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.     

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.     

Large-scale purification,dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK(2)) of Salmonella typhimurium

The maltose ATP-binding cassette (ABC) transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK(2)) and a periplasmic substrate binding protein. To further elucidate protein-protein interactions between the subunits, we have studied the dissociation and reassembly of the MalFGK(2) complex at the level of purified components in proteoliposomes. First, we optimized the yield in purified complex protein by taking advantage of a newly constructed expression plasmid that carries the malK, malF and malG genes in tandem orientation. Incorporated in proteoliposomes, the complex exhibited maltose binding protein/maltose-dependent ATPase activity with a V(max) of 1.25 micromol P(i)/min/mg and a K(m) of 0.1 mM. ATPase activity was sensitive to vanadate and enzyme IIA(Glc), a component of the enterobacterial glucose transport system. The proteoliposomes displayed maltose transport activity with an initial rate of 61 nmol/min/mg. Treatment of proteoliposomes with 6.6 M urea resulted in the release of medium-exposed MalK subunits concomitant with the complete loss of ATPase activity. By adding increasing amounts of purified MalK to urea-treated proteoliposomes, about 50% of vanadate-sensitive ATPase activity relative to the control could be recovered. Furthermore, the phenotype of MalKQ140K that exhibits ATPase activity in solution but not when associated with MalFG was confirmed by reassembly with MalK-depleted proteoliposomes.     

The bacterium Thermus thermophilus,like hyperthermophilic archaea,uses a two-step pathway for the synthesis of mannosylglycerate

The biosynthetic pathway for the synthesis of the compatible solute alpha-mannosylglycerate (MG) in the thermophilic bacterium Thermus thermophilus HB27 was identified based on the activities of recombinant mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). The sequences of homologous genes from the archaeon Pyrococcus horikoshii were used to identify MPGS and MPGP genes in T. thermophilus HB27 genome. Both genes were separately cloned and overexpressed in Escherichia coli, yielding 3 to 4 mg of pure recombinant protein per liter of culture. The molecular masses were 43.6 and 28.1 kDa for MPGS and MPGP, respectively. The recombinant MPGS catalyzed the synthesis of alpha-mannosyl-3-phosphoglycerate (MPG) from GDP-mannose and D-3-phosphoglycerate, while the recombinant MPGP catalyzed the dephosphorylation of MPG to MG. The recombinant MPGS had optimal activity at 80 to 90 degrees C and a pH optimum near 7.0; MPGP had maximal activity between 90 and 95 degrees C and at pH 6.0. The activities of both enzymes were strictly dependent on divalent cations; Mn(2+) was most effective for MPGS, while Mn(2+), Co(2+), Mg(2+), and to a lesser extent Ni(2+) activated MPGP. The organization of MG biosynthetic genes in T. thermophilus HB27 is different from the P. horikoshii operon-like structure, since the genes involved in the conversion of fructose-6-phosphate to GDP-mannose are not found immediately downstream of the contiguous MPGS and MPGP genes. The biosynthesis of MG in the thermophilic bacterium T. thermophilus HB27, proceeding through a phosphorylated intermediate, is similar to the system found in hyperthermophilic archaea.     

Metagenomes of complex microbial consortia derived from different soils as sources for novel genes conferring formation of carbonyls from short-chain polyols on Escherichia coli

Metagenomic DNA libraries from three different soil samples (meadow, sugar beet field, cropland) were constructed. The three unamplified libraries comprised approximately 1267000 independent clones and harbored approximately 4.05 Gbp of environmental DNA. Approximately 300000 recombinant Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from short-chain (C2 to C4) polyols such as 1,2-ethanediol, 2,3-butanediol, and a mixture of glycerol and 1,2-propanediol on indicator agar. Twenty-four positive E. COLI clones were obtained during the initial screen. Fifteen of them contained recombinant plasmids, designated pAK201-215, which conferred a stable carbonyl-forming phenotype on E. coli Sequencing revealed that the inserts of pAK201-215 encoded 26 complete and 14 incomplete predicted protein-encoding genes. Most of these genes were similar to genes with unknown functions from other microorganisms or unrelated to any other known gene. The further analysis was focused on the 7 plasmids (pAK204, pAK206, pAK208, and pAK210-213) recovered from the positive clones, which exhibited an NAD(H)-dependent alcohol oxidoreductase activity with polyols or the correlating carbonyls as substrates in crude extracts. Three genes (ORF6, ORF24, and ORF25) conferring this activity were identified during subcloning of the inserts of pAK204, pAK211, and pAK212. The sequences of the three deduced gene products revealed no significant similarities to known alcohol oxidoreductases, but contained putative glycine-rich regions, which are characteristic for binding of nicotinamide cofactors.