Versatile Expression and Secretion Vectors for Bacillus subtilis

Most expression systems are based on Escherichia coli as the host strain because of the large availability of all kinds of vector plasmids. However, aside from the obvious advantages of E. coli systems, serious problems can occur during the process of heterologous gene expression and purification. Therefore, low expression rates, formation of inclusion bodies, improper protein-folding, and/or toxicity problems might enforce changing the expression host. Here we describe the construction of two new vectors, pBSMuL1 and pBSMuL2, for overexpression and secretion of heterologous proteins in Bacillus subtilis as an alternative expression host. The new plasmids combine several advantages in comparison to available Bacillus expression systems: an appropriate multiple cloning site consisting of 13 unique restriction sites, one (pBSMuL1) or two (pBSMuL2) strong constitutive promoters, a high efficient signal sequence for protein secretion, and the possibility to express proteins as His-tagged fusions for easy detection and purification. We have demonstrated the applicability of the novel vector plasmids for the production and purification of the heterologous cutinase from Fusarium solani pisi.     

Euglena gracilis ribonucleotide reductase: the eukaryote class II enzyme and the possible antiquity of eukaryote B12 dependence

Ribonucleotide reductases provide the building blocks for DNA synthesis. Three classes of enzymes are known, differing widely in amino acid sequence but with similar structural motives and allosteric regulation. Class I occurs in eukaryotes and aerobic prokaryotes, class II occurs in aerobic and anaerobic prokaryotes, and class III occurs in anaerobic prokaryotes. The eukaryote Euglena gracilis contains a class II enzyme (Gleason, F. K., and Hogenkamp, H. P. (1970) J. Biol. Chem. 245, 4894-4899) and, thus, forms an exception. Class II enzymes depend on vitamin B(12) for their activity. We purified the reductase from Euglena cells, determined partial peptide sequences, identified its cDNA, and purified the recombinant enzyme. Its amino acid sequence and general properties, including its allosteric behavior, were similar to the class II reductase from Lactobacillus leichmannii. Both enzymes belong to a distinct small group of reductases that unlike all other homodimeric reductases are monomeric. They compensate the loss of the second polypeptide of dimeric enzymes by a large insertion in the monomeric chain. Data base searching and sequence comparison revealed a homolog from the eukaryote Dictyostelium discoideum as the closest relative to the Euglena reductase, suggesting that the class II enzyme was present in a common, B(12)-dependent, eukaryote ancestor.     

Heparan sulfate-related oligosaccharides in ternary complex formation with fibroblast growth factors 1 and 2 and their receptors

Biosynthesis of heparan sulfate (HS) is strictly regulated to yield products with cell/tissue-specific composition. Interactions between HS and a variety of proteins, including growth factors and morphogens, are essential for embryonic development and for homeostasis in the adult. Fibroblast growth factors (FGFs) and their various receptors (FRs) form ternary complexes with HS, as required for receptor signaling. Libraries of HS-related, radiolabeled oligosaccharides were generated by chemo-enzymatic modification of heparin and tested for affinity to immobilized FR ectodomains in the presence of FGF1 or FGF2. Experiments were designed to enable assessment of N-sulfated 8- and 10-mers with defined numbers of iduronic acid 2-O-sulfate and glucosamine 6-O-sulfate groups. FGF1 and FGF2 were found to require similar oligosaccharides in complex formation with FR1c-3c, FGF2 affording somewhat more efficient oligosaccharide recruitment than FGF1. FR4, contrary to FR1c-3c, bound oligosaccharides at physiological ionic conditions even in the absence of FGFs, and this interaction was further promoted by FGF1 but not by FGF2. In all systems studied, the stability of FGF-oligosaccharide-FR complexes correlated with the overall level of saccharide O-sulfation rather than on the precise distribution of sulfate groups.     

3-O-sulfated oligosaccharide structures are recognized by anti-heparan sulfate antibody HS4C3

Antibodies against heparan sulfate (HS) are useful tools to study the structural diversity of HS. They demonstrate the large sequential variation within HS and show the distribution of HS oligosaccharide sequences within their natural environment. We analyzed the distribution and the structural characteristics of the oligosaccharide epitope recognized by anti-HS antibody HS4C3. Biosynthetic and synthetic heparin-related oligosaccharide libraries were used in affinity chromatography, immunoprecipitation, and enzyme-linked immunosorbent assay to identify this epitope as a 3-O-sulfated motif with antithrombin binding capacity. The antibody binds weakly to any N-sulfated, 2-O- and 6-O-sulfated hexa- to octasaccharide fragment but strongly to the corresponding oligosaccharide when there is a 3-O-sulfated glucosamine residue present in the sequence. This difference was highlighted by affinity interaction and immunohistochemistry at salt concentrations from 500 mm. At physiological salt conditions the antibody strongly recognized basal lamina of epithelia and endothelia. At 500 mm salt conditions, when 3-O sulfation is required for binding, antibody recognition was more restricted and selective. Antibody HS4C3 bound similar tissue structures as antithrombin in rat kidney. Furthermore, antithrombin and antibody HS4C3 could compete with one another for binding to heparin. Antibody HS4C3 was also able to inhibit the anti-coagulant activities of heparin and Arixtra as demonstrated using the activated partial thromboplastin time clotting and the anti-factor Xa assays. In summary, antibody HS4C3 selectively detects 3-O-sulfated HS structures and interferes with the coagulation activities of heparin by association with the anti-thrombin binding pentasaccharide sequence.     

Filling a gap in the phylogeny of flatworms: relationships within the Rhabdocoela (Platyhelminthes), inferred from 18S ribosomal DNA sequences

The phylogeny of the Rhabdocoela, a species-rich taxon of free-living flatworms, is reconstructed based on complete 18S rDNA sequences. The analysis includes 62 rhabdocoels and 102 representatives of all major flatworm taxa. In total, 46 new sequences are used, 41 of them from rhabdocoel species, five from proseriates. Phylogenetic analysis was performed using maximum parsimony and Bayesian inference. Clade support was evaluated with parsimony jackknifing, Bremer support indices and Bayesian posterior probabilities. The resulting cladogram corroborates that the Rhabdocoela is monophyletic, but its sister group remains uncertain. The 'Dalyellioida' and the 'Typhloplanoida', both former rhabdocoel subtaxa, are polyphyletic. Within the Rhabdocoela the monophyletic Kalyptorhynchia, characterized by a muscular proboscis, forms the sister group of all other rhabdocoels. The Schizorhynchia is a monophyletic subtaxon of the Kalyptorhynchia, with the split proboscis as a synapomorphy. Except for the Dalyelliidae and the Typhloplanidae, both freshwater taxa, none of the 'families' previously included in the 'Typhloplanoida' and the 'Dalyellioida' appears to be monophyletic. As a result of this analysis, three existing and four new taxon names are formally defined following the rules of the Phylocode.     

A sensitive proximity ligation assay for active PSA

Prostate-specific antigen (PSA) is a widely used marker for prostate cancer. The utility of PSA tests is limited by their inability to differentiate prostate cancer from non-malignant conditions such as benign prostatic hyperplasia and prostatitis. In circulation, PSA occurs in various complexed and free forms, and specific determination of some of these can be used to improve the diagnostic accuracy of PSA tests. We have previously identified peptides that specifically bind to enzymatically active PSA and using such a peptide we have developed an immunopeptidometric assay for this form of PSA. However, the sensitivity of that assay is too low to measure active PSA at clinically important levels. Recently a novel sensitive immunoassay for analysis of proteins, termed the proximity ligation assay, has been established. Here we describe a sensitive implementation of the proximity ligation assay, which utilizes a PSA-binding peptide and antibody as probes to detect active PSA. The assay has a sensitivity of 0.07 microg/l, which is approximately ten-fold lower than that of our previous assay. It does not cross-react with inactive proPSA or the highly similar kallikrein hK2. Our results show that a highly sensitive immunopeptidometric assay can be developed using proximity ligation. This principle should facilitate establishment of specific assays for active forms of other proteases.     

Association of PTPN22 1858 single-nucleotide polymorphism with rheumatoid arthritis in a German cohort: higher frequency of the risk allele in male compared to female patients

The functional single-nucleotide polymorphism (SNP) of the gene PTPN22 is a susceptibility locus for rheumatoid arthritis (RA). The study presented here describes the association of the PTPN22 1858T allele with RA in a German patient cohort; 390 patients with RA and 349 controls were enrolled in the study. For 123 patients, clinical and radiographic documentation over 6 years was available from the onset of disease. Genotyping of the PTPN22 1858 SNP was performed using an restriction fragment length polymorphism PCR-based genotyping assay. The odds ratio to develop RA was 2.57 for carriers of the PTPN22 1858T allele (95% confidence interval (CI) 1.85–3.58, p < 0.001), and 5.58 for homozygotes (95% CI 1.85–16.79). The PTPN22 1858T allele was significantly associated not only with rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) positive RA, but also with RF and anti-CCP negative disease. The frequency of the PTPN22 1858T allele was increased disproportionately in male patients (53.8% compared to 33.0% in female patients, p < 0.001), and the resulting odds ratio for male carriers was increased to 4.47 (95% CI 2.5–8.0, p < 0.001). Moreover, within the male patient population, the rare allele was significantly associated with the HLA-DRB1 shared epitope (p = 0.01). No significant differences in disease activity or Larsen scores were detected. The results provide further evidence that the PTPN22 1858T allele is associated with RA irrespective of autoantibody production. The increased frequency of the risk allele in male patients and its association with the shared epitope indicate that the genetic contribution to disease pathogenesis might be more prominent in men.     

Combined supplementation of folic acid and vitamin E diminishes diabetes-induced embryotoxicity in rats

BACKGROUND: Oxidative stress and enhanced apoptosis may be involved in the induction of embryonic dysmorphogenesis in diabetic pregnancy. Administration of folic acid or vitamin E diminishes embryonic dysmorphogenesis. We aimed to evaluate the effect of combined treatment with folic acid and vitamin E on the disturbed development in embryos of diabetic rats. METHODS: Pregnant nondiabetic and diabetic rats were treated with daily injections of 15 mg/kg folic acid or with 5% vitamin E in the diet. A third group received combined treatment. Day 10 and day 11 embryos were evaluated for development and apoptotic profile. RESULTS: We found increased malformations, resorptions, and profound growth retardation in embryos of diabetic rats compared to control embryos. Vitamin E or folic acid alone, or the 2 compounds combined, normalized embryonic demise. Maternal diabetes caused decreased nuclear factor-kappaB (NF-kappaB) activity and B-cell lymphoma 2 (Bcl-2) protein level, and increased Bcl-2-associated x proteins (Bax) in embryos. Supplementation of vitamin E alone normalized the Bax protein level in a diabetic environment. Administration of folic acid to diabetic rats increased NF-kappaB activity and Bcl-2 protein level. Combined treatment normalized Bcl-2 and Bax protein level in a diabetic environment. CONCLUSIONS: Combined supplementation of folic acid and vitamin E to pregnant diabetic rats diminished diabetes-induced malformations and resorptions, concomitant with normalization of apoptotic protein levels. No treatment completely abolished the embryonic demise; therefore, other mechanisms than oxidative stress and apoptosis are likely to be involved in diabetic embryopathy.     

Heparan sulfate C5-epimerase is essential for heparin biosynthesis in mast cells

Biosynthesis of heparin, a mast cell-derived glycosaminoglycan with widespread importance in medicine, has not been fully elucidated. In biosynthesis of heparan sulfate (HS), a structurally related polysaccharide, HS glucuronyl C5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) residues. We have generated Hsepi-null mouse mutant mast cells, and we show that the same enzyme catalyzes the generation of IdoA in heparin and that 'heparin' lacking IdoA shows a distorted O-sulfation pattern.     

Ultraviolet radiation shapes seaweed communities

Stratospheric ozone depletion and the concomitant increase in irradiance of ultraviolet-B radiation (UVB) at the earth’s surface represent major threats to terrestrial and aquatic ecosystems. In costal rocky shore environments, seaweeds constitute a group of organisms of particular significance to ecosystem function. Thus, impairment of seaweed performance by UVB-exposure may result in severe changes in the functioning of coastal ecosystems. Here we present our view on how UVB radiation affects seaweed physiology and ecology and, thus, shapes the coastal environment by affecting the spatial, species and functional structure of seaweed communities.