immunoblot

Phosphorylation of serine 523 on 5-lipoxygenase in human B lymphocytes.

Phosphorylation of serine 523 on 5-lipoxygenase in human B lymphocytes.

Prostaglandins Leukot Essent Fatty Acids. 2015 Jun 24;

Authors: Mahshid Y, Markoutsa S, Dincbas-Renqvist V, Sürün D, Christensson B, Sander B, Björkholm M, Sorg BL, Rådmark O, Claesson HE

Abstract
The key enzyme in leukotriene (LT) biosynthesis is 5-lipoxygenase (5-LO), which is expressed in myeloid cells and in B lymphocytes. There are three phosphorylation sites on 5-LO (Ser271, Ser523 and Ser663). Protein kinase A (PKA) phosphorylates 5-LO on Ser523. In this report, we demonstrate by immunoblotting that native 5-LO in mantle B cell lymphoma (MCL) cells (Granta519, JEKO1, and Rec1) and in primary chronic B lymphocytic leukemia cells (B-CLL) is phosphorylated on Ser523. In contrast, we could not detect phosphorylation of 5-LO on Ser523 in human granulocytes or monocytes. Phosphorylated 5-LO was purified from Rec1 cells, using an ATP-agarose column, and the partially purified enzyme could be dephosphorylated with alkaline phosphatase. Incubation of Rec1 cells with 8-Br-cAMP or prostaglandin E2 stimulated phosphorylation at Ser523. Furthermore, FLAG-5LO was expressed in Rec1 cells, and the cells were cultivated in the presence of 8-Br-cAMP. The 5-LO protein from these cells was immunoprecipitated, first with anti-FLAG, followed by anti-pSer523-5-LO. The presence of 5-LO protein in the final precipitate further supported the finding that the protein recognized by the pSer523 antibody was 5-LO. Taken together, this study shows that 5-LO in B cells is phosphorylated on Ser523 and demonstrates for the first time a chemical difference between 5-LO in myeloid cells and B cells.

PMID: 26210919 [PubMed - as supplied by publisher]



via pubmed: lymphoma daily http://ift.tt/1LNhSq8
Pathology

http://www.patho.hku.hk/Pdip_webpage/message_director.html


Postgraduate Diploma in Molecular and Diagnostic Pathology (PDipMDPath)
     分子及診斷病理學深造文憑

Postgraduate Certificate in Molecular and Diagnostic Pathology (PCMDPath)
   分子及診斷病理學深造證書


Modules

PATH6001 Principles and Techniques of Molecular Pathology (24 hours)
           This module will cover the following topics: Introduction to molecular pathology and genetics; Principles of electrophoresis and immunoblotting; Principles of automated DNA sequencing and various methods of genotyping and mutation analysis; Human identity by DNA typing; Principles and applications of Quantitative-PCR; Basic concepts in conventional cytogenetics and molecular cytogenetics; In-situ hybridization techniques (ISH, FISH, CISH, SISH); Principles and applications of flow cytometry; Emerging technologies (epigenetics, gene expression profiling, stem cells); Laboratory management issues in molecular testing.

PATH6002 Clinical Applications of Molecular Testing (24 hours)
           This module provides an overview of the Principles of HPV testing, its clinical relevance and the various methods of genotyping; Hepatitis B virus infection – testing for viral load and HBV DNA mutants detection; Quantification of EBV DNA plasma for EBV associated diseases; BCR/ABL kinase mutation detection for chronic myelogenous leukaemia and related disorders; KRAS mutation detection for colorectal cancer; EGFR mutation in lung cancer; In-situ hybridization tests: EBER; Kappa/Lamda; HER2 amplification in breast cancer; FISH test for early bladder cancer detection; PCR for gene rearrangements and translocations for haematolymphoid malignancies and soft tissue tumors; RET and Menin gene mutation detection for Multiple endocrine neoplasia syndrome; c-kit mutation detection for gastrointestinal stromal tumors; Gene expression profiling for haematolymphoid malignancies;: Laboratory management issues in molecular testing.

PATH6003 Fundamentals of Genetic Testing for Hereditary Disorders (24 hours)
           This course provides a comprehensive introduction to Molecular Genetics; Genetic testing for Thalassemia; Genetic testing for familial colorectal cancer; Genetic testing for familial breast and ovarian cancer; Molecular genetics in Paediatrics; Molecular genetics in Neurological diseases; Laboratory management issues in molecular testing.

PATH6004 Chemical Pathology, Immunology, Diagnostic Haematology and Transfusion Medicine (40 hours)
           This module will cover topics on the Renal and Liver function tests; Cardiac Markers; Tumor markers; Clinical Toxicology; Expanded Newborn Screening; Pharmacogenetics, Drug Safety and Personalized Medicine; Biochemical diagnosis of adrenal disorders; Immunochemistry Tests; Autoantibody Tests; Diagnostic Haematology Test; Transfusion Medicine; Laboratory Management.

PATH6005 Essential Anatomical Pathology for Clinicians (40 hours)
           Topics include Haematolymphoid malignancies: relevance of classification; Tumors of the respiratory tract and pleural cavity; Tumors of the Central Nervous System; Urologic oncology; Tumors of the breast; Endocrine tumors; Soft tissue and bone tumors; Tumors of the liver and GI tract; Cytological, histopathological and molecular devices for diagnosis and management of gynaecological pathology; uterine pathology; ovarian tumors and endometriosis Squamous and glandular lesions of the cervix: cytology and histology; Gestational trophoblastic diseases; Renal biopsy procedure and specimen handling; Interpreting renal biopsies for glomerular, tubular, interstitial and vascular diseases; Classification of lupus nephritis; Banff classification of renal transplant rejection; Glomerular diseases in transplanted kidney; Laboratory management.

PATH6006 Project Report (48 hours)
           For the fulfillment of the Diploma course, candidates will need to submit a project report of at least 3000 words based on a clinical and/or laboratory project which should be conducted over a period of at least 3 months within the candidate’s own hospital or practice. It should reflect an application of the knowledge acquired from this course. The candidate is expected to design the project related to his work situation and have it conducted from his workplace. The choice of topic for the study should be discussed in advance with an identified supervisor who will give guidance in the writing of the project report.

PATH6007 Practical Course in Laboratory Methods (24 hours)
           This course provides practical sessions on Tissue processing, immunohistochemistry and histological analysis; Basic tissue culture techniques and flow cytometry analysis; Extraction methods for DNA, RNA, protein and electrophoresis; and Reverse transcription, polymerase chain reaction, DNA sequencing.

PATH6008 Molecular Microbiology and Infectious Diseases Update (40 hours)
           The Molecular Microbiology component includes the following topics:
           Clinical application in bacteriology - Bacterial pathogens: typical and atypical, mycobacterium species, MALDI-TOF-MS technology and application, and Molecular approaches for the diagnosis of sepsis, infections of gastrointestinal tract and central nervous system); Clinical application in virology - Viral pathogens: hepatitis, enteric and respiratory viruses; rapid quantitation of HIV and drug resistance determination; Clinical application in mycology and parasitology - Fungal pathogens, Parasitic pathogens, Limitations and future perspectives of genomic techniques; Clinical applications in epidemiological surveillance and outbreak management - Principles of molecular phylogenetic analysis and molecular typing method, Laboratory and epidemiological considerations for data interpretation and Limitations and future perspectives.
 
           Students are required to attend any of two of the six courses given under the Infectious Disease Update offered by the Department of Microbiology which include the following:
 
           • Infectious disease update and emerging infections
           • Infectious disease emergencies, indwelling device and surgical infections
           • Common problems in infectious diseases
           • Radiology and radionuclide imaging in ID; genitourinary medicine and HIV problems
           • Surprises in daily medical practice: tropical diseases in the developed world
           • Infections in immunocompromised hosts and common infective problems in general practice
         

PATH6009 Clinical Applications of Genetic Testing in Inherited Diseases and Genetic Counselling (24 hours)
           This course introduces the general principles of cytogenetics, biochemical genetics and molecular genetics in genetic testing and the various laboratory techniques for identification of disease-causing mutations. Through the practical sessions and group discussion, students will learn how to read a laboratory report. Topics will include principles of genetic counselling; ethical and legal aspects; biochemical diagnosis of acute IEM patients; SNP arrays in clinical practice; bioinformatics for mutation reporting; extended newborn screening for metabolic disease; inherited metabolic disease; endocrine disease and neuromuscular disease; neurogenetics and inherited bone disease; pharmacogenetics; constitutional cytogenetics; molecular cytogenetics; next-generation Sequencing – hardware, software and clinical application; practicum: Interpretation of reports; tutorials on inherited cardiac disease, mitochondrial disease,lysosomal storage disease, autistic spectrum disorder. Laboratory visits to genetic laboratories in Hong Kong.

Periodontitis in RA - the citrullinated enolase connection (P.5)

Bản gốc:

The most striking feature of this bacterium, in the context of autoimmunity in RA, is that P. gingivalis expresses its own unique citrullinating enzyme, PPAD. PPAD has no sequence homology with human PADs, and seems to differ in substratespecificity by showing a marked preference for the citrullination of carboxy-terminal arginines. Studies in our laboratory have shown that P. gingivalis contains a range of endogenous citrullinated proteins that are not present in other common oral pathogens. We have also, for the first time to our know ledge, demonstrated citrullination of human fibrinogen and human α-enolase by P. gingivalis. Citrullination of these proteins was dependent on PPAD, but also on arginine-gingipains, which proteolytically cleave proteins at arginine residues, thus generating short peptides containing carboxy-terminal arginine residues that are subsequently citrullinated by PPAD.

The enolase connection

we first identified citrullinated α-enolase as a candidate autoantigen by immunoblotting HL60 cell lysates with serum samples from patients with RA, and sequencing a reactive 47 kDa band by mass spectrometry.21 in a subsequent study, we screened serum from 102 patients with RA, 110 patients with other rheumatic diseases and 92 healthy controls for reactivity with 11 cyclic CEPs, covering 15 of the 17 arginine residues within α-enolase. We went on to map the immunodominant B-cell epitope(s) to the CeP-1 peptide, which corresponds to amino acids 5–21 in the full-length protein. CeP-1 contains two arginine residues replaced by citrulline, and we demonstrated that the second of these (citrulline 15) was most important for antibody recognition. Reactivity against the arginine-containing control peptide was similarly low in patients and controls. A quantitative enzyme-linked immunosorbent assay using CEP-1 has now become the basis of several published studies of over 3,000 serum samples, which have demonstrated an anti-CeP-1 antibody frequency of approximately 40% in patients with RA, and a disease-specificity of 97%.

Keep reading

Phosphorylation of serine 523 on 5-lipoxygenase in human B lymphocytes.

Phosphorylation of serine 523 on 5-lipoxygenase in human B lymphocytes.

Prostaglandins Leukot Essent Fatty Acids. 2015 Jun 24;

Authors: Mahshid Y, Markoutsa S, Dincbas-Renqvist V, Sürün D, Christensson B, Sander B, Björkholm M, Sorg BL, Rådmark O, Claesson HE

Abstract
The key enzyme in leukotriene (LT) biosynthesis is 5-lipoxygenase (5-LO), which is expressed in myeloid cells and in B lymphocytes. There are three phosphorylation sites on 5-LO (Ser271, Ser523 and Ser663). Protein kinase A (PKA) phosphorylates 5-LO on Ser523. In this report, we demonstrate by immunoblotting that native 5-LO in mantle B cell lymphoma (MCL) cells (Granta519, JEKO1, and Rec1) and in primary chronic B lymphocytic leukemia cells (B-CLL) is phosphorylated on Ser523. In contrast, we could not detect phosphorylation of 5-LO on Ser523 in human granulocytes or monocytes. Phosphorylated 5-LO was purified from Rec1 cells, using an ATP-agarose column, and the partially purified enzyme could be dephosphorylated with alkaline phosphatase. Incubation of Rec1 cells with 8-Br-cAMP or prostaglandin E2 stimulated phosphorylation at Ser523. Furthermore, FLAG-5LO was expressed in Rec1 cells, and the cells were cultivated in the presence of 8-Br-cAMP. The 5-LO protein from these cells was immunoprecipitated, first with anti-FLAG, followed by anti-pSer523-5-LO. The presence of 5-LO protein in the final precipitate further supported the finding that the protein recognized by the pSer523 antibody was 5-LO. Taken together, this study shows that 5-LO in B cells is phosphorylated on Ser523 and demonstrates for the first time a chemical difference between 5-LO in myeloid cells and B cells.

PMID: 26210919 [PubMed - as supplied by publisher]



via pubmed: cllsllupdate1 http://ift.tt/1LNhSq8
First de novo KCND3 mutation causes severe Kv4.3 channel dysfunction leading to early onset cerebellar ataxia, intellectual disability, oral apraxia and epilepsy.

PubMed: Related Articles

First de novo KCND3 mutation causes severe Kv4.3 channel dysfunction leading to early onset cerebellar ataxia, intellectual disability, oral apraxia and epilepsy.

BMC Med Genet. 2015;16(1):51

Authors: Smets K, Duarri A, Deconinck T, Ceulemans B, van de Warrenburg BP, Züchner S, Gonzalez MA, Schüle R, Synofzik M, Van der Aa N, De Jonghe P, Verbeek DS, Baets J

Abstract
BACKGROUND: Identification of the first de novo mutation in potassium voltage-gated channel, shal-related subfamily, member 3 (KCND3) in a patient with complex early onset cerebellar ataxia in order to expand the genetic and phenotypic spectrum.
METHODS: Whole exome sequencing in a cerebellar ataxia patient and subsequent immunocytochemistry, immunoblotting and patch clamp assays of the channel were performed.
RESULTS: A de novo KCND3 mutation (c.877_885dupCGCGTCTTC; p.Arg293_Phe295dup) was found duplicating the RVF motif and thereby adding an extra positive charge to voltage-gated potassium 4.3 (Kv4.3) in the voltage-sensor domain causing a severe shift of the voltage-dependence gating to more depolarized voltages. The patient displayed a severe phenotype with early onset cerebellar ataxia complicated by intellectual disability, epilepsy, attention deficit hyperactivity disorder, strabismus, oral apraxia and joint hyperlaxity.
CONCLUSIONS: We identified a de novo KCND3 mutation causing the most marked change in Kv4.3’s channel properties reported so far, which correlated with a severe and unique spinocerebellar ataxia (SCA) type 19/22 disease phenotype.

PMID: 26189493 [PubMed - as supplied by publisher] http://dlvr.it/BbgVD7