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Sample records for light-chain amyloidosis al

  1. 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

  2. AL (Light-Chain) Cardiac Amyloidosis: A Review of Diagnosis and Therapy.

    PubMed

    Falk, Rodney H; Alexander, Kevin M; Liao, Ronglih; Dorbala, Sharmila

    2016-09-20

    The amyloidoses are a group of protein-folding disorders in which ≥1 organ is infiltrated by proteinaceous deposits known as amyloid. The deposits are derived from 1 of several amyloidogenic precursor proteins, and the prognosis of the disease is determined both by the organ(s) involved and the type of amyloid. Amyloid involvement of the heart (cardiac amyloidosis) carries the worst prognosis of any involved organ, and light-chain (AL) amyloidosis is the most serious form of the disease. The last decade has seen considerable progress in understanding the amyloidoses. In this review, current and novel approaches to the diagnosis and treatment of cardiac amyloidosis are discussed, with particular reference to AL amyloidosis in the heart. PMID:27634125

  3. [Light Chain Amyloidosis: an Update for Treatment].

    PubMed

    Shen, Kai-Ni; Li, Jian

    2015-06-01

    Systemic light chain amyloidosis (AL amyloidosis) is the most common type of amyloidosis, in which deposition of misfolded monoclonal light chain secreted by underlying clonal plasma cells leads to organ dysfunction. Tissue biopsy of involved organ is needed to confirm the type of amyloid deposits, thus proper treatment could be applied. Laser microdissection followed by mass spectrometry, performed on formalin-fixed paraffin-embedded specimens, has been proven superior to traditional methods on accurate diagnosis of amyloidosis. Prognosis depends on the extent of cardiac involvement. The Mayo staging system using NT-ProBNP, cardiac troponin-T and free light chain, is the most robust method for risk stratification and treatment guidance. The introduction of autologous stem cell transplantation (auto-ASCT) resulted in long-term survival in responders, while treatment-related toxicity substantially limited the number of eligible candidates. Novel agents, especially bortezomib, thalidomide and lenalidomide hold promise to achieve comparable hematological responses with auto-ASCT, which might play significant role in treatment of recurrent or refractory AL amyloidosis. PMID:26117060

  4. [The concurrence of light-chain deposition disease, AL-amyloidosis, and cast nephropathy in a patient with multiple myeloma].

    PubMed

    Rekhtina, I G; Zakharova, E V; Stoliarevich, E S; Sinitsina, M N; Denisova, E N

    2015-01-01

    Despite of the fact that their clinical manifestations are similar, AL-amyloidosis (AL-A) and light chain deposition disease (LCDD) are individual nosological entities in view of considerable differences in their pathogenesis and pathomorphology. The paper describes a rare case of the concurrence of LCDD and AL-A in a patient with multiple myeloma. Clinically, there was dialysis-dependent renal failure, flail leg syndrome, myocardiopathy, and rhabdomyolysis. At the disease onset, his nephrobiopsy specimen could diagnose LCDD and myeloma or cast nephropathy. The disease was characterized by an aggressive course. Despite the administration of innovative agents, the patient had a short-term remission and died from disease progression. Autopsy additionally revealed amyloid deposition in the heart and kidney. The development of AL-A in the presence of prior LCDD may reflect the progression of the tumor and the appearance of an additional subclone of plasma cells that produce amyloidogenic light chains. The uncommonness of this case is that renal amyloid was found in the tubular casts and absent in the glomeruli, which may be considered as a special form--tubular AL-amyloidosis. PMID:26281203

  5. Increased Serum Free Light Chains Precede the Presentation of Immunoglobulin Light Chain Amyloidosis

    PubMed Central

    Weiss, Brendan M.; Hebreo, Joseph; Cordaro, Daniel V.; Roschewski, Mark J.; Baker, Thomas P.; Abbott, Kevin C.; Olson, Stephen W.

    2014-01-01

    Purpose Patients with immunoglobulin light chain amyloidosis (AL amyloidosis) generally present with advanced organ dysfunction and have a high risk of early death. We sought to characterize monoclonal immunoglobulin (M-Ig) light chains before clinical presentation of AL amyloidosis. Patients and Methods We obtained prediagnostic sera from 20 cases with AL amyloidosis and 20 healthy controls matched for age, sex, race, and age of serum sample from the Department of Defense Serum Repository. Serum protein electrophoresis with immunofixation and serum free light chain (FLC) analysis were performed on all samples. Results An M-Ig was detected in 100% of cases and 0% of controls (P < .001). The M-Ig was present in 100%, 80%, and 42% of cases at less than 4 years, 4 to 11 years, and more than 11 years before diagnosis, respectively. The median FLC differential (FLC-diff) was higher in cases compared with controls at all time periods, less than 4 years (174.8 v 0.3 mg/L; P < .001), 4 to 11 years (65.1 v 2.2 mg/L; P < .001), and more than 11 years (4.5 v 0.4 mg/L; P = .03) before diagnosis. The FLC-diff was greater than 23 mg/L in 85% of cases and 0% of controls (P < .001). The FLC-diff level increased more than 10% per year in 84% of cases compared with 16% of controls (P < .001). Conclusion Increase of FLCs, including within the accepted normal range, precedes the development of AL amyloidosis for many years. PMID:25024082

  6. Recurrent Syncope and Cardiac Arrest in a Patient with Systemic Light Chain Amyloidosis Treated with Bortezomib.

    PubMed

    Jaipaul, Navin; Pi, Alexander; Zhang, Zhiwei

    2016-05-10

    About 10-15% of patients with multiple myeloma develop light chain (AL) amyloidosis. AL amyloidosis is a systemic disease that may involve multiple organs, often including the heart. It may present clinically with bradyarrhythmia and syncope. The proteasome inhibitor bortezomib has been used with clinical efficacy in treating patients with AL amyloidosis but also implicated as a possible cause of cardiomyocyte injury. We report a case of a 48-year-old man with AL amyloidosis and increased frequency of syncope and cardiac arrest after starting bortezomib. The biologic and clinical plausibility of a heightened risk for cardiac arrest in patients with cardiac AL amyloidosis and history of syncope being treated with bortezomib is a possibility that is not well documented in the medical literature and warrants further investigation. PMID:27499835

  7. Recurrent Syncope and Cardiac Arrest in a Patient with Systemic Light Chain Amyloidosis Treated with Bortezomib

    PubMed Central

    Jaipaul, Navin; Pi, Alexander; Zhang, Zhiwei

    2016-01-01

    About 10-15% of patients with multiple myeloma develop light chain (AL) amyloidosis. AL amyloidosis is a systemic disease that may involve multiple organs, often including the heart. It may present clinically with bradyarrhythmia and syncope. The proteasome inhibitor bortezomib has been used with clinical efficacy in treating patients with AL amyloidosis but also implicated as a possible cause of cardiomyocyte injury. We report a case of a 48-year-old man with AL amyloidosis and increased frequency of syncope and cardiac arrest after starting bortezomib. The biologic and clinical plausibility of a heightened risk for cardiac arrest in patients with cardiac AL amyloidosis and history of syncope being treated with bortezomib is a possibility that is not well documented in the medical literature and warrants further investigation. PMID:27499835

  8. Cell Damage in Light Chain Amyloidosis: FIBRIL INTERNALIZATION, TOXICITY AND CELL-MEDIATED SEEDING.

    PubMed

    Marin-Argany, Marta; Lin, Yi; Misra, Pinaki; Williams, Angela; Wall, Jonathan S; Howell, Kyle G; Elsbernd, Laura R; McClure, Megan; Ramirez-Alvarado, Marina

    2016-09-16

    Light chain (AL) amyloidosis is an incurable human disease characterized by the misfolding, aggregation, and systemic deposition of amyloid composed of immunoglobulin light chains (LC). This work describes our studies on potential mechanisms of AL cytotoxicity. We have studied the internalization of AL soluble proteins and amyloid fibrils into human AC16 cardiomyocytes by using real time live cell image analysis. Our results show how external amyloid aggregates rapidly surround the cells and act as a recruitment point for soluble protein, triggering the amyloid fibril elongation. Soluble protein and external aggregates are internalized into AC16 cells via macropinocytosis. AL amyloid fibrils are shown to be highly cytotoxic at low concentrations. Additionally, caspase assays revealed soluble protein induces apoptosis, demonstrating different cytotoxic mechanisms between soluble protein and amyloid aggregates. This study emphasizes the complex immunoglobulin light chain-cell interactions that result in fibril internalization, protein recruitment, and cytotoxicity that may occur in AL amyloidosis. PMID:27462073

  9. Systemic immunoglobulin light-chain amyloidosis presenting hematochezia as the initial symptom.

    PubMed

    Kon, Tetsuo; Nakagawa, Naoki; Yoshikawa, Fumitsugu; Haba, Kazunao; Kitagawa, Nagako; Izumi, Michihiro; Kumazaki, Setsuo; Ishida, Satoshi; Aikawa, Ryuichi

    2016-08-01

    Immunoglobulin light-chain (AL) amyloidosis is characterized by the deposition of insoluble fibrils composed of immunoglobulin light chains secreted by monoclonal plasma cells. Given the recent advances in the therapy of AL amyloidosis, it is important to diagnose this disease as early as possible. Herein, we describe the case of a 62-year-old man with hepatitis C virus (HCV)-related cirrhosis presenting with hematochezia. Colonoscopy showed multiple submucosal hematomas within the region ranging from the transverse colon to the sigmoid colon. Kappa immunoglobulin light-chain amyloid deposition was also detected. Bone marrow examination revealed a monoclonal abnormal plasma cell population. Thus, the patient was diagnosed with systemic immunoglobulin light-chain amyloidosis. The hematochezia was conservatively managed. However, because of liver failure caused by liver cirrhosis, the patient developed massive pleural effusion and died of respiratory failure. Postmortem examination revealed amyloid deposition in the esophagus, stomach, duodenum, ileum, descending colon, pancreas, heart, and lung. In these organs, amyloid deposition was limited to the vascular wall. We concluded that AL amyloidosis can present hematochezia arising from submucosal hematoma in the large colon before other systemic symptoms appear.

  10. Systemic immunoglobulin light-chain amyloidosis presenting hematochezia as the initial symptom.

    PubMed

    Kon, Tetsuo; Nakagawa, Naoki; Yoshikawa, Fumitsugu; Haba, Kazunao; Kitagawa, Nagako; Izumi, Michihiro; Kumazaki, Setsuo; Ishida, Satoshi; Aikawa, Ryuichi

    2016-08-01

    Immunoglobulin light-chain (AL) amyloidosis is characterized by the deposition of insoluble fibrils composed of immunoglobulin light chains secreted by monoclonal plasma cells. Given the recent advances in the therapy of AL amyloidosis, it is important to diagnose this disease as early as possible. Herein, we describe the case of a 62-year-old man with hepatitis C virus (HCV)-related cirrhosis presenting with hematochezia. Colonoscopy showed multiple submucosal hematomas within the region ranging from the transverse colon to the sigmoid colon. Kappa immunoglobulin light-chain amyloid deposition was also detected. Bone marrow examination revealed a monoclonal abnormal plasma cell population. Thus, the patient was diagnosed with systemic immunoglobulin light-chain amyloidosis. The hematochezia was conservatively managed. However, because of liver failure caused by liver cirrhosis, the patient developed massive pleural effusion and died of respiratory failure. Postmortem examination revealed amyloid deposition in the esophagus, stomach, duodenum, ileum, descending colon, pancreas, heart, and lung. In these organs, amyloid deposition was limited to the vascular wall. We concluded that AL amyloidosis can present hematochezia arising from submucosal hematoma in the large colon before other systemic symptoms appear. PMID:27318996

  11. Immunoglobulin Light-Chain Amyloidosis: From Basics to New Developments in Diagnosis, Prognosis and Therapy.

    PubMed

    Muchtar, Eli; Buadi, Francis K; Dispenzieri, Angela; Gertz, Morie A

    2016-01-01

    Immunoglobulin amyloid light-chain (AL) amyloidosis is the most common form of systemic amyloidosis, where the culprit amyloidogenic protein is immunoglobulin light chains produced by marrow clonal plasma cells. AL amyloidosis is an infrequent disease, and since presentation is variable and often nonspecific, diagnosis is often delayed. This results in cumulative organ damage and has a negative prognostic effect. AL amyloidosis can also be challenging on the diagnostic level, especially when demonstration of Congo red-positive tissue is not readily obtained. Since as many as 31 known amyloidogenic proteins have been identified to date, determination of the amyloid type is required. While several typing methods are available, mass spectrometry has become the gold standard for amyloid typing. Upon confirming the diagnosis of amyloidosis, a pursuit for organ involvement is essential, with a focus on heart involvement, even in the absence of suggestive symptoms for involvement, as this has both prognostic and treatment implications. Details regarding initial treatment options, including stem cell transplantation, are provided in this review. AL amyloidosis management requires a multidisciplinary approach with careful patient monitoring, as organ impairment has a major effect on morbidity and treatment tolerability until a response to treatment is achieved and recovery emerges.

  12. Risk factors for venous thromboembolism in immunoglobulin light chain amyloidosis

    PubMed Central

    Bever, Katherine M.; Masha, Luke I.; Sun, Fangui; Stern, Lauren; Havasi, Andrea; Berk, John L.; Sanchorawala, Vaishali; Seldin, David C.; Sloan, J. Mark

    2016-01-01

    Patients with immunoglobulin light chain amyloidosis are at risk for both thrombotic and bleeding complications. While the hemostatic defects have been extensively studied, less is known about thrombotic complications in this disease. This retrospective study examined the frequency of venous thromboembolism in 929 patients with immunoglobulin light chain amyloidosis presenting to a single referral center, correlated risk of venous thromboembolism with clinical and laboratory factors, and examined complications of anticoagulation in this population. Sixty-five patients (7%) were documented as having at least one venous thromboembolic event. Eighty percent of these patients had events within one year prior to or following diagnosis. Lower serum albumin was associated with increased risk of VTE, with a hazard ratio of 4.30 (CI 1.60–11.55; P=0.0038) for serum albumin less than 3 g/dL compared to serum albumin greater than 4 g/dL. Severe bleeding complications were observed in 5 out of 57 patients with venous thromboembolism undergoing treatment with anticoagulation. Prospective investigation should be undertaken to better risk stratify these patients and to determine the optimal strategies for prophylaxis against and management of venous thromboembolism. PMID:26452981

  13. What is new in diagnosis and management of light chain amyloidosis?

    PubMed

    Palladini, Giovanni; Merlini, Giampaolo

    2016-07-14

    Light chain (AL) amyloidosis is caused by a usually small plasma cell clone producing a misfolded light chain that deposits in tissues. Survival is mostly determined by the severity of heart involvement. Recent studies are clarifying the mechanisms of cardiac damage, pointing to a toxic effect of amyloidogenic light chains and offering new potential therapeutic targets. The diagnosis requires adequate technology, available at referral centers, for amyloid typing. Late diagnosis results in approximately 30% of patients presenting with advanced, irreversible organ involvement and dying in a few months despite modern treatments. The availability of accurate biomarkers of clonal and organ disease is reshaping the approach to patients with AL amyloidosis. Screening of early organ damage based on biomarkers can help identify patients with monoclonal gammopathy of undetermined significance who are developing AL amyloidosis before they become symptomatic. Staging systems and response assessment based on biomarkers facilitate the design and conduction of clinical trials, guide the therapeutic strategy, and allow the timely identification of refractory patients to be switched to rescue therapy. Treatment should be risk-adapted. Recent studies are linking specific characteristics of the plasma cell clone to response to different types of treatment, moving toward patient-tailored therapy. In addition, novel anti-amyloid treatments are being developed that might be combined with anti-plasma cell chemotherapy. PMID:27053535

  14. Cardiac Amyloidosis

    MedlinePlus

    ... abbreviation AL stands for Amyloidosis formed from Light chains, and it is a disease of the bone ... proteins that make up antibodies, known as light chains. These light chains circulate in the blood and ...

  15. 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

  16. Phenotypic, transcriptomic, and genomic features of clonal plasma cells in light-chain amyloidosis.

    PubMed

    Paiva, Bruno; Martinez-Lopez, Joaquin; Corchete, Luis A; Sanchez-Vega, Beatriz; Rapado, Inmaculada; Puig, Noemi; Barrio, Santiago; Sanchez, Maria-Luz; Alignani, Diego; Lasa, Marta; García de Coca, Alfonso; Pardal, Emilia; Oriol, Alberto; Garcia, Maria-Esther Gonzalez; Escalante, Fernando; González-López, Tomás J; Palomera, Luis; Alonso, José; Prosper, Felipe; Orfao, Alberto; Vidriales, Maria-Belen; Mateos, María-Victoria; Lahuerta, Juan-Jose; Gutierrez, Norma C; San Miguel, Jesús F

    2016-06-16

    Immunoglobulin light-chain amyloidosis (AL) and multiple myeloma (MM) are 2 distinct monoclonal gammopathies that involve the same cellular compartment: clonal plasma cells (PCs). Despite the fact that knowledge about MM PC biology has significantly increased in the last decade, the same does not apply for AL. Here, we used an integrative phenotypic, molecular, and genomic approach to study clonal PCs from 24 newly diagnosed patients with AL. Through principal-component-analysis, we demonstrated highly overlapping phenotypic profiles between AL and both monoclonal gammopathy of undetermined significance and MM PCs. However, in contrast to MM, highly purified fluorescence-activated cell-sorted clonal PCs from AL (n = 9) showed almost normal transcriptome, with only 38 deregulated genes vs normal PCs; these included a few tumor-suppressor (CDH1, RCAN) and proapoptotic (GLIPR1, FAS) genes. Notwithstanding, clonal PCs in AL (n = 11) were genomically unstable, with a median of 9 copy number alterations (CNAs) per case, many of such CNAs being similar to those found in MM. Whole-exome sequencing (WES) performed in 5 AL patients revealed a median of 15 nonrecurrent mutations per case. Altogether, our results show that in the absence of a unifying mutation by WES, clonal PCs in AL display phenotypic and CNA profiles similar to MM, but their transcriptome is remarkably similar to that of normal PCs. PMID:27069257

  17. Burden of cytogenetically abnormal plasma cells in light chain amyloidosis and their prognostic relevance.

    PubMed

    Kim, Seon Young; Im, Kyongok; Park, Si Nae; Kim, Jung-Ah; Yoon, Sung-Soo; Lee, Dong Soon

    2016-05-01

    We performed cytoplasmic fluorescence in situ hybridization assays of light chain amyloidosis (AL). In total, 234 patients were enrolled: 28 patients with AL, 24 with monoclonal gammopathy of undetermined significance (MGUS), and 182 with multiple myeloma (MM). Chromosomal abnormalities were detected in 13 of 22 (59%) AL patients without MM. All 13 patients demonstrated IGH rearrangement, and t(11;14)/IGH-CCND1 was most frequent (32%). Chromosome gain was not observed in AL patients without MM. These findings were dissimilar to findings in MGUS patients, in whom trisomy 9 was the most frequent abnormality. Of 6 AL patients with MM, 5 (83%) patients had cytogenetic abnormalities: 1q gain (4/6, 67%), gains of chromosome 9 (3/6, 50%), IGH rearrangement and RB1 (13q) deletions (2/6 each, 33%). The percentage of clonal plasma cells among total plasma cells was variable (median, 75%; range, 16-100%) for AL patients without MM, which was lower than the results for MM patients (median 100%). The overall survival of AL patients without MM was not significantly different according to the presence of cytogenetic abnormalities (P=0.510). In summary, among Korean AL patients, IGH rearrangement was the most frequent cytogenetic abnormality and cytogenetic aberration patterns differ compared with MGUS and MM patients. PMID:27015231

  18. 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 ...

  19. Long term outcomes of cardiac transplant for immunoglobulin light chain amyloidosis: The Mayo Clinic experience

    PubMed Central

    Grogan, Martha; Gertz, Morie; McCurdy, Arleigh; Roeker, Lindsey; Kyle, Robert; Kushwaha, Sudhir; Daly, Richard; Dearani, Joseph; Rodeheffer, Richard; Frantz, Robert; Lacy, Martha; Hayman, Suzanne; McGregor, Christopher; Edwards, Brooks; Dispenzieri, Angela

    2016-01-01

    AIM: To determine the outcome of orthotopic heart transplantation (OHT) in immunoglobulin light chain (AL) amyloidosis. METHODS: The medical records of patients with AL who underwent orthotopic heart transplantation at the Mayo Clinic in Rochester Minnesota from 1992 to 2011 were reviewed. Patients met at least one of the following at: New York Heart Association class IV heart failure, ventricular thickness > 15 mm, ejection fraction < 40%. Selection guidelines for heart transplant included age < 60 years, absence of multiple myeloma and significant extra-cardiac organ involvement. Baseline characteristics including age, gender, organ involvement, and New York Heart Association functional class were recorded. Laboratory data, waiting time until heart transplant, and type of treatment of the underlying plasma cell disorder were recorded. Survival from the time of OHT was calculated using Kaplan-Meier survival curves. Survival of patients undergoing OHT for AL was compared to that of non-amyloid patients undergoing OHT during the same time period. RESULTS: Twenty-three patients (median age 53 years) with AL received OHT. There were no deaths in the immediate perioperative period. Twenty patients have died post OHT. For the entire cohort, the median overall survival was 3.5 years (95%CI: 1.2, 8.2 years). The 1-year survival post OHT was 77%, the 2-year survival 65%, and the 5-year survival 43%. The 5-year survival for non-amyloid patients undergoing OHT during the same era was 85%. Progressive amyloidosis contributed to death in twelve patients. Of those without evidence of progressive amyloidosis, the cause of death included complications of autologous hematopoietic stem cell transplantation for 3 patients, post-transplant lymphoproliferative disorder for 2 patients; and for the remaining one death was related to each of the following causes: acute rejection; cardiac vasculopathy; metastatic melanoma; myelodysplastic syndrome; and unknown. Eight patients had

  20. Light chain amyloidosis of the urinary bladder. A site restricted deposition of an externally produced immunoglobulin

    PubMed Central

    Livneh, A; Shtrasburg, S; Martin, B; Baniel, J; Gal, R; Pras, M

    2001-01-01

    Aims—To identify the amyloid protein in a patient with amyloidosis localised to the urinary bladder, and to see whether subtyping of the protein by sequence analysis increases the understanding of the selection of the urinary bladder as the site of amyloid deposition. Methods—A patient with gross haematuria and a congophilic mass in his urinary bladder was evaluated further. Characterisation of the amyloid protein was performed using conventional histological and immunohistochemical methods. Determination of the N-terminal amino acid sequence of the amyloid protein was performed using protein sequencers. Results—The patient's history, physical examination, and laboratory evaluation excluded the involvement of other organs, justifying a diagnosis of amyloidosis localised to the urinary bladder. Histological and immunological studies showed that the amyloid protein deposited in the urinary bladder of the patient was probably of the amyloid light chain type. No plasma cells or lymphocytes were seen in sections of the urinary bladder and lower ureter adjacent to the amyloid deposits. Molecular analysis showed the sequence NFMLTQPHSISGSPG, which assigned the amyloid protein to either the VλI or the VλVI immunoglobulin (Ig) light chain families. Conclusions—The findings suggest that the amyloid protein in this patient originated outside the urinary bladder. The heterogeneity of the Ig proteins in known cases of amyloidosis of the lower urinary tract suggests that the amino acid residues, which determine the Vλ subtyping, have no major role in restricting the deposited protein to the urinary bladder. Key Words: primary amyloidosis • urinary bladder • λ light chain • amino acid sequence PMID:11729210

  1. 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

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

    PubMed

    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

  3. Multiple myeloma-associated skin light chain amyloidosis: A case of misdiagnosis

    PubMed Central

    XIAO, LI; LIN, FENGXIA; XIAO, RONG; HU, CHUN; DENG, MINGYANG; LI, DAIQIANG; SHE, XIAOLING; LIU, FUYOU; SUN, LIN

    2016-01-01

    The present study reports the case of a 42-year-old male with multiple myeloma (MM)-associated skin light chain amyloidosis who presented with skin purpura as the initial symptom, which was misdiagnosis as Henoch-Schönlein purpura nephritis prior to admission to the Second Xiangya Hospital (Changsha, Hunan, China). The patient presented with purpura, papules petechiae and spontaneous ecchymosis, which was located scattered around the neck, chest and limbs, accompanied by a small amount of bleeding in the conjunctival and oral mucosa, and a swollen tongue. Upon laboratory examination, the serum immunological change showed increased serum immunoglobulin G and λ light chain levels, and a urine Bence Jones protein level of >1 g/24 h. This was accompanied with an abnormal result for immunofixation electrophoresis, and positive staining with Congo red showing apple-green birefringence in skin biopsy specimens. Thus, the patient was diagnosed with MM-associated skin amyloidosis with the initial symptom of skin purpura. Following treatment with chemotherapy consisting of prednisone and bortezomib, the skin lesions markedly improved. The present study indicates that the presentation of skin purpura in systemic amyloidosis associated with MM may be an important aid in the diagnosis and direct treatment of this disease in the clinic. PMID:27284363

  4. Immunoparesis status in immunoglobulin light chain amyloidosis at diagnosis affects response and survival by regimen type

    PubMed Central

    Muchtar, Eli; Dispenzieri, Angela; Kumar, Shaji K.; Dingli, David; Lacy, Martha Q.; Buadi, Francis K.; Hayman, Suzanne R.; Kapoor, Prashant; Leung, Nelson; Chakraborty, Rajshekhar; Russell, Stephen; Lust, John A.; Lin, Yi; Go, Ronald S.; Zeldenrust, Steven; Kyle, Robert A.; Rajkumar, S. Vincent; Gertz, Morie A.

    2016-01-01

    Clinical tools to guide in the appropriate treatment selection in immunoglobulin light chain (AL) amyloidosis are not well developed. We evaluated the response and outcome for various regimens at first-line treatment (n=681) and first progression (n=240) stratified by the immunoparesis status at diagnosis. Immunoparesis was assessed by the average relative difference of the uninvolved immunoglobulins, classifying patients into a negative average relative difference (i.e. significant immunoparesis) or a positive average relative difference (no/modest immunoparesis). Treatment was categorized as autologous stem cell transplant and four non-transplant regimens (melphalan-based; bortezomib-based, immunomodulatory drug-based and dexamethasone alone). Patients with significant immunoparesis who underwent stem cell transplant had a significantly lower rate of very good partial response or better response (58%), progression-free survival (median 30 months) and overall survival (108 months), compared to those without significant immunoparesis (80%, 127 months, median not reached, respectively; P<0.001 for all comparisons). Among the non-transplant regimens, melphalan resulted in an unfavorable progression-free survival (11 vs. 27 months; P<0.001) and overall survival (30 vs. 74 months; P=0.001) in patients with significant immunoparesis compared to those without significant immunoparesis. In contrast, no significant difference in outcomes between the immunoparesis groups was seen for those treated with bortezomib or immunomodulatory drugs. At first progression, immunoparesis status did not impact response or survival of any regimen. Melphalan at first-line provided poorer outcomes for patients with significant immunoparesis, while bortezomib or immunomodulatory drugs were more likely to overcome the adverse prognosis associated with significant immunoparesis. PMID:27479823

  5. Successful Treatment of Amyloid Light-chain Amyloidosis in a Charcot-Marie-Tooth Disease Patient with Lenalidomide, Cyclophosphamide, and Dexamethasone.

    PubMed

    Kikukawa, Yoshitaka; Hata, Hiroyuki; Ueda, Mitsuharu; Yamashita, Taro; Nasu, Singo; Ide, Kazuhiko; Ueno, Shikiko; Ando, Yukio; Mitsuya, Hiroaki; Okuno, Yutaka

    2016-01-01

    A 70-year-old woman with Charcot-Marie-Tooth disease (CMT) suffered from nephrotic syndrome and a renal biopsy revealed non-AA amyloid depositions that contained immunoglobulin light chain λ. Her serum λ free LC was elevated to 80.8 mg/L and she was diagnosed with primary amyloid light-chain (AL) amyloidosis. She was subsequently treated with lenalidomide, cyclophosphamide, and dexamethasone (RCD). After 14 cycles of RCD, she achieved complete remission. Her serum albumin levels gradually normalized to 3.1 g/dL. No exacerbation of neurologic symptoms related to CMT was observed. Thus, RCD may be a well-tolerated and effective regimen for treating AL amyloidosis in patients with CMT disease. PMID:27629972

  6. Bilateral Chronic Subdural Nontraumatic Hematoma in a Woman Affected by AL Amyloidosis

    PubMed Central

    Luongo, M.; Grassi, M.; Godano, U.

    2015-01-01

    Chronic subdural hematoma (CSDH) is a condition frequently seen in neurosurgical practice, especially among the elderly. It is often preceded by head injury, even a trivial trauma. Light chain (AL) amyloidosis is a disorder involving extracellular tissue deposition of misfolded native proteins called amyloids. The several types of amyloidosis differ by source of proteins, organ involvement, treatment, and prognosis. We describe the case of a 59-year-old woman affected by AL amyloidosis, harboring surgically treated bilateral chronic nontraumatic subdural hematoma, and we suggest a possible correlation between the clinical entities of CSDH and AL amyloidosis. PMID:26251797

  7. Amyloidosis.

    PubMed

    Pinney, Jennifer H; Hawkins, Philip N

    2012-05-01

    The term amyloid describes the deposition in the extracellular space of certain proteins in a highly characteristic, insoluble fibrillar form. Amyloidosis describes the various clinical syndromes that occur as a result of damage by amyloid deposits in tissues and organs throughout the body. The clinical significance of amyloid varies enormously, ranging from incidental asymptomatic deposits to localized disease through to rapidly fatal systemic forms that can affect multiple vital organs. Currently available therapy is focused on reducing the supply of the respective amyloid fibril precursor protein and supportive medical care, which together have greatly improved survival. Chemotherapy and anti-inflammatory treatment for the disorders that underlie AL and AA amyloidosis are guided by serial measurements of the respective circulating amyloid precursor proteins, i.e. serial serum free light chains in AL and serum amyloid A protein in AA type. Quality of life and prognosis of some forms of hereditary systemic amyloidosis can be improved by liver and other organ transplants. Various new therapies, ranging from silencing RNA, protein stabilizers to monoclonal antibodies, aimed at inhibiting fibril precursor supply, fibril formation or the persistence of amyloid deposits, are in development; some are already in clinical phase. PMID:22402917

  8. Abnormal FISH in patients with immunoglobulin light chain amyloidosis is a risk factor for cardiac involvement and for death

    PubMed Central

    Warsame, R; Kumar, S K; Gertz, M A; Lacy, M Q; Buadi, F K; Hayman, S R; Leung, N; Dingli, D; Lust, J A; Ketterling, R P; Lin, Y; Russell, S; Hwa, L; Kapoor, P; Go, R S; Zeldenrust, S R; Kyle, R A; Rajkumar, S V; Dispenzieri, A

    2015-01-01

    Importance of interphase fluorescent in situ hybridization (FISH) with cytoplasmic staining of immunoglobulin FISH (cIg-FISH) on bone marrow is not well understood in light chain amyloidosis (AL). This is in contrast with multiple myeloma where prognostic and treatment related decisions are dependent on cytogenetic testing. This retrospective study reviewed 401 AL patients with cIg-FISH testing performed at our institution between 2004 and 2012. Eighty-one percent of patients had an abnormal cIg-FISH. Common abnormalities involved translocations of chromosome 14q32 (52%), specifically: t(11;14) (43%), t(14;16) (3%) and t(4;14) (2%). Other common abnormalities include monosomy 13/deletion 13q (30%), trisomies 9 (20%), 15 (14%), 11 (10%) and 3 (10%). Median overall survival for this cohort of patients is 3.5 years. When plasma cell burden was greater than 10% trisomies predicted for worse survival (44 vs 19 months), and when it was ⩽10% t(11;14) predicted for worse survival (53 months vs not reached). Abnormal cIg-FISH was significantly associated with advanced cardiac involvement, and remained a prognostic factor on multivariate analysis. This large AL cohort demonstrates that abnormal FISH at diagnosis is prognostic for survival and advanced cardiac disease. Particularly, trisomies and t(11;14) affect survival when degree of plasma cell burden is considered. PMID:25933374

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

    PubMed

    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

  10. 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

  11. AL amyloidosis with temporal artery involvement simulates giant-cell arteritis.

    PubMed

    Audemard, Alexandra; Boutemy, Jonathan; Galateau-Salle, Françoise; Macro, Margaret; Bienvenu, Boris

    2012-03-01

    Light-chain (AL) amyloidosis may present with features suggesting vasculitis, including giant-cell arteritis (GCA). We describe a case of an 80-year-old man, who initially presented with bilateral jaw claudication, bi-temporal headache and arthralgia, however a temporal-artery biopsy then revealed AL amyloidosis. A diagnosis of AL amyloidosis complicating multiple myelome simulates GCA and polymyalgia rheumatica was established. The patient was successfully treated with melphalan and dexamethasone: the free kappa light chains decreased, the patient's jaw claudication and headache disappeared. Then we discuss similarities between GCA and AL amyloidosis and potential confusion in diagnosis. We suggest that, in patients with clinical features of GCA without any temporal-artery typical findings, specimens are stained with Congo red, which then results in a different diagnosis and treatment.

  12. Clinical benefits of bortezomib-containing regimens for newly diagnosed AL amyloidosis with severe cardiac impairment.

    PubMed

    Tsukune, Yutaka; Yahata, Yuriko; Sasaki, Makoto; Hiki, Makoto; Tsutsui, Miyuki; Hamano, Yasuharu; Itoh, Seigo; Miyazaki, Tetsuro; Dohi, Tomotaka; Maruyama, Masaki; Gotoh, Akihiko; Komatsu, Norio

    2016-08-01

    Cardiac amyloid light-chain amyloidosis (AL amyloidosis) is a rare disease with a very poor prognosis, associated with plasma cell dyscrasias such as monoclonal gammopathy of undetermined significance and multiple myeloma. Though bortezomib-containing regimens have achieved high hematologic response rates, there are still few reports describing the outcomes of Japanese patients. Six patients with severe cardiac AL amyloidosis were treated with bortezomib-containing regimens. Involved free light chain (iFLC) decreased immediately in most of these cases. However, the condition of heart failure and N-terminal pro-B-type natriuretic peptide (NT-proBNP) worsened in the early phase of this treatment and then improved several months later. At 29 months, the median duration of follow-up (2-47months), all patients remain alive except one who died of sudden cardiac arrest. Bortezomib-containing regimens are considered to be among the effective treatments for severe cardiac AL amyloidosis. PMID:27599413

  13. Clinical benefits of bortezomib-containing regimens for newly diagnosed AL amyloidosis with severe cardiac impairment.

    PubMed

    Tsukune, Yutaka; Yahata, Yuriko; Sasaki, Makoto; Hiki, Makoto; Tsutsui, Miyuki; Hamano, Yasuharu; Itoh, Seigo; Miyazaki, Tetsuro; Dohi, Tomotaka; Maruyama, Masaki; Gotoh, Akihiko; Komatsu, Norio

    2016-08-01

    Cardiac amyloid light-chain amyloidosis (AL amyloidosis) is a rare disease with a very poor prognosis, associated with plasma cell dyscrasias such as monoclonal gammopathy of undetermined significance and multiple myeloma. Though bortezomib-containing regimens have achieved high hematologic response rates, there are still few reports describing the outcomes of Japanese patients. Six patients with severe cardiac AL amyloidosis were treated with bortezomib-containing regimens. Involved free light chain (iFLC) decreased immediately in most of these cases. However, the condition of heart failure and N-terminal pro-B-type natriuretic peptide (NT-proBNP) worsened in the early phase of this treatment and then improved several months later. At 29 months, the median duration of follow-up (2-47months), all patients remain alive except one who died of sudden cardiac arrest. Bortezomib-containing regimens are considered to be among the effective treatments for severe cardiac AL amyloidosis.

  14. Induction Therapy with Bortezomib Followed by Bortezomib-High Dose Melphalan and Stem Cell Transplantation for Light Chain Amyloidosis: Results of a Prospective Clinical Trial.

    PubMed

    Sanchorawala, Vaishali; Brauneis, Dina; Shelton, Anthony C; Lo, Stephen; Sun, Fangui; Sloan, J Mark; Quillen, Karen; Seldin, David C

    2015-08-01

    The depth of hematologic response has been shown to correlate with survival and organ responses for patients with light chain (AL) amyloidosis. We conducted a prospective trial of 2 cycles of induction with bortezomib and dexamethasone on a twice a week schedule followed by conditioning with bortezomib and high-dose melphalan (HDM) and autologous stem cell transplantation (SCT). The objectives were hematologic responses, tolerability, and survival. Thirty-five patients were enrolled from 2010 to 2013. Of these, 30 proceeded with SCT, whereas 5 did not because of clinical deterioration during induction (n = 3) or complications after stem cell collection (n = 2). Two patients developed features of an autologous graft-versus-host disease-like syndrome post-SCT, which responded to steroids; no other unusual complications were seen. Treatment-related mortality occurred in 8.5% (3/35). Hematologic responses were achieved by 100% of the 27 assessable patients (63% complete response, 37% very good partial response [VGPR]) who completed the planned treatment. By intention-to-treat, hematologic responses occurred in 77% of patients (49% complete response, 29% VGPR). With a median follow-up of 36 months, the median overall survival and progression-free survival were not reached. In conclusion, incorporating bortezomib into induction and conditioning yielded a high rate of hematologic responses after HDM/SCT in patients with AL amyloidosis.

  15. Systemic kappaAL amyloidosis associated with bovine leukocyte adhesion deficiency.

    PubMed

    Taniyama, H; Yamamoto, S; Sako, T; Hirayama, K; Higuchi, H; Nagahata, H

    2000-01-01

    Histopathologic and immunohistochemical examinations were conducted on a 5-year-old Holstein-Friesian cow with systemic kappaAL amyloidosis associated with bovine leukocyte adhesion deficiency. Amyloid deposits were present in the perivascular and intercellular spaces of the visceral organs, such as the liver, kidneys, pancreas, adrenal glands, and upper alimentary tract. Amyloid was stained positively with Congo red with or without 5% potassium permanganate pretreatment and had green birefringence observed under polarized light. Immunohistochemically, amyloid reacted strongly against anti-bovine IgG (H+L) and anti-bovine kappa-light chain and reacted weakly against bovine X-light chain antibodies but was negative for anti-human amyloid AA antibody. This is the first description of AL amyloidosis immunohistochemically related to immunoglobulin kappa-light chains of precursor protein in cattle. PMID:10643989

  16. Bilateral kidney infarction due to primary Al amyloidosis: a first case report.

    PubMed

    Mihout, Fabrice; Joseph, Laure; Brocheriou, Isabelle; Leblond, Véronique; Varnous, Shaïda; Ronco, Pierre; Plaisier, Emmanuelle

    2015-05-01

    Primary Amyloid Light-chain (AL) amyloidosis is a rare form of plasma cell dyscrasia characterized by tissue deposition of monoclonal immunoglobulin light chain. Kidney involvement is the most frequent manifestation, and patients usually present with glomerular disease.We report an exceptional case of bilateral kidney infarcts caused by AL amyloidosis. A 34-years-old man presented with progressive dyspnea, loin pain, recurrent macroscopic hematuria, and acute kidney injury. Computed tomography showed bilateral kidney infarcts.The diagnosis of AL amyloidosis was established on the kidney biopsy with the characterization of major vascular amyloid deposits that selectively stained with antilambda light chain antibody. An amyloid restrictive cardiomyopathy was also present, responsible for the life-threatening conduction disturbance, but without patent cardioembolic disease. The patient then underwent emergency heart transplantation, followed by a conventional chemotherapy with bortezomib, melphalan, and dexamethasone. More than 3 years later, the patient has subnormal renal function, a well-functioning heart transplant, and a sustained hematologic response.In addition to the very uncommon presentation, this case illustrates the tremendous progress that has occurred in the management of severe forms of AL amyloidosis.

  17. Bilateral kidney infarction due to primary Al amyloidosis: a first case report.

    PubMed

    Mihout, Fabrice; Joseph, Laure; Brocheriou, Isabelle; Leblond, Véronique; Varnous, Shaïda; Ronco, Pierre; Plaisier, Emmanuelle

    2015-05-01

    Primary Amyloid Light-chain (AL) amyloidosis is a rare form of plasma cell dyscrasia characterized by tissue deposition of monoclonal immunoglobulin light chain. Kidney involvement is the most frequent manifestation, and patients usually present with glomerular disease.We report an exceptional case of bilateral kidney infarcts caused by AL amyloidosis. A 34-years-old man presented with progressive dyspnea, loin pain, recurrent macroscopic hematuria, and acute kidney injury. Computed tomography showed bilateral kidney infarcts.The diagnosis of AL amyloidosis was established on the kidney biopsy with the characterization of major vascular amyloid deposits that selectively stained with antilambda light chain antibody. An amyloid restrictive cardiomyopathy was also present, responsible for the life-threatening conduction disturbance, but without patent cardioembolic disease. The patient then underwent emergency heart transplantation, followed by a conventional chemotherapy with bortezomib, melphalan, and dexamethasone. More than 3 years later, the patient has subnormal renal function, a well-functioning heart transplant, and a sustained hematologic response.In addition to the very uncommon presentation, this case illustrates the tremendous progress that has occurred in the management of severe forms of AL amyloidosis. PMID:25929920

  18. 77 FR 6466 - Schedule for Rating Disabilities; AL Amyloidosis (Primary Amyloidosis)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-08

    ... hemic and lymphatic systems to include AL amyloidosis. This regulatory action is necessary to add AL... lymphatic systems, 38 CFR 4.117, by creating diagnostic code 7717. The schedule previously did not contain a... amyloidosis because it was the first available diagnostic code in the Hemic and Lymphatic Systems listed...

  19. [Cardiac amyloidosis].

    PubMed

    Hoyer, Caroline; Angermann, Christiane E; Knop, Stefan; Ertl, Georg; Störk, Stefan

    2008-03-15

    Amyloidoses are a heterogeneous group of multisystem disorders, which are characterized by an extracellular deposition of amyloid fibrils. Typically affected are the heart, liver, kidneys, and nervous system. More than half of the patients die due to cardiac involvement. Clinical signs of cardiac amyloidosis are edema of the lower limbs, hepatomegaly, ascites and elevated jugular vein pressure, frequently in combination with dyspnea. There can also be chest pain, probably due to microvessel disease. Dysfunction of the autonomous nervous system or arrhythmias may cause low blood pressure, dizziness, or recurrent syncope. The AL amyloidosis caused by the deposition of immunoglobulin light chains is the most common form. It can be performed by monoclonal gammopathy. The desirable treatment therapy consists of high-dose melphalan therapy twice followed by autologous stem cell transplantation. Due to the high peritransplantation mortality, selection of appropriate patients is mandatory. The ATTR amyloidosis is an autosomal dominant disorder caused by the amyloidogenic form of transthyretin, a plasmaprotein that is synthesized in the liver. Therefore, liver transplantation is the only curative therapy. The symptomatic treatment of cardiac amyloidosis is based on the current guidelines for chronic heart failure according to the patient's New York Heart Association (NYHA) state. Further types of amyloidosis with possible cardiac involvement comprise the senile systemic amyloidosis caused by the wild-type transthyretin, secondary amyloidosis after chronic systemic inflammation, and the beta(2)-microglobulin amyloidosis after long-term dialysis treatment. PMID:18344065

  20. 75 FR 65279 - Schedule for Rating Disabilities; AL Amyloidosis (Primary Amyloidosis)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-22

    ... the schedule of ratings for the hemic and lymphatic systems to include AL amyloidosis. This regulatory... diagnostic code in the Hemic and Lymphatic Systems listed in Sec. 4.117. VA proposes to assign a 100 percent... diagnostic code 7716, to read as follows: Sec. 4.117 Schedule of ratings--hemic and lymphatic systems....

  1. Malnutrition, pharmaconutrition and other considerations in AL amyloidosis, a rare disease with masquerading symptoms and usually delayed diagnosis.

    PubMed

    Franco-López, Ángeles; Culebras, Jesús M

    2015-06-01

    Amyloid Light-chain (AL) amyloidosis is a very rare disease. Nutritional and pharmaconutrional aspects are described. Nutrition repletion of malnourished AL patients is an essential strategy for improving treatment efficacy and clinical outcomes. Early diagnosis of AL amiloidosis is difficult to establish due to the fact that signs and symptoms appearing mimic other processes that delay the final correct histological diagnosis. Untreated patients with this disease have a dismal outcome, with a median survival of 10-14 months from diagnosis. The sooner the treatment is established the better the results are. Modern chemotherapeutical agents, based primarily in cyclophosphamide, bortethomid and dexametasone, produce a rapid, deep, and durable response in the majority of patients. Autologous stem cell transplantation remains restricted to selected patients who are generally without advanced cardiac amyloidosis.

  2. [Heart failure and arterial hypertension disclosing amyloidosis].

    PubMed

    Habbal, R; Noureddine, M; Hachim, K; Zahraoui, M; Azzouzi, L; Fadouach, S; Zaid, D; Chraibi, N

    1997-01-01

    Amyloidosis results from protein infiltration of the extracellular space of organs and tissues. Several amyloidosis proteins have been identified. Protein AL, (deriving from immunoglobulin light chain), protein AA and prealbumin are the most involved in this disease. When AL amyloidosis involves the heart, the illness is often terminal. Most clinical symptoms are heart failure and arrhythmia or block conduction. This case was characterised by the unusual combination of hypertension and amyloidosis. The diagnosis suggested by the echocardiographic but was confirmed by the damaged organ's biopsy. The present case concerns a young woman, who has hypertension and a pulmonary oedema. The echocardiographic scan showed a septal hypertrophy with a shining and granite-like aspect which is compatible with heart amyloidosis. Systolic and diastolic disorder with mitral and aortic regurgitation were also revealed. The kidney and rectum biopsies confirmed amyloidosis AL of the Kappa dysglobulinemia type, without extraosseous plasmocytoma. The heart and kidney failure symptoms disappeared after treatment with diuretics and ACE inhibitors.

  3. Rationale, application and clinical qualification for NT-proBNP as a surrogate end point in pivotal clinical trials in patients with AL amyloidosis

    PubMed Central

    Merlini, G; Lousada, I; Ando, Y; Dispenzieri, A; Gertz, M A; Grogan, M; Maurer, M S; Sanchorawala, V; Wechalekar, A; Palladini, G; Comenzo, R L

    2016-01-01

    Amyloid light-chain (LC) amyloidosis (AL amyloidosis) is a rare and fatal disease for which there are no approved therapies. In patients with AL amyloidosis, LC aggregates progressively accumulate in organs, resulting in organ failure that is particularly lethal when the heart is involved. A significant obstacle in the development of treatments for patients with AL amyloidosis, as well as for those with any disease that is rare, severe and heterogeneous, has been satisfying traditional clinical trial end points (for example, overall survival or progression-free survival). It is for this reason that many organizations, including the United States Food and Drug Administration through its Safety and Innovation Act Accelerated Approval pathway, have recognized the need for biomarkers as surrogate end points. The international AL amyloidosis expert community is in agreement that the N-terminal fragment of the pro-brain natriuretic peptide (NT-proBNP) is analytically validated and clinically qualified as a biomarker for use as a surrogate end point for survival in patients with AL amyloidosis. Underlying this consensus is the demonstration that NT-proBNP is an indicator of cardiac response in all interventional studies in which it has been assessed, despite differences in patient population, treatment type and treatment schedule. Furthermore, NT-proBNP expression is directly modulated by amyloidogenic LC-elicited signal transduction pathways in cardiomyocytes. The use of NT-proBNP will greatly facilitate the development of targeted therapies for AL amyloidosis. Here, we review the data supporting the use of NT-proBNP, a biomarker that is analytically validated, clinically qualified, directly modulated by LC and universally accepted by AL amyloidosis specialists, as a surrogate end point for survival. PMID:27416985

  4. Amyloidosis

    PubMed Central

    Khan, M; Falk, R

    2001-01-01

    Amyloidosis is not a single disease but a series of diseases in which there is extracellular deposition of a protein which, although it may be derived from different and unrelated sources, folds into a β pleated sheet.
There have recently been significant advances in elucidating the pathogenesis and in the treatment of this group of disorders. By identifying the source of precursor protein, treatment is aimed at eliminating or reducing the extent of deposition and is tailored for each patient.
Early diagnosis is required for the optimal effect of treatment on patient survival and quality of life. An increased awareness among physicians of the spectrum of the disease and tools to aid its diagnosis is of increasing importance.


Keywords: amyloidosis PMID:11677276

  5. Risk-adapted autologous stem cell transplantation with adjuvant dexamethasone +/- thalidomide for systemic light-chain amyloidosis: results of a phase II trial.

    PubMed

    Cohen, Adam D; Zhou, Ping; Chou, Joanne; Teruya-Feldstein, Julie; Reich, Lilian; Hassoun, Hani; Levine, Beth; Filippa, Daniel A; Riedel, Elyn; Kewalramani, Tarun; Stubblefield, Michael D; Fleisher, Martin; Nimer, Stephen; Comenzo, Raymond L

    2007-10-01

    High-dose melphalan (MEL) with autologous stem cell transplant (SCT) is an effective therapy for systemic AL amyloidosis (AL), but treatment-related mortality (TRM) has historically been high. We performed a phase II trial of risk-adapted SCT followed by adjuvant dexamethasone (dex) and thalidomide (thal) in an attempt to reduce TRM and improve response rates. Patients (n = 45) with newly diagnosed AL involving < or =2 organ systems were assigned to MEL 100, 140, or 200 mg/m(2) with SCT, based on age, renal function and cardiac involvement. Patients with persistent clonal plasma cell disease 3 months post-SCT received 9 months of adjuvant thal/dex (or dex if there was a history of deep vein thrombosis or neuropathy). Organ involvement was kidney (67%), heart (24%), liver/GI (22%) and peripheral nervous system (18%), with 31% having two organs involved. TRM was 4.4%. Thirty-one patients began adjuvant therapy, with 16 (52%) completing 9 months of treatment and 13 (42%) achieving an improvement in haematological response. By intention-to-treat, overall haematological response rate was 71% (36% complete response), with 44% having organ responses. With a median follow-up of 31 months, 2-year survival was 84% (95% confidence interval: 73%, 94%). Risk-adapted SCT with adjuvant thal/dex is feasible and results in low TRM and high haematological and organ response rates in AL patients. PMID:17897298

  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. Primary Systemic Al Amyloidosis Presenting as Temporal Arteritis

    PubMed Central

    Emmungil, Hakan; Kalfa, Melike; Başarık, Burcu; Kahraman, Hasip; Tanhan, Ferhat; Yaman, Banu; Öztürk, Atiye; Erdemir, Zehra; Kandiloğlu, Gülşen; İnal, Vedat; Kabasakal, Yasemin

    2014-01-01

    Temporal arteritis is most common vasculitis in elderly and imitated by miscellaneous disorders. Temporal artery biopsy is the gold standard test in the diagnosis of giant cell arteritis (GCA). Hereby, we describe a case of a 67-year-old man who presented initially with temporal arteritis; however, a lip biopsy then revealed AL amyloidosis. In this respect, temporal artery biopsy should be performed for definitive diagnosis of GCA particularly patients with systemic symptoms and treatment resistant. PMID:24551471

  8. An overview of the use of high-dose melphalan with autologous stem cell transplantation for the treatment of AL amyloidosis.

    PubMed

    Sanchorawala, V; Wright, D G; Seldin, D C; Dember, L M; Finn, K; Falk, R H; Berk, J; Quillen, K; Skinner, M

    2001-10-01

    Primary or AL amyloidosis results from a plasma cell dyscrasia in which fibrillar light chain protein deposition leads to organ failure and death. Standard treatment for AL amyloidosis has been oral melphalan and prednisone. However, this form of treatment modifies the natural history of this lethal disease only marginally, extending median survival from 13 months following diagnosis to 17 months. At Boston University Medical Center, we have developed treatment protocols using high-dose intravenous melphalan with autologous peripheral blood stem cell transplantation (HDM/SCT) to treat AL amyloidosis, and we have treated over 200 patients with HDM/SCT during the past six years. This extensive experience has shown that patients with AL amyloidosis, despite multisystem involvement and compromised organ function can tolerate this aggressive form of treatment. Furthermore, HDM/SCT results in durable hematologic responses in a substantial proportion of patients, and such responses are associated with clinical improvement, decreased amyloid-related organ dysfunction, and prolonged survival. However, toxicity from treatment is high (overall peri-transplant mortality, 14%), particularly for those patients with clinically significant cardiac involvement. For this reason, we believe a multidisciplinary management approach is essential when using HDM/SCT for treatment of AL amyloidosis. Based on our experience, we believe that HDM/SCT is the treatment of choice for patients with AL amyloidosis who have a good performance status and limited cardiac involvement at the time of diagnosis. HDM/SCT offers the best chance for hematologic remission, prolongation of survival, and reversal of amyloid-related disease. At the same time, we believe that HDM/SCT should continue to be examined in the context of clinical trials, directed at developing approaches to broaden the applicability of this therapy by minimizing toxicity and to increase the likelihood of complete hematologic responses

  9. Bortezomib-based induction for transplant ineligible AL amyloidosis and feasibility of later transplantation.

    PubMed

    Cornell, R F; Zhong, X; Arce-Lara, C; Atallah, E; Blust, L; Drobyski, W R; Fenske, T S; Pasquini, M C; Rizzo, J D; Saber, W; Hari, P N

    2015-07-01

    Recent studies support the use of bortezomib-based therapies in light chain amyloidosis (AL). We performed a retrospective analysis of the safety, efficacy and long-term survival (median follow-up 3 years) after bortezomib-based treatment in 28 consecutive patients with de novo AL deemed ineligible at initial presentation. The first 14 patients received bortezomib and dexamethasone (VD), and the second 14 patients received cyclophosphamide, bortezomib and dexamethasone (CVD; CyBorD). Both regimens were well tolerated with no treatment-related mortality. The overall hematological response (HR) rate was 93% in both the groups. Median time to response was shorter in the CVD group (39 days vs 96 days in the VD group; P=0.002). Hematological and organ responses induced with bortezomib-based therapy enabled 8 (33%) of initially transplant ineligible patients to undergo autologous hematopoietic stem cell transplantation (AHCT), including 4 patients with cardiac stage III or IV. Seven of the eight patients (88%) who underwent subsequent AHCT achieved sustained HR at a median of 33 months posttransplant. These data suggest that bortezomib-based induction followed by AHCT is a viable therapeutic strategy for transplant-ineligible AL. Larger, multicenter prospective trials are necessary to confirm our findings.

  10. 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

  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. Long-term follow-up from a phase 1/2 study of single-agent bortezomib in relapsed systemic AL amyloidosis.

    PubMed

    Reece, Donna E; Hegenbart, Ute; Sanchorawala, Vaishali; Merlini, Giampaolo; Palladini, Giovanni; Bladé, Joan; Fermand, Jean-Paul; Hassoun, Hani; Heffner, Leonard; Kukreti, Vishal; Vescio, Robert A; Pei, Lixia; Enny, Christopher; Esseltine, Dixie-Lee; van de Velde, Helgi; Cakana, Andrew; Comenzo, Raymond L

    2014-10-16

    CAN2007 was a phase 1/2 study of once- and twice-weekly single-agent bortezomib in relapsed primary systemic amyloid light chain amyloidosis (AL) amyloidosis. Seventy patients were treated, including 18 and 34 patients at the maximum planned doses on the once- and twice-weekly schedules. This prespecified final analysis provides mature response and long-term outcomes data after 3-year additional follow-up since the last report. In the once-weekly 1.6 mg/m(2) and twice-weekly 1.3 mg/m(2) bortezomib groups, final hematologic response rates were 68.8% and 66.7%; 80% of patients in each group sustained their response for ≥1 year. One-year progression-free rates were 72.2% and 76.8%. Median overall survival (OS) was 62.1 months and not reached; 4-year OS rates were 75.0% and 63.0%. Low baseline difference in κ/λ free light-chain level was associated with higher hematologic complete response rates and longer OS. At data cutoff, 40 (57%) patients had received subsequent therapy, including 19 (27%) retreated with bortezomib, 11 (58%) of whom achieved complete or partial hematologic responses. Four patients received prolonged bortezomib for between 3.5 and 5.6 years, with no new safety concerns, highlighting the feasibility of long-term therapy. Single-agent bortezomib produced durable hematologic responses and promising long-term OS in relapsed AL amyloidosis. This trial was registered at www.clinicaltrials.gov as #NCT00298766. PMID:25202139

  13. 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

  14. Interaction between glycosaminoglycans and immunoglobulin light chains.

    SciTech Connect

    Jiang, X.; Myatt, E.; Lykos, P.; Stevens, F. J.; Center for Mechanistic Biology and Biotechnology; Illinois Inst. of Tech.

    1997-01-01

    Amyloidosis is a pathological process in which normally soluble proteins polymerize to form insoluble fibrils (amyloid). Amyloid formation is found in a number of diseases, including Alzheimer's disease, adult-onset diabetes, and light-chain-associated amyloidosis. No pharmaceutical methods currently exist to prevent this process or to remove the fibrils from tissue. The search for treatment and prevention methods is hampered by a limited understanding of the biophysical basis of amyloid formation. Glycosaminoglycans (GAGs) are long, unbranched heteropolysaccharides composed of repeating disaccharide subunits and are known to associate with amyloid fibrils. The interaction of amyloid-associated free light chains with GAGs was tested by both size-exclusion high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments. The results indicated that heparin 16 000 and chondroitin sulfate B and C precipitated both human intact light chains and recombinant light chain variable domains. Although all light chains interacted with heparin, the strongest interactions were obtained with proteins that had formed amyloid. Molecular modeling indicated the possibility of interaction between heparin and the conserved saddle like surface of the light chain dimer opposite the complementarity-determining segments that form part of the antigen-binding site of a functional antibody. This suggestion might offer a new path to block the aggregation of amyloid-associated light chain proteins, by design of antagonists based on properties of GAG binding. A hexasaccharide was modeled as the basis for a possible antagonist.

  15. 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

  16. Presumptive service connection for disease associated with exposure to certain herbicide agents: AL amyloidosis. Final rule.

    PubMed

    2009-05-01

    This document amends the Department of Veterans Affairs (VA) adjudication regulations concerning presumptive service connection for a certain disease based on the most recent National Academy of Sciences (NAS) Institute of Medicine committee report, "Veterans and Agent Orange: Update 2006" (Update 2006). This amendment is necessary to implement a decision of the Secretary of Veterans Affairs that there is a positive association between exposure to herbicides used in the Republic of Vietnam during the Vietnam era and the subsequent development of AL amyloidosis. The intended effect of this amendment is to establish presumptive service connection for AL amyloidosis based on herbicide exposure. PMID:19507326

  17. Familial and primary (AL) cardiac amyloidosis: echocardiographically similar diseases with distinctly different clinical outcomes.

    PubMed Central

    Dubrey, S. W.; Cha, K.; Skinner, M.; LaValley, M.; Falk, R. H.

    1997-01-01

    OBJECTIVE: To determine whether patients with myocardial amyloidosis due either to AL (primary) amyloid or familial amyloid have distinguishing echocardiographic or electrocardiographic features; and to compare the prevalence of heart failure and survival in the two types of amyloidosis in relation to echocardiographic findings. DESIGN: Blinded group comparison of randomly selected cases of cardiac amyloidosis. SETTING: International referral centre for amyloid research and treatment. PATIENTS: 36 patients with cardiac amyloid heart disease, of whom 12 had familial and 24 had primary AL amyloidosis. RESULTS: Familial and AL echocardiograms were morphologically indistinguishable, with similar left ventricular wall thickness, mean (SD) 15.4 (2.3) nu 15.8 (2.5) mm, respectively; right ventricular wall thickness was also similar between amyloid types: 9.6 (2.8) nu 9.7 (6.5) mm, respectively. Doppler indices of left and right ventricular function, left ventricular volume, and ejection fraction were also similar. Low voltage electrocardiograms (< 0.5 mV) were more common in the AL (16/24, 67%) than in the familial group (4/12, 25%), P < 0.05. The one year survival for familial and AL forms was 92% (11/12) nu 38% (6/24), respectively, with virtually all deaths due to cardiac causes. CONCLUSIONS: Although cardiac involvement is echocardiographically indistinguishable, cardiac mortality is very different between the two forms of amyloidosis. Preservation of electrocardiographic voltage in familial amyloidosis suggests that the particular biochemical characteristics of distinct types of amyloid fibril have different pathological effects on the myocardium. This distinction becomes critical in the evaluation, treatment, and management of patients who have a diagnosis within the spectrum of the protein deposition diseases. Images PMID:9290406

  18. 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.

  19. 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.

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

    DOE PAGES

    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; et al

    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

  1. 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.

  2. Primary amyloidosis A. Immunohistochemical and biochemical characterization.

    PubMed Central

    Picken, M. M.; Pelton, K.; Frangione, B.; Gallo, G.

    1987-01-01

    Primary "idiopathic" amyloidosis is usually related to immunoglobulin light chain (AL) associated with immunocytic dyscrasias, while secondary "reactive" amyloidosis (AA) is related to serum amyloid A protein (SAA) and typically occurs with chronic inflammation, malignancy, or familial Mediterranean fever. In the present study, amyloid fibril protein extracted from frozen and paraffin-embedded tissue from a patient (CAR) with primary systemic amyloidosis proved to be AA protein by immunohistochemical, immunochemical, and amino terminal sequence. Extracts from both frozen and formalin-fixed paraffin-embedded kidney and spleen yielded similar monomers and dimers of the AA protein. The additional high-molecular-weight bands and a distinct 12,000-dalton fragment in the amyloid protein extracted from the formalin-fixed paraffin-embedded lung suggest that different processing of proteins, ie, by polymerization and/or degradation, may occur in different organs. Images Figure 1 Figure 2 Figure 3 PMID:3425691

  3. 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

  4. Serum-free light-chain assay: clinical utility and limitations.

    PubMed

    Bhole, Malini V; Sadler, Ross; Ramasamy, Karthik

    2014-09-01

    In the last decade, the introduction of the serum-free light-chain (sFLC) assay has been an important advance in the diagnosis and management of plasma cell dyscrasias, particularly monoclonal light-chain diseases. The immunoassay was developed to detect free light chains in serum by using anti-FLC antibodies which specifically recognised epitopes on light chains that were 'hidden' in intact immunoglobulins. Since its introduction in 2001, there have been several publications in the English language literature discussing the clinical utility as well as analytical limitations of the sFLC assay. These studies have highlighted both positive and negative aspects of the assay particularly with regard to its sensitivity and specificity and the technical challenges that can affect its performance. The contribution and significance of the sFLC assay in the management of light-chain myeloma, primary amyloid light-chain (AL) amyloidosis and non-secretory myeloma are well recognised and will be addressed in this review. The aim of this article is to also review the published literature with a view to providing a clear understanding of its utility and limitations in the diagnosis, prognosis and monitoring of plasma dyscrasias including intact immunoglobulin multiple myeloma (MM) and monoclonal gammopathy of unknown significance (MGUS). The increasing interest in using this assay in other haematological conditions will also be briefly discussed. PMID:24489083

  5. Serum-free light-chain assay: clinical utility and limitations.

    PubMed

    Bhole, Malini V; Sadler, Ross; Ramasamy, Karthik

    2014-09-01

    In the last decade, the introduction of the serum-free light-chain (sFLC) assay has been an important advance in the diagnosis and management of plasma cell dyscrasias, particularly monoclonal light-chain diseases. The immunoassay was developed to detect free light chains in serum by using anti-FLC antibodies which specifically recognised epitopes on light chains that were 'hidden' in intact immunoglobulins. Since its introduction in 2001, there have been several publications in the English language literature discussing the clinical utility as well as analytical limitations of the sFLC assay. These studies have highlighted both positive and negative aspects of the assay particularly with regard to its sensitivity and specificity and the technical challenges that can affect its performance. The contribution and significance of the sFLC assay in the management of light-chain myeloma, primary amyloid light-chain (AL) amyloidosis and non-secretory myeloma are well recognised and will be addressed in this review. The aim of this article is to also review the published literature with a view to providing a clear understanding of its utility and limitations in the diagnosis, prognosis and monitoring of plasma dyscrasias including intact immunoglobulin multiple myeloma (MM) and monoclonal gammopathy of unknown significance (MGUS). The increasing interest in using this assay in other haematological conditions will also be briefly discussed.

  6. Differential effects on light chain amyloid formation depend on mutations and type of glycosaminoglycans.

    PubMed

    Blancas-Mejía, Luis M; Hammernik, Jared; Marin-Argany, Marta; Ramirez-Alvarado, Marina

    2015-02-20

    Amyloid light chain (AL) amyloidosis is a protein misfolding disease where immunoglobulin light chains sample partially folded states that lead to misfolding and amyloid formation, resulting in organ dysfunction and death. In vivo, amyloid deposits are found in the extracellular space and involve a variety of accessory molecules, such as glycosaminoglycans, one of the main components of the extracellular matrix. Glycosaminoglycans are a group of negatively charged heteropolysaccharides composed of repeating disaccharide units. In this study, we investigated the effect of glycosaminoglycans on the kinetics of amyloid fibril formation of three AL cardiac amyloidosis light chains. These proteins have similar thermodynamic stability but exhibit different kinetics of fibril formation. We also studied single restorative and reciprocal mutants and wild type germ line control protein. We found that the type of glycosaminoglycan has a different effect on the kinetics of fibril formation, and this effect seems to be associated with the natural propensity of each AL protein to form fibrils. Heparan sulfate accelerated AL-12, AL-09, κI Y87H, and AL-103 H92D fibril formation; delayed fibril formation for AL-103; and did not promote any fibril formation for AL-12 R65S, AL-103 delP95aIns, or κI O18/O8. Chondroitin sulfate A, on the other hand, showed a strong fibril formation inhibition for all proteins. We propose that heparan sulfate facilitates the formation of transient amyloidogenic conformations of AL light chains, thereby promoting amyloid formation, whereas chondroitin sulfate A kinetically traps partially unfolded intermediates, and further fibril elongation into fibrils is inhibited, resulting in formation/accumulation of oligomeric/protofibrillar aggregates. PMID:25538238

  7. Serologically defined V region subgroups of human lambda light chains.

    PubMed

    Solomon, A; Weiss, D T

    1987-08-01

    The availability of numerous antisera prepared against lambda-type Bence Jones proteins and lambda chains of known amino acid sequence has led to the differentiation and classification of human lambda light chains into one of five V lambda subgroups. The five serologically defined subgroups, V lambda I, V lambda II, V lambda III, V lambda IV, and V lambda VI, correspond to the chemical classification that is based on sequence homologies in the first framework region (FR1). Proteins designated by sequence as lambda V react with specific anti-lambda II antisera and are thus included in the V lambda II subgroup classification. The isotypic nature of the five V lambda subgroups was evidenced through analyses of lambda-type light chains that were isolated from the IgG of normal individuals. Based on analyses of 116 Bence Jones proteins, the frequency of distribution of the lambda I, lambda II/V, lambda III, lambda IV, and lambda VI proteins in the normal lambda chain population is estimated to be 27%, 37%, 23%, 3%, and 10%, respectively. This distribution of V lambda subgroups was comparable to that found among 82 monoclonal Ig lambda proteins. Considerable V lambda intragroup antigenic heterogeneity was also apparent. At least two sub-subgroups were identified among each of the five major V lambda subgroups, implying the existence of multiple genes in the human V lambda genome. The V lambda classification of 54 Ig lambda proteins obtained from patients with primary or multiple myeloma-associated amyloidosis substantiated the preferential association of lambda VI light chains with amyloidosis AL and the predominance of the normally rare V lambda VI subgroup in this disease. PMID:3110284

  8. 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.

  9. 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

  10. Neurofilament light chain

    PubMed Central

    Lu, Ching-Hua; Macdonald-Wallis, Corrie; Gray, Elizabeth; Pearce, Neil; Petzold, Axel; Norgren, Niklas; Giovannoni, Gavin; Fratta, Pietro; Sidle, Katie; Fish, Mark; Orrell, Richard; Howard, Robin; Talbot, Kevin; Greensmith, Linda; Kuhle, Jens

    2015-01-01

    Objective: To test blood and CSF neurofilament light chain (NfL) levels in relation to disease progression and survival in amyotrophic lateral sclerosis (ALS). Methods: Using an electrochemiluminescence immunoassay, NfL levels were measured in samples from 2 cohorts of patients with sporadic ALS and healthy controls, recruited in London (ALS/control, plasma: n = 103/42) and Oxford (ALS/control, serum: n = 64/36; paired CSF: n = 38/20). NfL levels in patients were measured at regular intervals for up to 3 years. Change in ALS Functional Rating Scale–Revised score was used to assess disease progression. Survival was evaluated using Cox regression and Kaplan–Meier analysis. Results: CSF, serum, and plasma NfL discriminated patients with ALS from healthy controls with high sensitivity (97%, 89%, 90%, respectively) and specificity (95%, 75%, 71%, respectively). CSF NfL was highly correlated with serum levels (r = 0.78, p < 0.0001). Blood NfL levels were approximately 4 times as high in patients with ALS compared with controls in both cohorts, and maintained a relatively constant expression during follow-up. Blood NfL levels at recruitment were strong, independent predictors of survival. The highest tertile of blood NfL at baseline had a mortality hazard ratio of 3.91 (95% confidence interval 1.98–7.94, p < 0.001). Conclusion: Blood-derived NfL level is an easily accessible biomarker with prognostic value in ALS. The individually relatively stable levels longitudinally offer potential for NfL as a pharmacodynamic biomarker in future therapeutic trials. Classification of evidence: This report provides Class III evidence that the NfL electrochemiluminescence immunoassay accurately distinguishes patients with sporadic ALS from healthy controls. PMID:25934855

  11. AA-negative and Kappa-positive Amyloidosis in a Patient with Rheumatoid Arthritis.

    PubMed

    Ueno, Toshiharu; Sumida, Keiichi; Hoshino, Junichi; Suwabe, Tatsuya; Mise, Koki; Hazue, Ryo; Hayami, Noriko; Hiramatsu, Rikako; Kawada, Masahiro; Imafuku, Aya; Hasegawa, Eiko; Sawa, Naoki; Takaichi, Kenmei; Kinowaki, Keiichi; Ohashi, Kenichi; Fujii, Takeshi; Nishida, Aya; Ubara, Yoshifumi

    2016-01-01

    A 57-year-old Japanese woman with a 5-year history of rheumatoid arthritis (RA) was admitted to our hospital for an evaluation of nephrotic range proteinuria (4.8 g/day). A renal biopsy led to the diagnosis of amyloidosis according to strong positivity for Congo red staining and the detection of microfibrillar structures on electron microscopy that were negative for AA and positive for kappa light chain. Combination therapy with high-dose melphalan and autologous stem cell transplantation was performed according to the regimen for AL amyloidosis. Her proteinuria and RA subsided, but relapsed after 3 years. This is the first report regarding kappa light chain amyloidosis in an RA patient. PMID:27580556

  12. Soluble suppression of tumorigenicity 2 (sST2), but not galactin-3, adds to prognostication in patients with systemic AL amyloidosis independent of NT-proBNP and troponin T.

    PubMed

    Dispenzieri, Angela; Gertz, Morie A; Saenger, Amy; Kumar, Shaji K; Lacy, Martha Q; Buadi, Francis K; Dingli, David; Leung, Nelson; Zeldenrust, Steven; Hayman, Suzanne R; Kapoor, Prashant; Grogan, Martha; Hwa, Lisa; Russell, Stephen J; Go, Ronald S; Rajkumar, S Vincent; Kyle, Robert A; Jaffe, Allan

    2015-06-01

    The use of soluble cardiac biomarkers such as N-terminal pro-brain natriuretic peptide (NT-proBNP) and troponin has revolutionized prognostication for patients with AL amyloidosis. Soluble ST2 (sST2) and galectin-3 have also been reported to have prognostic value in other cardiac patient populations. We identified 502 patients with AL amyloidosis, who provided a research sample and consent to review their medical records between 1/1/2006-12/31/2010 within 90 days of their diagnosis. Samples were assayed for sST2 and galectin-3. Within this AL amyloidosis population, overall survival (OS) was 25.5 months (95% CI 18, 35.7 months). Receiver operating curve analyses were done to detect the best cut-points for sST2 and galectin-3 to predict both 1- and 5-year OS. The respective cut points for sST2 were 30 and 29.7 ng/mL, while the median sST2 for the entire population was 31 ng/mL (IQR 19.8, 53.6). The respective cut points for galectin-3 were 11 and 10.4 ng/mL while the median for the entire population was 16.6 ng/mL (IQR 11.5, 24.0). Although on univariate analysis, both sST2 and galectin-3 were prognostic, upon multivariate analysis, only sST2 was independent of troponin, NT-proBNP, serum immunoglobulin free light chain, and blood pressure. Not only did sST2 add to previously reported prognostication systems, but a novel prognostication 5-point system including sST2 was possible. The addition of sST2 - but not galectin-3 - to existing prognostication systems for patients with AL amyloidosis strengthens the ability to predict for death.

  13. Prognostic impact of cytogenetic aberrations in AL amyloidosis patients after high-dose melphalan: a long-term follow-up study.

    PubMed

    Bochtler, Tilmann; Hegenbart, Ute; Kunz, Christina; Benner, Axel; Kimmich, Christoph; Seckinger, Anja; Hose, Dirk; Goldschmidt, Hartmut; Granzow, Martin; Dreger, Peter; Ho, Anthony D; Jauch, Anna; Schönland, Stefan O

    2016-07-28

    Cytogenetic aberrations detected by interphase fluorescence in situ hybridization (iFISH) of plasma cells are routinely evaluated as prognostic markers in multiple myeloma. This long-term follow-up study aimed to assess the prognosis of systemic light chain amyloidosis (AL) patients treated with high-dose melphalan (HDM) chemotherapy and autologous stem cell transplantation, depending on iFISH results. Therefore, we analyzed a consecutive cohort of 123 AL patients recruited from 2003 to 2014. HDM was safe, with only 1 of 123 patients dying as a result of treatment-related mortality, and effective, with a complete remission (CR) rate of 34%. Translocation t(11;14) as the most prevalent aberration (59%) led to an improved CR rate after high-dose therapy (41.2% vs 20.0%; P = .02), translating into a prolonged hematologic event-free survival (hemEFS; median, 46.1 vs 28.1 months; P = .05) and a trend for better overall survival (median, not reached vs 93.7 months; P = .07). In multivariate analysis, t(11;14) was confirmed as a favorable prognostic factor regarding hemEFS along with lower values for the difference between involved and uninvolved free light chains. Conversely, deletion 13q14, gain of 1q21, and hyperdiploidy had no significant prognostic impact. The high-risk cytogenetic aberrations t(4;14), t(14;16), and del(17p13) conferred an unfavorable prognosis, although statistical significance was reached only for univariate CR analysis in this small group of 9 patients. Thus, t(11;14) positivity in HDM-treated AL patients conferred superior CR rates and hemEFS. In view of the reduced response of t(11;14) to bortezomib, this highlights the impact of therapy on the prognostic role of cytogenetic aberrations.

  14. Unusual Presentation of Light Chain Deposition Disease: A Case Report

    PubMed Central

    Uppal, Mayank; Amitabh, Vindu; Agrawal, Usha

    2016-01-01

    Light Chain Deposition Disease (LCDD) is a rare disease characterized by deposition of monoclonal non-amyloid light chains in multiple organs. We report an unusual histologic manifestation of LCDD in a 55-year-old female patient, who presented with nephrotic syndrome and an increased serum creatinine. This case of LCDD had features of cast nephropathy on biopsy which is diagnostic of myeloma kidney, when the patient was clinically asymptomatic. Serum electrophoresis showed no abnormal band. There was no other evidence of a B-cell clonal disorder or amyloidosis. Following chemotherapy, improvement in renal function correlated with a reduction in circulating light-chain levels. PMID:27437235

  15. Immunoglobulin-free light chain monomer-dimer patterns help to distinguish malignant from premalignant monoclonal gammopathies: a pilot study.

    PubMed

    Kaplan, Batia; Golderman, Sizilia; Aizenbud, Boris; Esev, Konstantin; Kukuy, Olga; Leiba, Merav; Livneh, Avi; Ben-Zvi, Ilan

    2014-09-01

    Multiple myeloma (MM) and AL amyloidosis (AL) are two malignant forms of monoclonal gammopathies. For the purposes of prognosis and treatment, it is important to distinguish these diseases from the premalignant forms of monoclonal gammopathies, such as monoclonal gammopathy of unknown significance (MGUS) and smoldering myeloma (SMM). Routine serum/urine tests for monoclonal protein are insufficient for differential diagnosis. Thus, invasive procedures, such as tissue aspiration or biopsy, are applied. In this study, we aimed at characterization of serum-free light chain (FLC) monomer-dimer patterns to distinguish the malignant from the premalignant forms of monoclonal gammopathies. A quantitative Western blotting was applied to estimate the FLC monomer and dimer levels in AL, MM, MGUS, and SMM patients, and in control subjects (healthy individuals and patients with AA amyloidosis). AL and MM patients displayed an abnormally increased dimerization of monoclonal FLC, accompanied by higher clonality values of FLC dimers, as compared to that of monomers. These abnormalities of FLC patterns were not observed in patients with MGUS, SMM, AA amyloidosis, and healthy individuals. Analysis of FLC patterns helped to differentiate AL and MM from MGUS and SMM, a goal difficult to achieve using routine serum tests. Also, our technique might serve as a complimentary diagnostic tool in the cases with suspected AL amyloidosis, where the diagnosis of MM is excluded, while the results of amyloid typing by routine immunohistochemical techniques are inconclusive. PMID:24866208

  16. Immunoglobulin synthesis in primary and myeloma amyloidosis.

    PubMed Central

    Preud'homme, J L; Ganeval, D; Grünfeld, J P; Striker, L; Brouet, J C

    1988-01-01

    Bone marrow cells from 14 patients with primary amyloidosis and two patients with myeloma amyloidosis were studied by immunofluorescence and biosynthesis experiments after incorporation of radioactive amino acids. Cells from four patients affected with non-myeloma secondary amyloidosis were also studied as controls. In primary amyloidosis, monoclonal plasma cell populations were demonstrated by immunofluorescence in virtually every case, even in patients without serum and urine monoclonal immunoglobulin and with a normal percentage of bone marrow plasma cells. Biosynthesis experiments showed the secretion of large amounts of free light chains, most often of the lambda type, in every primary or myeloma amyloidosis case and the presence of light chain fragments in almost all cases. Special features in certain patients were the synthesis of short gamma chains (two cases), assembly block at the HL half molecule level of a monoclonal IgA (one case) and secretion of decameric abnormally large kappa chains (one case). This is in contrast with non-myelomatous secondary amyloidosis where the distribution of bone marrow plasma cells was normal by immunofluorescence and where normal sized immunoglobulins were synthesized, without free light chain secretion and fragments. These data confirm that primary amyloidosis belongs to plasma cell dyscrasias and emphasize the role of free light chains and light chain fragments in the pathogenesis of amyloid deposition. PMID:3145161

  17. Does AL amyloidosis have a unique genomic profile? Gene expression profiling meta-analysis and literature overview.

    PubMed

    Kryukov, Fedor; Kryukova, Elena; Brozova, Lucie; Kufova, Zuzana; Filipova, Jana; Growkova, Katerina; Sevcikova, Tereza; Jarkovsky, Jiri; Hajek, Roman

    2016-10-15

    Immunoglobulin light chain amyloidosis (ALA) is a plasma cell dyscrasia characterized by deposition of amyloid fibrils in various organs and tissues. The current paper is devoted to clarify if ALA has a unique gene expression profile and to its pathogenetic argumentation. The meta-analysis of ALA patients vs. healthy donors, monoclonal gammopathy of undetermined significance, smoldering and multiple myeloma patients' cohorts have revealed molecular signature of ALA consists of 256 genes representing mostly ribosomal proteins and immunoglobulin regions. This signature appears pathogenetically supported and elucidates for the first time the role of ribosome dysfunction in ALA. In summary of our findings with literature overview, we hypothesize that ALA development is associated not only with changes in genes, coding amyloidogenic protein itself, but with post-transcriptional disbalance as well. Based on our data analysis in ALA, ribosome machinery is impaired and the affected link mainly involves translational initiation, elongation and co-translational protein folding. PMID:27288311

  18. 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. PMID:27467897

  19. Phage Display and Peptide Mapping of an Immunoglobulin Light Chain Fibril-Related Conformational Epitope†

    PubMed Central

    O’Nuallain, Brian; Allen, Amy; Ataman, Demet; Weiss, Deborah T.; Solomon, Alan; Wall, Jonathan S.

    2008-01-01

    Amyloid fibrils and partially unfolded intermediates can be distinguished serologically from native amyloidogenic precursor proteins or peptides. In this regard, we previously had reported that mAb 11-1F4, generated by immunizing mice with a thermally denatured variable domain (VL) fragment of the human κ4 Bence Jones protein Len, bound to a non-native conformational epitope located within the N-terminal 18 residues of fibrillar, as well as partially denatured, Ig light chains (O’Nuallain B. et al. (2006) Biochemistry 46, 1240–247). To define further the antibody binding site, we used random peptide phage display and epitope mapping of VL Len using wild-type and alanine-mutated Len peptides where it was shown that the antibody epitope was reliant on up to 10 of the first 15 residues of protein Len. Comparison of Vκ and Vλ N-terminal germline consensus sequences with protein Len and 11-1F4-binding phages indicated that this antibody’s cross-reactivity with light chains was related to an invariant proline at position(s) 7 and/or 8, bulky hydrophobic residues at positions 11 and 13, and additionally, to the ability to accommodate amino acid diversity at positions 1–4. Sequence alignments of the phage peptides revealed a central proline, often flanked by aromatic residues. Taken together, these results have provided evidence for the structural basis of the specificity of 11-1F4 for both κ and λ light chain fibrils. We posit that the associated binding site involves a rare type VI β-turn or touch-turn that is anchored by a cis-proline residue. The identification of an 11-1F4-related mimotope should facilitate development of pan-light chain fibril-reactive antibodies that could be used in the diagnosis and treatment of patients with AL amyloidosis. PMID:17944486

  20. A Case of Abdominal Aortic Retroperitoneal and Mesenteric Amyloid Light Chain Amyloidoma

    PubMed Central

    Yokota, Kazuhiro; Kishida, Dai; Kayano, Hidekazu; Yazaki, Masahide; Shimada, Yuki; Akiyama, Yuji

    2016-01-01

    We report the case of a Japanese woman with amyloid light chain (AL) amyloidoma in the abdominal aortic retroperitoneum and mesentery. Irregular soft tissue mass lesions with calcification in the abdominal aortic retroperitoneum and mesentery were initially detected by computed tomography at another hospital. The lesions gradually compressed the duodenum, causing symptoms of bowel obstruction. The patient was clinically diagnosed with retroperitoneal fibrosis, and prednisolone was administered at a dose of 40 mg/day. However, the lesions did not change in size and her symptoms continued. She was transferred to our hospital and underwent mesenteric biopsy for histopathology using abdominal laparotomy. The histopathological and immunohistological findings of the mesenteric specimen demonstrated lambda light chain deposition. Accordingly, the patient was finally diagnosed with AL amyloidoma with no evidence of systemic amyloidosis. After laparotomy, her general condition worsened because of complications of pneumonia and deep vein thrombosis. She died suddenly from acute myocardial infarction. We have concluded that abdominal aortic retroperitoneal and mesenteric AL amyloidoma may have very poor prognoses in accordance with previous reports. In addition, the size and location of AL amyloidoma directly influence the prognosis. We suggest that early histopathology is important for improving prognosis. PMID:27752386

  1. Recruitment of Light Chains by Homologous and Heterologous Fibrils Shows Distinctive Kinetic and Conformational Specificity.

    PubMed

    Blancas-Mejía, Luis M; Ramirez-Alvarado, Marina

    2016-05-31

    Light chain amyloidosis is a protein misfolding disease in which immunoglobulin light chains aggregate as insoluble fibrils that accumulate in extracellular deposits. Amyloid fibril formation in vitro has been described as a nucleation-polymerization, autocatalytic reaction in which nascent fibrils catalyze formation of new fibrils, recruiting soluble protein into the fibril. In this context, it is also established that preformed fibrils or "seeds" accelerate fibril formation. In some cases, seeds with a substantially different sequence are able to accelerate the reaction, albeit with a lower efficiency. In this work, we studied the recruitment and addition of monomers in the presence of seeds of five immunoglobulin light chain proteins, covering a broad range of protein stabilities and amyloidogenic properties. Our data reveal that in the presence of homologous or heterologous seeds, the fibril formation reactions become less stochastic than de novo reactions. The kinetics of the most amyloidogenic proteins (AL-T05 and AL-09) do not present significant changes in the presence of seeds. Amyloidogenic protein AL-103 presented fairly consistent acceleration with all seeds. In contrast, the less amyloidogenic proteins (AL-12 and κI) presented dramatic differential effects that are dependent on the kind of seed used. κI had a poor efficiency to elongate preformed fibrils. Together, these results indicate that fibril formation is kinetically determined by the conformation of the amyloidogenic precursor and modulated by the differential ability of each protein to either nucleate or elongate fibrils. We observe morphological and conformational properties of some seeds that do not favor elongation with some proteins, resulting in a delay in the reaction. PMID:27158939

  2. 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

  3. [Clinical usefulness of serum free light chain measurement in monoclonal gammopathy].

    PubMed

    Shimazaki, Chihiro; Murakami, Hirokazu; Sawamura, Morio; Matsuda, Masayuki; Kinoshita, Tomohiro; Hata, Hiroyuki; Sugiura, Isamu; Tsushita, Keitaroh; Nagura, Eiichi; Kosugi, Hiroshi; Itoh, Junji; Shimizu, Kazuyuki

    2010-04-01

    Recently, a highly sensitive assay (FREELITE) capable of measuring serum immunoglobulin-free light chains (FLC) was developed. An abnormal kappa/lambda ratio supports the presence of clonal plasma cell expansion. Using this assay, we measured serum and urine samples of 178 healthy volunteers, 184 patients with polyclonal gamma-globulinemia and 150 patients with monoclonal gamma-globulinemia. The diagnostic sensitivity of the FLC assays for monoclonal gammopathies was 88.0% and the specificity for healthy volunteers and polyclonal gammopathies was 96.1%. The minimal detection sensitivity of this assay for serum FLC was 0.3 mg/l and was greater than 100-fold more sensitive than serum protein electrophoresis (SPE). The combination of FLC with SPE and immunoelectrophoresis identified 99% of patients with monoclonal gammopathies. Effective treatment often leads to a more rapid reduction of the involved FLC level relative to the intact immunoglobulin or total light chain concentration because the half-life of FLC is <6 hours. These observations suggest that FREELITE is useful for diagnosis, disease monitoring and assessment of response to treatment in patients with monoclonal gammopathies such as multiple myeloma and AL amyloidosis.

  4. 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

  5. The morphologic spectrum and clinical significance of light chain proximal tubulopathy with and without crystal formation.

    PubMed

    Larsen, Christopher P; Bell, Jane M; Harris, Alexis A; Messias, Nidia C; Wang, Yihan H; Walker, Patrick D

    2011-11-01

    The renal diseases most frequently associated with myeloma include amyloidosis, monoclonal immunoglobulin deposition disease, and cast nephropathy. Less frequently reported is light chain proximal tubulopathy, a disease characterized by κ-restricted crystal deposits in the proximal tubule cytoplasm. Light chain proximal tubulopathy without crystal deposition is only loosely related to the typical light chain proximal tubulopathy, and little is known about this entity. A search was performed of the 10 081 native kidney biopsy samples processed by our laboratory over the past 2 years for cases that had light chain restriction limited to the proximal tubule cytoplasm. A total of 10 cases of light chain proximal tubulopathy without crystal deposition were found representing 3.1% of light chain-related diseases. Nine of these 10 showed λ-light chain restriction. Only three cases of light chain proximal tubulopathy with crystals were found accounting for 0.9% of light chain-related diseases. Two of these three were κ subtype. Plasma cell dyscrasia was unsuspected in seven of the 10 patients with light chain proximal tubulopathy without crystals at the time of renal biopsy. After the biopsy was reported, follow-up was available on 9/10 patients with 9/9 showing a plasma cell dyscrasia including 8/9 with multiple myeloma. We found that light chain proximal tubulopathy without crystal formation, despite being rarely described in the literature, is over three times more common than light chain proximal tubulopathy with crystal formation in our series. And given that it is often associated with previously unrecognized myeloma, it is a critically important diagnosis.

  6. [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.

  7. Primary amyloidosis

    MedlinePlus

    ... Swelling in the arms and legs Weak hand grip Weight loss Other symptoms that may occur with ... kidney involvement may lead to organ failure and death. Body-wide ( systemic ) amyloidosis can lead to death ...

  8. Amyloidosis of the renal pelvis: a harbinger of mammary carcinoma?

    PubMed

    Grigor, Thomas; Munro, Nicolas

    2015-01-01

    We describe a rare case of light chain immunoglobulin amyloid (AL) accumulation in the central and lower pole renal calyces. Our patient, a woman aged 60, presented with several episodes of gross haematuria. Radiological imaging detected a filling defect in the left renal pelvis. Rigid ureteroscopy showed a corresponding mucosal abnormality resembling transitional cell carcinoma. A definitive preoperative tissue diagnosis could not be reached. Laparoscopic-assisted left nephroureterectomy was indicated. Histopathological examination excluded malignancy, revealing congophilic deposits of submucosal amyloid. A constellation of findings confirmed localised or primary amyloidosis with an AL immunophenotype but no evidence of clonal B-cell disease in the amyloid-associated lymphoplasmacytic cell infiltrate. Investigation for systemic plasma cell dyscrasia and echocardiography and scintigraphy for visceral amyloid deposits were negative for systemic disease. At a follow-up period of 30 months, there is no recurrence. However, our patient was diagnosed with breast cancer 21 months ago. PMID:25596296

  9. Nuclear Imaging of Amyloidosis

    PubMed Central

    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. 123I-SAP proved to have high sensitivity in imaging of AA and AL amyloidosis in visceral organs. 99mTc-Aprotinin was found to be useful in detecting cardiac amyloidosis. A couple of classical radiotracers, such as 201Tl, 123I-mIBG, together with 111In-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. 67Ga-citrate and 99mTc-penta-DMSA. High sensitivity and specificity was achieved with β2-microglobulin labeled with 131I or 111In. Among PET tracers, 11C-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. PMID:25071873

  10. 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.

  11. Amyloidosis: an unusual cause of portal hypertension.

    PubMed

    Takayasu, Vilma; 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

  12. 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

  13. Amyloidosis and Kidney Disease

    MedlinePlus

    ... body has fewer red blood cells than normal. Dialysis-related Amyloidosis People who suffer from kidney failure ... weight loss [ Top ] What are the symptoms of dialysis-related amyloidosis? The symptoms of dialysis-related amyloidosis ...

  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. [18F]-NaF PET/CT imaging in cardiac amyloidosis.

    PubMed

    Van Der Gucht, Axel; Galat, Arnault; Rosso, Jean; Guellich, Aziz; Garot, Jérôme; Bodez, Diane; Plante-Bordeneuve, Violaine; Hittinger, Luc; Dubois-Randé, Jean-Luc; Evangelista, Eva; Sasanelli, Myriam; Chalaye, Julia; Meignan, Michel; Itti, Emmanuel; Damy, Thibaud

    2016-08-01

    Cardiac amyloidosis (CA) is recognized as a common cause of restrictive cardiomyopathy and heart failure due to the deposition of insoluble proteins in the myocardial interstitium. We emphasize the role of [18F]-sodium fluoride (NaF) PET/CT as a potential noninvasive tool to identify and differentiate the transthyretin-related cardiac amyloidosis from the light-chain cardiac amyloidosis. We report cases of a 73-year-old man and a 75-year-old woman followed in our center for congestive heart failure with marked alteration of the left ventricular ejection fraction due to familial transthyretin Val122Ile cardiac amyloidosis and light-chain cardiac amyloidosis, respectively, confirmed on endomyocardial biopsy.

  17. [The role of the assessment of heavy/light chain pairs of immunoglobulin in monoclonal gammopathies].

    PubMed

    Ščudla, Vlastimil; Pika, Tomáš; Minařík, Jiří

    2015-01-01

    The aim of the paper is to inform about the contribution of novel, highly sensitive analytic technique for the assessment of serum immunoglobulins (Hevylite), enabling separate quantitative assessment of heavy/light chain pairs of immunoglobulin (HLC), i. e. the monoclonal ("involved") and polyclonal ("noninvolved") isotype including their ratio (HLC-r) in monoclonal gammopathies. We particularly target the characteristics of this technique, the compari-son of its clinical contribution with standard methods used in the diagnostics, course and the detection of relapse and progression of the disease, as well as the stratification, assessment of therapeutic outcome and prognosis in monoclonal gammopathy of undetermined significance, multiple myeloma, Waldenström´s macroglobulinemia, systemic AL-amyloidosis and some non-Hodgkin lymphomas. Present results show that in comparison with existing routinely used techniques the Hevylite method enriches clinical practice with the assessment of serum levels of "uninvolved" Ig. It enables the evaluation of the depth of "immunoparesis", and the determination of HLC-r index that is needful for the stratification of MM into "risk cohorts". It also contributes to prognostic assessment and improvement of the evaluation of the depth of therapeutic response. In MGUS individuals the HLC-r index provides information about the risk of malignant transformation. We await the results of ongoing validation studies that are expected to provide specific indications for Hevylite technique for MG in routine practice.

  18. Prognostic Value of Late Gadolinium Enhancement Cardiovascular Magnetic Resonance in Cardiac Amyloidosis

    PubMed Central

    Fontana, Marianna; Pica, Silvia; Reant, Patricia; Abdel-Gadir, Amna; Treibel, Thomas A.; Banypersad, Sanjay M.; Maestrini, Viviana; Barcella, William; Rosmini, Stefania; Bulluck, Heerajnarain; Sayed, Rabya H.; Patel, Ketna; Mamhood, Shameem; Bucciarelli-Ducci, Chiara; Whelan, Carol J.; Herrey, Anna S.; Lachmann, Helen J.; Wechalekar, Ashutosh D.; Manisty, Charlotte H.; Schelbert, Eric B.; Kellman, Peter; Gillmore, Julian D.; Hawkins, Philip N.

    2015-01-01

    Background— The prognosis and treatment of the 2 main types of cardiac amyloidosis, immunoglobulin light chain (AL) and transthyretin (ATTR) amyloidosis, are substantially influenced by cardiac involvement. Cardiovascular magnetic resonance with late gadolinium enhancement (LGE) is a reference standard for the diagnosis of cardiac amyloidosis, but its potential for stratifying risk is unknown. Methods and Results— Two hundred fifty prospectively recruited subjects, 122 patients with ATTR amyloid, 9 asymptomatic mutation carriers, and 119 patients with AL amyloidosis, underwent LGE cardiovascular magnetic resonance. Subjects were followed up for a mean of 24±13 months. LGE was performed with phase-sensitive inversion recovery (PSIR) and without (magnitude only). These were compared with extracellular volume measured with T1 mapping. PSIR was superior to magnitude-only inversion recovery LGE because PSIR always nulled the tissue (blood or myocardium) with the longest T1 (least gadolinium). LGE was classified into 3 patterns: none, subendocardial, and transmural, which were associated with increasing amyloid burden as defined by extracellular volume (P<0.0001), with transitions from none to subendocardial LGE at an extracellular volume of 0.40 to 0.43 (AL) and 0.39 to 0.40 (ATTR) and to transmural at 0.48 to 0.55 (AL) and 0.47 to 0.59 (ATTR). Sixty-seven patients (27%) died. Transmural LGE predicted death (hazard ratio, 5.4; 95% confidence interval, 2.1–13.7; P<0.0001) and remained independent after adjustment for N-terminal pro-brain natriuretic peptide, ejection fraction, stroke volume index, E/E′, and left ventricular mass index (hazard ratio, 4.1; 95% confidence interval, 1.3–13.1; P<0.05). Conclusions— There is a continuum of cardiac involvement in systemic AL and ATTR amyloidosis. Transmural LGE is determined reliably by PSIR and represents advanced cardiac amyloidosis. The PSIR technique provides incremental information on outcome even after

  19. High-dose therapy with auto-SCT is feasible in high-risk cardiac amyloidosis.

    PubMed

    Kongtim, P; Qazilbash, M H; Shah, J J; Hamdi, A; Shah, N; Bashir, Q; Wang, M; Champlin, R; Manasanch, E E; Weber, D; Orlowski, R Z; Parmar, S

    2015-05-01

    Cardiac involvement in light-chain amyloidosis (AL) predicts poor prognosis and is associated with higher TRM and morbidity during high-dose therapy and auto-SCT (HDT-ASCT). We studied the outcomes of 30 patients with cardiac amyloidosis undergoing HDT-ASCT at our center between January 1998 and March 2012. The median age of the patients was 53 years (range, 36-74) with a median follow-up of 35 months (range, 0.4-97 months). Twenty-seven patients (90%) had more than one organ involved besides the heart with 37% with cardiac stage ⩾3. Melphalan-based conditioning regimen (140-200 mg/m(2)) was used for HDT-ASCT. One-year TRM is 10%. Three-year OS and EFS from HDT-ASCT was 83% and 56.8%, respectively. Cumulative incidence of relapse at 3 years was 38.5%. Negative factors affecting survival included age >60 years, lack of novel induction therapy and BM plasmacytosis >10%. We conclude that HDT-ASCT is well tolerated in patients with high-risk cardiac amyloidosis and can lead to improved overall outcomes.

  20. Dystrophic amyloidosis: a local complication of tissue damage with heterogeneous distribution.

    PubMed

    Looi, L M

    1991-08-01

    Seventeen consecutive patients with dystrophic amyloidosis are reported here (eight Chinese, three Indian, three Iban, two Malay and one Caucasian). Ten were females and seven males, with ages ranging from 12 to 80 years (mean of 48 years). Five instances of dystrophic amyloidosis occurred in areas of tissue damage in the cardiovascular system, including fibrotic cardiac valves and an atheromatous plaque. Three occurred in osteoarthritic joint tissue. Of note were three occurrences in endometriotic cyst walls, four in the fibrotic walls of epidermal cysts, one in a hernial sac and one at the edge of a skin ulcer. All deposits were congophilic and exhibited green-birefringence and permanganate-resistance. Immunohistochemistry did not reveal reactivity for AA protein or immunoglobulin lambda or kappa light-chains. AP protein was detected in 35% of cases. Our results show that, besides the usual sites of osteoarthritic joints and damaged heart valves, dystrophic amyloidosis can complicate other areas of chronic tissue damage and fibrosis such as walls of cysts and ulcers. While the pathogenesis and biochemical nature remain unresolved, immunohistochemistry indicates that neither AA nor AL proteins are present in the deposits, and suggests that a different amyloid protein is involved. PMID:1757071

  1. Four structural risk factors identify most fibril-forming kappa light chains.

    SciTech Connect

    Stevens, F. J.; Biosciences Division

    2000-09-01

    Antibody light chains (LCs) comprise the most structurally diverse family of proteins involved in amyloidosis. Many antibody LCs incorporate structural features that impair their stability and solubility, leading to their assembly into fibrils and to their subsequent pathological deposition when produced in excess during multiple myeloma and primary amyloidosis. The particular amino acid variations in antibody LCs that account for fibril formation and amyloidogenesis have not been identified. This study focuses on amyloidogenesis within the Kl family of human LCs. Reanalysis of the current database of primary structures of proteins from more than 100 patients who produced Kl LCS, 37 of which were amyloidogenic, reveals apparent structural features that may contribute to amyloidosis. These features include loss of conserved residues or the gain of particular residues through mutation at sites involving a repertoire of approximately 20% of the amino acid positions in the light chain variable domain (V{sub L}). Moreover, 80% of all K1 amyloidogenic V{sub L}s are identifiable by the presence of at least one of three single-site substitutions or the acquisition of an N-linked glycosylation site through mutations. These findings suggest that it is feasible to predict fibril propensity by analysis of primary structure.

  2. [Optimization of the immunohistochemical diagnosis of AL amyloidosis using novel antibodies].

    PubMed

    Mikhaleva, L M; Gioeva, Z V; Reken, K

    2015-01-01

    Цель — улучшение иммуногистохимической диагностики AL-амилоидоза путем создания новых пептидных антител, направленных к вариабельному и константному регионам каппа-легких цепей. Материал и методы. В научной литературе, а также в базе данных национального центра биотехнологической информации (NCBI) провели поиск всех амилоидогенных каппа-легких цепей. На основании полученных данных составлена цепь из наиболее часто встречающихся аминокислотных остатков, из которых и были выбраны пептидные регионы для иммунизации. Четыре антитела были генерированы в результате иммунизации кроликов двумя пептидами, которые соответствовали вариабельному и константному регионам каппа-легких цепей. Результаты. Специфичность полученных антител подтверждена на серии из 222 биоптатов, полученных у 193 пациентов с AA-, AL-амилоидозом, транстиретиновым и инсулиновым амилоидозом. Все новые поликлональные пептидные антитела дали позитивную окраску в случаях с AL-каппа-амилоидозом. Заключение. Созданные нами поликлональные антитела, направленные к вариабельному и константному регионам каппа-легких

  3. Natural history and outcome of light chain deposition disease

    PubMed Central

    Sayed, Rabya H.; Wechalekar, Ashutosh D.; Gilbertson, Janet A.; Bass, Paul; Mahmood, Shameem; Sachchithanantham, Sajitha; Fontana, Marianna; Patel, Ketna; Whelan, Carol J.; Lachmann, Helen J.; Hawkins, Philip N.

    2015-01-01

    Light chain deposition disease (LCDD) is characterized by the deposition of monotypic immunoglobulin light chains in the kidney, resulting in renal dysfunction. Fifty-three patients with biopsy-proven LCDD were prospectively followed at the UK National Amyloidosis Center. Median age at diagnosis was 56 years, and patients were followed for a median of 6.2 years (range, 1.1-14.0 years). Median renal survival from diagnosis by Kaplan-Meier analysis was 5.4 years, and median estimated patient survival was 14.0 years; 64% of patients were alive at censor. Sixty-two percent of patients required dialysis, and median survival from commencement of dialysis was 5.2 years. There was a strong association between hematologic response to chemotherapy and renal outcome, with a mean improvement in glomerular filtration rate (GFR) of 6.1 mL/min/year among those achieving a complete or very good partial hematologic response (VGPR) with chemotherapy, most of whom remained dialysis independent, compared with a mean GFR loss of 6.5 mL/min/year among those achieving only a partial or no hematologic response (P < .009), most of whom developed end-stage renal disease (ESRD; P = .005). Seven patients received a renal transplant, and among those whose underlying clonal disorder was in sustained remission, there was no recurrence of LCDD up to 9.7 years later. This study highlights the need to diagnose and treat LCDD early and to target at least a hematologic VGPR with chemotherapy, even among patients with advanced renal dysfunction, to delay progression to ESRD and prevent recurrence of LCDD in the renal allografts of those who subsequently receive a kidney transplant. PMID:26392598

  4. Light Chain Deposition Disease Presenting as Cholestatic Jaundice: A Case Report

    PubMed Central

    Kumar, Prasanna N.

    2012-01-01

    Light-chain deposition disease (LCDD) is characterized by tissue deposition of the immunoglobulin light chains in multiple organs. These deposits appear similar to amyloid on routine sections, but differ in their staining properties and ultrastructural appearance. The deposits of LCCD are non -Congophilic and do not exhibit a fibrillar ultrastructure; while, the proteinaceous substance seen in primary amyloidosis is Congo red positive and fibrillar. One of the most common organs to be involved in LCDD is the kidney. Earlier reports on cases of LCDD have mostly shown simultaneous liver and renal involvement, there are very few cases in the literature describing LCDD of the liver without renal involvement. This report describes a patient who presented with severe cholestatic jaundice and liver cell failure with normal renal function. PMID:22359728

  5. Structural basis of light chain amyloidogenicity: comparison of the thermodynamic properties, fibrillogenic potential and tertiary structural features of four Vlambda6 proteins.

    PubMed

    Wall, Jonathan S; Gupta, Vibha; Wilkerson, Matthew; Schell, Maria; Loris, Remy; Adams, Paul; Solomon, Alan; Stevens, Fred; Dealwis, Chris

    2004-01-01

    Primary (AL) amyloidosis results from the pathologic deposition of monoclonal light chains as amyloid fibrils. Studies of recombinant-derived variable region (VL) fragments of these proteins have shown an inverse relationship between thermodynamic stability and fibrillogenic potential. Further, ionic interactions within the VL domain were predicted to influence the kinetics of light chain fibrillogenicity, as evidenced from our analyses of a relatively stable Vlambda6 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 Vlambda6 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 immunoglobulin light chains.

  6. 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

  7. 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

  8. 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.

  9. [Hepatic amyloidosis as a rare differential diagnosis of progressive liver failure].

    PubMed

    Bettinger, Dominik; Lutz, Lisa; Schultheiß, Michael; Werner, Martin; Thimme, Robert; Neumann-Haefelin, Christoph

    2016-09-01

    Primary systemic amyloidosis is a rare disorder resulting in extracellular deposition of insoluble fibrils in different organs. Liver involvement has been reported. Since hepatic amyloidosis often presents clinically asymptomatic without specific laboratory or imaging hallmarks, diagnosis is challenging. However, cases of progressive hepatic failure due to liver amyloidosis have been reported. A 63 year old man presented with newly diagnosed ascites to our department. The patient reported occasional alcohol consumption. Viral hepatitis, genetic-metabolic causes as well as hepatic vascular disorders were excluded and ultrasound did not show any signs of liver cirrhosis or intraabdominal malignancy. Initially, alcoholic hepatitis was suspected. Due to the rapid deterioration of liver function, however, transjugular liver biopsy was performed showing light chain amyloidosis of kappa isotype. As diagnosis of hepatic amyloidosis is challenging, early liver biopsy is mandatory in patients with unexplained acute or chronic liver disease to exclude rare diseases with high mortality. PMID:27642740

  10. [Hepatic amyloidosis as a rare differential diagnosis of progressive liver failure].

    PubMed

    Bettinger, Dominik; Lutz, Lisa; Schultheiß, Michael; Werner, Martin; Thimme, Robert; Neumann-Haefelin, Christoph

    2016-09-01

    Primary systemic amyloidosis is a rare disorder resulting in extracellular deposition of insoluble fibrils in different organs. Liver involvement has been reported. Since hepatic amyloidosis often presents clinically asymptomatic without specific laboratory or imaging hallmarks, diagnosis is challenging. However, cases of progressive hepatic failure due to liver amyloidosis have been reported. A 63 year old man presented with newly diagnosed ascites to our department. The patient reported occasional alcohol consumption. Viral hepatitis, genetic-metabolic causes as well as hepatic vascular disorders were excluded and ultrasound did not show any signs of liver cirrhosis or intraabdominal malignancy. Initially, alcoholic hepatitis was suspected. Due to the rapid deterioration of liver function, however, transjugular liver biopsy was performed showing light chain amyloidosis of kappa isotype. As diagnosis of hepatic amyloidosis is challenging, early liver biopsy is mandatory in patients with unexplained acute or chronic liver disease to exclude rare diseases with high mortality.

  11. 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. PMID:26867784

  12. Amyloidosis and Waldenström's macroglobulinemia.

    PubMed

    Gertz, Morie A; Merlini, Giampaolo; Treon, Steven P

    2004-01-01

    Primary systemic amyloidosis is an immunoglobulin light chain disorder that is 1/5th as common as multiple myeloma. Amyloidosis is regularly seen in the practice of a hematologist and has recently undergone major advances in terms of the ability to evaluate responses as well as new therapeutic options that were not available when this topic was covered as an education session at the American Society of Hematology meeting 5 years ago. Waldenström macroglobulinemia (WM) is rarer than amyloidosis (1500 per year WM versus 3000 per year amyloid in the US), and recent consensus panels have established the definition of the disease, the diagnostic criteria, criteria for initiation of therapy and a new classification scheme. In this session, new developments in amyloid and macroglobulinemia, from suspicion of the diagnosis to treatment, are covered. In Section I, Dr. Morie Gertz answers four specific questions: (1) When should amyloidosis be suspected? (2) How does one heighten ones index of suspicion for amyloid? (3) How is the diagnosis confirmed and the type classified as primary? (4) What is the prognosis and how is it accurately assessed? Recent findings on cardiac biomarkers, presenting features and use of the free light chain assay are reviewed. Staging for amyloid and recently proposed criteria of response and progression are covered. In Section II, Dr. Giampaolo Merlini comprehensively reviews therapy of amyloidosis from the use of standard melphalan/prednisone to the recently described standard dose therapies including dexamethasone, thalidomide/dexamethasone, melphalan/dexamethasone and IV melphalan/dexamethasone. An extensive discussion of the role of high-dose therapy with stem cell reconstitution follows and includes patient selection, predictors of immediate morbidity and mortality, and survival expectation. Finally, a therapeutic strategy is proposed. In Section III, Drs. Steven Treon and Giampaolo Merlini review the most current information on WM. The

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2015-11-01

    derived from leukocyte cell-derived chemotaxin 2 amyloidosis as Ig light chain-derived amyloidosis to avoid harmful chemotherapy. PMID:25873265

  15. 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.

  16. Histomorphological patterns of renal amyloidosis: a correlation between histology and chemical type of amyloidosis.

    PubMed

    Looi, L M; Cheah, P L

    1997-07-01

    A retrospective study was conducted to investigate whether there was a correlation between the histological pattern of renal amyloidosis, the chemical type of amyloid protein involved and the clinical presentation. Eighteen consecutive cases of systemic amyloidosis that had renal biopsies processed and examined histopathologically at the Department of Pathology, Faculty of Medicine, University of Malaya, Kuala Lumpur were reviewed. The age range of patients was 25 to 64 yrs (mean, 46 yrs). The male:female ratio was 2.6:1. Three patients were Malay, 9 Chinese, 3 Indian, 1 Indonesian, 1 Iban, and 1 Bisaya. According to the predominant site of amyloid deposition, 14 cases showed a glomerular pattern and 4 a vascular pattern. 8 cases were designated as 2 anti-human amyloid-A (AA) amyloidosis on the basis of permanganate-sensitivity and immunoreactivity of deposits with anti-human AA protein antibody. Ten cases contained deposits that were permanganate-resistant and nonimmunoreactive for AA protein and were designated as AL in type. The histomorphologic pattern of renal amyloidosis did not provide a reliable means of differentiating AA from AL amyloidosis. The glomerular pattern tended to present with renal manifestations such as nephrotic syndrome and chronic renal failure, whereas the vascular pattern tended to present with nonrenal manifestations such as diarrhoea. These findings may have a bearing on the pathophysiology of amyloidosis and provide clues to appropriate management. PMID:9224755

  17. Anterior Orbit and Adnexal Amyloidosis

    PubMed Central

    Al Hussain, Hailah; Edward, Deepak P.

    2013-01-01

    Purpose: To describe six cases of anterior orbital and adnexal amyloidosis and to report on proteomic analysis to characterize the nature of amyloid in archived biopsies in two cases. Materials and Methods: The clinical features, radiological findings, pathology, and outcome of six patients with anterior orbit and adnexal amyloidosis were retrieved from the medical records. The biochemical nature of the amyloid was determined using liquid chromatography/mass spectroscopy archived paraffin-embedded tissue in two cases. Results: Of the six cases, three had unilateral localized anterior orbit and lacrimal gland involvement. Four of the six patients were female with an average duration of 12.8 years from the time of onset to presentation eyelid infiltration by amyloid caused ptosis in five cases. CT scan in patients with lacrimal gland involvement (n = 3) demonstrated calcified deformable anterior orbital masses and on pathological exmaintionamyloid and calcific deposits replaced the lacrimal gland acini. Ptosis repair was performed in three patients with good outcomes. One patient required repeated debulking of the mass and one patient had recurrenct disease. Proteomic analysis revealed polyclonal IgG-associated amyloid deposition in one patient and AL kappa amyloid in the second patient. Conclusion: Amyloidosis of the anterior orbit and lacrimal gland can present with a wide spectrum of findings with good outcomes after surgical excision. The nature of amyloid material can be precisely determined in archival pathology blocks using diagnostic proteomic analysis. PMID:24014979

  18. Restrictive cardiomyopathy in inherited ATTR amyloidosis (TTR-Ser23Asn) in a patient of German-Italian extraction

    PubMed Central

    Mueller, Iris I; Gawaz, Meinrad; Linke, Reinhold P; Zuern, Christine; Steiner, Dagmar; Altland, Klaus; Von Beckerath, Nicolas; Weig, Hans-Joerg

    2010-01-01

    Amyloidosis occurs when certain soluble proteins are transformed into amyloid fibrils in the extracellular space. Most common are the light-chain amyloidoses; less common is the AA-amyloidosis, which follows chronic inflammatory diseases, and the amyloidoses of transthyretin (TTR) origin. We report on a women of Italian-German origin with the mutation TTR (Ser23Asn). Whole body scintigraphy using TC99m-DPD showed end stage hereditary amyloidosis caused by ATTR with predominant tracer retention in the myocardium. Myocardial biopsies revealed the presence of amyloid by Congo red staining. Further immunohistochemical analysis showed ATTR amyloidosis. DNA sequencing revealed a point mutation of the transthyretin gene leading to a single amino acid substitution. The only effective treatment in patients with manifest cardiac ATTR amyloidosis is combined heart and liver transplantation. Our patient was placed on a list for this procedure, but unfortunately she died during the standby procedure due to urosepsis. PMID:22400056

  19. Restrictive cardiomyopathy in inherited ATTR amyloidosis (TTR-Ser23Asn) in a patient of German-Italian extraction.

    PubMed

    Mueller, Iris I; Gawaz, Meinrad; Linke, Reinhold P; Zuern, Christine; Steiner, Dagmar; Altland, Klaus; Von Beckerath, Nicolas; Weig, Hans-Joerg

    2010-01-01

    Amyloidosis occurs when certain soluble proteins are transformed into amyloid fibrils in the extracellular space. Most common are the light-chain amyloidoses; less common is the AA-amyloidosis, which follows chronic inflammatory diseases, and the amyloidoses of transthyretin (TTR) origin. We report on a women of Italian-German origin with the mutation TTR (Ser23Asn). Whole body scintigraphy using TC99m-DPD showed end stage hereditary amyloidosis caused by ATTR with predominant tracer retention in the myocardium. Myocardial biopsies revealed the presence of amyloid by Congo red staining. Further immunohistochemical analysis showed ATTR amyloidosis. DNA sequencing revealed a point mutation of the transthyretin gene leading to a single amino acid substitution. The only effective treatment in patients with manifest cardiac ATTR amyloidosis is combined heart and liver transplantation. Our patient was placed on a list for this procedure, but unfortunately she died during the standby procedure due to urosepsis.

  20. 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

  1. 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

  2. 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.

  3. 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).

  4. Wild-Type Transthyretin Cardiac Amyloidosis: Novel Insights From Advanced Imaging.

    PubMed

    Narotsky, David L; Castano, Adam; Weinsaft, Jonathan W; Bokhari, Sabahat; Maurer, Mathew S

    2016-09-01

    Amyloidosis is caused by extracellular deposition of abnormal protein fibrils, resulting in destruction of tissue architecture and impairment of organ function. The most common forms of systemic amyloidosis are light-chain and transthyretin-related (ATTR). ATTR can result from an autosomal dominant hereditary transmission of mutated genes in the transthyretin or from a wild-type form of disease (ATTRwt), previously known as senile cardiac amyloidosis. With the aging of the worldwide population, ATTRwt will emerge as the most common type of cardiac amyloidosis that clinicians encounter. Diagnosis of systemic amyloidosis is often delayed, either because of the false assumption that it is a rare disease, or because of misdiagnosis as a result of mistaking it with other conditions. Clinicians must integrate clinical clues from history, physical examination, and common diagnostic tests to raise suspicion for ATTRwt. The historical gold standard for diagnosis of cardiac amyloid is endomyocardial biopsy analysis with pathological distinction of precursor protein type, but this method often results in delayed diagnosis because of the limited availability of expertise to perform and interpret the endomyocardial biopsy specimen. Emerging noninvasive imaging modalities provide easier, accurate screening for ATTRwt. These modalities include advanced echocardiography, using strain imaging and the myocardial contraction fraction; nuclear scintigraphy, which can differentiate between ATTR and light-chain cardiac amyloid; and cardiac magnetic resonance imaging, using extracellular volume measurement, late gadolinium enhancement, and distinct T1 mapping. These novel approaches reveal insights into the prevalence, clinical course, morphological effects, and prognosis of ATTRwt. PMID:27568874

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

    SciTech Connect

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

    2013-06-21

    Highlights: •Polybasic peptide p5 binds human light chain amyloid extracts. •The binding of p5 with amyloid involves both glycosaminoglycans and fibrils. •Heparinase treatment led to a correlation between p5 binding and fibril content. •p5 binding to AL amyloid requires electrostatic interactions. -- Abstract: 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

  6. Insulin-derived amyloidosis

    PubMed Central

    Gupta, Yashdeep; Singla, Gaurav; Singla, Rajiv

    2015-01-01

    Amyloidosis is the term for diseases caused by the extracellular deposition of insoluble polymeric protein fibrils in tissues and organs. Insulin-derived amyloidosis is a rare, yet significant complication of insulin therapy. Insulin-derived amyloidosis at injection site can cause poor glycemic control and increased insulin dose requirements because of the impairment in insulin absorption, which reverse on change of injection site and/or excision of the mass. This entity should be considered and assessed by histopathology and immunohistochemistry, in patients with firm/hard local site reactions, which do not regress after cessation of insulin injection at the affected site. Search strategy: PubMed was searched with terms “insulin amyloidosis”. Full text of articles available in English was reviewed. Relevant cross references were also reviewed. Last search was made on October 15, 2014. PMID:25593849

  7. Spontaneous bilateral kidney rupture during autologous stem cell transplantation in a patient affected by amyloidosis

    PubMed Central

    Ferrannini, Michele; Vischini, Gisella; De Angelis, Gottardo; Giannakakis, Konstantinos; Arcese, William

    2011-01-01

    Kidney spontaneous rupture is not a recognized complication neither for amyloidosis nor of autologous stem cell transplantation (ASCT). A 46-year-old white woman, affected by nephrotic syndrome, was diagnosed as AL amyloidosis by renal biopsy. We report the singular case of a bilateral spontaneous kidney rupture during ASCT for AL with renal rescue. PMID:25984105

  8. Primary systemic amyloidosis as a real diagnostic challenge – case study

    PubMed Central

    Jerzykowska, Sonia; Gil, Lidia A.; Balcerzak, Andrzej; Pupek-Musialik, Danuta; Komarnicki, Mieczysław A.

    2014-01-01

    Primary amyloidosis (AL) is a rare variety of plasma cell dyscrasia, the diagnosis of which is often difficult to establish. Pathogenesis of amyloidosis involves extracellular deposition of insoluble protein fibrils in tissues, leading to insufficiency of affected organs. According to various sources, mean survival rate of patients with primary amyloidosis ranges from 12 to 24 months, making primary amyloidosis a disease with a very poor prognosis. Survival rate is significantly lowered in case of cardiac manifestation of amyloidosis (about 6 months survival in untreated patients). In recent years a considerable progress in AL treatment has been observed. Nowadays we are able not only to delay progression of amyloidosis, but also to improve the function of the affected organs. Unfortunately as first signs and symptoms of AL are usually nonspecific, the diagnosis of AL is often delayed, resulting in late introduction of optimal therapy. There are many diagnostic tests which can be used in diagnostic process of amyloidosis, i.e. electrophoresis, serum and urine immunofixation or affected organs and bone marrow biopsy. On establishing the diagnosis in a patient with suspected amyloidosis it should be remembered that particular diagnostic methods vary considerably in sensitivity. The aim of this paper is to present a case report of a 27-year-old patient with primary amyloidosis focusing on diagnostic aspect of this condition. On the basis of this case, the authors would like to emphasize the value of precise diagnostic process, with immunological techniques playing undoubtedly a crucial role. PMID:26155101

  9. 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

  10. Nodular pulmonary amyloidosis.

    PubMed Central

    Pinto, A; Rasmussen, S L; Campbell, T M; Lester, W M

    1984-01-01

    An elderly man had a 10-year history of multiple pulmonary nodules that he had refused to have investigated. He died of a ruptured abdominal aortic aneurysm. At autopsy the nodules were shown to consist of amyloid. There was no evidence of systemic amyloidosis. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 1 Fig. 2 PMID:6744167

  11. Serum free light chain assays not total light chain assays are the standard of care to assess Monoclonal Gammopathies.

    PubMed

    Tietsche de Moraes Hungria, Vania; Allen, Syreeta; Kampanis, Petros; Soares, Elyara Maria

    2016-01-01

    The diagnosis of Multiple Myeloma is a challenge to the physician due to the non-specific symptoms (anemia, bone pain and recurrent infections) that are commonplace in the elderly population. However, early diagnosis is associated with less severe disease, including fewer patients presenting with acute renal injury, pathological fractures and severe anemia. Since 2006, the serum free light chain test Freelite(®) has been included alongside standard laboratory tests (serum and urine protein electrophoresis, and serum and urine immunofixation) as an aid in the identification of monoclonal proteins, which are a cornerstone for the diagnosis of Multiple Myeloma. The serum free light chain assay recognizes the light chain component of the immunoglobulin in its free form with high sensitivity. Other assays that measure light chains in the free and intact immunoglobulin forms are sensitive, but unfortunately, due to the nomenclature used, these assays (total light chains) are sometimes used in place of the free light chain assay. This paper reviews the available literature comparing the two assays and tries to clarify hypothetical limitations of the total assay to detect Multiple Myeloma. Furthermore, we elaborate on our study comparing the two assays used in 11 Light Chain Multiple Myeloma patients at presentation and 103 patients taken through the course of their disease. The aim of this article is to provide a clear discrimination between the two assays and to provide information to physicians and laboratory technicians so that they can utilize the International Myeloma Working Group guidelines.

  12. Serum free light chain assays not total light chain assays are the standard of care to assess Monoclonal Gammopathies

    PubMed Central

    Tietsche de Moraes Hungria, Vania; Allen, Syreeta; Kampanis, Petros; Soares, Elyara Maria

    2016-01-01

    The diagnosis of Multiple Myeloma is a challenge to the physician due to the non-specific symptoms (anemia, bone pain and recurrent infections) that are commonplace in the elderly population. However, early diagnosis is associated with less severe disease, including fewer patients presenting with acute renal injury, pathological fractures and severe anemia. Since 2006, the serum free light chain test Freelite® has been included alongside standard laboratory tests (serum and urine protein electrophoresis, and serum and urine immunofixation) as an aid in the identification of monoclonal proteins, which are a cornerstone for the diagnosis of Multiple Myeloma. The serum free light chain assay recognizes the light chain component of the immunoglobulin in its free form with high sensitivity. Other assays that measure light chains in the free and intact immunoglobulin forms are sensitive, but unfortunately, due to the nomenclature used, these assays (total light chains) are sometimes used in place of the free light chain assay. This paper reviews the available literature comparing the two assays and tries to clarify hypothetical limitations of the total assay to detect Multiple Myeloma. Furthermore, we elaborate on our study comparing the two assays used in 11 Light Chain Multiple Myeloma patients at presentation and 103 patients taken through the course of their disease. The aim of this article is to provide a clear discrimination between the two assays and to provide information to physicians and laboratory technicians so that they can utilize the International Myeloma Working Group guidelines. PMID:26969773

  13. [The renal pathology in light chain deposition disease].

    PubMed

    Giannakakis, K N; Faraggiana, T

    2005-01-01

    Light Chain Deposition Disease (LCDD) is a relatively frequent renal disease associated with dysproteinemia. Although the light chain deposits can be widespread, the kidney is the most frequently involved organ, and renal involvement can dominate the clinical condition. The morphological features of LCDD can be recognized by light microscopy; however, the diagnosis can be made certain only by immunofluorescence microscopy, using antisera to kappa and lambda chains, and by electron microscopy.

  14. Specific dimerization of the light chains of human immunoglobulin.

    PubMed

    Stevenson, G T; Straus, D

    1968-07-01

    1. The light chains of human immunoglobulin were allowed to dimerize in vitro on removal of the dispersing agents acetic acid or urea. 2. On electrophoresis in polyacrylamide gel at pH8.8 the dimers yielded up to nine regularly spaced bands. This approximates to the number of electrophoretic components known to occur among the monomers. 3. Single electrophoretic components of the dimers were isolated from the gel, dissociated into monomers, and subjected as such to electrophoresis in urea-containing gels. Each gave two adjacent bands. 4. Similarly, after all the light chains as monomers had been subjected to electrophoresis in urea-containing gels, single electrophoretic components were isolated and allowed to dimerize. When examined now as dimers in the absence of urea, each component gave two adjacent bands. 5. These findings are explicable on the following basis. (a) The dimerization of the light chains is specific, at least inasmuch as it occurs between monomers of the same electrophoretic mobilities. (b) With the buffer constant, different light chains undergo different changes in net charge on being transferred from urea-containing to urea-free solution; in this way two different chains of the same initial charge can acquire a charge difference of 1. 6. Experiments with Bence-Jones proteins and other homogeneous light chains gave results substantiating the conclusions (a) and (b). PMID:4174431

  15. Myosin light chain genes in the turkey (Meleagris gallopavo).

    PubMed

    Chaves, L D; Ostroski, B J; Reed, K M

    2003-01-01

    Myosin light chains associate with the motor protein myosin and are believed to play a role in the regulation of its actin-based ATPase activity. Myosin light chain cDNA clones from the turkey (Meleagris gallopavo) were isolated and sequenced. One sequence corresponded to an alternative transcript, the skeletal muscle essential light chain (MYL1 isoform 1) and a second to the smooth muscle isoform of myosin light chain (MYL6). The DNA and predicted amino acid sequences of both light chain genes were compared to that of the chicken. Based on the cDNA sequence, oligonucleotide primers were designed to amplify genomic DNA from six of the seven introns of the MYL1 gene. Approximately 5 kb of DNA was sequenced (introns and 3' UTR) and evaluated for the presence of single nucleotide polymorphisms (SNPs). SNPs were verified by sequencing common intron regions from multiple individuals and three polymorphisms were used to genotype pedigreed families. MYL1 is assigned to a turkey linkage group that corresponds to a region of chicken chromosome 7 (GGA7). The results of this study provide genomic reagents for comparative studies of avian muscle components and muscle biology.

  16. 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.

  17. 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.

  18. Multifocal fibrosclerosis and renal amyloidosis.

    PubMed Central

    Hamilton, D. V.; Thiru, S.

    1983-01-01

    A 34-year-old female with a 14-year history of multifocal fibrosclerosis developed the nephrotic syndrome due to renal amyloidosis. Although the association between chronic inflammatory processes and amyloidosis is well known, this is the first report of amyloidosis occurring in a patient with multifocal fibrosclerosis. Other interesting features of this case are the involvement of subcutaneous adipose tissue in the inflammatory-fibrotic process and the presence of features of multifocal fibrosclerosis in the patient's twin sister. Images Fig. 1 PMID:6622334

  19. Targeted disruption of the porcine immunoglobulin kappa light chain locus.

    PubMed

    Ramsoondar, J; Mendicino, M; Phelps, C; Vaught, T; Ball, S; Monahan, J; Chen, S; Dandro, A; Boone, J; Jobst, P; Vance, A; Wertz, N; Polejaeva, I; Butler, J; Dai, Y; Ayares, D; Wells, K

    2011-06-01

    Inactivation of the endogenous pig immunoglobulin (Ig) loci, and replacement with their human counterparts, would produce animals that could alleviate both the supply and specificity issues of therapeutic human polyclonal antibodies (PAbs). Platform genetics are being developed in pigs that have all endogenous Ig loci inactivated and replaced by human counterparts, in order to address this unmet clinical need. This report describes the deletion of the porcine kappa (κ) light chain constant (Cκ) region in pig primary fetal fibroblasts (PPFFs) using gene targeting technology, and the generation of live animals from these cells via somatic cell nuclear transfer (SCNT) cloning. There are only two other targeted loci previously published in swine, and this is the first report of a targeted disruption of an Ig light chain locus in a livestock species. Pigs with one targeted Cκ allele (heterozygous knockout or ±) were bred together to generate Cκ homozygous knockout (-/-) animals. Peripheral blood mononuclear cells (PBMCs) and mesenteric lymph nodes (MLNs) from Cκ -/- pigs were devoid of κ-containing Igs. Furthermore, there was an increase in lambda (λ) light chain expression when compared to that of wild-type littermates (Cκ +/+). Targeted inactivation of the Ig heavy chain locus has also been achieved and work is underway to inactivate the pig lambda light chain locus.

  20. Corneal amyloidosis associated with keratoconus.

    PubMed

    Stern, G A; Knapp, A; Hood, C I

    1988-01-01

    Nodular, gray-white, central corneal opacities which extended from the subepithelial zone through the anterior four fifths of the stroma developed in a 50-year-old man with a longstanding history of hard contact lens wear for keratoconus. Results of histopathologic analysis of the corneal button obtained at the time of penetrating keratoplasty disclosed that the opacities were composed of amyloid. Corneal amyloidosis is rarely found in association with keratoconus. Although there were some similarities in the pattern of amyloid deposition to that seen in primary familial amyloidosis of the cornea, the authors believe that their patient is more likely to have had a secondary amyloidosis. Corneal amyloidosis should be considered in keratoconus patients with development of unusual forms of central corneal opacification. PMID:3278260

  1. [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.

  2. Dual immunoglobulin light chain B cells: Trojan horses of autoimmunity?

    PubMed

    Pelanda, Roberta

    2014-04-01

    Receptor editing, a major mechanism of B cell tolerance, can also lead to allelic inclusion at the immunoglobulin light chain loci and the development of B cells that coexpress two different immunoglobulin light chains and, therefore, two antibody specificities. Most allelically included B cells express two κ chains, although rare dual-λ cells are also observed. Moreover, these cells typically coexpress an autoreactive and a nonautoreactive antibody. Thus, allelically included B cells could operate like 'Trojan horses': expression and function of the nonautoreactive antigen receptors might promote their maturation, activation, and terminal differentiation into effector cells that also express and secrete autoantibodies. Indeed, dual-κ B cells are greatly expanded into effector B cell subsets in some autoimmune mice, thus indicating they might play an important role in disease.

  3. Is 18F-flutemetamol PET/CT able to reveal cardiac amyloidosis?

    PubMed

    Lhommel, Renaud; Sempoux, Christine; Ivanoiu, Adrian; Michaux, Lucienne; Gerber, Bernhard

    2014-08-01

    We report here the case of a 56-year-old man with well-documented cardiac light chain amyloidosis successfully imaged with F-flutemetamol PET/CT in 2011. A matched pair healthy volunteer was imaged in the same conditions, and no specific heart uptake could be detected. The present report suggests the potential benefit of this new pharmaceutical in this indication, as it has been recently reported for C-Pittsburgh compound B, but with the main advantage to benefit from the more practical half-life of F. Further prospective trials would, however, be required to define the potential impact of F-flutemetamol PET/CT in cardiac amyloidosis.

  4. Cysteine Racemization on IgG Heavy and Light Chains

    PubMed Central

    Zhang, Qingchun; Flynn, Gregory C.

    2013-01-01

    Under basic pH conditions, the heavy chain 220-light chain 214 (H220-L214) disulfide bond, found in the flexible hinge region of an IgG1, can convert to a thioether. Similar conditions also result in racemization of the H220 cysteine. Here, we report that racemization occurs on both H220 and L214 on an IgG1 with a λ light chain (IgG1λ) but almost entirely on H220 of an IgGl with a κ light chain (IgG1κ) under similar conditions. Likewise, racemization was detected at significant levels on H220 and L214 on endogenous human IgG1λ but only at the H220 position on IgG1κ. Low but measurable levels of d-cysteines were found on IgG2 cysteines in the hinge region, both with monoclonal antibodies incubated under basic pH conditions and on antibodies isolated from human serum. A simplified reaction mechanism involving reversible β-elimination on the cysteine is presented that accounts for both base-catalyzed racemization and thioether formation at the hinge disulfide. PMID:24142697

  5. Upper Gastrointestinal Bleeding from Gastric Amyloidosis in a Patient with Smoldering Multiple Myeloma

    PubMed Central

    Gjeorgjievski, Mihajlo; Purohit, Treta; Amin, Mitual B.; Kurtin, Paul J.; Cappell, Mitchell S.

    2015-01-01

    Amyloidosis is a common complication of patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and multiple myeloma (MM). This proteinaceous material can be deposited intercellularly in any organ system, including the gastrointestinal (GI) tract. In the GI tract, amyloidosis affects the duodenum most commonly, followed by the stomach and colorectum. Gastric amyloidosis causes symptoms of nausea, vomiting, early satiety, abdominal pain, and GI bleeding. A case of upper GI bleeding from gastric amyloidosis is presented in a patient with SMM. Esophagogastroduodenoscopy (EGD) revealed a gastric mass. Endoscopic biopsies revealed amyloid deposition in the lamina propria, consistent with gastric amyloidosis. Liquid chromatography tandem mass spectrometry performed on peptides extracted from Congo red-positive microdissected areas of paraffin-embedded stomach specimens revealed a peptide profile consistent with AL- (lambda-) type amyloidosis. Based on this and multiple other case reports, we recommend that patients with GI bleeding and MGUS, SMM, or MM undergo EGD and pathologic examination of endoscopic biopsies of identified lesions using Congo red stains for amyloidosis for early diagnosis and treatment. PMID:26366309

  6. Upper Gastrointestinal Bleeding from Gastric Amyloidosis in a Patient with Smoldering Multiple Myeloma.

    PubMed

    Gjeorgjievski, Mihajlo; Purohit, Treta; Amin, Mitual B; Kurtin, Paul J; Cappell, Mitchell S

    2015-01-01

    Amyloidosis is a common complication of patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and multiple myeloma (MM). This proteinaceous material can be deposited intercellularly in any organ system, including the gastrointestinal (GI) tract. In the GI tract, amyloidosis affects the duodenum most commonly, followed by the stomach and colorectum. Gastric amyloidosis causes symptoms of nausea, vomiting, early satiety, abdominal pain, and GI bleeding. A case of upper GI bleeding from gastric amyloidosis is presented in a patient with SMM. Esophagogastroduodenoscopy (EGD) revealed a gastric mass. Endoscopic biopsies revealed amyloid deposition in the lamina propria, consistent with gastric amyloidosis. Liquid chromatography tandem mass spectrometry performed on peptides extracted from Congo red-positive microdissected areas of paraffin-embedded stomach specimens revealed a peptide profile consistent with AL- (lambda-) type amyloidosis. Based on this and multiple other case reports, we recommend that patients with GI bleeding and MGUS, SMM, or MM undergo EGD and pathologic examination of endoscopic biopsies of identified lesions using Congo red stains for amyloidosis for early diagnosis and treatment. PMID:26366309

  7. Role of myosin light chain and myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability in vitro and in vivo.

    PubMed

    Wu, Fan; Guo, Xiaohua; Xu, Jing; Wang, Weiju; Li, Bingling; Huang, Qiaobing; Su, Lei; Xu, Qiulin

    2016-03-01

    We have previously reported that advanced glycation end products activated Rho-associated protein kinase and p38 mitogen-activated protein kinase, causing endothelial hyperpermeability. However, the mechanisms involved were not fully clarified. Here, we explored the role of myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability. Myosin light chain phosphorylation significantly increased by advanced glycation end products in endothelial cells in a time- and dose-dependent manner, indicating that myosin light chain phosphorylation is involved in the advanced glycation end product pathway. Advanced glycation end products also induced myosin phosphatase-targeting subunit 1 phosphorylation, and small interfering RNA knockdown of the receptor for advanced glycation end products, or blocking myosin light chain kinase with its inhibitor, ML-7, or small interfering RNA abated advanced glycation end product-induced myosin light chain phosphorylation. Advanced glycation end product-induced F-actin rearrangement and endothelial hyperpermeability were also diminished by inhibition of receptor for advanced glycation end product or myosin light chain kinase signalling. Moreover, inhibiting myosin light chain kinase with ML-7 or blocking receptor for advanced glycation end product with its neutralizing antibody attenuated advanced glycation end product-induced microvascular hyperpermeability. Our findings suggest a novel role for myosin light chain and myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability.

  8. 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.

  9. 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.

  10. Amyloidosis in Retinal Neurodegenerative Diseases.

    PubMed

    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

  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. A case report of hereditary apolipoprotein A-I amyloidosis associated with a novel APOA1 mutation and variable phenotype.

    PubMed

    Tougaard, Birgitte G; Pedersen, Katja Venborg; Krag, Søren Rasmus; Gilbertson, Janet A; Rowczenio, Dorota; Gillmore, Julian D; Birn, Henrik

    2016-09-01

    Apolipoprotein A-I (apo A-I) amyloidosis is a non-AL, non-AA, and non-transthyretin type of amyloidosis associated with mutations in the APOA1 gene inherited in an autosomal dominant fashion. It is a form of systemic amyloidosis, but at presentation, can also mimic localized amyloidosis. The renal presentation generally involves interstitial and medullary deposition of apo A-I amyloid protein. We describe the identification of apo A-I amyloidosis by mass spectrometry in a 52-year old male, with no family history of amyloidosis, presenting with nephrotic syndrome and associated with heterozygosity for a novel APOA1 mutation (c.220 T > A) which encodes the known amyloidogenic Trp50Arg variant. Renal amyloid deposits in this case were confined to the glomeruli alone, and the patient developed progressive renal impairment. One year after diagnosis, the patient had a successful kidney transplant from an unrelated donor. Pathogenic mutations in the APOA1 gene are generally associated with symptoms of amyloidosis. In this family however, genotyping of family members identified several unaffected carriers suggesting a variable disease penetrance, which has not been described before in this form of amyloidosis and has implications when counselling those with APOA1 mutations. PMID:27240838

  13. A case report of hereditary apolipoprotein A-I amyloidosis associated with a novel APOA1 mutation and variable phenotype.

    PubMed

    Tougaard, Birgitte G; Pedersen, Katja Venborg; Krag, Søren Rasmus; Gilbertson, Janet A; Rowczenio, Dorota; Gillmore, Julian D; Birn, Henrik

    2016-09-01

    Apolipoprotein A-I (apo A-I) amyloidosis is a non-AL, non-AA, and non-transthyretin type of amyloidosis associated with mutations in the APOA1 gene inherited in an autosomal dominant fashion. It is a form of systemic amyloidosis, but at presentation, can also mimic localized amyloidosis. The renal presentation generally involves interstitial and medullary deposition of apo A-I amyloid protein. We describe the identification of apo A-I amyloidosis by mass spectrometry in a 52-year old male, with no family history of amyloidosis, presenting with nephrotic syndrome and associated with heterozygosity for a novel APOA1 mutation (c.220 T > A) which encodes the known amyloidogenic Trp50Arg variant. Renal amyloid deposits in this case were confined to the glomeruli alone, and the patient developed progressive renal impairment. One year after diagnosis, the patient had a successful kidney transplant from an unrelated donor. Pathogenic mutations in the APOA1 gene are generally associated with symptoms of amyloidosis. In this family however, genotyping of family members identified several unaffected carriers suggesting a variable disease penetrance, which has not been described before in this form of amyloidosis and has implications when counselling those with APOA1 mutations.

  14. Myosin light chain kinase (MLCK) regulates cell migration in a myosin regulatory light chain phosphorylation-independent mechanism.

    PubMed

    Chen, Chen; Tao, Tao; Wen, Cheng; He, Wei-Qi; Qiao, Yan-Ning; Gao, Yun-Qian; Chen, Xin; Wang, Pei; Chen, Cai-Ping; Zhao, Wei; Chen, Hua-Qun; Ye, An-Pei; Peng, Ya-Jing; Zhu, Min-Sheng

    2014-10-10

    Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration.

  15. 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

    2016-07-01

    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. PMID:27292537

  16. 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

    2016-07-01

    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.

  17. 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. PMID:26793847

  18. 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.

  19. 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.

  20. Single Nodular Pulmonary Amyloidosis: Case Report

    PubMed Central

    Ko, Young Chun; Jeong, Jong Pil; Park, Chan Woo; Seo, Seok Ho; Kim, Jong Taek; Park, Dae Won; Bak, Cheol Min; Moon, Seung Ki; Jo, Shin Hyoung; Kim, Se Mi; Jung, Ah Lon

    2015-01-01

    Amyloidosis is defined as the presence of extra-cellular deposits of an insoluble fibrillar protein, amyloid. The pulmonary involvement of amyloidosis is usually classified as tracheobronchial, parenchymal nodular, or diffuse alveolar septal. A single nodular lesion can mimic various conditions, including malignancy, pulmonary tuberculosis, and fungal infection. To date, only one case of nodular pulmonary amyloidosis has been reported in Korea, a case involving multiple nodular lesions. Here, we report and discuss the case of a patient having single nodular amyloidosis. PMID:26508930

  1. Variable domain structure of {kappa}IV human light chain len : high homology to the murine light chain McPC603.

    SciTech Connect

    Huang, D.-B.; Chang, C.-H.; Ainsworth, C.; Johnson, G.; Solomon, A.; Stevens, F. J.; Schiffer, M.; Center for Mechanistic Biology and Biotechnology; Univ. of Tennessee Medical Center

    1997-12-01

    Antibody light chains of the {kappa} subgroup are the predominant light chain component in human immune responses and are used almost exclusively in the antibody repertoire of mice. Human {kappa} light chains comprise four subgroups. To date, all crystallographic studies of human {kappa} light chains were carried out on proteins of the {kappa}I subgroup. The light chain produced by multiple myeloma patient Len, was of the {kappa}IV subgroup, it differed by only one residue from the germ-line gene encoded protein. The variable domain fragment of the light chain was crystallized from ammonium sulfate in space group C222{sub 1}. The crystal structure was determined by molecular replacement and refined at 1.95 Angstrom resolution to an R-factor of 0.15. Protein Len has six additional residues in its CDR1 segment compared to the {kappa}I proteins previously characterized. The {kappa}IV variable domain. Len, differs in only 23 of 113 residues from murine {kappa} light chain McPC603. The RMS deviation upon superimposing their {alpha}-carbons was 0.69 Angstrom. The CDR1 segment of the human and murine variable domains have the same length and conformation although their amino acid sequences differ in 5 out of 17 residues. Structural features were identified that could account for the significantly higher stability of the human {kappa}IV protein relative to its murine counterpart. This human {kappa}IV light chain structure is the closest human homolog to a murine light chain and can be expected to facilitate detailed structural comparisons necessary for effective humanization of murine antibodies.

  2. Light-chain binding sites on renal brush-border membranes

    SciTech Connect

    Batuman, V.; Dreisbach, A.W.; Cyran, J.

    1990-05-01

    Immunoglobulin light chains are low-molecular-weight proteins filtered at the renal glomerulus and catabolized within the proximal tubular epithelium. Excessive production and urinary excretion of light chains are associated with renal dysfunction. They also interfere with proximal renal tubule epithelial functions in vitro. We studied the binding of 125I-labeled kappa- and lambda-light chains, obtained from the urine of multiple myeloma patients, to rat and human renal proximal tubular brush-border membranes. Light-chain binding to brush borders was also demonstrated immunologically by flow cytometry. Computer analysis of binding data was consistent with presence of a single class of low-affinity, high-capacity, non-cooperative binding sites with relative selectivity for light chains on both rat and human kidney brush-border membranes. The dissociation constants of light chains ranged from 1.6 X 10(-5) to 1.2 X 10(-4) M, and maximum binding capacity ranged from 4.7 +/- 1.3 X 10(-8) to 8.0 +/- 0.9 X 10(-8) (SD) mol/mg protein at 25 degrees C. Kappa- and lambda-light chains competed with each other for binding with comparable affinity constants. Competition by albumin and beta-lactoglobulin, however, was much weaker, suggesting relative site selectivity for light chains. These binding sites probably function as endocytotic receptors for light chains and possibly other low-molecular-weight proteins.

  3. 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

  4. Systemic transthyretin amyloidosis in a patient with bent spine syndrome.

    PubMed

    Rezania, Kourosh; Pytel, Peter; Smit, Laurel J; Mastrianni, James; Dina, Michelle A; Highsmith, W Edward; Dogan, Ahmet

    2013-06-01

    Wild-type and mutant transthyretin (TTR) are implicated in systemic amyloidosis (ATTR). Myopathy is a rare complication of ATTR amyloidosis, however no patient with bent spine syndrome secondary to ATTR amyloidosis has been reported so far. We present the first case of bent spine syndrome in a patient with wild-type ATTR amyloidosis who also had concomitant Alzheimer's disease.

  5. AA amyloidosis in vaccinated growing chickens.

    PubMed

    Murakami, T; Inoshima, Y; Sakamoto, E; Fukushi, H; Sakai, H; Yanai, T; Ishiguro, N

    2013-01-01

    Systemic amyloid-A (AA) amyloidosis in birds occurs most frequently in waterfowl such as Pekin ducks. In chickens, AA amyloidosis is observed as amyloid arthropathy. Outbreaks of systemic amyloidosis in flocks of layers are known to be induced by repeated inflammatory stimulation, such as those resulting from multiple vaccinations with oil-emulsified bacterins. Outbreaks of fatal AA amyloidosis were observed in growing chickens in a large scale poultry farm within 3 weeks of vaccination with multiple co-administered vaccines. This study documents the histopathological changes in tissues from these birds. Amyloid deposits were also observed at a high rate in the tissues of apparently healthy chickens. Vaccination should therefore be considered as a potential risk factor for the development of AA amyloidosis in poultry.

  6. Primary breast amyloidosis presenting as microcalcifications only.

    PubMed

    Shim, Youngsub; Kim, Min Jung; Ryu, Han Suk; Park, Sung Hee

    2013-01-01

    Amyloidosis is a rare disease characterized by the formation of pathological protein deposits in organs or tissues. It is typically a systemic disease which can occur in a localized form. Amyloidosis of the breast is uncommon. Common mammographic findings of breast amyloidosis are multiple nodules with or without calcifications. We report a case of primary localized breast amyloidosis presenting suspicious microcalcifications on mammography without associated masses. Mammography in a 72-year-old woman displayed multiple, linearly distributed, irregular and rod-like calcifications in the subareolar area of the left breast. The patient underwent surgical excision under mammo-guided needle localization and the pathology was confirmed to be breast amyloidosis. PMID:24043964

  7. Primary Breast Amyloidosis Presenting as Microcalcifications Only

    PubMed Central

    Shim, Youngsub; Kim, Min Jung; Ryu, Han Suk

    2013-01-01

    Amyloidosis is a rare disease characterized by the formation of pathological protein deposits in organs or tissues. It is typically a systemic disease which can occur in a localized form. Amyloidosis of the breast is uncommon. Common mammographic findings of breast amyloidosis are multiple nodules with or without calcifications. We report a case of primary localized breast amyloidosis presenting suspicious microcalcifications on mammography without associated masses. Mammography in a 72-year-old woman displayed multiple, linearly distributed, irregular and rod-like calcifications in the subareolar area of the left breast. The patient underwent surgical excision under mammo-guided needle localization and the pathology was confirmed to be breast amyloidosis. PMID:24043964

  8. The ferritin light-chain homologue promoter in Aedes aegypti.

    PubMed

    Pham, D Q-D; Chavez, C A

    2005-06-01

    Promoters that direct the expression of antipathogenic molecules to primary sites of pathogenic invasions provide a means to interfere with these invasions. Thus, they have the potential to be used in mosquito control. However, exogenous elements are known to lower the fitness of most insects, and given the ability of insects to evolve rapidly, all currently known promoters could be rendered useless. As transgenic mosquitoes may be a major component in the fight against mosquito-borne diseases, the identification of new mosquito promoters is needed. The promoter of the Aedes aegypti ferritin light-chain homologue (LCH) gene, a gene whose expression is induced in gut tissues during blood feeding has been identified and mapped. Transfection data indicate that the ferritin LCH promoter is a strong promoter. DNase I footprinting data and Transfac analyses suggest that the ferritin LCH promoter contains putative GATA, E2F, NIT2, TATA and DPE sites. These data together provide the first detailed map of a known ferritin LCH gene.

  9. Cargo selection by specific kinesin light chain 1 isoforms

    PubMed Central

    Woźniak, Marcin J; Allan, Victoria J

    2006-01-01

    Kinesin-1 drives the movement of diverse cargoes, and it has been proposed that specific kinesin light chain (KLC) isoforms target kinesin-1 to these different structures. Here, we test this hypothesis using two in vitro motility assays, which reconstitute the movement of rough endoplasmic reticulum (RER) and vesicles present in a Golgi membrane fraction. We generated GST-tagged fusion proteins of KLC1B and KLC1D that included the tetratricopeptide repeat domain and the variable C-terminus. We find that preincubation of RER with KLC1B inhibits RER motility, whereas KLC1D does not. In contrast, Golgi fraction vesicle movement is inhibited by KLC1D but not KLC1B reagents. Both RER and vesicle movement is inhibited by preincubation with the GST-tagged C-terminal domain of ubiquitous kinesin heavy chain (uKHC), which binds to the N-terminal domain of uKHC and alters its interaction with microtubules. We propose that although the TRR domains are required for cargo binding, it is the variable C-terminal region of KLCs that are vital for targeting kinesin-1 to different cellular structures. PMID:17093494

  10. The immunoglobulin light chain locus of the turkey, Meleagris gallopavo.

    PubMed

    Bao, Yonghua; Wu, Sun; Zang, Yunlong; Wang, Hui; Song, Xiangfeng; Xu, Chunyang; Xie, Bohong; Guo, Yongchen

    2012-06-15

    To date, most jawed vertebrate species encode more than one immunoglobulin light (IgL) chain isotypes. It has been shown that several bird species (chickens, white Pekin or domestic duck, and zebra finches) exclusively express lambda isotype. We analyze here the genomic organization of another bird species turkey IgL genes based on the recently released genome data. The turkey IgL locus located on chromosome 17 spans approximately 75.2kb and contains a single functional V(λ) gene, twenty V(λ) pseudogenes, and a single functional J(λ)-C(λ) block. These data suggest that the genomic organization of bird IgL chain genes seems to be conserved. Ten cDNA clones from turkey Igλ chain containing almost full-length V(λ), J(λ) and C(λ) segments were acquired. The comparison of V(λ) cDNA sequences to all the germline V(λ) segments suggests that turkey species may be generating IgL chain diversity by gene conversion and somatic hypermutation like the chicken. This study provides insights into the immunoglobulin light chain genes in another bird species.

  11. Localized axillary milia en plaque: a rare cutaneous case presentation of systemic amyloidosis.

    PubMed

    Dickison, P; Howard, V; Wylie, B; Smith, S D

    2016-10-01

    Systemic AL amyloidosis is known to be associated with plasma cell dyscrasias, including multiple myeloma. The cutaneous manifestations of systemic AL amyloidosis are varied, but typically include waxy plaques or subcutaneous nodules. We report a woman who presented with bilateral eruptions of hyperpigmented plaques in her axillae, which were diagnosed as milia en plaque. She had a history of multiple myeloma, for which she was under the care of a haematologist. This is the first documented case, to our knowledge, of an eruption in the axillae being milia en plaque.

  12. Localized axillary milia en plaque: a rare cutaneous case presentation of systemic amyloidosis.

    PubMed

    Dickison, P; Howard, V; Wylie, B; Smith, S D

    2016-10-01

    Systemic AL amyloidosis is known to be associated with plasma cell dyscrasias, including multiple myeloma. The cutaneous manifestations of systemic AL amyloidosis are varied, but typically include waxy plaques or subcutaneous nodules. We report a woman who presented with bilateral eruptions of hyperpigmented plaques in her axillae, which were diagnosed as milia en plaque. She had a history of multiple myeloma, for which she was under the care of a haematologist. This is the first documented case, to our knowledge, of an eruption in the axillae being milia en plaque. PMID:27663153

  13. Recurrent Light Chain Proximal Tubulopathy in a Kidney Allograft.

    PubMed

    Angioi, Andrea; Amer, Hatem; Fervenza, Fernando C; Sethi, Sanjeev

    2016-09-01

    We describe a rare case of light chain proximal tubulopathy developing in a kidney transplant 12 months following transplantation. The patient was known to have a monoclonal gammopathy of undetermined significance (MGUS) for more than 15 years. A kidney biopsy done to determine the cause of decline in kidney transplant function showed light chain proximal tubulopathy characterized by numerous eosinophilic and fuchsinophilic granules in proximal tubular epithelial cells, which stained for κ light chains on pronase-based immunofluorescence studies. Electron microscopy confirmed the diagnosis and showed numerous amorphous and geometrically shaped inclusions in proximal tubular epithelial cells. Evaluation of free light chains revealed markedly elevated κ light chains and bone marrow biopsy showed 5% to 10% κ light chain-restricted plasma cells. Retrospective evaluation of the native kidney biopsy performed 15 years earlier also showed numerous fuchsinophilic granules in proximal tubules that stained brightly for κ light chains on pronase-based immunofluorescence studies. The patient was treated with a regimen of bortezomib and dexamethasone with good partial hematologic response and improvement of kidney function. To summarize, we describe a case of recurrent light chain proximal tubulopathy in the transplant, which is an unusual but important cause of decreased kidney function in the setting of a monoclonal gammopathy. PMID:27321964

  14. 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....

  15. 21 CFR 866.5550 - Immunoglobulin (light chain specific) immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 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....

  16. 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....

  17. 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....

  18. 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....

  19. Tracheobronchial amyloidosis and confocal endomicroscopy.

    PubMed

    Newton, Richard C; Kemp, Samuel V; Yang, Guang-Zhong; Darzi, Ara; Sheppard, Mary N; Shah, Pallav L

    2011-01-01

    Tracheobronchial amyloidosis is one of many causes of endobronchial stenosis and nodularity, the concrete diagnosis of which currently requires the finding of apple-green birefringence from endobronchial biopsies. Bronchoscopic probe-based confocal endomicroscopy (pCLE) is a novel optical biopsy technique which provides real-time images of the lattice structure of the bronchial basement membrane - a finding lost in malignancy. This case study outlines the imperfect, essentially palliative management of this rare disease, and shows for the first time the unusual dappled in vivo pCLE images of amyloid-affected endobronchium.

  20. 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

  1. Structure and diversity of Mexican axolotl lambda light chains.

    PubMed

    André, S; Guillet, F; Charlemagne, J; Fellah, J S

    2000-11-01

    We report here the structure of cDNA clones encoding axolotl light chains of the lambda type. A single IGLC gene and eight different potential IGLV genes belonging to four different families were detected. The axolotl Cgamma domain has several residues or stretches of residues that are typically conserved in mammalian, avian, and Xenopus Cgamma, but the KATLVCL stretch, which is well conserved in the Cgamma and T-cell receptor Cbeta domains of many vertebrate species, is not well conserved. All axolotl Vgamma sequences closely match several human and Xenopus Vgamma-like sequences and, although the axolotl Cgamma and Vgamma sequences are very like their tetrapod homologues, they are not closely related to nontetrapod L chains. Southern blot experiments suggested the presence of a single IGLC gene and of a limited number of IGLV genes, and analysis of IGLV-J junctions clearly indicated that at least three of the IGLJ segments can associate with IGLV1, IGLV2, or IGLV3 subgroup genes. The overall diversity of the axolotl Vgamma CDR3 junctions seems to be of the same order as that of mammalian Vgamma chains. However, a single IGLV4 segment was found among the 45 cDNAs analyzed. This suggests that the axolotl IGL locus may have a canonical tandem structure, like the mammalian IGK or IGH loci. Immunofluorescence, immunoblotting, and microsequencing experiments strongly suggested that most, if not all L chains are of the gamma type. This may explain in part the poor humoral response of the axolotl. PMID:11132150

  2. [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.

  3. 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.

  4. A second immunoglobulin light chain isotype in the rainbow trout.

    PubMed

    Partula, S; Schwager, J; Timmusk, S; Pilström, L; Charlemagne, J

    1996-01-01

    A novel immunoglobulin (Ig) light chain isotype, termed IgL2, has been isolated from trout lymphoid tissues both by reverse transcription - polymerase chain reaction (PCR) and screening of cDNA libraries. The CL domain of the new isotype shares only 29% residues with a recently cloned trout IgL isotype, termed IgL1, which has some similarities to Ckappa and Clambda isotype domains of several vertebrate species. Using anchored PCR, a VL element rearranged to CL2 was isolated. It is a member of a new VL family (VL2) of which four members were sequenced. These differ in the sequence of CDR1 and CDR2 but are remarkably similar in CDR3, i. e., at the junction between VL and JL segments. VL elements are rearranged to novel JL elements which differ from those described for VL1-CL1 rearrangements. Two cDNA clones contained JL-CL2 segments but no VL segments. The JL segments were preceded by typical rearrangements signal sequences [RSS, nonamer-23 base pair (bp) spacer-heptamer]. Further upstream of RSS were located two to three near identical 53 bp repeats, each of which included a 16 bp sequence similar to KI and KII sequences located at similar places in human and mouse Jk1 genes. These sequences are believed to act as binding sites for the protein KLP, which could be a transcriptional factor involved in the synthesis of germline Jk transcripts. Their phylogenic conservation in vertebrates suggests that they have an important role in B-cell differentiation. Remarkably, an RNA species of about 0.7 kilobase is the predominant IgL mRNA in trout spleen and coincides in size with JLCL2 transcripts. Genomic DNA blot analysis indicates that the trout L2 locus has a cluster-like organization similar to the trout L1 locus and the IgL locus of several teleost fish. A phylogenic analysis of VL2 and CL2 corroborates their low similarity to other vertebrate IgL chains and suggests an ancient diversification of the IgL locus. PMID:8881036

  5. Involvement of myosin light-chain kinase in endothelial cell retraction

    SciTech Connect

    Wysolmerski, R.B.; Lagunoff, D. )

    1990-01-01

    Permeabilized bovine pulmonary artery endothelial cell monolayers were used to investigate the mechanism of endothelial cell retraction. Postconfluent endothelial cells permeabilized with saponin retracted upon exposure to ATP and Ca{sup 2+}. Retraction was accompanied by thiophosphorylation of 19,000-Da myosin light chains when adenosine 5'-(gamma-({sup 35}S)thio)triphosphate was included in the medium. Both retraction and thiophosphorylation of myosin light chains exhibited a graded quantitative dependence on Ca{sup 2+}. When permeabilized monolayers were extracted in buffer D containing 100 mM KCl and 30 mM MgCl2 for 30 min, the cells failed to retract upon exposure to ATP and Ca{sup 2+}, and no thiophosphorylation of myosin light chains occurred. The ability both to retract and to thiophosphorylate myosin light chains was restored by the addition to the permeabilized, extracted cells of myosin light-chain kinase and calmodulin together but not by either alone. These studies indicate that endothelial cell retraction, as does smooth muscle contraction, depends on myosin light-chain kinase phosphorylation of myosin light chains.

  6. Pneumatosis intestinalis due to gastrointestinal amyloidosis: A case report & review of literature

    PubMed Central

    Khalid, Filza; Kaiyasah, Hadiel; Binfadil, Wafa; Majid, Maiyasa; Hazim, Wessam; ElTayeb, Yousif

    2016-01-01

    Introduction Pneumatosis intestinalis (PI) is not a disease but a radiological finding with a poorly understood pathogenesis. It can be divided into primary/idiopathic (15%) or secondary (85%) Kim et al. 2007, based on the factors thought to play a role in its development. Amongst the rare causes of secondary PI is gastrointestinal (GI) amyloidosis. Presentation of the case We report a case of a 46-year-old gentleman who presented with a one month history of acute on chronic abdominal pain, associated with one episode of melena. Upon further investigation, he was found to have pneumoperitoneum. He was taken to the operating theatre, where he was noted to have features of pneumatosis intestinalis of the small bowel with no evidence of bowel perforation. Postoperatively, he underwent an upper GI endoscopy with biopsies that revealed GI amyloidosis. Discussion One of the rare causes that can lead to secondary PI is GI amyloidosis as proven in our case. Patients with symptomatic gastrointestinal amyloidosis usually present with one of four syndromes: gastrointestinal bleeding, malabsorption, protein-losing gastroenteropathy, and, less often, gastrointestinal dysmotility. Conclusion GI amyloidosis is a rare cause of secondary pneumatosis intestinalis. The presentation of the disease varies from patient to patient, therefore, the management should be tailored accordingly. PMID:27085104

  7. 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.

  8. 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. PMID:27113164

  9. Primary sjögren syndrome manifested as localized cutaneous nodular amyloidosis.

    PubMed

    Wey, Shiow-Jiuan; Chen, Yi-Ming; Lai, Po-Ju; Chen, Der-Yuan

    2011-10-01

    Localized cutaneous nodular amyloidosis (LCNA) is the rarest type of cutaneous amyloidosis. Typically presenting as waxy nodules on the lower extremities, it demonstrates localized deposition of AL-type amyloid in immunohistologic study and is often associated with focal plasma cell proliferation. Sjögren syndrome, an autoimmune lymphoproliferative disorder, is characterized by keratoconjunctivitis sicca and xerostomia with lymphocytic infiltration of exocrine glands. As shown in case reports, the association of LCNA with Sjögren syndrome is considerable. Herein, we report a 78-year-old woman with LCNA, who was further surveyed and diagnosed with Sjögren syndrome. In light of the significant relation between these 2 diseases, further examination for coexistence of Sjögren syndrome in addition to systemic amyloidosis is well warranted. Prompt identification of an underlying Sjögren syndrome in LCNA with polyclonal immunoglobulin amyloid may have important therapeutic consequences.

  10. Amyloidosis: Modern Cross-sectional Imaging.

    PubMed

    Czeyda-Pommersheim, Ferenc; Hwang, Misun; Chen, Sue Si; Strollo, Diane; Fuhrman, Carl; Bhalla, Sanjeev

    2015-01-01

    Amyloidosis is a rare diverse condition caused by the pathologic extracellular deposition of abnormal insoluble proteins throughout the body. It may exist as a primary disease or, more commonly, may be secondary to a wide variety of pathologic processes ranging from chronic infection or inflammation to malignancy. Hereditary forms also exist. On the basis of the structure of the protein deposits, more than two dozen subtypes of amyloidosis have been described. A single organ or multiple organ systems may be affected. The radiologic manifestations of amyloidosis are varied and often nonspecific, making amyloidosis a diagnostic challenge for the radiologist. In the chest, the lungs, mediastinum, pleura, and heart may be involved. Lung involvement may manifest as diffuse reticulonodular interstitial thickening, consolidations, or solitary or multiple parenchymal nodules that may calcify, cavitate, and slowly enlarge. Pleural involvement most commonly manifests as pleural effusions. Tracheobronchial involvement may exhibit concentric airway thickening, mural and intraluminal nodules, submucosal calcification, and airway obstruction. Mediastinal and hilar lymph nodes may enlarge and frequently calcify. At cardiac magnetic resonance (MR) imaging, the left ventricular wall is typically thickened, with associated diastolic dysfunction. Delayed contrast material-enhanced cardiac MR imaging typically shows global transmural or subendocardial enhancement. The pathophysiology, classification, treatment, and prognosis of amyloidosis are reviewed, followed by case examples of the appearance of thoracic and cardiac amyloidosis on chest radiographs, computed tomographic (CT) images, and cardiac MR images. PMID:26230754

  11. Clathrin light chains: arrays of protein motifs that regulate coated-vesicle dynamics.

    PubMed

    Brodsky, F M; Hill, B L; Acton, S L; Näthke, I; Wong, D H; Ponnambalam, S; Parham, P

    1991-06-01

    Polymerization of clathrin triskelions into clathrin coats and subsequent disassembly by the heat shock protein hsc70 control receptor-mediated pathways of intracellular transport. The clathrin light chains are major regulatory elements in these processes. These polypeptides consist of linear arrays of functional domains with distinctive sequence motifs. Comparison of unicellular and multicellular eukaryotes reveals differences in the numbers of clathrin light chains and in the functional domains they contain. PMID:1909824

  12. 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.

  13. Immunoglobulin light chain immunohistochemistry revisited, with emphasis on reactive follicular hyperplasia versus follicular lymphoma.

    PubMed

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

    2010-05-01

    The identification of monotypic light chains is an important adjunct to the diagnosis of B-cell lymphoma, yet to reliably perform it on formalin-fixed paraffin sections is often difficult. We have evaluated a new set of monoclonal antibodies to kappa and lambda light chains 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 of the appropriate light chain was seen in each of the 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 was also seen in 6 additional cases (10%) in which flow cytometry was 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.

  14. Light Chain Escape in 3 Cases: Evidence of Intraclonal Heterogeneity in Multiple Myeloma from a Single Institution in Poland.

    PubMed

    Kraj, Maria; Kruk, Barbara; Endean, Kelly; Warzocha, Krzysztof; Budziszewska, Katarzyna; Dąbrowska, Monika

    2015-01-01

    We report three cases of light chain escape (LCE) at a single institution in Poland, including an interesting case of biclonal monoclonal gammopathy of undetermined significance (MGUS) that satisfied the criteria for progression to light chain multiple myeloma (LCMM) with a rapid rise in serum free light chain (FLC) levels, following steroidal treatment for simultaneous temporal artery inflammation and polymyalgia rheumatica (PMR). In the three cases discussed, progression of the disease by light chain escape was associated with rapid and severe renal impairment, highlighting the necessity for prompt detection of such free light chain-only producing clones in order to prevent the possible development of irreversible end-organ damage. Interestingly, monitoring of these three patients by serum free light chain assay (sFLC) and retrospective heavy/light chain analysis (HLC) detected this clonal evolution prior to clinical relapse and suggests that these assays represent important additional tools for more accurate monitoring of multiple myeloma patients. PMID:26881153

  15. Light Chain Escape in 3 Cases: Evidence of Intraclonal Heterogeneity in Multiple Myeloma from a Single Institution in Poland

    PubMed Central

    Kraj, Maria; Kruk, Barbara; Endean, Kelly; Warzocha, Krzysztof; Budziszewska, Katarzyna; Dąbrowska, Monika

    2015-01-01

    We report three cases of light chain escape (LCE) at a single institution in Poland, including an interesting case of biclonal monoclonal gammopathy of undetermined significance (MGUS) that satisfied the criteria for progression to light chain multiple myeloma (LCMM) with a rapid rise in serum free light chain (FLC) levels, following steroidal treatment for simultaneous temporal artery inflammation and polymyalgia rheumatica (PMR). In the three cases discussed, progression of the disease by light chain escape was associated with rapid and severe renal impairment, highlighting the necessity for prompt detection of such free light chain-only producing clones in order to prevent the possible development of irreversible end-organ damage. Interestingly, monitoring of these three patients by serum free light chain assay (sFLC) and retrospective heavy/light chain analysis (HLC) detected this clonal evolution prior to clinical relapse and suggests that these assays represent important additional tools for more accurate monitoring of multiple myeloma patients. PMID:26881153

  16. 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

  17. [Osteoarticular amyloidosis caused by dialysis].

    PubMed

    Orzincolo, C; Bedani, P L; Scutellari, P N; Cardona, P; Farinelli, A; Vita, G

    1988-01-01

    The accumulation of amyloid in the bone and joint system has recently been recognized as a peculiar disease in patients undergoing long-term hemodialysis (5 years at least), especially in those who use cuprophan membranes. The pathology of amyloidosis is characterized by deposits of amyloid (beta 2-microglobulin mainly) in the bone, in the synovia, and in pericapsular soft tissues. The skeleton of 46 long-term hemodialysis patients (19 males and 27 females) was studied by X-ray: bone and joint abnormalities due to amyloid deposition were observed in 45% of cases. The shoulder, hip, and wrist were the most frequently involved joints. Destructive spondyloarthropathy was present in 15% of cases. The radiographic patterns of AOD are generally divided into axial and peripheral lesions. In the appendicular skeleton abnormalities include: well-defined lytic areas (geodes), pathologic fractures, marginal erosions, and periarticular soft tissue swelling. Destructive spondyloarthropathy is frequently present in the cervical spine (85% of our cases), and is characterized by narrowing of the intervertebral space, marginal erosion, and subchondral bone sclerosis of the vertebral body.

  18. 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.

  19. 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

  20. 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. PMID:27594885

  1. [Systemic amyloidosis secondary to xanthogranulomatous pyelonephritis].

    PubMed

    Almirall, J; López, T; Sáez, A; Gratacós, J; Prats, J

    2001-01-01

    Secondary amyloidosis is a relatively common pathology in which chronic infectious diseases are common causes, especially infected bronchiectasis, osteomyelitis or chronic ulcers. The association of xanthogranulomatous pyelonephritis and systemic amyloidosis is extremely rare. To our knowledge, despite innumerable cases of xanthogranulomatous pyelonephritis reported in the literature, this association has been reported in only 8 previous cases. Patients usually complain of fever, back or flank pain and urinary tract symptoms. A long lasting evolution of the process is frequent. We report a 70 year old patient who developed amyloidosis secondary to xanthogranulomatous pyelonephritis. As well as the rarity of this association, this case is exceptional in its clinical presentation, without any urinary tract symptoms that could suggest the diagnosis.

  2. Transthyretin Cardiac Amyloidosis in Black Americans.

    PubMed

    Shah, Keyur B; Mankad, Anit K; Castano, Adam; Akinboboye, Olakunle O; Duncan, Phillip B; Fergus, Icilma V; Maurer, Mathew S

    2016-06-01

    Transthyretin-related cardiac amyloidosis is a progressive infiltrative cardiomyopathy that mimics hypertensive and hypertrophic heart disease and often goes undiagnosed. In the United States, the hereditary form disproportionately afflicts black Americans, who when compared with whites with wild-type transthyretin amyloidosis, a phenotypically similar condition, present with more advanced disease despite having a noninvasive method for early identification (genetic testing). Although reasons for this are unclear, this begs to consider the inadequate access to care, societal factors, or a biological basis. In an effort to improve awareness and explore unique characteristics, we review the pathophysiology, epidemiology, and therapeutic strategies for transthyretin amyloidosis and highlight diagnostic pitfalls and clinical pearls for identifying patients with amyloid heart disease.

  3. Transthyretin Cardiac Amyloidosis in Black Americans.

    PubMed

    Shah, Keyur B; Mankad, Anit K; Castano, Adam; Akinboboye, Olakunle O; Duncan, Phillip B; Fergus, Icilma V; Maurer, Mathew S

    2016-06-01

    Transthyretin-related cardiac amyloidosis is a progressive infiltrative cardiomyopathy that mimics hypertensive and hypertrophic heart disease and often goes undiagnosed. In the United States, the hereditary form disproportionately afflicts black Americans, who when compared with whites with wild-type transthyretin amyloidosis, a phenotypically similar condition, present with more advanced disease despite having a noninvasive method for early identification (genetic testing). Although reasons for this are unclear, this begs to consider the inadequate access to care, societal factors, or a biological basis. In an effort to improve awareness and explore unique characteristics, we review the pathophysiology, epidemiology, and therapeutic strategies for transthyretin amyloidosis and highlight diagnostic pitfalls and clinical pearls for identifying patients with amyloid heart disease. PMID:27188913

  4. Surgical management of advanced ocular adnexal amyloidosis.

    PubMed

    Patrinely, J R; Koch, D D

    1992-06-01

    Ocular adnexal amyloidosis is characterized by amyloid deposition within the deep connective tissue layers of the eyelids, conjunctiva, and anterior orbit. Management of advanced cases has traditionally been unsatisfactory, with either no surgery offered because of fear of hemorrhage or an en bloc resection performed of the entire involved area. We present two cases of advanced periorbital amyloidosis successfully managed by preserving the anatomic planes of the eyelids and meticulously debulking the deposits with a spooned curette. Lax eyelid tendons and aponeuroses were simultaneously repaired, and no sacrifice of eyelid tissues was necessary. One patient remained asymptomatic for 2 years after surgery before developing early reaccumulation in the lower eyelids. The other patient required additional eyelid debulking and ptosis revision 8 months after surgery, but was in stable condition at follow-up 2 years after surgery. This technique offers safe, easily repeatable, nondestructive treatment for advanced periocular amyloidosis.

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

    PubMed

    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.

  6. 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

  7. Spontaneous pancreatic islet amyloidosis in 40 baboons.

    PubMed

    Hubbard, G B; Steele, K E; Davis, K J; Leland, M M

    2002-04-01

    Spontaneous amyloidosis occurs in many nonhuman primate species but remains difficult to diagnose and treat. Nonhuman primates continue to offer promise as animal models in which to study amyloidosis in humans. Amyloidosis was not diagnosed clinically but was found histologically in four male and 36 female baboons. The baboons averaged 18 years of age at death (range, 7-28 years). Clinical signs, if present, were hyperglycemia and cachexia. Blood glucose values were elevated in 12 of 30 baboons with available clinical pathology data. Four baboons had been clinically diagnosed as diabetic and three were treated with insulin. Amyloid was found in the islets of Langerhans of the pancreas in 40 baboons; 35 baboons had amyloid only in the islets of Langerhans. Amyloid was found in nonislet tissue of baboons as follows: five, nonislet pancreas; four, intestine and adrenal; three, kidney; two, prostate and spleen; and one each, lymph node, liver, gall bladder, stomach, tongue, urinary bladder, and salivary gland. Sections of paraffin-embedded tissues were evaluated for amyloid with hematoxylin and eosin (HE) and congo red (CR) staining, and using immunohistochemistry for human islet amyloid polypeptide (IAPP), calcitonin gene-related peptide (CGRP), glucagon, pancreatic polypeptide (PP), somatostatin (SS), and porcine insulin. Islet amyloid was positive with HE in 40 baboons, with CR in 39 baboons, and with IAPP and CGRP in 35 baboons. IAPP and CGRP only stained islet amyloid. PP, SS, glucagon, and porcine insulin did not stain amyloid. Islet amyloidosis in the baboon appears to be difficult to diagnose clinically, age-related, and similar to islet amyloidosis in other species. The baboon may be a good model for the study of islet amyloidosis in humans.

  8. Abnormal scintigraphic evolution in AA hepatic amyloidosis

    SciTech Connect

    Lomena, F.; Rosello, R.; Pons, F.; Grau, M.; Garcia, A.; Catafau, A.; Setoain, J.

    1988-03-01

    A patient with AA amyloidosis secondary to ankylosing spondylitis showed intense liver uptake of Tc-99m MDP on bone imaging. The biopsy showed hepatic amyloid deposition. A repeat bone scan with Tc-99m MDP 1 year later was negative, although the clinical signs and liver function tests of the patient had not changed. A mechanism might exist, other than the affinity of amyloid to calcium, which would explain the extraosseous uptake of pyrophosphates and diphosphonates in organs and soft tissues affected by systemic amyloidosis.

  9. Regulatory and essential light chains of myosin rotate equally during contraction of skeletal muscle.

    PubMed

    Borejdo, Julian; Ushakov, Dmitry S; Akopova, Irina

    2002-06-01

    Myosin head consists of a globular catalytic domain and a long alpha-helical regulatory domain. The catalytic domain is responsible for binding to actin and for setting the stage for the main force-generating event, which is a "swing" of the regulatory domain. The proximal end of the regulatory domain contains the essential light chain 1 (LC1). This light chain can interact through the N and C termini with actin and myosin heavy chain. The interactions may inhibit the motion of the proximal end. In consequence the motion of the distal end (containing regulatory light chain, RLC) may be different from the motion of the proximal end. To test this possibility, the angular motion of LC1 and RLC was measured simultaneously during muscle contraction. Engineered LC1 and RLC were labeled with red and green fluorescent probes, respectively, and exchanged with native light chains of striated muscle. The confocal microscope was modified to measure the anisotropy from 0.3 microm(3) volume containing approximately 600 fluorescent cross-bridges. Static measurements revealed that the magnitude of the angular change associated with transition from rigor to relaxation was less than 5 degrees for both light chains. Cross-bridges were activated by a precise delivery of ATP from a caged precursor. The time course of the angular change consisted of a fast phase followed by a slow phase and was the same for both light chains. These results suggest that the interactions of LC1 do not inhibit the angular motion of the proximal end of the regulatory domain and that the whole domain rotates as a rigid body.

  10. Growth Hormone and Cerebral Amyloidosis.

    PubMed

    Benvenga, S; Guarneri, F

    2016-08-01

    Great interest has recently been focused on a paper reporting characteristic deposits of amyloid-β protein associated with Alzheimer's disease in brains of adults who died of Creutzfeldt-Jakob disease. As they had contracted such disease after treatment with prion-contaminated human growth hormone extracted from cadaver-derived pituitaries, the authors have suggested that interhuman transmission of Alzheimer's disease had occurred. Our previous research led us to find that amyloid-forming peptides share amino acid sequence homology, summarized by a motif. Here, we probed the amino acid sequence of human growth hormone for such a motif, and found that 2 segments fit the motif and are potentially amyloid-forming. This finding was confirmed by Aggrescan, another well-known software for the prediction of amyloidogenic peptides. Our results, taken together with data from the literature that are missing in the aforementioned paper and associated commentaries, minimize the contagious nature of the iatrogenically-acquired coexistence of Creutzfeldt-Jakob disease and Alzheimer's disease. In particular, the above mentioned paper misses literature data on intratumoral amyloidosis in growth hormone- and prolactin-secreting adenomas, tumors relatively frequent in adults, which are often silent. It cannot be excluded that some pituitaries used to extract growth hormone contained clinically silent microadenomas, a fraction of which containing amyloid deposits, and patients might had received a fraction of growth hormone (with or without prolactin) that already was an amyloid seed. The intrinsic amyloidogenicity of growth hormone, in the presence of contaminating prion protein (and perhaps prolactin as well) and amyloid-β contained in some cadavers' pituitaries, may have led to the observed co-occurring of Creutzfeldt-Jakob disease and Alzheimer's disease. PMID:27214308

  11. 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.

  12. Effect of nucleotides and actin on the orientation of the light chain-binding domain in myosin subfragment 1.

    PubMed

    Smyczynski, C; Kasprzak, A A

    1997-10-28

    The X-ray structure of myosin head (S1) reveals the presence of a long alpha-helical structure that supports both the essential and the regulatory light chains. It has been proposed that small structural changes in the catalytic domain of S1 are amplified by swinging the long alpha-helix (the "lever arm") to produce approximately 11 nm steps. To probe the spatial position of the putative lever in various S1 states, we have measured, by fluorescence resonance energy transfer (FRET), the effect of nucleotides and actin on the distances between Cys-177 of the essential light chain A1 (which is attached to the alpha-helix) and three loci in the catalytic domain. Cys-177 (donor) was labeled with 1,5-IAEDANS. The trinitrophenylated ADP analog (TNP-ADP, acceptor) was used to measure the distance to the active site. Lys-553 at the actin-binding site, labeled with a fluorescein derivative, and Lys-83 modified with trinitrobenzenesulfonic acid served as two other acceptors. FRET measurements were performed for S1 alone, for its complexes with MgADP and MgATP, for the analogs of the transition state of the ATPase reaction, S1.ADP.BeFx, S1.ADP.AlF4, and S1.ADP.VO4, and for acto-S1 in the absence and in the presence of ADP. When the transition state and acto-S1 complexes were formed, the change in the Cys-177 --> Lys-83 distance was <1.1 A, for the distance Cys-177 --> Lys-553, the change was +/-2.5 A. These distance changes correspond to rotations by <10 degrees and approximately 25 degrees, respectively. For the Cys-177 --> TNP-ADP the interprobe separation decreased by approximately 6 A in the presence of BeFx and AlF4- but only 1.9 A in the presence of vanadate; we do not interpret the 6 A change as resulting from the lever rotation. Using the coordinates of the acto-S1 complex, we have computed the expected changes in these distances resulting from rotation of the lever. These changes were much greater than the ones observed. The above results are inconsistent with models

  13. 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

  14. Primary diffuse tracheo-bronchial amyloidosis

    PubMed Central

    Antunes, Maria L.; Da Luz, J. M. Vieira

    1969-01-01

    A case of diffuse tracheo-bronchial amyloidosis in a 62-year-old woman thought to have chronic bronchitis is reported. The immunological mechanism in the pathogenesis of this condition is considered and the need for bronchological investigation in chronic bronchial disorders is stressed. Images PMID:5810372

  15. 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...

  16. Structure of the light chain-binding domain of myosin V

    PubMed Central

    Terrak, Mohammed; Rebowski, Grzegorz; Lu, Renne C.; Grabarek, Zenon; Dominguez, Roberto

    2005-01-01

    Myosin V is a double-headed molecular motor involved in organelle transport. Two distinctive features of this motor, processivity and the ability to take extended linear steps of ≈36 nm along the actin helical track, depend on its unusually long light chain-binding domain (LCBD). The LCBD of myosin V consists of six tandem IQ motifs, which constitute the binding sites for calmodulin (CaM) and CaM-like light chains. Here, we report the 2-Å resolution crystal structure of myosin light chain 1 (Mlc1p) bound to the IQ2–IQ3 fragment of Myo2p, a myosin V from Saccharomyces cerevisiae. This structure, combined with FRET distance measurements between probes in various CaM–IQ complexes, comparative sequence analysis, and the previously determined structures of Mlc1p-IQ2 and Mlc1p-IQ4, allowed building a model of the LCBD of myosin V. The IQs of myosin V are distributed into three pairs. There appear to be specific cooperative interactions between light chains within each IQ pair, but little or no interaction between pairs, providing flexibility at their junctions. The second and third IQ pairs each present a light chain, whether CaM or a CaM-related molecule, bound in a noncanonical extended conformation in which the N-lobe does not interact with the IQ motif. The resulting free N-lobes may engage in protein–protein interactions. The extended conformation is characteristic of the single IQ of myosin VI and is common throughout the myosin superfamily. The model points to a prominent role of the LCBD in the function, regulation, and molecular interactions of myosin V. PMID:16120677

  17. Localized amyloidosis masquerading as nasopharyngeal tumor: a review.

    PubMed

    Panda, Naresh K; Saravanan, Karuppiah; Purushotaman, Gilbert Pragache; Gurunathan, Ramesh Kumar; Mahesha, Vankalakunti

    2007-01-01

    Amyloidosis comprises a diverse collection of disease characterized by the presence of amorphous extracellular eosinophilic deposits of unique protein fibrils that gives apple green birefringence under polarized light after staining with Congo red. Head and neck region is the commonest site for localized form of amyloidosis. We report a case of a 43-year-old man with localized amyloidosis of nasopharynx with oropharyngeal extension and its management, along with a review of relevant literatures.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. Nodular pulmonary amyloidosis associated with asbestos exposure.

    PubMed

    Hiroshima, K; Ohwada, H; Ishibashi, M; Yamamoto, N; Tamiya, N; Yamaguchi, Y

    1996-01-01

    A 71 year old man was admitted for the purpose of diagnosis of a right solitary pulmonary nodule. The size of the nodule was 18 x 18 mm in diameter 2 years ago, but it has become large, 25 x 25 mm in diameter. The nodule was resected by thoracotomy. Microscopically, eosinophilic amorphous, acellular substances were surrounded by inflammatory infiltrates. It stained with Congo red stain and showed green birefringence with polarizing microscopy. Amyloid fibrils were observed electron microscopically. Asbestos bodies were observed in the lung parenchyma around the nodule. This case shows that a nodule in nodular pulmonary amyloidosis can grow gradually and suggests the possibility of asbestos fibers as one of the etiologic factors in nodular pulmonary amyloidosis. PMID:10846552

  3. Hereditary Transthyretin Amyloidosis in Eight Chinese Families

    PubMed Central

    Meng, Ling-Chao; Lyu, He; Zhang, Wei; Liu, Jing; Wang, Zhao-Xia; Yuan, Yun

    2015-01-01

    Background: Mutations of transthyretin (TTR) cause the most common type of autosomal-dominant hereditary systemic amyloidosis, which occurs worldwide. To date, more and more mutations in the TTR gene have been reported. Some variations in the clinical presentation are often observed in patients with the same mutation or the patients in the same family. The purpose of this study was to find out the clinicopathologic and genetic features of Chinese patients with hereditary TTR amyloidosis. Methods: Clinical and necessary examination materials were collected from nine patients of eight families with hereditary TTR amyloidosis at Peking University First Hospital from January 2007 to November 2014. Sural nerve biopsies were taken for eight patients and skin biopsies were taken in the calf/upper arm for two patients, for light and electron microscopy examination. The TTR genes from the nine patients were analyzed. Results: The onset age varied from 23 to 68 years. The main manifestations were paresthesia, proximal and/or distal weakness, autonomic dysfunction, cardiomyopathy, vitreous opacity, hearing loss, and glossohypertrophia. Nerve biopsy demonstrated severe loss of myelinated fibers in seven cases and amyloid deposits in three. One patient had skin amyloid deposits which were revealed from electron microscopic examination. Genetic analysis showed six kinds of mutations of TTR gene, including Val30Met, Phe33Leu, Ala36Pro, Val30Ala, Phe33Val, and Glu42Gly in exon 2. Conclusions: Since the pathological examinations of sural nerve were negative for amyloid deposition in most patients, the screening for TTR mutations should be performed in all the adult patients, who are clinically suspected with hereditary TTR amyloidosis. PMID:26521788

  4. A Familial Poikiloderma-Like Cutaneous Amyloidosis

    PubMed Central

    Unni, Mahesh; Ankad, Balachandra; Naidu, Varna; Sudakar Rao, KM

    2014-01-01

    Familial poikiloderma-like cutaneous amyloidosis(FPLCA) is a rare, generalized but genetic dyschromic skin disorder characterized by amyloid deposits in dermis due to defective DNA repair secondary to sunlight damage. Clinically, it presents with diffuse brownish pigmentation with hypo-pigmented macules and many brownish scattered lichenoid papules with normal developmental milestones. The condition is autosomal dominant with incomplete penetrance. We are here reporting a rare familial case of FPLCA with a review of the literature PMID:25484425

  5. A novel antibody light chain dimer: Implications for T-cell receptor structure

    SciTech Connect

    Schiffer, M.; Chang, Chong-Hwan; Solomon, A.; Stevens, F.J.

    1989-01-01

    The dimeric structures of antibody light chains produced in patients with multiple myeloma (Bence Jones proteins) have for some time been studied chemically and crystallographically as models of the antigen binding fragment (Fab) of an antibody. The conformational concordance of Fabs and a Bence Jones dimer was demonstrated by the initial immunoglobulin crystallographic structures. We have recently described the structure of a second intact light chain, the lambda-type protein Loc. The Loc protein exhibits an unanticipated protruding arrangement of its complementarity-determining residues. Grooves on each side of the protrusion may function as separate binding sites. In this report, we examine the Loc structure and its intracrystalline interactions in more detail and consider aspects of this structure that may possess implications for models of a nonantibody constituent of the immunoglobulin superfamily, the T-cell antigen receptor. 26 refs., 3 figs., 1 tab.

  6. 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. PMID:22471437

  7. 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.

  8. Immunoglobulin heavy chain/light chain pairs (HLC, Hevylite™) assays for diagnosing and monitoring monoclonal gammopathies.

    PubMed

    Kraj, Maria

    2014-01-01

    Immunofixation (IFE) is a standard method for detecting monoclonal immunoglobulins and characterizing its isotype. Recently clonality can also be determined by using immunoglobulin (Ig) heavy chain/light chain immunoassays - HLC, HevyliteTM. HLC separately measures in pairs light chain types of each intact Ig class generating ratios of monoclonal Ig/uninvolved polyclonal Ig concentrations. Studies have shown that HLC and IFE are complementary methods. HLC assays quantify monoclonal proteins and identify monoclonality. It is possible to predict prognosis in multiple myeloma and to monitor response to treatment using HLC ratio. HLC ratio may serve as a parameter for myeloma induced immunoparesis and serve as a new marker for validating remission depth and relapse probabilities.

  9. 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.

  10. EPR and CD spectroscopy of fast myosin light chain conformation during binding of trifluoperazine.

    PubMed

    Huang, W; Wilson, G J; Brown, L J; Lam, H; Hambly, B D

    1998-10-15

    The conformations of isolated rabbit fast myosin light chains (LCs) were modified using trifluoperazine (TFP), the hydrophobic calmodulin inhibitor. CD spectroscopy showed that TFP altered secondary structural content of the LCs, with half-maximal effects at TFP concentrations of approximately 14-50 microM, which is within the range required to alter muscle fiber contraction in both agonistic and antagonistic ways [Kurebayashi, N. & Ogawa, Y. (1988) J. Physiol. 403, 407-424]. EPR spectroscopy provided structural information from paramagnetic probes on C-terminal domain surfaces. In the absence of TFP, tauR (rotational correlation time) was 1.6 ns for both alkali light chains (ALCs) and 1.8 ns for light chain 2 (LC2). This was faster than expected for proteins of this size (approximately 10 ns). TFP progressively recruited the probes into populations with tauR sevenfold to 12-fold slower, with half-maximal effects at a TFP concentration of approximately 370-800 microM. The differences probably indicate that CD spectroscopy detects changes in protein conformation due to 'specific' TFP binding at the LC hydrophobic core, while less specific binding at higher TFP concentrations is required to effect conformational changes on the protein surfaces near the paramagnetic probes. TFP binding was generally not cooperative. Comparative sequence analysis between calmodulin, troponin C, and myosin LCs indicated considerable conservation between residues expected to bind TFP.

  11. Tumor Stiffness Is Unrelated to Myosin Light Chain Phosphorylation in Cancer Cells

    PubMed Central

    Fry, Madeline; Greene, Madelyne; Chernaya, Olga; Hu, Wen-Yang; Chew, Teng-Leong; Mahmud, Nadim; Kadkol, Shrihari S.; Glover, Sarah; Prins, Gail; Strakova, Zuzana; de Lanerolle, Primal

    2013-01-01

    Many tumors are stiffer than their surrounding tissue. This increase in stiffness has been attributed, in part, to a Rho-dependent elevation of myosin II light chain phosphorylation. To characterize this mechanism further, we studied myosin light chain kinase (MLCK), the main enzyme that phosphorylates myosin II light chains. We anticipated that increases in MLCK expression and activity would contribute to the increased stiffness of cancer cells. However, we find that MLCK mRNA and protein levels are substantially less in cancer cells and tissues than in normal cells. Consistent with this observation, cancer cells contract 3D collagen matrices much more slowly than normal cells. Interestingly, inhibiting MLCK or Rho kinase did not affect the 3D gel contractions while blebbistatin partially and cytochalasin D maximally inhibited contractions. Live cell imaging of cells in collagen gels showed that cytochalasin D inhibited filopodia-like projections that formed between cells while a MLCK inhibitor had no effect on these projections. These data suggest that myosin II phosphorylation is dispensable in regulating the mechanical properties of tumors. PMID:24224004

  12. 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

  13. 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.

  14. 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.

  15. Analysis of heavy and light chain pairings indicates that receptor editing shapes the human antibody repertoire.

    PubMed

    de Wildt, R M; Hoet, R M; van Venrooij, W J; Tomlinson, I M; Winter, G

    1999-01-22

    In the bone marrow, diversity in the primary antibody repertoire is created by the combinatorial rearrangement of different gene segments and by the association of different heavy and light chains. During the secondary response in the germinal centres, antibodies are diversified by somatic mutation and possibly by further rearrangements, or "receptor editing". Here, we have analysed the pairings of heavy and light chain variable domains (VH and VL) in 365 human IgG+ B cells from peripheral blood, and established that these pairings are largely random. The repertoire is dominated by a limited number of pairings of segments and folds. Among these pairings we identified two identical mutated heavy chains in combination with two different mutated light chains (one kappa and one lambda). This shows that receptor editing occurs in the human periphery and that the same antibody lineage can be subjected to both receptor editing and somatic hypermutation. This suggests that receptor editing may be used together with somatic mutation for the affinity maturation of antibodies. We also propose that receptor editing has shaped variable gene segment use and the evolution of V gene families.

  16. Production and characterization of monoclonal antibodies specific for kappa and. gamma. light chain types of porcine immunoglobulins

    SciTech Connect

    McCauley, I.; Kim, Y.B.

    1986-03-05

    It has been difficult to raise specific antisera to the light chain types of pigs because of the difficulty in isolating sufficient pure material from polyclonal immunoglobulin. The authors have taken an approach based upon the characterization of a number of monoclonal antibodies (MoAb) raised against porcine IgG in order to obtain antisera specific for light chain types. Spleen cells from mice immunized with porcine IgG were fused with myeloma P3x63-Ag 653. Hybridomas were screened by an ELISA technique against pure porcine light chains coated on microtiter plates. Five clones specific for light chains were isolated. MoAb from these clones have been characterized by sequential immunoprecipitation of /sup 125/I labelled light chains. The pattern of reactivities show that the MoAb can be classified into two mutually exclusive groups, each of which precipitate approximately equal amounts of the labelled light chains. The type specificity of these groups has been determined by utilizing the cross-reaction between anti-human kappa and ..gamma.. with porcine light chains and the groups of MoAb in sequential immunoprecipitations. The MoAb were used in an immunofluorescence study of porcine B lymphocytes. The anti-..gamma.. MoAb stained 57% and the anti-kappa, 43% of total B lymphocytes.

  17. 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. PMID:22448530

  18. 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.

  19. Thirteen is enough: the myosins of Dictyostelium discoideum and their light chains

    PubMed Central

    Kollmar, Martin

    2006-01-01

    Background Dictyostelium discoideum is one of the most famous model organisms for studying motile processes like cell movement, organelle transport, cytokinesis, and endocytosis. Members of the myosin superfamily, that move on actin filaments and power many of these tasks, are tripartite proteins consisting of a conserved catalytic domain followed by the neck region consisting of a different number of so-called IQ motifs for binding of light chains. The tails contain functional motifs that are responsible for the accomplishment of the different tasks in the cell. Unicellular organisms like yeasts contain three to five myosins while vertebrates express over 40 different myosin genes. Recently, the question has been raised how many myosins a simple multicellular organism like Dictyostelium would need to accomplish all the different motility-related tasks. Results The analysis of the Dictyostelium genome revealed thirteen myosins of which three have not been described before. The phylogenetic analysis of the motor domains of the new myosins placed Myo1F to the class-I myosins and Myo5A to the class-V myosins. The third new myosin, an orphan myosin, has been named MyoG. It contains an N-terminal extension of over 400 residues, and a tail consisting of four IQ motifs and two MyTH4/FERM (myosin tail homology 4/band 4.1, ezrin, radixin, and moesin) tandem domains that are separated by a long region containing an SH3 (src homology 3) domain. In contrast to previous analyses, an extensive comparison with 126 class-VII, class-X, class-XV, and class-XXII myosins now showed that MyoI does not group into any of these classes and should not be used as a model for class-VII myosins. The search for calmodulin related proteins revealed two further potential myosin light chains. One is a close homolog of the two EF-hand motifs containing MlcB, and the other, CBP14, phylogenetically groups to the ELC/RLC/calmodulin (essential light chain/regulatory light chain) branch of the tree

  20. 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

  1. 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.

  2. Myosin light chain kinase-dependent microvascular hyperpermeability in thermal injury.

    PubMed

    Huang, Qiaobing; Xu, Wenjuan; Ustinova, Elena; Wu, Mack; Childs, Ed; Hunter, Felicia; Yuan, Sarah

    2003-10-01

    Although the critical role of systemic inflammatory edema in the development of multiple organ failure in patients with massive burns has been fully recognized, the precise mechanisms responsible for the accumulation of blood fluid and proteins in tissues remote from the burn wound are poorly understood. The aim of this study was to test the hypothesis that circulating factors released during thermal injury cause microvascular leakage by triggering endothelial cell contraction and barrier dysfunction. A third-degree scald burn was induced in rats on the dorsal skin covering 25% total body surface area. The microcirculation and transvascular flux of albumin were observed in the rat mesentery using intravital fluorescence microscopy. The direct effect of circulating factors on microvascular barrier function was assessed by measuring the apparent permeability coefficient of albumin in isolated rat mesenteric venules during perfusion of plasma freshly withdrawn from burned rats. The in vivo study showed that the transvenular flux of albumin was significantly increased over a 6-h period with a maximal response seen at 3 h postburn. Importantly, perfusion of noninjured venules with burn plasma induced a time-dependent increase in albumin permeability. Pharmacological inhibition of protein kinase C, Src tyrosine kinases, or mast cell activation did not significantly affect the hyperpermeability response; however, blockage of myosin light chain phosphorylation with the myosin light chain kinase inhibitor ML-7 greatly attenuated the burn-induced increase in venular permeability in a dose-related pattern. The results support a role for endogenous circulating factors in microvascular leakage during burns. Myosin light chain phosphorylation-dependent endothelial contractile response may serve as an end-point effector leading to microvascular barrier dysfunction. PMID:14501951

  3. High sensitivity and specificity of elevated cerebrospinal fluid kappa free light chains in suspected multiple sclerosis.

    PubMed

    Hassan-Smith, G; Durant, L; Tsentemeidou, A; Assi, L K; Faint, J M; Kalra, S; Douglas, M R; Curnow, S J

    2014-11-15

    Cerebrospinal fluid (CSF) analysis is routinely used in the diagnostic work-up of multiple sclerosis (MS), by detecting CSF-specific oligoclonal bands (OCB). More recently, several studies have reported CSF free light chains (FLC) as an alternative. We show that absolute CSF κFLC concentrations were highly sensitive - more than OCB testing - and specific for clinically isolated syndrome, relapsing remitting and primary progressive MS. Measurement of κFLC alone was sufficient. Our results suggest that CSF κFLC levels measured by nephelometry, if validated in a larger series, are a preferred test to OCB analysis in the diagnostic work-up of patients suspected of having MS.

  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. PMID:26682113

  5. 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.

  6. Characterization of DLC-A and DLC-B, two families of cytoplasmic dynein light chain subunits.

    PubMed Central

    Gill, S R; Cleveland, D W; Schroer, T A

    1994-01-01

    Cytoplasmic dynein is a minus-end-directed, microtubule-dependent motor composed of two heavy chains (approximately 530 kDa), three intermediate chains (approximately 74 kDa), and a family of approximately 52-61 kDa light chains. Although the approximately 530 kDa subunit contains the motor and microtubule binding domains of the complex, the functions of the smaller subunits are not known. Using two-dimensional gel electrophoresis and proteolytic mapping, we show here that the light chains are composed of two major families, a higher M(r) family (58, 59, 61 kDa; dynein light chain group A [DLC-A]) and lower M(r) family (52, 53, 55, 56 kDa; dynein light chain group B [DLC-B]). Dissociation of the cytoplasmic dynein complex with potassium iodide reveals that all light chain polypeptides are tightly associated with the approximately 530 kDa heavy chain, whereas the approximately 74 kDa intermediate chain polypeptides are more readily extracted. Treatment with alkaline phosphatase alters the mobility of four of the light chain polypeptides, indicating that these subunits are phosphorylated. Sequencing of a cDNA clone encoding one member of the DLC-A family reveals a predicted globular structure that is not homologous to any known protein but does contain numerous potential phosphorylation sites and a consensus nucleotide-binding motif. Images PMID:7949421

  7. Anti-cardiolipin/beta-2 glycoprotein activities co-exist on human anti-DNA antibody light chains.

    PubMed

    Kumar, Sanjeev; Nagl, Sylvia; Kalsi, J K; Ravirajan, C T; Athwal, Dee; Latchman, David S; Pearl, Laurence H; Isenberg, David A

    2003-12-01

    We have recently shown that the human anti-DNA antibodies B3 and 33H11 also bind cardiolipin and that the anti-autoantigen activity resides predominantly on their lambda light chains. We now show that the two auto-antibodies possess strong reactivity to the plasma-protein 2-Glycoprotein I (beta2-GPI) also. Utilizing chain shuffling experiments involving an unrelated anti-p185 antibody 4D5 with insignificant reactivity to cardiolipin or to beta2-GPI, we now demonstrate that hybrid Fabs with constituent light chain, but not the heavy chain, of B3 or 33H11, exhibit anti-cardiolipin activity. Furthermore, the constructs possessing the auto-antibody-derived light chain also exhibited significant reactivity to beta2-GPI. The results suggest that anti-DNA, anti-cardiolipin and anti-beta2-GPI activities co-exist on the light chains of the antibodies studied and, importantly, these activities could be transferred to antibody constructs by their light chains alone. Computer-generated models of the three-dimensional structures of the auto-antibodies and their hybrids, suggest predominant interaction of their light chains with domain IV of beta2-GPI.

  8. 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

  9. Unusual Combination of Tracheobronchopathia Osteochondroplastica and AA Amyloidosis

    PubMed Central

    Kırbaş, Gökhan; Dağlı, Canan Eren; Tanrıkulu, Abdullah Çetin; Yıldız, Fetin; Bükte, Yaşar; Kıyan, Esen

    2009-01-01

    Tracheobronchopathia osteochondroplastica (TO) is a rare disorder of unknown cause characterized by the presence of multiple submucosal osseous and/or cartilaginous nodules that protrude into the lumen of the trachea and large bronchi. A simultaneous diagnosis of TO and amyloidosis is rarely reported. In this report, a case initially suspected to be asthma bronchiole that could not be treated, was radiologically diagnosed as TO, and also secondary amyloidosis is presented. A 53 years, man patient reported a 3 years history of dyspnea. Pulmonary function tests (PFTs) showed an obstructive pattern. Chest X-rays revealed right middle lobe atelectasis. FOB and CT detected nodular lesions in the trachea and in the anterior and lateral walls of the main bronchi. AA amyloidosis was confirmed by endobronchial biopsy. In the abdominal fat pad biopsy, amyloidosis was not detected. Asthma bronchiole was excluded by PFTs. This case illustrates that it is possible for TO and amyloidosis to masquerade as asthma. TO and amyloidosis should be suspected in patients of older ages with asthma and especially with poorly treated asthmatic patients. Although nodular lesions in the anterior and lateral tracheobronchial walls are typical for TO, a biopsy should be obtained to exclude amyloidosis. PMID:19881980

  10. Neurologic complications of primary systemic amyloidosis.

    PubMed

    Kelly, John J

    2006-01-01

    Amyloid is a botanical term that denotes a waxy, amorphous, eosinophilic material and is used to describe similar pathological findings in humans. The deposits in primary systemic amyloidosis are derived from monoclonal serum proteins in plasma cell dyscrasia, which are degraded locally in tissues and deposited in insoluble sheets that damage organs. The cause of organ damage in amyloidosis is unclear but most likely due to the direct toxic effects of amyloid. Symptoms of the disorder include neuropathy, myopathy, and cardiac or renal insufficiency; there is often multiple-organ involvement. Congestive heart failure and nephrotic syndrome are predominant causes of death. Differential difficulties exist in diagnosing the disorder, and familial amyloid polyneuropathy directly mimics the disease. Diagnostic tools include electromyography, laboratory testing for abnormalities in serum and urine, and histological investigation of appropriate tissue. The median survival of patients is 2 years. However, melphalan and prednisone treatment for at least 1 year has resulted in increased survival rates. There have also been reports of benefit from high-dose chemotherapy followed by peripheral blood stem cell transplantation. Without early therapy, however, the disease has a dismal prognosis, and peripheral neuropathy usually persists or worsens despite therapy and improvement in other organs.

  11. CaMKII in addition to MLCK contributes to phosphorylation of regulatory light chain in cardiomyocytes.

    PubMed

    Eikemo, Hilde; Moltzau, Lise Román; Hussain, Rizwan I; Nguyen, Cam H T; Qvigstad, Eirik; Levy, Finn Olav; Skomedal, Tor; Osnes, Jan-Bjørn

    2016-02-26

    The aim was to identify kinase activities involved in the phosphorylation of regulatory light chain (RLC) in situ in cardiomyocytes. In electrically stimulated rat cardiomyocytes, phosphatase inhibition by calyculin A unmasked kinase activities evoking an increase of phosphorylated RLC (P-RLC) from about 16% to about 80% after 80 min. The phosphorylation rate in cardiomyocytes was reduced by about 40% by the myosin light chain kinase (MLCK) inhibitor, ML-7. In rat ventricular muscle strips, calyculin A induced a positive inotropic effect that correlated with P-RLC levels. The inotropic effect and P-RLC elevation were abolished by ML-7 treatment. The kinase activities phosphorylating RLC in cardiomyocytes were reduced by about 60% by the non-selective kinase inhibitor staurosporine and by about 50% by the calmodulin antagonist W7. W7 eliminated the inhibitory effect of ML-7, suggesting that the cardiac MLCK is Ca(2+)/calmodulin (CaM)-dependent. The CaM-dependent kinase II (CaMKII) inhibitor KN-93 attenuated the calyculin A-induced RLC phosphorylation by about 40%, indicating a contribution from CaMKII. The residual phosphorylation in the presence of W7 indicated that also CaM-independent kinase activities might contribute. RLC phosphorylation was insensitive to protein kinase C inhibition. In conclusion, in addition to MLCK, CaMKII phosphorylates RLC in cardiomyocytes. Involvement of other kinases cannot be excluded.

  12. The Roadblock light chains are ubiquitous components of cytoplasmic dynein that form homo- and heterodimers.

    PubMed

    Nikulina, Karina; Patel-King, Ramila S; Takebe, Sachiko; Pfister, K Kevin; King, Stephen M

    2004-04-01

    The Roadblock/LC7 class of light chains associate with the intermediate chains at the base of the soluble dynein particle. In mammals, there are two Roadblock isoforms (Robl1 and Robl2), one of which (Robl2) is differentially expressed in a tissue-dependent manner and is especially prominent in testis. Here we define the alpha helical content of Robl and demonstrate using both the yeast two-hybrid system and in vitro biochemistry that Robl1 and Robl2 are capable of forming homo- and heterodimers. This is the first report of heterodimer formation by any cytoplasmic dynein component, and it further enlarges the number of potential cytoplasmic dynein isoforms available for binding specific cellular cargoes. In addition, we have generated an antibody that specifically recognizes Robl light chains and shows a 5-10 fold preference for Robl2 over Robl1. Using this antibody, we show that Robl is a ubiquitous cytoplasmic dynein component, being found in samples purified from brain, liver, kidney, and testis. Immunofluorescence analysis reveals that Robl is present in punctate organelles in rat neuroblastoma cells. In testis, Robl is found in Leydig cells, spermatocytes, and sperm flagella.

  13. Endothelial cell substrate stiffness influences neutrophil transmigration via myosin light chain kinase-dependent cell contraction.

    PubMed

    Stroka, Kimberly M; Aranda-Espinoza, Helim

    2011-08-11

    A vast amount of work has been dedicated to the effects of shear flow and cytokines on leukocyte transmigration. However, no studies have explored the effects of substrate stiffness on transmigration. Here, we investigated important aspects of endothelial cell contraction-mediated neutrophil transmigration using an in vitro model of the vascular endothelium. We modeled blood vessels of varying mechanical properties using fibronectin-coated polyacrylamide gels of varying physiologic stiffness, plated with human umbilical vein endothelial cell (HUVEC) monolayers, which were activated with tumor necrosis factor-α. Interestingly, neutrophil transmigration increased with increasing substrate stiffness below the endothelium. HUVEC intercellular adhesion molecule-1 expression, stiffness, cytoskeletal arrangement, morphology, and cell-substrate adhesion could not account for the dependence of transmigration on HUVEC substrate stiffness. We also explored the role of cell contraction and observed that large holes formed in endothelium on stiff substrates several minutes after neutrophil transmigration reached a maximum. Further, suppression of contraction through inhibition of myosin light chain kinase normalized the effects of substrate stiffness by reducing transmigration and eliminating hole formation in HUVECs on stiff substrates. These results provide strong evidence that neutrophil transmigration is regulated by myosin light chain kinase-mediated endothelial cell contraction and that this event depends on subendothelial cell matrix stiffness. PMID:21652678

  14. Clathrin light chains' role in selective endocytosis influences antibody isotype switching.

    PubMed

    Wu, Shuang; Majeed, Sophia R; Evans, Timothy M; Camus, Marine D; Wong, Nicole M L; Schollmeier, Yvette; Park, Minjong; Muppidi, Jagan R; Reboldi, Andrea; Parham, Peter; Cyster, Jason G; Brodsky, Frances M

    2016-08-30

    Clathrin, a cytosolic protein composed of heavy and light chain subunits, assembles into a vesicle coat, controlling receptor-mediated endocytosis. To establish clathrin light chain (CLC) function in vivo, we engineered mice lacking CLCa, the major CLC isoform in B lymphocytes, generating animals with CLC-deficient B cells. In CLCa-null mice, the germinal centers have fewer B cells, and they are enriched for IgA-producing cells. This enhanced switch to IgA production in the absence of CLCa was attributable to increased transforming growth factor β receptor 2 (TGFβR2) signaling resulting from defective endocytosis. Internalization of C-X-C chemokine receptor 4 (CXCR4), but not CXCR5, was affected in CLCa-null B cells, and CLC depletion from cell lines affected endocytosis of the δ-opioid receptor, but not the β2-adrenergic receptor, defining a role for CLCs in the uptake of a subset of signaling receptors. This instance of clathrin subunit deletion in vertebrates demonstrates that CLCs contribute to clathrin's role in vivo by influencing cargo selectivity, a function previously assigned exclusively to adaptor molecules. PMID:27540116

  15. Structural and Thermodynamic Characterization of a Cytoplasmic Dynein Light Chain-Intermediate Chain Complex

    SciTech Connect

    Williams,J.; Roulhac, P.; Roy, A.; Vallee, R.; Fitzgerald, M.; Hendrickson, W.

    2007-01-01

    Cytoplasmic dynein is a microtubule-based motor protein complex that plays important roles in a wide range of fundamental cellular processes, including vesicular transport, mitosis, and cell migration. A single major form of cytoplasmic dynein associates with membranous organelles, mitotic kinetochores, the mitotic and migratory cell cortex, centrosomes, and mRNA complexes. The ability of cytoplasmic dynein to recognize such diverse forms of cargo is thought to be associated with its several accessory subunits, which reside at the base of the molecule. The dynein light chains (LCs) LC8 and TcTex1 form a subcomplex with dynein intermediate chains, and they also interact with numerous protein and ribonucleoprotein partners. This observation has led to the hypothesis that these subunits serve to tether cargo to the dynein motor. Here, we present the structure and a thermodynamic analysis of a complex of LC8 and TcTex1 associated with their intermediate chain scaffold. The intermediate chains effectively block the major putative cargo binding sites within the light chains. These data suggest that, in the dynein complex, the LCs do not bind cargo, in apparent disagreement with a role for LCs in dynein cargo binding interactions.

  16. Cloning and characterization of a dynein light chain gene from Puccinia striiformis f. sp. tritici.

    PubMed

    Liu, Jie; Zhang, Qiong; Chang, Qing; Wang, Qiuling; Han, Lina; Liu, Jia; Li, Man; Zhuang, Hua; Kang, Zhensheng

    2014-07-01

    Stripe rust is one of the most serious wheat diseases worldwide. The fungus Puccinia striiformis f. sp. tritici (Pst), the causal agent of this disease, is an obligate biotrophic basidiomycete fungus. Numerous studies have shown that dyneins play important roles during fungal growth and propagation. However, knowledge is limited regarding the function of dyneins in Pst. In this study, we cloned the dynein light chain gene PsDLC1 from Pst and characterized its expression. The function of PsDLC1 was determined by heterologous mutant complementation. Expression of PsDLC1 in Aspergillus nidulans partially complemented the defects of the ΔnudG mutant, indicating that PsDLC1 belongs to the dynein light chain LC8 family. In addition, PsDLC1 was identified in Pst using virus-induced gene silencing (VIGS). Knockdown of PsDLC1 produces no significant effect on Pst growth and development, indicating that PsDLC1 is unnecessary for Pst infection of wheat.

  17. 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. PMID:27548633

  18. 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.

  19. [Secondary amyloidosis (AA-type) due to localized cutaneous vasculitis].

    PubMed

    Esteve, V; Ribera, L; Ponz, E; Almirall, J; López, T; Martínez Ocaña, J C; Ibeas, J; Rodríguez Jornet, A; Andreu, X; García, M

    2007-01-01

    We report a case of a 49 year old man, diagnosed soon after the outcome of casual proteinuria, of AA-type amyloidosis in relation to small and medium vessel cutaneous vasculitis without systemic involvement. This combination is a rare entity and only two cases of cutaneous hypersensibility vasculitis complicated with AA-type amyloidosis had been reported. We describe the results of the use of several immunosuppressive drugs during four years follow up with temporally total remission of the disease. PMID:18045042

  20. 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

  1. Mitral valve involvement as a predominant feature of cardiac amyloidosis

    PubMed Central

    Viswanathan, Girish; Williams, James; Slinn, Simon; Campbell, Philip

    2010-01-01

    Cardiac involvement in systemic amyloidosis carries poor prognosis with a median survival of 5 months.1 The authors report an unusual presentation of cardiac amyloidosis in the form of predominant mitral regurgitation. The patient responded very well to medical therapy with subsequent improvement of mitral valve dysfunction. The authors would like to highlight this multisystem involvement and the presence of a complex overlap of systemic features. PMID:22767536

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

    PubMed

    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

  3. ADMA Levels Correlate with Proteinuria, Secondary Amyloidosis, and Endothelial Dysfunction

    PubMed Central

    Yilmaz, Mahmut Ilker; Sonmez, Alper; Saglam, Mutlu; Qureshi, Abdul R.; Carrero, Juan Jesus; Caglar, Kayser; Eyileten, Tayfun; Cakir, Erdinc; Oguz, Yusuf; Vural, Abdulgaffar; Yenicesu, Mujdat; Lindholm, Bengt; Stenvinkel, Peter; Axelsson, Jonas

    2008-01-01

    Asymmetric dimethyl-arginine (ADMA), a residue of the proteolysis of arginine-methylated proteins, is a potent inhibitor of nitric oxide synthesis. The increased protein turnover that accompanies proteinuric secondary amyloidosis may increase circulating levels of ADMA, and this may contribute to endothelial dysfunction. We performed a cross-sectional study of 121 nondiabetic proteinuric patients with normal GFR (including 39 patients with nephrotic-range proteinuria and secondary amyloidosis) and 50 age-, sex-, and BMI–matched healthy controls. The proteinuric patients had higher levels of serum ADMA, symmetric dimethyl-arginine (SDMA), high-sensitivity C-reactive protein (hsCRP), and insulin resistance (homeostasis model assessment index) than controls. Compared with controls, brachial artery flow-mediated dilatation (FMD), serum L-Arginine, and the L-Arginine/ADMA ratio were significantly lower among proteinuric patients, suggesting greater endothelial dysfunction. When patients with secondary amyloidosis were compared with patients with glomerulonephritis who had similar levels of proteinuria, those with amyloidosis had higher ADMA and SDMA levels and lower L-Arginine/ADMA ratios and FMD measurements (P < 0.001 for all). Finally, even after adjusting for confounders, ADMA level correlated with both proteinuria and the presence of secondary amyloidosis, and was an independent predictor of FMD. We propose that ADMA synthesis may be increased in chronic kidney disease, especially in secondary amyloidosis, and this may explain part of the mechanism by which proteinuria increases cardiovascular morbidity and mortality. PMID:18199801

  4. 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.

  5. Proliferative glomerulonephritis with monoclonal immunoglobulin deposition disease: The utility of routine staining with immunoglobulin light chains

    PubMed Central

    Gowda, K. K.; Nada, R.; Ramachandran, R.; Joshi, K.; Tewari, R.; Kohli, H. S.; Jha, V.; Gupta, K. L.

    2015-01-01

    Proliferative glomerulonephritis occurring as a consequence of monoclonal glomerular deposits of IgG is uncommon. It is a form of renal involvement in monoclonal gammopathy that mimics immune complex glomerulonephritis. Here, we report the first series of proliferative glomerulonephritis with monoclonal IgG deposits (PGNMID) from the Indian subcontinent highlighting use of light chain immunofluorescence (IF) in routine renal biopsy interpretation. We retrieved 6 patients diagnosed as proliferative glomerulonephritis with monoclonal IgG deposits (PGNMID) out of 160 biopsies (3.7%) with membranoproliferative patterns over 5 1/2 years (2009–2014), one of whom had recurrence 6 months post-renal transplant. Four (67%) patients presented with rapidly progressive renal failure and two (33%) with nephrotic syndrome. None of these patients had overt multiple myeloma. The predominant histologic pattern was membranoproliferative with all the biopsies showing IgG3 Kappa deposits on IF. The deposits were primarily subendothelial on electron microscopy. PMID:26664209

  6. 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.

  7. 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

  8. 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.

  9. PKC-mediated cerebral vasoconstriction: Role of myosin light chain phosphorylation versus actin cytoskeleton reorganization.

    PubMed

    El-Yazbi, Ahmed F; Abd-Elrahman, Khaled S; Moreno-Dominguez, Alejandro

    2015-06-15

    Defective protein kinase C (PKC) signaling has been suggested to contribute to abnormal vascular contraction in disease conditions including hypertension and diabetes. Our previous work on agonist and pressure-induced cerebral vasoconstriction implicated PKC as a major contributor to force production in a myosin light chain (LC20) phosphorylation-independent manner. Here, we used phorbol dibutyrate to selectively induce a PKC-dependent constriction in rat middle cerebral arteries and delineate the relative contribution of different contractile mechanisms involved. Specifically, we employed an ultra-sensitive 3-step western blotting approach to detect changes in the content of phosphoproteins that regulate myosin light chain phosphatase (MLCP) activity, thin filament activation, and actin cytoskeleton reorganization. Data indicate that PKC activation evoked a greater constriction at a similar level of LC20 phosphorylation achieved by 5-HT. PDBu-evoked constriction persisted in the presence of Gö6976, a selective inhibitor of Ca(2+)-dependent PKC, and in the absence of extracellular Ca(2+). Biochemical evidence indicates that either + or - extracellular Ca(2+), PDBu (i) inhibits MLCP activity via the phosphorylation of myosin targeting subunit of myosin phosphatase (MYPT1) and C-kinase potentiated protein phosphatase-1 inhibitor (CPI-17), (ii) increases the phosphorylation of paxillin and heat shock protein 27 (HSP27), and reduces G-actin content, and (iii) does not change the phospho-content of the thin filament proteins, calponin and caldesmon. PDBu-induced constriction was more sensitive to disruption of actin cytoskeleton compared to inhibition of cross-bridge cycling. In conclusion, this study provided evidence for the pivotal contribution of cytoskeletal actin polymerization in force generation following PKC activation in cerebral resistance arteries. PMID:25931148

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

    PubMed

    Del Coso, Juan; 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.

  11. Loss of tubular creatinine secretion as the only sign of tubular proximal cell dysfunction in light chain proximal tubulopathy

    PubMed Central

    Stehlé, Thomas; Vignon, Marguerite; Flamant, Martin; Figueres, Marie-Lucile; Rabant, Marion; Rodenas, Anita; Noël, Laure-Hélène; Arnulf, Bertrand; Vidal-Petiot, Emmanuelle

    2016-01-01

    Abstract Light chain proximal tubulopathy (LCPT) is a rare disease, characterized by cytoplasmic inclusions of light chain (usually kappa) immunoglobulins. Clinical presentation is usually a Fanconi syndrome. The proximal tubular dysfunction can be incomplete, and exceptional cases of LCPT without any tubular dysfunction have even been described. Here, we report a case of LCPT in which the only sign of proximal tubulopathy is the absence of secretion of creatinine, as assessed by the simultaneous measurement of renal clearance of creatinine and 51CrEDTA. The loss of tubular creatinine secretion as a sign of tubular proximal cell dysfunction ought to be identified in patients with light chain proximal tubulopathy as it leads to a clinically relevant underestimation of GFR by the creatinine-derived equations. The prevalence and prognostic significance of this particular proximal tubular damage in LCPT remain to be determined. PMID:27367983

  12. Purification and characterization of a stimulator of plasmin generation from the antiangiogenic agent Neovastat: identification as immunoglobulin kappa light chain.

    PubMed

    Boivin, Dominique; Provençal, Mathieu; Gendron, Sébastien; Ratel, David; Demeule, Michel; Gingras, Denis; Béliveau, Richard

    2004-11-15

    We have recently shown that Neovastat, an antiangiogenic extract from shark cartilage, stimulates the in vitro activation of plasminogen by facilitating the tissue-type plasminogen activator (tPA)-dependent conversion of plasminogen to plasmin. In this report, we describe the purification and characterization of the stimulatory molecules. Neovastat was subjected to a three-step purification procedure including gel filtration, preparative isoelectric focusing, and preparative SDS-PAGE. Two 28-kDa proteins with pIs of approximately 4.5 and 6.5 were purified to apparent homogeneity and identified as immunoglobulin (Ig) kappa light chains by N-terminal microsequencing. Ig light chains do not directly stimulate the activity of tPA or plasmin, suggesting a mechanism of action involving an interaction with plasminogen. Kinetic analysis showed that both Ig light chains accelerate the in vitro tPA-dependent conversion of plasminogen in plasmin by increasing the affinity of tPA for plasminogen by 32- and 38-fold (Km decrease from 456 nM to 12-14 nM). Shark Ig light chains also stimulated the degradation of fibrin by the tPA/plasminogen system in an in vitro assay. A direct interaction between Ig light chains and plasminogen (KA=4.0-5.5 x 10(7) M(-1); KD=18-25 nM) and with tPA (KA=2.8 x 10(7) M(-1); KD=36 nM) was demonstrated using real time binding measured by surface plasmon resonance. Ig light chain is the first molecule associated with the antiangiogenic activity of Neovastat to be purified and identified. PMID:15488468

  13. Amyloidosis in black-footed cats (Felis nigripes).

    PubMed

    Terio, K A; O'Brien, T; Lamberski, N; Famula, T R; Munson, L

    2008-05-01

    A high prevalence of systemic amyloidosis was documented in the black-footed cat (Felis nigripes) based on a retrospective review of necropsy tissues (n = 38) submitted as part of ongoing disease surveillance. Some degree of amyloid deposition was present in 33 of 38 (87%) of the examined cats, and amyloidosis was the most common cause of death (26/38, 68%). Amyloid deposition was most severe in the renal medullary interstitium (30/33, 91%) and glomeruli (21/33, 63%). Other common sites included the splenic follicular germinal centers (26/31, 84%), gastric lamina propria (9/23, 39%), and intestinal lamina propria (3/23, 13%). Amyloid in all sites stained with Congo red, and in 13 of 15 (87%) cats, deposits had strong immunoreactivity for canine AA protein by immunohistochemistry. There was no association with concurrent chronic inflammatory conditions (P = .51), suggesting that amyloidosis was not secondary to inflammation. Adrenal cortical hyperplasia, a morphologic indicator of stress that can predispose to amyloid deposition, was similarly not associated (P = .09) with amyloidosis. However, adrenals were not available from the majority of cats without amyloidosis; therefore, further analysis of this risk factor is warranted. Heritability estimation suggested that amyloidosis might be familial in this species. Additionally, tissues from a single free-ranging black-footed cat had small amounts of amyloid deposition, suggesting that there could be a predilection for amyloidosis in this species. Research to identify the protein sequence of serum amyloid A (SAA) in the black-footed cat is needed to further investigate the possibility of an amyloidogenic SAA in this species. PMID:18487501

  14. Amyloidosis in black-footed cats (Felis nigripes).

    PubMed

    Terio, K A; O'Brien, T; Lamberski, N; Famula, T R; Munson, L

    2008-05-01

    A high prevalence of systemic amyloidosis was documented in the black-footed cat (Felis nigripes) based on a retrospective review of necropsy tissues (n = 38) submitted as part of ongoing disease surveillance. Some degree of amyloid deposition was present in 33 of 38 (87%) of the examined cats, and amyloidosis was the most common cause of death (26/38, 68%). Amyloid deposition was most severe in the renal medullary interstitium (30/33, 91%) and glomeruli (21/33, 63%). Other common sites included the splenic follicular germinal centers (26/31, 84%), gastric lamina propria (9/23, 39%), and intestinal lamina propria (3/23, 13%). Amyloid in all sites stained with Congo red, and in 13 of 15 (87%) cats, deposits had strong immunoreactivity for canine AA protein by immunohistochemistry. There was no association with concurrent chronic inflammatory conditions (P = .51), suggesting that amyloidosis was not secondary to inflammation. Adrenal cortical hyperplasia, a morphologic indicator of stress that can predispose to amyloid deposition, was similarly not associated (P = .09) with amyloidosis. However, adrenals were not available from the majority of cats without amyloidosis; therefore, further analysis of this risk factor is warranted. Heritability estimation suggested that amyloidosis might be familial in this species. Additionally, tissues from a single free-ranging black-footed cat had small amounts of amyloid deposition, suggesting that there could be a predilection for amyloidosis in this species. Research to identify the protein sequence of serum amyloid A (SAA) in the black-footed cat is needed to further investigate the possibility of an amyloidogenic SAA in this species.

  15. 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

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

    PubMed

    Del Coso, Juan; 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

  17. Light chain editing in kappa-deficient animals: a potential mechanism of B cell tolerance

    PubMed Central

    1994-01-01

    The genetic organization of the kappa and lambda light chain loci permits multiple, successive rearrangement attempts at each allele. Multiple rearrangements allow autoreactive B cells to escape clonal deletion by editing their surface receptors. Editing may also facilitate efficient B cell production by salvaging cells with nonproductive light chain (L chain) rearrangements. To study receptor editing of kappa L chains, we have characterized B cells from mice hemizygous for the targeted inactivation of kappa (JCkD/wt) which have an anti-DNA heavy chain transgene, 3H9. Hybridomas from JCkD/wt mice exhibited an increased frequency of rearrangements to downstream Jk segments (such as Jk5) compared with most surveys from normal mice, consistent with receptor editing by sequential kappa locus rearrangements in JCkD/wt. We observed an even higher frequency of rearrangements to Jk5 in 3H9 JCkD/wt animals compared with nontransgenic JCkD/wt, consistent with editing of autoreactive kappa in 3H9 JCkD/wt. We also recovered a large number of 3H9 JCkD/wt lines with Vk12/13-Jk5 rearrangements and could demonstrate by PCR and Southern analysis that up to three quarters of these lines underwent multiple kappa rearrangements. To investigate editing at the lambda locus, we used homozygous kappa-deficient animals (JCkD/JCkD and 3H9 JCkD/JCkD). The frequencies of V lambda 1 and V lambda 2 rearrangements among splenic hybridomas in 3H9 JCkD/JCkD were reduced by 75% whereas V lambda X was increased 5-10-fold, compared with nontransgenic JCkD/JCkD animals. This indicates that V lambda 1 and V lambda 2 are negatively regulated in 3H9 JCkD/JCkD, consistent with earlier studies that showed that the 3H9 heavy chain, in combination with lambda 1 binds DNA. As successive lambda rearrangements to V lambda X do not inactivate V lambda 1, the consequence of lambda editing in 3H9 JCkD/JCkD would be failed allelic exclusion at lambda. However, analysis of 18 3H9 JCkD/JCkD hybridomas with V lambda 1

  18. 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

  19. 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

  20. Survey on spontaneous systemic amyloidosis in aging mice.

    PubMed

    Majeed, S K

    1993-02-01

    The incidence of systemic amyloidosis in CD-1 mice (Charles River, caesarian derived) obtained from long term studies over more than a 15-year period is reported. The survey included samples of visceral organs, peripheral and central nervous tissues, bone and bone marrow. The total incidence in all mice of this survey did not show any clear evidence of a difference between males and females. Amyloidosis deposits were mainly seen in the stomach (glandular), heart, small intestines, kidney, liver, spleen, thyroid, parathyroid, adrenals, salivary glands and ovaries, but not in the brain, spinal cord, bone or bone marrow. The survey showed that amyloidosis in CD-1 mice was spontaneous, systemic and it is age-related. Amyloid deposition was extracellular and it stained positively with Congo Red and also stained positively with Oil Red O and Alcian blue. In general, amyloidosis in CD-1 mice, was higher in comparison with B6C3F (cross between C57BL/C6 NCRLB and C3H/HEN NCRLB, (bred by Charles River), CFLP strain (hysterectomy derived strain of Swiss origin) and MAGF: TIF (SPF). This survey also showed that spontaneous systemic amyloidosis in CD-1 mice, was one of the major factors contributory to death in aging CD-1 mice. PMID:8457241

  1. Evolving landscape in the management of transthyretin amyloidosis.

    PubMed

    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-TTR(Rx), 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. Monitoring serum free light chains in patients with multiple myeloma who achieved negative immunofixation after allogeneic stem cell transplantation.

    PubMed

    Mösbauer, Ulrike; Ayuk, Francis; Schieder, Heike; Lioznov, Michael; Zander, Axel R; Kröger, Nicolaus

    2007-02-01

    Monitoring of serum free immunoglobulin light chains (FLC) in 26 myeloma patients who achieved immunofixation negativity after allografting showed a decrease of FLC at a median of 128 days before immunofixation negativity. In patients who subsequently relapsed, a 25% increase of FLC was observed at a median of 98 days before immunofixation positivity.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27069035

  4. Effects of myosin light chain phosphorylation on length-dependent myosin kinetics in skinned rat myocardium.

    PubMed

    Pulcastro, Hannah C; Awinda, Peter O; Breithaupt, Jason J; Tanner, Bertrand C W

    2016-07-01

    Myosin force production is Ca(2+)-regulated by thin-filament proteins and sarcomere length, which together determine the number of cross-bridge interactions throughout a heartbeat. Ventricular myosin regulatory light chain-2 (RLC) binds to the neck of myosin and modulates contraction via its phosphorylation state. Previous studies reported regional variations in RLC phosphorylation across the left ventricle wall, suggesting that RLC phosphorylation could alter myosin behavior throughout the heart. We found that RLC phosphorylation varied across the left ventricle wall and that RLC phosphorylation was greater in the right vs. left ventricle. We also assessed functional consequences of RLC phosphorylation on Ca(2+)-regulated contractility as sarcomere length varied in skinned rat papillary muscle strips. Increases in RLC phosphorylation and sarcomere length both led to increased Ca(2+)-sensitivity of the force-pCa relationship, and both slowed cross-bridge detachment rate. RLC-phosphorylation slowed cross-bridge rates of MgADP release (∼30%) and MgATP binding (∼50%) at 1.9 μm sarcomere length, whereas RLC phosphorylation only slowed cross-bridge MgATP binding rate (∼55%) at 2.2 μm sarcomere length. These findings suggest that RLC phosphorylation influences cross-bridge kinetics differently as sarcomere length varies and support the idea that RLC phosphorylation could vary throughout the heart to meet different contractile demands between the left and right ventricles. PMID:26763941

  5. Sequence analysis of the myosin regulatory light chain gene of the vestimentiferan Riftia pachyptila.

    PubMed

    Ravaux, J; Hassanin, A; Deutsch, J; Gaill, F; Markmann-Mulisch, U

    2001-01-24

    We have isolated and characterized a cDNA (DNA complementary to RNA) clone (Rf69) from the vestimentiferan Riftia pachyptila. The cDNA insert consists of 1169 base pairs. The aminoacid sequence deduced from the longest reading frame is 193 residues in length, and clearly characterized it as a myosin regulatory light chain (RLC). The RLC primary structure is described in relation to its function in muscle contraction. The comparison with other RLCs suggested that Riftia myosin is probably regulated through its RLC either by phosphorylation like the vertebrate smooth muscle myosins, and/or by Ca2+-binding like the mollusk myosins. Riftia RLC possesses a N-terminal extension lacking in all other species besides the earthworm Lumbricus terrestris. Aminoacid sequence comparisons with a number of RLCs from vertebrates and invertebrates revealed a relatively high identity score (64%) between Riftia RLC and the homologous gene from Lumbricus. The relationships between the members of the myosin RLCs were examined by two phylogenetic methods, i.e. distance matrix and maximum parsimony. The resulting trees depict the grouping of the RLCs according to their role in myosin activity regulation. In all trees, Riftia RLC groups with RLCs that depend on Ca2+-binding for myosin activity regulation. PMID:11223252

  6. Kinesin's light chains inhibit the head- and microtubule-binding activity of its tail.

    PubMed

    Wong, Yao Liang; Rice, Sarah E

    2010-06-29

    Kinesin-1 is a microtubule-based motor comprising two heavy chains (KHCs) and two light chains (KLCs). Motor activity is precisely regulated to avoid futile ATP consumption and to ensure proper intracellular localization of kinesin-1 and its cargoes. The KHC tail inhibits ATPase activity by interacting with the enzymatic KHC heads, and the tail also binds microtubules. Here, we present a role for the KLCs in regulating both the head- and microtubule-binding activities of the kinesin-1 tail. We show that KLCs reduce the affinity of the head-tail interaction over tenfold and concomitantly repress the tail's regulatory activity. We also show that KLCs inhibit tail-microtubule binding by a separate mechanism. Inhibition of head-tail binding requires steric and electrostatic factors. Inhibition of tail-microtubule binding is largely electrostatic, pH dependent, and mediated partly by a highly negatively charged linker region between the KHC-interacting and cargo-binding domains of the KLCs. Our data support a model wherein KLCs promote activation of kinesin-1 for cargo transport by simultaneously suppressing tail-head and tail-microtubule interactions. KLC-mediated inhibition of tail-microtubule binding may also influence diffusional movement of kinesin-1 on microtubules, and kinesin-1's role in microtubule transport/sliding. PMID:20547877

  7. 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

  8. Functions of Myosin Light Chain-2 (MYL2) In Cardiac Muscle and Disease

    PubMed Central

    Sheikh, Farah; Lyon, Robert C.; Chen, Ju

    2015-01-01

    Myosin light chain-2 (MYL2, also called MLC-2) is an ∼19 kDa sarcomeric protein that belongs to the EF-hand calcium binding protein superfamily and exists as three major isoforms encoded by three distinct genes in mammalian striated muscle. Each of the three different MLC-2 genes (MLC-2f; fast twitch skeletal isoform, MLC-2v; cardiac ventricular and slow twitch skeletal isoform, MLC-2a; cardiac atrial isoform) has a distinct developmental expression pattern in mammals. Genetic loss-of-function studies in mice demonstrated an essential role for cardiac isoforms of MLC-2, MLC-2v and MLC-2a, in cardiac contractile function during early embryogenesis. In the adult heart, MLC-2v function is regulated by phosphorylation, which displays a specific expression pattern (high in epicardium and low in endocardium) across the heart. These data along with new data from computational models, genetic mouse models, and human studies have revealed a direct role for MLC-2v phosphorylation in cross-bridge cycling kinetics, calcium-dependent cardiac muscle contraction, cardiac torsion, cardiac function and various cardiac diseases. This review focuses on the regulatory functions of MLC-2 in the embryonic and adult heart, with an emphasis on phosphorylation-driven actions of MLC-2v in adult cardiac muscle, which provide new insights into mechanisms regulating myosin cycling kinetics and human cardiac diseases. PMID:26074085

  9. Regulatory and structural motifs of chicken gizzard myosin light chain kinase.

    PubMed Central

    Olson, N J; Pearson, R B; Needleman, D S; Hurwitz, M Y; Kemp, B E; Means, A R

    1990-01-01

    The amino acid sequence for chicken smooth muscle myosin light chain kinase (smMLCK) was deduced from a full-length cDNA. This has allowed definition of both the complete sequence of the inactive 64-kDa proteolytic fragment, which contains the pseudosubstrate autoregulatory sequence, and of the active 61-kDa Ca2+/calmodulin-independent fragment, which lacks the autoregulatory domain. Comparison of the two sequences shows that the autoregulatory domain extends from Asn-780 to Arg-808. The peptide Leu-774 to Ser-787 does not inhibit smMLCK, whereas peptides of similar or shorter length from the pseudosubstrate region (Ser-787 to Val-807) are potent inhibitors. These data define the autoregulatory region as being contained within and probably identical to the pseudosubstrate domain. The catalytic and regulatory regions are flanked by several copies of 100-amino acid segments containing one of two consensus motifs. These motifs are absent from mammalian skeletal muscle MLCK or from Dictyostelium discoideum MLCK but are present in the Caenorhabditis elegans unc-22 gene product and the titin molecule of skeletal muscle myofibrils. These results indicate that the amino acid sequence of smMLCK encodes multiple functional motifs in addition to the catalytic domain. PMID:2315320

  10. Cloning of immunoglobulin kappa light chain genes from mouse liver and myeloma MOPC 173.

    PubMed

    Steinmetz, M; Zachau, H G; Mach, B

    1979-07-25

    The organization of the kappa chain constant region gene was compared in DNA from an immunoglobulin-producing mouse myeloma (MOPC 173) and from liver. In situ hybridization using the Southern blotting technique revealed constant region gene-containing EcoRI-DNA fragments of 14 and 20 kb in the myeloma tissue whereas one EcoRI-DNA fragment with a length of 15 kb was found in liver DNA. After enrichment by RPC-5 chromatography and preparative electrophoresis the 14 kb fragment from MOPC 173 DNA and the 15 kb fragment from liver DNA were cloned in the bacteriophage lambda vector Charon 4A using in vitro packaging. Extensive characterization of the two fragments by restriction endonuclease mapping, in situ hybridization, and electron microscopy (R-loop and heteroduplex) showed that both fragments contain the constant region but no MOPC 173 variable region gene. Both fragments are homologous over a length of 12.5 kb including the constant region but differ from one another starting about 2.7 kb from the 5' end of the constant region gene. This indicates that the 14 kb EcoRI-DNA fragment from the myeloma tissue clearly resulted from somatic DNA rearrangement although it does not seem to carry the MOPC 173 variable region gene. These observations suggest that somatic DNA rearrangement of immunoglobulin light chain genes can involve both homologous chromosomes.Images

  11. Prognostic value of serum heavy/light chain ratios in patients with POEMS syndrome.

    PubMed

    Wang, Chen; Su, Wei; Cai, Qian-Qian; Cai, Hao; Ji, Wei; Di, Qian; Duan, Ming-Hui; Cao, Xin-Xin; Zhou, Dao-Bin; Li, Jian

    2016-07-01

    POEMS syndrome is a rare plasma cell dyscrasia. Serum concentrations of the monoclonal protein in this disorder are typically low, and inapplicable to monitor disease activity in most cases, resulting in limited practical and prognostic values. Novel immunoassays measuring isotype-specific heavy/light chain (HLC) pairs showed its utility in disease monitoring and outcome prediction in several plasma cell dyscrasias. We report results of HLC measurements in 90 patients with POEMS syndrome. Sixty-six patients (73%; 95% confidence interval, 63-82%) had an abnormal HLC ratio at baseline. It could stratify the risk of disease relapse and was strongly associated with worse progression-free survival in a multivariate analysis (P = 0.021; hazard ratio [HR] 6.89, 95% CI 1.34-35.43). After therapy, HLC ratios improved, with 43 patients (48%) remaining abnormal. The post-therapeutic HLC ratio, if abnormal, also remained as an independent prognostic factor associated with worse progression-free survival (P = 0.019; HR 4.30, 95% CI 1.27-14.56). These results suggest the prognostic utility of HLC ratios in clinical management of POEMS patients.

  12. Mn2+ activates skinned smooth muscle cells in the absence of myosin light chain phosphorylation.

    PubMed

    Hoar, P E; Kerrick, W G

    1988-08-01

    Two effects of Mn2+ on skinned fibers from chicken gizzard smooth muscle were observed, dependent on the presence or absence of dithiothreitol (DTT) reducing agent. One involves protein oxidation (in the absence of DTT) with production of a "latch"-like state, and the other involves direct Mn2+ activation of contractile proteins. Cells activated by Mn2+ in the presence of ATP and the absence of Ca2+, Mg2+ and DTT did not relax when transferred to normal relaxing solutions. In contrast, when 5 mM DTT was included in the Mn2+ contracting solution to prevent protein oxidation by Mn2+, the cells still contracted when exposed to Mn2+, but relaxed rapidly when the Mn2+ was removed. In the presence of DTT both the Mn2+ activation and the relaxation following removal of Mn2+ were more rapid than normal Ca2+-activated contractions and relaxations. The skinned fibers activated by Mn2+ in the absence of DTT showed little active shortening unless DTT was added. This rigor-like state is probably due to oxidation of contractile proteins since the cells relaxed when exposed to a relaxing solution containing DTT (50 mM) and then contracted again in response to Ca2+ and relaxed normally. The Mn2+ activation was not associated with myosin light chain phosphorylation, in contrast to Ca2+-activated contractions. PMID:3186428

  13. Urine immunofixation electrophoresis remains important and is complementary to serum free light chain.

    PubMed

    Levinson, Stanley S

    2011-11-01

    Articles have debated whether or not urine analysis remains valuable for identifying monoclonal gammopathies. A general impression is that the newer serum free light chain (FLC) assay is more analytically sensitive, more quantitative and simpler to perform. Many laboratory directors may have seized on the idea of eliminating urine analysis because it is a tedious procedure and requires expert interpretation while most laboratories can perform automated serum FLC assay. Others have concluded that urine immunofixation electrophoresis (IFE) optimizes the diagnostic sensitivity and should be included when there is a clinical indication. Here, I show that papers faulting urine analysis often used inappropriate urine methodology and this helps explain why there was misinterpretation. Moreover, the literature, shows urine IFE is often more sensitive for identifying low-level monoclonal FLC than the serum assay because urine IFE is as sensitive when performed appropriately and generally more specific. Besides, the reference range for serum FLC assay is unclear which is a great problem in assessing response to treatment and in identifying diseases when there is low concentration monoclonal FLC. I conclude that urine IFE remains important and is complementary to serum FLC assay, although the best algorithms for use remains to be elucidated.

  14. AMPK regulates mitotic spindle orientation through phosphorylation of myosin regulatory light chain.

    PubMed

    Thaiparambil, Jose T; Eggers, Carrie M; Marcus, Adam I

    2012-08-01

    The proper orientation of the mitotic spindle is essential for mitosis; however, how these events unfold at the molecular level is not well understood. AMP-activated protein kinase (AMPK) regulates energy homeostasis in eukaryotes, and AMPK-null Drosophila mutants have spindle defects. We show that threonine(172) phosphorylated AMPK localizes to the mitotic spindle poles and increases when cells enter mitosis. AMPK depletion causes a mitotic delay with misoriented spindles relative to the normal division plane and a reduced number and length of astral microtubules. AMPK-depleted cells contain mitotic actin bundles, which prevent astral microtubule-actin cortex attachments. Since myosin regulatory light chain (MRLC) is an AMPK downstream target and mediates actin function, we investigated whether AMPK signals through MRLC to control spindle orientation. Mitotic levels of serine(19) phosphorylated MRLC (pMRLC(ser19)) and spindle pole-associated pMRLC(ser19) are abolished when AMPK function is compromised, indicating that AMPK is essential for pMRLC(ser19) spindle pole activity. Phosphorylation of AMPK and MRLC in the mitotic spindle is dependent upon calcium/calmodulin-dependent protein kinase kinase (CamKK) activity in LKB1-deficient cells, suggesting that CamKK regulates this pathway when LKB1 function is compromised. Taken together, these data indicate that AMPK mediates spindle pole-associated pMRLC(ser19) to control spindle orientation via regulation of actin cortex-astral microtubule attachments.

  15. Conventional Kinesin Holoenzymes Are Composed of Heavy and Light Chain Homodimers†

    PubMed Central

    DeBoer, Scott R.; You, YiMei; Szodorai, Anita; Kaminska, Agnieszka; Pigino, Gustavo; Nwabuisi, Evelyn; Wang, Bin; Estrada-Hernandez, Tatiana; Kins, Stefan; Brady, Scott T.; Morfini, Gerardo

    2009-01-01

    Conventional kinesin is a major microtubule-based motor protein responsible for anterograde transport of various membrane-bounded organelles (MBO) along axons. Structurally, this molecular motor protein is a tetrameric complex composed of two heavy (kinesin-1) chains and two light chain (KLC) subunits. The products of three kinesin-1 (kinesin-1A, -1B, and -1C, formerly KIF5A, -B, and -C) and two KLC (KLC1, KLC2) genes are expressed in mammalian nervous tissue, but the functional significance of this subunit heterogeneity remains unknown. In this work, we examine all possible combinations among conventional kinesin subunits in brain tissue. In sharp contrast with previous reports, immunoprecipitation experiments here demonstrate that conventional kinesin holoenzymes are formed of kinesin-1 homodimers. Similar experiments confirmed previous findings of KLC homodimerization. Additionally, no specificity was found in the interaction between kinesin-1s and KLCs, suggesting the existence of six variant forms of conventional kinesin, as defined by their gene product composition. Subcellular fractionation studies indicate that such variants associate with biochemically different MBOs and further suggest a role of kinesin-1s in the targeting of conventional kinesin holoenzymes to specific MBO cargoes. Taken together, our data address the combination of subunits that characterize endogenous conventional kinesin. Findings on the composition and subunit organization of conventional kinesin as described here provide a molecular basis for the regulation of axonal transport and delivery of selected MBOs to discrete subcellular locations. PMID:18361505

  16. 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.

  17. Developmental Expression of IL-2-Receptor Light Chain (CD25) in the Chicken Embryo

    PubMed Central

    Fedecka-Bruner, Barbara; Penninger, Josef; Vaigot, Pierre; Lehmann, Anne; Martínez-A., Carlos

    1991-01-01

    Thymocyte differentiation obeys the same fundamental principles in mammals as in avian species. This parallelism does not only affect the developmentally controlled acquisition of CD3, 4, 8, and TcR isotype expression, but also concerns CD25, the light chain of the interleukin-2 receptor (IL-2R). On chicken thymocytes, surface CD25, which is recognized by the monoclonal antibody INN Ch16, is first observed during day 11 of embryonic life, and peaks at day 14, when it is expressed by about one-third of all lymphoid cells. CD25 is found on subsets of all ,thymocyte populations as defined by TcRαβ, TcRγδ, 2, CD4, and CD8 expression, cortical or medullary localization, and is also present on a subset of intrathymic nurse-cell lymphocytes. These findings suggest phylogenetic conservation of the IL-2/IL-2R-triggered differentiation pathway previously described for mammalian species, thus under-lining its probable functional importance. PMID:1840381

  18. 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

  19. Dynein light chain regulates axonal trafficking and synaptic levels of Bassoon.

    PubMed

    Fejtova, Anna; Davydova, Daria; Bischof, Ferdinand; Lazarevic, Vesna; Altrock, Wilko D; Romorini, Stefano; Schöne, Cornelia; Zuschratter, Werner; Kreutz, Michael R; Garner, Craig C; Ziv, Noam E; Gundelfinger, Eckart D

    2009-04-20

    Bassoon and the related protein Piccolo are core components of the presynaptic cytomatrix at the active zone of neurotransmitter release. They are transported on Golgi-derived membranous organelles, called Piccolo-Bassoon transport vesicles (PTVs), from the neuronal soma to distal axonal locations, where they participate in assembling new synapses. Despite their net anterograde transport, PTVs move in both directions within the axon. How PTVs are linked to retrograde motors and the functional significance of their bidirectional transport are unclear. In this study, we report the direct interaction of Bassoon with dynein light chains (DLCs) DLC1 and DLC2, which potentially link PTVs to dynein and myosin V motor complexes. We demonstrate that Bassoon functions as a cargo adapter for retrograde transport and that disruption of the Bassoon-DLC interactions leads to impaired trafficking of Bassoon in neurons and affects the distribution of Bassoon and Piccolo among synapses. These findings reveal a novel function for Bassoon in trafficking and synaptic delivery of active zone material.

  20. Dynein light chain interaction with the peroxisomal import docking complex modulates peroxisome biogenesis in yeast.

    PubMed

    Chang, Jinlan; Tower, Robert J; Lancaster, David L; Rachubinski, Richard A

    2013-10-15

    Dynein is a large macromolecular motor complex that moves cargo along microtubules. A motor-independent role for the light chain of dynein, Dyn2p, in peroxisome biology in Saccharomyces cerevisiae was suggested from its interaction with Pex14p, a component of the peroxisomal matrix protein import docking complex. Here we show that cells of the yeast Yarrowia lipolytica deleted for the gene encoding the homologue of Dyn2p are impaired in peroxisome function and biogenesis. These cells exhibit compromised growth on medium containing oleic acid as the carbon source, the metabolism of which requires functional peroxisomes. Their peroxisomes have abnormal morphology, atypical matrix protein localization, and an absence of proteolytic processing of the matrix enzyme thiolase, which normally occurs upon its import into the peroxisome. We also show physical and genetic interactions between Dyn2p and members of the docking complex, particularly Pex17p. Together, our results demonstrate a role for Dyn2p in the assembly of functional peroxisomes and provide evidence that Dyn2p acts in cooperation with the peroxisomal matrix protein import docking complex to effect optimal matrix protein import.

  1. The regulation of RhoGEF Lfc by dynein light chain Tctex-1

    NASA Astrophysics Data System (ADS)

    Balan, Marc

    Lfc is a guanine nucleotide exchange factor (GEF) that activates the small GTPase RhoA, and its GEF activity is tightly regulated through protein-protein interactions, phosphorylation, and cellular localization. Lfc is anchored to microtubules through its interaction with the dynein light chain Tctex-1, which results in inhibition of Lfc's GEF activity. Here we present a crystallographic structure of Tctex-1 in complex with Lfc with residues 143-155 of Lfc bound at the Tctex-1 dimer interface. Structural alignment of our structure with Tctex-1 in complex with the dynein intermediate chain (DIC) shows the binding site of the DIC peptide and Lfc substantially overlap. Biochemical evidence, NMR perturbations assays and intrinsic fluorescence provide structural validation and support an extension of the Lfc binding site to the andalpha;-helices that may accommodate additional contact points with Tctex-1. We postulate a potential mechanism for Lfcandrsquo;s recruitment to the microtubules through a tripartite complex with Tctex-1 and DIC.

  2. 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

  3. Dictyostelium discoideum myosin: Isolation and characterization of cDNAs encoding the essential light chain

    SciTech Connect

    Chisholm, R.L.; Rushforth, A.M.; Pollenz, R.S.; Kuczmarski, E.R.; Tafuri, S.R.

    1988-02-01

    The authors used an antibody specific for Dictyostelium discoideum myosin to screen a lambdagt11 cDNA expression library to obtain cDNA clones which encode the Dictyostelium essential myosin light chain (EMLC). The amino acid sequence predicted from the sequence of the cDNA clone showed 31.5% identity with the amino acid sequence of the chicken EMLC. Comparisons of the Dictyostelium EMLC, a nonmuscle cell type, with EMLC sequences from similar MLCs of skeletal- and smooth-muscle origin, showed distinct regions of homology. Much of the observed homology was localized to regions corresponding to consensus Ca/sup 2 +/-binding of E-F hand domains. Southern blot analysis suggested that the Dictyostelium genome contains a single gene encoding the EMLC. Examination of the pattern of EMLC mRNA expression showed that a significant increase in EMLC message levels occurred during the first few hours of development, coinciding with increased actin expression and immediately preceding the period of maximal chemotactic activity.

  4. Regulatory Light Chain Mutations Associated with Cardiomyopathy Affect Myosin Mechanics and Kinetics

    PubMed Central

    Greenberg, Michael J.; Watt, James D.; Jones, Michelle; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R.

    2009-01-01

    The myosin regulatory light chain (RLC) wraps around the alpha helical neck region of myosin. This neck region has been proposed to act as a lever arm, amplifying small conformational changes in the myosin head to generate motion. The RLC serves an important structural role, supporting the myosin neck region and a modulatory role, tuning the kinetics of the actin myosin interaction. Given the importance of the RLC, it is not surprising that mutations of the RLC can lead to familial hypertrophic cardiomyopathy (FHC), the leading cause of sudden cardiac death in people under 30. Population studies identified two FHC mutations located near the cationic binding site of the RLC, R58Q and N47K. Although these mutations are close in sequence, they differ in clinical presentation and prognosis with R58Q showing a more severe phenotype. We examined the molecular based changes in myosin that are responsible for the disease phenotype by purifying myosin from transgenic mouse hearts expressing mutant myosins and examining actin filament sliding using the in vitro motility assay. We found that both R58Q and N47K showed reductions in force compared to the wild type that could result in compensatory hypertrophy. Furthermore, we observed a higher ATPase rate and an increased activation at submaximal calcium levels for the R58Q myosin that could lead to decreased efficiency and incomplete cardiac relaxation, potentially explaining the more severe phenotype for the R58Q mutation. PMID:18929571

  5. Interhead fluorescence energy transfer between probes attached to translationally equivalent sites on the regulatory light chains of scallop myosin.

    PubMed

    Chantler, P D; Tao, T

    1986-11-01

    Interhead fluorescence energy transfer studies between probes located at translationally equivalent sites on the two heads of scallop myosin indicates that the distance between such sites is no less than 50 A. Regulatory light chains, possessing either one (Mercenaria, chicken gizzard) or two (Loligo, rabbit skeletal) sulfhydryl groups, were modified either with 1,5-IAEDANS (N'-iodoacetyl-N'-(1-sulfo-5-n-naphthyl)ethylenediamine), as energy transfer donor, or with IAF (5-(iodoacetamido)fluorescein) or DABMI (4-dimethylaminophenylazophenyl-4'-maleimide), as energy transfer acceptor. The sulfhydryl groups on these light chains are located at different positions within the regulatory light-chain primary sequence; this enables one to probe a variety of locations, with respect to regulatory light-chain topology, on each myosin head. These independently modified regulatory light chains were added back to desensitized scallop myosin under a variety of conditions, including biphasic re-addition, the aim being to maximize the number of interhead energy transfer couples present. The efficiency of energy transfer was determined on the same samples by both steady-state and time-decay techniques. Results obtained by these two techniques were in good agreement with each other and indicated that the efficiency of energy transfer did not exceed 20% in any of the hybrids studied. Transfer efficiencies were invariant, irrespective of the presence or absence of MgATP, calcium or actin, either separately or in combination. Results using heavy meromyosin at low ionic strength were identical. It is shown that these results, in conjunction with the results of recent crosslinking studies performed on comparable myosin hybrids, may place certain restrictions on the configurations of the two heads of myosin.

  6. [Laryngeal amyloidosis: a clinical case and review of literature].

    PubMed

    Fries, S; Pasche, P; Brunel, C; Schweizer, V

    2015-09-30

    Amyloidosis consists of different forms of systemic or isolated organ lesions characterised by fibrillary protein deposits in extra-cellular tissue. The isolated involvement of the larynx is the most frequent form in the ENT sphere. We present a clinical case of a 67 year-old woman addressed for a sub-acute laryngitis resistant to conservative treatment, and finally diagnosed with laryngeal amyloidosis. We reviewed its physiopathology, the scientific literature as well as the different possibilities of management. Laryngeal amyloidosis is rare. A thorough additional work-up for the research of multifocal or systemic forms is highly recommended. The treatment aims at a minimal invasive endoscopic surgery with functional organ preservation.

  7. 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.

  8. Alopecia in Systemic Amyloidosis: Trichoscopic-Pathologic Correlation

    PubMed Central

    Miteva, Mariya; Wei, Erin; Milikowski, Clara; Tosti, Antonella

    2015-01-01

    Alopecia in systemic amyloidosis is very rare and has been described as individual cases of diffuse nonscarring alopecia and a case of alopecia universalis. We report the trichoscopic findings in alopecia associated with systemic amyloidosis. The most prominent feature was a salmon colored halo (0.3-1 mm in diameter) surrounding the follicular ostia. Other features included broken hairs and black dots. The salmon colored halo correlated on pathology with the perifollicular deposition of amyloid. The horizontal sections showed that the sebaceous glands were preserved which supports the nonscarring pattern of the alopecia. PMID:26903748

  9. UNUSUAL PRESENTATION OF GENERALIZED MACULAR AMYLOIDOSIS IN A YOUNG ADULT

    PubMed Central

    Kudur, Mohan H; B, Sathish Pai; H, Sripathi; Prabhu, Smitha

    2008-01-01

    Macular amyloidosis is a common problem seen dermatology out-patient department. Generalized macular amyloidosis presenting with a poikilodermatous appearance is rare. In our case, an 18-year-old male presented with generalized hypopigmented macules with a poikilodermatous appearance of 10-year duration. His developmental milestones were normal with negative family history of similar complaints. Histopathology of hyperpigmented lesions revealed hyperkeratosis and acanthosis of epidermis and hypopigmented lesion showing only hyperkeratosis. Both lesions were showing the deposition of amorphous, hazy material in the tips of papillary dermis with perivascular inflammatory infiltrate. Congo red staining of the amorphous material was positive for amyloid. PMID:19882037

  10. [Hepatic amyloidosis as cause of severe intrahepatic cholestasis].

    PubMed

    Gavilán, J C; Bermúdez, F J; Márquez, A; Sánchez-Carrillo, J J; González-Santos, P

    2003-01-01

    The liver is frequently involved by amyloidosis, but hyperbilirubinemia and liver failure are uncommon features. A mild elevation of the serum alkaline phosphatase value and, less frequently, hepatomegaly are the most common findings. Usually the patients have no symptoms related with the liver involvement; the clinical manifestation and the long term prognosis depends on the renal and cardiac disease. We report an unusual clinical presentation of primary amyloidosis in a previously asymptomatic 65 years old woman who was admitted to the hospital because of ictericia and ascitis mimicking a drug induced acute hepatic failure.

  11. A Heterologous Reporter Defines the Role of the Tetanus Toxin Interchain Disulfide in Light-Chain Translocation

    PubMed Central

    Zuverink, Madison; Chen, Chen; Przedpelski, Amanda; Blum, Faith C.

    2015-01-01

    Botulinum neurotoxins (BoNTs) and tetanus toxin (TeNT) are the most potent toxins for humans and elicit unique pathologies due to their ability to traffic within motor neurons. BoNTs act locally within motor neurons to elicit flaccid paralysis, while retrograde TeNT traffics to inhibitory neurons within the central nervous system (CNS) to elicit spastic paralysis. BoNT and TeNT are dichain proteins linked by an interchain disulfide bond comprised of an N-terminal catalytic light chain (LC) and a C-terminal heavy chain (HC) that encodes an LC translocation domain (HCT) and a receptor-binding domain (HCR). LC translocation is the least understood property of toxin action, but it involves low pH, proteolysis, and an intact interchain disulfide bridge. Recently, Pirazzini et al. (FEBS Lett 587:150–155, 2013, http://dx.doi.org/10.1016/j.febslet.2012.11.007) observed that inhibitors of thioredoxin reductase (TrxR) blocked TeNT and BoNT action in cerebellar granular neurons. In the current study, an atoxic TeNT LC translocation reporter was engineered by fusing β-lactamase to the N terminus of TeNT [βlac-TeNT(RY)] to investigate LC translocation in primary cortical neurons and Neuro-2a cells. βlac-TeNT(RY) retained the interchain disulfide bond, showed ganglioside-dependent binding to neurons, required acidification to promote βlac translocation, and was sensitive to auranofin, an inhibitor of thioredoxin reductase. Mutation of βlac-TeNT(RY) at C439S and C467S eliminated the interchain disulfide bond and inhibited βlac translocation. These data support the requirement of an intact interchain disulfide for LC translocation and imply that disulfide reduction is a prerequisite for LC delivery into the host cytosol. The data also support a model that LC translocation proceeds from the C to the N terminus. βlac-TeNT(RY) is the first reporter system to measure translocation by an AB single-chain toxin in intact cells. PMID:25895970

  12. Microinjection of antibodies to the calpactin I light chain in MDBK cells causes precipition of the cytoskeletal calpactin I complex without affecting the distribution of related proteins.

    PubMed

    Glenney, J R

    1990-01-01

    The calpactin I complex is composed of two heavy chain (39K) and two light chain (11K) subunits. The heavy chain is a member of a protein family that includes lipocortins, endonexin and chromobindins while the light chain is a member of the S100 family (7 distinct members are known). We have found that the kidney epithelial cell line MDBK expresses four members of the heavy chain family and two members of the light chain protein family. Antibodies to the light chain of calpactin I were found to cause the precipitation of injected antibody together with the associated heavy chain without apparent effect on the distribution of related proteins. This suggests a differential targeting of various members of the calpactin heavy and light chain families even within the same cell.

  13. 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. PMID:26116789

  14. Elevated neurofilament light chain (NFL) mRNA levels in prediabetic peripheral neuropathy.

    PubMed

    Celikbilek, Asuman; Tanik, Nermin; Sabah, Seda; Borekci, Elif; Akyol, Lutfi; Ak, Hakan; Adam, Mehmet; Suher, Murat; Yilmaz, Neziha

    2014-06-01

    Evidence suggests that peripheral nerve injury occurs during the early stages of disease with mild glycemic dysregulation. Two proteins, neuron-specific enolase (NSE) and neurofilament light chain (NFL), have been examined previously as possible markers of neuronal damage in the pathophysiology of neuropathies. Herein, we aimed to determine the potential value of circulatory NSE and NFL mRNA levels in prediabetic patients and in those with peripheral neuropathy. This prospective clinical study included 45 prediabetic patients and 30 age- and sex-matched controls. All prediabetic patients were assessed with respect to diabetes-related microvascular complications, such as peripheral neuropathy, retinopathy and nephropathy. mRNA levels of NSE and NFL were determined in the blood by real-time polymerase chain reaction. NSE mRNA levels were similar between prediabetic and control groups (p > 0.05), whereas NFL mRNA levels were significantly higher in prediabetics than in controls (p < 0.001). NSE mRNA levels did not significantly differ between prediabetic patients with and without peripheral neuropathy (p > 0.05), while NFL mRNA levels were significantly higher in prediabetics with peripheral neuropathy than in those without (p = 0.038). According to correlation analysis, NFL mRNA levels were positively correlated with the Douleur Neuropathique 4 questionnaire score in prediabetic patients (r = 0.302, p = 0.044). This is the first study to suggest blood NFL mRNA as a surrogate marker for early prediction of prediabetic peripheral neuropathy, while NSE mRNA levels may be of no diagnostic value in prediabetic patients.

  15. 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.

  16. 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.

  17. Early Reduction of Serum-Free Light Chains Associates with Renal Recovery in Myeloma Kidney

    PubMed Central

    Cockwell, Paul; Stringer, Stephanie; Bradwell, Arthur; Cook, Mark; Gertz, Morie A.; Dispenzieri, Angela; Winters, Jeffrey L.; Kumar, Shaji; Rajkumar, S. Vincent; Kyle, Robert A.; Leung, Nelson

    2011-01-01

    Myeloma kidney is the major cause of severe irreversible renal failure in patients with multiple myeloma. This tubulointerstitial injury is a direct consequence of high concentrations of circulating monoclonal free light chains (FLCs) produced by a clonal expansion of plasma cells. Early reduction of serum FLCs associates with renal recovery, but the target threshold of reduction to facilitate renal recovery is unknown. To determine the relationship between the achieved FLC reduction and renal recovery, we identified 39 patients with biopsy-proven myeloma kidney, the majority of whom had severe renal failure at presentation (median estimated GFR 9 ml/min per 1.73 m2). In a multivariable analysis incorporating demographic, hematologic, and renal variables, only the achieved FLC reduction significantly predicted renal recovery (P = 0.003). The relationship between renal recovery and FLC reduction was linear with no absolute threshold for FLC reduction. A 60% reduction in FLCs by day 21 associated with recovery of renal function for 80% of the population. Patient survival strongly associated with renal recovery: the median survival was 42.7 months (range 0 to 80) among those who recovered function compared with 7.8 months (range 0 to 54) among those who did not (P < 0.02). Cox-regression analysis demonstrated that the first presentation of myeloma, the kappa isotype of FLC, and renal recovery were independent predictors of survival. In conclusion, recovery of renal function in myeloma kidney depends on early reduction of serum FLCs, and this recovery associates with a significant survival advantage. PMID:21511832

  18. 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.

  19. [Role of phosphatidylinositol 3-kinase and myosin light chain kinase during the activation of thrombin receptors].

    PubMed

    Han, Yue; Gao, Hai-Li; Zhang, Wei; Bai, Xia; Dai, Lan; Sheng, Wen-Hong; Sun, Ai-Ning; Wu, De-Pei; Wang, Zhao-Yue; Ruan, Chang-Geng

    2009-06-01

    The objective of study was to compare the influences of wortmannin on platelet aggregation and platelet membrane surface glycoproteins GPIb expression after thrombin receptor activation, and to investigate the role of phosphatidylinositol 3-kinase (PI3-K) and myosin light chain kinase (MLCK) in the course of thrombin receptor activation. Peptide SFLLRN (PAR1-AP) and AYPGKF (PAR4-AP) were used for stimulating platelet, and the changes of platelet aggregation and GPIb were analyzed with 100 nmol/L wortmannin (inhibitor of PI3-K) and 10 micromol/L wortmannin (inhibitor of MLCK). The results indicated that the platelet activation was influenced by either concentration of wortmannin in response to PAR stimulation. Platelet aggregation was apparently inhibited by 10 micromol/L wortmannin through both PAR peptides, and was slightly inhibited by 100 nmol/L wortmannin only under PAR1-AP activation. In addition, GPIbalpha internalization was partly inhibited by 100 nmol/L wortmannin in response to PAR1 (p < 0.05 at 1, 2, 5 min) and PAR4 (p < 0.05 at 2, 5, 10 min) activation. Meanwhile, 10 micromol/L wortmannin induced little change for GPIbalpha centralisation in the course of PAR activation, with a delayed restoration of surface GPIbalpha observed under PAR1-AP activation, and no change of GPIbalpha redistribution existed under PAR4-AP activation. It is concluded that the different roles of PI3-K and MLCK exist in the course of thrombin receptor activation. PI3-K accelerates the short course of GPIb centralisation for two PAR signal pathways, while MLCK inhibits the restoration of GPIbalpha in PAR1 pathway. PMID:19549383

  20. 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. PMID:24162233

  1. 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

  2. Factor VII Light Chain-Targeted Lidamycin Shows Intensified Therapeutic Efficacy for Liver Cancer

    PubMed Central

    Liu, Xiujun; Xu, Shuangshuang; Li, Caihong; Zhang, Yang; Yang, Jie; Zheng, Junnian

    2012-01-01

    Abstract The overexpression of tissue factor (TF) observed in numerous cancer cells and clinical samples of human cancers makes TF an ideal target for cancer therapy. The purpose of this study is to develop a TF-targeting energized fusion protein hlFVII-LDP-AE, which is composed of a human Factor VII light chain (hlFVII) as the targeting domain conjugated to the cytotoxic antibiotic lidamycin (LDM, LDP-AE) as the effector domain. The potential efficacy of hlFVII-LDP-AE for cancer therapy was tested in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays and in vivo with a BALB/c nude mouse xenograft model of human liver cancer line HepG2. The inhibitory concentration (IC50) value of hlFVII-LDP-AE varied from 0.15 to 0.64 nM for the various human tumor lines. hlFVII-LDP-AE showed a tumor growth inhibition rate of 90.6% at the dose of 0.6 mg/kg in in vivo animal experiments. The mechanism through which hlFVII-LDP-AE inhibits tumor growth also was determined by Hoechst 33342 staining and Tdt-mediated dUTP nick-end labeling (TUNEL) assay. hlFVII-LDP-AE causes tumor cell death through inducing chromatin condensation and cleavage of genomic DNA. These findings suggest that the hlFVII-LDP-AE protocol is efficacious and tolerated in the mouse model of human liver cancer HepG2 and has clinical applicability for treating cancer patients. PMID:22651685

  3. 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

    2016-09-01

    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.

  4. [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. PMID:7864303

  5. 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

  6. Experimental murine amyloidosis: a model system for studying amyloid formation.

    PubMed Central

    Baumal, R.; Wilson, B.; Pass, E.

    1975-01-01

    Myeloma-associated and casein-induced murine amyloidosis were used as models to study the role of lymphocytes and macrophages in amyloid formation. Amyloidosis occurred rarely and in small amounts in Balb/C mice with immunoglobulin (Ig)-producing myeloma tumours but large amounts could be induced by injections of casein. Fluorescent staining of both forms of amyloid deposits by means of anti-casein- and anti-myeloma-amyloid antibodies indicated that they either crossreacted or coexisted. Nor abnormality of Ig biosynthesis was detected in amyloidosis, suggesting that abnormal degradation was responsible for production of the Ig form of amyloid. Although spleen lymphocytes of casein-injected mice with amyloidosis demonstrated diminished cellular immunologic responses, this did not indicate generalized immunologic incompetence. The non-Ig form of amyloid in casein-injected mice was shown to be produced by macrophages, and a technique was developed for increasing the yield of amyloid-containing cells. Images FIG. 1 FIG. 2 FIG. 3 FIG. 6 FIG. 7 FIG. 8 PMID:1080430

  7. [Secondary monoclonal gammopathy after bone marrow autotransplantation as a cause of worse renal function in light chain immunoglobulin deposition disease].

    PubMed

    Rekhtina, I G; Mendeleeva, L P; Stolyarevich, E S; Gal'tseva, I V; Povilaitite, P E; Biryukova, L S

    2016-01-01

    The paper describes a clinical case of a female woman with nephropathy due to light chain deposition disease caused by secretion of κ Bence-Jones protein. Complete immunochemical remission was achieved after induction therapy using a bortezomib + cyclophosphamide + dexamethasone regimen. Renal function remained unchanged (glomerular filtration rate 16 ml/min), there was a reduction in proteinuria from 5.8 to 2.6 g/day. High-dose melphalan (200 mg/m2) chemotherapy with peripheral blood stem cell autotransplantation was performed as consolidation of remission. A year posttransplantation, there was no secretion of κ light chains; however, monoclonal IgG lambda emerged in a quantity of 3.2 g/l. At the same period, nephrotic syndrome became progressive (daily proteinuria 12 g) and dialysis-dependent renal failure developed. A repeat renal biopsy specimen revealed changes, suggesting that there was a decrease in renal deposits of κ light chains. Simultaneously with this, the obvious negative trend as progressive nephrosclerosis and fixation of IgG and λ light chains in the glomeruli (in the sclerotic areas) cause IgGλ monoclonal protein to be involved in the genesis of further kidney injury. Attention is also paid to different characteristics of capillary wall deposits by density (according to the electron microscopic findings), which may point to their different qualitative composition and possibly different formation duration. Papaprotein Gλ disappeared after a year without therapy, suggesting its reactivity. The findings confirm that worse renal function is caused by the action of paraprotein Gλ due to secondary (after autologous hematopoietic stem cells transplantation) monoclonal gammopathy.

  8. 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

  9. Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain

    PubMed Central

    Guhathakurta, Piyali; Prochniewicz, Ewa; Thomas, David D.

    2015-01-01

    We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40–45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC’s location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders. PMID:25825773

  10. Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain.

    PubMed

    Guhathakurta, Piyali; Prochniewicz, Ewa; Thomas, David D

    2015-04-14

    We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40-45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC's location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders. PMID:25825773

  11. [Secondary monoclonal gammopathy after bone marrow autotransplantation as a cause of worse renal function in light chain immunoglobulin deposition disease].

    PubMed

    Rekhtina, I G; Mendeleeva, L P; Stolyarevich, E S; Gal'tseva, I V; Povilaitite, P E; Biryukova, L S

    2016-01-01

    The paper describes a clinical case of a female woman with nephropathy due to light chain deposition disease caused by secretion of κ Bence-Jones protein. Complete immunochemical remission was achieved after induction therapy using a bortezomib + cyclophosphamide + dexamethasone regimen. Renal function remained unchanged (glomerular filtration rate 16 ml/min), there was a reduction in proteinuria from 5.8 to 2.6 g/day. High-dose melphalan (200 mg/m2) chemotherapy with peripheral blood stem cell autotransplantation was performed as consolidation of remission. A year posttransplantation, there was no secretion of κ light chains; however, monoclonal IgG lambda emerged in a quantity of 3.2 g/l. At the same period, nephrotic syndrome became progressive (daily proteinuria 12 g) and dialysis-dependent renal failure developed. A repeat renal biopsy specimen revealed changes, suggesting that there was a decrease in renal deposits of κ light chains. Simultaneously with this, the obvious negative trend as progressive nephrosclerosis and fixation of IgG and λ light chains in the glomeruli (in the sclerotic areas) cause IgGλ monoclonal protein to be involved in the genesis of further kidney injury. Attention is also paid to different characteristics of capillary wall deposits by density (according to the electron microscopic findings), which may point to their different qualitative composition and possibly different formation duration. Papaprotein Gλ disappeared after a year without therapy, suggesting its reactivity. The findings confirm that worse renal function is caused by the action of paraprotein Gλ due to secondary (after autologous hematopoietic stem cells transplantation) monoclonal gammopathy. PMID:27296267

  12. CaMKII and at least two unidentified kinases phosphorylate regulatory light chain in non-contracting cardiomyocytes.

    PubMed

    Eikemo, Hilde; Moltzau, Lise Román; Nguyen, Cam H T; Levy, Finn Olav; Skomedal, Tor; Osnes, Jan-Bjørn

    2016-08-12

    In cardiac tissue, regulatory light chain (RLC, myosin light chain 2) phosphorylation (Ser(15)) leads to modulation of muscle contraction through Ca(2+)-sensitization. To elucidate which kinases that are involved in the basal (diastolic phase) RLC phosphorylation, we studied non-contracting adult rat cardiomyocytes. RLC kinase activities in situ were unmasked by maximally inhibiting myosin light chain phosphatase (MLCP) by calyculin A in the absence and presence of various protein kinase inhibitors. Surprisingly MLCK did not contribute to the phosphorylation of RLC in the non-contracting cardiomyocytes. Two kinase activity groups were revealed by different sensitivities to staurosporine. The fraction with the highest sensitivity to staurosporine was inhibited by KN-93, a selective CaMKII inhibitor, producing a 23% ± 7% reduction in RLC phosphorylation. Calmodulin antagonism (W7) and reduction in Ca(2+) (EGTA) combined with low concentration of staurosporine caused a larger decrease in RLC phosphorylation than staurosporine alone. These data strongly suggest that in addition to CaMKII, there is another Ca(2+)/calmodulin-dependent kinase and a Ca(2+)/calmodulin-independent kinase phosphorylating RLC. Thus the RLC phosphorylation seems to be ensured by redundant kinase activities.

  13. Expression of heavy chain-only antibodies can support B-cell development in light chain knockout chickens.

    PubMed

    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; Leighton, Philip A

    2016-09-01

    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.

  14. TCTEX1D4 Interactome in Human Testis: Unraveling the Function of Dynein Light Chain in Spermatozoa

    PubMed Central

    Freitas, Maria João; Korrodi-Gregório, Luís; Morais-Santos, Filipa; da Cruz e Silva, Edgar

    2014-01-01

    Abstract Studies were designed to identify the TCTEX1D4 interactome in human testis, with the purpose of unraveling putative protein complexes essential to male reproduction and thus novel TCTEX1D4 functions. TCTEX1D4 is a dynein light chain that belongs to the DYNT1/TCTEX1 family. In spermatozoa, it appears to be important to sperm motility, intraflagellar transport, and acrosome reaction. To contribute to the knowledge on TCTEX1D4 function in testis and spermatozoa, a yeast two-hybrid assay was performed in testis, which allowed the identification of 40 novel TCTEX1D4 interactors. Curiously, another dynein light chain, TCTEX1D2, was identified and its existence demonstrated for the first time in human spermatozoa. Immunofluorescence studies proved that TCTEX1D2 is an intra-acrosomal protein also present in the midpiece, suggesting a role in cargo movement in human spermatozoa. Further, an in silico profile of TCTEX1D4 revealed that most TCTEX1D4 interacting proteins were not previously characterized and the ones described present a very broad nature. This reinforces TCTEX1D4 as a dynein light chain that is capable of interacting with a variety of functionally different proteins. These observations collectively contribute to a deeper molecular understanding of the human spermatozoa function. PMID:24606217

  15. Myosin light chain kinase steady-state kinetics: comparison of smooth muscle myosin II and nonmuscle myosin IIB as substrates

    PubMed Central

    Alcala, Diego B.; Haldeman, Brian D.; Brizendine, Richard K.; Krenc, Agata K.; Baker, Josh E.; Rock, Ronald S.; Cremo, Christine R.

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat/Km) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions. PMID:27528075

  16. Suppression of uninvolved immunoglobulins defined by heavy/light chain pair suppression is a risk factor for progression of MGUS

    PubMed Central

    Katzmann, JA; Clark, R; Kyle, RA; Larson, DR; Therneau, TM; Melton, LJ; Benson, JT; Colby, CL; Dispenzieri, A; Landgren, O; Kumar, S; Bradwell, AR; Cerhan, JR; Rajkumar, SV

    2013-01-01

    We hypothesized that the suppression of uninvolved immunoglobulin in monoclonal gammopathy of undetermined significance (MGUS) as detected by suppression of the isotype-specific heavy and light chain (HLC-pair suppression) increases the risk of progression to malignancy. This approach required quantitation of individual heavy/light chains (for example, IgGλ in IgGκ MGUS patients). Of 1384 MGUS patients from Southeastern Minnesota seen at the Mayo Clinic from 1960 to 1994, baseline serum samples obtained within 30 days of diagnosis were available in 999 persons. We identified HLC-pair suppression in 27% of MGUS patient samples compared with 11% of patients with suppression of uninvolved IgG, IgA or IgM. HLC-pair suppression was a significant risk factor for progression (hazard ratio (HR), 2.3; 95% confidence interval (CI) 1.5–3.7; P<0.001). On multivariate analysis, HLC-pair suppression was an independent risk factor for progression to malignancy in combination with serum M-spike size, heavy chain isotype and free light chain ratio (HR, 1.8; 95% CI, 1.1–3.00; P = 0.018). The finding that HLC-pair suppression predicts progression in MGUS and occurs several years before malignant transformation has implications for myeloma biology. PMID:22781594

  17. TCTEX1D4 interactome in human testis: unraveling the function of dynein light chain in spermatozoa.

    PubMed

    Freitas, Maria João; Korrodi-Gregório, Luís; Morais-Santos, Filipa; Cruz e Silva, Edgar da; Fardilha, Margarida

    2014-04-01

    Studies were designed to identify the TCTEX1D4 interactome in human testis, with the purpose of unraveling putative protein complexes essential to male reproduction and thus novel TCTEX1D4 functions. TCTEX1D4 is a dynein light chain that belongs to the DYNT1/TCTEX1 family. In spermatozoa, it appears to be important to sperm motility, intraflagellar transport, and acrosome reaction. To contribute to the knowledge on TCTEX1D4 function in testis and spermatozoa, a yeast two-hybrid assay was performed in testis, which allowed the identification of 40 novel TCTEX1D4 interactors. Curiously, another dynein light chain, TCTEX1D2, was identified and its existence demonstrated for the first time in human spermatozoa. Immunofluorescence studies proved that TCTEX1D2 is an intra-acrosomal protein also present in the midpiece, suggesting a role in cargo movement in human spermatozoa. Further, an in silico profile of TCTEX1D4 revealed that most TCTEX1D4 interacting proteins were not previously characterized and the ones described present a very broad nature. This reinforces TCTEX1D4 as a dynein light chain that is capable of interacting with a variety of functionally different proteins. These observations collectively contribute to a deeper molecular understanding of the human spermatozoa function.

  18. Restricted Immunoglobulin Variable Region (Ig V) Gene Expression Accompanies Secondary Rearrangements of Light Chain Ig V Genes in Mouse Plasmacytomas

    PubMed Central

    Diaw, Lena; Siwarski, David; Coleman, Allen; Kim, Jennifer; Jones, Gary M.; Dighiero, Guillaume; Huppi, Konrad

    1999-01-01

    The many binding studies of monoclonal immunoglobulin (Ig) produced by plasmacytomas have found no universally common binding properties, but instead, groups of plasmacytomas with specific antigen-binding activities to haptens such as phosphorylcholine, dextrans, fructofuranans, or dinitrophenyl. Subsequently, it was found that plasmacytomas with similar binding chain specificities not only expressed the same idiotype, but rearranged the same light (VL) and heavy (VH) variable region genes to express a characteristic monoclonal antibody. In this study, we have examined by enzyme-linked immunosorbent assay five antibodies secreted by silicone-induced mouse plasmacytomas using a broader panel of antigens including actin, myosin, tubulin, single-stranded DNA, and double-stranded DNA. We have determined the Ig heavy and light chain V gene usage in these same plasmacytomas at the DNA and RNA level. Our studies reveal: (a) antibodies secreted by plasmacytomas bind to different antigens in a manner similar to that observed for natural autoantibodies; (b) the expressed Ig heavy genes are restricted in V gene usage to the VH-J558 family; and (c) secondary rearrangements occur at the light chain level with at least three plasmacytomas expressing both κ and λ light chain genes. These results suggest that plasmacytomas use a restricted population of B cells that may still be undergoing rearrangement, thereby bypassing the allelic exclusion normally associated with expression of antibody genes. PMID:10562316

  19. Enhanced paracellular transport of insulin can be achieved via transient induction of myosin light chain phosphorylation.

    PubMed

    Taverner, Alistair; Dondi, Ruggero; Almansour, Khaled; Laurent, Floriane; Owens, Siân-Eleri; Eggleston, Ian M; Fotaki, Nikoletta; Mrsny, Randall J

    2015-07-28

    The intestinal epithelium functions to effectively restrict the causal uptake of luminal contents but has been demonstrated to transiently increase paracellular permeability properties to provide an additional entry route for dietary macromolecules. We have examined a method to emulate this endogenous mechanism as a means of enhancing the oral uptake of insulin. Two sets of stable Permeant Inhibitor of Phosphatase (PIP) peptides were rationally designed to stimulate phosphorylation of intracellular epithelial myosin light chain (MLC) and screened using Caco-2 monolayers in vitro. Apical application of PIP peptide 640, designed to disrupt protein-protein interactions between protein phosphatase 1 (PP1) and its regulator CPI-17, resulted in a reversible and non-toxic transient reduction in Caco-2 monolayer trans-epithelial electric resistance (TEER) and opening of the paracellular route to 4kDa fluorescent dextran but not 70kDa dextran in vitro. Apical application of PIP peptide 250, designed to impede MYPT1-mediated regulation of PP1, also decreased TEER in a reversible and non-toxic manner but transiently opened the paracellular route to both 4 and 70kDa fluorescent dextrans. Direct injection of PIP peptides 640 or 250 with human insulin into the lumen of rat jejunum caused a decrease in blood glucose levels that was PIP peptide and insulin dose-dependent and correlated with increased pMLC levels. Systemic levels of insulin suggested approximately 3-4% of the dose injected into the intestinal lumen was absorbed, relative to a subcutaneous injection. Measurement of insulin levels in the portal vein showed a time window of absorption that was consistent with systemic concentration-time profiles and approximately 50% first-pass clearance by the liver. Monitoring the uptake of a fluorescent form of insulin suggested its uptake occurred via the paracellular route. Together, these studies add validation to the presence of an endogenous mechanism used by the intestinal

  20. Diagnostic value of immunoglobulin κ light chain gene rearrangement analysis in B-cell lymphomas

    PubMed Central

    KOKOVIC, IRA; NOVAKOVIC, BARBARA JEZERSEK; NOVAKOVIC, SRDJAN

    2015-01-01

    Analysis of the immunoglobulin κ light chain (IGK) gene is an alternative method for B-cell clonality assessment in the diagnosis of mature B-cell proliferations in which the detection of clonal immunoglobulin heavy chain (IGH) gene rearrangements fails. The aim of the present study was to evaluate the added value of standardized BIOMED-2 assay for the detection of clonal IGK gene rearrangements in the diagnostic setting of suspected B-cell lymphomas. With this purpose, 92 specimens from 80 patients with the final diagnosis of mature B-cell lymphoma (37 specimens), mature T-cell lymphoma (26 specimens) and reactive lymphoid proliferation (29 specimens) were analyzed for B-cell clonality. B-cell clonality analysis was performed using the BIOMED-2 IGH and IGK gene clonality assays. The determined sensitivity of the IGK assay was 67.6%, while the determined sensitivity of the IGH assay was 75.7%. The sensitivity of combined IGH+IGK assay was 81.1%. The determined specificity of the IGK assay was 96.2% in the group of T-cell lymphomas and 96.6% in the group of reactive lesions. The determined specificity of the IGH assay was 84.6% in the group of lymphomas and 86.2% in the group of reactive lesions. The comparison of GeneScan (GS) and heteroduplex pretreatment-polyacrylamide gel electrophoresis (HD-PAGE) methods for the analysis of IGK gene rearrangements showed a higher efficacy of GS analysis in a series of 27 B-cell lymphomas analyzed by both methods. In the present study, we demonstrated that by applying the combined IGH+IGK clonality assay the overall detection rate of B-cell clonality was increased by 5.4%. Thus, we confirmed the added value of the standardized BIOMED-2 IGK assay for assessment of B-cell clonality in suspected B-cell lymphomas with inconclusive clinical and cyto/histological diagnosis. PMID:25501347

  1. 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

  2. [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.

  3. Binding of nascent collagen by amyloidogenic light chains and amyloid fibrillogenesis in monolayers of human fibrocytes.

    PubMed

    Harris, D L; King, E; Ramsland, P A; Edmundson, A B

    2000-01-01

    Light (L) chain dimers expressed by multiple myeloma cells and collected as Bence-Jones proteins from the urine of human subjects were tested for their ability to form deposits in fibroblast monolayer cell cultures. Bence-Jones proteins from subjects with primary amyloidosis associated with L chains were shown to form fibrillar deposits by the in vitro assay introduced in this report. Filaments interspersed with nascent collagen could be detected after only 48 h. Deposition of L chains continued over a period of 72 h culminating in the appearance of dense fibrils with widths of 80-100 A and a variety of lengths. Formation of amyloid-like fibrils was accompanied by interference with the maturation of the collagen produced by the fibroblast cells. Fibrils composed of the Mcg lambda-type L chain were deposited between collagen fibers, thus expanding them laterally and leading to their partial disintegration. Mature collagen was completely missing from fibroblast monolayers exposed to the Sea lambda chain and the Jen kappa chain. Collagen with the characteristic striped pattern matured normally in control samples, such as those not dosed with amyloid precursors or those treated with a non-amyloidogenic Bence-Jones protein (e.g., the Hud lambda chain dimer). By immunochemical techniques using fluorescein- and gold-labeled anti-L chain antibodies, amyloidogenic L chains were shown to decorate the strands of nascent collagen. This observation suggests that amyloidogenic L chains are concentrated in the extracellular matrix by monovalent antigen-antibody type reactions. The capacity of the Mcg L chain dimer to bind collagen-derived sequences was tested by soaking crystals with a collagenase substrate, PZ-Pro-Leu-Gly-Pro-D-Arg. Difference Fourier analysis at 2.7 A resolution indicated that the PZ-peptide is a site-filling ligand. It could not be removed from the active site by perfusion of the crystal with ammonium sulfate crystallizing media. Similar experiments with the

  4. Combined pulmonary involvement in hereditary lysozyme amyloidosis with associated pulmonary sarcoidosis: a case report.

    PubMed

    McCarthy, Cormac; Deegan, Alexander P; Garvey, John F; McDonnell, Timothy J

    2013-01-01

    Sarcoidosis is a multisystem inflammatory disorder of unknown cause which can affect any organ system. Autosomal dominant lysozyme amyloidosis is a very rare form of hereditary amyloidosis. The Arg64 variant is extraordinarily rare with each family showing a particular pattern of organ involvement, however while Sicca syndrome, gastrointestinal involvement and renal failure are common, lymph node involvement is very rare. In this case report we describe the first reported case of sarcoidosis in association with hereditary lysozyme amyloidosis. PMID:24351625

  5. Combined use of free light chain and heavy/light chain ratios allow diagnosis and monitoring of patients with monoclonal gammopathies: Experience of a single institute, with three exemplar case reports

    PubMed Central

    Gagliardi, Alfredo; Carbone, Claudio; Russo, Angela; Cuccurullo, Rosanna; Lucania, Anna; Cioppa, Paola Della; Misso, Gabriella; Caraglia, Michele; Tommasino, Catello; Mastrullo, Lucia

    2016-01-01

    Monoclonal gammopathies are characterized by serum monoclonal component (MC) plus an intact immunoglobulin and a free light chain (FLC), or a combination of both. The measurement of FLC with Freelite® is the standard practice recommended by International Myeloma Working Group guidelines. Recently, Hevylite® heavy/light chains (HLC) assays were introduced to specifically target junctional epitopes between the heavy and light chains of intact immunoglobulins, allowing the independent quantification of the involved (MC) and uninvolved (polyclonal immunoglobulin background) HLC isotype. Between January 2012 and March 2014, 90 patients were examined: 49 multiple myeloma (MM), 6 smoldering MM (SMM) and 35 monoclonal gammopathy of undetermined significance (MGUS). Of these 90 patients, 300 samples were collected at different times. The diagnostic and monitoring contribution of Hevylite A and G assays was assessed in all 90 patients examined. Additionally, 3 representative cases were selected. The Hevylite absolute values and ratio demonstrated high sensitivity and specificity with respect to serum protein electrophoresis and serum immunofixation. The combined use of Hevylite A and G with Freelite was particularly useful in dubious cases with more than one MC or with co-migrating components, as well as in the course of monitoring to assess the independent change of FLC and HLC, possibly reflecting the presence of clonal heterogeneity in the cohort. From this study, it can be concluded that FLC and HLC are independent, useful markers to monitor the MC and to assess with greater specificity and sensitivity the effect of therapy, thereby providing clinical support. Further studies are required to assess the prognostic potential of Hevylite in MGUS and SMM.

  6. Combined use of free light chain and heavy/light chain ratios allow diagnosis and monitoring of patients with monoclonal gammopathies: Experience of a single institute, with three exemplar case reports

    PubMed Central

    Gagliardi, Alfredo; Carbone, Claudio; Russo, Angela; Cuccurullo, Rosanna; Lucania, Anna; Cioppa, Paola Della; Misso, Gabriella; Caraglia, Michele; Tommasino, Catello; Mastrullo, Lucia

    2016-01-01

    Monoclonal gammopathies are characterized by serum monoclonal component (MC) plus an intact immunoglobulin and a free light chain (FLC), or a combination of both. The measurement of FLC with Freelite® is the standard practice recommended by International Myeloma Working Group guidelines. Recently, Hevylite® heavy/light chains (HLC) assays were introduced to specifically target junctional epitopes between the heavy and light chains of intact immunoglobulins, allowing the independent quantification of the involved (MC) and uninvolved (polyclonal immunoglobulin background) HLC isotype. Between January 2012 and March 2014, 90 patients were examined: 49 multiple myeloma (MM), 6 smoldering MM (SMM) and 35 monoclonal gammopathy of undetermined significance (MGUS). Of these 90 patients, 300 samples were collected at different times. The diagnostic and monitoring contribution of Hevylite A and G assays was assessed in all 90 patients examined. Additionally, 3 representative cases were selected. The Hevylite absolute values and ratio demonstrated high sensitivity and specificity with respect to serum protein electrophoresis and serum immunofixation. The combined use of Hevylite A and G with Freelite was particularly useful in dubious cases with more than one MC or with co-migrating components, as well as in the course of monitoring to assess the independent change of FLC and HLC, possibly reflecting the presence of clonal heterogeneity in the cohort. From this study, it can be concluded that FLC and HLC are independent, useful markers to monitor the MC and to assess with greater specificity and sensitivity the effect of therapy, thereby providing clinical support. Further studies are required to assess the prognostic potential of Hevylite in MGUS and SMM. PMID:27698801

  7. AA amyloidosis in the renal allograft: a report of two cases and review of the literature.

    PubMed

    Rojas, Rebecca; Josephson, Michelle A; Chang, Anthony; Meehan, Shane M

    2012-04-01

    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.

  8. Chronic eyelid swelling as an initial manifestation of myeloma-associated amyloidosis.

    PubMed

    Chee, Elaine; Kim, Yoon-Duck; Lee, Jung Hye; Woo, Kyung In

    2013-01-01

    Orbital amyloidosis is uncommon and difficult to diagnose due to their variable clinical presentations. The authors report a case of a patient who presents with chronic eyelid swelling as an initial manifestation of myeloma-associated amyloidosis. This patient was also found to have retrobulbar infiltration with no visual impairment. The authors also describe the first documentation of the atypical necrotic appearance of amyloidosis in the involved eyelid tissues. Myeloma-associated amyloidosis can present as chronic, nonspecific periorbital swelling, hence a biopsy of the affected tissues is important in preventing a delay in the correct diagnosis.

  9. 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. PMID:8869690

  10. The importance of screening for serum free light chains in suspected cases of multiple myeloma and their impact on the kidney

    PubMed Central

    Talbot, B; Wright, D; Basnayake, K

    2014-01-01

    Multiple myeloma (MM) is the second most common haematological malignancy in the UK. We present a case series of three patients with light chain only myeloma who had normal serum protein electrophoretic studies at screening and were diagnosed using serum and urine free light chain assessment. This series reiterates the importance of thorough and robust screening for MM in patients presenting with renal disease. We review the up to date literature and we highlight the need to screen patients for MM with a combination of serum electrophoresis/immunofixation and either urinary or serum free light chain measurement and to maintain a high index of suspicion regardless of the presence or absence of proteinuria. We also discuss the emerging role of the serum free light chain assay. PMID:25326567

  11. 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.

  12. Myosin light chain phosphorylation in sup 32 P-labeled rabbit aorta stimulated by phorbol 12,13-dibutyrate and phenylephrine

    SciTech Connect

    Singer, H.A.; Oren, J.W.; Benscoter, H.A. )

    1989-12-15

    The mechanism(s) of force development in vascular smooth muscle following pharmacological activation of protein kinase C by phorbol esters are not known. In this study, we examined the myosin light chain phosphorylation response following stimulation by phorbol 12,13-dibutyrate (PDB) or phenylephrine in rabbit aorta which had been incubated with 32PO4 in order to label ATP pools. Through tryptic phosphopeptide mapping of myosin light chain from intact tissue and comparison to controls using purified components, we inferred that Ca2+-dependent force stimulated by PDB was associated with small increases in serine-19 phosphorylation, consistent with a contractile mechanism involving indirect activation of myosin light chain kinase. Additional residues, consistent with the in vitro substrate specificity of protein kinase C, were also observed to be phosphorylated in response to PDB and represented proportionately a larger fraction of the total phosphorylated myosin light chain in Ca2+-depleted tissues. Stimulation by an alpha 1-adrenergic agonist (phenylephrine) resulted in phosphorylation of residues which were consistent with an activation mechanism involving myosin light chain kinase only. These results indicate that in rabbit aorta the contractile effects of PDB may be partially mediated by Ca2+-dependent activation of myosin light chain kinase. However, the data do not rule out a component of the PDB-stimulated contractile response which is independent of myosin light chain phosphorylation on the serine-19 residue. In addition, activation by a more physiological stimulus, phenylephrine, does not result in protein kinase C-mediated myosin light chain phosphorylation.

  13. Quantitative analysis of the free energy coupling in the system calmodulin, calcium, smooth muscle myosin light chain kinase.

    PubMed

    Mamar-Bachi, A; Cox, J A

    1987-12-01

    Interactions between Ca2+, calmodulin and turkey gizzard myosin light chain kinase have been studied by equilibrium gel filtration and analyzed in terms of the theory of free energy coupling as formulated by Huang and King for calmodulin-regulated systems (Current Topics in Cellular Regulation 27, 1966-1971, 1985). Direct binding studies revealed that upon interaction with the enzyme, calmodulin acquires strong positive cooperativity in Ca2+-binding. The determination of the Ca2+-binding constants is inherently approximative due to the apparent homotropic cooperativity; therefore a statistical chi 2 analysis was carried out to delimit the formation-, and subsequently the stoichiometric Ca2+-binding constants. Whereas the first two stoichiometric Ca2+-binding constants of enzyme-bound CaM do not differ or are at the upmost 10-fold higher than those in free calmodulin, the third Ca2+ ion binds with an at least 70-fold and more likely 3000-fold higher affinity constant. The binding constant for the fourth Ca2+ is only 5-fold higher than the corresponding one in free calmodulin, thus creating a plateau at 3 bound Ca2+ in the isotherm. Direct binding of Ca2+-free calmodulin to myosin light chain kinase at 10(-7) M free Ca2+ yielded a l/l stoichiometry and an affinity constant of 2.2 x 10(5) M-1. It is thus anticipated that in resting smooth muscle ([Ca2+] less than or equal to 10(-7) M) more than half of the enzyme is bound to metal-free calmodulin. Analysis of the enzymatic activation of myosin light chain kinase at different concentrations of calmodulin and Ca2+ revealed that this Ca2+-free complex is inactive and that activation is concomitant with the formation of the enzyme.calmodulin.Ca3 complex.

  14. [Effect of myosin alkali light chains on myosin subfragment 1 interaction with actin in solution and in ghost muscle fiber].

    PubMed

    Levistkiĭ, D I; Borovikov, Iu S; Nikolaeva, O P; Golitsyna, N L; Poglazov, B F

    1990-09-01

    At low ionic strength (7-25 mM) Mg2(+)-ATPase of myosin subfragment 1 (S1) isoforms containing alkali light chain A1 [S1(A1)] is activated by actin 1.5-2.5 times as strongly as Mg2(+)-ATPase of S1 isoforms containing alkali light chain A2[S1(A2)]. Data from analytical ultracentrifugation suggest that at low ionic strength in the absence of ATP in solution S1(A1) displays a higher affinity for F-actin than S1(A2). Such a higher affinity of S1(A1) for F-actin was also demonstrated by experiments, in which the interaction of S1 isoforms fluorescently labeled by 1.5-IAEDANS with F-actin of ghost fibers (single glycerinated muscle fibers containing F-actin but devoid of myosin) was studied. Using polarization microfluorimetry, it was shown that the interaction of both S1 isoforms with ghost fiber F-actin induces similar changes in the parameters of polarized tryptophan fluorescence. At the same time the mobility of the fluorescent probe, 1.5-IAEDANS, specifically attached to the SH-group of Cys-374 in the C-terminal region of action is markedly decreased by S1(A1) and is only slightly affected by S1(A2). The data obtained suggest that S1(A1) and S1(A2) interact with the C-terminal region of the actin molecule in different ways, i.e. S1(A1) is attached more firmly than S1(A2). This may be due to the existence of contacts between the alkali light chain of A1 of S1(A1) and the C-terminal region of actin as well as to the absence of such contacts in the case of S1(A2).

  15. β-Arrestin Regulation of Myosin Light Chain Phosphorylation Promotes AT1aR-mediated Cell Contraction and Migration

    PubMed Central

    Simard, Elie; Kovacs, Jeffrey J.; Miller, William E.; Kim, Jihee; Grandbois, Michel; Lefkowitz, Robert J.

    2013-01-01

    Over the last decade, it has been established that G-protein-coupled receptors (GPCRs) signal not only through canonical G-protein-mediated mechanisms, but also through the ubiquitous cellular scaffolds β-arrestin-1 and β-arrestin-2. Previous studies have implicated β-arrestins as regulators of actin reorganization in response to GPCR stimulation while also being required for membrane protrusion events that accompany cellular motility. One of the most critical events in the active movement of cells is the cyclic phosphorylation and activation of myosin light chain (MLC), which is required for cellular contraction and movement. We have identified the myosin light chain phosphatase Targeting Subunit (MYPT-1) as a binding partner of the β-arrestins and found that β-arrestins play a role in regulating the turnover of phosphorylated myosin light chain. In response to stimulation of the angiotensin Type 1a Receptor (AT1aR), MLC phosphorylation is induced quickly and potently. We have found that β-arrestin-2 facilitates dephosphorylation of MLC, while, in a reciprocal fashion, β-arrestin 1 limits dephosphorylation of MLC. Intriguingly, loss of either β-arrestin-1 or 2 blocks phospho-MLC turnover and causes a decrease in the contraction of cells as monitored by atomic force microscopy (AFM). Furthermore, by employing the β-arrestin biased ligand [Sar1,Ile4,Ile8]-Ang, we demonstrate that AT1aR-mediated cellular motility involves a β-arrestin dependent component. This suggests that the reciprocal regulation of MLC phosphorylation status by β-arrestins-1 and 2 causes turnover in the phosphorylation status of MLC that is required for cell contractility and subsequent chemotaxic motility. PMID:24255721

  16. Familial Amyloidosis Cutis Dyschromica in Three Siblings: Report from Indonesia

    PubMed Central

    Hermawan, Melyawati; Rihatmadja, Rahadi; Sirait, Sondang Pandjaitan

    2014-01-01

    Amyloidosis cutis dyschromica (ACD) is an extremely rare type of primary cutaneous amyloidosis. To date there are fewer than 40 published cases worldwide; some were reported affecting several family members. Its resemblance to other common pigmentation disorders makes it rarely recognized at first sight. Our patient, the 12-year-old firstborn son of non-consanguineous parents presented with generalized mottled pigmentation starting from lower extremities. His siblings suffered from similar condition. The clue for diagnosis is the amyloid deposition in the papillary dermis. The etiology of ACD is still unknown, but genetic factors and ultraviolet radiation are implicated. It is proposed that disturbance of keratinocyte repair following ultraviolet radiation results in amyloid deposition. The treatment remains a challenge. Oral acitretin treatment, thought to repair keratinization defect, gave a slight improvement in our case. Our is the first case of ACD reported in Indonesia. PMID:25386328

  17. Systemic amyloidosis: unusual presentation mistaken for a recurrent scabies infection.

    PubMed

    Haley, Erin M; Nabatian, Adam S; Kopp, Sandra A; Falasca, Gerald F; Haupt, Helen M; Halpern, Analisa V

    2014-06-01

    We report the case of a 63-year-old woman with a history of undifferentiated connective-tissue disease, polyarthritis, and bilateral carpal tunnel syndrome who presented with generalized pruritus and erythematous and excoriated papules on the trunk and extremities. Empiric scabies treatment was unsuccessful. Patch testing and T-cell receptor gene rearrangement studies were unremarkable. The patient was found to have mild interstitial lung disease and hypogammaglobulinemia. Eventually a diagnosis of primary systemic amyloidosis was made after she developed frank lingual hypertrophy despite normal initial serum protein electrophoresis and negative abdominal fat pad aspiration. Diagnosis was confirmed with lingual biopsy. This case demonstrates an unusual presentation of primary systemic amyloidosis consisting of arthritis and intense debilitating pruritus without primary skin lesions for a full year prior to diagnosis of multiple myeloma. The patient responded to treatment with chemotherapy and corticosteroids.

  18. Renal Light Chain Deposition Associated with the Formation of Intracellular Crystalline Inclusion Bodies in Podocytes: A Rare Case Report.

    PubMed

    Wang, Yuan-da; Dong, Zhe-yi; Zhang, Xue-guang; Zhang, Wei; Yin, Zhong; Qiu, Qiang; Chen, Xiang-mei

    2016-01-01

    We herein report the case of an elderly woman with bone pain and proteinuria as the main clinical manifestations. The patient was diagnosed with the IgG κ type of multiple myeloma. Her renal pathology consisted of widespread κ light chain protein deposition associated with the formation of large quantities of rod-like crystals in podocytes. This phenomenon is very rare. We explored the significance of this crystal formation via a detailed and descriptive analysis and also performed a literature review, thus providing data to increase the available information about this type of disease.

  19. TNF causes changes in glomerular endothelial permeability and morphology through a Rho and myosin light chain kinase-dependent mechanism.

    PubMed

    Xu, Chang; Wu, Xiaoyan; Hack, Bradley K; Bao, Lihua; Cunningham, Patrick N

    2015-12-01

    A key function of the endothelium is to serve as a regulated barrier between tissue compartments. We have previously shown that tumor necrosis factor (TNF) plays a crucial role in lipopolysaccharide (LPS)-induced acute kidney injury, in part by causing injury to the renal endothelium through its receptor TNFR1. Here, we report that TNF increased permeability to albumin in primary culture mouse renal endothelial cells, as well as human glomerular endothelial cells. This process occurred in association with changes in the actin cytoskeleton and was associated with gaps between previously confluent cells in culture and decreases in the tight junction protein occludin. This process was dependent on myosin light chain activation, as seen by its prevention with Rho-associated kinase and myosin light chain kinase (MLCK) inhibitors. Surprisingly, permeability was not blocked by inhibition of apoptosis with caspase inhibitors. Additionally, we found that the renal glycocalyx, which plays an important role in barrier function, was also degraded by TNF in a Rho and MLCK dependent fashion. TNF treatment caused a decrease in the size of endothelial fenestrae, dependent on Rho and MLCK, although the relevance of this to changes in permeability is uncertain. In summary, TNF-induced barrier dysfunction in renal endothelial cells is crucially dependent upon the Rho/MLCK signaling pathway.

  20. Reconstitution of heavy chain and light chain 1 in cardiac subfragment-1 from hyperthyroid and euthyroid rabbit hearts.

    PubMed

    Ueda, S; Yamaoki, K; Nagai, R; Yazaki, Y

    1983-01-01

    It is now established that cardiac myosin from hyperthyroid rabbit hearts (TXM) exhibits high Ca2+ ATPase activity. The high Ca2+ ATPase activity of TXM was completely retained in cardiac myosin subfragment-1 (S-1) (1.33 +/- 0.04 mumol Pi/mg per min; euthyroid, 0.51 +/- 0.04). Cardiac S-1 from hyperthyroid and euthyroid rabbits (TXS-1 and NS-1) had the same pattern in SDS-polyacrylamide gel electrophoresis. The possible influence of heavy and light chains of TXM on increasing the ATPase activity was examined by reconstitution in the S-1 preparation. Crosswise reconstitution was performed using cardiac S-1 heavy chain (90,000 daltons) and light chain 1 (LC1) (27,000 daltons) from hyperthyroid and euthyroid hearts. Reconstitution was verified by using radiolabeled LC1. More than 95% of S-1 was recovered with full ATPase activity. When TXS-1 was reconstituted with LC1 from euthyroid hearts, the reconstituted molecule retained high ATPase activity. On the other hand, NS-1 reconstituted with LC1 from hyperthyroid hearts failed to increase the ATPase activity. The ATPase activity of S-1 was determined by the source of the heavy chain. These results suggest that the high Ca2+ ATPase activity of cardiac myosin and S-1 from hyperthyroid animals arises from the molecular alteration of the heavy chain induced by thyroxine administration. PMID:6304826

  1. Quantification of β region IgA paraproteins - should we include immunochemical "heavy/light chain" measurements? Counterpoint.

    PubMed

    Paolini, Lucia

    2016-06-01

    Serum protein electrophoresis (SPE), serum immunofixation (s-IFE), free light chain measurement (FLC) and nephelometric measurements of total immunoglobulin in serum (IgTot) are some of the laboratory tests required for the management of plasma cell proliferative disorders. The monoclonal protein is usually visible on SPE as a spike (M-spike) in the γ region and the derived densitogram is used to quantify it relative to serum total protein concentration. IgA M-protein, however, often migrates in the β region on SPE and its quantification can be masked by other serum proteins that migrate in this region. The immunoassay Hevylite™ (heavy/light chain, HLC) seems to solve this problem: it quantifies the involved/uninvolved isotype, calculating the ratio IgAκ/IgAλ, considered indicative of clonal proliferation. However, this test seems redundant in the case of artifacts on SPE such as obvious hemolysis or lipemia, or if the IgA M-spike is clearly visible in the β region. In conclusion whereas the IgA HLC assay does not represent an alternative to SPE and s-IFE in the diagnostic patient workup, it may prove to be an alternative to SPE, s-IFE and total IgA quantification in risk stratification and evaluation of response to therapy in patients affected by MM and other monoclonal plasma proliferative disorders.

  2. European trial of free light chain removal by extended haemodialysis in cast nephropathy (EuLITE): A randomised control trial

    PubMed Central

    Hutchison, Colin A; Cook, Mark; Heyne, Nils; Weisel, Katja; Billingham, Lucinda; Bradwell, Arthur; Cockwell, Paul

    2008-01-01

    Background Renal failure is a frequent complication of multiple myeloma and when severe is associated with a greatly increased morbidity and mortality. The principal cause of severe renal failure is cast nephropathy, a direct consequence of high concentrations of monoclonal free light chains (FLCs) in patients' sera. FLC removal by extended haemodialysis, using a high cut-off dialyser, has recently been described as a novel therapeutic option. Methods The EUropean trial of free LIght chain removal by exTEnded haemodialysis in cast nephropathy (EuLITE) trial is a prospective, randomised, multicentre, open label clinical trial to investigate the clinical benefits of FLC removal haemodialysis in patients with cast nephropathy, dialysis dependent acute renal failure and de novo multiple myeloma. Recruitment commenced in May 2008. In total, 90 patients will be recruited. Participants will be randomised, centrally, upon enrolment, to either trial chemotherapy and FLC removal haemodialysis or trial chemotherapy and standard high flux haemodialysis. Trial chemotherapy consists of bortezomib, doxorubicin and dexamethasone. FLC removal haemodialysis is undertaken with two Gambro HCO 1100 dialysers in series using an intensive treatment schedule. The primary outcome for the study is independence of dialysis at 3 months. Secondary outcomes are: duration of dialysis, reduction in serum FLC concentrations; myeloma response and survival. Hypothesis FLC removal haemodialysis will increase the rate of renal recovery in patients with severe renal failure secondary to cast nephropathy in de novo multiple myeloma. Trial registration ISRCTN45967602 PMID:18822172

  3. The importance of complete tissue homogenization for accurate stoichiometric measurement of myosin light chain phosphorylation in airway smooth muscle.

    PubMed

    Wang, Lu; Paré, Peter D; Seow, Chun Y

    2015-02-01

    The standard method for measuring the phosphorylation of the regulatory myosin light chain (MLC20) in smooth muscle is extraction of the light chain using a urea extraction buffer, urea-glycerol gel electrophoresis of the soluble portion of the extract (supernatant) and Western blot analysis. The undissolved portion of the tissue during extraction (the pellet) is usually discarded. Because the pellet contains a finite amount of MLC20, omission of the pellet could result in inaccurate measurement of MLC20 phosphorylation. In this study we compared the level of tracheal smooth muscle MLC20 phosphorylation in the supernatant alone, with that in the complete tissue homogenate (supernatant and pellet) using the standard method. The supernatant fraction showed the well-known double bands representing phosphorylated and un-phosphorylated MLC20. The dissolved pellet fraction showed varying amounts of un-phosphorylated and phosphorylated MLC20. There was a small but statistically significant overestimation of the percent MLC20 phosphorylation if the pellet was not taken into consideration. The overestimation was 7% ± 2% (mean ± SEM) (p < 0.05) in unstimulated muscle and 2% ± 1% (p < 0.05) in acetylcholine (10(-6) mol/L) stimulated muscle. This finding suggests that for accurate estimation of the stoichiometry of MLC20 phosphorylation it is necessary to consider the contribution from the pellet portion of the muscle tissue homogenate.

  4. 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.

  5. TNF causes changes in glomerular endothelial permeability and morphology through a Rho and myosin light chain kinase-dependent mechanism.

    PubMed

    Xu, Chang; Wu, Xiaoyan; Hack, Bradley K; Bao, Lihua; Cunningham, Patrick N

    2015-12-01

    A key function of the endothelium is to serve as a regulated barrier between tissue compartments. We have previously shown that tumor necrosis factor (TNF) plays a crucial role in lipopolysaccharide (LPS)-induced acute kidney injury, in part by causing injury to the renal endothelium through its receptor TNFR1. Here, we report that TNF increased permeability to albumin in primary culture mouse renal endothelial cells, as well as human glomerular endothelial cells. This process occurred in association with changes in the actin cytoskeleton and was associated with gaps between previously confluent cells in culture and decreases in the tight junction protein occludin. This process was dependent on myosin light chain activation, as seen by its prevention with Rho-associated kinase and myosin light chain kinase (MLCK) inhibitors. Surprisingly, permeability was not blocked by inhibition of apoptosis with caspase inhibitors. Additionally, we found that the renal glycocalyx, which plays an important role in barrier function, was also degraded by TNF in a Rho and MLCK dependent fashion. TNF treatment caused a decrease in the size of endothelial fenestrae, dependent on Rho and MLCK, although the relevance of this to changes in permeability is uncertain. In summary, TNF-induced barrier dysfunction in renal endothelial cells is crucially dependent upon the Rho/MLCK signaling pathway. PMID:26634902

  6. Crystal structure of human dynein light chain Dnlc2A: Structural insights into the interaction with IC74

    SciTech Connect

    Liu Junfeng; Wang Zhanxin; Wang Xinquan; Tang Qun; An Xiaomin; Gui Lulu; Liang Dongcai . E-mail: dcliang@sun5.ibp.ac.cn

    2006-10-27

    The human light chain of the motor protein dynein, Dnlc2A, is also a novel TGF-{beta}-signaling component, which is altered with high frequency in epithelial ovarian cancer. It is an important mediator of dynein and the development of cancer, owing to its ability to bind to the dynein intermediate light chain (DIC) IC74 and to regulate TGF-{beta}-dependent transcriptional events. Here we report the 2.1-A crystal structure of Dnlc2A using single anomalous diffraction. The proteins form a homodimer in solution and interact mainly through the helix {alpha}{sub 2}, strand {beta}{sub 3}, and the loop following this strand in each protein to generate a 10-stranded {beta}-sheet core. The surface of the {beta}-sheet core is mainly positively charged and predicted (by software PPI-Pred) to be the site that interacts with other partners. At the same time, the residues 79-82, 88, and 90 of each molecule formed two holes in the core. Residue 89 of each molecule, which is crucial for the DIC binding function of Dnlc2A, is within the holes. On the basis of these observations, we propose that the homodimer is the structural and functional unit maintained by hydrogen bonding interactions and hydrophobic packing, and that the patch of the surface of the {beta}-sheet core is the main area of interaction with other partners. Furthermore, the two holes would be the key sites to interact with IC74.

  7. Development and application of in vivo molecular traps reveals that dynein light chain occupancy differentially affects dynein-mediated processes.

    PubMed

    Varma, Dileep; Dawn, Amrita; Ghosh-Roy, Anindya; Weil, Sarah J; Ori-McKenney, Kassandra M; Zhao, Yanqiu; Keen, James; Vallee, Richard B; Williams, John C

    2010-02-23

    The ability to rapidly and specifically regulate protein activity combined with in vivo functional assays and/or imaging can provide unique insight into underlying molecular processes. Here we describe the application of chemically induced dimerization of FKBP to create nearly instantaneous high-affinity bivalent ligands capable of sequestering cellular targets from their endogenous partners. We demonstrate the specificity and efficacy of these inducible, dimeric "traps" for the dynein light chains LC8 (Dynll1) and TcTex1 (Dynlt1). Both light chains can simultaneously bind at adjacent sites of dynein intermediate chain at the base of the dynein motor complex, yet their specific function with respect to the dynein motor or other interacting proteins has been difficult to dissect. Using these traps in cultured mammalian cells, we observed that induction of dimerization of either the LC8 or TcTex1 trap rapidly disrupted early endosomal and lysosomal organization. Dimerization of either trap also disrupted Golgi organization, but at a substantially slower rate. Using either trap, the time course for disruption of each organelle was similar, suggesting a common regulatory mechanism. However, despite the essential role of dynein in cell division, neither trap had a discernable effect on mitotic progression. Taken together, these studies suggest that LC occupancy of the dynein motor complex directly affects some, but not all, dynein-mediated processes. Although the described traps offer a method for rapid inhibition of dynein function, the design principle can be extended to other molecular complexes for in vivo studies.

  8. [EXPRESSION OF THE LIGHT CHAINS OF IMMUNOGLOBULINS IN NORMAL B-CELLS AND SOME B-CELL LYMPHOMAS].

    PubMed

    Khudoleeva, O A; Vorobjev, I A

    2015-01-01

    The quantitative method of determining the level of expression of immunoglobulin light chains on uncompensated data was suggested and used to examine disorders in light chain expression in various B-cell tumors. The average level of expression of the lambda isotype was 4 times higher than the level of expression of kappa isotype. The level of surface and cytoplasmic expression of LC IG varied within wide limits for different people, but there was a high degree of correlation between the levels of expression of kappa and lambda isotypes LC IG as well as between expression of the surface and cytoplasmic forms of each in isotype the same individual. In the majority of B-cell non-Hodgkin's lymphomas correlation between the expression of LC IG on the surface and in the cytoplasm of the cells was diminished. Expression of LC IG in CLL was significantly reduced on the surface of the cells and to a lesser extent--in the cytoplasm. In the case of marginal zone cell lymphoma, LC IG expression level was reduced on the surface of circulating cells and to a lesser extent--in the cytoplasm. In the case of mantle cell lymphoma and DLBCL, expression level of LC IG on the cell surface and in the cytoplasm was the same as in normal B-cells. However, in some cases DLBCL, no LC IG was expressed both on the surface and in the cytoplasm. PMID:26863766

  9. 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. PMID:27085355

  10. 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 ...

  11. 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

  12. Buccal mucosa graft urethroplasty in a case of urethral amyloidosis presenting with long anterior urethral stricture

    PubMed Central

    Kurbatov, Dmitry; Stojanovic, Borko; Dubskiy, Sergey; Lepetukhin, Alex; Djordjevic, Miroslav L.

    2015-01-01

    Urethral amyloidosis is a rare condition, but clinically relevant because it can mimic urothelial carcinoma. We report a case of localized urethral amyloidosis presenting with a long anterior urethral stricture. We used extensive grafts of buccal mucosa for standard augmentation urethroplasty, with a successful outcome at the 2-year follow-up. PMID:26600896

  13. Beta-2-glycoprotein specificity of human anti-phospholipid antibody resides on the light chain: a novel mechanism for acquisition of cross-reactivity by an autoantibody.

    PubMed

    Kumar, Sanjeev; Nagl, Sylvia; Kalsi, Jatinderpal K; Ravirajan, Chelliah T; Athwal, Dee; Latchman, David S; Pearl, Laurence H; Isenberg, David A

    2005-01-01

    We have recently shown that the anti-cardiolipin activity of human anti-phospholipid antibody UK4 (lambda) resides on its heavy chain. We now show that UK4 possesses strong reactivity to the plasma-protein beta2-Glycoprotein I (beta2-GPI) also. Utilizing chain shuffling experiments involving an unrelated anti-p185 antibody 4D5 (kappa) with no reactivity to beta2-GPI, we now demonstrate that both the constructs possessing the auto-antibody-derived light chain exhibited significant binding to beta2-GPI. However, the construct possessing UK4 heavy chain in association with 4D5 light chain, exhibited no anti-beta2-GPI activity. Furthermore, there was a low increase (approximately 10%) in the binding of UK4 to cardiolipin in the presence of beta2-GPI. The results demonstrate that anti-beta2-GPI activity resides on UK4 light chain and, importantly, this activity could be transferred to a novel antibody construct via the light chain alone. Computer-generated models of the three-dimensional structures of UK4 and its hybrids, suggest predominant interaction of UK4 light chain with domain IV of beta2-GPI. Molecular docking experiments highlight a number of potential sites on beta2-GPI for interaction of UK4 and indicate as to how beta2-GPI recognition may occur primarily via the autoantibody light chain. The study provides first demonstration of the occurrence of anti-phospholipid and anti-beta2-GPI activities separately on heavy and light chains of an autoantibody. The possible mechanisms that such antibodies may employ to recognise their antigens, are discussed.

  14. Serum amyloid A and protein AA: molecular mechanisms of a transmissible amyloidosis.

    PubMed

    Westermark, Gunilla T; Westermark, Per

    2009-08-20

    Systemic AA-amyloidosis is a complication of chronic inflammatory diseases and the fibril protein AA derives from the acute phase reactant serum AA. AA-amyloidosis can be induced in mice by an inflammatory challenge. The lag phase before amyloid develops can be dramatically shortened by administration of a small amount of amyloid fibrils. Systemic AA-amyloidosis is transmissible in mice and may be so in humans. Since transmission can cross species barriers it is possible that AA-amyloidosis can be induced by amyloid in food, e.g. foie gras. In mice, development of AA-amyloidosis can also be accelerated by other components with amyloid-like properties. A new possible risk factor may appear with synthetically made fibrils from short peptides, constructed for tissue repair.

  15. 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

  16. 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

  17. Neuregulin1-β decreases interleukin-1β-induced RhoA activation, myosin light chain phosphorylation, and endothelial hyperpermeability.

    PubMed

    Wu, Limin; Ramirez, Servio H; Andrews, Allison M; Leung, Wendy; Itoh, Kanako; Wu, Jiang; Arai, Ken; Lo, Eng H; Lok, Josephine

    2016-01-01

    Neuregulin-1 (NRG1) is an endogenous growth factor with multiple functions in the embryonic and postnatal brain. The NRG1 gene is large and complex, transcribing more than twenty transmembrane proteins and generating a large number of isoforms in tissue and cell type-specific patterns. Within the brain, NRG1 functions have been studied most extensively in neurons and glia, as well as in the peripheral vasculature. Recently, NRG1 signaling has been found to be important in the function of brain microvascular endothelial cells, decreasing IL-1β-induced increases in endothelial permeability. In the current experiments, we have investigated the pathways through which the NRG1-β isoform acts on IL-1β-induced endothelial permeability. Our data show that NRG1-β increases barrier function, measured by transendothelial electrical resistance, and decreases IL-1β-induced hyperpermeability, measured by dextran-40 extravasation through a monolayer of brain microvascular endothelial cells plated on transwells. An investigation of key signaling proteins suggests that the effect of NRG1-β on endothelial permeability is mediated through RhoA activation and myosin light chain phosphorylation, events which affect filamentous actin morphology. In addition, AG825, an inhibitor of the erbB2-associated tyrosine kinase, reduces the effect of NRG1-β on IL-1β-induced RhoA activation and myosin light chain phosphorylation. These data add to the evidence that NRG1-β signaling affects changes in the brain microvasculature in the setting of neuroinflammation. We propose the following events for neuregulin-1-mediated effects on Interleukin-1 β (IL-1β)-induced endothelial hyperpermeability: IL-1β leads to RhoA activation, resulting in an increase in phosphorylation of myosin light chain (MLC). Phosphorylation of MLC is known to result in actin contraction and alterations in the f-actin cytoskeletal structure. These changes are associated with increased endothelial permeability

  18. 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.

  19. 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. PMID:25140811

  20. 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.

  1. 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

  2. Induction of light chain replacement in human plasma cells by caffeine is independent from both the upregulation of RAG protein expression and germ line transcription.

    PubMed

    Tachibana, H; Haruta, H; Ueda, K; Chiwata, T; Yamada, K

    2000-02-25

    When some human plasma cell lines are cultured with concanavalin A, the original light chain is replaced with another light chain which results from secondary VJ recombination (light chain shifting). We examined various intracellular factors involved in the induction of light chain shifting. Light chain shifting can be induced upon treatment with agents with phosphatase inhibitory activity such as caffeine and okadaic acid. Although the plasma cells used express both RAG-1 and RAG-2, the expression level of these proteins was not affected by caffeine or okadaic acid. Transcription of the germ line locus, which correlates to the locus activation for rearrangement, is also not influenced by phosphatase inhibition. However, the amount of signal broken-ended DNA intermediates generated during V(D)J rearrangement was shown to increase upon caffeine or okadaic acid treatment. The inhibitory activity of caffeine on phosphatase was the same as okadaic acid. However, caffeine exhibited much higher activity for VJ coding joint formation than okadaic acid. Therefore, although phosphatase inhibition might act, in part, on a mechanism by which V(D)J recombinase activity is regulated within the human plasma cells, other factor(s) are probably also involved in the process.

  3. Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody.

    PubMed

    Vlasak, Josef; Bussat, Marie C; Wang, Shiyi; Wagner-Rousset, Elsa; Schaefer, Mark; Klinguer-Hamour, Christine; Kirchmeier, Marc; Corvaïa, Nathalie; Ionescu, Roxana; Beck, Alain

    2009-09-15

    Despite technological advances, detection of deamidation in large proteins remains a challenge and the use of orthogonal methods is needed for unequivocal assignment. By a combination of cation-exchange separation, papain digestion, and a panel of mass spectrometry techniques we identified asparagine deamidation in light chain complementarity determining region 1 (CDR1) of a humanized IgG1 monoclonal antibody. The reaction yields both Asp and isoAsp, which were assigned by Edman degradation and by isoAsp detection using protein isoaspartate methyltransferase. The deamidated antibody variants were less potent in antigen binding compared to the nondegraded antibody. Changes in near-UV CD spectra, susceptibility to papain cleavage in an adjacent CDR2 loop, and the tendency of the newly formed isoAsp to undergo isomerization suggest local perturbations in the structure of the isoAsp-containing antibody.

  4. MRC OX-2 antigen: a lymphoid/neuronal membrane glycoprotein with a structure like a single immunoglobulin light chain.

    PubMed Central

    Clark, M J; Gagnon, J; Williams, A F; Barclay, A N

    1985-01-01

    The MRC OX-2 antigen is a rat cell surface glycoprotein of mol. wt. 41 000-47 000 found on neurones, thymocytes, B cells, follicular dendritic cells and endothelium. We now report the amino sequence for this antigen as deduced from the nucleotide sequence of cDNA clones detected by use of an oligonucleotide probe. The sequence contains 248 amino acid residues of which 202 residues are likely to be outside the cell with two domains that show homology with immunoglobulins. The N-terminal domain fits best with Ig V domains and Thy-1 antigen while the C-terminal part is like an Ig C domain. Thus the structure overall is similar to an Ig light chain or the T cell receptor beta chain. Three glycosylation sites are identified on each of the MRC OX-2 antigen domains. Images Fig. 1. Fig. 2. PMID:2862025

  5. Cardiac myosin light chain phosphorylation and inotropic effects of a biased ligand, TRV120023, in a dilated cardiomyopathy model

    PubMed Central

    Tarigopula, Madhusudhan; Davis, Robert T.; Mungai, Paul T.; Ryba, David M.; Wieczorek, David F.; Cowan, Conrad L.; Violin, Jonathan D.; Wolska, Beata M.; Solaro, R. John

    2015-01-01

    Aims Therapeutic approaches to treat familial dilated cardiomyopathy (DCM), which is characterized by depressed sarcomeric tension and susceptibility to Ca2+-related arrhythmias, have been generally unsuccessful. Our objective in the present work was to determine the effect of the angiotensin II type 1 receptor (AT1R) biased ligand, TRV120023, on contractility of hearts of a transgenic mouse model of familial DCM with mutation in tropomyosin at position 54 (TG-E54K). Our rationale is based on previous studies, which have supported the hypothesis that biased G-protein-coupled receptor ligands, signalling via β-arrestin, increase cardiac contractility with no effect on Ca2+ transients. Our previous work demonstrated that the biased ligand TRV120023 is able to block angiotensin-induced hypertrophy, while promoting an increase in sarcomere Ca2+ response. Methods and results We tested the hypothesis that the depression in cardiac function associated with DCM can be offset by infusion of the AT1R biased ligand, TRV120023. We intravenously infused saline, TRV120023, or the unbiased ligand, losartan, for 15 min in TG-E54K and non-transgenic mice to obtain left ventricular pressure–volume relations. Hearts were analysed for sarcomeric protein phosphorylation. Results showed that the AT1R biased ligand increases cardiac performance in TG-E54K mice in association with increased myosin light chain-2 phosphorylation. Conclusion Treatment of mice with an AT1R biased ligand, acting via β-arrestin signalling, is able to induce an increase in cardiac contractility associated with an increase in ventricular myosin light chain-2 phosphorylation. AT1R biased ligands may prove to be a novel inotropic approach in familial DCM. PMID:26045475

  6. Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis

    PubMed Central

    Wong, Raymond; Fabian, Lacramioara; Forer, Arthur; Brill, Julie A

    2007-01-01

    Background Phosphatidylinositol 4,5-bisphosphate (PIP2) is required for successful completion of cytokinesis. In addition, both PIP2 and phosphoinositide-specific phospholipase C (PLC) have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP2 to yield diacylglycerol (DAG) and inositol trisphosphate (IP3), which in turn induces calcium (Ca2+) release from the ER. Several studies suggest PIP2 must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM). In addition, the pathway by which PIP2 regulates cytokinesis remains to be elucidated. Results Here we compared the effects of U73122 and the structurally unrelated PLC inhibitor ET-18-OCH3 (edelfosine) on cytokinesis in crane-fly and Drosophila spermatocytes. Our data show that the effects of U73122 are indeed via PLC because U73122 and ET-18-OCH3 produced similar effects on cell morphology and actin cytoskeleton organization that were distinct from those caused by NEM. Furthermore, treatment with the myosin light chain kinase (MLCK) inhibitor ML-7 caused cleavage furrow regression and loss of both F-actin and phosphorylated myosin regulatory light chain from the contractile ring in a manner similar to treatment with U73122 and ET-18-OCH3. Conclusion We have used multiple inhibitors to examine the roles of PLC and MLCK, a predicted downstream target of PLC regulation, in cytokinesis. Our results are consistent with a model in which PIP2 hydrolysis acts via Ca2+ to activate myosin via MLCK and thereby control actin dynamics during constriction of the contractile ring. PMID:17509155

  7. Genomic clone for sandbar shark lambda light chain: generation of diversity in the absence of gene rearrangement.

    PubMed Central

    Hohman, V S; Schuchman, D B; Schluter, S F; Marchalonis, J J

    1993-01-01

    While the general structure of immunoglobulin chains has remained relatively unchanged throughout evolution, the organization of the genes encoding these molecules differs substantially. To understand how the rearranging immunoglobulin system arose, it is necessary to examine living representatives of the most early vertebrate phyla. Elasmo-branches, which include the sharks, skates, and rays, are the most primitive phylogenetic class of vertebrates from which immunoglobulin DNA sequences have been obtained. In the sandbar shark (Carcharhinus plumbeus), the genes are arranged in individual clusters in which a single variable (V), joining (J), and constant (C) region gene, along with upstream regulatory elements, span a distance of approximately 4.4 kb or approximately 5.8 kb. We report the complete sequence of a genomic clone encoding sandbar shark lambda light chain. A unique finding of our study is that the V and J genes are fused in the germ line. Three additional clones have been shown by DNA sequencing to also have fused V and J genes. The four clones have complementarity-determining regions 3 of various lengths and amino acid sequence variability similar to the products of rearranged genes. Furthermore, analysis by polymerase chain reaction technology revealed an additional 26 genomic clones demonstrating fusion of the V and J segments. Therefore, VJ fusion is the prominent organizational feature of sandbar shark immunoglobulin light chain genes. This finding raises questions concerning the necessity of recombination to produce an antibody repertoire capable of reacting against a diverse array of antigens. Images Fig. 1 Fig. 2 Fig. 3 PMID:8234330

  8. Immunoglobulin heavy/light chain analysis enhances the detection of residual disease and monitoring of multiple myeloma patients

    PubMed Central

    Batinić, Josip; Perić, Zinaida; Šegulja, Dragana; Last, James; Prijić, Sanja; Dubravčić, Klara; Volarić, Lidija; Sertić, Dubravka; Radman, Ivo; Bašić-Kinda, Sandra; Matišić, Danica; Batinić, Drago; Labar, Boris; Nemet, Damir

    2015-01-01

    Aim To evaluate the clinical utility of incorporating a novel heavy/light chain immunoassay (HLC) into the existing methods for the assessment of multiple myeloma (MM) patients. Methods Convenience sera samples from 90 previously treated IgG and IgA MM patients in different disease stages were analyzed. The study was conducted in Clinical Hospital Center Zagreb between 2011 and 2013. The collected sera were analyzed by standard laboratory techniques (serum protein electrophoresis, quantification of total immunoglobulins, serum immunofixation, serum free light chain [FLC] assay) and HLC assay. Results HLC ratios outside the normal range were found in 58 of 90 patients, including 28 out of 61 patients with total immunoglobulin measurements within the normal range and 5 out of 23 patients in complete response. Both elevated HLC isotype level and abnormal HLC ratio correlated with the parameters of tumor burden, including percentage of plasma cells in the bone marrow (P < 0.001 and P = 0.002, respectively) and an abnormal serum FLC ratio (for both P < 0.001). In addition, abnormal HLC isotype level correlated with serum beta-2-microglobulin level (P = 0.038). In terms of prognosis, abnormal HLC isotype level and abnormal HLC ratio were significantly associated with shorter overall survival (P < 0.001 and P = 0.002, respectively). Interestingly, suppression of the uninvolved (polyclonal) isotype pair, but not other non-myeloma immunoglobulin isotypes, was also associated with a shorter overall survival (P = 0.021). In a multivariate analysis, an abnormal HLC ratio and β2-microglobulin level >3.5mg/L were independent risk factors for survival. Conclusion The new HLC assay has greater sensitivity in detecting monoclonal protein, correlates with tumor burden markers, and affects patients' outcome. PMID:26088851

  9. Anti-nociceptive effect of a conjugate of substance P and light chain of botulinum neurotoxin type A.

    PubMed

    Mustafa, Golam; Anderson, Ethan M; Bokrand-Donatelli, Yvonne; Neubert, John K; Caudle, Robert M

    2013-11-01

    Neuropathic pain is a debilitating condition resulting from damage to sensory transmission pathways in the peripheral and central nervous system. A potential new way of treating chronic neuropathic pain is to target specific pain-processing neurons based on their expression of particular receptor molecules. We hypothesized that a toxin-neuropeptide conjugate would alter pain by first being taken up by specific receptors for the neuropeptide expressed on the neuronal cells. Then, once inside the cell the toxin would inhibit the neurons' activity without killing the neurons, thereby providing pain relief without lesioning the nervous system. In an effort to inactivate the nociceptive neurons in the trigeminal nucleus caudalis in mice, we targeted the NK1 receptor (NK1R) using substance P (SP). The catalytically active light chain of botulinum neurotoxin type A (LC/A) was conjugated with SP. Our results indicate that the conjugate BoNT/A-LC:SP is internalized in cultured NK1R-expressing neurons and also cleaves the target of botulinum toxin, a component-docking motif necessary for release of neurotransmitters called SNAP-25. The conjugate was next tested in a murine model of Taxol-induced neuropathic pain. An intracisternal injection of BoNT/A-LC:SP decreased thermal hyperalgesia as measured by the operant orofacial nociception assay. These findings indicate that conjugates of the light chain of botulinum toxin are extremely promising agents for use in suppressing neuronal activity for extended time periods, and that BoNT/A-LC:SP may be a useful agent for treating chronic pain.

  10. Molecular characterization and expression analysis of four isotypes of immunoglobulin light chain genes in orange-spotted grouper, Epinephelus coioides.

    PubMed

    Wu, Ming-Shan; Cheng, Chao-An; Lin, Chih-Hung; Lee, Chiou-Yueh; Tseng, Shih-Jou; Tzeng, Chyng-Shyan; Chang, Chi-Yao

    2013-03-01

    To date, many immunoglobulin (Ig) genes have been identified in diverse teleost species, but the contributions of different types of light chain (IgL) to the immune response remain unclear. Screening of a stimulated kidney cDNA library from orange-spotted grouper (Osg, Epinephelus coioides) resulted in the identification of 26 full Ig light chain (OsgIgL) coding sequences. These 26 OsgIgLs encoded peptides from 235 to 248 amino acid residues and could be grouped into five variable (V(L)) and four constant (C(L)) isotypes. The C(L) regions contained three conserved cysteine residues that may participate in intra- or inter-chain disulfide bond formation. The four C(L) isotypes could be sub-grouped into two serological types: κ (C(L)-I, C(L)-II and C(L)-III) and σ (C(L)-IV), by phylogenetic analysis. The OsgIgL genes were found to be expressed in various tissues, with greatest levels of expression observed in the head-kidney and spleen. The major expression type was C(L)-I, which comprised 92% and 91% of total OsgIgL gene expression in the head-kidney and spleen, respectively. Transcription of all four C(L) isotypes was differentially affected in response to various immunostimulators, including lipopolysaccharide (LPS), poly I:C and grouper iridovirus (GIV). Induction of OsgIgL genes in response to immunostimulators was particularly dramatic in the spleen, suggesting this organ holds particular importance for the regulation of OsgIgL expression. Furthermore, vaccination of grouper with formalin-inactivated GIV also induced differential patterns of expression in all four OsgIgL isotypes. In summary, the significant and diverse patterns of transcriptional induction observed for OsgIgL isotypes in the spleen and head-kidney imply that each isotype may have unique roles in the immune response.

  11. Evidence indicating independent assortment of framework and complementarity-determining segments of the variable regions of rabbit light chains. Delineation of a possible J minigene.

    PubMed

    Kabat, E A; Wu, T T; Bilofsky, H

    1980-07-01

    Amino acid sequences of rabbit light chains show considerable evidence of independent assortment of framework (FR) and complementarity-determining (CDR) segments. This suggests that they are coded for by independent genetic units (minigaenes) and that individual light chains are assembled somatically by recombining these units. Identical FR sets with multiple members generally comprise chains with different specificities, whereas identical CDR sets tend to have chains of a single specificity. A J segment, which, by analogy with mouse light chains, is made up of the last two residues of CDR3 plus all of FR4, contained 18 different sets and could contribute to diversity generated by CDR3. The longest segment, FR3, had a very large number of sets. Evidence is presented showing that the number of sets could be substantially reduced by permitting FR3 to be formed by two independently assorting segments comprising residues 57-68 and 69-88.

  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. Light Chain Deposition Disease in an Older Adult Patient Successfully Treated with Long-term Administration of Bortezomib, Melphalan and Prednisone.

    PubMed

    Hiyamuta, Hiroto; Yamada, Shunsuke; Matsukuma, Yuta; Tsuchimoto, Akihiro; Nakano, Toshiaki; Taniguchi, Masatomo; Masutani, Kosuke; Yoshimoto, Goichi; Muta, Tsuyoshi; Akashi, Koichi; Kitazono, Takanari; Tsuruya, Kazuhiko

    2016-01-01

    A 70-year-old woman was admitted to our hospital because of fatigue and renal dysfunction and was diagnosed with light chain deposition disease (LCDD) with multiple organ involvement (kidney, thyroid gland, heart and eyes). After chemotherapy with bortezomib, cyclophosphamide and dexamethasone, hepatobiliary enzyme levels increased abruptly. A liver biopsy showed light chain deposition in Disse spaces. After two years of treatment with bortezomib, melphalan and prednisone (VMP) administered at shorter intervals relative to regular cycles, the patient showed a hematological and organ response. This case indicates that a relatively low dose intensity VMP regimen is preferable for elderly patients with LCDD with multiple organ involvement. PMID:27181540

  14. Serum transthyretin levels in senile systemic amyloidosis: effects of age, gender and ethnicity.

    PubMed

    Buxbaum, Joel; Koziol, James; Connors, Lawreen H

    2008-12-01

    Serum transthyretin (TTR) levels are reduced in familial amyloidotic polyneuropathy (FAP). A single study of patients with senile systemic amyloidosis (SSA) in Sweden found that those individuals also had a significantly lower mean serum TTR concentration than age- and gender-matched controls. To determine if the same phenomenon prevailed in an ethnically more heterogeneous population, we compared the serum TTR levels, as determined by ELISA, in 45 documented SSA patients with congestive heart failure, 20 AL patients with congestive heart failure and population controls. Serum TTR concentrations in the controls were influenced in a statistically significant manner by age, gender and ethnicity. Although it is unlikely that such differences are clinically relevant, they must be considered when assessing the meaning of serum TTR concentrations in any clinically defined population. The serum concentrations in patients with SSA did not differ from age, gender and ethnically matched controls or from a group of AL patients with significant clinical cardiac involvement. We also compared TTR concentrations in 12 African-Americans carrying the TTR V122I allele with those in 826 African-Americans who were homozygous wild type at the TTR locus. The TTR V122I carriers had significantly lower serum TTR concentrations than appropriate controls even though the majority of such individuals had not reached the age of clinical or anatomic risk, i.e. over 60. Thus, as in carriers of other TTR mutations the serum TTR level is lower than normal, despite having a much later appearance of clinical disease. PMID:19065297

  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. Proteomics and mass spectrometry in the diagnosis of renal amyloidosis

    PubMed Central

    Picken, Maria M.

    2015-01-01

    The amyloidoses are a ‘group’ of disorders, all of which are associated with deposits that display similar staining and ultrastructural features and are toxic to tissues. Many proteins—currently 31 protein types and many more variants—have been shown to undergo such transformations. Among the various currently known amyloidoses, there are marked differences with regard to their pathogenesis and incidence, while the associated clinical picture is frequently overlapping. However, the therapies that are currently available are amyloid-type specific. The diagnosis of amyloidosis thus involves two steps: (i) a generic diagnosis, followed by (ii) an amyloid type-specific diagnosis or ‘amyloid typing’. Immunofluorescence in frozen sections or immunohistochemistry (IHC) in paraffin sections has traditionally been used in the typing of amyloid. However, IHC of amyloid differs significantly from IHC in other areas of surgical pathology; both caution and experience are necessary for its interpretation. The rationale for the application of proteomic methods to amyloid typing lies in the relative abundance of amyloid proteins in tissue where, frequently, it is the ‘dominant’ protein. Proteomic techniques include the following steps: sample preparation, protein extraction and digestion into peptide fragments, followed by their subsequent separation and measurement by mass spectrometry (MS) and protein identification by informatics. The advantages as well as the limitations of both methods—immunohistochemistry and MS-based proteomics—are discussed. The current recommendations for the application of proteomics in renal amyloidosis are summarized. PMID:26613021

  17. Online registry for mutations in hereditary amyloidosis including nomenclature recommendations.

    PubMed

    Rowczenio, Dorota M; Noor, Islam; Gillmore, Julian D; Lachmann, Helen J; Whelan, Carol; Hawkins, Philip N; Obici, Laura; Westermark, Per; Grateau, Gilles; Wechalekar, Ashutosh D

    2014-09-01

    Hereditary systemic amyloidosis comprises a group of rare monogenic diseases inherited in an autosomal dominant fashion. It is associated with mutations in genes encoding eight different proteins, including transthyretin, apolipoprotein AI, apolipoprotein AII, lysozyme, fibrinogen A α-chain, cystatin C, gelsolin and beta-2-microglobulin. With support from the EU FP6 EURAMY project we have designed an online registry of genes and mutations in hereditary amyloidosis including their associated clinical phenotypes, with a view to having a single free online portal for the collection and distribution of this information. Users can search the registry by either mutation, phenotype or authors who have published or submitted mutations. It provides a submission form for reporting newly identified mutations. We also wanted to introduce nomenclature which complies with recommendations set out by Human Genome Variation Society and HUGO Gene Nomenclature Committee for description of new and known genetic variants. We hope this registry would be a useful and convenient tool for the medical and scientific community. PMID:25044787

  18. Primary Systemic Amyloidosis with Unusual Dermatological Manifestations: A Rare Case Report

    PubMed Central

    Vyas, Kapil; Morgaonkar, Manjaree; Gupta, Savera; Jain, Suresh Kumar

    2016-01-01

    Amyloidosis is a group of heterogeneous diseases characterized by pathological deposition of proteinaceous substance extracellularly in various tissues. The clinical presentation depends on the site of amyloid deposition, with predominant involvement of mesenchymal elements and cutaneous findings in 30–40% of patients in case of primary systemic amyloidosis. We present a case of idiopathic primary systemic amyloidosis presenting with an unusual finding of nodulo-ulcerative lesion over tongue along with multiple skin-colored nodules, mimicking squamous cell carcinoma of tongue with secondary cutaneous metastasis, as well as lacking the classical presentation of purpura, macroglossia, waxy papules, and plaques. PMID:27057028

  19. Cardiac Amyloidosis: Typical Imaging Findings and Diffuse Myocardial Damage Demonstrated by Delayed Contrast-Enhanced MRI

    SciTech Connect

    Sueyoshi, Eijun Sakamoto, Ichiro; Okimoto, Tomoaki; Hayashi, Kuniaki; Tanaka, Kyouei; Toda, Genji

    2006-08-15

    Amyloidosis is a rare systemic disease. However, involvement of the heart is a common finding and is the most frequent cause of death in amyloidosis. We report the sonographic, scintigraphic, and MRI features of a pathologically proven case of cardiac amyloidosis. Delayed contrast-enhanced MR images, using an inversion recovery prepped gradient-echo sequence, revealed diffuse enhancement in the wall of both left and right ventricles. This enhancement suggested expansion of the extracellular space of the myocardium caused by diffuse myocardial necrosis secondary to deposition of amyloid.

  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. [The effect of phosphorylation of myosin light chains on the structural state of tropomyosin in thin filaments, decorated with heavy meromyosin].

    PubMed

    Vorovikov, Iu S; Szczesna, D; Kakol, I

    1989-06-01

    The structural state of tropomyosin (TM) modified by 5-(iodoacetamidoethyl)-aminonaphthalene-1-sulfonate (1.5-IAEDANS) upon F-actin decoration with myosin subfragment 1 (S1) and heavy meromyosin (HMM) in glycerinated myosin- and troponin-free muscle fibers was studied. HMM preparations contained native phosphorylated myosin light chains, while S1 preparations did not. The changes in the polarized fluorescence of 1.5-IAEDANS-TM during the F-actin interaction with S1 were independent of light chains phosphorylation and Ca2+ concentration, but were dependent on these factors during the F-actin interaction with HMM. The binding of myosin heads to F-actin is supposed to initiate conformational changes in TM which are accompanied by changes in the flexibility and molecular arrangement of TM. In the presence of light chains, the structural changes in TM depend on light chains phosphorylation and Ca2+ concentration. The conformational changes in TM seem to be responsible for the mechanisms of coupling of the myosin and tropomyosin modulation system during the actin-myosin interaction in skeletal muscles.

  3. Generation of immunoglobulin light chain gene diversity in Raja erinacea is not associated with somatic rearrangement, an exception to a central paradigm of B cell immunity

    PubMed Central

    1995-01-01

    In all vertebrate species examined to date, rearrangement and somatic modification of gene segmental elements that encode portions of the antigen-combining sites of immunoglobulins are integral components of the generation of antibody diversity. In the phylogenetically primitive cartilaginous fishes, gene segments encoding immunoglobulin heavy and light chain loci are arranged in multiple clusters, in which segmental elements are separated by only 300-400 bp. In some cases, segmental elements are joined in the germline of nonlymphoid cells (joined genes). Both genomic library screening and direct amplification of genomic DNA have been used to characterize at least 89 different type I light chain gene clusters in the skate, Raja. Analyses of predicted nucleotide sequences and predicted peptide structures are consistent with the distribution of genes into different sequence groups. Predicted amino acid sequence differences are preferentially distributed in complementarity-determining versus framework regions, and replacement-type substitutions exceed neutral substitutions. When specific germline sequences are related to the sequences of individual cDNAs, it is apparent that the joined genes are expressed and are potentially somatically mutated. No evidence was found for the presence of any type I light chain gene in Raja that is not germline joined. The type I light chain gene clusters in Raja appear to represent a novel gene system in which combinatorial and junctional diversity are absent. PMID:7790811

  4. Nomenclature of amyloid and amyloidosis. WHO-IUIS Nomenclature Sub-Committee.

    PubMed Central

    1993-01-01

    This classification of amyloid and amyloidosis is based on the amyloid fibril proteins, followed by a designation of the fibril protein precursor. Additional information includes the protein type or variant (where applicable) and the clinical diagnosis. PMID:8440029

  5. Regulation of cell wall synthesis by the clathrin light chain is essential for viability in Schizosaccharomyces pombe.

    PubMed

    de León, Nagore; Sharifmoghadam, Mohammad Reza; Hoya, Marta; Curto, M-Ángeles; Doncel, Cristina; Valdivieso, M-Henar

    2013-01-01

    The regulation of cell wall synthesis by the clathrin light chain has been addressed. Schizosaccharomyces pombe clc1Δ mutant was inviable in the absence of osmotic stabilization; when grown in sorbitol-supplemented medium clc1Δ cells grew slowly, formed aggregates, and had strong defects in morphology. Additionally, clc1Δ cells exhibited an altered cell wall composition. A mutant that allowed modulating the amount of Clc1p was created to analyze in more detail the dependence of cell wall synthesis on clathrin. A 40% reduction in the amount of Clc1p did not affect acid phosphatase secretion and bulk lipid internalization. Under these conditions, β(1,3)glucan synthase activity and cell wall synthesis were reduced. Also, the delivery of glucan synthases to the cell surface, and the secretion of the Eng1p glucanase were defective. These results suggest that the defects in the cell wall observed in the conditional mutant were due to a defective secretion of enzymes involved in the synthesis/remodelling of this structure, rather than to their endocytosis. Our results show that a reduction in the amount of clathrin that has minor effects on general vesicle trafficking has a strong impact on cell wall synthesis, and suggest that this is the reason for the lethality of clc1Δ cells in the absence of osmotic stabilization. PMID:23977061

  6. A Differentiation-dependent Splice Variant of Myosin Light Chain Kinase, MLCK1, Regulates Epithelial Tight Junction Permeability*

    PubMed Central

    Clayburgh, Daniel R.; Rosen, Shari; Witkowski, Edwina D.; Wang, Fengjun; Blair, Stephanie; Dudek, Steven; Garcia, Joe G. N.; Alverdy, John C.; Turner, Jerrold R.

    2005-01-01

    Activation of Na+-nutrient cotransport leads to increased tight junction permeability in intestinal absorptive (villus) enterocytes. This regulation requires myosin II regulatory light chain (MLC) phosphorylation mediated by MLC kinase (MLCK). We examined the spatiotemporal segregation of MLCK isoform function and expression along the crypt-villus axis and found that long MLCK, which is expressed as two alternatively spliced isoforms, accounts for 97 ± 4% of MLC kinase activity in interphase intestinal epithelial cells. Expression of the MLCK1 isoform is limited to well differentiated enterocytes, both in vitro and in vivo, and this expression correlates closely with development of Na+-nutrient cotransport-dependent tight junction regulation. Consistent with this role, MLCK1 is localized to the perijunctional actomyosin ring. Furthermore, specific knockdown of MLCK1 using siRNA reduced tight junction permeability in monolayers with active Na+-glucose cotransport, confirming a functional role for MLCK1. These results demonstrate unique physiologically relevant patterns of expression and subcellular localization for long MLCK isoforms and show that MLCK1 is the isoform responsible for tight junction regulation in absorptive enterocytes. PMID:15507455

  7. A differentiation-dependent splice variant of myosin light chain kinase, MLCK1, regulates epithelial tight junction permeability.

    PubMed

    Clayburgh, Daniel R; Rosen, Shari; Witkowski, Edwina D; Wang, Fengjun; Blair, Stephanie; Dudek, Steven; Garcia, Joe G N; Alverdy, John C; Turner, Jerrold R

    2004-12-31

    Activation of Na(+)-nutrient cotransport leads to increased tight junction permeability in intestinal absorptive (villus) enterocytes. This regulation requires myosin II regulatory light chain (MLC) phosphorylation mediated by MLC kinase (MLCK). We examined the spatiotemporal segregation of MLCK isoform function and expression along the crypt-villus axis and found that long MLCK, which is expressed as two alternatively spliced isoforms, accounts for 97 +/- 4% of MLC kinase activity in interphase intestinal epithelial cells. Expression of the MLCK1 isoform is limited to well differentiated enterocytes, both in vitro and in vivo, and this expression correlates closely with development of Na(+)-nutrient cotransport-dependent tight junction regulation. Consistent with this role, MLCK1 is localized to the perijunctional actomyosin ring. Furthermore, specific knockdown of MLCK1 using siRNA reduced tight junction permeability in monolayers with active Na(+)-glucose cotransport, confirming a functional role for MLCK1. These results demonstrate unique physiologically relevant patterns of expression and subcellular localization for long MLCK isoforms and show that MLCK1 is the isoform responsible for tight junction regulation in absorptive enterocytes.

  8. 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

  9. Four things to know about myosin light chains as reporters for non-muscle myosin-2 dynamics in live cells.

    PubMed

    Heissler, Sarah M; Sellers, James R

    2015-02-01

    The interplay between non-muscle myosins-2 and filamentous actin results in cytoplasmic contractility which is essential for eukaryotic life. Concomitantly, there is tremendous interest in elucidating the physiological function and temporal localization of non-muscle myosin-2 in cells. A commonly used method to study the function and localization of non-muscle myosin-2 is to overexpress a fluorescent protein (FP)-tagged version of the regulatory light chain (RLC) which binds to the myosin-2 heavy chain by mass action. Caveats about this approach include findings from recent studies indicating that the RLC does not bind exclusively to the non-muscle myosin-2 heavy chain. Rather, it can also associate with the myosin heavy chains of several other classes as well as other targets than myosin. In addition, the presence of the FP moiety may compromise myosin's enzymatic and mechanical performance. This and other factors to be discussed in this commentary raise questions about the possible complications in using FP-RLC as a marker for the dynamic localization and regulatory aspects of non-muscle myosin-2 motor functions in cell biological experiments.

  10. Resonance Assignments and Secondary Structure Analysis of Dynein Light Chain 8 by Magic-angle Spinning NMR Spectroscopy

    SciTech Connect

    Sun, Shangjin; Butterworth, Andrew H.; Paramasivam, Sivakumar; Yan, Si; Lightcap, Christine M.; Williams, John C.; Polenova, Tatyana E.

    2011-08-04

    Dynein light chain LC8 is the smallest subunit of the dynein motor complex and has been shown to play important roles in both dynein-dependent and dynein-independent physiological functions via its interaction with a number of its binding partners. It has also been linked to pathogenesis including roles in viral infections and tumorigenesis. Structural information for LC8-target proteins is critical to understanding the underlying function of LC8 in these complexes. However, some LC8-target interactions are not amenable to structural characterization by conventional structural biology techniques owing to their large size, low solubility, and crystallization difficulties. Here, we report magic-angle spinning (MAS) NMR studies of the homodimeric apo-LC8 protein as a first effort in addressing more complex, multi-partner, LC8-based protein assemblies. We have established site-specific backbone and side-chain resonance assignments for the majority of the residues of LC8, and show TALOS+-predicted torsion angles ø and ψ in close agreement with most residues in the published LC8 crystal structure. Data obtained through these studies will provide the first step toward using MAS NMR to examine the LC8 structure, which will eventually be used to investigate protein–protein interactions in larger systems that cannot be determined by conventional structural studies.

  11. The effect of anti-inflammatory properties of ferritin light chain on lipopolysaccharide-induced inflammatory response in murine macrophages.

    PubMed

    Fan, Yumei; Zhang, Jie; Cai, Linlin; Wang, Shengnan; Liu, Caizhi; Zhang, Yongze; You, Linhao; Fu, Yujian; Shi, Zhenhua; Yin, Zhimin; Luo, Lan; Chang, Yanzhong; Duan, Xianglin

    2014-11-01

    Ferritin light chain (FTL) reduces the free iron concentration by forming ferritin complexes with ferritin heavy chain (FTH). Thus, FTL competes with the Fenton reaction by acting as an antioxidant. In the present study, we determined that FTL influences the lipopolysaccharide (LPS)-induced inflammatory response. FTL protein expression was regulated by LPS stimulation in RAW264.7 cells. To investigate the role of FTL in LPS-activated murine macrophages, we established stable FTL-expressing cells and used shRNA to silence FTL expression in RAW264.7 cells. Overexpression of FTL significantly decreased the LPS-induced production of tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), nitric oxide (NO) and prostaglandin E2 (PGE2). Additionally, overexpression of FTL decreased the LPS-induced increase of the intracellular labile iron pool (LIP) and reactive oxygen species (ROS). Moreover, FTL overexpression suppressed the LPS-induced activation of MAPKs and nuclear factor-κB (NF-κB). In contrast, knockdown of FTL by shRNA showed the reverse effects. Therefore, our results indicate that FTL plays an anti-inflammatory role in response to LPS in murine macrophages and may have therapeutic potential for treating inflammatory diseases.

  12. 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

  13. Pseudo-Peritoneal Carcinomatosis Presentation of a Crystal-Storing Histiocytosis With an Unmutated Monoclonal κ Light Chain.

    PubMed

    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

    2015-08-01

    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 Vk3-2001/Jk201 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.

  14. Top-Down Targeted Proteomics Reveals Decrease in Myosin Regulatory Light-Chain Phosphorylation That Contributes to Sarcopenic Muscle Dysfunction.

    PubMed

    Gregorich, Zachery R; Peng, Ying; Cai, Wenxuan; Jin, Yutong; Wei, Liming; Chen, Albert J; McKiernan, Susan H; Aiken, Judd M; Moss, Richard L; Diffee, Gary M; Ge, Ying

    2016-08-01

    Sarcopenia, the loss of skeletal muscle mass and function with advancing age, is a significant cause of disability and loss of independence in the elderly and thus, represents a formidable challenge for the aging population. Nevertheless, the molecular mechanism(s) underlying sarcopenia-associated muscle dysfunction remain poorly understood. In this study, we employed an integrated approach combining top-down targeted proteomics with mechanical measurements to dissect the molecular mechanism(s) in age-related muscle dysfunction. Top-down targeted proteomic analysis uncovered a progressive age-related decline in the phosphorylation of myosin regulatory light chain (RLC), a critical protein involved in the modulation of muscle contractility, in the skeletal muscle of aging rats. Top-down tandem mass spectrometry analysis identified a previously unreported bis-phosphorylated proteoform of fast skeletal RLC and localized the sites of decreasing phosphorylation to Ser14/15. Of these sites, Ser14 phosphorylation represents a previously unidentified site of phosphorylation in RLC from fast-twitch skeletal muscle. Subsequent mechanical analysis of single fast-twitch fibers isolated from the muscles of rats of different ages revealed that the observed decline in RLC phosphorylation can account for age-related decreases in the contractile properties of sarcopenic fast-twitch muscles. These results strongly support a role for decreasing RLC phosphorylation in sarcopenia-associated muscle dysfunction and suggest that therapeutic modulation of RLC phosphorylation may represent a new avenue for the treatment of sarcopenia. PMID:27362462

  15. Utility of Serum Free Light Chain Measurements in Multiple Myeloma Patients Not Achieving Complete Response to Therapy

    PubMed Central

    Moustafa, Muhamad Alhaj; Rajkumar, S. Vincent; Dispenzieri, Angela; Gertz, Morie A.; Lacy, Martha Q.; Buadi, Francis K.; Hwa, Yi Lisa; Dingli, David; Kapoor, Prashant; Hayman, Suzanne R.; Lust, John A.; Kyle, Robert A.; Kumar, Shaji K.

    2015-01-01

    Normalization of the serum free light chain ratio (FLCr) with the absence of bone marrow monoclonal plasma cells following achievement of a complete response (CR) to therapy denotes a stringent CR in multiple myeloma (MM), and is associated with improved overall survival (OS). However, its value in patients achieving

  16. Tarantula myosin free head regulatory light chain phosphorylation stiffens N-terminal extension, releasing it and blocking its docking back.

    PubMed

    Alamo, Lorenzo; Li, Xiaochuan Edward; Espinoza-Fonseca, L Michel; Pinto, Antonio; Thomas, David D; Lehman, William; Padrón, Raúl

    2015-08-01

    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 length 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. 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

  17. 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

  18. Axonemal dynein light chain-1 locates at the microtubule-binding domain of the γ heavy chain

    PubMed Central

    Ichikawa, Muneyoshi; Saito, Kei; Yanagisawa, Haru-aki; Yagi, Toshiki; Kamiya, Ritsu; Yamaguchi, Shin; Yajima, Junichiro; Kushida, Yasuharu; Nakano, Kentaro; Numata, Osamu; Toyoshima, Yoko Y.

    2015-01-01

    The outer arm dynein (OAD) complex is the main propulsive force generator for ciliary/flagellar beating. In Chlamydomonas and Tetrahymena, the OAD complex comprises three heavy chains (α, β, and γ HCs) and >10 smaller subunits. Dynein light chain-1 (LC1) is an essential component of OAD. It is known to associate with the Chlamydomonas γ head domain, but its precise localization within the γ head and regulatory mechanism of the OAD complex remain unclear. Here Ni-NTA-nanogold labeling electron microscopy localized LC1 to the stalk tip of the γ head. Single-particle analysis detected an additional structure, most likely corresponding to LC1, near the microtubule-binding domain (MTBD), located at the stalk tip. Pull-down assays confirmed that LC1 bound specifically to the γ MTBD region. Together with observations that LC1 decreased the affinity of the γ MTBD for microtubules, we present a new model in which LC1 regulates OAD activity by modulating γ MTBD's affinity for the doublet microtubule. PMID:26399296

  19. 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

  20. 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

  1. Diphosphorylated but not monophosphorylated myosin II regulatory light chain localizes to the midzone without its heavy chain during cytokinesis.

    PubMed

    Kondo, Tomo; Isoda, Rieko; Uchimura, Takashi; Sugiyama, Mutsumi; Hamao, Kozue; Hosoya, Hiroshi

    2012-01-13

    Myosin II is activated by the monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). Its ATPase activity is further enhanced by MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC). As these phosphorylated MRLCs are colocalized with their heavy chains at the contractile ring in dividing cells, we believe that the phosphorylated MRLC acts as a subunit of the activated myosin II during cytokinesis. However, the distinct role(s) of 1P- and 2P-MRLC during cytokinesis has not been elucidated. In this study, a monoclonal antibody (4F12) specific for 2P-MRLC was raised and used to examine the roles of 2P-MRLC in cultured mammalian cells. Our confocal microscopic observations using 4F12 revealed that 2P-MRLC localized to the contractile ring, and, unexpectedly, to the midzone also. Interestingly, 2P-MRLC did not colocalize with 1P-MRLC, myosin II heavy chain, and F-actin at the midzone. These results suggest that 2P-MRLC has a role different from that of 1P-MRLC at the midzone, and is not a subunit of myosin II. PMID:22166199

  2. 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

  3. 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

  4. 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.

  5. Chlamydia trachomatis inclusion membrane protein CT850 interacts with the dynein light chain DYNLT1 (Tctex1).

    PubMed

    Mital, Jeffrey; Lutter, Erika I; Barger, Alexandra C; Dooley, Cheryl A; Hackstadt, Ted

    2015-06-26

    Chlamydia trachomatis actively subverts the minus-end directed microtubule motor, dynein, to traffic along microtubule tracks to the Microtubule Organizing Center (MTOC) where it remains within a membrane bound replicative vacuole for the duration of its intracellular development. Unlike most substrates of the dynein motor, disruption of the dynactin cargo-linking complex by over-expression of the p50 dynamitin subunit does not inhibit C. trachomatis transport. A requirement for chlamydial protein synthesis to initiate this process suggests that a chlamydial product supersedes a requirement for p50 dynamitin. A yeast 2-hybrid system was used to screen the chlamydia inclusion membrane protein CT850 against a HeLa cell cDNA library and identified an interaction with the dynein light chain DYNLT1 (Tctex1). This interaction was at least partially dependent upon an (R/K-R/K-X-X-R/K) motif that is characteristic of DYNLT1 binding domains. CT850 expressed ectopically in HeLa cells localized at the MTOC and this localization is similarly dependent upon the predicted DYNLT1 binding domain. Furthermore, DYNLT1 is enriched at focal concentrations of CT850 on the chlamydial inclusion membrane that are known to interact with dynein and microtubules. Depletion of DYNLT1 disrupts the characteristic association of the inclusion membrane with centrosomes. Collectively, the results suggest that CT850 interacts with DYNLT1 to promote appropriate positioning of the inclusion at the MTOC.

  6. Slow motility in hair cells of the frog amphibian papilla: Myosin light chain-mediated shape change

    PubMed Central

    Farahbakhsh, Nasser A.; Narins, Peter M.

    2008-01-01

    Using video, fluorescence and confocal microscopy, quantitative analysis and modeling, we investigated intracellular processes mediating the calcium/calmodulin (Ca2+/CaM)-dependent slow motility in hair cells dissociated from the rostral region of amphibian papilla, one of the two auditory organs in frogs. The time course of shape changes in these hair cells during the period of pretreatment with several specific inhibitors, as well as their response to the calcium ionophore, ionomycin, were recorded and compared. These cells respond to ionomycin with a tri-phasic shape change: an initial phase of iso-volumetric length decrease; a period of concurrent shortening and swelling; and the final phase of increase in both length and volume. We found that both the myosin light chain kinase inhibitor, ML-7, and antagonists of the multifunctional Ca2+/CaM-dependent kinases, KN-62 and KN-93, inhibit the iso-volumetric shortening phase of the response to ionomycin. The type 1 protein phosphatase inhibitors, calyculin A and okadaic acid induce minor shortening on their own, but do not significantly alter the phase 1 response. However, they appear to counter effects of the inhibitors of Ca2+/CaM-dependent kinases. We hypothesize that an active actomyosin-based process mediates the iso-volumetric shortening in the frog rostral amphibian papillar hair cells. PMID:18534795

  7. Slow motility in hair cells of the frog amphibian papilla: myosin light chain-mediated shape change.

    PubMed

    Farahbakhsh, Nasser A; Narins, Peter M

    2008-07-01

    Using video, fluorescence and confocal microscopy, quantitative analysis and modeling, we investigated intracellular processes mediating the calcium/calmodulin (Ca(2+)/CaM)-dependent slow motility in hair cells dissociated from the rostral region of amphibian papilla, one of the two auditory organs in frogs. The time course of shape changes in these hair cells during the period of pretreatment with several specific inhibitors, as well as their response to the calcium ionophore, ionomycin, were recorded and compared. These cells respond to ionomycin with a tri-phasic shape change: an initial phase of iso-volumetric length decrease; a period of concurrent shortening and swelling; and the final phase of increase in both length and volume. We found that both the myosin light chain kinase inhibitor, ML-7, and antagonists of the multifunctional Ca(2+)/CaM-dependent kinases, KN-62 and KN-93, inhibit the iso-volumetric shortening phase of the response to ionomycin. The type 1 protein phosphatase inhibitors, calyculin A and okadaic acid induce minor shortening on their own, but do not significantly alter phase 1 response. However, they appear to counter effects of the inhibitors of Ca(2+)/CaM-dependent kinases. We hypothesize that an active actomyosin-based process mediates the iso-volumetric shortening in the frog rostral amphibian papillar hair cells.

  8. Resonance Assignments and Secondary Structure Analysis of Dynein Light Chain 8 by Magic Angle Spinning NMR Spectroscopy

    PubMed Central

    Sun, Shangjin; Butterworth, Andrew H.; Paramasivam, Sivakumar; Yan, Si; Lightcap, Christine M.; Williams, John C.; Polenova, Tatyana

    2012-01-01

    Dynein light chain LC8 is the smallest subunit of the dynein motor complex and has been shown to play important roles in both dynein dependent and dynein independent physiological functions via its interaction with a number of its binding partners. It has also been linked to pathogenesis including roles in viral infections and tumorigenesis. Structural information for LC8-target proteins is critical to understanding the underlying function of LC8 in these complexes. However, some LC8-target interactions are not amenable for structural characterization by conventional structural biology techniques due to their large size, low solubility and crystallization difficulties. Here, we report magic angle spinning (MAS) NMR studies of the homodimeric apo-LC8 protein as a first effort in addressing more complex, multi-partner LC8-based protein assemblies. We have established site-specific backbone and side chain resonance assignments for the majority of the residues of LC8, and show TALOS+ predicted torsion angles ϕ and ψ in close agreement with most residues in the published LC8 crystal structure. Data obtained through these studies will provide the first step toward using MAS NMR to examine the LC8 structure, which will eventually be used to investigate protein-protein interactions in larger systems, which cannot be determined by conventional structural studies. PMID:23243318

  9. Direct reduction of antigen receptor expression in polyclonal B cell populations developing in vivo results in light chain receptor editing.

    PubMed

    Shen, Shixue; Manser, Tim

    2012-01-01

    Secondary Ab V region gene segment rearrangement, termed receptor editing, is a major mechanism contributing to B lymphocyte self-tolerance. However, the parameters that determine whether a B cell undergoes editing are a current subject of debate. We tested the role that the level of BCR expression plays in the regulation of receptor editing in a polyclonal population of B cells differentiating in vivo. Expression of a short hairpin RNA for κ L chain RNA in B cells resulted in reduction in levels of this RNA and surface BCRs. Strikingly, fully mature and functional B cells that developed in vivo and efficiently expressed the short hairpin RNA predominantly expressed BCRs containing λ light chains. This shift in L chain repertoire was accompanied by inhibition of development, increased Rag gene expression, and increased λ V gene segment-cleavage events at the immature B cell stage. These data demonstrated that reducing the translation of BCRs that are members of the natural repertoire at the immature B cell stage is sufficient to promote editing.

  10. Dynein Light Chain 1 Regulates Dynamin-mediated F-Actin Assembly during Sperm Individualization in DrosophilaD⃞

    PubMed Central

    Ghosh-Roy, Anindya; Desai, Bela S.; Ray, Krishanu

    2005-01-01

    Toward the end of spermiogenesis, spermatid nuclei are compacted and the clonally related spermatids individualize to become mature and active sperm. Studies in Drosophila showed that caudal end-directed movement of a microfilament-rich structure, called investment cone, expels the cytoplasmic contents of individual spermatids. F-actin dynamics plays an important role in this process. Here we report that the dynein light chain 1 (DLC1) of Drosophila is involved in two separate cellular processes during sperm individualization. It is enriched around spermatid nuclei during postelongation stages and plays an important role in the dynein-dynactin–dependent rostral retention of the nuclei during this period. In addition, DDLC1 colocalizes with dynamin along investment cones and regulates F-actin assembly at this organelle by retaining dynamin along the cones. Interestingly, we found that this process does not require the other subunits of cytoplasmic dynein-dynactin complex. Altogether, these observations suggest that DLC1 could independently regulate multiple cellular functions and established a novel role of this protein in F-actin assembly in Drosophila. PMID:15829565

  11. 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.

  12. High prevalence of immunoglobulin light chain gene aberrations as revealed by FISH in multiple myeloma and MGUS.

    PubMed

    Türkmen, Seval; Binder, Anastasia; Gerlach, Antje; Niehage, Sylke; Theodora Melissari, Maria; Inandiklioglu, Nihal; Dörken, Bernd; Burmeister, Thomas

    2014-08-01

    Multiple myeloma (MM) is a malignant B-cell neoplasm characterized by an uncontrolled proliferation of aberrant plasma cells in the bone marrow. Chromosome aberrations in MM are complex and represent a hallmark of the disease, involving many chromosomes that are altered both numerically and structurally. Nearly half of the cases are nonhyperdiploid and show IGH translocations with the following partner genes: CCND1, FGFR3 and MMSET, MAF, MAFB, and CCND3. The remaining 50% are grouped into a hyperdiploid group that is characterized by multiple trisomies involving chromosomes 3, 5, 7, 9, 11, 15, 19, and 21. In this study, we analyzed the immunoglobulin light chain kappa (IGK, 2p12) and lambda (IGL, 22q11) loci in 150 cases, mostly with MM but in a few cases monoclonal gammopathy of undetermined significance (MGUS), without IGH translocations. We identified aberrations in 27% (= 40 patients) including rearrangements (12%), gains (12%), and deletions (4.6%). In 6 of 18 patients with IGK or/and IGL rearrangements, we detected a MYC rearrangement which suggests that MYC is the translocation partner in the majority of these cases. PMID:24729354

  13. A new method to specifically label thiophosphorylatable proteins with extrinsic probes. Labeling of serine-19 of the regulatory light chain of smooth muscle myosin.

    PubMed

    Facemyer, K C; Cremo, C R

    1992-01-01

    We present a new method to specifically and stably label proteins by attaching extrinsic probes to amino acids that are thiophosphorylated by protein kinases and ATP gamma S. The method was demonstrated for labeling of a thiophosphorylatable serine of the isolated regulatory light chain of smooth muscle myosin. We stoichiometrically blocked the single thiol (Cys-108) either by forming a reversible intermolecular disulfide bond or by reacting with iodoacetic acid. The protein was stoichiometrically thiophosphorylated at Ser-19 by myosin light chain kinase and ATP gamma S. The nucleophilic sulfur of the protein phosphorothioate was coupled at pH 7.9 and 25 degrees C to the fluorescent haloacetate [3H]-5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1- sulfonic acid ([3H]IAEDANS) by displacement of the iodide. Typical labeling efficiencies were 70-100%. The labeling was specific for the thiophosphorylated Ser-19, as determined from the sequences of two labeled peptides isolated from a tryptic digest of the labeled protein. [3H]IAEDANS attached to the thiophosphorylated Ser-19 was stable at pH 3-10 at 25 degrees C, and to boiling in high concentrations of reductant. The labeled light chains were efficiently exchanged for unlabeled regulatory light chains of the whole myosin molecule. The resulting labeled myosin had normal ATPase activities in the absence of actin, indicating that the modification of Ser-19 and the exchange of the labeled light chain into myosin did not significantly disrupt the protein. The labeled myosin partially retained the elevated actin-activated Mg(2+)-ATPase activity which is characteristic of thiophosphorylated myosin. This indicates that labeling of the thiophosphate group with [3H]IAEDANS did not completely disrupt the functional properties of the thiophosphorylated protein in the presence of actin.

  14. Tissue distribution of amyloid deposits in Abyssinian cats with familial amyloidosis.

    PubMed

    DiBartola, S P; Tarr, M J; Benson, M D

    1986-07-01

    The tissue distribution of amyloid deposits was studied in 15 related Abyssinian cats with familial amyloidosis. There was interstitial medullary amyloidosis in the kidneys of all 15 cats but only 11 had detectable glomerular involvement. The thyroid glands, stomach and colon were affected in all cats examined. Most of the cats also had amyloid deposits in the small intestine, spleen, heart, adrenals, pancreas, liver, lymph nodes and bladder. In 50 per cent or fewer of the cats examined, there was involvement of the parathyroids, lung and gonads. The central nervous system was not involved in any of the 3 cats evaluated. In 8 of the cats, no concurrent inflammatory disease could be detected. The tissue distribution of amyloid deposits resembled that found in other breeds of domestic cats with systemic amyloidosis. Despite the wide tissue distribution of amyloid deposits, clinical signs were related to renal amyloidosis. Familial amyloidosis in the Abyssinian cat may represent a valuable spontaneous animal model for the study of Familial Mediterranean Fever in man and the pathogenesis of reactive amyloidosis in general. PMID:3734172

  15. Very long term studies of the seeding of beta-amyloidosis in primates.

    PubMed

    Ridley, R M; Baker, H F; Windle, C P; Cummings, R M

    2006-09-01

    Cerebral beta-amyloidosis was found in 16/18 marmosets aged <10 yrs and 8/9 marmosets aged >10 yrs, injected intracerebrally with human or marmoset brain homogenate containing beta-amyloid 1-8 years previously. It was found in only 2/12 marmosets aged <10 yrs and 1/15 marmosets aged >10 yrs, injected with synthetic Abeta-peptides, CSF, or brain tissue which did not contain beta-amyloid. Cerebral beta-amyloidosis was found in 0/11 uninjected marmosets aged <10 yrs and in 5/29 uninjected marmosets aged >10 yrs. The beta-amyloidosis comprised small and large vessel angiopathy and some plaques throughout cortex and was qualitatively similar in injected marmosets and, when present, in uninjected marmosets. Of those injected marmosets which were positive, the amount of beta-amyloidosis was unrelated to age or incubation times but the 3 injected marmosets without beta-amyloidosis had incubation times of <3.5 years. We conclude that beta-amyloid, or associated factors, can initiate or accelerate the process of cerebral amyloidosis in primates.

  16. 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

  17. Mechanisms of Islet Amyloidosis Toxicity in Type 2 Diabetes

    PubMed Central

    Abedini, Andisheh; Schmidt, Ann Marie

    2014-01-01

    Amyloid formation by the neuropancreatic hormone, islet amyloid polypeptide (IAPP or amylin), one of the most amyloidogenic sequences known, leads to islet amyloidosis in type 2 diabetes and to islet transplant failure. Under normal conditions, IAPP plays a role in the maintenance of energy homeostasis by regulating several metabolic parameters, such as satiety, blood glucose levels, adiposity and body weight. The mechanisms of IAPP amyloid formation, the nature of IAPP toxic species and the cellular pathways that lead to pancreatic β-cell toxicity are not well characterized. Several mechanisms of toxicity, including receptor and non-receptor-mediated events, have been proposed. Analogs of IAPP have been approved for the treatment of diabetes and are under investigation for the treatment of obesity. PMID:23337872

  18. Spontaneous resolution of multiple nodular pulmonary AA amyloidosis.

    PubMed

    Fukatsu, Hirotoshi; Miyoshi, Haruka; Ishiki, Kuniharu

    2010-01-01

    A 62-year-old man presented with a two-week history of dry cough. A chest computed tomography (CT) showed three nodular masses of soft tissue density without calcification or cavitary formation in the right lung. F-18 fluorodeoxyglucose PET/CT scan revealed high FDG uptake in two out of three pulmonary nodules. Transbronchial lung biopsy specimens consisted of amorphous eosinophilic deposits that were demonstrated to be amyloid because they were positive for Congo Red staining. After oxidation with permanganate solution, the Congo Red staining disappeared, indicating that this amyloid was amyloid A protein-derived type. There was no evidence of any systemic diseases. We diagnosed the patient as having multiple nodular pulmonary AA amyloidosis. The patient was conservatively managed without treatment, and the pulmonary nodules disappeared spontaneously three months later. PMID:21048364

  19. Primary localized cutaneous amyloidosis with lichen and poikiloderma-like lesions and an excellent response to systemic acitretin*

    PubMed Central

    MA, Han; Su, Xiangyang; Zhu, Guoxing; Yin, Songchao; Lu, Chun; Lai, Wei

    2016-01-01

    Primary localized cutaneous amyloidosis is a skin-limited amyloidosis that does not involve internal organs. It is clinically subclassified into 3 general categories and some rare variants. However, there is considerable overlap within the classification. Though there are a variety of therapeutic measures, the treatment is often unsatisfactory, particularly when the disease is severe and extensive. We describe a rare case of primary localized cutaneous amyloidosis with lichen and poikiloderma-like lesions that showed an excellent response to systemic acitretin.

  20. Serum Free Light Chains

    MedlinePlus

    ... changes in the ratio of kappa and lambda production, which indicate an excess of one clone of ... test to detect abnormal monoclonal protein (M-protein) production and to calculate a kappa/lambda free light ...

  1. Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier.

    PubMed

    Ashokkumar, Balasubramaniem; Nabokina, Svetlana M; Ma, Thomas Y; Said, Hamid M

    2009-09-01

    The reduced folate carrier (RFC) is a major folate transport system in mammalian cells. RFC is highly expressed in the intestine and believed to play a role in folate absorption. Studies from our laboratory and others have characterized different aspects of the intestinal folate absorption process, but little is known about possible existence of accessory protein(s) that interacts with RFC and influences its physiology and/or cell biology. We investigated this issue by employing a bacterial two-hybrid system to screen a BacterioMatch II human intestinal cDNA library using the large intracellular loop between transmembrane domains 6 and 7 of the human RFC (hRFC) as bait. Our screening has resulted in the identification of dynein light chain road block-1 (DYNLRB1) as an interacting partner with hRFC. Existence of a direct protein-protein interaction between hRFC and DYNLRB1 was confirmed by in vitro pull-down assay and in vivo mammalian two-hybrid luciferase assay and coimmunoprecipitation analysis. Furthermore, confocal imaging of live human intestinal epithelial HuTu-80 cells demonstrated colocalization of DYNLRB1 with hRFC. Coexpression of DYNLRB1 with hRFC led to a significant (P < 0.05) increase in folate uptake. On the other hand, inhibiting the endogenous DYNLRB1 with gene-specific small interfering RNA or pharmacologically with a specific inhibitor (vanadate) led to a significant (P < 0.05) decrease in folate uptake. This study demonstrates for the first time the identification of DYNLRB1 as an interacting protein partner with hRFC. Furthermore, DYNLRB1 appears to influence the function and cell biology of hRFC.

  2. Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle.

    PubMed

    Hong, Feng; Brizendine, Richard K; Carter, Michael S; Alcala, Diego B; Brown, Avery E; Chattin, Amy M; Haldeman, Brian D; Walsh, Michael P; Facemyer, Kevin C; Baker, Josh E; Cremo, Christine R

    2015-10-01

    Smooth muscle myosin (SMM) light chain kinase (MLCK) phosphorylates SMM, thereby activating the ATPase activity required for muscle contraction. The abundance of active MLCK, which is tightly associated with the contractile apparatus, is low relative to that of SMM. SMM phosphorylation is rapid despite the low ratio of MLCK to SMM, raising the question of how one MLCK rapidly phosphorylates many SMM molecules. We used total internal reflection fluorescence microscopy to monitor single molecules of streptavidin-coated quantum dot-labeled MLCK interacting with purified actin, actin bundles, and stress fibers of smooth muscle cells. Surprisingly, MLCK and the N-terminal 75 residues of MLCK (N75) moved on actin bundles and stress fibers of smooth muscle cell cytoskeletons by a random one-dimensional (1-D) diffusion mechanism. Although diffusion of proteins along microtubules and oligonucleotides has been observed previously, this is the first characterization to our knowledge of a protein diffusing in a sustained manner along actin. By measuring the frequency of motion, we found that MLCK motion is permitted only if acto-myosin and MLCK-myosin interactions are weak. From these data, diffusion coefficients, and other kinetic and geometric considerations relating to the contractile apparatus, we suggest that 1-D diffusion of MLCK along actin (a) ensures that diffusion is not rate limiting for phosphorylation, (b) allows MLCK to locate to areas in which myosin is not yet phosphorylated, and (c) allows MLCK to avoid getting "stuck" on myosins that have already been phosphorylated. Diffusion of MLCK along actin filaments may be an important mechanism for enhancing the rate of SMM phosphorylation in smooth muscle.

  3. The essential light chain N-terminal extension alters force and fiber kinetics in mouse cardiac muscle.

    PubMed

    Miller, Mark S; Palmer, Bradley M; Ruch, Stuart; Martin, Lisa A; Farman, Gerrie P; Wang, Yuan; Robbins, Jeffrey; Irving, Thomas C; Maughan, David W

    2005-10-14

    The functional significance of the actin-binding region at the N terminus of the cardiac myosin essential light chain (ELC) remains elusive. In a previous experiment, the endogenous ventricular ELC was replaced with a protein containing a 10-amino acid deletion at positions 5-14 (ELC1vDelta5-14, referred to as 1vDelta5-14), a region that interacts with actin. 1vDelta5-14 mice showed no discernable mutant phenotype in skinned ventricular strips. However, because the myofilament lattice swells upon skinning, the mutant phenotype may have been concealed by the inability of the ELC to reach the actin-binding site. Using the same mouse model, we repeated earlier measurements and performed additional experiments on skinned strips osmotically compressed to the intact lattice spacing as determined by x-ray diffraction. 1vDelta5-14 mice exhibited decreased maximum isometric tension without a change in calcium sensitivity. The decreased force was most evident in 5-6-month-old mice compared with 13-15-month-old mice and may account for the greater ventricular wall thickness in young 1vDelta5-14 mice compared with age-matched controls. No differences were observed in unloaded shortening velocity at maximum calcium activation. However, 1vDelta5-14 mice exhibited a significant difference in the frequency at which minimum complex modulus amplitude occurred, indicating a change in cross-bridge kinetics. We hypothesize that the ELC N-terminal extension interaction with actin inhibits the reversal of the power stroke, thereby increasing isometric force. Our results strongly suggest that an interaction between residues 5-14 of the ELC N terminus and the C-terminal residues of actin enhances cardiac performance.

  4. A shrimp pacifastin light chain-like inhibitor: molecular identification and role in the control of the prophenoloxidase system.

    PubMed

    Sangsuriya, Pakkakul; Charoensapsri, Walaiporn; Chomwong, Sudarat; Senapin, Saengchan; Tassanakajon, Anchalee; Amparyup, Piti

    2016-01-01

    Pacifastin is a recently classified family of serine proteinase inhibitors that play essential roles in various biological processes, including in the regulation of the melanization cascade. Here, a novel pacifastin-related gene, termed PmPacifastin-like, was identified from a reverse suppression subtractive hybridization (SSH) cDNA library created from hemocytes of the prophenoloxidase PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon. The full-length sequences of PmPacifastin-like and its homologue LvPacifastin-like from the Pacific white shrimp Litopenaeus vannamei were determined. Sequence analysis revealed that both sequences contained thirteen conserved pacifastin light chain domains (PLDs), followed by two putative kunitz domains. Expression analysis demonstrated that the PmPacifastin-like transcript was expressed in all tested shrimp tissues and larval developmental stages, and its expression responded to Vibrio harveyi challenge. To gain insight into the functional roles of PmPacifastin-like protein, the in vivo RNA interference experiment was employed; the results showed that PmPacifastin-like depletion strongly increased PO activity. Interestingly, suppression of PmPacifastin-like also down-regulated the expression of the proPO-activating enzyme PmPPAE2 transcript; the PmPacifastin-like transcript was down-regulated after the PmproPO1/2 transcripts were silenced. Taken together, these results suggest that PmPacifastin-like is important in the shrimp proPO system and may play an essential role in shrimp immune defense against bacterial infection. These results also expand the knowledge of how pacifastin-related protein participates in the negative regulation of the proPO system in shrimp.

  5. Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle

    PubMed Central

    Hong, Feng; Brizendine, Richard K.; Carter, Michael S.; Alcala, Diego B.; Brown, Avery E.; Chattin, Amy M.; Haldeman, Brian D.; Walsh, Michael P.; Facemyer, Kevin C.; Baker, Josh E.

    2015-01-01

    Smooth muscle myosin (SMM) light chain kinase (MLCK) phosphorylates SMM, thereby activating the ATPase activity required for muscle contraction. The abundance of active MLCK, which is tightly associated with the contractile apparatus, is low relative to that of SMM. SMM phosphorylation is rapid despite the low ratio of MLCK to SMM, raising the question of how one MLCK rapidly phosphorylates many SMM molecules. We used total internal reflection fluorescence microscopy to monitor single molecules of streptavidin-coated quantum dot–labeled MLCK interacting with purified actin, actin bundles, and stress fibers of smooth muscle cells. Surprisingly, MLCK and the N-terminal 75 residues of MLCK (N75) moved on actin bundles and stress fibers of smooth muscle cell cytoskeletons by a random one-dimensional (1-D) diffusion mechanism. Although diffusion of proteins along microtubules and oligonucleotides has been observed previously, this is the first characterization to our knowledge of a protein diffusing in a sustained manner along actin. By measuring the frequency of motion, we found that MLCK motion is permitted only if acto–myosin and MLCK–myosin interactions are weak. From these data, diffusion coefficients, and other kinetic and geometric considerations relating to the contractile apparatus, we suggest that 1-D diffusion of MLCK along actin (a) ensures that diffusion is not rate limiting for phosphorylation, (b) allows MLCK to locate to areas in which myosin is not yet phosphorylated, and (c) allows MLCK to avoid getting “stuck” on myosins that have already been phosphorylated. Diffusion of MLCK along actin filaments may be an important mechanism for enhancing the rate of SMM phosphorylation in smooth muscle. PMID:26415568

  6. 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

  7. Interleukin-18 facilitates neutrophil transmigration via myosin light chain kinase-dependent disruption of occludin, without altering epithelial permeability.

    PubMed

    Lapointe, Tamia K; Buret, Andre G

    2012-02-01

    Compromised epithelial barrier function and tight junction alterations are hallmarks of a number of gastrointestinal disorders, including inflammatory bowel disease (IBD). Increased levels of IL-18 have been observed in mucosal samples from Crohn's disease and ulcerative colitis patients. Remarkably, several reports have demonstrated that immunological or genetic blockage of IL-18 ameliorates the severity of colitis in multiple in vivo models of IBD. Nevertheless, the effects of IL-18 on intestinal epithelial barrier function remain unclear. We hypothesized that IL-18 could disrupt intestinal epithelial barrier structure and function, thus contributing to tissue damage in the context of IBD. The aims of the present study were to determine the effects of IL-18 on epithelial barrier structure and function and to characterize the mechanisms involved in these modulatory properties. Human colonic epithelial Caco-2 monolayers were coincubated with IL-18 for 24 h and processed for immunocytochemistry, immunoblotting, quantitative PCR, and permeability measurements (transepithelial resistance, FITC-dextran fluxes, and bacterial translocation). Our findings indicate that IL-18 selectively disrupts tight junctional occludin, without affecting the distribution pattern of claudin-4, claudin-5, zonula occludens-1, or E-cadherin. This effect coincided with a significant increase in myosin light chain kinase (MLCK) protein levels and activity. Pharmacological inhibition of MLCK and NF-κB prevented IL-18-induced loss of occludin. Although too subtle to alter paracellular permeability, these fine changes correlated with an MLCK-dependent increase in neutrophil transepithelial migration. In conclusion, our data suggest that IL-18 may potentiate inflammation in the context of IBD by facilitating neutrophil transepithelial migration via MLCK-dependent disruption of tight junctional occludin.

  8. Non–Muscle Myosin Light Chain Kinase Isoform Is a Viable Molecular Target in Acute Inflammatory Lung Injury

    PubMed Central

    Mirzapoiazova, Tamara; Moitra, Jaideep; Moreno-Vinasco, Liliana; Sammani, Saad; Turner, Jerry R.; Chiang, Eddie T.; Evenoski, Carrie; Wang, Ting; Singleton, Patrick A.; Huang, Yong; Lussier, Yves A.; Watterson, D. Martin; Dudek, Steven M.; Garcia, Joe G. N.

    2011-01-01

    Acute lung injury (ALI) and mechanical ventilator-induced lung injury (VILI), major causes of acute respiratory failure with elevated morbidity and mortality, are characterized by significant pulmonary inflammation and alveolar/vascular barrier dysfunction. Previous studies highlighted the role of the non–muscle myosin light chain kinase isoform (nmMLCK) as an essential element of the inflammatory response, with variants in the MYLK gene that contribute to ALI susceptibility. To define nmMLCK involvement further in acute inflammatory syndromes, we used two murine models of inflammatory lung injury, induced by either an intratracheal administration of lipopolysaccharide (LPS model) or mechanical ventilation with increased tidal volumes (the VILI model). Intravenous delivery of the membrane-permeant MLC kinase peptide inhibitor, PIK, produced a dose-dependent attenuation of both LPS-induced lung inflammation and VILI (∼50% reductions in alveolar/vascular permeability and leukocyte influx). Intravenous injections of nmMLCK silencing RNA, either directly or as cargo within angiotensin-converting enzyme (ACE) antibody–conjugated liposomes (to target the pulmonary vasculature selectively), decreased nmMLCK lung expression (∼70% reduction) and significantly attenuated LPS-induced and VILI-induced lung inflammation (∼40% reduction in bronchoalveolar lavage protein). Compared with wild-type mice, nmMLCK knockout mice were significantly protected from VILI, with significant reductions in VILI-induced gene expression in biological pathways such as nrf2-mediated oxidative stress, coagulation, p53-signaling, leukocyte extravasation, and IL-6–signaling. These studies validate nmMLCK as an attractive target for ameliorating the adverse effects of dysregulated lung inflammation. PMID:20139351

  9. Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments.

    PubMed

    Kampourakis, Thomas; Sun, Yin-Biao; Irving, Malcolm

    2016-05-24

    Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease.

  10. Molecular cloning, characterisation and mRNA expression analysis of the sheep myosin light chain 1 gene.

    PubMed

    Zhang, Chunlan; Wang, Guizhi; Ji, Zhibin; Liu, Zhaohua; Hou, Lei; Liu, Guanqing; Wang, Jianmin

    2015-09-10

    The complete cDNA sequence of the sheep MYL1 (Myosin light chain 1) gene was cloned using RT-PCR, 5' RACE and 3' RACE. We obtained two alternatively spliced isoforms of the MYL1 gene, MYL1a and MYL1b, which are 849 and 1046bp in length and encode proteins composed of 150 and 192 amino acid residues, respectively. And the GenBank accession numbers of MYL1a and MYL1b full-length cDNA sequences that we cloned are KJ700419 and KJ710701, respectively. Neither protein was predicted to have a signal peptide, but both were predicted to have several N-glycosylation and phosphorylation sites. More than half of the secondary structure of these proteins was predicted to be α-helical. The human MYL2 protein (1m8q.1.C) is the most similar in tertiary structure. Sequence alignment showed that the sheep MYL1a protein shares more than 92% amino acid sequence similar with Mus musculus, Homo sapiens, Rattus norvegicus, Sus scrofa and Gallus gallus and that the MYL1b protein shares more than 93% amino acid sequence similar with M. musculus, H. sapiens, R. norvegicus, Bos taurus and Oryctolagus cuniculus. Transcription profile analyses of various tissues indicated that the sheep MYL1a and MYL1b mRNAs were highly but differentially expressed in the longissimus dorsi. Moreover, the expression levels of these genes in the longissimus dorsi differed between Dorper and Small-tailed Han sheep. These results serve as a foundation for further investigations of the function of the sheep MYL1 gene. PMID:25911560

  11. Phosphorylated Myosin Light Chain 2 (p-MLC2) as a Molecular Marker of Antemortem Coronary Artery Spasm.

    PubMed

    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 AND 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

  12. Immunoglobulin Free Light Chains and GAGs Mediate Multiple Myeloma Extracellular Vesicles Uptake and Secondary NfκB Nuclear Translocation

    PubMed Central

    Di Noto, Giuseppe; Chiarini, Marco; Paolini, Lucia; Mazzoldi, Elena Laura; Giustini, Viviana; Radeghieri, Annalisa; Caimi, Luigi; Ricotta, Doris

    2014-01-01

    Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasma cells in the bone marrow. Monoclonal plasma cells often secrete high amounts of immunoglobulin free light chains (FLCs) that could induce tissue damage. Recently, we showed that FLCs are internalized in endothelial and myocardial cell lines and secreted in extracellular vesicles (EVs). MM serum derived EVs presented phenotypic differences if compared with monoclonal gammopathy of undetermined significance (MGUS) serum derived EVs suggesting their involvement in MM pathogenesis or progression. To investigate the effect of circulating EVs on endothelial and myocardial cells, we purified MM and MGUS serum derived EVs with differential ultracentrifugation protocols and tested their biological activity. We found that MM and MGUS EVs induced different proliferation and internalization rates in endothelial and myocardial cells, thus we tried to find specific targets in MM EVs docking and processing. Pre-treatment of EVs with anti-FLCs antibodies or heparin blocked the MM EVs uptake, highlighting that FLCs and glycosaminoglycans are involved. Indeed, only MM EVs exposure induced a strong nuclear factor kappa B nuclear translocation that was completely abolished after anti-FLCs antibodies and heparin pre-treatment. The protein tyrosine kinase c-src is present on MM circulating EVs and redistributes to the cell plasma membrane after MM EVs exposure. The anti-FLCs antibodies and heparin pre-treatments were able to block the intracellular re-distribution of the c-src kinase and the subsequent c-src kinase containing EVs production. Our results open new insights in EVs cellular biology and in MM therapeutic and diagnostic approaches. PMID:25386176

  13. Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments

    PubMed Central

    Kampourakis, Thomas; Sun, Yin-Biao; Irving, Malcolm

    2016-01-01

    Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease. PMID:27162358

  14. 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

  15. Berberine ameliorates severe acute pancreatitis‑induced intestinal barrier dysfunction via a myosin light chain phosphorylation‑dependent pathway.

    PubMed

    Liang, Hong-Yin; Chen, Tao; Yan, Hong-Tao; Huang, Zhu; Tang, Li-Jun

    2014-05-01

    Berberine is a traditional drug used to treat gastrointestinal disorders in China and has been demonstrated to attenuate intestinal barrier dysfunction in certain animal models. However, the effects of berberine on pancreatitis-induced intestinal barrier dysfunction are yet to be fully elucidated. This study aimed to investigate the effect of berberine pretreatment on the attenuation of intestinal barrier dysfunction induced by severe acute pancreatitis (SAP). A total of 36 rats were randomly divided into Sham, SAP and SAP plus berberine groups. Pancreatitis was induced using retrograde injection of 3% Na-taurocholate into the pancreatic duct. Histological examinations of the pancreas were performed and intestinal barrier dysfunction was characterized by histological measurements and the assessment of serum diamine oxidase activity and endotoxin levels. Zonula occludens-1 and occludin mRNA and protein expression, as well as myosin light chain (MLC) phosphorylation, were assessed. SAP rat models were successfully established. Berberine treatment was found to have no significant effect on the histological changes in the pancreas, but was observed to ameliorate the intestinal mucosal barrier damage and membrane permeability associated with SAP. Although berberine exerted minimal effects on tight junction proteins in the ilea of SAP rats, it was observed to significantly inhibit SAP-induced MLC phosphorylation. To the best of our knowledge, this is the first study to demonstrate that berberine attenuates SAP‑induced intestinal barrier dysfunction in vivo. In addition, this study shows that the effect of berberine on intestinal barrier function may be associated with the inhibition of SAP‑induced upregulation of MLC phosphorylation.

  16. Kinesin Light Chain 1 Suppression Impairs Human Embryonic Stem Cell Neural Differentiation and Amyloid Precursor Protein Metabolism

    PubMed Central

    Killian, Rhiannon L.; Flippin, Jessica D.; Herrera, Cheryl M.; Almenar-Queralt, Angels; Goldstein, Lawrence S. B.

    2012-01-01

    The etiology of sporadic Alzheimer disease (AD) is largely unknown, although evidence implicates the pathological hallmark molecules amyloid beta (Aβ) and phosphorylated Tau. Work in animal models suggests that altered axonal transport caused by Kinesin-1 dysfunction perturbs levels of both Aβ and phosphorylated Tau in neural tissues, but the relevance of Kinesin-1 dependent functions to the human disease is unknown. To begin to address this issue, we generated human embryonic stem cells (hESC) expressing reduced levels of the kinesin light chain 1 (KLC1) Kinesin-1 subunit to use as a source of human neural cultures. Despite reduction of KLC1, undifferentiated hESC exhibited apparently normal colony morphology and pluripotency marker expression. Differentiated neural cultures derived from KLC1-suppressed hESC contained neural rosettes but further differentiation revealed obvious morphological changes along with reduced levels of microtubule-associated neural proteins, including Tau and less secreted Aβ, supporting the previously established connection between KLC1, Tau and Aβ. Intriguingly, KLC1-suppressed neural precursors (NPs), isolated using a cell surface marker signature known to identify cells that give rise to neurons and glia, unlike control cells, failed to proliferate. We suggest that KLC1 is required for normal human neural differentiation, ensuring proper metabolism of AD-associated molecules APP and Tau and for proliferation of NPs. Because impaired APP metabolism is linked to AD, this human cell culture model system will not only be a useful tool for understanding the role of KLC1 in regulating the production, transport and turnover of APP and Tau in neurons, but also in defining the essential function(s) of KLC1 in NPs and their progeny. This knowledge should have important implications for human neurodevelopmental and neurodegenerative diseases. PMID:22272245

  17. Proline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics.

    PubMed

    Belvitch, Patrick; Adyshev, Djanybek; Elangovan, Venkateswaran R; Brown, Mary E; Naureckas, Caitlin; Rizzo, Alicia N; Siegler, Jessica H; Garcia, Joe G N; Dudek, Steven M

    2014-09-01

    Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characterize a proline-rich region of nmMLCK previously identified as the possible site of interaction between nmMLCK and cortactin. A mutant nmMLCK construct deficient in proline residues at the putative sites of cortactin binding (amino acids 973, 976, 1019, 1022) was generated. Co-immunoprecipitation studies in human lung EC transfected with wild-type or mutant nmMLCK demonstrated similar levels of cortactin interaction at baseline and after stimulation with the barrier-enhancing agonist, sphingosine 1-phosphate (S1P). In contrast, binding studies utilizing recombinant nmMLCK fragments containing the wild-type or proline-deficient sequence demonstrated a two-fold increase in cortactin binding (p<0.01) to the mutant construct. Immunofluorescent microscopy revealed an increased stress fiber density in ECs expressing GFP-labeled mutant nmMLCK at baseline (p=0.02) and after thrombin (p=0.01) or S1P (p=0.02) when compared to wild-type. Mutant nmMLCK demonstrated an increase in kinase activity in response to thrombin (p<0.01). Kymographic analysis demonstrated an increased EC membrane retraction distance and velocity (p<0.01) in response to the barrier disrupting agent thrombin in cells expressing the mutant vs. the wild-type nmMLCK construct. These results provide evidence that critical prolines within nmMLCK (amino acids 973, 976, 1019, 1022) regulate cytoskeletal and membrane events associated with pulmonary endothelial barrier function. PMID:25072537

  18. 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.

  19. 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. PMID:11745150

  20. Kinetic and Motor Functions Mediated by Distinct Regions of the Regulatory Light Chain of Smooth Muscle Myosin1,2

    PubMed Central

    Ni, Shaowei; Hong, Feng; Brewer, Paul D.; Ikebe, Mitsuo; Onishi, Hirofumi; Baker, Jonathan E.; Facemyer, Kevin C.; Cremo, Christine R.

    2009-01-01

    To understand the importance of selected regions of the regulatory light chain (RLC) for phosphorylation-dependent regulation of smooth muscle myosin (SMM), we expressed three heavy meromyosins (HMMs) containing the following RLC mutants; K12E in a critical region of the phosphorylation domain, GTDP95-98/AAAA in the central hinge, and R160C a putative binding residue for phosphorylated S19. Single-turnover actin-activated Mg2+-ATPase (Vmax and Katpase) and in vitro actin sliding velocities were examined for both unphosphorylated (up-) and phosphorylated (p-) states. Turnover rates for the upstate (0.007-0.030 s-1) and velocities (no motion) for all constructs were not significantly different from the up-wild type (WT) indicating that they were completely turned off. The apparent binding constants for actin in the presence of ATP (Katpase) were too weak to measure as expected for fully regulated constructs. For p-HMM containing GTDP/AAAA, we found that both ATPase and motility were normal. The data suggest that the native sequence in the central hinge between the two lobes of the RLC is not required for turning the HMM off and on both kinetically and mechanically. For p-HMM containing R160C, all parameters were normal, suggesting that R160C is not involved in coordination of the phosphorylated S19. For p-HMM containing K12E, the Vmax was 64% and actin sliding velocity was ∼50% of WT, suggesting that K12 is an important residue for the ability to sense or to promote the conformational changes required for kinetic and mechanical activation. PMID:19635597

  1. Renal leukocyte chemotactic factor 2 (LECT2) amyloidosis in First Nations people in Northern British Columbia, Canada: a report of 4 cases.

    PubMed

    Hutton, Holly L; DeMarco, Mari L; Magil, Alex B; Taylor, Paul

    2014-11-01

    Leukocyte chemotactic factor 2 (LECT2) amyloidosis is a recently identified type of amyloidosis that may represent an underdiagnosed cause of chronic kidney disease. LECT2 amyloidosis typically is reported as being renal limited and, in the United States, more prevalent in Hispanic patients. We add to the epidemiologic data of this condition by describing 4 First Nations people from Northern British Columbia, Canada, who presented with slowly progressive chronic kidney disease that was found to be due to LECT2 amyloidosis.

  2. The nondigestible disaccharide epilactose increases paracellular Ca absorption via rho-associated kinase- and myosin light chain kinase-dependent mechanisms in rat small intestines.

    PubMed

    Suzuki, Takuya; Nishimukai, Megumi; Takechi, Maki; Taguchi, Hidenori; Hamada, Shigeki; Yokota, Atsushi; Ito, Susumu; Hara, Hiroshi; Matsui, Hirokazu

    2010-02-10

    We previously showed that epilactose, a nondigestible disaccharide, increased calcium (Ca) absorption in the small intestines of rats. Here, we explored the mechanism(s) underlying the epilactose-mediated promotion of Ca absorption in a ligated intestinal segment of anesthetized rats. The addition of epilactose to the luminal solution increased Ca absorption and chromium (Cr)-EDTA permeability, a paracellular indicator, with a strong correlation (R = 0.93) between these changes. Epilactose induced the phosphorylation of myosin regulatory light chains (MLCs), which is known to activate the paracellular route, without any change in the association of tight junction proteins with the actin cytoskeleton. The epilactose-mediated promotion of the Ca absorption was suppressed by specific inhibitors of myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK). These results indicate that epilactose increases paracellular Ca absorption in the small intestine of rats through the induction of MLC phosphorylation via MLCK- and ROCK-dependent mechanisms.

  3. Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle.

    PubMed

    Poetter, K; Jiang, H; Hassanzadeh, S; Master, S R; Chang, A; Dalakas, M C; Rayment, I; Sellers, J R; Fananapazir, L; Epstein, N D

    1996-05-01

    The muscle myosins and hexomeric proteins consisting of two heavy chains and two pairs of light chains, the latter called essential (ELC) and regulatory (RLC). The light chains stabilize the long alpha helical neck of the myosin head. Their function in striated muscle, however, is only partially understood. We report here the identification of distinct missense mutations in a skeletal/ventricular ELC and RLC, each of which are associated with a rare variant of cardiac hypertrophy as well as abnormal skeletal muscle. We show that myosin containing the mutant ELC has abnormal function, map the mutant residues on the three-dimensional structure of myosin and suggest that the mutations disrupt the stretch activation response of the cardiac papillary muscles.

  4. A Toxoplasma gondii class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility.

    PubMed

    Bookwalter, Carol S; Kelsen, Anne; Leung, Jacqueline M; Ward, Gary E; Trybus, Kathleen M

    2014-10-31

    Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors.

  5. Dynamin-1-like protein (Dnm1L) interaction with kinesin light chain 1 (KLC1) through the tetratricopeptide repeat (TPR) domains.

    PubMed

    Jang, Won Hee; Jeong, Young Joo; Choi, Sun Hee; Kim, Sang-Jin; Urm, Sang-Hwa; Seog, Dae-Hyun

    2014-01-01

    Kinesin light chain 1 (KLC1) mediates binding of KIF5 motor to specific cargo. Using the yeast two-hybrid screening, we found that mitochondrial fission protein dynamin-1-like protein (Dnm1L) interacted with KLC1, but not KIF5. Dnm1L and KLC1 were co-localized in cultured cells. These results suggest that KLC1 may play a potential role in post-fission mitochondrial transport.

  6. Role of kinesin light chain-2 of kinesin-1 in the traffic of Na,K-ATPase-containing vesicles in alveolar epithelial cells

    PubMed Central

    Trejo, Humberto E.; Lecuona, Emilia; Grillo, Doris; Szleifer, Igal; Nekrasova, Oksana E.; Gelfand, Vladimir I.; Sznajder, Jacob I.

    2010-01-01

    Recruitment of the Na,K-ATPase to the plasma membrane of alveolar epithelial cells results in increased active Na+ transport and fluid clearance in a process that requires an intact microtubule network. However, the microtubule motors involved in this process have not been identified. In the present report, we studied the role of kinesin-1, a plus-end microtubule molecular motor that has been implicated in the movement of organelles in the Na,K-ATPase traffic. We determined by confocal microscopy and biochemical assays that kinesin-1 and the Na,K-ATPase are present in the same membranous cellular compartment. Knockdown of kinesin-1 heavy chain (KHC) or the light chain-2 (KLC2), but not of the light chain-1 (KLC1), decreased the movement of Na,K-ATPase-containing vesicles when compared to sham siRNA-transfected cells (control group). Thus, a specific isoform of kinesin-1 is required for microtubule-dependent recruitment of Na,K-ATPase to the plasma membrane, which is of physiological significance—Trejo, H. E., Lecuona, E., Grillo, D., Szleifer, I., Nekrasova, O. E., Gelfand, V. I., Sznajder, J. I. Role of kinesin light chain-2 of kinesin-1 in the traffic of Na,K-ATPase-containing vesicles in alveolar epithelial cells. PMID:19773350

  7. Immunoglobulin heavy/light chain ratios improve paraprotein detection and monitoring, identify residual disease and correlate with survival in multiple myeloma patients

    PubMed Central

    Ludwig, H; Milosavljevic, D; Zojer, N; Faint, J M; Bradwell, A R; Hübl, W; Harding, S J

    2013-01-01

    The novel heavy/light chain (HLC) assay was used for the detection and measurement of monoclonal immunoglobulins, response evaluation and prognostication. This test allows identification and quantification of the different light chain types of each immunoglobulin class (for example, IgGκ and IgGλ) and enables calculation of ratios of monoclonal/polyclonal immunoglobulin (HLC ratio). Sequential sera of 156 patients with IgG or IgA myeloma started on first-line therapy and followed for a median of 46.1 months were analyzed. Results were compared with those obtained with conventional techniques (serum protein electrophoresis (SPEP), immunofixation electrophoresis (IFE), nephelometry (NEPH), and the free light chain test (FLC)). Our data show that the HLC assay allowed quantification of monoclonal proteins not accurately measurable by SPEP or NEPH. When both HLC and FLC testing were applied for response assessment, clonal excess was noted in 14/31 patients with complete response (CR). HLC ratio indicated presence of disease in 8/31 patients who achieved CR and, in sequential studies indicated evolving relapse in three patients before IFE became positive. Highly abnormal HLC ratios at presentation were significantly associated with shorter overall survival (40.5 months vs median not reached, P=0.016). Multivariate analysis revealed HLC ratio (P=0.03) and β2-microglobulin (P<0.01) as independent risk factors for survival. PMID:22955329

  8. The novel zinc finger protein dASCIZ regulates mitosis in Drosophila via an essential role in dynein light-chain expression.

    PubMed

    Zaytseva, Olga; Tenis, Nora; Mitchell, Naomi; Kanno, Shin-ichiro; Yasui, Akira; Heierhorst, Jörg; Quinn, Leonie M

    2014-02-01

    The essential zinc finger protein ASCIZ (also known as ATMIN, ZNF822) plays critical roles during lung organogenesis and B cell development in mice, where it regulates the expression of dynein light chain (DYNLL1/LC8), but its functions in other species including invertebrates are largely unknown. Here we report the identification of the Drosophila ortholog of ASCIZ (dASCIZ) and show that loss of dASCIZ function leads to pronounced mitotic delays with centrosome and spindle positioning defects during development, reminiscent of impaired dynein motor functions. Interestingly, similar mitotic and developmental defects were observed upon knockdown of the DYNLL/LC8-type dynein light chain Cutup (Ctp), and dASCIZ loss-of-function phenotypes could be suppressed by ectopic Ctp expression. Consistent with a genetic function of dASCIZ upstream of Ctp, we show that loss of dASCIZ led to reduced endogenous Ctp mRNA and protein levels and dramatically reduced Ctp-LacZ reporter gene activity in vivo, indicating that dASCIZ regulates development and mitosis as a Ctp transcription factor. We speculate that the more severe mitotic defects in the absence of ASCIZ in flies compared to mice may be due to redundancy with a second, ASCIZ-independent, Dynll2 gene in mammals in contrast to a single Ctp gene in Drosophila. Altogether, our data demonstrate that ASCIZ is an evolutionary highly conserved transcriptional regulator of dynein light-chain levels and a novel regulator of mitosis in flies.

  9. Activation of smooth muscle myosin light chain kinase by calmodulin. Role of LYS(30) and GLY(40).

    PubMed

    Van Lierop, Jacquelyn E; Wilson, David P; Davis, Jonathan P; Tikunova, Svetlana; Sutherland, Cindy; Walsh, Michael P; Johnson, J David

    2002-02-22

    Calmodulin (CaM)-dependent myosin light chain kinase (MLCK) plays a key role in activation of smooth muscle contraction. A soybean isoform of CaM, SCaM-4 (77% identical to human CaM) fails to activate MLCK, whereas SCaM-1 (90.5% identical to human CaM) is as effective as CaM. We exploited this difference to gain insights into the structural requirements in CaM for activation of MLCK. A chimera (domain I of SCaM-4 and domains II-IV of SCaM-1) behaved like SCaM4, and analysis of site-specific mutants of SCaM-1 indicated that K30E and G40D mutations were responsible for the reduction in activation of MLCK. Competition experiments showed that SCaM-4 binds to the CaM-binding site of MLCK with high affinity. Replacement of CaM in skinned smooth muscle by exogenous CaM or SCaM-1, but not SCaM-4, restored Ca(2+)-dependent contraction. K30E/M36I/G40D SCaM-1 was a poor activator of contraction, but site-specific mutants, K30E, M36I and G40D, each restored Ca(2+)-induced contraction to CaM-depleted skinned smooth muscle, consistent with their capacity to activate MLCK. Interpretation of these results in light of the high-resolution structures of (Ca(2+))(4)-CaM, free and complexed with the CaM-binding domain of MLCK, indicates that a surface domain containing Lys(30) and Gly(40) and residues from the C-terminal domain is created upon binding to MLCK, formation of which is required for activation of MLCK. Interactions between this activation domain and a region of MLCK distinct from the known CaM-binding domain are required for removal of the autoinhibitory domain from the active site, i.e., activation of MLCK, or this domain may be required to stabilize the conformation of (Ca(2+))(4)-CaM necessary for MLCK activation.

  10. Systemic Amyloid A Amyloidosis in Island Foxes (Urocyon littoralis): Severity and Risk Factors.

    PubMed

    Gaffney, P M; Witte, C; Clifford, D L; Imai, D M; O'Brien, T D; Trejo, M; Liberta, F; Annamalai, K; Fändrich, M; Masliah, E; Munson, L; Sigurdson, C J

    2016-05-01

    Systemic amyloid A (AA) amyloidosis is highly prevalent (34%) in endangered island foxes (Urocyon littoralis) and poses a risk to species recovery. Although elevated serum AA (SAA) from prolonged or recurrent inflammation predisposes to AA amyloidosis, additional risk factors are poorly understood. Here we define the severity of glomerular and medullary renal amyloid and identify risk factors for AA amyloidosis in 321 island foxes necropsied from 1987 through 2010. In affected kidneys, amyloid more commonly accumulated in the medullary interstitium than in the glomeruli (98% [n= 78 of 80] vs 56% [n= 45], respectively;P< .0001), and medullary deposition was more commonly severe (19% [n= 20 of 105]) as compared with glomeruli (7% [n= 7];P= .01). Univariate odds ratios (ORs) of severe renal AA amyloidosis were greater for short- and long-term captive foxes as compared with free-ranging foxes (ORs = 3.2, 3.7, respectively; overall P= .05) and for females as compared with males (OR = 2.9;P= .05). Multivariable logistic regression revealed that independent risk factors for amyloid development were increasing age class (OR = 3.8;P< .0001), San Clemente Island subspecies versus San Nicolas Island subspecies (OR = 5.3;P= .0003), captivity (OR = 5.1;P= .0001), and nephritis (OR = 2.3;P= .01). The increased risk associated with the San Clemente subspecies or captivity suggests roles for genetic as well as exogenous risk factors in the development of AA amyloidosis. PMID:26419399

  11. Impaired angiogenesis in a transgenic mouse model of cerebral amyloidosis.

    PubMed

    Paris, Daniel; Patel, Nikunj; DelleDonne, Anthony; Quadros, Amita; Smeed, Robert; Mullan, Michael

    2004-08-01

    Abeta peptides are naturally occurring peptides, which are thought to play a key role in the pathophysiology of Alzheimer's disease (AD). In AD cases, levels of soluble and insoluble Abeta peptides increase in the brain as well as in the cerebrovasculature, a phenomenon that does not occur in extra-cranial vessels. There are frequently anomalies in the cerebrovasculature in AD, and despite increases in several pro-angiogenic factors in AD brain, evidence for increased vascularity is lacking; in fact there is evidence to the contrary. It has also been recently shown that Abeta peptides may have profound anti-angiogenic effects in vitro and in vivo. We therefore investigated whether there is evidence for altered angiogenesis in the vasculature in a transgenic mouse model of Abeta amyloidosis (Tg APPsw line 2576). In vitro, the formation of capillary-like structures on a reconstituted extracellular matrix by endothelial cells isolated from Tg APPsw is impaired. Ex vivo, the sprouting of new capillaries from arterial explants (over expressing Abeta) isolated from 9-month-old Tg APPsw is reduced compared to arterial explants isolated from control littermates. In addition, Tg APPsw mice show a reduction in vascular density in the cortex and hippocampus compared to control littermates. Altogether, our data suggest that the over expression of APPsw in the vasculature may oppose angiogenesis.

  12. Primary localized amyloidosis presenting as diffuse amorphous calcified mass in both orbits: case report.

    PubMed

    Gonçalves, Allan Christian Pieroni; Moritz, Rodrigo Bernal da Costa; Monteiro, Mário Luiz Ribeiro

    2011-01-01

    Primary localized amyloidosis is rare in the orbit. We report the case of a 63-year-old woman that presented with bilateral proptosis and ophthalmoplegia. A computed tomography scan revealed an infiltrative amorphous and markedly calcified mass in both orbits while a magnetic resonance scan showed a heterogeneous hypointense signal on T2-weighted images. A biopsy was performed through an anterior orbitotomy. Microscopy revealed extracellular amorphous and eosinophilic hyaline material which stained pink with Congo red and displayed green birefringence on polarized microscopy, leading to a diagnosis of amyloidosis. The results of the systemic workup were completely normal. A two-year follow-up period without any treatment disclosed no worsening of the condition. While calcification of nonvascular orbital lesions has often been regarded as suggestive of malignant disease, our case is a reminder that it can also be a characteristic presenting sign of orbital amyloidosis.

  13. Topical application of ankaferd hemostat in a patient with gastroduodenal amyloidosis complicated with gastrointestinal bleeding.

    PubMed

    Beyazit, Yavuz; Onder, Fatih Oguz; Torun, Serkan; Tas, Adnan; Purnak, Tugrul; Tenlik, Ilyas; Turhan, Nesrin

    2013-10-01

    Amyloidosis rarely manifests itself as gastrointestinal hemorrhage, especially in the absence of systemic involvement. Despite urgent endoscopic and/or pharmacological therapy, bleeding due to gastric amyloidosis usually recurs after a short period and has considerable morbidity and mortality rates, even in patients undergoing gastrointestinal surgery. For this reason, there is a need for a therapeutic armamentarium for such cases that is effective, easily applicable and has minimal side effects. In this respect, ankaferd blood stopper (ABS) offers a well tolerated and effective alternative approach for these patients. Herein we would like to report a 77-year-old man who had massive bleeding from a gastric ulcer complicating primary gastroduodenal amyloidosis, in whom topical ABS was successfully applied. PMID:23751610

  14. Transthyretin Cardiac Amyloidosis: Pathogenesis, Treatments, and Emerging Role in Heart Failure with Preserved Ejection Fraction

    PubMed Central

    Ton, Van-Khue; Mukherjee, Monica; Judge, Daniel P

    2014-01-01

    Transthyretin (TTR) amyloidosis causes heart failure from cardiac deposition of TTR amyloid fibrils, the by-product of TTR homotetramer disassembly. Wild-type (WT) TTR deposition leads to senile amyloidosis, predominantly manifesting with cardiomyopathy. Missense mutations in the TTR gene result in familial TTR amyloidosis. Certain mutations are more likely to affect the heart, while others cause more neurologic involvement. Extracellular fibril deposition triggers intracellular stress response, upregulation of the inflammatory cascades, apoptosis, and organ dysfunction. Recent studies suggest that TTR cardiac amyloid may be a significant contributor to the pathogenesis of heart failure with preserved ejection fraction (HFpEF). Summarized in this review are the molecular pathways underlying the cellular toxicity of TTR amyloid fibrils and the emerging therapies aimed at TTR tetramer stabilization, abrogation of TTR synthesis in the liver, or inhibition of amyloidogenesis. PMID:25628512

  15. Transthyretin Ile 122: Gene frequency and risk of cardiac amyloidosis in African Americans

    SciTech Connect

    Buxbaum, J. |; Pastore, R.; Yaghoubian, R.

    1994-09-01

    Point mutations in the protein transthyretin (TTR) cause familial amyloidosis involving mainly the heart and peripheral nerves. To determine the TTR Ile 122 gene frequency in African Americans, the allele encoding TTR Ile 122 was identified by PCR-Primer Introduced Restriction Analysis of TTR exon 4 and FokI digestion on 1659 control DNA samples from 1659 African Americans: 64 were heterozygous and one homozygous (allele frequency .02). To determine the risk to gene carriers of developing cardiac amyloidosis, immunohistochemical and genetic studies were performed on amyloid-containing samples identified among 52,370 autopsy samples examined over 32 years. In autopsy samples from patients over age 60, isolated cardiac amyloidosis was more common in African Americans (1.6%) than in White Americans (.40%), suggesting a genetic risk factor. All 29 samples from African Americans were immunohistochemically TTR-positive, and 5 were heterozygous for the TTR Ile 122 allele (17%), as compared with 5/108 heterozygotes (4.6%) among age-matched (over age 60) African American controls without amyloidosis from the same autopsy study (one-tailed P value < .03). Among the 29 amyloid-positive samples were 16 with 1+ or 2+ deposition and 13 with 3+ or 4+ deposition. All 5 TTR Ile 122 heterozygotes had 3+ or 4+ deposition. None of 15 samples from White Americans with cardiac amyloidosis was TTR Ile 122-positive. Extrapolating from the 5/29 samples from which we were able to obtain genetic data, we estimate that 9/54 samples containing cardiac amyloid were TTR Ile 122 heterozygotes. We conclude that heterozygosity for TTR Ile 122 is associated with a greater frequency (relative risk 5.8) and greater severity of cardiac amyloid deposition than in non-carriers of the TTR Ile 122 gene. Of over 40 TTR point mutations associated with amyloidosis described to date, TTR Ile 122 appears to be the most common worldwide and to have the lowest penetrance in causing disease.

  16. Cerebral amyloidosis associated with cognitive decline in autosomal dominant Alzheimer disease

    PubMed Central

    Wang, Fen; Gordon, Brian A.; Ryman, Davis C.; Ma, Shengmei; Xiong, Chengjie; Hassenstab, Jason; Goate, Alison; Fagan, Anne M.; Cairns, Nigel J.; Marcus, Daniel S.; McDade, Eric; Ringman, John M.; Graff-Radford, Neill R.; Ghetti, Bernardino; Farlow, Martin R.; Sperling, Reisa; Salloway, Steve; Schofield, Peter R.; Masters, Colin L.; Martins, Ralph N.; Rossor, Martin N.; Jucker, Mathias; Danek, Adrian; Förster, Stefan; Lane, Christopher A.S.; Morris, John C.; Bateman, Randall J.

    2015-01-01

    Objective: To investigate the associations of cerebral amyloidosis with concurrent cognitive performance and with longitudinal cognitive decline in asymptomatic and symptomatic stages of autosomal dominant Alzheimer disease (ADAD). Methods: Two hundred sixty-three participants enrolled in the Dominantly Inherited Alzheimer Network observational study underwent neuropsychological evaluation as well as PET scans with Pittsburgh compound B. One hundred twenty-one participants completed at least 1 follow-up neuropsychological evaluation. Four composite cognitive measures representing global cognition, episodic memory, language, and working memory were generated using z scores from a battery of 13 standard neuropsychological tests. General linear mixed-effects models were used to investigate the relationship between baseline cerebral amyloidosis and baseline cognitive performance and whether baseline cerebral amyloidosis predicts cognitive change over time (mean follow-up 2.32 years ± 0.92, range 0.89–4.19) after controlling for estimated years from expected symptom onset, APOE ε4 allelic status, and education. Results: In asymptomatic mutation carriers, amyloid burden was not associated with baseline cognitive functioning but was significantly predictive of longitudinal decline in episodic memory. In symptomatic mutation carriers, cerebral amyloidosis was correlated with worse baseline performance in multiple cognitive composites and predicted greater decline over time in global cognition, working memory, and Mini-Mental State Examination. Conclusions: Cerebral amyloidosis predicts longitudinal episodic memory decline in presymptomatic ADAD and multidomain cognitive decline in symptomatic ADAD. These findings imply that amyloidosis in the brain is an indicator of early cognitive decline and provides a useful outcome measure for early assessment and prevention treatment trials. PMID:26245925

  17. Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains

    PubMed Central

    1992-01-01

    Recent biochemical studies of p190, a calmodulin (CM)-binding protein purified from vertebrate brain, have demonstrated that this protein, purified as a complex with bound CM, shares a number of properties with myosins (Espindola, F. S., E. M. Espreafico, M. V. Coelho, A. R. Martins, F. R. C. Costa, M. S. Mooseker, and R. E. Larson. 1992. J. Cell Biol. 118:359-368). To determine whether or not p190 was a member of the myosin family of proteins, a set of overlapping cDNAs encoding the full-length protein sequence of chicken brain p190 was isolated and sequenced. Verification that the deduced primary structure was that of p190 was demonstrated through microsequence analysis of a cyanogen bromide peptide generated from chick brain p190. The deduced primary structure of chicken brain p190 revealed that this 1,830-amino acid (aa) 212,509-D) protein is a member of a novel structural class of unconventional myosins that includes the gene products encoded by the dilute locus of mouse and the MYO2 gene of Saccharomyces cerevisiae. We have named the p190-CM complex "myosin-V" based on the results of a detailed sequence comparison of the head domains of 29 myosin heavy chains (hc), which has revealed that this myosin, based on head structure, is the fifth of six distinct structural classes of myosin to be described thus far. Like the presumed products of the mouse dilute and yeast MYO2 genes, the head domain of chicken myosin-V hc (aa 1-764) is linked to a "neck" domain (aa 765-909) consisting of six tandem repeats of an approximately 23-aa "IQ-motif." All known myosins contain at least one such motif at their head-tail junctions; these IQ-motifs may function as calmodulin or light chain binding sites. The tail domain of chicken myosin-V consists of an initial 511 aa predicted to form several segments of coiled-coil alpha helix followed by a terminal 410-aa globular domain (aa, 1,421-1,830). Interestingly, a portion of the tail domain (aa, 1,094-1,830) shares 58% amino acid

  18. Renal amyloidosis in leprosy, an infrequent cause of nephrotic syndrome in Europe.

    PubMed

    Sanz-Martín, Noelia; Samillán-Sosa, Kelly Del Rocío; De Miguel, Julio; Martínez-Miguel, Patricia

    2016-01-01

    Leprosy is a chronic infectious disease caused by Mycobacterium leprae The main clinical manifestations involve the skin and the peripheral nervous system. Several types of nephropathy have been described in leprosy. One frequent form of renal involvement is amyloidosis, especially in patients with lepromatous leprosy. In these patients, end-stage renal disease is an important contributor to morbidity and mortality. Here, we present the case of a patient with nephrotic syndrome caused by secondary amyloidosis, chronic peripheral neuropathy and a history of leprosy. The patient was correctly treated in her youth, which is the best way to avoid renal pathology, but she developed a nephrotic syndrome years later. PMID:27489069

  19. Apolipoprotein AI mutation Arg-60 causes autosomal dominant amyloidosis.

    PubMed Central

    Soutar, A K; Hawkins, P N; Vigushin, D M; Tennent, G A; Booth, S E; Hutton, T; Nguyen, O; Totty, N F; Feest, T G; Hsuan, J J

    1992-01-01

    A mutation in the gene for apolipoprotein AI (apoAI) was identified in an English family with autosomal dominant non-neuropathic systemic amyloidosis. The plasma of all affected individuals contained a variant apoAI with one additional charge, as well as normal apoAI. The propositus was heterozygous; the coding region of his apoAI gene contained both the normal sequence and a single-base substitution changing the codon for residue 60 of the mature protein from CTG (leucine) to CGG (arginine). Allele-specific oligonucleotide hybridization showed that the other affected individuals were also heterozygotes and that there was concordance of the mutant allele with the presence of variant plasma apoAI. Amyloid fibrils isolated from the spleen of the propositus consisted of proteins that ran as a doublet with an apparent mass of approximately 10 kDa in SDS/PAGE and a trace band at 28 kDa. Electrospray mass spectrometry of the purified 10-kDa material revealed components with mass corresponding to the N-terminal 88, 92, 93, and 94 residues of apoAI each with substitution of arginine for leucine. These observations were confirmed by direct protein sequencing and laser desorption time-of-flight mass analysis. No material with the normal apoAI sequence was detected. The trace band at 28 kDa yielded the N-terminal sequence of mature apoAI, indicating that intact or minimally degraded apoAI was also present in the fibril preparation. Discovery of this mutation and the detailed characterization of the apoAI fragments that form the amyloid fibrils open additional avenues for investigation of amyloidogenesis. Images PMID:1502149

  20. Clasmatodendrosis and β-amyloidosis in aging hippocampus.

    PubMed

    Mercatelli, Raffaella; Lana, Daniele; Bucciantini, Monica; Giovannini, Maria Grazia; Cerbai, Francesca; Quercioli, Franco; Zecchi-Orlandini, Sandra; Delfino, Giovanni; Wenk, Gary L; Nosi, Daniele

    2016-04-01

    Alterations of the tightly interwoven neuron/astrocyte interactions are frequent traits of aging, but also favor neurodegenerative diseases, such as Alzheimer disease (AD). These alterations reflect impairments of the innate responses to inflammation-related processes, such as β-amyloid (Aβ) burdening. Multidisciplinary studies, spanning from the tissue to the molecular level, are needed to assess how neuron/astrocyte interactions are influenced by aging. Our study addressed this requirement by joining fluorescence-lifetime imaging microscopy/phasor multiphoton analysis with confocal microscopy, implemented with a novel method to separate spectrally overlapped immunofluorescence and Aβ autofluorescence. By comparing data from young control rats, chronically inflamed rats, and old rats, we identified age-specific alterations of neuron/astrocyte interactions in the hippocampus. We found a correlation between Aβ aggregation (+300 and +800% of aggregated Aβ peptide in chronically inflamed and oldvs.control rats, respectively) and fragmentation (clasmatodendrosis) of astrocyte projections (APJs) (+250 and +1300% of APJ fragments in chronically inflamed and oldvs.control rats, respectively). Clasmatodendrosis, in aged rats, associates with impairment of astrocyte-mediated Aβ clearance (-45% of Aβ deposits on APJs, and +33% of Aβ deposits on neurons in oldvs.chronically inflamed rats). Furthermore, APJ fragments colocalize with Aβ deposits and are involved in novel Aβ-mediated adhesions between neurons. These data define the effects of Aβ deposition on astrocyte/neuron interactions as a key topic in AD biology.-Mercatelli, R., Lana, D., Bucciantini, M., Giovannini, M. G., Cerbai, F., Quercioli, F., Zecchi-Orlandini, S., Delfino, G., Wenk, G. L., Nos, D. Clasmatodendrosis and β-amyloidosis in aging hippocampus. PMID:26722005

  1. Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein.

    PubMed

    Timofeeva, Anna M; Buneva, Valentina N; Nevinsky, Georgy A

    2015-10-01

    Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP-Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27 kDa). Seventy-two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty-two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7-9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease-like and three thiol protease-like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca(2+), Mg(2+), Mn(2+), Ni(2+), Zn(2+), Cu(2+), and Co(2+) was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti-MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti-MBP abzymes, which can attack MBP of myelin-proteolipid sheath of axons and play an important role in MS and SLE pathogenesis.

  2. High serum-free light chain levels and their rapid reduction in response to therapy define an aggressive multiple myeloma subtype with poor prognosis.

    PubMed

    van Rhee, Frits; Bolejack, Vanessa; Hollmig, Klaus; Pineda-Roman, Mauricio; Anaissie, Elias; Epstein, Joshua; Shaughnessy, John D; Zangari, Maurizio; Tricot, Guido; Mohiuddin, Abid; Alsayed, Yazan; Woods, Gail; Crowley, John; Barlogie, Bart

    2007-08-01

    Serum-free light chain (SFLC) levels are useful for diagnosing nonsecretory myeloma and monitoring response in light-chain-only disease, especially in the presence of renal failure. As part of a tandem autotransplantation trial for newly diagnosed multiple myeloma, SFLC levels were measured at baseline, within 7 days of starting the first cycle, and before both the second induction cycle and the first transplantation. SFLC baseline levels higher than 75 mg/dL (top tertile) identified 33% of 301 patients with higher near-complete response rate (n-CR) to induction therapy (37% vs 20%, P = .002) yet inferior 24-month overall survival (OS: 76% vs 91%, P < .001) and event-free survival (EFS: 73% vs 90%, P < .001), retaining independent prognostic significance for both EFS (HR = 2.40, P = .008) and OS (HR = 2.43, P = .016). Baseline SFLC higher than 75 mg/dL was associated with light-chain-only secretion (P < .001), creatinine level 176.8 microM (2 mg/dL) or higher (P < .001), beta-2-microglobulin 297.5 nM/L (3.5 mg/L) or higher (P < .001), lactate dehydrogenase 190 U/L or higher (P < .001), and bone marrow plasmacytosis higher than 30% (P = .003). Additional independent adverse implications were conferred by top-tertile SFLC reductions before cycle 2 (OS: HR = 2.97, P = .003; EFS: HR = 2.56, P = .003) and before transplantation (OS: HR = 3.31, P = .001; EFS: HR = 2.65, P = .003). Unlike baseline and follow-up analyses of serum and urine M-proteins, high SFLC levels at baseline-reflecting more aggressive disease-and steeper reductions after therapy identified patients with inferior survival.

  3. Expression of v-rel induces mature B-cell lines that reflect the diversity of avian immunoglobulin heavy- and light-chain rearrangements.

    PubMed Central

    Barth, C F; Humphries, E H

    1988-01-01

    The infection of newly hatched chickens with reticuloendotheliosis virus strain T (REV-T) and a nonimmunosuppressive helper virus, chicken syncytial virus, induces rapidly metastatic B-cell lymphomas. In vivo analysis of these tumors with monoclonal antibodies detected the expression of the B-cell surface markers immunoglobulin M (IgM), CIa, Bu2, and CLA-1, but not IgG, Bu1, or a T-cell surface marker, CT-1. Cell lines derived from tumors exhibited the same pattern of staining, suggesting that expression of cell surface markers does not change during in vitro cell line development. All cell lines examined synthesized IgM in varying amounts. Northern (RNA blot) analysis confirmed abundant expression of v-rel mRNA, and Southern analysis revealed rearrangement of both heavy- and light-chain immunoglobulin loci. Analysis of the light-chain