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Receptors interacting with the constant domain of immunoglobulins (Igs) have a number of important functions in vertebrates. They facilitate phagocytosis by opsonization, are key components in antibody-dependent cellular cytotoxicity as well as activating cells to release granules. In mammals, four major types of classical Fc receptors (FcRs) for IgG have been identified, one high-affinity receptor for IgE, one for both IgM and IgA, one for IgM and one for IgA. All of these receptors are related in structure and all of them, except the IgA receptor, are found in primates on chromosome 1, indicating that they originate from a common ancestor by successive gene duplications. The number of Ig isotypes has increased gradually during vertebrate evolution and this increase has likely been accompanied by a similar increase in isotype-specific receptors. To test this hypothesis we have performed a detailed bioinformatics analysis of a panel of vertebrate genomes. The first components to appear are the poly-Ig receptors (PIGRs), receptors similar to the classic FcRs in mammals, so called FcRL receptors, and the FcR gamma chain. These molecules are not found in cartilagous fish and may first appear within bony fishes, indicating a major step in Fc receptor evolution at the appearance of bony fish. In contrast, the receptor for IgA is only found in placental mammals, indicating a relatively late appearance. The IgM and IgA/M receptors are first observed in the monotremes, exemplified by the platypus, indicating an appearance during early mammalian evolution. Clearly identifiable classical receptors for IgG and IgE are found only in marsupials and placental mammals, but closely related receptors are found in the platypus, indicating a second major step in Fc receptor evolution during early mammalian evolution, involving the appearance of classical IgG and IgE receptors from FcRL molecules and IgM and IgA/M receptors from PIGR.

Serine proteases are among the most abundant granule constituents of several hematopoietic cell lineages including mast cells, neutrophils, cytotoxic T cells and NK cells. These proteases are stored in their active form in the cytoplasmic granules and in mammals are encoded from four different chromosomal loci: the chymase locus, the met-ase locus, the T cell tryptase and the mast cell tryptase locus. In order to study their appearance during vertebrate evolution we have performed a bioinformatic analysis of related genes and gene loci from a large panel of metazoan animals from sea urchins to placental mammals for three of these loci: the chymase, met-ase and granzyme A/K loci. Genes related to mammalian granzymes A and K were the most well conserved and could be traced as far back to cartilaginous fish. Here, the granzyme A and K genes were found in essentially the same chromosomal location from sharks to humans. However in sharks, no genes clearly identifiable as members of the chymase or met-ase loci were found. A selection of these genes seemed to appear with bony fish, but sometimes in other loci. Genes related to mammalian met-ase locus genes were found in bony fish. Here, the most well conserved member was complement factor D. However, genes distantly related to the neutrophil proteases were also identified in this locus in several bony fish species, indicating that this locus is also old and appeared at the base of bony fish. In fish, a few of the chymase locus-related genes were found in a locus with bordering genes other than the mammalian chymase locus and some were found in the fish met-ase locus. This indicates that a convergent evolution rather than divergent evolution has resulted in chymase locus-related genes in bony fish.

Chymases are chymotrypsin-like serine proteases that are found in large amounts in mast cell granules. So far, the extended cleavage specificities of eight such chymases have been determined, and four of these were shown to have a strong preference for acidic amino acids at position P2'. These enzymes have basic amino acids in positions 143 and 192 (Arg and Lys, respectively). We therefore hypothesized that Arg143 and Lys192 of human chymase mediate the preference for acidic amino acids at position P2' of substrates. In order to address this question, we performed site-directed mutagenesis of these two positions in human chymase. Analysis of the extended cleavage specificities of two single mutants (Arg143 -> Gln and Lys192 -> Met) and the combined double mutant revealed an altered specificity for P2' amino acids, whereas all other positions were essentially unaffected. A weakened preference for acidic amino acids at position P2' was observed for the two single mutants, whereas the double mutant lacked this preference. Therefore, we conclude that positions 143 and 192 in human chymase contribute to the strong preference for negatively charged amino acids at position P2'. This is the first time that a similar combined effect has been shown to influence the cleavage specificity, apart from position P1, among the chymases. Furthermore, the conservation of the preference for acidic P2' amino acids for several mast cell chymases clearly indicates that other substrates than angiotensin I may be major in vivo targets for these enzymes.

Antibodies against type II collagen (CII) are essential for development of collagen-induced arthritis (CIA), but how and where the B-cell response to CII is initiated is not fully known. We show here that naive DBA/1 mice display naturally reactive IgM and IgG anti-CII producing B cells prior to immunization. The CII-reactive B cells were observed in the spleen and recognized as marginal zone (MZ) B cells. After CII immunization, CII-specific B cells expanded rapidly in the spleen, in contrast to the lymph nodes, with the initial response derived from MZ B cells and later by follicular (FO) B cells. This was evident despite that the MZ B cells were subject to stringent tolerance mechanisms by having a greater Fc gamma receptor IIb expression than the FO B cells. Further, the MZ B cells migrated to the FO areas upon immunization, possibly providing antigen and activating FO T cells and subsequently FO B cells. Thus, around CIA onset increased numbers of IgG anti-CII producing FO B cells was seen in the spleen, which was dominated by IgG2a- and IgG2b-positive cells. These data demonstrate that CII-reactive MZ B cells are present before and expand after CII immunization, suggesting an initiating role of MZ B cells in the development of CIA.

Thrombin is a serine protease of the chymotrypsin family that acts both as a procoagulant and as an anticoagulant by cleaving either factor VIII, factor V and fibrinogen or protein C, respectively. Numerous previous studies have shown that electropositive regions at a distance from the active site, so called exosites, are of major importance for the cleavage by human thrombin. Upstream of all the known major cleavage sites for thrombin in factor VIII, factor V and fibrinogen are clusters of negatively charged amino acids. To study the importance of these sites for the interaction with the exosites and thereby the cleavage by thrombin, we have developed a new type of recombinant substrate. We have compared the cleavage rate of the minimal cleavage site, involving only 8-9 amino acids (typically the P4-P4' positions) surrounding the cleavage site, with the substrates also containing the negatively charged regions upstream of the cleavage sites. The results showed that addition of these regions enhanced the cleavage rate by more than fifty fold. However, the enhancement was highly dependent on the sequence of the actual cleavage site. A minimal site that showed poor activity by itself could be cleaved as efficiently as an optimal cleavage site when presented together with these negatively charged regions. Whereas sites conforming closely to the optimal site were only minimally enhanced by the addition of these regions. The possibility to mimic this interaction for the sites in factor V and factor VIII by recombinant substrates, which do not have the same folding as the full size target, indicates that the enhancement was primarily dependent on a relatively simple electrostatic interaction. However, the situation was very different for fibrinogen and protein C where other factors than only charge is of major importance.

We provide the genome sequences of the type strains of the polychlorophenol-degrading Mycobacterium chlorophenolicum (DSM43826), the degrader of chlorinated aliphatics Mycobacterium chubuense (DSM44219) and Mycobacterium obuense (DSM44075) that has been tested for use in cancer immunotherapy. The genome sizes of M. chlorophenolicum, M. chubuense and M. obuense are 6.93, 5.95 and 5.58 Mbps with GC-contents of 68.4, 69.2 and 67.9%, respectively. Comparative genomic analysis revealed that 3254 genes are common and we predicted approximately 250 genes acquired through horizontal gene transfer from different sources including proteobacteria. The data also showed that the biodegrading Mycobacterium spp. NBB4, also referred to as M. chubuense NBB4, is distantly related to the M. chubuense type strain and should be considered as a separate species, we suggest it to be named M. ethylenense NBB4. Among different categories we identified genes with potential roles in: biodegradation of aromatic compounds, and copper homeostasis. These are the first non-pathogenic Mycobacterium spp. found harboring genes involved in copper homeostasis. These findings would therefore provide insight into the role of this group of Mycobacterium spp. in bioremediation as well as the evolution of copper homeostasis within the Mycobacterium genus.

Mycobacterium phlei, a nontuberculosis mycobacterial species, was first described in 1898-1899. We present the complete genome sequence for the IV, phlei CCUG21000(T) type strain and the draft genomes for four additional strains. The genome size for all five is 5.3 Mb with 69.4% Guanine-Cytosine content. This is approximate to 0.35 Mbp smaller than the previously reported M. phlei RIVM draft genome. The size difference is attributed partly to large bacteriophage sequence fragments in the M. phlei RIVM genome. Comparative analysis revealed the following: 1) A CRISPR system similar to Type 1E (cas3) in M. phiei RIVM; 2) genes involved in polyamine metabolism and transport (potAD, potT) that are absent in other mycobacteria, and 3) strain specific variations in the number of sigma-factor genes. Moreover, M. phlei has as many as 82 mce (mammalian cell entry) homologs and many of the horizontally acquired genes in M. phlei are present in other environmental bacteria including mycobacteria that share similar habitat. Phylogenetic analysis based on 693 Mycobacterium core genes present in all complete mycobacterial genomes suggested that its closest neighbor is Mycobacterium smegmatis JS623 and Mycobacterium rhodesiae NBB3, while it is more distant to M. smegmatis mc2 155.

Background Deadenylation regulates RNA function and fate. Poly(A)-specific ribonuclease (PARN) is a deadenylase that processes mRNAs and non-coding RNA. Little is known about the biological significance of germline mutations in PARN. Methods We identified mutations in PARN in patients with haematological and neurological manifestations. Genomic, biochemical and knockdown experiments in human marrow cells and in zebrafish have been performed to clarify the role of PARN in the human disease. Results We identified large monoallelic deletions in PARN in four patients with developmental delay or mental illness. One patient in particular had a severe neurological phenotype, central hypomyelination and bone marrow failure. This patient had an additional missense mutation on the non-deleted allele and severely reduced PARN protein and deadenylation activity. Cells from this patient had impaired oligoadenylation of specific H/ACA box small nucleolar RNAs. Importantly, PARN-deficient patient cells manifested short telomeres and an aberrant ribosome profile similar to those described in some variants of dyskeratosis congenita. Knocking down PARN in human marrow cells and zebrafish impaired haematopoiesis, providing further evidence for a causal link with the human disease. Conclusions Large monoallelic mutations of PARN can cause developmental/mental illness. Biallelic PARN mutations cause severe bone marrow failure and central hypomyelination.

We describe the design, synthesis and biological evaluation of conformationally-locked 5'-acyl sulfamoyl adenosine derivatives as new parasitic inhibitors against Trypanosoma and Leishmania. The conformationally-locked (3'-endo, North-type) nucleosides have been synthesized by covalently attaching a 4'-CH2-O-2' bridge (Fig. 2) across C2'-C4' of adenosine in order to reduce the conformational flexibility of the pentose ring. This is designed to decrease the entropic penalty for complex formation with the target protein, which may improve free-energy of stabilization of the complex leading to improved potency. Conformationally-locked 5'-acyl sulfamoyl adenosine derivatives (16-22) were tested against parasitic protozoans for the first time in this work, and showed potent inhibition of Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma rhodesiense and Leishmania infantum with IC50 = 0.25-0.51 mu M. In particular, the potent 5'-pentanyl acyl sulfamoyl adenosine derivative 17 (IC50 = 0.25 mu M) against intracellular L. infantum amastigotes and Trypanosoma subspecies is interesting in view of its almost insignificant cytotoxicity in murine macrophage host cells (CC50 >4 mu M) and in diploid human fibroblasts MRC-5 cell lines (CC50 4 mu M). This work also suggests that variable alkyl chain length of the acyl group on the acylsulfamoyl side chain at 5' can modulate the toxicity of 5'-O-sulfamoylnucleoside analogues. This conformationally-locked sulfamoyl adenosine scaffold presents some interesting possibilities for further drug design and lead optimization.

The conformationally-locked carbocyclic nucleosides carbaLNA ("jcLNA") (Gagnon et al., Biochemistry, 2010, 49, 10166; Srivastava et al., J. Am. Chem. Soc., 2007, 129, 8362; Xu et al., J. Org. Chem., 2009, 74, 6534; Zhou and Chattopadhyaya, J. Org. Chem., 2010, 75, 2341; Zhou et al., J. Org. Chem., 2009, 74, 118) are chemically engineered by fusing a carbocyclic ring at the C2' to C4' chiral centres in a stereospecific manner at the alpha-face of the pentose-sugar of the native nucleosides. The benefit of the chemically-modified oligonucleotides with the jcLNA scaffold has been shown to be their uniquely enhanced nuclease resistance in the blood serum as well as their improved RNase H recruitment capability to cleave the target RNA in the hybrid antisense-RNA duplex when used as an antisense agent, compared to those of locked nucleic acid (LNA) modified counterparts. Herein we report the relative inhibition efficiency of HIV-1 by jcLNA modified siRNAs targeting TAR region compared to those of the LNA counterparts, in that the former were found to exhibit improved silencing efficiency and displayed enhanced stability in human serum with negligible cytotoxicity compared to those of the latter. A single jcLNA substitution as the 3'-overhang of the guide strand displayed near native-like IC50 value (of 4.01 +/- 0.87 nM compared to the nearly two-fold higher IC50 value of 7.15 +/- 1.57 nM for LNA modified counterparts, and of the native siRNA of 1.84 +/- 0.16 nM) and significantly higher hp value for the stability in serum (11.9 h for jcLNA, 6.8 h for LNA and 3.0 h for native), thereby showing that the efficiency of jcLNA-modified-siRNAs is supported by stability without compromising the native-like efficiency and target RNA recognition and subsequent down-regulation. Amongst all the modified siRNAs so far used to target HIV-1 TAR region, the best IC50 value was obtained for the doubly-modified siRNA in which jcLNA substitution was introduced both at position 1 and 20 of the antisense strand (T-1 + T-20, i.e. jcLNA11 which showed IC50 value of 0.54 +/- 0.14 nM). The IC50 of this doubly-modified siRNA was more than three-fold lower than that of the native and two-fold lower than that of LNA modified counterpart, i.e. LNA12: IC50: 1.13 +/- 0.27 nM. Hence the strategy to chemically modify the native siRNAs by substitution with the jcLNA can be considered as a significant development, leading to both enhanced siRNA efficiency and serum stability over that of the native.