Diplopod hemocyanin sequence and the phylogenetic position of the Myriapoda

Hemocyanins are copper-containing respiratory proteins of the Arthropoda that have so far been thoroughly investigated only in the Chelicerata and the Crustacea but have remained unstudied until now in the Myriapoda. Here we report the first sequence of a myriapod hemocyanin. The hemocyanin of Spirostreptus sp. (Diplopoda: Spirostreptidae) is composed of two distinct subunits that are arranged in a 6 x 6 native molecule. The cloned hemocyanin subunit cDNA codes of for a polypeptide of 653 amino acids (75.5 kDa) that includes a signal peptide of 18 amino acids. The sequence closely resembles that of the chelicerate hemocyanins. Molecular phylogenetic analyses reject with high statistical confidence the integrity of the Tracheata (i.e., Myriapoda + Insecta) but give conflicting results on the position of the myriapod hemocyanin. While distance matrix and maximum-likelihood methods support a basal position of the Spirostreptus hemocyanin with respect to the other hemocyanins, parsimony analysis suggests a sister group relationship with the chelicerate hemocyanins. The latter topology is also supported by a unique shared deletion of an alpha-helix. A common ancestry of Myriapoda and Chelicerata should be seriously considered.     

Molecular evolution of the arthropod hemocyanin superfamily

Arthropod hemocyanins are members of a protein superfamily that also comprises the arthropod phenoloxidases (tyrosinases), crustacean pseudohemocyanins (cryptocyanins), and insect storage hexamerins. The evolution of these proteins was inferred by neighbor-joining, maximum-parsimony, and maximum-likelihood methods. Monte Carlo shuffling approaches provided evidence against a discernible relationship of the arthropod hemocyanin superfamily and molluscan hemocyanins or nonarthropodan tyrosinases. Within the arthropod hemocyanin superfamily, the phenoloxidase probably emerged early in the (eu-)arthropod stemline and thus form the most likely outgroup. The respiratory hemocyanins evolved from these enzymes before the radiation of the extant euarthropodan subphyla. Due to different functional constraints, replacement rates greatly vary between the clades. Divergence times were thus estimated assuming local molecular clocks using several substitution models. The results were consistent and indicated the separation of the cheliceratan and crustacean hemocyanins close to 600 MYA. The different subunit types of the multihexameric cheliceratan hemocyanin have a rather conservative structure and diversified in the arachnidan stemline between 550 and 450 MYA. By contrast, the separation of the crustacean (malacostracan) hemocyanin subunits probably occurred only about 200 MYA. The nonrespiratory pseudohemocyanins evolved within the Decapoda about 215 MYA. The insect hemocyanins and storage hexamerins emerged independently from the crustacean hemocyanins. The time of divergence of the insect proteins from the malacostracan hemocyanins was estimated to be about 430-440 MYA, providing support for the notion that the Hexapoda evolved from the same crustacean lineage as the Malacostraca.     

Isolation of glucocardiolipins from Geobacillus stearothermophilus NRS 2004/3a

Glucose-substituted cardiolipins account for about 4 mol% of total phospholipid extracted from exponentially grown cells of Geobacillus stearothermophilus NRS 2004/3a. Individual glucocardiolipin species exhibited differences in fatty acid substitution, with iso-C(15:0) and anteiso-C(17:0) prevailing. The compounds were purified to homogeneity by a novel protocol and precharacterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.     

Confidence intervals for genotype relative risks and allele frequencies from the case parent trio design for candidate-gene studies

OBJECTIVES: Confidence intervals for genotype relative risks, for allele frequencies and for the attributable risk in the case parent trio design for candidate-gene studies are proposed which can be easily calculated from the observed familial genotype frequencies. METHODS: Likelihood theory and the delta method were used to derive point estimates and confidence internals. We used Monte Carlo simulations to show the validity of the formulae for a variety of given modes of inheritance and allele frequencies and illustrated their usefulness by applying them to real data. RESULTS: Generally these formulae were found to be valid for 'sufficiently large' sample sizes. For smaller sample sizes the estimators for genotype relative risks tended to be conservative whereas the estimator for attributable risk was found to be anti-conservative for moderate to high allele frequencies. CONCLUSIONS: Since the proposed formulae provide quantitative information on the individual and epidemiological relevance of a genetic variant they might be a useful addition to the traditional statistical significance level of TDT results.     

Galiellalactone and its biogenetic precursors as chemotaxonomic markers of the Sarcosomataceae (Ascomycota)

(-)-Galiellalactone is a hexaketide metabolite with interesting pharmacological activities which was detected in four strains of Galiella rufa (Sarcosomataceae, Ascomycota) and in two unidentified fungi shown by their 18S rDNA sequences also to belong to the Sarcosomataceae. These were a wood-inhabiting apothecial species from Chile and an endophytic isolate from Cistus salviifolius (Sardinia). Other members of the family (Urnula helvelloides, one Strumella coryneoidea isolate) produced no galiellalactone but merely hexaketides structurally related to galiellalactone precursors, whereas a third group of species (Sarcosoma latahensis, Strumella griseola, one S. coryneoidea isolate) lacked hexaketide production altogether. Despite thorough screening programmes, galiellalactone and its precursors have not yet been found in any fungus outside the Sarcosomataceae and may thus be a chemotaxonomic marker of the family, supporting its current phylogenetic definition. Two pentaketide derivatives of the 6-pentyl-alpha-pyrone type were found in all G. rufa strains as well as in A111-95 and the hexaketide-producing S. coryneoidea isolate.     

Analysis of immunoglobulin light chain rearrangements in the salivary gland and blood of a patient with Sjögren's syndrome

Patients with Sj?gren's syndrome (SS) have characteristic lymphocytic infiltrates of the salivary glands. To determine whether the B cells accumulating in the salivary glands of SS patients represent a distinct population and to delineate their potential immunopathologic impact, individual B cells obtained from the parotid gland and from the peripheral blood were analyzed for immunoglobulin light chain gene rearrangements by PCR amplification of genomic DNA. The productive immunoglobulin light chain repertoire in the parotid gland of the SS patient was found to be restricted, showing a preferential usage of particular variable lambda chain genes (V lambda 2E) and variable kappa chain genes (V kappa A27). Moreover, clonally related V(L) chain rearrangements were identified; namely, V kappa A27-J kappa 5 and V kappa A19-J kappa 2 in the parotid gland, and V lambda 1C-J lambda 3 in the parotid gland and the peripheral blood. V kappa and V lambda rearrangements from the parotid gland exhibited a significantly elevated mutational frequency compared with those from the peripheral blood (P < 0.001). Mutational analysis revealed a pattern of somatic hypermutation similar to that found in normal donors, and a comparable impact of selection of mutated rearrangements in both the peripheral blood and the parotid gland. These data indicate that there is biased usage of V(L) chain genes caused by selection and clonal expansion of B cells expressing particular V(L) genes. In addition, the data document an accumulation of B cells bearing mutated V(L) gene rearrangements within the parotid gland of the SS patient. These results suggest a role of antigen-activated and selected B cells in the local autoimmune process in SS.     

Respiratory organs in wolf spiders: morphometric analysis of lungs and tracheae in Pardosa lugubris (L.) (Arachnida, Araneae, Lycosidae)

The respiratory system of the wolf spider Pardosa lugubris consists of a pair of well-developed lungs and four unbranched tube tracheae. We used stereological morphometric methods to investigate the morphological diffusing capacity of the lungs and of the walls of the tracheae ('lateral diffusing capacity'). We examined three groups of female P. lugubris with different mean body masses. The barrier thickness of the gas-exchange epithelium of the lungs was 0.17 microm for the total diffusion barrier and the calculated oxygen diffusing capacity (D(O2)) for the lungs was between 12.9 and 13.4 microl min(-1)g(-1)kPa(-1). Measured metabolic rates compared with the D(O2) of the lungs result in necessary oxygen partial pressure differences of 0.2 kPa during rest and 2.1 kPa during maximum measured activity. The diffusion barrier of the entire tracheal walls was 0.31-0.50 microm and the calculated lateral D(O2) was 0.05-0.2 microl min(-1)g(-1)kPa(-1). Therefore, tracheae are of no importance for the overall oxygen exchange. However, they might be of some importance in local oxygen supply or in overall carbon dioxide release. The comparison with the respiratory system of the jumping spider Salticus scenicus reveals that the lungs have very similar mass-specific D(O2) in both species, and that, in addition, jumping spiders possess a much better developed tracheal system.     

Evidence for local inflammation in complex regional pain syndrome type 1

BACKGROUND: The pathophysiology of complex regional pain syndrome type 1 (CRPS 1) is still a matter of debate. Peripheral afferent, efferent and central mechanisms are supposed. Based on clinical signs and symptoms (e.g. oedema, local temperature changes and chronic pain) local inflammation is suspected. AIM: To determine the involvement of neuropetides, cytokines and eicosanoids as locally formed mediators of inflammation. METHODS: In this study, nine patients with proven CRPS 1 were included. Disease activity and impairment was determined by means of a Visual Analogue Scale, the McGill Pain Questionnaire, the difference in volume and temperature between involved and uninvolved extremities, and the reduction in active range of motion of the involved extremity. Venous blood was sampled from and suction blisters made on the involved and uninvolved extremities for measurement of cytokines interleukin (IL)-6, II-1beta and tumour necrosis factor-alpha (TNF-alpha), the neuropetides NPY and CRGP, and prostaglandin E2RESULTS: The patients included in this study did have a moderate to serious disease activity and impairment. In plasma, no changes of mediators of inflammation were observed. In blister fluid, however, significantly higher levels of IL-6 and TNF-alpha in the involved extremity were observed in comparison with the uninvolved extremity. CONCLUSIONS: This is the first time that involvement of mediators of inflammation in CRPS 1 has been so clearly and directly demonstrated. This observation opens new approaches for the succesful use and development of immunosuppressives in CRPS 1.     

Regulation of succinoglycan and galactoglucan biosynthesis in Sinorhizobium meliloti

Sinorhizobium meliloti (Rhizobium meliloti) 2011 has the ability to produce the two acidic exopolysaccharides succinoglycan (EPS I) and galactoglucan (EPS II). EPS I is a branched heteropolysaccharide composed of octasaccharide repeating units, whereas EPS II is a linear heteropolysaccharide consisting of disaccharide subunits. The exo-exs and exp gene clusters are involved in the biosynthesis of EPSI and EPSII, respectively. EPSI and EPSII biosynthesis genes are differentially expressed resulting in a complex regulation of EPS production in S. meliloti. The phosphate concentration was identified as an important factor affecting the expression of exp genes.     

The consensus concept for thermostability engineering of proteins: further proof of concept

Previously, we calculated a consensus amino acid sequence from 13 homologous fungal phytases. A synthetic gene was constructed and recombinantly expressed. Surprisingly, consensus phytase-1 was 15-26 degrees C more thermostable than all parent phytases used in its design [Lehmann et al. (2000)Protein Eng., 13, 49-57]. In the present study, inclusion of six further phytase sequences in the amino acid sequence alignment resulted in the replacement of 38 amino acid residues in either one or both of the new consensus phytases-10 and -11. Since consensus phytase-10, again, was 7.4 degrees C more thermostable than consensus phytase-1, the thermostability effects of most of the 38 amino acid substitutions were tested by site-directed mutagenesis. Both stabilizing and destabilizing mutations were identified, but all affected the stability of the enzyme by <3 degrees C. The combination of all stabilizing amino acid exchanges in a multiple mutant of consensus phytase-1 increased the unfolding temperature from 78.0 to 88.5 degrees C. Likewise, back-mutation of four destabilizing amino acids and introduction of an additional stabilizing amino acid in consensus phytase-10 further increased the unfolding temperature from 85.4 to 90.4 degrees C. The thermostabilization achieved is the result of a combination of slight improvements from multiple amino acid exchanges rather than being the effect of a single or of just a few dominating mutations that have been introduced by chance. The present findings support the general validity of the consensus concept for thermostability engineering of proteins.