IgM Heavy Chain Research Articles

The IgM CH2 domain as covalently linked homodimerization module for the generation of fusion proteins with dual specificity

Dimeric assembly of antibody fragments and other therapeutic molecules can result in increased binding and improved bioactivity. Here, we investigated the use of the IgM heavy chain domain 2 (MHD2) as covalently linked homodimerization module. Fusion of single-chain fragment variable (scFv) molecules directed against epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 to the N- and/or C-terminus of the MHD2, respectively, resulted in molecules with single or dual specificity for tumor cells. Bispecific tetravalent molecules were further generated by fusing a bispecific single-chain diabody directed against EGFR and epithelial cell adhesion molecule to the N-terminus of the MHD2. By combining an anti-EGFR scFv with a single-chain derivative of tumor necrosis factor, a tetravalent bifunctional fusion protein was produced. This fusion protein exhibited improved TNF activity, also mimicking the membrane-bound form of TNF, as shown by the activation of TNFR2-mediated cell killing. Furthermore, the scFv moiety allowed for an antigen-dependent delivery of TNF to EGFR-positive cells and an improved stimulatory TNF action on these cells. Thus, we established the MHD2 as a versatile module for the generation of bispecific and bifunctional fusion proteins.

Genome-wide Analysis of Pre-mRNA 3′ End Processing Reveals a Decisive Role of Human Cleavage Factor I in the Regulation of 3′ UTR Length

Through alternative polyadenylation, human mRNAs acquire longer or shorter 3' untranslated regions, the latter typically associated with higher transcript stability and increased protein production. To understand the dynamics of polyadenylation site usage, we performed transcriptome-wide mapping of both binding sites of 3' end processing factors CPSF-160, CPSF-100, CPSF-73, CPSF-30, Fip1, CstF-64, CstF-64τ, CF I(m)25, CF I(m)59, and CF I(m)68 and 3' end processing sites in HEK293 cells. We found that although binding sites of these factors generally cluster around the poly(A) sites most frequently used in cleavage, CstF-64/CstF-64τ and CFI(m) proteins have much higher positional specificity compared to CPSF components. Knockdown of CF I(m)68 induced a systematic use of proximal polyadenylation sites, indicating that changes in relative abundance of a single 3' end processing factor can modulate the length of 3' untranslated regions across the transcriptome and suggesting a mechanism behind the previously observed increase in tumor cell invasiveness upon CF I(m)68 knockdown.

Detection of weak receptor–ligand interactions using IgM and J‐chain‐based fusion proteins

Detection of weak receptor–ligand interactions using <scp>I</scp>g<scp>M</scp> and <scp>J</scp>‐chain‐based fusion proteins

Case study interpretation-Portland: Case 2

Case study interpretation-Portland: Case 2

Proliferative glomerulonephritis with monoclonal IgM deposits without Waldenström’s macroglobulinemia: case report and review of the literature

A 38-year-old man was presented with nephrotic syndrome associated with hematuria and mild hypocomplementemia. Renal biopsy revealed lobular mesangial proliferation, thickened capillary walls, and intraluminal protein thrombi. Immunofluorescence showed marked and mild depositions of immunoglobulin (Ig) M heavy chains and complement C3, respectively, in a peripheral lobular pattern. On light chain staining, only kappa (κ) light and IgM heavy chains showed a similar pattern. Electron microscopy showed fine granular electrondense deposits in subendothelial areas and numerous globular deposits (varying size and density) in glomerular capillary lumens. Serum levels of Ig κ, but not of free κ, light chains were significantly increased. Bone marrow aspiration revealed a normocellular marrow. Waldenström's macroglobulinemia and cryoglobulinemia were ruled out. Clinically, steroid therapy was ineffective and proteinuria persisted. This report demonstrates that glomerular deposition of monoclonal IgM-κ can produce membranoproliferative- like changes in the glomeruli. This condition may be recognized as proliferative glomerulonephritis with monoclonal IgM deposits similar to the recently recognized proliferative glomerulonephritis with monoclonal IgG deposits.

Molecular characterization of IgM, IgZ and IgD heavy chain genes in orange-spotted grouper(Epinephelus coioides)

In the present study,the membrane-bound immunoglobulin M(mIgM),mIgD and secretory IgZ(sIgZ) have been identified by using RACE-PCR from the orange-spotted grouper,Epinephelus coioides.The mIgM and sIgZ are comparable with their counterparts in other teleost in terms of their heavy chain cDNA sequences and gene structures.The constant region of the mIgM had three CH domains,i.e.,CH1,CH2,CH3,and two transmembrane domains,TM1 and TM2.The TM1 exon is spliced into the regular donor site at the 3’ terminus of the CH3 domain.When comparing the amino acid sequence of mIgM with those in teleost fish,it is found that the grouper mIgM CH region shares highest identity with that in the oliver flounder(Paralichthys olivaceus),with CH1,CH2,CH3 having 53% 78% identity with those in the flounder,respectively.The complete transcript of the grouper mIgD contains 3 375 bp,including a 3 006 bp open reading frame,and the constant region of the mIgD consists serially of a μ1 domain,seven δ domains(δ1 to δ7),and a TM region.The degree of amino acid sequence identity,in domain-by-domain comparison with other teleost fishes,reveals an overall higher identity between the IgD in the grouper and that in mandarin fish(Siniperca chuatsi),with 1 to 7 having 75.5%,75.8%,65.4%,76.6%,88.1%,90.6%,82.8% identity,and the transmembrane domain having 82.7% identity,respectively.The sIgZ has a four exon structure in genome,with the introns being 222,129,and 458 bp in length,respectively.The semi-quantitative RT-PCR was performed to examine the expressions of IgZ,IgM,and IgD in various organs / tissues of the grouper.Transcripts encoding the mIgM were detected in all examined samples.IgD was mainly detected in pronephros,kidney,spleen and thymus,with week expression in intestine.IgZ was expressed in lymphoid tissues such as pronephros,kidney and spleen,with relatively low expression detected in gills,thymus and heart.

Full-length cDNA cloning of IgM heavy chain of cobiaRachycentron canadiumand quantitative analysis in different tissues

Full-length cDNA of IgM haevy chain of cobia (Rachycentron canadium Linnaeus) was ampli-fied by the techniques of Rapid Amplification of cDNA ends (RACE) and PCR, and expression of target gene was analyzed by the method of real time RT-PCR in different tissues. The full-length cDNA of μ of cobia is 1 933 bp, with a 5?terminus untranslated region (UTR) of 26 bp and a 3?terminus UTR of 164 bp, carried an open reading frame (ORF) of 1 743 bp, encoding 580 amino acids with estimated molecular weight of 64.83 ku and the theoretical isoelectric point of 6.6. The similarity of the deduced Igμ amino acid sequences of cobia was less than 30%, when compared with those of other animals. Moreover, similarities of the conservative CH4 region of cobia,to other fish were 64%-71%. But, to other living things, similarity was only 31%-33%, having a strong specificity. Clustal X analysis indicated that there existed conserved amino acid sites (cysteines, tryptophans) and two E-box motifs GKGLEW at framework region two and YYCAR at framework region three, respectively in the IgM heavy chain of cobia. Ig μ gene was ex-pressed in all the tissues but kidney in healthy cobia and the expression with the highest quantity was pre-sent in intestine by real time RT-PCR. The target gene was respectively expressed in all tissues of cobia after stimulation by intraperitoneal injection with Vibrio carchariae for 48 h, 96 h and 192 h. The expres-sion of Igμ gene was significantly increased in heart, gill and intestine after 48 h. Increased expression of gene in spleen, stomach, brain was after 0 h and in liver, head kidney and kidney was after 192 h. Conclu-sion could be drawn that mucosal immune defended against foreign pathogens as the first barrier, followed by the liver, spleen, kidney, head kidney immune response, which indicated that immune system plays an important role in defense against foreign pathogens for a long time.

Long-term stimulation of trout head kidney cells with the cytokines MCSF, IL-2 and IL-6: Gene expression dynamics

The production of salmonid leukocyte cell lines from primary cell cultures has been attempted on several occasions, however, to date only monocyte/macrophage like cell lines exist (e.g. RTS-11 and SHK-1 cells). With the increasing number of cytokines discovered in fish in recent years, many of which are growth factors for leukocytes, we now have the possibility of using these molecules to promote leukocyte development and differentiation in culture. We have generated stable cell lines transfected with a variety of plasmids expressing cytokines (Interleukin (IL)-2, IL-6 and Macrophage Colony Stimulating Factor (MCSF)), in order to produce conditioned media rich in these cytokines. The cytokine-conditioned media were used to assess their activity and ability to support the growth of primary head kidney (HK) leukocyte cultures. Here, we describe a series of experiments aimed to determine which cell population(s) of primary HK cultures is supported and will grow in conditioned media containing MCSF, IL-2 or IL-6. For a period of 5 weeks, cells were incubated at 22 °C and media were changed every 3–4 days. Samples were taken at different time points, from freshly isolated HK cells (T0), one week post-stimulation (1-WPS), 3-WPS and 5-WPS for RNA extraction. A variety of cell lineage markers (MCSF Receptor 2 (MCSFR2) for macrophages, CD4 and CD8a for T cells and IgM heavy chain for B cells) were then analysed by real-time qPCR to study the cell population dynamics as influenced by the different recombinant cytokines in the cultures. We show here that whilst MCSF appears to drive macrophage differentiation and maintenance, IL-2 and IL-6 seem to preferentially drive lymphocyte differentiation.

Characterization of the immunoglobulin repertoire of the spiny dogfish (Squalus acanthias)

The cartilaginous fish (chimeras, sharks, skates and rays) are the oldest group relative to mammals in which an adaptive immune system founded upon immunoglobulins has been found. In this manuscript we characterize the immunoglobulins of the spiny dogfish (Squalus acanthias) at both the molecular and expressed protein levels. Despite the presence of hundreds of IgM clusters in this species the serum levels of this isotype are comparatively low. However, analysis of cDNA sequences and serum protein suggests microheterogeneity in the IgM heavy chains and supports the proposal that different clusters are preferentially used in the two forms (monomer or pentamer) of this isotype. We also found that the IgNAR isotype in this species exists in a previously unknown multimeric format in serum. Finally, we identified a new form of the IgW isotype (the shark IgD orthologue), in which the leader is spliced directly to the first constant domain, resulting in a molecule lacking an antigen-binding domain.

Zebrafish immunoglobulin IgD: Unusual exon usage and quantitative expression profiles with IgM and IgZ/T heavy chain isotypes

The zebrafish is an emerging model for comparative immunology and biomedical research. In contrast to the five heavy chain isotype system of mice and human (IgD, IgM, IgA, IgG, IgE), zebrafish harbor gene segments for IgD, IgM, and novel heavy chain isotype called IgZ/T which appears restricted to bony fishes. The purpose of this study was to design and validate a suite of quantitative real time RT-PCR protocols to measure IgH expression in a vertebrate model which has considerable promise for modeling both pathogenic infection and chronic conditions leading to immune dysfunction. Specific primers were designed and following verification of their specificty, relative expression levels of IgD, IgM, and IgZ/T were measured in triplicate for zebrafish raised under standard laboratory conditions. During embryonic stages, low levels of each heavy chain isotype (IgH) were detected with each increasing steadily between 2 and 17 weeks post fertilization. Overall IgM>IgZ>IgD throughout zebrafish development with the copy number of IgM being several fold higher than that of IgD or IgZ/T. IgD exon usage was also characterized, as its extremely long size and presence of a stop codon in the second IgD exon in zebrafish, raised questions as to how this antibody might be expressed. Zebrafish IgD was found to be a chimeric immunoglobulin, with the third IgD exon spliced to the first IgM constant exon thereby circumventing the first and second IgD exons. Collectively, the qRT-PCR results represent the first comparative profile of IgD, IgM, IgZ/T expression over the lifespan of any fish species and the primers and assay parameters reported should prove useful in enabling researchers to rapidly quantify changes in IgH expression in zebrafish models of disease where altered IgH expression is manifested.

A monoclonal antibody distinguishes between two IgM heavy chain isotypes in Atlantic salmon and brown trout: Protein characterization, 3D modeling and epitope mapping

Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) possess two distinct subpopulations of IgM which can be separated by anion exchange chromatography. Accordingly, there are two isotypic μ genes in these species, related to ancestral tetraploidy. In the present work it was verified by mass spectrometry that IgM of peak 1 (subpopulation 1) have heavy chains previously designated as μB type whereas IgM of peak 2 (subpopulation 2) have heavy chains of μA type. Two adjacent cysteine residues are present near the C-terminal part of μB, in contrast to one cysteine residue in μA. Salmon IgM of both peak 1 and peak 2 contain light chains of the two most common isotypes: IgL1 and IgL3. In contrast to salmon and brown trout, IgM of rainbow trout (Oncorhynchus mykiss) is eluted in a single peak when subjected to anion exchange chromatography. Surprisingly, a monoclonal antibody MAb4C10 against rainbow trout IgM, reacted with μA in salmon, whereas in brown trout it reacted with μB. It is plausible to assume that DNA has been exchanged between the paralogous A and B loci during evolution while maintaining the two sub-variants, with and without the extra cysteine. MAb4C10 was conjugated to magnetic beads and used to separate cells, demonstrating that μ transcripts residing from captured cells were primarily of A type in salmon and B type in brown trout. An analysis of amino acid substitutions in μA and μB of salmon and brown trout indicated that the third constant domain is essential for MAb4C10 binding. This was supported by 3D modeling and was finally verified by studies of MAb4C10 reactivity with a series of recombinant μ3 constructs.

M protein deposition in the skin: a rare manifestation of Waldenström macroglobulinemia

Waldenstrom macroglobulinemia (WM) is defined by diffuse and nodular bone marrow infiltration of lymphoplasmacytic lymphoma as defined by the World Health Organization (WHO), and by a monoclonal IgM serum paraprotein (macroglobulin) [1]. Clinical symptoms at presentation are frequently unspecific, including fatigue, weakness, and weight loss. Clinical symptoms such as hyperviscosity syndrome, peripheral neuropathy, and glomerular disease are often related to the IgM paraprotein, both in form of circulating IgM and IgM deposition in tissue. The skin constitutes an unusual site of manifestation of WM, both for tumor infiltrates and IgM paraprotein deposition. A 70-year-old male patient was hospitalized in April 2009 with progressive peripheral neuropathy and poor general condition. Further examination revealed small, non-pruritic, soft indolent wart-like papules on both knees. As noted by the patient, the lesions were easily scratched off, bled slightly, leaving scabs and causing additional papules to appear during healing. Laboratory findings included slight anemia with a hemoglobin level of 8.2 mmol/l (normal 8.4–10.9 mmol/l) and a hematocrit reading of 0.379 l/l (normal 0.400–0.530 l/l). The RBC count was 4.22 9 10/l (normal 4.50–5.90 9 10/l) with 3% reticulocytes (normal 6–18%). The thrombocyte count and the differential white blood count were within the normal range. Renal, hepatic and cardial functions were also normal, and there was no splenomegaly or lymphadenopathy. The total protein level was 94.8 g/l (normal 66.0–87.0 g/l), and quantitative immunoglobulin findings were as follows: IgM 23.70 g/l (normal 0.40–2.80 g/l), j-light chains 5.63 g/l (normal 1.50–3.90 g/l), free j-light chains 284.00 mg/l (normal 3.30–19.40 mg/l), free k-light chains 13.90 mg/l (normal 5.71–26.30 mg/l) and a free j-light chains/k-light chains ratio of 20.43 (normal 0.26–1.65). Serum electrophoresis presented an elevated IgM gradient. Serum immunofixation provided a clear precipitation pattern of monoclonal IgM j-type immunoglobulin. The b2 microglobulin level was 1.7 mg/l (normal \2.0 mg/l). Bone marrow microscopy revealed atypical lymphoplasmocytic cells. Immunohistochemistry demonstrated low proliferation and B cell nature of the lymphocytes (25% of all hematopoietic cells) beside a mature CD138 positive plasma cell population (15%) with monotypic restriction for j-light chain and IgM heavy chain (Fig. 1). Given these findings, the diagnosis of Waldenstrom’s macroglobulinemia was established. Skin sections displayed multifocal nodular strongly eosinophilic, PASpositive and Elastica van Gieson, Congo red, and Alcian blue-negative homogeneous deposits in the upper portion of the dermis. Immunohistochemistry presented a faint monotypic staining for IgM and j-light chains, manifesting as multifocal nodular depositions of monoclonal immunoglobulin (Fig. 1). A diagnosis of macroglobulinemia cutis presenting as IgM storage papules was therefore established. To suppress disease activity, the patient received four cycles of Rituximab-CHOP, during which the cutaneous lesions disappeared. B. Oberschmid U. Siebolts (&) A. Beller C. Wickenhauser Institute of Pathology, University of Leipzig, Leipzig, Germany e-mail: udo.siebolts@medizin.uni-leipzig.de

A Study on the Presence of Ferritin-binding Proteins in Fetal Horse Plasma.

In mammal circulation, ferritin-binding proteins (FBPs) are thought to be involved in clearance of circulating ferritin after complex formation with it through receptor-mediated uptake. However, there is no report on fetal FBP in fetal circulation. Although iron concentrations of fetal horse plasma were higher than those of adult horse plasma, plasma ferritin concentrations and ferritin-binding activities were found to be significantly lower in fetus than in adult. FBPs were purified from fetal or adult horse plasma on horse spleen ferritin-Sepharose 4B affinity column. Partially affinity-purified fetal horse plasma FBPs were mainly separated into 65 and 41 kDa bands in addition to minor bands with higher molecular masses ranged from 102 to 140 kDa on SDS-PAGE under reducing condition. The adult horse plasma FBPs were separated into 74, 54 and 28 kDa bands, and the 74 and 54 kDa bands reacted with antibodies specific for horse IgM and IgG heavy chains, respectively, by immunoblotting analyses. On the other hand, no antibodies to horse immunoglobulin classes detected any bands in fetal horse plasma FBPs. The affinity-purified adult and fetal horse plasma FBPs did not contain fibrinogen as a plasma specific FBP, probably due to its lower affinity to the ligand ferritin. These results demonstrate the presence of FBPs which are different from adult horse plasma FBPs including anti-ferritin autoantibodies in fetal plasma.

Decrease in antibody somatic hypermutation frequency under extreme, extended spaceflight conditions

Somatic hypermutation diversifies antibody binding sites by introducing point mutations in the variable domains of rearranged immunoglobulin genes. In this study, we analyzed somatic hypermutation in variable heavy-chain (VH) domains of specific IgM antibodies of the urodele amphibian Pleurodeles waltl, immunized either on Earth or onboard the Mir space station. To detect somatic hypermutation, we aligned the variable domains of IgM heavy-chain transcripts with the corresponding VH gene. We also quantified NF-κB and activation-induced cytidine deaminase transcripts. Results were compared with those obtained using control animals immunized on Earth. Our data show that, as in most species of ectotherms, somatic hypermutation in P. waltl exhibits a mutational bias toward G and C bases. Furthermore, we show for the first time that somatic hypermutation occurs in space following immunization but at a lower frequency. This decrease is not due to a decrease in food intake or of the B-cell receptor/antigen interaction or to the absence of the germinal center-associated nuclear protein. It likely results from the combination of several spaceflight-associated changes, such as the severe reduction in T-cell activation, important perturbations of the cytoskeleton, and changes in the distribution of lymphocyte subpopulations and adhesion molecule expression.

Lymphocytes with T-cell-like properties express the Fas ligand in the Japanese flounder Paralichthys olivaceus

In this study, we aimed to identify the leukocyte population that expresses Fas ligand (FasL) in the Japanese flounder ( Paralichthys olivaceus). The transcriptional activity of FasL was examined for the first time in the fish leukocytes. Transcription of the FasL gene in flounder leukocytes was significantly increased by phytohemagglutinin (PHA) treatment. All the leukocyte populations we tested possessed binding activity for PHA, but this was especially high in the lymphocyte population. However, the lymphocytes consisted of two subsets showing heterogeneity with respect to PHA binding, with the high-binding subset being surface IgM-negative. We also found that only the lymphocyte population showed a significant increase in the expression of the FasL gene after stimulation with PHA. In addition, only the lymphocyte subset showing high binding to PHA showed conspicuous expression of the FasL gene. This subset also had a CD3γ/δ (+), CD8α (+) and IgM heavy-chain (−) phenotype. These results suggested that lymphocytes with T-cell-like properties are FasL-expressing cells in the Japanese flounder.

Molecular cloning of IgT from Atlantic salmon, and analysis of the relative expression of τ, μ and δ in different tissues

In the present study, IgT genes of Atlantic salmon were cloned and characterised. Analysis of our sequence data as well as ESTs reported to the databases revealed three distinct IgT heavy chain sub-variants in salmon, as opposed to two of IgM and IgD. The IgT sub-variants in salmon are 76–80% identical to each other, and 75–82% identical to the reported rainbow trout sequences, whereas the similarity to the orthologous molecules in zebrafish, grass carp, mandarin fish, and grouper is 25–41%. The heavy chains of both secreted and membrane anchored forms of salmon IgT include four constant Ig domains, τ1–τ4. This parallels the IgM heavy chains in elasmobranch fish and higher vertebrates, but differs from IgM in teleost fish where the membrane anchored form include only three constant Ig domains, μ1–μ3. The similarity between τ1 and μ1 in salmon is relatively high (52%) when compared to the remaining part of the molecules (τ2–τ4 and μ2–μ4 are 13–24% similar). To compare τ, μ and δ expressions in different tissues (head kidney, thymus, spleen, gill, skin, hind gut, brain and muscle) of Atlantic salmon, RT-qPCR assays were designed and evaluated. The analyses revealed that IgM transcripts are most abundant (up to 200 times more than IgD) followed by IgT (up to 20 times more than IgD) in most tissues. Highest expression of IgM, IgT, and IgD was in head kidney and spleen.

CDNA cloning of the immunoglobulin heavy chain genes in banded houndshark Triakis scyllium

In this study, cDNAs encoding the secreted forms of the immunoglobulin (Ig) heavy chains of IgM, IgNAR, and IgW were cloned from the banded houndshark Triakis scyllium. Two clones for the IgM heavy chains encoded 569 and 570 amino acids, whose conserved (C) region showed 47–70% amino acid identities to those reported in other cartilaginous fish. Four clones for the IgNAR encoded 673–670 amino acids with conserved Ig-superfamily domains. The IgNAR C region showed 56–69% amino acid identities to those so far reported. High-throughput sequencing revealed that in most of the IgNAR sequences, the two variable regions (CDR1 and CDR3) each possess a cysteine residue. Three types of IgW were identified; one contained Ig-superfamily domains that are in other cartilaginous fish, one lacks the 3rd domain in the constant region, and one lacks the 3rd to 5th domains. Despite these differences, the IgW isoforms clustered with IgWs of other cartilaginous fishes and the C regions showed 47–89% amino acid identities. mRNAs for IgM and IgNAR were detected in various tissues, while IgW mRNA was mainly detected in pancreas. The banded hounded shark also has IgM, IgW and IgNAR as well as the other cartilaginous fish with unique IgW isoform.

IgM Expression on Paraffin Sections Distinguishes Primary Cutaneous Large B-cell Lymphoma, Leg Type From Primary Cutaneous Follicle Center Lymphoma

In the World Health Organization (WHO) 2008 classification 2 main types of primary cutaneous large B-cell lymphomas (PCLBCLs) are distinguished: primary cutaneous follicle center lymphoma (PCFCL) and primary cutaneous large B-cell lymphoma, leg type (PCBCL-LT). PCFCL has a 5-year overall survival rate of 95%, and PCBCL-LT of approximately 50%. Expression profiling studies have shown higher RNA expression of the IgM heavy chain in PCBCL-LT compared with PCFCL. To find out whether this difference could also be demonstrated at the protein level, we performed immunohistochemical staining for B-cell receptor heavy and light chains on skin biopsies from 53 patients with PCFCL and 40 patients with PCBCL-LT. All 40 cases of PCBCL-LT consistently showed cytoplasmic staining for IgM, in 18 of them with coexpression of IgD. In contrast, only 5 of the PCFCL cases showed cytoplasmic staining for IgM and/or IgD, including all 3 PCFCLs presenting on the leg. Hence, staining for IgM on paraffin-embedded sections seems to be an additional tool for differentiating between the 2 entities in clinical pathology practice. Analogous to other nodal and extranodal large B-cell lymphomas, expression of IgM in PCLBCL seems to be related to an activated B cell-like phenotype. Finally, the expression of IgM (and IgD) in this type of lymphoma might imply defective class switch recombination.

Evolution of the Antarctic teleost immunoglobulin heavy chain gene

Notothenioid teleosts underwent major modifications of their genome to adapt to the cooling of the Antarctic environment. In order to identify specific features of the Antarctic teleost immunoglobulin, transcripts encoding the constant region of the IgM heavy chain from 13 Antarctic and non-Antarctic notothenioid species were sequenced. The primary mRNA splicing for the membrane form was found to be atypical in the majority of Antarctic species, because it led to exclusion of two entire constant exons, and to inclusion of 39-nucleotide exons encoding an unusually long Extracellular Membrane-Proximal Domain (EMPD). Genomic DNA analysis revealed that each 39-nucleotide exon fell within a long sequence that was the reverse complement of an upstream region. Deduced amino acid sequence analysis lead to the identification of cysteine encoding codons in the 39-nucleotide exons, but not in the respective sequence counterpart, suggesting that these residues might play an important role in the folding of the EMPD.

UK Myeloma Forum (UKMF) and Nordic Myeloma Study Group (NMSG): guidelines for the investigation of newly detected M‐proteins and the management of monoclonal gammopathy of undetermined significance (MGUS)

Avon Haematology Unit, Bristol Haematology and Oncology Centre, Bristol, Department of Haematology, St Helier Hospital, Carshalton, Surrey, UK, Department of Haematology, Sahlgrenska University Hospital, Gothenburg, Sweden, Department of Medicine, Malmo University Hospital, Malmo, Sweden, Division of Immunity and Infection, University of Birmingham, Birmingham, Department of Clinical Biochemistry, Frenchay Hospital, Bristol, Department Haematology, University College, London, Glasgow Western Infirmary, Glasgow, UK, Department of Haematology, NTNU/St Olavs Hospital, Trondheim, Department of Haematology, Ulleval University Hospital, Oslo, Norway, Department of Haematology Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark, and Myeloma UK.