Separation and analysis of Bacillus subtilis respiratory chain complexes.

Bacillus subtilis is a Gram-positive bacterium with a respiratory chain embedded in the cytoplasmic membrane. The respiratory chain is bifurcated after menaquinol into a cytochrome b6c + caa3 branch and a branch with up to three quinol oxidases. The complexes that generate the proton gradient are b6c, associated with caa3 and aa3 oxidase. The b6c and caa3 complexes form a supercomplex, and it is proposed to form respiratory strings in the membrane. There is still information missing about the quinol branch and if the primary oxidase quinol aa3 is associated with the electron donor complexes. It is unclear whether succinate quinone reductase (SQR) can form associations with the quinol branch or the cytochrome branch. In this paper, we show the separation of an almost pure b6c complex associated with cytochromes c550 and c551. We obtained a b6c + caa3 supercomplex of 600 kDa and SQR, aa3, and NADH dehydrogenase by dodecyl maltoside solubilization and separation of the respiratory chain components by ionic exchange chromatography. We found that aa3 does not associate with other complexes. SQR was associated with the b6c complex in a mutant lacking aa3. This association could facilitate electron transfer from SQR to menaquinone-7. The lack of associations between the abundant quinol oxidase aa3 and other complexes is a feature we cannot explain yet.

Solid-state fermentation increases secretome complexity in Aspergillus brasiliensis.

Aspergillus is used for the industrial production of enzymes and organic acids, mainly by submerged fermentation (SmF). However, solid-state fermentation (SSF) offers several advantages over SmF. Although differences related to lower catabolite repression and substrate inhibition, as well as higher extracellular enzyme production in SSF compared to SmF have been shown, the mechanisms undelaying such differences are still unknown. To explain some differences among SSF and SmF, the secretome of Aspergillus brasiliensis obtained from cultures in a homogeneous physiological state with high glucose concentrations was analyzed. Of the regulated proteins produced by SmF, 74% were downregulated by increasing the glucose concentration, whereas all those produced by SSF were upregulated. The most abundant and upregulated protein found in SSF was the transaldolase, which could perform a moonlighting function in fungal adhesion to the solid support. This study evidenced that SSF: (i) improves the kinetic parameters in relation to SmF, (ii) prevents the catabolite repression, (iii) increases the branching level of hyphae and oxidative metabolism, as well as the concentration and diversity of secreted proteins, and (iv) favors the secretion of typically intracellular proteins that could be involved in fungal adhesion. All these differences can be related to the fact that molds are more specialized to growth in solid materials because they mimic their natural habitat.

Biodegradation of recalcitrant compounds and phthalates by culturable bacteria isolated from Liometopum apiculatum microbiota.

Liometopum apiculatum is a species of ants widely distributed in arid and semi-arid ecosystems where there is a relative food shortage compared with tropical ecosystems. L. apiculatum has established an ecological balance involving symbiotic interactions, which have allowed them to survive through mechanisms that are still unknown. Therefore, the aim of this study was to explore the metabolic potential of isolated bacteria from L. apiculatum using enzymatic activity assay and substrate assimilation. Results revealed a complex bacteria consortium belonging to Proteobacteria, Firmicutes, and Actinobacteria phylum. Most of the isolated bacteria showed activities associated with biopolymers degradation, from them Exiguobacterium and B. simplex showed the highest amylolytic activity (27 U/mg protein), while A. johnsonii and B. pumulis showed the highest cellulolytic and xylanolytic activities (1 and 2.9 U/mg protein, respectively). By other hand, some microorganisms such as S. ficaria, E. asburiae, P. agglomerans, A. johnsonii, S. rubidaea, S. marcescens, S. warneri, and M. hydrocarbonoxydans were able to grow up to 1000 mg/L of phthalates esters. These results not only revealed the important contribution of the symbionts in L apiculatum ants feeding habits, but also have shown a promising source of enzymes with potential biotechnological applications such as lignocellulosic biomass hydrolysis and bioremediation processes.

Protein complexes associated with ß-catenin differentially influence the differentiation profile of neonatal and adult CD8+ T cells.

The canonical Wnt signaling pathway is a master cell regulator involved in CD8+ T cell proliferation and differentiation. In human CD8+ T cells, this pathway induces differentiation into memory cells or a "stem cell memory like" population, which is preferentially present in cord blood. To better understand the role of canonical Wnt signals in neonatal or adult blood, we compared the proteins associated with ß-catenin, in nonstimulated and Wnt3a-stimulated human neonatal and adult naive CD8+ T cells. Differentially recruited proteins established different complexes in adult and neonatal cells. In the former, ß-catenin-associated proteins were linked to cell signaling and immunological functions, whereas those of neonates were linked to proliferation and metabolism. Wnt3a stimulation led to the recruitment and overexpression of Wnt11 in adult cells and Wnt5a in neonatal cells, suggesting a differential connexion with planar polarity and Wnt/Ca2+ noncanonical pathways, respectively. The chromatin immunoprecipitation polymerase chain reaction ß-catenin was recruited to a higher level on the promoters of cell renewal genes in neonatal cells and of differentiation genes in those of adults. We found a preferential association of ß-catenin with CBP in neonatal cells and with p300 in the adult samples, which could be involved in a higher self-renewal capacity of the neonatal cells and memory commitment in those of adults. Altogether, our results show that different proteins associated with ß-catenin during Wnt3a activation mediate a differential response of neonatal and adult human CD8+ T cells.

Microbiota of edible Liometopum apiculatum ant larvae reveals potential functions related to their nutritional value.

Edible insects, due to their high nutritive value, are currently considered as a potential renewable source for food and feed production. Liometopum apiculatum ants are widely distributed in arid and semi-arid ecosystems and their larvae (escamoles) are considered as a delicacy, however the microbial importance in L. apiculatum nutritional ecology is unknown. The aim of this research was to characterize the microorganisms associated with both L. apiculatum larvae and the reproductive adult ants using the 16S rRNA gene sequencing and culturomics approaches. The obligate endosymbionts were also investigated through microscopic analysis. The most abundant Phylum identified by sequencing in the larvae was Firmicutes while in adult ants was Proteobacteria. Interestingly, the culturomics results showed 15 genera corresponding to the bacteria identified by sequencing analysis. Particularly, it was observed a large population of nitrogen-fixing bacteria, which could be linked with the high protein content in escamoles. Endosymbionts were detected in bacteoriocytes, these bacteria are related with vitamins and essential amino acids biosynthesis, and both compounds contributing to the high nutritional value of escamoles. This is the first report of the microorganisms present in the escamolera ant ensuring their safety as food and opening new areas of nutritional ecological and food processing.

Distribution of isoforms of ferredoxin-NADP+ reductase (FNR) in cyanobacteria in two growth conditions.

Ferredoxin-NADP+ reductase (FNR) transfers reducing equivalents between ferredoxin and NADP(H) in the photosynthetic electron transport chains of chloroplasts and cyanobacteria. In most cyanobacteria, FNR is coded by a single petH gene. The structure of FNR in photosynthetic organisms can be constituted by FAD-binding and NADPH-binding domains (FNR-2D), or by these and an additional N-terminal domain (FNR-3D). In this article, biochemical evidence is provided supporting the induction of FNR-2D by iron or combined nitrogen deficiency in the cyanobacteria Synechocystis PCC 6803 and Anabaena variabilis ATCC 29413. In cell extracts of these cyanobacteria, most of FNR was associated to phycobilisomes (PBS) or phycocyanin (PC), and the rest was found as free enzyme. Free FNR activity increased in both cyanobacteria under iron stress and during diazotrophic conditions in A. variabilis. Characterization of FNR from both cyanobacteria showed that the PBS-associated enzyme was FNR-3D and the free enzyme was mostly a FNR-2D isoform. Predominant isoforms in heterocysts of A. variabilis were FNR-2D; where its N-terminal sequence lacked an initial (formyl)methionine. This means that FNR-3D is targeted to thylakoid membrane, and anchored to PBS, and FNR-2D is found as a soluble protein in the cytoplasm, when iron or fixed nitrogen deficiencies prevail in the environment. Moreover, given that Synechocystis and Anabaena variabilis are dissimilar in genotype, phenotype and ecology, the presence of these two-domain proteins in these species suggests that the mechanism of FNR induction is common among cyanobacteria regardless of their habitat and morphotype.

The composition of the Bacillus subtilis aerobic respiratory chain supercomplexes.

Bacillus subtilis has a bifurcated respiratory chain composed of a cytochrome branch and a quinol oxidase branch. The respiratory complexes of this bacterium have been elucidated mostly by the analysis of the genome and by the isolation of individual complexes. The supramolecular organization of this respiratory chain is not known. In this work, we have analyzed the organization of the supercomplex in membranes isolated from B. subtilis grown in aerobic conditions in a medium with 3 % succinate. We used two different native electrophoretic techniques, clear native electrophoresis (CNE) and blue native electrophoresis (BNE). Using a heme-specific stain and Coomassie blue stain with in-gel activity assays followed by mass spectrometry, we identified the proteins resolved in both the first and second dimensions of the electrophoreses to detect the supercomplexes. We found that complexes b ( 6 ) c and caa ( 3 ) form a very high molecular mass supercomplex with the membrane-bound cytochrome c ( 550 ) and with ATP synthase. Most of the ATP synthase was found as a monomer. Succinate dehydrogenase was identified within a high molecular band between F(0)F(1) and F(1) and together with nitrate reductase. The type-2 NADH dehydrogenase was detected within a low molecular mass band. Finally, the quinol oxidase aa ( 3 ) seems to migrate as an oligomer of high molecular mass.

Hox B4 as potential marker of non-differentiated cells in human cervical cancer cells.

BACKGROUND: Cervical cancer (CC) is a common malignancy in women worldwide. Cervical tumorigenesis involves a multistep process in which accumulations of genetic alterations are present. Homeotic genes, such as HOX gene re-expression, have been reported in a wide variety of tumors. METHODS: In order to know the role of HOX B4 gene expression in CC, in the present study, two-dimensional polyacrylamide gel electrophoresis, matrix-assisted laser desorption/ionization, and time-of-flight mass spectrometry were used for differential screening of protein expression in CC. Immunohistochemical analysis was performed on the cervical tissue microarray (TMA) to detect the Hox B4 protein. RESULTS: Hox B4 peptide was detected among 15 increased spots differentially observed in CC. Using TMA, Hox B4 protein was also immunodetected in the nuclei of cervical epithelial tumor cells, while in normal cervical epithelium, it was absent. Interestingly, it was possible to detect the Hox B4 protein in the precursor lesions. CONCLUSIONS: Hox B4 protein is present in the precursor lesions as CC cells, suggesting that Hox B4 could be a protein related to the neoplastic state (non-differentiated cells) of human cervical epithelium.

In guinea pig sperm, aldolase A forms a complex with actin, WAS, and Arp2/3 that plays a role in actin polymerization.

Glycolytic enzymes have, in addition to their role in energy production, other functions in the regulation of cellular processes. Aldolase A has been reported to be present in sperm, playing a key role in glycolysis; however, despite its reported interactions with actin and WAS, little is known about a non-glycolytic role of aldolase A in sperm. Here, we show that in guinea pig spermatozoa, aldolase A is tightly associated to cytoskeletal structures where it interacts with actin, WAS, and Arp2/3. We show that aldolase A spermatozoa treatment increases their polymerized actin levels. In addition, we show that there is a direct correlation between the levels of polymerized actin and the levels of aldolase A-actin interaction. Our results suggest that aldolase A functions as a bridge between filaments of actin and the actin-polymerizing machinery.

Analysis of human serum from women affected by cervical lesions.

Cervical cancer is one of the first causes of death in Mexican women population. The plasma proteome has a wide dynamic range concentrations of different protein and their alterations reflect the physiological state of the individual's health. The aim of this study was to characterize the 2D-PAGE serum patterns from healthy women and with different levels of cervical lesions. Changes in haptoglobin, apolipoproteins, and transthyretin, when comparing the serum from healthy women and serum from patients with different levels of cervical lesion were found. The Western blot analysis showed increasing concentrations of metalloproteinases (MMP's), proteins with important biological roles in tumor development and metastasis. Protein profiles in conjunction with MS, bioinformatics, and Western blot analysis, allow us to compile information for the acquisition of results to proposed candidates biomarkers of cervical cancer among Mexican women population.

Cloning of a cDNA encoding a cystatin from grain amaranth (Amaranthus hypochondriacus) showing a tissue-specific expression that is modified by germination and abiotic stress.

A cDNA, encoding a cysteine protease inhibitor (AhCPI), was isolated from an immature seed cDNA library of grain amaranth (Amaranthus hypochondriacus L.) and characterized. It encoded a polypeptide of 247 amino acids (aa), including a putative N-terminal signal peptide. Other relevant regions found in its sequence included the G and PW conserved aa motifs, the consensus LARFAV sequence for phytocystatins and the reactive site QVVAG. The predicted aa sequence for AhCPI showed a significant homology to other plant cystatins. Gene expression analyses indicated that AhCPI was constitutively expressed in mature seeds, and gradually decreased during germination. In vegetative tissues, AhCPI was expressed in the radicle and hypocotyls of seedlings and in the stems and roots of young plantlets. Its expression in roots and stems increased substantially in response to water deficit, salinity-, cold- and heat-stress, whereas heat-stress induced a rapid and transient accumulation of AhCPI transcripts in leaves. The results obtained were suggestive of multiple roles for AhCPI in grain amaranth, acting as a regulator of seed germination and as a protective agent against diverse types of abiotic stress, which induced this gene in a tissue- and stress-specific manner. The work herewith described reports a novel, and apparently, single cystatin protein in which, in agreement with other plant model systems, could have a regulatory role in germination, and further expands previous findings linking the accumulation of protease inhibitors, mostly of the serine proteinase type, with protection against (a)biotic stress in A. hypochondriacus.

The presence of multidomain linkers determines the bundle-shape structure of the phycobilisome of the cyanobacterium Gloeobacter violaceus PCC 7421.

The complete genome sequence of Gloeobacter violaceus [Nakamura et al. (2003a, b) DNA Res 10:37-45, 181-201] allows us to understand better the structure of the phycobilisomes (PBS) of this cyanobacterium. Genomic analysis revealed peculiarities in these PBS: the presence of genes for two multidomain linker proteins, a core membrane linker with four repetitive sequences (REP domains), the absence of rod core linkers, two sets of phycocyanin (PC) alpha and beta subunits, two copies of a rod PC associated linker (CpcC), and two rod cap associated linkers (CpcD). Also, there is one ferredoxin-NADP(+) oxidoreductase with only two domains. The PBS proteins were investigated by gel electrophoresis, amino acid sequencing and peptide mass fingerprinting (PMF). The two unique multidomain linkers contain three REP domains with high similarity and these were found to be in tandem and were separated by dissimilar Arms. One of these, with a mass of 81 kDa, is found in heavy PBS fragments rich in PC. We propose that it links six PC hexamers in two parallel rows in the rods. The other unique linker has a mass of 91 kDa and is easily released from the heavy fragments of PBS. We propose that this links the rods to the core. The presence of these multidomain linkers could explain the bundle shaped rods of the PBS. The presence of 4 REP domains in the core membrane linker protein (129 kDa) was established by PMF. This core linker may hold together 16 AP trimers of the pentacylindrical core, or alternatively, a tetracylindrical core of the PBS of G. violaceus.

Isolation and properties of a Kunitz-type protein inhibitor obtained from Pithecellobium dulce seeds.

We report for the first time the isolation and characterization of a protease inhibitor from the seeds of Pithecellobium dulce, which is a Leguminosae tree native to Mexico. The purification of the P. dulce trypsin inhibitor (PDTI) was a direct process. After its extraction (pH 8.0) and precipitation (80% (NH(4))(2)SO(4)), the pH was adjusted to 4.0, the supernatant was loaded onto a CM-Sepharose column, and a single peak of trypsin inhibitory activity was eluted (CM-TIA). The main component of CM-TIA was PDTI, a protein composed of two polypeptide chains joined by disulfide bridge(s), with a pI of 4.95 and a molecular weight determined by electrospray mass spectrometry of 19 614 Da. The N-terminal sequence of PDTI has the highest similarity with the seed inhibitor of Acacia confusa. PDTI lacks chymotrypsin inhibitory activity. A low rate of cytotoxicity of CM-TIA toward RINm5F cells contrasted with a high rate of the active fraction G75-TIA (gel filtration chromatography; LC(50) of 0.04 mg/mL).