt (21,000-22,000). These mol. wt abnormalities were detected by electrophoresis in sodium dodecyl sulfate 10% polyacrylamide gels (SDS-PAGE) after reduction with 2-mercaptoethanol. The mol. wt of three of the heavier kappa or lambda chains was also estimated by filtration through a Sephadex G100 column and by sedimentation equilibrium. All three methods indicated a mol. wt increase of about 15-25% as compared with the usual mol. wt. The distribution of the high mol. wt chains among all L-chains examined was found to be 11 out of 62 kappa chains (17.7%) and 23 out of 43 lambda chains (53%) (P less than 0.001). A preferential association of such L-chains with H-chains producing multiple bands in SDS-PAGE (P less than 0.01) and an association between multiple L-chain and multiple H-chain band (P less than 0.05) were also observed. In contrast, no abnormal L-chain was found in immunoglobulins from normal subjects. Spontaneous degradation of the normal H-chains sometimes yielded fragments of 30,000 mol. wt. These fragments were easily distinguishable from abnormal L-chains. The nature of extra mol. wt in heavy L-chains was investigated for the presence of carbohydrate moiety. Four large and three normal size L-chains were examined for amino-sugar and sialic acid content. A small amount (one residue per molecule) of amino-sugar was detected only in two normal and two heavy L-chains, whereas sialic acid was only found in the heaviest (27,000-30,000) L-chains (Lh) and in small percentage (one or two residues per molecule). Total sugar estimation in one Lh chain indicated a proportion not exceeding

  20. Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

    SciTech Connect

    Colson, Brett A.; Locher, Matthew R.; Bekyarova, Tanya; Patel, Jitandrakumar R.; Fitzsimons, Daniel P.; Irving, Thomas C.; Moss, Richard L.

    2010-05-25

    Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca{sup 2+} sensitivity of force (pCa{sub 50}), PKA treatment has been shown to decrease pCa{sub 50}, presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca{sup 2+}-independent force and maximum Ca{sup 2+}-activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I{sub 1,1}/I{sub 1,0}) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d{sub 1,0}) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I{sub 1,1}/I{sub 1,0} and, as hypothesized, treatment with MLCK also increased I{sub 1,1}/I{sub 1,0}, which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by {approx}2 nm ({Delta} 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca{sup 2+} sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

  1. Mining the antibodyome for HIV-1-neutralizing antibodies with next-generation sequencing and phylogenetic pairing of heavy/light chains.

    PubMed

    Zhu, Jiang; Ofek, Gilad; Yang, Yongping; Zhang, Baoshan; Louder, Mark K; Lu, Gabriel; McKee, Krisha; Pancera, Marie; Skinner, Jeff; Zhang, Zhenhai; Parks, Robert; Eudailey, Joshua; Lloyd, Krissey E; Blinn, Julie; Alam, S Munir; Haynes, Barton F; Simek, Melissa; Burton, Dennis R; Koff, Wayne C; Mullikin, James C; Mascola, John R; Shapiro, Lawrence; Kwong, Peter D

    2013-04-16

    Next-generation sequencing of antibody transcripts from HIV-1-infected individuals with broadly neutralizing antibodies could provide an efficient means for identifying somatic variants and characterizing their lineages. Here, we used 454 pyrosequencing and identity/divergence grid sampling to analyze heavy- and light-chain sequences from donor N152, the source of the broadly neutralizing antibody 10E8. We identified variants with up to 28% difference in amino acid sequence. Heavy- and light-chain phylogenetic trees of identified 10E8 variants displayed similar architectures, and 10E8 variants reconstituted from matched and unmatched phylogenetic branches displayed significantly lower autoreactivity when matched. To test the generality of phylogenetic pairing, we analyzed donor International AIDS Vaccine Initiative 84, the source of antibodies PGT141-145. Heavy- and light-chain phylogenetic trees of PGT141-145 somatic variants also displayed remarkably similar architectures; in this case, branch pairings could be anchored by known PGT141-145 antibodies. Altogether, our findings suggest that phylogenetic matching of heavy and light chains can provide a means to approximate natural pairings.

  2. Mining the antibodyome for HIV-1–neutralizing antibodies with next-generation sequencing and phylogenetic pairing of heavy/light chains

    PubMed Central

    Zhu, Jiang; Ofek, Gilad; Yang, Yongping; Zhang, Baoshan; Louder, Mark K.; McKee, Krisha; Pancera, Marie; Skinner, Jeff; Zhang, Zhenhai; Parks, Robert; Eudailey, Joshua; Lloyd, Krissey E.; Blinn, Julie; Alam, S. Munir; Haynes, Barton F.; Simek, Melissa; Burton, Dennis R.; Koff, Wayne C.; Mullikin, James C.; Mascola, John R.; Shapiro, Lawrence; Kwong, Peter D.; Becker, Jesse; Benjamin, Betty; Blakesley, Robert; Bouffard, Gerry; Brooks, Shelise; Coleman, Holly; Dekhtyar, Mila; Gregory, Michael; Guan, Xiaobin; Gupta, Jyoti; Han, Joel; Hargrove, April; Ho, Shi-ling; Johnson, Taccara; Legaspi, Richelle; Lovett, Sean; Maduro, Quino; Masiello, Cathy; Maskeri, Baishali; McDowell, Jenny; Montemayor, Casandra; Mullikin, James; Park, Morgan; Riebow, Nancy; Schandler, Karen; Schmidt, Brian; Sison, Christina; Stantripop, Mal; Thomas, James; Thomas, Pam; Vemulapalli, Meg; Young, Alice

    2013-01-01

    Next-generation sequencing of antibody transcripts from HIV-1–infected individuals with broadly neutralizing antibodies could provide an efficient means for identifying somatic variants and characterizing their lineages. Here, we used 454 pyrosequencing and identity/divergence grid sampling to analyze heavy- and light-chain sequences from donor N152, the source of the broadly neutralizing antibody 10E8. We identified variants with up to 28% difference in amino acid sequence. Heavy- and light-chain phylogenetic trees of identified 10E8 variants displayed similar architectures, and 10E8 variants reconstituted from matched and unmatched phylogenetic branches displayed significantly lower autoreactivity when matched. To test the generality of phylogenetic pairing, we analyzed donor International AIDS Vaccine Initiative 84, the source of antibodies PGT141–145. Heavy- and light-chain phylogenetic trees of PGT141–145 somatic variants also displayed remarkably similar architectures; in this case, branch pairings could be anchored by known PGT141–145 antibodies. Altogether, our findings suggest that phylogenetic matching of heavy and light chains can provide a means to approximate natural pairings. PMID:23536288

  3. Intestinal amyloidosis: two cases with different patterns of clinical and imaging presentation.

    PubMed

    Mainenti, Pier-Paolo; Segreto, Sabrina; Mancini, Marcello; Rispo, Antonio; Cozzolino, Immacolata; Masone, Stefania; Rinaldi, Ciro-Roberto; Nardone, Gerardo; Salvatore, Marco

    2010-05-28

    The involvement of the small bowel in systemic forms of amyloidosis may be diffuse or very rarely focal. Some cases of focal amyloidomas of the duodenum and jejunum without extraintestinal manifestations have been reported. The focal amyloidomas consisted of extensive amyloid infiltration of the entire intestinal wall thickness. Radiological barium studies, ultrasound and computed tomography (CT) patterns of diffuse small bowel amyloidosis have been described: the signs are non-specific and may include small-bowel dilatation, symmetric bowel wall thickening, mesenteric infiltration, and mesenteric adenopathy. No data are available about the positron emission tomography (PET)/CT and magnetic resonance imaging (MRI) patterns of intestinal amyloidosis. We report two cases of small bowel amyloidosis: the former characterized by focal deposition of amyloid proteins exclusively within blood vessel walls of the terminal ileum, the latter characterized by diffuse intestinal involvement observed on MRI and PET/CT studies.

  4. Intestinal amyloidosis: Two cases with different patterns of clinical and imaging presentation

    PubMed Central

    Mainenti, Pier Paolo; Segreto, Sabrina; Mancini, Marcello; Rispo, Antonio; Cozzolino, Immacolata; Masone, Stefania; Rinaldi, Ciro Roberto; Nardone, Gerardo; Salvatore, Marco

    2010-01-01

    The involvement of the small bowel in systemic forms of amyloidosis may be diffuse or very rarely focal. Some cases of focal amyloidomas of the duodenum and jejunum without extraintestinal manifestations have been reported. The focal amyloidomas consisted of extensive amyloid infiltration of the entire intestinal wall thickness. Radiological barium studies, ultrasound and computed tomography (CT) patterns of diffuse small bowel amyloidosis have been described: the signs are non-specific and may include small-bowel dilatation, symmetric bowel wall thickening, mesenteric infiltration, and mesenteric adenopathy. No data are available about the positron emission tomography (PET)/CT and magnetic resonance imaging (MRI) patterns of intestinal amyloidosis. We report two cases of small bowel amyloidosis: the former characterized by focal deposition of amyloid proteins exclusively within blood vessel walls of the terminal ileum, the latter characterized by diffuse intestinal involvement observed on MRI and PET/CT studies. PMID:20503459

  5. Association of Skin with the Pathogenesis and Treatment of Neurodegenerative Amyloidosis

    PubMed Central

    Clos, Audra L.; Kayed, Rakez; Lasagna-Reeves, Cristian A.

    2012-01-01

    Amyloidosis are a large group of conformational diseases characterized by abnormal protein folding and assembly which results in the accumulation of insoluble protein aggregates that may accumulate systemically or locally in certain organs or tissue. In local amyloidosis, amyloid deposits are restricted to a particular organ or tissue. Alzheimer’s, Parkinson’s disease, and amyotrophic lateral sclerosis are some examples of neurodegenerative amyloidosis. Local manifestation of protein aggregation in the skin has also been reported. Brain and skin are highly connected at a physiological and pathological level. Recently several studies demonstrated a strong connection between brain and skin in different amyloid diseases. In the present review, we discuss the relevance of the “brain–skin connection” in different neurodegenerative amyloidosis, not only at the pathological level, but also as a strategy for the treatment of these diseases. PMID:22319507

  6. Recombinant immunoglobulin variable domains generated from synthetic genes provide a system for in vitro characterization of light-chain amyloid proteins.

    PubMed Central

    Stevens, P. W.; Raffen, R.; Hanson, D. K.; Deng, Y. L.; Berrios-Hammond, M.; Westholm, F. A.; Murphy, C.; Eulitz, M.; Wetzel, R.; Solomon, A.

    1995-01-01

    The primary structural features that render human monoclonal light chains amyloidogenic are presently unknown. To gain further insight into the physical and biochemical factors that result in the pathologic deposition of these proteins as amyloid fibrils, we have selected for detailed study three closely homologous protein products of the light-chain variable-region single-gene family VkIV. Two of these proteins, REC and SMA, formed amyloid fibrils in vivo. The third protein, LEN, was excreted by the patient at levels of 50 g/day with no indication of amyloid deposits. Sequences of amyloidogenic proteins REC and SMA differed from the sequence of the nonpathogenic protein LEN at 14 and 8 amino acid positions, respectively, and these amino acid differences have been analyzed in terms of the three-dimensional structure of the LEN dimer. To provide a replenishable source of these human proteins, we constructed synthetic genes coding for the REC, SMA, and LEN variable domains and expressed these genes in Escherichia coli. Immunochemical and biophysical comparisons demonstrated that the recombinant VkIV products have tertiary structural features comparable to those of the patient-derived proteins. This well-defined set of three clinically characterized human kIV light chains, together with the capability to produce these kIV proteins recombinantly, provide a system for biophysical and structural comparisons of two different amyloidogenic light-chain proteins and a nonamyloidogenic protein of the same subgroup. This work lays the foundation for future investigations of the structural basis of light-chain amyloidogenicity. PMID:7795526

  7. Tarantula Myosin Free Head Regulatory Light Chain Phosphorylation Stiffens N-terminal Extension Releasing it and Blocking its Docking Back

    PubMed Central

    Alamo, Lorenzo; Li, Xiaochuan (Edward); Espinoza-Fonseca, L. Michel; Pinto, Antonio; Thomas, David D.; Lehman, William; Padrón, Raúl

    2015-01-01

    Molecular dynamics simulations of smooth and striated muscle myosin regulatory light chain (RLC) N-terminal extension (NTE) showed that diphosphorylation induces a disorder-to-order transition. Our goal here was to further explore the effects of mono- and diphosphorylation on the straightening and rigidification of the tarantula myosin RLC NTE. For that we used MD simulations followed by persistence length analysis to explore the consequences of secondary and tertiary structure changes occurring on RLC NTE following phosphorylation. Static and dynamic persistence lengths analysis of tarantula RLC NTE peptides suggest that diphosphorylation produces an important 24-fold straightening and a 16-fold rigidification of the RLC NTE, while monophosphorylation has a less profound effect. This new information on myosin structural mechanics, not fully revealed by previous EM and MD studies, add support to a cooperative phosphorylation-dependent activation mechanism as proposed for the tarantula thick filament. Our results suggest that the RLC NTE straightening and rigidification after Ser45 phosphorylation leads to a release of the constitutively Ser35 monophosphorylated free head swaying away from the thick filament shaft in the relaxed state. This is so because the stiffened diphosphorylated RLC NTE would hinder the docking back of the free head after swaying away, becoming released and mobile and unable to recover its original interacting position on activation. PMID:26038302

  8. During cooled storage the extender influences processed autophagy marker light chain 3 (LC3B) of stallion spermatozoa.

    PubMed

    Bolaños, J M Gallardo; Morán, A Miró; da Silva, C M Balao; Dávila, M Plaza; Muñoz, P Martín; Aparicio, I M; Tapia, J A; Ferrusola, C Ortega; Peña, F J

    2014-02-01

    To investigate the role of the processed autophagy marker light chain 3 (LC3B) protein in sperm survival in stallion semen processing during cooled storage, split ejaculates were diluted in two different extenders, KMT and INRA 96, and LC3B processing and sperm quality evaluated during incubation at 5°C for five days. After 3 days of incubation there was a drop in total motility in both extenders, although the percentage of progressive motile sperm was greater (P<0.05) in samples extended in INRA96. On Day 5 of cooled storage all sperm parameters decreased significantly independent of the extender, however, samples extended in INRA 96 maintained motility values while those extended in KMT had a further decrease in motility compared with data collected on Day 3 of incubation. The percentage of live sperm decreased over the time of incubation, but only in samples incubated in KMT. The extender had a marked effect in LC3B processing during cooled storage. Spermatozoa maintained in KMT extender did not exhibit LC3B processing, while in spermatozoa incubated in INRA96 there was an increase (P<0.01) in LC3B processing after 5 days of cooled storage. Stallion spermatozoa experience LC3B turnover during cooled storage, however, the extent depends on the extender used. Apparently LC3B turnover is associated with enhanced survival.

  9. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase

    PubMed Central

    Bhargava, Amol; Cotton, James A.; Dixon, Brent R.; Gedamu, Lashitew; Yates, Robin M.; Buret, Andre G.

    2015-01-01

    Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections. PMID:26334299

  10. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase.

    PubMed

    Bhargava, Amol; Cotton, James A; Dixon, Brent R; Gedamu, Lashitew; Yates, Robin M; Buret, Andre G

    2015-01-01

    Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.

  11. Pseudo-Peritoneal Carcinomatosis Presentation of a Crystal-Storing Histiocytosis With an Unmutated Monoclonal κ Light Chain

    PubMed Central

    Aline-Fardin, Aude; Bender, Sebastien; Fabiani, Bettina; Buob, David; Brahimi, Said; Verpont, Marie Christine; Mothy, Mohamad; Ronco, Pierre; Boffa, Jean Jacques; Aucouturier, Pierre; Garderet, Laurent

    2015-01-01

    Abstract Crystal-storing histiocytosis (CSH) is a rare complication of monoclonal gammopathies caused by accumulation of crystalline material inside macrophages, and it may result in a variety of clinical manifestations depending on the involved organs. Although immunoglobulin κ light chains (LCs) seem to be the most frequent pathogenic component, very few molecular data are currently available. A 69-year-old man presented with a very poor performance status. Remarkable features were mesenteric lymph node enlargement and proteinuria, including a monoclonal κ LC. Light and electron microscopy studies revealed the presence of crystals within macrophages in the lymph nodes, bone marrow, and kidney, leading to the diagnosis of CSH. The pathogenic κ LC variable domain sequence was identical to the germline Vk3-20∗01/Jk2∗01 gene segments, without any somatic mutation, suggesting an extra-follicular B cell proliferation. The patient was successfully treated with 4 cycles of bortezomib and dexamethasone. After a 12-month follow-up, he remains in hematological and renal remission. CSH may present as pseudo-peritoneal carcinomatosis and relate to a monoclonal κ LC encoded by an unmutated gene. Bortezomib-based therapy proved efficacious in this case. PMID:26266355

  12. Microtubule-associated protein light chain 3 is involved in melanogenesis via regulation of MITF expression in melanocytes

    PubMed Central

    Yun, Woo Jin; Kim, Eun-Young; Park, Ji-Eun; Jo, Soo Youn; Bang, Seung Hyun; Chang, Eun-Ju; Chang, Sung Eun

    2016-01-01

    Although autophagy plays a role in melanogenesis by regulating melanosome degradation and biogenesis in melanocytes, a detailed understanding of the regulatory functions of autophagy factors is lacking. Here, we report a mechanistic link between microtubule-associated protein light chain 3 (LC3) activation and melanogenesis. We observed high expression of LC3 in melanosome-associated pigment-rich melanocytic nevi of sun-exposed skin, as indicated by patterns of melanosomal protein MART1 expression. Rapamycin-induced autophagy significantly increased the melanin index, tyrosinase activity and expression of several proteins linked to melanosome biogenesis, including microphthalmia transcription factor (MITF), pre-melanosome protein and tyrosinase, in Melan-a melanocytes. siRNA-mediated knockdown of LC3, but not beclin-1 or ATG5, decreased melanin content and tyrosinase activity. LC3 knockdown also markedly inhibited MITF expression and subsequent rapamycin-induced melanosome formation. More importantly, LC3 knockdown suppressed α-MSH-mediated melanogenesis by attenuating cAMP response element-binding protein (CREB) phosphorylation and MITF expression in Melan-a cells via decreased extracellular signal-regulated kinase (ERK) activity. Overexpression of constitutively active ERK reversed the effect of LC3 knockdown on CREB phosphorylation and MITF expression. These findings demonstrate that LC3 contributes to melanogenesis by increasing ERK-dependent MITF expression, thereby providing a mechanistic insight into the signaling network that links autophagy to melanogenesis. PMID:26814135

  13. The role of the N-terminus of the myosin essential light chain in cardiac muscle contraction

    PubMed Central

    Kazmierczak, Katarzyna; Xu, Yuanyuan; Jones, Michelle; Guzman, Georgianna; Hernandez, Olga M.; Kerrick, W. Glenn L.; Szczesna-Cordary, Danuta

    2011-01-01

    Summary To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43 amino acid N-terminal truncation mutant (Tg-Δ43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle and the ELC protein distribution in Tg-Δ43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Δ43 mice demonstrate reduced force per cross-sectional area of muscle, which is likely caused by a reduced number of force generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Δ43 mice and the mutant hearts develop a phenotype of non-pathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Δ43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin. PMID:19361417

  14. Performance goals for immunoglobulins and serum free light chain measurements in plasma cell dyscrasias can be based on biological variation.

    PubMed

    Hansen, Charlotte Toftmann

    2016-06-01

    Measurements of immunoglobulins and serum free light chains (sFLC) are frequently used in patients with monoclonal plasma cell dyscrasia (PCD). For optimum patient care, well-defined performance standards or goals for the measured concentrations of immunoglobulins and sFLC are required. Generally, data based on biological variation is a good and reliable method for setting desirable performance standards; this also applies for the measurements of paraprotein and sFLC. The benefits of this approach are several. Among others, it is independent of the clinician, and it provides us with information about reference change value and index of individuality. Several studies on biological variation of both immunoglobulins and sFLC have been published, and mostly the studies are well performed. The studies normally show small within-subject biological variation resulting in strict analytical goals, which in most cases are difficult to meet. Nevertheless, we still need further information on biological variation of immunoglobulins and sFLC in patients with PCD and in the elderly, which are the main target populations for the two measurands. Furthermore, to improve data on biological variation of immunoglobulins and sFLC, studies accounting for number of individuals, samples, and replicates, as well as time length of the studies are needed.

  15. Cyclical compressive stress induces differentiation of rat primary mandibular condylar chondrocytes through phosphorylated myosin light chain II.

    PubMed

    Liu, Limin; Chen, Lin; Mai, Zhihui; Peng, Zhuli; Yu, Kafung; Liu, Guanqi; Ai, Hong

    2016-11-01

    The role of myosin light chain II (MLC‑II) in cellular differentiation of rat mandibular condylar chondrocytes (MCCs) induced by cyclical uniaxial compressive stress (CUCS) remains unclear. In the current study, a four‑point bending system was used to apply CUCS to primary cultured MCCs from rats. It was identified that CUCS stimulated features of cellular differentiation including morphological alterations, cytoskeleton rearrangement and overproduction of proteoglycans. Furthermore, CUCS promoted runt‑related transcription factor‑2 (RUNX2) expression at mRNA (P<0.01) and protein levels (P<0.05) and elevated alkaline phosphatase (ALP) activity (P<0.01), which are both markers of osteogenic differentiation. Under conditions of stress, western blotting indicated that the ratio of phosphorylated MLC‑II to total MLC‑II was increased significantly (P<0.05). Silencing MLC‑II by RNA interference reduced ALP activity (P<0.01), and eliminated RUNX2 mRNA expression (P<0.01). Addition of the MLC kinase inhibitor, ML‑7, reduced the CUCS‑associated upregulation of RUNX2 expression (P<0.01) and ALP activity (P<0.01). The data indicated that CUCS promoted cellular differentiation of rat primary MCCs, and this was suggested to be via the phosphorylation of MLC‑II.

  16. The human myosin light chain kinase (MLCK) from hippocampus: Cloning, sequencing, expression, and localization to 3qcen-q21

    SciTech Connect

    Potier, M.C.; Rossier, J.; Turnell, W.G.; Pekarsky, Y.; Gardiner, K.

    1995-10-10

    Myosin light chain kinase (MLCK), a key enzyme in muscle contraction, has been shown by immunohistology to be present in neurons and glia. We describe here the cloning of the cDNA for human MLCK from hippocampus, encoding a protein sequence 95% similar to smooth muscle MLCKs but less than 60% similar to skeletal muscle MLCKs. The cDNA clone detected two RNA transcripts in human frontal and entorhinal cortex, in hippocampus, and in jejunum, one corresponding to MLCK and the other probably to telokin, the carboxy-terminal 154 codons of MLCK expressed as an independent protein in smooth muscle. Levels of expression were lower in brain compared to smooth muscle. We show that within the protein sequence, a motif of 28 or 24 residues is repeated five times, the second repeat ending with the putative methionine start codon. These repeats overlap with a second previously reported module of 12 residues repeated five times in the human sequence. In addition, the acidic C-terminus of all MLCKs from both brain and smooth muscle resembles the C-terminus of tubulins. The chromosomal localization of the gene for human MLCK is shown to be at 3qcen-q21, as determined by PCR and Southern blotting using two somatic cell hybrid panels. 33 refs., 8 figs.

  17. Immunoglobulin light chain (IGL) genes in torafugu: Genomic organization and identification of a third teleost IGL isotype

    PubMed Central

    Fu, Xi; Zhang, Fengjun; Watabe, Shugo; Asakawa, Shuichi

    2017-01-01

    Here, we report a genome-wide survey of immunoglobulin light chain (IGL) genes of torafugu (Takifugu rubripes) revealing multi-clusters spanning three separate chromosomes (v5 assembly) and 45 scaffolds (v4 assembly). Conventional sequence similarity searches and motif scanning approaches based on recombination signal sequence (RSS) motifs were used. We found that three IGL isotypes (L1, L2, and L3) exist in torafugu and that several loci for each isotype are present. The transcriptional orientations of the variable IGL (VL) segments were found to be either the same (in the L2 isotype) or opposite (in the L1 and L3 isotypes) to the IGL joining (JL) and constant (CL) segments, suggesting they can undergo rearrangement by deletion or inversion when expressed. Alignments of expressed sequence tags (ESTs) to corresponding germline gene segments revealed expression of the three IGL isotypes in torafugu. Taken together, our findings provide a genomic framework for torafugu IGL genes and show that the IG diversity of this species could be attributed to at least three distinct chromosomal regions. PMID:28098239

  18. Free Light Chains and Intrathecal B Cells Activity in Multiple Sclerosis: A Prospective Study and Meta-Analysis

    PubMed Central

    Passerini, Gabriella; Sangalli, Francesca; Moiola, Lucia; Colombo, Bruno; Locatelli, Massimo; Comi, Giancarlo

    2016-01-01

    Background. The presence of CSF oligoclonal bands (OBs) is an independent prognostic factor for multiple sclerosis (MS), but the difficulties in the standardization of the test and the interlaboratory variation in reporting have contributed to its limited use in the diagnosis of the disease. Standard nephelometric assays to measure free light chains (FLC) levels have been recently developed and the test may improve the detection of intrathecal B cells activity. Methods. The presence of OBs, kappa and lambda FLC levels, and standard indices of intrathecal inflammation were assessed in 100 consecutive patients, including patients with MS, clinically isolated syndromes (CIS), other inflammatory diseases of the CNS, and other noninflammatory diseases. Results. Both KFLC and LFLC correlated strongly with the presence of OCBs and with all common tests for intrathecal inflammation (p < 0.001 for all comparisons). KFLC and LFLC were significantly different in patients with MS and CIS compared to the other groups (p < 0.001 and p < 0.001, resp.) and had a better diagnostic accuracy than all the other tests (area under the curve 82.3 % for KFLC index and 79.3 % for LFLC index). Conclusion. Nephelometric assays for KFLC in CSF reliably detect intrathecal immunoglobulin synthesis and discriminate MS patients. PMID:28116160

  19. Masticatory (;superfast') myosin heavy chain and embryonic/atrial myosin light chain 1 in rodent jaw-closing muscles.

    PubMed

    Reiser, Peter J; Bicer, Sabahattin; Chen, Qun; Zhu, Ling; Quan, Ning

    2009-08-01

    Masticatory myosin is widely expressed among several vertebrate classes. Generally, the expression of masticatory myosin has been associated with high bite force for a carnivorous feeding style (including capturing/restraining live prey), breaking down tough plant material and defensive biting in different species. Masticatory myosin expression in the largest mammalian order, Rodentia, has not been reported. Several members of Rodentia consume large numbers of tree nuts that are encased in very hard shells, presumably requiring large forces to access the nutmeat. We, therefore, tested whether some rodent species express masticatory myosin in jaw-closing muscles. Myosin isoform expression in six Sciuridae species was examined, using protein gel electrophoresis, immunoblotting, mass spectrometry and RNA analysis. The results indicate that masticatory myosin is expressed in some Sciuridae species but not in other closely related species with similar diets but having different nut-opening strategies. We also discovered that the myosin light chain 1 isoform associated with masticatory myosin heavy chain, in the same four Sciuridae species, is the embryonic/atrial isoform. We conclude that rodent speciation did not completely eliminate masticatory myosin and that its persistent expression in some rodent species might be related to not only diet but also to feeding style.

  20. Monoclonal antibodies to kinesin heavy and light chains stain vesicle- like structures, but not microtubules, in cultured cells

    PubMed Central

    1989-01-01

    Kinesin, a microtubule-activated ATPase and putative motor protein for the transport of membrane-bounded organelles along microtubules, was purified from bovine brain and used as an immunogen for the production of murine monoclonal antibodies. Hybridoma lines that secreted five distinct antikinesin IgGs were cloned. Three of the antibodies reacted on immunoblots with the 124-kD heavy chain of kinesin, while the other two antibodies recognized the 64-kD light chain. When used for immunofluorescence microscopy, the antibodies stained punctate, cytoplasmic structures in a variety of cultured mammalian cell types. Consistent with the identification of these structures as membrane- bounded organelles was the observation that cells which had been extracted with Triton X-100 before fixation contained little or no immunoreactive material. Staining of microtubules in the interphase cytoplasm or mitotic spindle was never observed, nor were associated structures, such as centrosomes and primary cilia, labeled by any of the antibodies. Nevertheless, in double-labeling experiments using antibodies to kinesin and tubulin, kinesin-containing particles were most abundant in regions where microtubules were most highly concentrated and the particles often appeared to be aligned on microtubules. These results constitute the first direct evidence for the association of kinesin with membrane-bounded organelles, and suggest a molecular mechanism for organelle motility based on transient interactions of organelle-bound kinesin with the microtubule surface. PMID:2522455

  1. Effectiveness of haemodiafiltration with ultrafiltrate regeneration in the reduction of light chains in multiple myeloma with renal failure.

    PubMed

    Pendón-Ruiz de Mier, María Victoria; Alvarez-Lara, María Antonia; Ojeda-López, Raquel; Martín-Malo, Alejandro; Carracedo, Julia; Caballero-Villarraso, Javier; Alonso, Corona; Aljama, Pedro

    2013-11-13

    Acute kidney failure in multiple myeloma (MM) occurs in 12%-20% of patients and is a poor prognostic factor for patient survival. Recent studies have shown that dialysis with a High-Cut-Off membrane (HCO) removes free light chains (FLC) effectively although with significant albumin loss. Other adsorption-based techniques, such as haemodiafiltration with ultrafiltrate regeneration by adsorption in resin (SUPRA-HFR), have not been studied. We present three cases of MM, all haemodialysis-dependent since diagnosis. Two cases were IgG kappa and one was IgA lambda. All patients were treated with chemotherapy and SUPRA-HFR. The aim of this study was to evaluate the effectiveness of SUPRA-HFR in the reduction of FLC and its effect on albumin. We collected blood samples pre- and post-dialysis, and ultrafiltrate (UF) samples pre- and post-resin 5 minutes into the session and 5 minutes from the end. The mean reduction rate of FLC in blood per session in the three patients was 53% and 63% (kappa) and 38% (lambda). In the UF, the mean FLC reduction rate was close to 99%, both at the start and at the end of dialysis, without the removal of albumin. With the results obtained we can conclude that this technique achieves an effective reduction of FLC, which is maintained throughout the session, without resin saturation and without albumin loss. Therefore, SUPRA-HFR is effective as an adjunctive therapy for MM.

  2. Clathrin light chains are required for the gyrating-clathrin recycling pathway and thereby promote cell migration.

    PubMed

    Majeed, Sophia R; Vasudevan, Lavanya; Chen, Chih-Ying; Luo, Yi; Torres, Jorge A; Evans, Timothy M; Sharkey, Andrew; Foraker, Amy B; Wong, Nicole M L; Esk, Christopher; Freeman, Theresa A; Moffett, Ashley; Keen, James H; Brodsky, Frances M

    2014-05-23

    The clathrin light chain (CLC) subunits participate in several membrane traffic pathways involving both clathrin and actin, through binding the actin-organizing huntingtin-interacting proteins (Hip). However, CLCs are dispensable for clathrin-mediated endocytosis of many cargoes. Here we observe that CLC depletion affects cell migration through Hip binding and reduces surface expression of β1-integrin by interference with recycling following normal endocytosis of inactive β1-integrin. CLC depletion and expression of a modified CLC also inhibit the appearance of gyrating (G)-clathrin structures, known mediators of rapid recycling of transferrin receptor from endosomes. Expression of the modified CLC reduces β1-integrin and transferrin receptor recycling, as well as cell migration, implicating G-clathrin in these processes. Supporting a physiological role for CLC in migration, the CLCb isoform of CLC is upregulated in migratory human trophoblast cells during uterine invasion. Together, these studies establish CLCs as mediating clathrin-actin interactions needed for recycling by G-clathrin during migration.

  3. Dynein light chain binding to a 3′-untranslated sequence mediates parathyroid hormone mRNA association with microtubules

    PubMed Central

    Epstein, Eyal; Sela-Brown, Alin; Ringel, Israel; Kilav, Rachel; King, Stephen M.; Benashski, Sharon E.; Yisraeli, Joel K.; Silver, Justin; Naveh-Many, Tally

    2000-01-01

    The 3′-untranslated region (UTR) of mRNAs binds proteins that determine mRNA stability and localization. The 3′-UTR of parathyroid hormone (PTH) mRNA specifically binds cytoplasmic proteins. We screened an expression library for proteins that bind the PTH mRNA 3′-UTR, and the sequence of 1 clone was identical to that of the dynein light chain LC8, a component of the dynein complexes that translocate cytoplasmic components along microtubules. Recombinant LC8 binds PTH mRNA 3′-UTR, as shown by RNA electrophoretic mobility shift assay. We showed that PTH mRNA colocalizes with microtubules in the parathyroid gland, as well as with a purified microtubule preparation from calf brain, and that this association was mediated by LC8. To our knowledge, this is the first report of a dynein complex protein binding an mRNA. The dynein complex may be the motor that is responsible for transporting mRNAs to specific locations in the cytoplasm and for the consequent is asymmetric distribution of translated proteins in the cell. PMID:10683380

  4. A Chlamydomonas Homologue of the Putative Murine t Complex Distorter Tctex-2 Is an Outer Arm Dynein Light Chain

    PubMed Central

    Patel-King, Ramila S.; Benashski, Sharon E.; Harrison, Alistair; King, Stephen M.

    1997-01-01

    Molecular analysis of a 19,000-Mr protein from the Chlamydomonas flagellum reveals that it is homologous to the t complex–encoded protein Tctex-2, which is a candidate for one of the distorter products that cause the extreme transmission ratio distortion (meiotic drive) of the murine t complex. The 19,000-Mr protein is extracted from the axoneme with 0.6 M NaCl and comigrates with the outer dynein arm in sucrose density gradients. This protein also is specifically missing in axonemes prepared from a mutant that does not assemble the outer arm. These data raise the possibility that Tctex-2 is a sperm flagellar dynein component. Combined with the recent identification of Tctex-1 (another distorter candidate) as a light chain of cytoplasmic dynein, these results lead to a biochemical model for how differential defects in spermiogenesis that result in the phenomenon of meiotic drive might be generated in wild-type vs t-bearing sperm. PMID:9166408

  5. Structures of Clostridium Botulinum Neurotoxin Serotype A Light Chain Complexed with Small-Molecule Inhibitors Highlight Active-Site Flexibility

    SciTech Connect

    Silvaggi,N.; Boldt, G.; Hixon, M.; Kennedy, J.; Tzipori, S.; Janda, K.; Allen, K.

    2007-01-01

    The potential for the use of Clostridial neurotoxins as bioweapons makes the development of small-molecule inhibitors of these deadly toxins a top priority. Recently, screening of a random hydroxamate library identified a small-molecule inhibitor of C. botulinum Neurotoxin Serotype A Light Chain (BoNT/A-LC), 4-chlorocinnamic hydroxamate, a derivative of which has been shown to have in vivo efficacy in mice and no toxicity. We describe the X-ray crystal structures of BoNT/A-LC in complexes with two potent small-molecule inhibitors. The structures of the enzyme with 4-chlorocinnamic hydroxamate or 2,4-dichlorocinnamic hydroxamate bound are compared to the structure of the enzyme complexed with L-arginine hydroxamate, an inhibitor with modest affinity. Taken together, this suite of structures provides surprising insights into the BoNT/A-LC active site, including unexpected conformational flexibility at the S1' site that changes the electrostatic environment of the binding pocket. Information gained from these structures will inform the design and optimization of more effective small-molecule inhibitors of BoNT/A-LC.

  6. Myosin light chain phosphatase activation is involved in the hydrogen sulfide-induced relaxation in mouse gastric fundus.

    PubMed

    Dhaese, Ingeborg; Lefebvre, Romain A

    2009-03-15

    The relaxant effect of hydrogen sulfide (H(2)S) in the vascular tree is well established but its influence and mechanism of action in gastrointestinal smooth muscle was hardly investigated. The influence of H(2)S on contractility in mouse gastric fundus was therefore examined. Sodium hydrogen sulfide (NaHS; H(2)S donor) was administered to prostaglandin F(2alpha) (PGF(2alpha))-contracted circular muscle strips of mouse gastric fundus, before and after incubation with interfering drugs. NaHS caused a concentration-dependent relaxation of the pre-contracted mouse gastric fundus strips. The K(+) channels blockers glibenclamide, apamin, charybdotoxin, 4-aminopyridin and barium chloride had no influence on the NaHS-induced relaxation. The relaxation by NaHS was also not influenced by L-NAME, ODQ and SQ 22536, inhibitors of the cGMP and cAMP pathway, by nerve blockers capsazepine, omega-conotoxin and tetrodotoxin or by several channel and receptor blockers (ouabain, nifedipine, 2-aminoethyl diphenylborinate, ryanodine and thapsigargin). The myosin light chain phosphatase (MLCP) inhibitor calyculin-A reduced the NaHS-induced relaxation, but the Rho-kinase inhibitor Y-27632 had no influence. We show that NaHS is able to relax PGF(2alpha)-contracted mouse gastric fundus strips. The results suggest that in the mouse gastric fundus, H(2)S causes relaxation at least partially via activation of MLCP.

  7. Myosin light chain-2 mutation affects flight, wing beat frequency, and indirect flight muscle contraction kinetics in Drosophila

    PubMed Central

    1992-01-01

    We have used a combination of classical genetic, molecular genetic, histological, biochemical, and biophysical techniques to identify and characterize a null mutation of the myosin light chain-2 (MLC-2) locus of Drosophila melanogaster. Mlc2E38 is a null mutation of the MLC-2 gene resulting from a nonsense mutation at the tenth codon position. Mlc2E38 confers dominant flightless behavior that is associated with reduced wing beat frequency. Mlc2E38 heterozygotes exhibit a 50% reduction of MLC-2 mRNA concentration in adult thoracic musculature, which results in a commensurate reduction of MLC-2 protein in the indirect flight muscles. Indirect flight muscle myofibrils from Mlc2E38 heterozygotes are aberrant, exhibiting myofilaments in disarray at the periphery. Calcium-activated Triton X-100-treated single fiber segments exhibit slower contraction kinetics than wild type. Introduction of a transformed copy of the wild type MLC-2 gene rescues the dominant flightless behavior of Mlc2E38 heterozygotes. Wing beat frequency and single fiber contraction kinetics of a representative rescued line are not significantly different from those of wild type. Together, these results indicate that wild type MLC-2 stoichiometry is required for normal indirect flight muscle assembly and function. Furthermore, these results suggest that the reduced wing beat frequency and possibly the flightless behavior conferred by Mlc2E38 is due in part to slower contraction kinetics of sarcomeric regions devoid or partly deficient in MLC-2. PMID:1469046

  8. Vincristine promotes migration and invasion of colorectal cancer HCT116 cells through RhoA/ROCK/ Myosin light chain pathway.

    PubMed

    Jin, X; Liu, K; Jiao, B; Wang, X; Huang, S; Ren, W; Zhao, K

    2016-10-31

    Vincristine is an antitumor vinca alkaloid isolated from vinca rosea, and is a medication used to treat a number of types of cancer. In this study, we investigated the impact of vincristine on oncogenic phenotypes of human colorectal cancer HCT116 cells. MTT assay demonstrated that vincristine showed a obviously inhibitory effect on cell growth compared to non-treated cells. However, Transwell assay showed that vincristine promoted migration and invasion of HCT116 cells in vitro in a concentration-dependent manner between 0.5 and 15 μM vincristine treatment, whereas cell growth showed no remarkable difference within the same concentration range. Additionally, Western blot analysis showed that vincristine significantly elevated RhoA activity and Myosin light chain (MLC) phosphorylation, suggesting the involvement of RhoA/ROCK pathway in the vincristine-induced enhancement of cellular motility. Furthermore, we found that both the siRNA for RhoA and ROCK inhibitor Y27632 attenuated the phosphorylation of MLC, as well as vincristine-induced migration and invasion. These data indicate that vincristine enhanced migration and invasion of HCT116 cells possibly through stimulating RhoA/ROCK/MLC signaling pathway.

  9. Neurofilament light chain level in cerebrospinal fluid can differentiate Parkinson's disease from atypical parkinsonism: Evidence from a meta-analysis.

    PubMed

    Sako, Wataru; Murakami, Nagahisa; Izumi, Yuishin; Kaji, Ryuji

    2015-05-15

    A reliable test that facilitates the accurate diagnosis of Parkinson's and disorders will help with both, clinical management and therapeutic research. In this context, neurofilament light chain (NFL) is candidate for a biomarker in cerebrospinal fluid (CSF). A comprehensive literature search yielded 4 eligible studies. We expressed between-group difference of NFL concentration in CSF as the standardized mean difference. Four studies involved 166 Parkinson's disease (PD), 116 multiple system atrophy (MSA) and 73 progressive supranuclear palsy (PSP) patients. Patients with MSA showed higher concentration of NFL concentration in CSF than those with PD (standardized mean difference=1.60, P<0.0001). These studies were homogeneous (P=0.17). NFL in CSF in PSP was significantly elevated relative to PD with homogeneous studies (standardized mean difference=2.04, P<0.0001; P=0.99). The present meta-analysis suggested that NFL concentration in CSF in MSA and PSP was significantly increased relative to PD, and that this could help us to separate PD from atypical parkinsonian syndromes.

  10. Evaluation of Newcastle disease virus immunoassays for waterfowl using a monoclonal antibody specific for the duck immunoglobulin light chain.

    PubMed

    Kothlow, Sonja; Haüslaigner, Rafaela; Kaspers, Bernd; Grund, Christian

    2008-06-01

    In the present study a monoclonal antibody (mAb 14A3) was tested for its reactivity against serum immunoglobulin Y (IgY) of several waterfowl species, and subsequently for its applicability as anti-species antibody in common immunoassays. Western blot analyses demonstrated its broad cross-reactivity with the serum IgY light chain of different duck species: Muscovy duck (Cairina moschata), Mallard (Anas platyrhynchos), white-winged wood duck (Asarcornis scutulatus), common pintail (Dafila acuta). Reactivity was also evident with IgY of two swan species--mute swan (Cygnus olor) and black-necked swan (Sthenelides melanocoryphus)--and two goose species--domestic goose (Anser anser var. domestica) and red-breasted goose (Rufibrenta ruficollis). Applying the mAb for Newcastle disease virus (avian paramyxovirus serotype 1 [APMV-1]) test systems, its functionality within indirect immunoassays was evaluated. Using APMV-1-positive sera of domestic geese and Muscovy ducks, mAb 14A3 facilitated specific staining of APMV-1-infected cells in an immunofluorescence test. In addition, it proved to be functional in an indirect enzyme-linked immunosorbent assay (ELISA) and a western blot assay. Thus, the analysed mAb represents an attractive and versatile reagent that offers the opportunity to develop serological tests for waterfowl, allowing a high sample throughput using the ELISA technique or the fine analysis of humoral immune responses using the western blot.

  11. Interactions of Yeast Dynein with Dynein Light Chain and Dynactin: GENERAL IMPLICATIONS FOR INTRINSICALLY DISORDERED DUPLEX SCAFFOLDS IN MULTIPROTEIN ASSEMBLIES.

    PubMed

    Jie, Jing; Löhr, Frank; Barbar, Elisar

    2015-09-25

    Intrinsically disordered protein (IDP) duplexes composed of two IDP chains cross-linked by bivalent partner proteins form scaffolds for assembly of multiprotein complexes. The N-terminal domain of dynein intermediate chain (N-IC) is one such IDP that forms a bivalent scaffold with multiple dynein light chains including LC8, a hub protein that promotes duplex formation of diverse IDP partners. N-IC also binds a subunit of the dynein regulator, dynactin. Here we characterize interactions of a yeast ortholog of N-IC (N-Pac11) with yeast LC8 (Dyn2) or with the intermediate chain-binding subunit of yeast dynactin (Nip100). Residue level changes in Pac11 structure are monitored by NMR spectroscopy, and binding energetics are monitored by isothermal titration calorimetry (ITC). N-Pac11 is monomeric and primarily disordered except for a single α-helix (SAH) at the N terminus and a short nascent helix, LH, flanked by the two Dyn2 recognition motifs. Upon binding Dyn2, the only Pac11 residues making direct protein-protein interactions are in and immediately flanking the recognition motifs. Dyn2 binding also orders LH residues of Pac11. Upon binding Nip100, only Pac11 SAH residues make direct protein-protein interactions, but LH residues at a distant sequence position and L1 residues in an adjacent linker are also ordered. The long distance, ligand-dependent ordering of residues reveals new elements of dynamic structure within IDP linker regions.

  12. A mutation in the atrial-specific myosin light chain gene (MYL4) causes familial atrial fibrillation

    PubMed Central

    Orr, Nathan; Arnaout, Rima; Gula, Lorne J.; Spears, Danna A.; Leong-Sit, Peter; Li, Qiuju; Tarhuni, Wadea; Reischauer, Sven; Chauhan, Vijay S.; Borkovich, Matthew; Uppal, Shaheen; Adler, Arnon; Coughlin, Shaun R.; Stainier, Didier Y. R.; Gollob, Michael H.

    2016-01-01

    Atrial fibrillation (AF), the most common arrhythmia, is a growing epidemic with substantial morbidity and economic burden. Mechanisms underlying vulnerability to AF remain poorly understood, which contributes to the current lack of highly effective therapies. Recognizing mechanistic subtypes of AF may guide an individualized approach to patient management. Here, we describe a family with a previously unreported syndrome characterized by early-onset AF (age <35 years), conduction disease and signs of a primary atrial myopathy. Phenotypic penetrance was complete in all mutation carriers, although complete disease expressivity appears to be age-dependent. We show that this syndrome is caused by a novel, heterozygous p.Glu11Lys mutation in the atrial-specific myosin light chain gene MYL4. In zebrafish, mutant MYL4 leads to disruption of sarcomeric structure, atrial enlargement and electrical abnormalities associated with human AF. These findings describe the cause of a rare subtype of AF due to a primary, atrial-specific sarcomeric defect. PMID:27066836

  13. Abnormalities of the Duo/Rac-1/PAK1 Pathway Drive Myosin Light Chain Phosphorylation in Frontal Cortex in Schizophrenia

    PubMed Central

    Rubio, María D.; Haroutunian, Vahram; Meador-Woodruff, James H.

    2012-01-01

    BACKGROUND Recent studies on GTPases have suggested that reduced Duo and Cdc42 transcript expression is involved in dendritic spine loss in schizophrenia. In murine models, Duo and Cdc42 phosphorylate PAK1, which modifies the activity of regulatory myosin light chain (MLC) and cofilin by altering their phosphorylation. Therefore, we hypothesized that in schizophrenia abnormal Duo and Cdc42 expression result in changes in MLC and/or cofilin phosphorylation, which may alter actin cytoskeleton dynamics underlying dendritic spine maintenance. METHODS We performed Western blot protein expression analysis in postmortem brains from patients diagnosed with schizophrenia and a comparison group. We focused our studies in the anterior cingulate cortex (ACC) (n=33 comparison group; n=36 schizophrenia) and dorsolateral prefrontal cortex (DLPFC) (n=29 comparison group; n=35 schizophrenia). RESULTS In both ACC and DLPFC, we found a reduction of Duo expression and PAK1 phosphorylation in schizophrenia. Cdc42 protein expression was decreased in ACC, but not in DLPFC. In ACC, we observed decreased PAK1 phosphorylation and increased MLC (pMLC) phosphorylation, while in DLPFC pMLC remained unchanged. DISCUSSION These data suggest a novel mechanism that may underlie dendritic spine loss in schizophrenia. The increase in pMLC seen in ACC may be associated with dendritic spine shrinkage. The lack of an effect on pMLC in DLPFC suggests that in schizophrenia PAK1 downstream pathways are differentially affected in these cortical areas. PMID:22458949

  14. Cutaneous amyloidosis associated with autoimmune hepatitis-primary biliary cirrhosis overlap syndrome.

    PubMed

    González-Moreno, Emmanuel I; Cámara-Lemarroy, Carlos R; Borjas-Almaguer, David O; Martínez-Cabriales, Sylvia A; Paz-Delgadillo, Jonathan; Gutiérrez-Udave, Rodrigo; Ayala-Cortés, Ana S; Ocampo-Candiani, Jorge; Cortéz-Hernández, Carlos A; Maldonado-Garza, Héctor J

    2015-01-01

    Cutaneous amyloidosis is a rare disease characterized by the deposition of amyloid in the dermis. It can be primary or secondary, depending on associated diseases. It has been linked to various autoimmune diseases, including primary biliary cirrhosis. We present the case of a patient with an autoimmune hepatitis-primary biliary cirrhosis overlap syndrome with concomitant cutaneous amyloidosis, a very unusual association, and discuss similar cases and possible pathophysiological implications.

  15. Induction of cerebral beta-amyloidosis: intracerebral versus systemic Abeta inoculation.

    PubMed

    Eisele, Yvonne S; Bolmont, Tristan; Heikenwalder, Mathias; Langer, Franziska; Jacobson, Laura H; Yan, Zheng-Xin; Roth, Klaus; Aguzzi, Adriano; Staufenbiel, Matthias; Walker, Lary C; Jucker, Mathias

    2009-08-04

    Despite the importance of the aberrant polymerization of Abeta in the early pathogenic cascade of Alzheimer's disease, little is known about the induction of Abeta aggregation in vivo. Here we show that induction of cerebral beta-amyloidosis can be achieved in many different brain areas of APP23 transgenic mice through the injection of dilute Abeta-containing brain extracts. Once the amyloidogenic process has been exogenously induced, the nature of the induced Abeta-deposition is determined by the brain region of the host. Because these observations are reminiscent of a prion-like mechanism, we then investigated whether cerebral beta-amyloidosis also can be induced by peripheral and systemic inoculations or by the intracerebral implantation of stainless steel wires previously coated with minute amounts of Abeta-containing brain extract. Results reveal that oral, intravenous, intraocular, and intranasal inoculations yielded no detectable induction of cerebral beta-amyloidosis in APP23 transgenic mice. In contrast, transmission of cerebral beta-amyloidosis through the Abeta-contaminated steel wires was demonstrated. Notably, plasma sterilization, but not boiling of the wires before implantation, prevented the induction of beta-amyloidosis. Our results suggest that minute amounts of Abeta-containing brain material in direct contact with the CNS can induce cerebral beta-amyloidosis, but that systemic cellular mechanisms of prion uptake and transport to the CNS may not apply to Abeta.

  16. Serum Free Light Chains

    MedlinePlus

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  17. Testicular amyloidosis in hamsters experimentally infected with Leishmania donovani.

    PubMed Central

    Gonzalez, J. L.; Gallego, E.; Castaño, M.; Rueda, A.

    1983-01-01

    Thirty hamsters were inoculated intraperitoneally with Leishmania donovani. Testes were examined grossly and histologically by light and electron microscopy. Progressive testicular atrophy developed. Spermatogenic cells of the seminiferous tubules showed vacuolar degeneration and decreased in number leading to a total azoospermia in the final weeks of the pathological process. Lymphoplasmocytic infiltrates with macrophages containing leishmanias appeared in the intertubular space. Amyloid deposits in the intertubular space and tubular basement membrane were identified by optical and ultrastructural methods. It has been suggested that testicular amyloidosis may have a pathogenic mechanism related to a dysfunction of plasma cells and stimulation of the reticuloendothial system, due to the antigenic character of the parasite. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6639870

  18. Phosphorylation Regulates Interaction of 210-kDa Myosin Light Chain Kinase N-terminal Domain with Actin Cytoskeleton.

    PubMed

    Vilitkevich, E L; Khapchaev, A Y; Kudryashov, D S; Nikashin, A V; Schavocky, J P; Lukas, T J; Watterson, D M; Shirinsky, V P

    2015-10-01

    High molecular weight myosin light chain kinase (MLCK210) is a multifunctional protein involved in myosin II activation and integration of cytoskeletal components in cells. MLCK210 possesses actin-binding regions both in the central part of the molecule and in its N-terminal tail domain. In HeLa cells, mitotic protein kinase Aurora B was suggested to phosphorylate MLCK210 N-terminal tail at serine residues (Dulyaninova, N. G., and Bresnick, A. R. (2004) Exp. Cell Res., 299, 303-314), but the functional significance of the phosphorylation was not established. We report here that in vitro, the N-terminal actin-binding domain of MLCK210 is located within residues 27-157 (N27-157, avian MLCK210 sequence) and is phosphorylated by cAMP-dependent protein kinase (PKA) and Aurora B at serine residues 140/149 leading to a decrease in N27-157 binding to actin. The same residues are phosphorylated in a PKA-dependent manner in transfected HeLa cells. Further, in transfected cells, phosphomimetic mutants of N27-157 showed reduced association with the detergent-stable cytoskeleton, whereas in vitro, the single S149D mutation reduced N27-157 association with F-actin to a similar extent as that achieved by N27-157 phosphorylation. Altogether, our results indicate that phosphorylation of MLCK210 at distinct serine residues, mainly at S149, attenuates the interaction of MLCK210 N-terminus with the actin cytoskeleton and might serve to regulate MLCK210 microfilament cross-linking activity in cells.

  19. Structure of a Yeast Dyn2-Nup159 Complex and Molecular Basis for Dynein Light Chain-Nuclear Pore Interaction*

    PubMed Central

    Romes, Erin M.; Tripathy, Ashutosh; Slep, Kevin C.

    2012-01-01

    The nuclear pore complex gates nucleocytoplasmic transport through a massive, eight-fold symmetric channel capped by a nucleoplasmic basket and structurally unique, cytoplasmic fibrils whose tentacles bind and regulate asymmetric traffic. The conserved Nup82 complex, composed of Nsp1, Nup82, and Nup159, forms the unique cytoplasmic fibrils that regulate mRNA nuclear export. Although the nuclear pore complex plays a fundamental, conserved role in nuclear trafficking, structural information about the cytoplasmic fibrils is limited. Here, we investigate the structural and biochemical interactions between Saccharomyces cerevisiae Nup159 and the nucleoporin, Dyn2. We find that Dyn2 is predominantly a homodimer and binds arrayed sites on Nup159, promoting the Nup159 parallel homodimerization. We present the first structure of Dyn2, determined at 1.85 Å resolution, complexed with a Nup159 target peptide. Dyn2 resembles homologous metazoan dynein light chains, forming homodimeric composite substrate binding sites that engage two independent 10-residue target motifs, imparting a β-strand structure to each peptide via antiparallel extension of the Dyn2 core β-sandwich. Dyn2 recognizes a highly conserved QT motif while allowing sequence plasticity in the flanking residues of the peptide. Isothermal titration calorimetric analysis of the comparative binding of Dyn2 to two Nup159 target sites shows similar affinities (18 and 13 μm), but divergent thermal binding modes. Dyn2 homodimers are arrayed in the crystal lattice, likely mimicking the arrayed architecture of Dyn2 on the Nup159 multivalent binding sites. Crystallographic interdimer interactions potentially reflect a cooperative basis for Dyn2-Nup159 complex formation. Our data highlight the determinants that mediate oligomerization of the Nup82 complex and promote a directed, elongated cytoplasmic fibril architecture. PMID:22411995

  20. Recombinant enterokinase light chain with affinity tag: expression from Saccharomyces cerevisiae and its utilities in fusion protein technology.

    PubMed

    Choi, S I; Song, H W; Moon, J W; Seong, B L

    2001-12-20

    Enterokinase and recombinant enterokinase light chain (rEK(L)) have been used widely to cleave fusion proteins with the target sequence of (Asp)(4)-Lys. In this work, we show that their utility as a site-specific cleavage agent is compromised by sporadic cleavage at other sites, albeit at low levels. Further degradation of the fusion protein in cleavage reaction is due to an intrinsic broad specificity of the enzyme rather than to the presence of contaminating proteases. To offer facilitated purification from fermentation broth and efficient removal of rEK(L) after cleavage reaction, thus minimizing unwanted cleavage of target protein, histidine affinity tag was introduced into rEK(L). Utilizing the secretion enhancer peptide derived from the human interleukin 1 beta, the recombinant EK(L) was expressed in Saccharomyces cerevisiae and efficiently secreted into culture medium. The C-terminal His-tagged EK(L) was purified in a single-step procedure on nickel affinity chromatography. It retained full enzymatic activity similar to that of EK(L), whereas the N-terminal His-tagged EK(L) was neither efficiently purified nor had any enzymatic activity. After cleavage reaction of fusion protein, the C-terminal His-tagged EK(L) was efficiently removed from the reaction mixture by a single passage through nickel-NTA spin column. The simple affinity tag renders rEK(L) extremely useful for purification, post-cleavage removal, recovery, and recycling and will broaden the utility and the versatility of the enterokinase for the production of recombinant proteins.

  1. Elevation of serum immunoglobulin free light chains during the pre-clinical period of rheumatoid arthritis: impact on mortality

    PubMed Central

    Deng, Xiaoli; Crowson, Cynthia S.; Rajkumar, S. Vincent; Dispenzieri, Angela; Larson, Dirk R.; Therneau, Terry M.; Matteson, Eric L.; Kyle, Robert A.; Katzmann, Jerry; Gabriel, Sherine E.; Davis, John M.

    2015-01-01

    Objective Immunoglobulin free light chains (FLCs) represent biomarkers of B-cell activity in rheumatoid arthritis (RA) and are associated with all-cause mortality in the general population. Our objective was to evaluate the relationships of serum FLCs to pre-clinical disease, RA characteristics, and mortality in RA compared to non-RA subjects. Methods A population-based study in Olmsted County, Minnesota, was performed by cross-linking a large cohort in the general population having available serum FLC measurements with established RA incidence and prevalence cohorts. Serum κ, λ, and total FLCs and their trends relative to RA incidence were compared between RA and non-RA subjects. Regression models were used to determine the associations between FLCs, disease characteristics and mortality, testing for differential effects of FLCs on mortality in RA. Results Among 16,609 subjects, 270 fulfilled the criteria for RA at the time of FLC measurement. Mean total FLCs were significantly higher in RA compared to non-RA subjects (4.2 vs. 3.3 mg/dL; p<0.001). FLCs became elevated 3 – 5 years before the clinical onset of RA and remained elevated during follow-up. Polyclonal FLCs were found to predict higher mortality in persons with RA, though elevation to the highest decile had a relatively lower impact on mortality in RA compared to non-RA subjects. Conclusions Elevation of serum FLCs precedes the development of RA and may be useful in monitoring B-cell activity and disease progression. FLCs are confirmed predictors of mortality, though the highest elevations of FLCs have a lower impact on mortality in RA than the general population. PMID:25593227

  2. Structural And Biochemical Studies of Botulinum Neurotoxin Serotype C1 Light Chain Protease: Implications for Dual Substrate Specificity

    SciTech Connect

    Jin, R.; Sikorra, S.; Stegmann, C.M.; Pich, A.; Binz, T.; Brunger, A.T.

    2009-06-01

    Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote a-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the a-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.

  3. Phosphorylation of myosin regulatory light chain eliminates force-dependent changes in relaxation rates in skeletal muscle.

    PubMed Central

    Patel, J R; Diffee, G M; Huang, X P; Moss, R L

    1998-01-01

    The rate of relaxation from steady-state force in rabbit psoas fiber bundles was examined before and after phosphorylation of myosin regulatory light chain (RLC). Relaxation was initiated using diazo-2, a photolabile Ca2+ chelator that has low Ca2+ binding affinity (K(Ca) = 4.5 x 10(5) M(-1)) before photolysis and high affinity (K(Ca) = 1.3 x 10(7) M(-1)) after photolysis. Before phosphorylating RLC, the half-times for relaxation initiated from 0.27 +/- 0.02, 0.51 +/- 0.03, and 0.61 +/- 0.03 Po were 90 +/- 6, 140 +/- 6, and 182 +/- 9 ms, respectively. After phosphorylation of RLC, the half-times for relaxation from 0.36 +/- 0.03 Po, 0.59 +/- 0.03 Po, and 0.65 +/- 0.02 Po were 197 +/- 35 ms, 184 +/- 35 ms, and 179 +/- 22 ms. This slowing of relaxation rates from steady-state forces less than 0.50 Po was also observed when bundles of fibers were bathed with N-ethylmaleimide-modified myosin S-1, a strongly binding cross-bridge derivative of S1. These results suggest that phosphorylation of RLC slows relaxation, most likely by slowing the apparent rate of transition of cross-bridges from strongly bound (force-generating) to weakly bound (non-force-generating) states, and reduces or eliminates Ca2+ and cross-bridge activation-dependent changes in relaxation rates. PMID:9449336

  4. Structure-function analysis of dynein light chain 1 identifies viable motility mutants in bloodstream-form Trypanosoma brucei.

    PubMed

    Ralston, Katherine S; Kisalu, Neville K; Hill, Kent L

    2011-07-01

    The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that is receiving increasing attention as a potential drug target and as a system for studying flagellum biology. RNA interference (RNAi) knockdown is widely used to test the requirement for a protein in flagellar motility and has suggested that normal flagellar motility is essential for viability in bloodstream-form trypanosomes. However, RNAi knockdown alone provides limited functional information because the consequence is often loss of a multiprotein complex. We therefore developed an inducible system that allows functional analysis of point mutations in flagellar proteins in T. brucei. Using this system, we identified point mutations in the outer dynein light chain 1 (LC1) that allow stable assembly of outer dynein motors but do not support propulsive motility. In procyclic-form trypanosomes, the phenotype of LC1 mutants with point mutations differs from the motility and structural defects of LC1 knockdowns, which lack the outer-arm dynein motor. Thus, our results distinguish LC1-specific functions from broader functions of outer-arm dynein. In bloodstream-form trypanosomes, LC1 knockdown blocks cell division and is lethal. In contrast, LC1 point mutations cause severe motility defects without affecting viability, indicating that the lethal phenotype of LC1 RNAi knockdown is not due to defective motility. Our results demonstrate for the first time that normal motility is not essential in bloodstream-form T. brucei and that the presumed connection between motility and viability is more complex than might be interpreted from knockdown studies alone. These findings open new avenues for dissecting mechanisms of flagellar protein function and provide an important step in efforts to exploit the potential of the flagellum as a therapeutic target in African sleeping sickness.

  5. Urinary free light chains may help to identify infection in patients with elevated systemic inflammation due to rheumatic disease.

    PubMed

    Bramlage, Carsten P; Froelich, Britta; Wallbach, Manuel; Minguet, Joan; Grupp, Clemens; Deutsch, Cornelia; Bramlage, Peter; Müller, Gerhard A; Koziolek, Michael

    2017-04-01

    The risk of infection in patients with rheumatic diseases is elevated, but a clear marker to differentiate the cause of the systemic inflammation is missing. We assessed the ability urinary immunoglobulin free light chains (FLCs) to indicate the presence of infection in patients with rheumatic disease. We performed a retrospective analysis of patients with rheumatic disease attending the Georg-August University Hospital in Goettingen, Germany, from January 2011 to December 2013. Subjects were included if they had urine levels of κ and λ FLCs available. A reference group of patients without autoimmune disease, but with documented infection, was constructed. A total of 1500 patients had their urinary FLCs quantified during the study period. Of the 382 patients with rheumatic disease, 172 (45%) displayed no systemic inflammation, 162 (42%) had inflammation due to the underlying disease activity, and 48 (13%) had inflammation due to a confirmed infection. Urinary FLC concentrations were much higher in patients with rheumatic diseases and infection (κ 68.8 ± 81.8 mg/L, λ 31.4 ± 53.5 mg/L) compared to those with inflammation due to rheumatic disease activity (κ 22.7 ± 26.3 mg/L, λ 8.1 ± 9.1 mg/L, κ p < 0.001, λ p = 0.004). Urinary κ FLCs demonstrated good ability to predict infection, with a sensitivity of 63% and specificity of 84%. Urinary λ FLCs gave similar values, with a sensitivity of 65% and specificity of 81%. FLCs may be useful for distinguishing inflammation due to rheumatic disease activity from that due to the additional presence of infection. The ability to quantify these proteins in urine provides a simple alternative to the use of blood.

  6. The significance and predictive value of free light chains in the urine of patients with chronic inflammatory rheumatic disease.

    PubMed

    Bramlage, Carsten Paul; Froelich, Britta; Wallbach, Manuel; Minguet, Joan; Grupp, Clemens; Deutsch, Cornelia; Bramlage, Peter; Koziolek, Michael; Müller, Gerhard Anton

    2016-12-01

    In patients with rheumatic diseases, reliable markers for determining disease activity are scarce. One potential parameter is the level of immunoglobulin free light chains (FLCs), which is known to be elevated in the blood of patients with certain rheumatic diseases. Few studies have quantified FLCs in urine, a convenient source of test sample, in patients with different rheumatic diseases. We carried out a retrospective analysis of patients with rheumatic disease attending the University hospital of Goettingen, Germany. Subjects were included if they had urine levels of both κ and λ FLCs available and did not have myeloma. Data regarding systemic inflammation and kidney function were recorded, and FLC levels were correlated with inflammatory markers. Of the 382 patients with rheumatic disease, 40.1 % had chronic polyarthritis, 21.2 % connective tissue disease, 18.6 % spondyloarthritis and 15.7 % vasculitis. Elevated levels of κ FLCs were found for 84 % of patients and elevated λ for 52.7 %. For the patients with rheumatoid arthritis, FLCs correlated with C-reactive protein (κ, r = 0.368, p < 0.001; λ, r = 0.398, p < 0.001) and erythrocyte sedimentation rate (κ, r = 0.692, p < 0.001; λ, r = 0.612, p < 0.001). Patients being treated with rituximab displayed FLC levels similar to those of the reference group. There were clear elevations in both κ and λ FLCs in patients with rheumatic disease, but not in κ/λ ratio. The correlation between FLCs and inflammatory markers in patients with rheumatoid arthritis demonstrates their potential for predicting disease activity.

  7. Biphasic regulation of myosin light chain phosphorylation by p21-activated kinase modulates intestinal smooth muscle contractility.

    PubMed

    Chu, Ji; Pham, Ngoc T; Olate, Nicole; Kislitsyna, Karina; Day, Mary-Clare; LeTourneau, Phillip A; Kots, Alexander; Stewart, Randolph H; Laine, Glen A; Cox, Charles S; Uray, Karen

    2013-01-11

    Supraphysiological mechanical stretching in smooth muscle results in decreased contractile activity. However, the mechanism is unclear. Previous studies indicated that intestinal motility dysfunction after edema development is associated with increased smooth muscle stress and decreased myosin light chain (MLC) phosphorylation in vivo, providing an ideal model for studying mechanical stress-mediated decrease in smooth muscle contraction. Primary human intestinal smooth muscle cells (hISMCs) were subjected to either control cyclical stretch (CCS) or edema (increasing) cyclical stretch (ECS), mimicking the biophysical forces in non-edematous and edematous intestinal smooth muscle in vivo. ECS induced significant decreases in phosphorylation of MLC and MLC phosphatase targeting subunit (MYPT1) and a significant increase in p21-activated kinase (PAK) activity compared with CCS. PAK regulated MLC phosphorylation in an activity-dependent biphasic manner. PAK activation increased MLC and MYPT1 phosphorylation in CCS but decreased MLC and MYPT1 phosphorylation in hISMCs subjected to ECS. PAK inhibition had the opposite results. siRNA studies showed that PAK1 plays a critical role in regulating MLC phosphorylation in hISMCs. PAK1 enhanced MLC phosphorylation via phosphorylating MYPT1 on Thr-696, whereas PAK1 inhibited MLC phosphorylation via decreasing MYPT1 on both Thr-696 and Thr-853. Importantly, in vivo data indicated that PAK activity increased in edematous tissue, and inhibition of PAK in edematous intestine improved intestinal motility. We conclude that PAK1 positively regulates MLC phosphorylation in intestinal smooth muscle through increasing inhibitory phosphorylation of MYPT1 under physiologic conditions, whereas PAK1 negatively regulates MLC phosphorylation via inhibiting MYPT1 phosphorylation when PAK activity is increased under pathologic conditions.

  8. Comparison of Orientation and Rotational Motion of Skeletal Muscle Cross-bridges Containing Phosphorylated and Dephosphorylated Myosin Regulatory Light Chain*

    PubMed Central

    Midde, Krishna; Rich, Ryan; Marandos, Peter; Fudala, Rafal; Li, Amy; Gryczynski, Ignacy; Borejdo, Julian

    2013-01-01

    Calcium binding to thin filaments is a major element controlling active force generation in striated muscles. Recent evidence suggests that processes other than Ca2+ binding, such as phosphorylation of myosin regulatory light chain (RLC) also controls contraction of vertebrate striated muscle (Cooke, R. (2011) Biophys. Rev. 3, 33–45). Electron paramagnetic resonance (EPR) studies using nucleotide analog spin label probes showed that dephosphorylated myosin heads are highly ordered in the relaxed fibers and have very low ATPase activity. This ordered structure of myosin cross-bridges disappears with the phosphorylation of RLC (Stewart, M. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 430–435). The slower ATPase activity in the dephosporylated moiety has been defined as a new super-relaxed state (SRX). It can be observed in both skeletal and cardiac muscle fibers (Hooijman, P., Stewart, M. A., and Cooke, R. (2011) Biophys. J. 100, 1969–1976). Given the importance of the finding that suggests a novel pathway of regulation of skeletal muscle, we aim to examine the effects of phosphorylation on cross-bridge orientation and rotational motion. We find that: (i) relaxed cross-bridges, but not active ones, are statistically better ordered in muscle where the RLC is dephosporylated compared with phosphorylated RLC; (ii) relaxed phosphorylated and dephosphorylated cross-bridges rotate equally slowly; and (iii) active phosphorylated cross-bridges rotate considerably faster than dephosphorylated ones during isometric contraction but the duty cycle remained the same, suggesting that both phosphorylated and dephosphorylated muscles develop the same isometric tension at full Ca2+ saturation. A simple theory was developed to account for this fact. PMID:23319584

  9. Phosphorylated Myosin Light Chain 2 (p-MLC2) as a Molecular Marker of Antemortem Coronary Artery Spasm

    PubMed Central

    Li, Liliang; Li, Yuhua; Lin, Junyi; Jiang, Jieqing; He, Meng; Sun, Daming; Zhao, Ziqin; Shen, Yiwen; Xue, Aimin

    2016-01-01

    Background It is not uncommon that only mild coronary artery stenosis is grossly revealed after a system autopsy. While coronary artery spasm (CAS) is the suspected mechanism of these deaths, no specific biomarker has been identified to suggest antemortem CAS. Material/Methods To evaluate the potential of using phosphorylated myosin light chain 2 (p-MLC2) as a diagnostic marker of antemortem CAS, human vascular smooth muscle cells (VSMCs) were cultured and treated with common vasoconstrictors, including prostaglandins F2α (PGF2α), acetylcholine (ACh), and 5-hydroxy tryptamine (5-HT). The p-MLC2 level was examined in the cultured cells using Western blot analysis and in a rat model of spasm provocation tests using immunohistochemistry (IHC). Effects of increased p-MLC2 level on VSMCs contractile activities were assessed in vitro using confocal immunofluorescence assay. Four fatal cases with known antemortem CAS were collected and subject to p-MLC2 detection. Results The p-MLC2 was significantly increased in VSMCs after treatments with vasoconstrictors and in the spasm provocation tests. Myofilament was well-organized and densely stained in VSMCs with high p-MLC2 level, but disarrayed in VSMCs with low p-MLC2 level. Three of the 4 autopsied cases showed strongly positive staining of p-MLC2 at the stenosed coronary segment and the adjacent interstitial small arteries. The fourth case was autopsied at the 6th day after death and showed negative-to-mild positive staining of p-MLC2. Conclusions p-MLC2 might be a useful marker for diagnosis of antemortem CAS. Autopsy should be performed as soon as possible to collect coronary arteries for detection of p-MLC2. PMID:27643564

  10. The LC7 Light Chains of Chlamydomonas Flagellar Dyneins Interact with Components Required for Both Motor Assembly and Regulation

    PubMed Central

    DiBella, Linda M.; Sakato, Miho; Patel-King, Ramila S.; Pazour, Gregory J.; King, Stephen M.

    2004-01-01

    Members of the LC7/Roadblock family of light chains (LCs) have been found in both cytoplasmic and axonemal dyneins. LC7a was originally identified within Chlamydomonas outer arm dynein and associates with this motor's cargo-binding region. We describe here a novel member of this protein family, termed LC7b that is also present in the Chlamydomonas flagellum. Levels of LC7b are reduced ∼20% in axonemes isolated from strains lacking inner arm I1 and are ∼80% lower in the absence of the outer arms. When both dyneins are missing, LC7b levels are diminished to <10%. In oda9 axonemal extracts that completely lack outer arms, LC7b copurifies with inner arm I1, whereas in ida1 extracts that are devoid of I1 inner arms it associates with outer arm dynein. We also have observed that some LC7a is present in both isolated axonemes and purified 18S dynein from oda1, suggesting that it is also a component of both the outer arm and inner arm I1. Intriguingly, in axonemal extracts from the LC7a null mutant, oda15, which assembles ∼30% of its outer arms, LC7b fails to copurify with either dynein, suggesting that it interacts with LC7a. Furthermore, both the outer arm γ heavy chain and DC2 from the outer arm docking complex completely dissociate after salt extraction from oda15 axonemes. EDC cross-linking of purified dynein revealed that LC7b interacts with LC3, an outer dynein arm thioredoxin; DC2, an outer arm docking complex component; and also with the phosphoprotein IC138 from inner arm I1. These data suggest that LC7a stabilizes both the outer arms and inner arm I1 and that both LC7a and LC7b are involved in multiple intradynein interactions within both dyneins. PMID:15304520

  11. A shrimp pacifastin light chain-like inhibitor: molecular identification and role in the control of the prophenoloxidase system.

    PubMed