Abundance dynamics and functional role of predaceous <Emphasis Type="Italic">Leptodora kindtii</Emphasis> in the Curonian Lagoon

The abundance and distribution of predatory cladoceran Leptodora kindtii was investigated in the estuarine lagoon (Curonian Lagoon, SE Baltic Sea). Three hydrodynamically different parts of the lagoon were selected, representing transitory oligohaline, intermediate freshwater and stagnant freshwater sites. L. kindtii was least abundant at the oligohaline site, never occurring at salinities greater than 4 psu. At the two freshwater sites, the abundance of L. kindtii varied from a low of <0.1 up to 2.2 indv. L⁻¹ during peak abundance. Two peaks of L. kindtii abundance were observed with timing differences between stations: at the stagnant site the population of L. kindtii peaked two weeks earlier relative to the more hydrodynamically active sites, likely due to a 2°C higher May temperature. The small body size of L. kindtii in the lagoon (seasonal mean 2.68±0.6 mm) shows high fish predation pressure and predicts small cladocerans, juvenile copepods and rotifers being in the preferred prey size range. The calculated L. kindtii daily consumption during the population peak was as high as 100% of the daily zooplankton production, which implies high potential of this predator to shape the grazing zooplankton community in the lagoon.     

Expression and Characterization of a Bifidobacterium adolescentis Beta-Mannanase Carrying Mannan-Binding and Cell Association Motifs

The gene encoding β-mannanase (EC 3.2.1.78) BaMan26A from the bacterium Bifidobacterium adolescentis (living in the human gut) was cloned and the gene product characterized. The enzyme was found to be modular and to contain a putative signal peptide. It possesses a catalytic module of the glycoside hydrolase family 26, a predicted immunoglobulin-like module, and two putative carbohydrate-binding modules (CBMs) of family 23. The enzyme is likely cell attached either by the sortase mechanism (LPXTG motif) or via a C-terminal transmembrane helix. The gene was expressed in Escherichia coli without the native signal peptide or the cell anchor. Two variants were made: one containing all four modules, designated BaMan26A-101K, and one truncated before the CBMs, designated BaMan26A-53K. BaMan26A-101K, which contains the CBMs, showed an affinity to carob galactomannan having a dissociation constant of 0.34 μM (8.8 mg/liter), whereas BaMan26A-53K did not bind, showing that at least one of the putative CBMs of family 23 is mannan binding. For BaMan26A-53K, k_cat was determined to be 444 s⁻¹ and K_m 21.3 g/liter using carob galactomannan as the substrate at the optimal pH of 5.3. Both of the enzyme variants hydrolyzed konjac glucomannan, as well as carob and guar gum galactomannans to a mixture of oligosaccharides. The dominant product from ivory nut mannan was found to be mannotriose. Mannobiose and mannotetraose were produced to a lesser extent, as shown by high-performance anion-exchange chromatography. Mannobiose was not hydrolyzed, and mannotriose was hydrolyzed at a significantly lower rate than the longer oligosaccharides.     

Human osteoclast-poor osteopetrosis with hypogammaglobulinemia due to TNFRSF11A (RANK) mutations

Autosomal-Recessive Osteopetrosis (ARO) comprises a heterogeneous group of bone diseases for which mutations in five genes are known as causative. Most ARO are classified as osteoclast-rich, but recently a subset of osteoclast-poor ARO has been recognized as due to a defect in TNFSF11 (also called RANKL or TRANCE, coding for the RANKL protein), a master gene driving osteoclast differentiation along the RANKL-RANK axis. RANKL and RANK (coded for by the TNFRSF11A gene) also play a role in the immune system, which raises the possibility that defects in this pathway might cause osteopetrosis with immunodeficiency. From a large series of ARO patients we selected a Turkish consanguineous family with two siblings affected by ARO and hypogammaglobulinemia with no defects in known osteopetrosis genes. Sequencing of genes involved in the RANKL downstream pathway identified a homozygous mutation in the TNFRSF11A gene in both siblings. Their monocytes failed to differentiate in vitro into osteoclasts upon exposure to M-CSF and RANKL, in keeping with an osteoclast-intrinsic defect. Immunological analysis showed that their hypogammaglobulinemia was associated with impairment in immunoglobulin-secreting B cells. Investigation of other patients revealed a defect in both TNFRSF11A alleles in six additional, unrelated families. Our results indicate that TNFRSF11A mutations can cause a clinical condition in which severe ARO is associated with an immunoglobulin-production defect.     

Structure of the O polysaccharides and serological classification of Pseudomonas syringae pv. porri from genomospecies 4.

Strains of Pseudomonas syringae pv. porri are characterized by a number of pathovar-specific phenotypic and genomic characters and constitute a highly homogeneous group. Using monoclonal antibodies, they all were classified in a novel P. syringae serogroup O9. The O polysaccharides (OPS) isolated from the lipopolysaccharides (LPS) of P. syringae pv. porri NCPPB 3365 and NCPPB 3364T possess multiple oligosaccharide O repeats, some of which are linear and composed of l-rhamnose (l-Rha), whereas the major O repeats are branched with l-rhamnose in the main chain and GlcNAc in side chains (structures 1 and 2). Both branched O repeats, which differ in the position of substitution of one of the Rha residues and in the site of attachment of GlcNAc, were found in the two strains studied, O repeat 1 being major in strain NCPPB 3365 and 2 in strain NCPPB 3364T. [formula: see text]. The relationship between OPS chemotype and serotype on one hand and the genomic characters of P. syringae pv. porri and other pathovars delineated in genomospecies 4 on the other hand is discussed.     

Baseline NT-Pro-BNP Levels and Arrhythmia Recurrence in Outpatients Undergoing Elective Cardioversion of Persistent Atrial Fibrillation: A Survival Analysis

Background

Atrial fibrillation (AF) is the most common arrhythmia encountered in clinical practice. Elective electrical cardioversion is often performed in patients with persistent AF to attempt sinus rhythm (SR) restoration. However, AF recurrences are frequent after successful cardioversion and several predictors have been identified.

Aim of the study

The present study was designed to prospectively analyse the correlation between NT-pro-BNP levels and AF recurrence in consecutive patients referred for electrical cardioversion of persistent atrial fibrillation.

Results

Forty consecutive patients referred for elective cardioversion of AF were enrolled in the study. Cardioversion restored sinus rhythm in 34/40 patients but 2 of them presented an early recurrence of AF before discharge. Patients were then followed for 6 months to assess AF recurrence. Cox regression analysis was performed using the parameters found predictive on univariate survival analysis (NT-pro-BNP quartiles, beta-blockers). The only independent predictor of AF recurrence on Cox-regression analysis was a level of NT-pro-BNP in the fourth quartile (HR 3.21 95%CI 1.26-8.14, p=0.014). On receiver operating curve (ROC) analysis, NT-pro-BNP levels above 1707 pg/ml had a specificity of 92% (and a sensitivity of 36%) in predicting atrial fibrillation recurrence by 6 months.

Conclusions

Baseline NT-pro-BNP levels are an independent predictor of AF recurrence at 6 months follow-up in candidates for elective direct current cardioversion.     

Protection induced by Plasmodium falciparum MSP1(42) is strain-specific, antigen and adjuvant dependent, and correlates with antibody responses

Vaccination with Plasmodium falciparum MSP1(42)/complete Freund's adjuvant (FA) followed by MSP1(42)/incomplete FA is the only known regimen that protects Aotus nancymaae monkeys against infection by erythrocytic stage malaria parasites. The role of adjuvant is not defined; however complete FA cannot be used in humans. In rodent models, immunity is strain-specific. We vaccinated Aotus monkeys with the FVO or 3D7 alleles of MSP1(42) expressed in Escherichia coli or with the FVO allele expressed in baculovirus (bv) combined with complete and incomplete FA, Montanide ISA-720 (ISA-720) or AS02A. Challenge with FVO strain P. falciparum showed that suppression of cumulative day 11 parasitemia was strain-specific and could be induced by E. coli expressed MSP1(42) in combination with FA or ISA-720 but not with AS02A. The coli42-FVO antigen induced a stronger protective effect than the bv42-FVO antigen, and FA induced a stronger protective effect than ISA-720. ELISA antibody (Ab) responses at day of challenge (DOC) were strain-specific and correlated inversely with c-day 11 parasitemia (r = -0.843). ELISA Ab levels at DOC meeting a titer of at least 115,000 ELISA Ab units identified the vaccinees not requiring treatment (noTx) with a true positive rate of 83.3% and false positive rate of 14.3 %. Correlation between functional growth inhibitory Ab levels (GIA) and cumulative day 11 parasitemia was weaker (r = -0.511), and was not as predictive for a response of noTx. The lowest false positive rate for GIA was 30% when requiring a true positive rate of 83.3%. These inhibition results along with those showing that antigen/FA combinations induced a stronger protective immunity than antigen/ISA-720 or antigen/AS02 combinations are consistent with protection as ascribed to MSP1-specific cytophilic antibodies. Development of an effective MSP1(42) vaccine against erythrocytic stage P. falciparum infection will depend not only on antigen quality, but also upon the selection of an optimal adjuvant component.     

Suramin and suramin analogues inhibit merozoite surface protein-1 secondary processing and erythrocyte invasion by the malaria parasite Plasmodium falciparum

Malarial merozoites invade erythrocytes; and as an essential step in this invasion process, the 42-kDa fragment of Plasmodium falciparum merozoite surface protein-1 (MSP142) is further cleaved to a 33-kDa N-terminal polypeptide (MSP133) and an 19-kDa C-terminal fragment (MSP119) in a secondary processing step. Suramin was shown to inhibit both merozoite invasion and MSP142 proteolytic cleavage. This polysulfonated naphthylurea bound directly to recombinant P. falciparum MSP142 (Kd = 0.2 microM) and to Plasmodium vivax MSP142 (Kd = 0.3 microM) as measured by fluorescence enhancement in the presence of the protein and by isothermal titration calorimetry. Suramin bound only slightly less tightly to the P. vivax MSP133 (Kd = 1.5 microM) secondary processing product (fluorescence measurements), but very weakly to MSP119 (Kd approximately 15 mM) (NMR measurements). Several residues in MSP119 were implicated in the interaction with suramin using NMR measurements. A series of symmetrical suramin analogues that differ in the number of aromatic rings and substitution patterns of the terminal naphthylamine groups was examined in invasion and processing assays. Two classes of analogue with either two or four bridging rings were found to be active in both assays, whereas two other classes without bridging rings were inactive. We propose that suramin and related compounds inhibit erythrocyte invasion by binding to MSP1 and by preventing its cleavage by the secondary processing protease. The results indicate that enzymatic events during invasion are suitable targets for drug development and validate the novel concept of an inhibitor binding to a macromolecular substrate to prevent its proteolysis by a protease.