Characterization of the HslU chaperone affinity for HslV protease

The HslVU complex is a bacterial two-component ATP-dependent protease, consisting of HslU chaperone and HslV peptidase. Investigation of protein-protein interactions using SPR in Escherichia coli HslVU and the protein substrates demonstrates that HslU and HslV have moderate affinity (Kd = 1 microM) for each other. However, the affinity of HslU for HslV fivefold increased (Kd approximately 0.2 microM) after binding with the MBP approximately SulA protein indicating the formation of a "ternary complex" of HslV-HslU-MBP approximately SulA. The molecular interaction studies also revealed that HslU strongly binds to MBP approximately SulA with 10(-9) M affinity but does not associate with nonstructured casein. Conversely, HslV does not interact with the MBP-SulA whereas it strongly binds with casein (Kd = 0.2 microM) requiring an intact active site of HslV. These findings provide evidence for "substrate-induced" stable HslVU complex formation. Presumably, the binding of HslU to MBP approximately SulA stimulates a conformational change in HslU to a high-affinity form for HslV.     

Lactoferrampin, an antimicrobial peptide of bovine lactoferrin, exerts its candidacidal activity by a cluster of positively charged residues at the C-terminus in combination with a helix-facilitating N-terminal part

The antimicrobial activity of bovine lactoferrin (bLF) is attributed to lactoferricin, which is situated in the N1-domain of bLF. Recently, another antimicrobial domain consisting of residues 268-284, designated lactoferrampin (LFampin), has been identified in the N1-domain of bLF, which exhibited antimicrobial activity against Candida albicans and several bacteria. In the present study, the candidacidal activity of a series of peptides spanning this antimicrobial domain was investigated in relation to the charge and the capacity to form a helical conformation in hydrophobic environments. C-Terminal truncation of LFampin resulted in a drastic decrease in candidacidal activity. Positively charged residues clustered at the C-terminal side of the LFampin domain appeared to be crucial for the candidacidal activity. The ability to adopt helical conformations did not change when LFampin was truncated at the C-terminal side. N-Terminally truncated LFampin peptides, truncated up to the sequence 270-284, were more reluctant to adopt a helical conformation. Therefore, we conclude that the C-terminal part of LFampin 265-284, which is the most active peptide, is crucial for its candidacidal activity, due to the presence of clustered positive charges, and that the N-terminal part is essential for activity as it facilitates helix formation.     

Bigger is not always better: when brains get smaller

Many studies assume that an increase in brain size is beneficial. However, the costs of producing and maintaining a brain are high, and we argue that brain size should be secondarily reduced by natural selection whenever the costs outweigh the benefits. Our results confirm this by showing that brain size is subject to bidirectional selection. Relative to the ancestral state, brain size in bats has been reduced in fast flyers, while it has increased in manoeuvrable flyers adapted to flight in complex habitats. This study emphasizes that brain reduction and enlargement are equally important, and they should both be considered when investigating brain size evolution.     

Crystal structure of homoserine transacetylase from Haemophilus influenzae reveals a new family of alpha/beta-hydrolases

Homoserine transacetylase catalyzes one of the required steps in the biosynthesis of methionine in fungi and several bacteria. We have determined the crystal structure of homoserine transacetylase from Haemophilus influenzae to a resolution of 1.65 A. The structure identifies this enzyme to be a member of the alpha/beta-hydrolase structural superfamily. The active site of the enzyme is located near the end of a deep tunnel formed by the juxtaposition of two domains and incorporates a catalytic triad involving Ser143, His337, and Asp304. A structural basis is given for the observed double displacement kinetic mechanism of homoserine transacetylase. Furthermore, the properties of the tunnel provide a rationale for how homoserine transacetylase catalyzes a transferase reaction vs hydrolysis, despite extensive similarity in active site architecture to hydrolytic enzymes.     

Immunodominance of poxviral-specific CTL in a human trial of recombinant-modified vaccinia Ankara

Many recombinant poxviral vaccines are currently in clinical trials for cancer and infectious diseases. However, these agents have failed to generate T cell responses specific for recombinant gene products at levels comparable with T cell responses associated with natural viral infections. The recent identification of vaccinia-encoded CTL epitopes, including a new epitope described in this study, allows the simultaneous comparison of CTL responses specific for poxviral and recombinant epitopes. We performed detailed kinetic analyses of CTL responses in HLA-A*0201 patients receiving repeated injections of recombinant modified vaccinia Ankara encoding a string of melanoma tumor Ag epitopes. The vaccine-driven CTL hierarchy was dominated by modified vaccinia Ankara epitope-specific responses, even in patients who had not received previous smallpox vaccination. The only recombinant epitope that was able to impact on the CTL hierarchy was the melan-A26-35 analog epitope, whereas responses specific for the weaker affinity epitope NY-ESO-1(157-165) failed to be expanded above the level detected in prevaccination samples. Our results demonstrate that immunodominant vaccinia-specific CTL responses limit the effectiveness of poxviruses in recombinant vaccination strategies and that more powerful priming strategies are required to overcome immunodominance of poxvirus-specific T cell responses.     

Crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis

Around 80 enzymes are implicated in the generic starch and sucrose pathways. One of these enzymes is sucrose phosphorylase, which reversibly catalyzes the conversion of sucrose and orthophosphate to d-Fructose and alpha-d-glucose 1-phosphate. Here, we present the crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis (BiSP) refined at 1.77 A resolution. It represents the first 3D structure of a sucrose phosphorylase and is the first structure of a phosphate-dependent enzyme from the glycoside hydrolase family 13. The structure of BiSP is composed of the four domains A, B, B', and C. Domain A comprises the (beta/alpha)(8)-barrel common to family 13. The catalytic active-site residues (Asp192 and Glu232) are located at the tips of beta-sheets 4 and 5 in the (beta/alpha)(8)-barrel, as required for family 13 members. The topology of the B' domain disfavors oligosaccharide binding and reduces the size of the substrate access channel compared to other family 13 members, underlining the role of this domain in modulating the function of these enzymes. It is remarkable that the fold of the C domain is not observed in any other known hydrolases of family 13. BiSP was found as a homodimer in the crystal, and a dimer contact surface area of 960 A(2) per monomer was calculated. The majority of the interactions are confined to the two B domains, but interactions between the loop 8 regions of the two barrels are also observed. This results in a large cavity in the dimer, including the entrance to the two active sites.     

Influenza hemagglutinins outside of the contact zone are necessary for fusion pore expansion

Current models for membrane fusion in diverse biological processes are focused on the local action of fusion proteins present in the contact zone where the proteins anchored in one membrane might interact directly with the other membrane. Are the fusion proteins outside of the contact zone just bystanders? Here we assess the role of these "outsider" proteins in influenza virus hemagglutinin-mediated fusion between red blood cells and either hemagglutinin-expressing cells or viral particles. To selectively inhibit or enhance the actions of hemagglutinin outsiders, the antibodies that bind to hemagglutinin and proteases that cleave it were conjugated to polystyrene microspheres too large to enter the contact zone. We also involved hemagglutinin outsiders into interactions with additional red blood cells. We find the hemagglutinin outsiders to be necessary and sufficient for fusion. Interfering with the activity of the hemagglutinin outsiders inhibited fusion. Selective conversion of hemagglutinin outsiders alone into fusion-competent conformation was sufficient to achieve fusion. The discovered functional role of fusion proteins located outside of the contact zone suggests a tempting analogy to mechanisms by which proteins mediate membrane fission from outside of the fission site.     

Papillary thyroid carcinoma and its variants in fine needle aspiration smears. A cytomorphologic study with special reference to tall cell variant

OBJECTIVE: To study the fine needle aspiration (FNA) cytologic features of papillary thyroid carcinoma (PTC) with special reference to its tall cell variant (TCV), which is the most aggressive of the variants. STUDY DESIGN: Fifty-four PTC cases were classified into variants, and the frequency of well-known morphologic criteria was determined. Four parameters were quantitatively analyzed based on a study of 200 consecutive neoplastic follicular cells: shape of cells, color of cytoplasm, intranuclear cytoplasmic inclusion (INCI) and nuclear grooves. RESULTS: The PTC cases included 6 TCV (> or = 30% tall cells), 8 cases with a significant tall cell component (sig. TCC) having 10-29% tall cells, 17 usual variant (UV), 17 follicular variant (FV) and 6 miscellaneous variants. TCV differed significantly from UV and FV in having a higher tall cell count, higher count of cells with reddish cytoplasm and INCI, and higher frequency of cases with lymphocytic infiltration. PTC (with significant tall cell component [TCC]) differed significantly from TCV with regard to tall cell count and lymphocytic infiltration, from UV with respect to tall cell count and monolayered sheets, and from FV with respect to tall cells, INCI, grooved nuclei, acinar formation, fire-flare appearance and giant cells. CONCLUSION: TCV was cytologically distinct from other variants. The biologic behavior of PTC cases with significant TCC, which morphologically seem to be a group intermediate between TCV on the one hand and UV and FV on the other, however, needs to be carefully monitored.     

Improved mass spectrometric proteomic profiling of the secretome of rat vascular endothelial cells

Serum albumin contamination of cells cultured in vitro significantly impedes the mass spectrometric analysis of proteins secreted by the cells. Here we report a novel washing and culturing technique for rat vascular endothelial cells that considerably reduces the concentration of the commonly used additive for cell culture, bovine serum albumin (BSA), in the secretome of these cells. Cells are rinsed stringently and cultured for 24 h in serum-free media without appreciably impeding cell growth or viability. The percentage of BSA scans identified by tandem mass spectrometry (LC-MS/MS) in stringently rinsed cells (average 13.2%) was significantly lower than either the moderately rinsed or no rinse cell treatments (average 35.2% and 45.2% respectively). Furthermore, the stringent wash treatment allowed the confident identification of a larger portion of the secretome of rat endothelial cells by LC-MS/MS.     

Heterotachy processes in rhodophyte-derived secondhand plastid genes: Implications for addressing the origin and evolution of dinoflagellate plastids

Serial transfer of plastids from one eukaryotic host to another is the key process involved in evolution of secondhand plastids. Such transfers drastically change the environment of the plastids and hence the selection regimes, presumably leading to changes over time in the characteristics of plastid gene evolution and to misleading phylogenetic inferences. About half of the dinoflagellate protists species are photosynthetic and unique in harboring a diversity of plastids acquired from a wide range of eukaryotic algae. They are therefore ideal for studying evolutionary processes of plastids gained through secondary and tertiary endosymbioses. In the light of these processes, we have evaluated the origin of 2 types of dinoflagellate plastids, containing the peridinin or 19'-hexanoyloxyfucoxanthin (19'-HNOF) pigments, by inferring the phylogeny using "covarion" evolutionary models allowing the pattern of among-site rate variation to change over time. Our investigations of genes from secondary and tertiary plastids derived from the rhodophyte plastid lineage clearly reveal "heterotachy" processes characterized as stationary covarion substitution patterns and changes in proportion of variable sites across sequences. Failure to accommodate covarion-like substitution patterns can have strong effects on the plastid tree topology. Importantly, multigene analyses performed with probabilistic methods using among-site rate and covarion models of evolution conflict with proposed single origin of the peridinin- and 19'-HNOF-containing plastids, suggesting that analysis of secondhand plastids can be hampered by convergence in the evolutionary signature of the plastid DNA sequences. Another type of sequence convergence was detected at protein level involving the psaA gene. Excluding the psaA sequence from a concatenated protein alignment grouped the peridinin plastid with haptophytes, congruent with all DNA trees. Altogether, taking account of complex processes involved in the evolution of dinoflagellate plastid sequences (both at the DNA and amino acid level), we demonstrate the difficulty of excluding independent, tertiary origin for both the peridinin and 19'-HNOF plastids involving engulfment of haptophyte-like algae. In addition, the refined topologies suggest the red algal order, Porphyridales, as the endosymbiont ancestor of the secondary plastids in cryptophytes, haptophytes, and heterokonts.