Functional recycling of C2 domains throughout evolution: A comparative study of synaptotagmin, protein kinase C and phospholipase C by sequence, structural and modelling approaches

The C2 domain is one of the most frequent and widely distributed calcium-binding motifs. Its structure comprises an eight-stranded beta-sandwich with two structural types as if the result of a circular permutation. Combining sequence, structural and modelling information, we have explored, at different levels of granularity, the functional characteristics of several families of C2 domains. At the coarsest level,the similarity correlates with key structural determinants of the C2 domain fold and, at the finest level, with the domain architecture of the proteins containing them, highlighting the functional diversity between the various subfamilies. The functional diversity appears as different conserved surface patches throughout this common fold. In some cases, these patches are related to substrate-binding sites whereas in others they correspond to interfaces of presumably permanent interaction between other domains within the same polypeptide chain. For those related to substrate-binding sites, the predictions overlap with biochemical data in addition to providing some novel observations. For those acting as protein-protein interfaces' our modelling analysis suggests that slight variations between families are a result of not only complementary adaptations in the interfaces involved but also different domain architecture. In the light of the sequence and structural genomic projects, the work presented here shows that modelling approaches along with careful sub-typing of protein families will be a powerful combination for a broader coverage in proteomics. (C) 2003 Elsevier Ltd. All rights reserved.

Comparative proteomic analysis to study molecular events during gonad development in mice

Sex determination represents a critical bifurcation in the road of embryonic development. It is based on a finely regulated network of gene activity, as well as protein-protein interactions and activation or silencing of signaling pathways. Despite the identification of a number of critical genes, many aspects of the molecular cascade that drives the differentiation of the embryonic gonad into either a testis or an ovary remain poorly understood. To identify new proteins involved in this cascade, we employed two-dimensional gel electrophoresis and mass spectrometry to compare the protein expression profiles of fetal mouse testes and ovaries. Three proteins, hnRPA1, TRA1, and HSC71, were found to be expressed in a male-specific manner and this expression was confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization. Moreover, HSC71 was found to be hyperphosphorylated in male compared to female gonads, emphasizing the advantage of the proteomic approach in allowing the detection of posttranslational modifications.

Comparative transcriptome analyses of Pseudomonas aeruginosa

One of the hallmarks of bacterial survival is their ability to adapt rapidly to changing environmental conditions. Niche adaptation is a response to the signals received that are relayed, often to regulators that modulate gene expression. In the post-genomic era, DNA microarrays are used to study the dynamics of gene expression on a global scale. Numerous studies have used Pseudomonas aeruginosa--a Gram-negative environmental and opportunistic human pathogenic bacterium--as the model organism in whole-genome transcriptome analysis. This paper reviews the transcriptome studies that have led to immense advances in our understanding of the biology of this intractable human pathogen. Comparative analysis of 23 P. aeruginosa transcriptome studies has led to the identification of a unique set of genes that are signal specific and a core set that is differentially regulated. The 303 genes in the core set are involved in bacterial homeostasis, making them attractive therapeutic targets.

Functional genomic and molecular analyses of transcripts related to glycerol synthesis and trafficking in rainbow smelt (Osmerus mordax) using cold temperature-induced models of glycerol production

The rainbow smelt (Osmerus mordax) is an anadromous teleost that produces type II antifreeze protein (AFP) and accumulates modest urea and high glycerol levels in plasma and tissues as adaptive cryoprotectant mechanisms in sub-zero temperatures. It is known that glyceroneogenesis occurs in liver via a branch in glycolysis and gluconeogenesis and is activated by low temperature; however, the precise mechanisms of glycerol synthesis and trafficking in smelt remain to be elucidated. The objective of this thesis was to provide further insight using functional genomic techniques [e.g. suppression subtractive hybridization (SSH) cDNA library construction, microarray analyses] and molecular analyses [e.g. cloning, quantitative reverse transcription - polymerase chain reaction (QPCR)]. Novel molecular mechanisms related to glyceroneogenesis were deciphered by comparing the transcript expression profiles of glycerol (cold temperature) and non-glycerol (warm temperature) accumulating hepatocytes (Chapter 2) and livers from intact smelt (Chapter 3). Briefly, glycerol synthesis can be initiated from both amino acids and carbohydrate; however carbohydrate appears to be the preferred source when it is readily available. In glycerol accumulating hepatocytes, levels of the hepatic glucose transporter (GLUT2) plummeted and transcript levels of a suite of genes (PEPCK, MDH2, AAT2, GDH and AQP9) associated with the mobilization of amino acids to fuel glycerol synthesis were all transiently higher. In contrast, in glycerol accumulating livers from intact smelt, glycerol synthesis was primarily fuelled by glycogen degradation with higher PGM and PFK (glycolysis) transcript levels. Whether initiated from amino acids or carbohydrate, there were common metabolic underpinnings. Increased PDK2 (an inhibitor of PDH) transcript levels would direct pyruvate derived from amino acids and / or DHAP derived from G6P to glycerol as opposed to oxidation via the citric acid cycle. Robust LIPL (triglyceride catabolism) transcript levels would provide free fatty acids that could be oxidized to fuel ATP synthesis. Increased cGPDH (glyceroneogenesis) transcript levels were not required for increased glycerol production, suggesting that regulation is more likely by post-translational modification. Finally, levels of a transcript potentially encoding glycerol-3-phosphatase, an enzyme not yet characterized in any vertebrate species, were transiently higher. These comparisons also led to the novel discoveries that increased G6Pase (glucose synthesis) and increased GS (glutamine synthesis) transcript levels were part of the low temperature response in smelt. Glucose may provide increased colligative protection against freezing; whereas glutamine could serve to store nitrogen released from amino acid catabolism in a non-toxic form and / or be used to synthesize urea via purine synthesis-uricolysis. Novel key aspects of cryoprotectant osmolyte (glycerol and urea) trafficking were elucidated by cloning and characterizing three aquaglyceroporin (GLP)-encoding genes from smelt at the gene and cDNA levels in Chapter 4. GLPs are integral membrane proteins that facilitate passive movement of water, glycerol and urea across cellular membranes. The highlight was the discovery that AQP10ba transcript levels always increase in posterior kidney only at low temperature. This AQP10b gene paralogue may have evolved to aid in the reabsorption of urea from the proximal tubule. This research has contributed significantly to a general understanding of the cold adaptation response in smelt, and more specifically to the development of a working scenario for the mechanisms involved in glycerol synthesis and trafficking in this species.

Angiomatoid fibrous histiocytoma: comparison of fluorescence in situ hybridization and reverse transcription polymerase chain reaction as adjunct diagnostic modalities

Angiomatoid fibrous histiocytoma (AFH) is a rare soft tissue neoplasm of intermediate biologic potential and uncertain differentiation, most often arising in the extremities of children and young adults. Although it has characteristic histologic features of a lymphoid cuff surrounding nodules of ovoid cells with blood-filled cystic cavities, diagnosis is often difficult due to its morphologic heterogeneity and lack of specific immunoprofile. Angiomatoid fibrous histiocytoma is associated with recurrent chromosomal translocations, leading to characteristic EWSR1-CREB1, EWSR1-ATF1, and, rarely, FUS-ATF1 gene fusions; fluorescence in situ hybridization (FISH), detecting EWSR1 or FUS rearrangements, and reverse transcription-polymerase chain reaction (RT-PCR) for EWSR1-CREB1 and EWSR1-ATF1 fusion transcripts have become routine ancillary tools. We present a large comparative series of FISH and RT-PCR for AFH. Seventeen neoplasms (from 16 patients) histologically diagnosed as AFH were assessed for EWSR1 rearrangements or EWSR1-CREB1 and EWSR1-ATF1 fusion transcripts. All 17 were positive for either FISH or RT-PCR or both. Of 16, 14 (87.5%) had detectable EWSR1-CREB1 or EWSR1-ATF1 fusion transcripts by RT-PCR, whereas 13 (76.5%) of 17 had positive EWSR1 rearrangement with FISH. All 13 of 13 non-AFH control neoplasms failed to show EWSR1-CREB1 or EWSR1-ATF1 fusion transcripts, whereas EWSR1 rearrangement was present in 2 of these 13 cases (which were histopathologically myoepithelial neoplasms). This study shows that EWSR1-CREB1 or EWSR1-ATF1 fusions predominate in AFH (supporting previous reports that FUS rearrangement is rare in AFH) and that RT-PCR has a comparable detection rate to FISH for AFH. Importantly, cases of AFH can be missed if RT-PCR is not performed in conjunction with FISH, and RT-PCR has the added advantage of specificity, which is crucial, as EWSR1 rearrangements are present in a variety of neoplasms in the histologic differential diagnosis of AFH, that differ in behavior and treatment.

Comparative Analysis of Genus Bifidobacterium: Insight into its Host Adaptation

Bifidobacteria is amongst one of the health promoting bacteria. The role of this important probiotic genera can be elucidated by understanding its genome. Comparative analysis of the whole genus of these bacteria can reveal their adaptation to a diverse host range. This study comprises of four research projects. In the first study, a reference library for genus Bifidobacterium was prepared. The core genes in each genus were selected based on a newly proposed statistical definition of core genome. Comparative analysis of Bifidobacterium with another probiotic genus Lactobacillus revealed the metabolic characteristics of genus Bifidobacterium. The second study investigated the immunomodulatory role of a B. bifidum strain TMC3115. The analysis of TMC3115 provided insights into its extracellular structures which might have their role in host interaction and immunomodulation. The study highlighted the variability among these genomes just not on species level but also on strain level in terms of host interaction. The last two studies aim to inspect the relationship between bifidobacteria and its host diet. Bifidobacteria, are both host- and niche-specific. Such adaptation of bifidobacterial species is considered relevant to the intestinal microecosystem and hosts’ oligosaccharides. Many species should have co-evolved with their hosts, but the phylogeny of Bifidobacterium is dissimilar to that of host animals. The discrepancy could be linked to the niche-specific evolution due to hosts’ dietary carbohydrates. The distribution of carbohydrate-active enzymes, in particular glycoside hydrolases (GHs) that metabolize unique oligosaccharides was examined. When bifidobacterial species were classified by their distribution of GH genes, five groups arose according to their hosts’ feeding behaviour. The distribution of GH genes was only weakly associated with the phylogeny of the host animals or with genomic features such as genome size. Thus, the hosts’ dietary pattern is the key determinant of the distribution and evolution of GH genes.

Comparative Cytogenetics Of Physalaemus albifrons And Physalaemus Cuvieri Species Groups (anura, leptodactylidae).

Recently, Physalaemus albifrons (Spix, 1824) was relocated from the Physalaemus cuvieri group to the same group as Physalaemus biligonigerus (Cope, 1861), Physalaemus marmoratus (Reinhardt & Lütken, 1862) and Physalaemus santafecinus Barrio, 1965. To contribute to the analysis of this proposition, we studied the karyotypes of Physalaemus albifrons, Physalaemus santafecinus and three species of the Physalaemus cuvieri group. The karyotype of Physalaemus santafecinus was found to be very similar to those of Physalaemus biligonigerus and Physalaemus marmoratus, which were previously described. A remarkable characteristic that these three species share is a conspicuous C-band that extends from the pericentromeric region almost to the telomere in the short arm of chromosome 3. This characteristic is not present in the Physalaemus albifrons karyotype and could be a synapomorphy of Physalaemus biligonigerus, Physalaemus marmoratus and Physalaemus santafecinus. The karyotype of Physalaemus santafecinus is also similar to those of Physalaemus marmoratus and Physalaemus biligonigerus owing to the presence of several terminal C-bands and the distal localization of the NOR in a small metacentric chromosome. In contrast, the Physalaemus albifrons karyotype has no terminal C-bands and its NOR is located interstitially in the long arm of submetacentric chromosome 8. The NOR-bearing chromosome of Physalaemus albifrons very closely resembles those found in Physalaemus albonotatus (Steindachner, 1864), Physalaemus cuqui Lobo, 1993 and some populations of Physalaemus cuvieri Fitzinger, 1826. Additionally, the Physalaemus albifrons karyotype has an interstitial C-band in chromosome 5 that has been exclusively observed in species of the Physalaemus cuvieri group. Therefore, we were not able to identify any chromosomal feature that supports the reallocation of Physalaemus albifrons.

New Microsatellite Markers For Wild And Commercial Species Of Passiflora (passifloraceae) And Cross-amplification.

• We developed the first microsatellites for Passiflora setacea and characterized new sets of markers for P. edulis and P. cincinnata, enabling further genetic diversity studies to support the conservation and breeding of passion fruit species. • We developed 69 microsatellite markers and, in conjunction with assessments of cross-amplification using primers available from the literature, present 43 new polymorphic microsatellite loci for three species of Passiflora. The mean number of alleles per locus was 3.1, and the mean values of the expected and observed levels of heterozygosity were 0.406 and 0.322, respectively. • These microsatellite markers will be valuable tools for investigating the genetic diversity and population structure of wild and commercial species of passion fruit (Passiflora spp.) and may be useful for developing conservation and improvement strategies by contributing to the understanding of the mating system and hybridization within the genus.

Comparative Study Of Planned And Unplanned Excisions For The Treatment Of Soft Tissue Sarcoma Of The Extremities.

Unplanned excision of soft tissue sarcomas is common because benign soft tissue lesions are very frequent. This study evaluated the impact of unplanned resections on overall survival, local recurrence and distant metastasis in patients with soft tissue sarcomas of the extremities. In total, 52 patients who were diagnosed with soft tissue sarcomas between May 2001 and March 2011 were analyzed in a retrospective study. Of these patients, 29 (55.8%) had not undergone previous treatment and the remaining 23 (44.2%) patients had undergone prior resection of the tumor without oncological planning. All subsequent surgical procedures were performed at the same cancer referral center. The follow-up ranged from 6 to 122 months, with a mean of 39.89 months. Age, lesion size and depth, histological grade, surgical margins, overall survival, local and distant recurrence and adjuvant therapies were compared. Residual disease was observed in 91.3% of the re-resected specimens in the unplanned excision group, which exhibited greater numbers of superficial lesions, low histological grades and contaminated surgical margins compared with the re-resected specimens in the planned excision group. No differences were observed in local recurrence and 5-year overall survival between the groups, but distant metastases were significantly associated with planned excision after adjustment for the variables. There was no difference between patients undergoing unplanned excision and planned excision regarding local recurrence and overall survival. The planned excision group had a higher risk of distant metastasis, whereas there was a high rate of residual cancer in the unplanned excision group.

Comparative Metagenomic Analysis Of Coral Microbial Communities Using A Reference-independent Approach.

By comparing the SEED and Pfam functional profiles of metagenomes of two Brazilian coral species with 29 datasets that are publicly available, we were able to identify some functions, such as protein secretion systems, that are overrepresented in the metagenomes of corals and may play a role in the establishment and maintenance of bacteria-coral associations. However, only a small percentage of the reads of these metagenomes could be annotated by these reference databases, which may lead to a strong bias in the comparative studies. For this reason, we have searched for identical sequences (99% of nucleotide identity) among these metagenomes in order to perform a reference-independent comparative analysis, and we were able to identify groups of microbial communities that may be under similar selective pressures. The identification of sequences shared among the metagenomes was found to be even better for the identification of groups of communities with similar niche requirements than the traditional analysis of functional profiles. This approach is not only helpful for the investigation of similarities between microbial communities with high proportion of unknown reads, but also enables an indirect overview of gene exchange between communities.

Comparative In vitro Mechanical Evaluation Of Techniques Using A 2.0 mm Locking Fixation System For Simulated Fractures Of The Mandibular Body.

To perform a comparative evaluation of the mechanical resistance of simulated fractures of the mandibular body which were repaired using different fixation techniques with two different brands of 2.0 mm locking fixation systems. Four aluminum hemimandibles with linear sectioning simulating a mandibular body fracture were used as the substrates and were fixed using the two techniques and two different brands of fixation plate. These were divided into four groups: groups I and II were fixed with one four-hole plate, with four 6 mm screws in the tension zone and one four-hole plate, with four 10 mm screws in the compression zone; and groups III and IV were fixed with one four-hole plate with four 6 mm screws in the neutral zone. Fixation plates manufactured by Tóride were used for groups I and III, and by Traumec for groups II and IV. The hemimandibles were submitted to vertical, linear load testing in an Instron 4411 servohydraulic mechanical testing unit, and the load/displacement (3 mm, 5 mm and 7 mm) and the peak loads were measured. Means and standard deviations were evaluated applying variance analysis with a significance level of 5%. The only significant difference between the brands was seen at displacements of 7 mm. Comparing the techniques, groups I and II showed higher mechanical strength than groups III and IV, as expected. For the treatment of mandibular linear body fracture, two locking plates, one in the tension zone and another in the compression zone, have a greater mechanical strength than a single locking plate in the neutral zone.

Genomic And Chemical Insights Into Biosurfactant Production By The Mangrove-derived Strain Bacillus Safensis Ccma-560.

Many Bacillus species can produce biosurfactant, although most of the studies on lipopeptide production by this genus have been focused on Bacillus subtilis. Surfactants are broadly used in pharmaceutical, food and petroleum industry, and biological surfactant shows some advantages over the chemical surfactants, such as less toxicity, production from renewable, cheaper feedstocks and development of novel recombinant hyperproducer strains. This study is aimed to unveil the biosurfactant metabolic pathway and chemical composition in Bacillus safensis strain CCMA-560. The whole genome of the CCMA-560 strain was previously sequenced, and with the aid of bioinformatics tools, its biosurfactant metabolic pathway was compared to other pathways of closely related species. Fourier transform infrared (FTIR) and high-resolution TOF mass spectrometry (MS) were used to characterize the biosurfactant molecule. B. safensis CCMA-560 metabolic pathway is similar to other Bacillus species; however, some differences in amino acid incorporation were observed, and chemical analyses corroborated the genetic results. The strain CCMA-560 harbours two genes flanked by srfAC and srfAD not present in other Bacillus spp., which can be involved in the production of the analogue gramicidin. FTIR and MS showed that B. safensis CCMA-560 produces a mixture of at least four lipopeptides with seven amino acids incorporated and a fatty acid chain with 14 carbons, which makes this molecule similar to the biosurfactant of Bacillus pumilus, namely, pumilacidin. This is the first report on the biosurfactant production by B. safensis, encompassing the investigation of the metabolic pathway and chemical characterization of the biosurfactant molecule.

[comparative Study Between Fast And Slow Induction Of Propofol Given By Target-controlled Infusion: Expected Propofol Concentration At The Effect Site. Randomized Controlled Trial].

studies have shown that rate of propofol infusion may influence the predicted propofol concentration at the effect site (Es). The aim of this study was to evaluate the Es predicted by the Marsh pharmacokinetic model (ke0 0.26min(-1)) in loss of consciousness during fast or slow induction. the study included 28 patients randomly divided into two equal groups. In slow induction group (S), target-controlled infusion (TCI) of propofol with plasma, Marsh pharmacokinetic model (ke0 0.26min(-1)) with target concentration (Tc) at 2.0-μg.mL(-1) were administered. When the predicted propofol concentration at the effect site (Es) reached half of Es value, Es was increased to previous Es + 1μg.mL(-1), successively, until loss of consciousness. In rapid induction group (R), patients were induced with TCI of propofol with plasma (6.0μg.ml(-1)) at Es, and waited until loss of consciousness. in rapid induction group, Tc for loss of consciousness was significantly lower compared to slow induction group (1.67±0.76 and 2.50±0.56μg.mL(-1), respectively, p=0.004). the predicted propofol concentration at the effect site for loss of consciousness is different for rapid induction and slow induction, even with the same pharmacokinetic model of propofol and the same balance constant between plasma and effect site.

Comparative Study Of Transgenic And Non-transgenic Maize (zea Mays) Flours Commercialized In Brazil, Focussing On Proteomic Analyses.

Genetically modified foods are a major concern around the world due to the lack of information concerning their safety and health effects. This work evaluates differences, at the proteomic level, between two types of crop samples: transgenic (MON810 event with the Cry1Ab gene, which confers resistance to insects) and non-transgenic maize flour commercialized in Brazil. The 2-D DIGE technique revealed 99 differentially expressed spots, which were collected in 2-D PAGE gels and identified via mass spectrometry (nESI-QTOF MS/MS). The abundance of protein differences between the transgenic and non-transgenic samples could arise from genetic modification or as a result of an environmental influence pertaining to the commercial sample. The major functional category of proteins identified was related to disease/defense and, although differences were observed between samples, no toxins or allergenic proteins were found.

Application Of Laser Microdissection Icp-ms For High Resolution Elemental Mapping In Mouse Brain Tissue: A Comparative Study With Laser Ablation Icp-ms.

Mapping of elements in biological tissue by laser induced mass spectrometry is a fast growing analytical methodology in life sciences. This method provides a multitude of useful information of metal, nonmetal, metalloid and isotopic distribution at major, minor and trace concentration ranges, usually with a lateral resolution of 12-160 µm. Selected applications in medical research require an improved lateral resolution of laser induced mass spectrometric technique at the low micrometre scale and below. The present work demonstrates the applicability of a recently developed analytical methodology - laser microdissection associated to inductively coupled plasma mass spectrometry (LMD ICP-MS) - to obtain elemental images of different solid biological samples at high lateral resolution. LMD ICP-MS images of mouse brain tissue samples stained with uranium and native are shown, and a direct comparison of LMD and laser ablation (LA) ICP-MS imaging methodologies, in terms of elemental quantification, is performed.