Identification of the disulfide bonds in the recombinant somatomedin B domain of human vitronectin

The NH(2)-terminal somatomedin B (SMB) domain (residues 1-44) of human vitronectin contains eight Cys residues organized into four disulfide bonds and is required for the binding of type 1 plasminogen activator inhibitor (PAI-1). In the present study, we map the four disulfide bonds in recombinant SMB (rSMB) and evaluate their functional importance. Active rSMB was purified from transformed Escherichia coli by immunoaffinity chromatography using a monoclonal antibody that recognizes a conformational epitope in SMB (monoclonal antibody 153). Plasmon surface resonance (BIAcore) and competitive enzyme-linked immunosorbent assays demonstrate that the purified rSMB domain and intact urea-activated vitronectin have similar PAI-1 binding activities. The individual disulfide linkages present in active rSMB were investigated by CNBr cleavage, partial reduction and S-alkylation, mass spectrometry, and protein sequencing. Two pairs of disulfide bonds at the NH(2)-terminal portion of active rSMB were identified as Cys(5)-Cys(9) and Cys(19)-Cys(21). Selective reduction/S-alkylation of these two disulfide linkages caused the complete loss of PAI-1 binding activity. The other two pairs of disulfide bonds in the COOH-terminal portion of rSMB were identified as Cys(25)-Cys(31) and Cys(32)-Cys(39) by protease-generated peptide mapping of partially reduced and S-alkylated rSMB. These results suggest a linear uncrossed pattern for the disulfide bond topology of rSMB that is distinct from the crossed pattern present in most small disulfide bond-rich proteins.     

Inhibition of food intake by central injection of anti-orexin antibody in fasted rats

It has been shown that intracerebroventricular injection of synthetic orexins stimulated food intake in rats. This pharmacological evidence suggests that orexins may have a role for the central regulation of feeding. In the present study, we investigated the hypothesis of whether endogenous orexins indeed play a vital role in feeding behavior. An anti-orexin polyclonal antibody was used throughout the study. First, we examined the specificity of the antibody to orexin by Western blot analysis and immunohistochemistry. Next, the effects of central injection of the orexin antibody on food intake in 24-h-fasted rats were evaluated. Western blot analysis revealed that the orexin antibody detected synthetic orexin-A. Immunohistochemical study showed that orexin-positive neurons were identified only in the lateral hypothalamic area, in agreement with previous reports. Neither control antibody nor the orexin antibody preabsorbed with excess amount of orexin-A detected neurons, indicating that the orexin antibody is specific. Intracisternal but not intraperitoneal injection of the orexin antibody dose-dependently suppressed feeding. All these results suggest that immunoneutralization of endogenous orexins in the brain reduced food intake. In other words, we suggest that endogenous brain orexin may have a physiologically relevant action on feeding behavior.     

Comparison of sensitization to crude and purified house dust mite allergens in inbred mice

To establish a murine model for house dust mite allergy to purified mite allergens, we studied the immune response to two major mite allergens, native Dermatophagoides farinae 1 (nDer f 1) and recombinant Der f 2 (rDer f 2), and crude mite extract in four mouse strains, A/J, BALB/c, C57BL/6, and C3H/He. Mice were immunized with mite extract, nDer f 1 or rDer f 2, three times at 2-week intervals. Then mice were examined to determine status of sensitization to the antigen. Anti-mite extract IgE production was induced in all strains, and plasma IgE concentration did not differ much among the four strains. In contrast, IgE response to nDer f 1 and rDer f 2 indicated an intra-strain difference. The A/J mice had high responses to both antigens, whereas BALB/c did not respond to rDer f 2. The C57BL/6 and C3H/He mice had moderate to low IgE responses to nDer f 1 and rDer f 2. Immediate airway constriction was provoked by inhalation of mite extract or rDer f 2 in sensitized mice, and the degree of the immediate response was almost proportional to antigen-specific IgE concentration. We concluded that immunization of inbred mice with nDer f 1 and rDer f 2 achieved sensitization to mite allergens. Among the four strains, A/J mice with H-2a haplotype were the highest responder to mite allergens.     

Constraint-based, homology model of the extracellular domain of the epithelial Na+ channel α subunit reveals a mechanism of channel activation by proteases

The epithelial Na(+) channel (ENaC) mediates Na(+) transport across high resistance epithelia. This channel is assembled from three homologous subunits with the majority of the protein's mass found in the extracellular domains. Acid-sensing ion channel 1 (ASIC1) is homologous to ENaC, but a key functional domain is highly divergent. Here we present molecular models of the extracellular region of α ENaC based on a large data set of mutations that attenuate inhibitory peptide binding in combination with comparative modeling based on the resolved structure of ASIC1. The models successfully rationalized the data from the peptide binding screen. We engineered new mutants that had not been tested based on the models and successfully predict sites where mutations affected peptide binding. Thus, we were able to confirm the overall general fold of our structural models. Further analysis suggested that the α subunit-derived inhibitory peptide affects channel gating by constraining motions within two major domains in the extracellular region, the thumb and finger domains.     

Binding and Functional Properties of Five Extrinsic Proteins in Oxygen-evolving Photosystem II from a Marine Centric Diatom,Chaetoceros gracilis

Oxygen-evolving photosystem II (PSII) isolated from a marine centric diatom, Chaetoceros gracilis, contains a novel extrinsic protein (Psb31) in addition to four red algal type extrinsic proteins of PsbO, PsbQ′, PsbV, and PsbU. In this study, the five extrinsic proteins were purified from alkaline Tris extracts of the diatom PSII by anion and cation exchange chromatographic columns at different pH values. Reconstitution experiments in various combinations with the purified extrinsic proteins showed that PsbO, PsbQ′, and Psb31 rebound directly to PSII in the absence of other extrinsic proteins, indicating that these extrinsic proteins have their own binding sites in PSII intrinsic proteins. On the other hand, PsbV and PsbU scarcely rebound to PSII alone, and their effective bindings required the presence of all of the other extrinsic proteins. Interestingly, PSII reconstituted with Psb31 alone considerably restored the oxygen evolving activity in the absence of PsbO, indicating that Psb31 serves as a substitute in part for PsbO in supporting oxygen evolution. A significant difference found between PSIIs reconstituted with Psb31 and with PsbO is that the oxygen evolving activity of the former is scarcely stimulated by Cl⁻ and Ca²⁺ ions but that of the latter is largely stimulated by these ions, although rebinding of PsbV and PsbU activated oxygen evolution in the absence of Cl⁻ and Ca²⁺ ions in both the former and latter PSIIs. Based on these results, we proposed a model for the association of the five extrinsic proteins with intrinsic proteins in diatom PSII and compared it with those in PSIIs from the other organisms.     

Nit1 and Fhit tumor suppressor activities are additive

The fragile histidine triad gene (human FHIT, mouse Fhit) has been shown to act as a tumor suppressor gene. Nit1 and Fhit form a fusion protein, encoded by the NitFhit gene in flies and worms, suggesting that mammalian Nit1 and Fhit proteins, which are encoded by genes on different chromosomes in mammals, may function in the same signal pathway(s). A previous study showed that Nit1 deficiency in knockout mice confers a cancer prone phenotype, as does Fhit deficiency. We have now assessed the tumor susceptibility of Fhit^?/?Nit1^?/? mice and observed that double knockout mice develop more spontaneous and carcinogen‐induced tumors than Fhit^?/? mice, suggesting that the extent of tumor susceptibility due to Nit1 and Fhit deficiency is additive, and that Nit1 and Fhit affect distinct signal pathways in mammals. Nit1, like Fhit, is present in cytoplasm and mitochondria but not nuclei. Because Fhit deficiency affects responses to replicative and oxidative stress, we sought evidence for Nit1 function in response to such stresses in tissues and cultured cells: when treated with hydroxyurea, the normal kidney‐derived double‐deficient cells appear not to activate the pChk2 pathway and when treated with H₂O₂, show little evidence of DNA damage, compared with wild type and Fhit^?/? cells. The relevance of Nit1 deficiency to human cancers was examined in human esophageal cancer tissues, and loss of Nit1 expression was observed in 48% of esophageal adenocarcinomas. J. Cell. Biochem. 107: 1097–1106, 2009. © 2009 Wiley‐Liss, Inc.     

Involvement of MEK-ERK signaling pathway in the inhibition of cell growth by troglitazone in human pancreatic cancer cells

Tif6p (eIF6) is necessary for 60S biogenesis, rRNA maturation and must be released from 60S to permit 80S assembly and translation. We characterized Tif6p interactors. Tif6p is mostly on 66S-60S pre-ribosomes, partly free. Tif6p complex(es) contain nucleo-ribosomal factors and Asc1p. Surprisingly, Tif6p particle contains the low-abundance endonuclease Sen34p. We analyzed Sen34p role on rRNA/tRNA synthesis, in vivo. Sen34p depletion impairs tRNA splicing and causes unexpected 80S accumulation. Accordingly, Sen34p overexpression causes 80S decrease and increased polysomes which suggest increased translational efficiency. With delayed kinetics, Sen34p depletion impairs rRNA processing. We conclude that Sen34p is absolutely required for tRNA splicing and that it is a rate-limiting element for efficient translation. Finally, we confirm that Tif6p accompanies 27S pre-rRNA maturation to 25S rRNA and we suggest that Sen34p endonuclease in Tif6p complex may affect also rRNA maturation.     

Construction of a virtual Mycobacterium tuberculosis consensus genome and its application to data from a next generation sequencer

Background

Although Mycobacterium tuberculosis isolates are consisted of several different lineages and the epidemiology analyses are usually assessed relative to a particular reference genome, M. tuberculosis H37Rv, which might introduce some biased results. Those analyses are essentially based genome sequence information of M. tuberculosis and could be performed in sillico in theory, with whole genome sequence (WGS) data available in the databases and obtained by next generation sequencers (NGSs). As an approach to establish higher resolution methods for such analyses, whole genome sequences of the M. tuberculosis complexes (MTBCs) strains available on databases were aligned to construct virtual reference genome sequences called the consensus sequence (CS), and evaluated its feasibility in in sillico epidemiological analyses.

Results

The consensus sequence (CS) was successfully constructed and utilized to perform phylogenetic analysis, evaluation of read mapping efficacy, which is crucial for detecting single nucleotide polymorphisms (SNPs), and various MTBC typing methods virtually including spoligotyping, VNTR, Long sequence polymorphism and Beijing typing. SNPs detected based on CS, in comparison with H37Rv, were utilized in concatemer-based phylogenetic analysis to determine their reliability relative to a phylogenetic tree based on whole genome alignment as the gold standard. Statistical comparison of phylogenic trees based on CS with that of H37Rv indicated the former showed always better results that that of later. SNP detection and concatenation with CS was advantageous because the frequency of crucial SNPs distinguishing among strain lineages was higher than those of H37Rv. The number of SNPs detected was lower with the consensus than with the H37Rv sequence, resulting in a significant reduction in computational time. Performance of each virtual typing was satisfactory and accorded with those published when those are available.

Conclusions

These results indicated that virtual CS constructed from genome sequence data is an ideal approach as a reference for MTBC studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1368-9) contains supplementary material, which is available to authorized users.