Multi-scale calculation and global-fit analysis of hydrodynamic properties of biological macromolecules: determination of the overall conformation of antibody IgG molecules

We present a scheme, based on existing and newly developed computational tools, for the determination of the overall conformation of biological macromolecules composed by domains or subunits, using from such structural determination easily available solution properties. In a multi-scale approach, atomic-level structures are used to provide simple shapes for the subunits, which are put together in a coarse grained model, with a few parameters that determine the overall shape of the macromolecule. Computer programs, like those in the HYDRO suite that evaluate the properties of either atomic or coarse-grained models. In this paper we present a new scheme for a global fit of multiple properties, implemented in a new computer program, HYDROFIT, which interfaces with the programs of the HYDRO suite to find an optimum, best-fitting structure in a robust but simple way. The determination of the overall structure of the native antibody IgG3, bearing a long hinge, and that of the hingeless mutant m15 is presented to test and confirm the validity of this simple, systematic and efficient scheme.     

Feeding habits of dolphinfish (Coryphaena hippurus) in the southeastern Gulf of California,Mexico

The objective of the study was to identify key elements of the feeding ecology of the dolphinfish, which is a high tropic predator important for sport and artisanal fishing in the Mexican Pacific. Feeding habits were investigated during the years 2000–2003. This species is seasonal in the southern Gulf of California and probably remains there because of the abundant prey. The contents of 232 dolphinfish stomachs were analyzed, identifying 98 prey species, although only eight of these were well‐represented in the diet. The most important prey by weight was the fish Hemiramphus saltator, however by number and frequency of occurrence was the crustacean Hemisquilla californiensis. No differences in the diet were found between males and females, although there was an ontogenic diet shift between seasons. There was no relationship between dolphinfish size and prey size, because dolphinfish fed preferentially on prey with an average size of 4.7 cm.     

Valoración de la utilidad del test de estimulación intraarterial con calcio en el diagnóstico de localización del hiperinsulinismo endógeno

Background and objectiveThe aim of this study was to assess the utility of arterial calcium stimulation with hepatic venous sampling (ASVS) in the localization of tumors in patients with endogenous hyperinsulinism not detected with other methods.Patients and methodsWe performed a retrospective study of 26 patients admitted to our hospital for hypoglycemia who underwent ASVS because the source of hyperinsulinism was not clearly identified by other imaging techniques. The histopathological result in patients who underwent a surgical procedure was considered the reference for statistical study of the accuracy of this technique. Statistical analysis was performed by comparing proportions with the chi-squared test with Yates’ correction for contingency tables, and Cohen′s kappa coefficient as a measure of interrater agreement between two observations.ResultsSurgery was performed in 17 patients, 13 with positive ASVS and the remaining four with negative results. An insulinoma was removed in 12 patients, and 10 of these were detected in the ASVS. A total of 76.9 % of positive ASVS tests corresponded to a histological diagnosis of insulinoma, and 83% of these insulinomas were positive in ASVS. This association was statistically significant (chi cuadrado = 7.340; p = 0.012). Two of three patients with nesidioblastosis had a positive response in the ASVS. A good and statistically significant agreement was obtained between histopathologic diagnosis and ASVS results (κ=0.556, p = 0.007).ConclusionsASVS is a useful procedure in the localization diagnosis of endogenous hyperinsulinism not detected by other imaging tests. This technique allows tumors in the pancreatic gland to be identified and may be useful in the choice of the surgical technique to be used.     

Immunoproteomics of the active degradome to identify biomarkers for Trichomonas vaginalis

Trichomonas vaginalis, a sexually transmitted parasite, has many cysteine proteinases (CPs); some are involved in trichomonal pathogenesis, express during infection, and antibodies against CPs have been detected in patient sera. The goal of this study was to identify the antigenic proteinases of T. vaginalis as potential biomarkers for trichomonosis. The proteases detected when T. vaginalis protein extracts are incubated without protease inhibitors, the trichomonad‐active degradome, and the immunoproteome were obtained by using 2‐DE, 2‐D‐zymograms, 2‐D‐Western blot (WB) assays with trichomonosis patient sera, and MS analysis. Forty‐nine silver‐stained spots were detected in the region of 200–21 kDa of parasite protease‐resistant extracts. A similar proteolytic pattern was observed in the 2‐D zymograms. Nine CPs were identified in the 30 kDa region (TvCP1, TvCP2, TvCP3, TvCP4, TvCP4‐like, TvCP12, TvCPT, TvLEGU‐1, and another legumain‐like CP). The major reactive spots to T. vaginalis‐positive patient sera by 2‐D‐WB corresponded to four papain‐like (TvCP2, TvCP4, TvCP4‐like, TvCPT), and one legumain‐like (TvLEGU‐1) CPs. The genes of TvCP4, TvCPT, and TvLEGU‐1 were cloned, sequenced, and expressed in Escherichia coli. Purified recombinant CPs were recognized by culture‐positive patient sera in 1‐D‐WB assays. These data show that some CPs could be potential biomarkers for serodiagnosis of trichomonosis.     

In vitro antiamyloidogenic properties of 1,4-naphthoquinones

The aim of this study is to find out whether several 1,4-naphthoquinones (1,4-NQ) can interact with the amyloidogenic pathway of the amyloid precursor protein processing, particularly targeting at β-secretase (BACE), as well as at β-amyloid peptide (Aβ) aggregation and disaggregating preformed Aβ fibrils. Compounds bearing hydroxyl groups at the quinoid (2) or benzenoid rings (5, 6) as well as some 2- and 3-aryl derivatives (11-15) showed BACE inhibitory activity, without effect on amyloid aggregation or disaggregation. The halogenated compounds 8 and 10 were selective for the inhibition of amyloid aggregation. On the other hand, 1,4-naphthoquinone (1), 6-hydroxy-1,4-naphthoquinone (4) and 2-(3,4-dichlorophenyl)-1,4-naphthoquinone (26) did not show any BACE inhibitory activity but were active on amyloid aggregation and disaggregation preformed Aβ fibrils. Juglone (5-hydroxy-1,4-naphthoquinone (3), and 3-(p-hydroxyphenyl)-5-methoxy-1,4-napththoquinone (19) were active on all the three targets. Therefore, we suggest that 1,4-NQ derivatives, specially 3 and 19, should be explored as possible drug candidates or lead compounds for the development of drugs to prevent amyloid aggregation and neurotoxicity in Alzheimer’s disease.     

Protection against Pseudomonas aeruginosa lung infection in mice by recombinant OprF-pulsed dendritic cell immunization

Background

Porcine reproductive and respiratory syndrome (PRRS) has now been widely recognized as an economically important disease. The objective of this study was to compare the molecular and biological characteristics of porcine reproductive and respiratory syndrome virus (PRRSV) field isolates in China to those of the modified live virus (MLV) PRRS vaccine and its parent strain (ATCC VR2332).

Results

Five genes (GP2, GP3, GP4, GP5 and NSP2) of seven isolates of PRRSV from China, designated LS-4, HM-1, HQ-5, HQ-6, GC-2, GCH-3 and ST-7/2008, were sequenced and analyzed. Phylogenetic analyses based on the nucleotide sequence of the ORF2-5 and NSP2 showed that the seven Chinese isolates belonged to the same genetic subgroup and were related to the North American PRRSV genotype. Comparative analysis with the relevant sequences of another Chinese isolate (BJ-4) and North American (VR2332 and MLV) viruses revealed that these isolates have 80.8-92.9% homology with VR-2332, and 81.3-98.8% identity with MLV and 80.7-92.9% with BJ-4. All Nsp2 nonstructural protein of these seven isolates exhibited variations (a 29 amino acids deletion) in comparison with other North American PRRSV isolates. Therefore, these isolates were novel strain with unique amino acid composition. However, they all share more than 97% identity with other highly pathogenic Chinese PRRSV strains. Additionally, there are extensive amino acid (aa) mutations in the GP5 protein and the Nsp2 protein when compared with the previous isolates.

Conclusions

These results might be useful to study the genetic diversity of PRRSV in China and to track the infection sources as well as for vaccines development.     

SEC14-like protein 1 interacts with cholinergic transporters

Trafficking of the vesicular acetylcholine transporter (VAChT) to synaptic vesicles has the potential to regulate storage and release of acetylcholine. We used the C-terminal tail of the vesicular acetylcholine transporter as bait for the screening of a brain cDNA library by yeast-two hybrids. Here we report an interaction uncovered in this screening with SEC14L1, a mammalian SEC14-like protein that may function as a phospholipid transfer protein. The interaction of VAChT and SEC14L1 occurred through the GOLD domain found in the latter and was confirmed in mammalian cells. In addition, we also found that SEC14L1 co-immunoprecipitates with the high affinity choline transporter (CHT1), but not with synaptophysin or synaptotagmin. In cultured cells SEC14L1 was predominantly found in the cytosol with little or no localization in defined organelles. In contrast, overexpression of VAChT or CHT1 with SEC14L1 recruited the latter to large intracellular organelles similar to vesicles or vesicle aggregates. Finally, we find that overexpression of SEC14L1 modestly decreases high affinity choline transport activity. We suggest that interaction of cholinergic transporters with proteins containing the GOLD domain may be relevant for transporter function.     

The inner cavity of Escherichia coli DegP protein is not essential for molecular chaperone and proteolytic activity

The Escherichia coli DegP protein is an essential periplasmic protein for bacterial survival at high temperatures. DegP has the unusual property of working as a chaperone below 28 degrees C, but efficiently degrading unfolded proteins above 28 degrees C. Monomeric DegP contains a protease domain and two PDZ domains. It oligomerizes into a hexameric cage through the staggered association of trimers. The active sites are located in a central cavity that is only accessible laterally, and the 12 PDZ domains act as mobile sidewalls that mediate opening and closing of the gates. As access to the active sites is restricted, DegP is an example of a self-compartmentalized protease. To determine the essential elements of DegP that maintain the integrity of the hexameric cage, we constructed several deletion mutants of DegP that formed trimers rather than hexamers. We found that residues 39 to 78 within the LA loops, as well as the PDZ2 domains are essential for the integrity of the DegP hexamer. In addition, we asked whether an enclosed cavity or cage of specific dimensions is required for the protease and chaperone activities in DegP. Both activities were maintained in the trimeric DegP mutants without an enclosed cavity and in deletion DegP mutants with significantly reduced dimensions of the cage. We conclude that the functional unit for the protease and chaperone activities of DegP is a trimer and that neither a cavity of specific dimensions nor the presence of an enclosed cavity appears to be essential for the protease and chaperone activities of DegP.