Isolation and comparison of the IgM heavy chain constant regions from Australian (Trichosurus vulpecula) and American (Monodelphis domestica) marsupials

cDNAs encoding IgM heavy chain constant region (Cμ) were isolated from two metatherians (marsupials) — the Australian common brushtail possum (Trichosurus vulpecula) and the South American grey short-tailed opossum (Monodelphis domestica). Analysis of the sequences suggested that they correspond to the secreted form of Cμ in both species. The domain size and structure of the marsupial Cμ sequences were compared with other Cμ sequences and a high degree of conservation throughout vertebrate evolution was observed. Amino acid sequence comparisons revealed a marked level of sequence similarity between the two marsupial sequences (79%), relatively high similarity between the marsupials and eutherians (63%), and lower similarities between marsupials and birds (45%), marsupials and amphibians (47%), marsupials and reptiles (45%) and marsupials and fish (37%). These data allow the incorporation of metatherians into the study of mammalian IgM evolution.     

Characterisation of echidna IgM provides insights into the time of divergence of extant mammals

The immunobiology of monotremes is poorly understood. In this paper, we describe the characterisation of the heavy chain of IgM from Tachyglossus aculeatus, the short-beaked echidna. The echidna heavy chain constant region of IgM (Cμ) was isolated from a spleen cDNA library using a Trichosurus vulpecula probe. It has approximately 46.5% amino acid identity to marsupial and eutherian Cμs, and approximately 30% amino acid identity with Cμs from birds and reptiles. Phylogenetic analysis of mammalian Cμ provides strong support for the Theria hypothesis, with a sister grouping of the eutherians and marsupials to the exclusion of the monotremes. Cμ sequences suggest that monotremes and therians separated approximately 170 million years ago (mya), marsupials and eutherians separated approximately 130 mya, and Australian and American marsupials separated approximately 65 mya.     

Cloning and structural analysis of IgM (μ chain) and the heavy chain V region repertoire in the marsupial Monodelphis domestica

To address the question of the Ig isotype repertoire of non placental mammals, we have examined the Ig expression in the marsupial Monodelphis domestica (grey short tailed opossum). Screening of an opossum spleen cDNA library has previously led to the isolation of full length clones for opossum IgG (γ chain), IgE ( chain) and IgA (α chain). We now present the isolation of several cDNA clones encoding the entire constant regions of the opossum IgM (μ chain). A comparative analysis of the amino acid sequences for IgM from various animal species showed that opossum IgM, within the various animals studied, is the most divergent member of its Ig class. However, it still conforms to the general structure of IgM in other vertebrates. Four Ig classes have now been identified in opossum and only one isotype is apparently present within each Ig class, IgM, IgG, IgA and IgE. Opossum has previously been shown to have a limited VH region diversity, with only two V gene families. Both of these belong to the group III of mammalian VH sequences. This limitation in variability is to some extent compensated for by a large variation in D, P and N regions, both in size and in sequence. However, evidence for the expression of only two functional J segments has so far been detected, which indicates a rather limited diversity also of the J segments in the opossum.     

Karyotype relationships between distantly related marsupials from South America and Australia

Reciprocal chromosome painting and G-banding were used to compare the karyotypes of three Australian marsupials (Sminthopsis crassicaudata, Macropus eugenii, Trichosurus vulpecula) and one South American marsupial (Monodelphis domestica). The results revealed only a limited number of rearrangements between these species and that the four karyotypes can be described as different combinations of fifteen conserved segments. Five chromosomes are totally conserved between M. domestica (pairs 1, 2, 5, 8 and the X) and the presumed 2n = 14 Australian ancestral karyotype, while M. domestica pairs 3 and 6 and 4 and 7 would have been involved in fusion/fission rearrangements. Chromosome comparisons are presented in a chromosome homology map. Although the species studied diverged 70 million years ago, the karyotype of Monodelphis domestica is highly conserved in relation to those of Australian marsupials. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nucleotide sequences and characterization of haemolysin genes from Aeromonas hydrophila and Aeromonas sobria

Extracellular haemolysin is thought to be one of the important virulence factors in Aeromonas infection. Two extracellular haemolysin genes (AHH3 and AHH4) from Aeromonas hydrophila strain 28SA, one (AHH5) from A. hydrophila strain AH-1 and one (ASA1) from Aeromonas sobria strain 33 were cloned into cosmid and plasmid vector DNA in Escherichia coli. The nucleotide sequences of the open reading frames of AHH3 and AHH4 are both 1476 basepairs (bp), whereas AHH5 and ASA1 are 1455 and 1467 bp in length, respectively. The deduced amino acid sequences of AHH3, AHH4, AHH5 and the previously reported aerolysin from A. hydrophila showed a significant degree of sequence homology of over 90% each. The amino acid identity of the ASA1 haemolysin and those from A. hydrophila and Aeromonas trota aerolysins ranged from 58–68%. From DNA hybridization analysis using our cloned haemolysin genes as probes, we found that the AHH5 and ASA1 DNA probes hybridized with about 31 and 75% strains of motile Aeromonas species, respectively. The activity of haemolysins of cloned genes were different in medium agar containing various erythrocytes.     

Molecular cloning of the brushtail possum (Trichosurus vulpecula) immunglobulin E heavy chain constant region

The immunobiology of marsupial IgE is poorly understood. As a first step towards the development of immunological reagents for marsupials and to obtain a further understanding of immunoglobulin evolution, a brushtail possum (Trichosurus vulpecula) mesenteric lymph node cDNA library was screened for the heavy chain constant region of IgE (Cε), using a partial Cε probe from the American marsupial, Monodelphis domestica. The cDNA sequence for T. vulpecula Cε was determined and found to be most similar to the M. domestica Cε sequence [(76%) at the amino acid level]. T. vulpecula Cε has amino acid sequence similarities ranging from 43–52% with various eutherian Cε sequences. The secondary structure of T. vulpecula Cε, based on loops formed by internal disulfide bonds, more closely resembles rodent Cε than the American marsupial sequence.     

Intraspecific variation in the distribution of the interstitial telomeric (TTAGGG)n sequences in Micoureus demerarae (Marsupialia: Didelphidae)

The distribution of the telomeric sequence (TTAGGG)_n was studied in chromosomes of Micoureus demerarae (2n=14), a South American marsupial, by fluorescence in-situ hybridization (FISH). The telomeric repeat sequence was present at both ends of all chromosomes, but also various interstitial telomeric sequences (ITS) were detected in the pericentromeric heterochromatic regions. Intraspecific differences in the number of ITS (2 to 8) were observed without intraindividual variation. The presence of telomere-like sequences in the same regions of constitutive heterochromatin suggest that these segments are not necessarily remnants of true telomeres resulting from chromosome rearrangements but could be part of the satellite DNA.     

SCN5A variants in Japanese patients with left ventricular noncompaction and arrhythmia

Left ventricular noncompaction (LVNC) is a genetically heterogenous disorder. Mutations in the human cardiac sodium channel alpha-subunit gene (SCN5A) are involved in the pathophysiology of cardiac arrhythmias and cardiomyopathies. This study was performed to compare the frequency of SCN5A variants in LVNC patients with or without arrhythmias, and to investigate the relationship between variants and disease severity. DNA was isolated from the peripheral blood of 62 Japanese probands with LVNC, comprising 17 familial cases and 45 sporadic cases. Blood samples were screened for variants in SCN5A using single-strand conformational polymorphism analysis (SSCP) and DNA sequencing. Seven variants, rs6599230:G > A, c.453C > T, c.1141-3C > A, rs1805124:A > G (p.H558R), rs1805125:C > T (p.P1090L), c.3996C > T, and rs1805126:T > C were identified in 7 familial and 12 sporadic cases. The frequency of SCN5A variants was significantly higher in the patients with arrhythmias than those without (50% vs 7%: P = 0.0003), suggesting these variants represent a risk factor for arrhythmia and supporting the hypothesis that genes encoding ion channels are involved in LVNC pathophysiology. The LVNC patients with heart failure also had high occurence of SCN5A variants, suggesting the presence of SCN5A variants and/or arrhythmias increase the severity of LVNC.     

Studies on the structural and biological functions of the Cμ3 and Cμ4 domains of IgM

The development of methods for the production of intact Cμ3 and Cμ4 domains of IgM have made possible the assessment of some of their structural and biological functions. Antiserum against Fcμ fragment detected both domains and illustrated their complete antigenic non-identity. Circular dichroism spectroscopy and the retention of antigenicity indicated that both domains had retained most of their native structure. No interaction of the type Cμ3—Cμ3, Cμ4—Cμ4 or Cμ3—Cμ4 could be detected under non-dissociating conditions by analytical ultracentrifugation or molecular exclusion chromatography experiments. These results lead us to believe that the transmission of effector messages between the Fab and Fc parts of IgM takes place through structural changes at the quaternary level.

C[unk]1-fixation experiments with IgM and several of its fragments and domains show that (a) the Cμ4 domain contains the C[unk]1-fixing site; (b) the high C[unk]1-fixing capacity of IgM or Fc₅μ cannot be explained on the basis of a simple accumulative model of complement fixing domains; (c) the C[unk]1-fixing site is independent of the native structure of the Cμ4 domain; (d) the C[unk]1-fixing site does not contain carbohydrate.

Examination of the IgM receptor on the surface of human T lymphocytes show that (a) Cμ4 domain is primarily responsible for the reaction and Cμ3 domain has very little affinity; (b) native structure is essential for the reaction because reduction and alkylation of the Cμ4 domain destroyed both its original conformation and affinity for this receptor; (c) IgM and Fc₅μ had a much greater affinity for the receptor than monomeric subunits: (d) carbohydrate on Cμ4 domain is not involved in the affinity reaction.

     

Specific patterns of histone marks accompany X chromosome inactivation in a marsupial

The inactivation of one of the two X chromosomes in female placental mammals represents a remarkable example of epigenetic silencing. X inactivation occurs also in marsupial mammals, but is phenotypically different, being incomplete, tissue-specific and paternal. Paternal X inactivation occurs also in the extraembryonic cells of rodents, suggesting that imprinted X inactivation represents a simpler ancestral mechanism. This evolved into a complex and random process in placental mammals under the control of the XIST gene, involving notably variant and modified histones. Molecular mechanisms of X inactivation in marsupials are poorly known, but occur in the absence of an XIST homologue. We analysed the specific pattern of histone modifications using immunofluorescence on metaphasic chromosomes of a model kangaroo, the tammar wallaby. We found that all active marks are excluded from the inactive X in marsupials, as in placental mammals, so this represents a common feature of X inactivation throughout mammals. However, we were unable to demonstrate the accumulation of inactive histone marks, suggesting some fundamental differences in the molecular mechanism of X inactivation between marsupial and placental mammals. A better understanding of the epigenetic mechanisms underlying X inactivation in marsupials will provide important insights into the evolution of this complex process. Edda Koina and Julie Chaumeil contributed equally to this work.     

Effect of nitrofurans and NO generators on biofilm formation by Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia 370

Antibacterial drugs in the nitrofuran series, such as nitrofurazone, furazidin, nitrofurantoin and nifuroxazide, as well as the nitric oxide generators sodium nitroprusside and isosorbide mononitrate in concentrations that do not suppress bacterial growth, were shown to increase the capacity of pathogenic bacteria Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia 370 to form biofilms. At 25–100 μg/ml, nitrofurans 2–2.5-fold enhanced biofilm formation of P. aeruginosa PAO1, and NO donors 3–6-fold. For B. cenocepacia 370, the enhancement was 2–5-fold (nitrofurans) and 4.5-fold (sodium nitroprusside), respectively.     

Ongoing hypermutation in the Ig V(D)J gene segments and c-myc proto-oncogene of an AIDS lymphoma segregates with neoplastic B cells at different sites: implications for clonal evolution

To investigate the role of somatic Ig hypermutation in the evolution of AIDS-associated B cell lymphomas, we analyzed the Ig V(D)J and c-myc genes expressed by neoplastic B cells in two extranodal sites, testis and orbit, and clonally related cells in the bone marrow. Testis and orbit B cells expressed differentially mutated but collinear V_HDJ_H, VκJκ and c-myc gene sequences. Shared mutations accounted for 10.2%, 8.4%, and 4.3% of the overall V_HDJ_H, VκJκ, and c-myc gene sequences. Tumor-site specific V_HDJ_H, VκJκ, and c-myc mutations were comparable in frequency, and a single point-mutation gave rise to an EcoRI site in the testis c-myc DNA. Both shared and tumor site-specific V_HDJ_H, VκJκ, and c-myc mutations displayed predominance of transitions over transversions. The “neoplastic” V_HDJ_H sequence was expressed by about 10⁻⁵ cells in the bone marrow, and contained two of the three orbital, but none of the testicular V_HDJ_H mutations. The nature and distribution of the Ig V(D)J mutations found in the κ chain suggested a selection by antigen in testis and orbit. Our data suggest that, in AIDS-associated B cell lymphomas, the Ig hypermutation machinery targets V_HDJ_H, VκJκ, and c-myc genes with comparable efficiency and modalities.     

Molecular cloning of the cDNA encoding the constant region of the immunoglobulin A heavy chain (Cα) from a marsupial: Trichosurus vulpecula (common brushtail possum)

A cDNA encoding the brushtail possum immunoglobulin A heavy chain constant region (Cα) was isolated by screening a mesenteric lymph node cDNA library with a porcine Cα exon 3 probe. The larger of the two positive clones isolated (Tv4a) consisted of 1325 bp of possum cDNA that included an open reading frame of 1191 bp. Its deduced amino acid sequence had a high degree of sequence identity with known eutherian Cα sequences. This clone appears to encode the entire possum IgA heavy chain constant region. The possum Cα sequence had a nucleotide sequence identity of 57.7% with porcine Cα, 51% with mouse Cα, 46.7% with dog Cα and 45.9% with human Cα2. The corresponding amino acid identities were 46.7, 45.6, 49.4 and 49%, respectively.     

Interaction of Leishmania mexicana promastigotes with the plasminogen–plasmin system

The binding of human plasminogen and plasmin to the promastigote form of Leishmania mexicana was investigated. L. mexicana was capable to bind both molecules, the binding being inhibited by -aminocaproic acid. Scatchard plot analysis revealed a dissociation constant (K_d) value of 2.4±0.8 μM and 0.9±0.1×10⁴ binding sites per cell for plasminogen and a K_d value of 1.2±0.4 μM and 1.6±0.2×10⁵ binding sites per cell for plasmin. C-terminal lysine residues are involved in plasminogen binding to cells, since carboxypeptidase B treatment reduced this binding by 34%. Ligand blotting analysis showed a group of proteins, with molecular masses between 105 and 115 kDa, capable to interact with plasminogen. Zymogram analysis showed that the protease activity acquired by L. mexicana, due to the interaction with either plasminogen or plasmin, comprises an important fraction of the total protease activity at pH 7.7. Plasminogen activation by tissue-type plasminogen activator (t-PA) was enhanced by the presence of L. mexicana promastigotes. These results raise the question whether the interaction of L. mexicana with components of the fibrinolytic system is involved in the virulence of the parasite.