Changes in polyamine, total protein and total carbohydrate content and peroxidase activity during the lifetime of chrysanthemum faroe

The aim of the present work was to evaluate the changes in polyamine (PA) content, peroxidase (POX) activity and levels of total protein and total soluble carbohydrates throughout the lifetime of leaves and inflorescences of chrysantemum 'Faroe' treated with gibberellic acidd (GA3) (used in production practices) and kept at room temperature and cold storage. The treatments were composed of four doses of GA3 (0, 15, 30 and 45 mg L-1) applied at the beggining of flower bud formation (28 days after transplanting of seedlings). After harvesting, the stems (95% of the expanded ligule) were stored at 10ºC and 95% relative humidity for 48 hrs, or kept at room temperature. For biochemical analysis samples of leaves and inflorescences were collected at the 4th, 8th, 12th and 16th day after harvest. The application of GA3 in the field and cold storage increased the content of PAs. There was an increase in POX activity in leaves and inflorescences during postharvest and this increase was related to oxidation of the PAs studied. The amount of proteins and carbohydrates in chrysantemum 'Faroe' decreased during the storage at 25ºC and under cold conditions.

Cultivation and bromatological analysis of the medicinal mushroom Ganoderma lucidum (Curt.: Fr.) P. Karst cultivated in agricultural waste

The objective of the study was to evaluate the production of two strains of Ganoderma lucidum on agricultural waste and carry out bromatological analyses of the basidiomata obtained from the cultivation. The experiment was carried out at the Mushroom Module at the School of Agronomic Sciences of the São Paulo State University (FCA/UNESP - Botucatu, SP, Brazil) and two strains were used (GLM-09/01 and GLM-10/02) which were cultivated on waste, oat straw, bean straw, brachiaria grass straw, Tifton grass straw and eucalyptus sawdust under two situations: with (20%) and without (0%) supplementation with wheat bran. All the waste was taken from dumps of agricultural activities in Botucatu-SP. Both treatments were carried out in 10 repetitions, totaling 200 packages. The mushrooms cultivation took 90 days. Next, the biological efficiency of the treatments and the bromatological analysis of the basidiomata were evaluated. The biological efficiency (BE) values (%) varied from 0.0 to 6.7%. In the mushroom bromatological analyses, the results ranged from 8.7 to 13.7%, from 2.0 to 6.7%, from 0.83 to 1.79% and from 38.8 to 54.5%, for total protein, ethereal extract, ash and crude fiber, respectively. Thus, we conclude that the substrates which presented the greater yield were the brachiaria straw, 20% in both strains tested (GLM-09/01 and GLM-10/02) and the bean straw, 20% in the strain GLM-10/02. The mushrooms showed high levels of ethereal extract, fibers and ashes and a low level of proteins.

Characterization of Glycation Sites on Human Serum Albumin using Mass Spectrometry

The modification of proteins by reducing sugars is a process that occurs naturally in the body. This process, which is known as glycation, has been linked to many of the chronic complications encountered during diabetes. Glycation has also been linked to changes in the binding of human serum albumin (HSA) to several drugs and small solutes in the body. While these effects are known, there is little information that explains why these changes in binding occur. The goal of this project was to obtain qualitative and quantitative information about glycation that occurs on HSA. The first section of this dissertation examined methods that could be used to quantify and identify glycation that occurs on HSA. The extent of glycation that occurred on HSA was quantified using oxygen-18 labeling mass spectrometry and the glycation sites were identified by observing the mass-to-charge (m/z) shifts that occurred in glycated HSA. This initial investigation revealed that oxygen-18 labeling based quantitation can be improved over previous methods if a relative comparison is done with oxygen-18 labeled peptides in a control HSA sample. Similarly, the process of making m/z shift-based assignments could be improved if only the peptides that were unique to the glycated HSA samples were used with internal calibration. These techniques were used in subsequent chapters for the assignment of early and late-stage glycation products on HSA. The regions of HSA that contained the highest amount of modification were identified, quantified, and ranked in order of their relative abundance. Of the commonly reported glycation sites, the N-terminus was found to have the highest extent of modification, followed by lysines 525, 199, and 439. The relative amount of modification on lysine 281, with respect to the aforementioned residues, varied with different degrees of glycation. The oxygen-18 labeling approach used for this analysis was novel because it allowed for the simultaneous quantification of all glycation-related modifications that were occurring on HSA. As such, several arginine residues were also found to have high amounts of modification on glycated HSA.

Pressure Dependence of N-15 Chemical Shifts in Model Peptides Ac-Gly-Gly-X-Ala-NH2

High pressure NMR spectroscopy has developed into an important tool for studying conformational equilibria of proteins in solution. We have studied the amide proton and nitrogen chemical shifts of the 20 canonical amino acids X in the random-coil model peptide Ac-Gly-Gly-X-Ala-NH2, in a pressure range from 0.1 to 200 MPa, at a proton resonance frequency of 800 MHz. The obtained data allowed the determination of first and second order pressure coefficients with high accuracy at 283 K and pH 6.7. The mean first and second order pressure coefficients <B-1(15N)> and <B-2(15N)> for nitrogen are 2.91 ppm/GPa and -2.32 ppm/GPa(2), respectively. The corresponding values <B-1(1H)> and <B-2(1H)> for the amide protons are 0.52 ppm/GPa and -0.41 ppm/GPa(2). Residual dependent (1)J(1H15N)-coupling constants are shown.

PHENOLOGY AND FRUIT TRAITS OF ARCHONTOPHOENIX CUNNINGHAMIANA, AN INVASIVE PALM TREE IN THE ATLANTIC FOREST OF BRAZIL

The Australian palm Archontophoenix cunninghamiana was introduced into Brazil as an ornamental species, and became a dangerous invader of remnant Atlantic forest patches, demanding urgent management actions that require careful planning. Its fruits are greatly appreciated by generalist birds and its sudden eradication could be as harmful as its permanence in the native community. Our hypothesis was that A. cunninghamiana phenology and fruit traits would have facilitated the invasion process. Hence the aim of the study was to characterize the reproductive phenology of the palm by registering flowering and fruiting events, estimating fruit production, and evaluating fruit nutritional levels. Phenological observations were carried out over 12 months and analyzed statistically. Fruit traits and production were estimated. Pulp nutritional levels were determined by analyzing proteins, lipids, and carbohydrates. Results showed constant flowering and fruiting throughout the year with a weak reproductive seasonality. On average, 3651 fruits were produced per bunch mainly in the summer. Fruit analysis revealed low nutrient contents, especially of proteins and lipids compared with other Brazilian native palm species. We concluded that the abundant fruit production all year round, and fruit attractivity mainly due to size and color, :may act positively on the reproductive performance and effective dispersion of A. cunninghamiana. As a management procedure which would add quality to frugivore food resources we suggest the replacement of A. cunninghamiana by the native palm Euterpe edulis, especially in gardens and parks near to Atlantic forest fragments.

Proteomic Analysis of Chloroplast-to-Chromoplast Transition in Tomato Reveals Metabolic Shifts Coupled with Disrupted Thylakoid Biogenesis Machinery and Elevated Energy-Production Components

A comparative proteomic approach was performed to identify differentially expressed proteins in plastids at three stages of tomato (Solanum lycopersicum) fruit ripening (mature-green, breaker, red). Stringent curation and processing of the data from three independent replicates identified 1,932 proteins among which 1,529 were quantified by spectral counting. The quantification procedures have been subsequently validated by immunoblot analysis of six proteins representative of distinct metabolic or regulatory pathways. Among the main features of the chloroplast-to-chromoplast transition revealed by the study, chromoplastogenesis appears to be associated with major metabolic shifts: (1) strong decrease in abundance of proteins of light reactions (photosynthesis, Calvin cycle, photorespiration) and carbohydrate metabolism (starch synthesis/degradation), mostly between breaker and red stages and (2) increase in terpenoid biosynthesis (including carotenoids) and stress-response proteins (ascorbate-glutathione cycle, abiotic stress, redox, heat shock). These metabolic shifts are preceded by the accumulation of plastid-encoded acetyl Coenzyme A carboxylase D proteins accounting for the generation of a storage matrix that will accumulate carotenoids. Of particular note is the high abundance of proteins involved in providing energy and in metabolites import. Structural differentiation of the chromoplast is characterized by a sharp and continuous decrease of thylakoid proteins whereas envelope and stroma proteins remain remarkably stable. This is coincident with the disruption of the machinery for thylakoids and photosystem biogenesis (vesicular trafficking, provision of material for thylakoid biosynthesis, photosystems assembly) and the loss of the plastid division machinery. Altogether, the data provide new insights on the chromoplast differentiation process while enriching our knowledge of the plant plastid proteome.

Vascular dementia: Different forms of vessel disorders contribute to the development of dementia in the elderly brain

The diagnosis of vascular dementia (VaD) describes a group of various vessel disorders with different types of vascular lesions that finally contribute to the development of dementia. Most common forms of VaD in the elderly brain are subcortical vascular encephalopathy, strategic infarct dementia, and the multi infarct encephalopathy. Hereditary forms of VaD are rare. Most common is the cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Sporadic forms of VaD are caused by degenerative vessel disorders such as atherosclerosis, small vessel disease (SVD) including small vessel arteriosclerosis, arteriolosclerosis, and lipohyalinosis, and cerebral amyloid angiopathy (CAA). Less frequently inflammatory vessel disorders and tumor-associated vessel lesions (e. g. angiocentric T-cell or angiotropic large cell lymphoma) can cause symptoms of dementia. Here, we review and discuss the impact of vessel disorders to distinct vascular brain tissue lesions and to the development of dementia in elderly individuals. The impact of coexisting neurodegenerative pathology in the elderly brain to VaD as well as the correlation between SVD and CAA expansion in the brain parenchyma with that of Alzheimer's disease (AD)-related pathology is highlighted. We conclude that "pure" VaD is rare and most frequently caused by infarctions. However, there is a significant contribution of vascular lesions and vessel pathology to the development of dementia that may go beyond tissue damage due to vascular lesions. Insufficient blood blow and alterations of the perivascular drainage mechanisms of the brain may also lead to a reduced protein clearance from extracellular space and subsequent increase of proteins in the brain parenchyma, such as the amyloid beta-protein, and foster, thereby, the development of AD-related neurodegeneration. As such, it seems to be important for clinical practice to consider treatment of potentially coexisting AD pathology in cognitively impaired patients with vascular lesions. (C) 2012 Elsevier Inc. All rights reserved.

Comparative Genome Analysis of Trichophyton rubrum and Related Dermatophytes Reveals Candidate Genes Involved in Infection

The major cause of athlete's foot is Trichophyton rubrum, a dermatophyte or fungal pathogen of human skin. To facilitate molecular analyses of the dermatophytes, we sequenced T. rubrum and four related species, Trichophyton tonsurans, Trichophyton equinum, Microsporum canis, and Microsporum gypseum. These species differ in host range, mating, and disease progression. The dermatophyte genomes are highly colinear yet contain gene family expansions not found in other human-associated fungi. Dermatophyte genomes are enriched for gene families containing the LysM domain, which binds chitin and potentially related carbohydrates. These LysM domains differ in sequence from those in other species in regions of the peptide that could affect substrate binding. The dermatophytes also encode novel sets of fungus-specific kinases with unknown specificity, including nonfunctional pseudokinases, which may inhibit phosphorylation by competing for kinase sites within substrates, acting as allosteric effectors, or acting as scaffolds for signaling. The dermatophytes are also enriched for a large number of enzymes that synthesize secondary metabolites, including dermatophyte-specific genes that could synthesize novel compounds. Finally, dermatophytes are enriched in several classes of proteases that are necessary for fungal growth and nutrient acquisition on keratinized tissues. Despite differences in mating ability, genes involved in mating and meiosis are conserved across species, suggesting the possibility of cryptic mating in species where it has not been previously detected. These genome analyses identify gene families that are important to our understanding of how dermatophytes cause chronic infections, how they interact with epithelial cells, and how they respond to the host immune response. IMPORTANCE Athlete's foot, jock itch, ringworm, and nail infections are common fungal infections, all caused by fungi known as dermatophytes (fungi that infect skin). This report presents the genome sequences of Trichophyton rubrum, the most frequent cause of athlete's foot, as well as four other common dermatophytes. Dermatophyte genomes are enriched for four gene classes that may contribute to the ability of these fungi to cause disease. These include (i) proteases secreted to degrade skin; (ii) kinases, including pseudokinases, that are involved in signaling necessary for adapting to skin; (iii) secondary metabolites, compounds that act as toxins or signals in the interactions between fungus and host; and (iv) a class of proteins (LysM) that appear to bind and mask cell wall components and carbohydrates, thus avoiding the host's immune response to the fungi. These genome sequences provide a strong foundation for future work in understanding how dermatophytes cause disease.

An in vitro study showing the three-dimensional microenvironment influence over the behavior of head and neck squamous cell carcinoma

Objectives: The Head and Neck Squamous Cell Carcinoma (HNSCC) ranks sixth worldwide. The mechanisms of growth, invasion and metastasis of this pathology are extensively studied and generally related to specific variations in signaling pathways like the PI3K-Akt; however most of these competent studies have been performed bidimensionally, which may hide important questions. This study sought to analyze the influence of the microenvironment upon the behavior of HNSCC. Study Design: The status of pAkt, NF-kappa B and Cyclin D1 proteins was accessed through immunofluorescence and western blot methods in HNSCC cell lines originating from tongue, pharynx and metastatic lymph node when submitted to a three-dimensional culture model utilizing a matrix system. A bidimensional culture model (monolayer) was used as control. Results: The HNSCC cell lines cultured three-dimensionally exhibited a growth pattern characterized by small isolated islands, different from the control group. When the three-dimensional model was applied, two of the studied cell lines showed the same expression pattern as the bidimensional model regarding nuclear or cytoplasmatic localization, as well as reduction of all protein levels; however, the cell line originated from tongue, which specially has the epidermal growth factor receptor constitutively activated, demonstrated nuclear translocation of pAkt and also an increase in the levels of Cyclin D1. Conclusions: The results suggest the influence of the microenvironment upon the behavior of HNSCC cells due to the changed expression of proteins related to tumor growth and cellular invasion. Furthermore, intrinsically genetic conditions also played important roles over the cells, despite the culture model employed.

Cellular Infiltrates and NF kappa B Subunit c-Rel Signaling in Kidney Allografts of Patients With Clinical Operational Tolerance

Background. Nuclear factor kappa B (NF kappa B) plays a potential role in tolerance by orchestrating onset and resolution of inflammation and regulatory T cell differentiation through subunit c-Rel. We characterized cellular infiltrates and expression of NF kappa B1, c-Rel and its upstream regulators phosphatidylinositol 3-kinase/RAC-alpha serine/threonine kinase, in allograft biopsies from patients with spontaneous clinical operational tolerance (COT). Methods. Paraffin-fixed kidney allograft biopsies from 40 patients with COT (n=4), interstitial rejection (IR; n=12), borderline changes (BC; n=12), and long-term allograft function without rejection (NR; n=12) were used in the study. Cellular infiltrates and immunohistochemical expression of key proteins of the NF kappa B pathway were evaluated in the cortical tubulointerstitium and in cellular infiltrates using digital image analysis software. Results were given as mean +/- SEM. Results. Biopsies from patients with COT exhibited a comparable amount of cellular infiltrate to IR, BC, and NR (COT, 191 +/- 81; IR, 291 +/- 62; BC, 178 +/- 45; and NR, 210 +/- 42 cells/mm(2)) but a significantly higher proportion of forkhead box P3-positive cells (COT, 11%+/- 1.7%; IR, 3.5%+/- 0.70%; BC, 3.4%+/- 0.57%; and NR, 3.7%+/- 0.78% of infiltrating cells; P=0.02). c-Rel expression in cellular infiltrates was significantly elevated in IR, BC, and NR when analyzing the number of positive cells per mm(2) (P=0.02) and positive cells per infiltrating cells (P=0.04). In contrast, tubular PI3K and c-Rel expression were significantly higher in IR and BC but not in NR compared with COT (P=0.03 and P=0.006, respectively). With RAC-alpha serine-threonine kinase, similar tendencies were observed (P=0.2). Conclusions. Allografts from COT patients show significant cellular infiltrates but a distinct expression of proteins involved in the NF kappa B pathway and a higher proportion of forkhead box P3-positive cells.

Proteome analysis of spinal cord during the clinical course of monophasic experimental autoimmune encephalomyelitis

The induction of autoimmune encephalomyelitis (EAE) in Lewis rats results in a period of exacerbation followed by complete recovery. Therefore, this model is widely used for studying the evolution of multiple sclerosis. In the present investigation, differentially expressed proteins in the spinal cord of Lewis rats during the evolution of EAE were assessed using the combination of 2DE and MALDI-TOF MS. The majority of the differentially expressed proteins were identified during the acute phase of EAE, in relation to naive control animals. On the other hand, recovered rats presented a similar protein expression pattern in comparison with the naive ones. This observation can be explained, at least in part, by the intense catabolism existent in acute phase due to nervous tissue damage. In recovered rats, we have described the upregulation of proteins that are apparently involved in the recovery of damaged tissue, such as light and medium neurofilaments, glial fibrillary acidic protein, tubulins subunits, and quaking protein. These proteins are involved mainly in cell growth, myelination, and remyelination as well as in astrocyte and oligodendrocyte maturation. The present study has demonstrated that the inflammatory response, characterized by an increase of the proliferative response and infiltration of autoreactive T lymphocytes in the central nervous system, occurs simultaneously with neurodegeneration.

Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells

Background: The bone morphogenetic proteins (BMPs) belong to a unique group of proteins that includes the growth factor TGF-beta. BMPs play important roles in cell differentiation, cell proliferation, and inhibition of cell growth. They also participate in the maturation of several cell types, depending on the microenvironment and interactions with other regulatory factors. Depending on their concentration gradient, the BMPs can attract various types of cells and act as chemotactic, mitogenic, or differentiation agents. BMPs can interfere with cell proliferation and the formation of cartilage and bone. In addition, BMPs can induce the differentiation of mesenchymal progenitor cells into various cell types, including chondroblasts and osteoblasts. The aim of this study was to analyze the effects of treatment with rhBMP-2 on the proliferation of canine mesenchymal stem cells (cMSCs) and the tumor suppression properties of rhBMP-2 in canine osteocarcoma (OST) cells. Osteosarcoma cell lines were isolated from biopsies and excisions of animals with osteosarcoma and were characterized by the Laboratory of Biochemistry and Biophysics, Butantan Institute. The mesenchymal stem cells were derived from the bone marrow of canine fetuses (cMSCs) and belong to the University of Sao Paulo, College of Veterinary Medicine (FMVZ-USP) stem cell bank. After expansion, the cells were cultured in a 12-well Transwell system; cells were treated with bone marrow mesenchymal stem cells associated with rhBMP2. Expression of the intracytoplasmic and nuclear markers such as Caspase-3, Bax, Bad, Bcl-2, Ki-67, p53, Oct3/4, Nanog, Stro-1 were performed by flow citometry. Results: We evaluated the regenerative potential of in vitro treatment with rhBMP-2 and found that both osteogenic induction and tumor regression occur in stem cells from canine bone marrow. rhBMP-2 inhibits the proliferation capacity of OST cells by mechanisms of apoptosis and tumor suppression mediated by p53. Conclusion: We propose that rhBMP-2 has great therapeutic potential in bone marrow cells by serving as a tumor suppressor to increase p53 and the pro-apoptotic proteins Bad and Bax, as well as by increasing the activity of phosphorylated caspase 3. Study design: Canine bone marrow mesenchymal stem cells associated with rhBMP2 in canine osteosarcoma treatment: "in vitro" study

Combined effect of AMPK/PPAR agonists and exercise training in mdx mice functional performance

The present investigation was undertaken to test whether exercise training (ET) associated with AMPK/PPAR agonists (EM) would improve skeletal muscle function in mdx mice. These drugs have the potential to improve oxidative metabolism. This is of particular interest because oxidative muscle fibers are less affected in the course of the disease than glycolitic counterparts. Therefore, a cohort of 34 male congenic C57Bl/10J mdx mice included in this study was randomly assigned into four groups: vehicle solution (V), EM [AICAR (AMPK agonist, 50 mg/Kg-1.day-1, ip) and GW 1516 (PPAR delta agonist, 2.5 mg/Kg-1.day-1, gavage)], ET (voluntary running on activity wheel) and EM+ET. Functional performance (grip meter and rotarod), aerobic capacity (running test), muscle histopathology, serum creatine kinase (CK), levels of ubiquitined proteins, oxidative metabolism protein expression (AMPK, PPAR, myoglobin and SCD) and intracellular calcium handling (DHPR, SERCA and NCX) protein expression were analyzed. Treatments started when the animals were two months old and were maintained for one month. A significant functional improvement (p<0.05) was observed in animals submitted to the combination of ET and EM. CK levels were decreased and the expression of proteins related to oxidative metabolism was increased in this group. There were no differences among the groups in the intracellular calcium handling protein expression. To our knowledge, this is the first study that tested the association of ET with EM in an experimental model of muscular dystrophy. Our results suggest that the association of ET and EM should be further tested as a potential therapeutic approach in muscular dystrophies.

Adhesion of Trypanosoma cruzi Trypomastigotes to Fibronectin or Laminin Modifies Tubulin and Paraflagellar Rod Protein Phosphorylation

Background: The unicellular parasite Trypanosoma cruzi is the causative agent of Chagas disease in humans. Adherence of the infective stage to elements of the extracellular matrix (ECM), as laminin and fibronectin, is an essential step in host cell invasion. Although members of the gp85/TS, as Tc85, were identified as laminin and fibronectin ligands, the signaling events triggered on the parasite upon binding to these molecules are largely unexplored. Methodology/Principal Findings: Viable infective parasites were incubated with laminin, fibronectin or bovine serum albumin for different periods of time and the proteins were separated by bidimensional gels. The phosphoproteins were envisaged by specific staining and the spots showing phosphorylation levels significantly different from the control were excised and identified by MS/MS. The results of interest were confirmed by immunoblotting or immunoprecipitation and the localization of proteins in the parasite was determined by immunofluorescence. Using a host cell-free system, our data indicate that the phosphorylation contents of T. cruzi proteins encompassing different cellular functions are modified upon incubation of the parasite with fibronectin or laminin. Conclusions/Significance: Herein it is shown, for the first time, that paraflagellar rod proteins and alpha-tubulin, major structural elements of the parasite cytoskeleton, are predominantly dephosphorylated during the process, probably involving the ERK1/2 pathway. It is well established that T. cruzi binds to ECM elements during the cell infection process. The fact that laminin and fibronectin induce predominantly dephosphorylation of the main cytoskeletal proteins of the parasite suggests a possible correlation between cytoskeletal modifications and the ability of the parasite to internalize into host cells.

Dynamic complex formation between HD-GYP, GGDEF and PilZ domain proteins regulates motility in Xanthomonas campestris

RpfG is a member of a class of wide spread bacterial two-component regulators with an HD-GYP cyclic di-GMP phosphodiesterase domain. In the plant pathogen Xanthomonas campestris, RpfG together with the sensor kinase RpfC regulates multiple factors as a response to the cell-to-cell Diffusible Signalling Factor (DSF). A dynamic physical interaction of RpfG with two diguanylate cyclase (GGDEF) domain proteins controls motility. Here we show that, contrary to expectation, regulation of motility by the GGDEF domain proteins does not depend upon their cyclic di-GMP synthetic activity. Furthermore we show that the complex of RpfG and GGDEF domain proteins recruits a specific PilZ domain adaptor protein, and this complex then interacts with the pilus motor proteins PilU and PiIT. The results support a model in which DSF signalling influences motility through the highly regulated dynamic interaction of proteins that affect pilus action. A specific motif that we identify to be required for HD-GYP domain interaction is conserved in a number of GGDEF domain proteins, suggesting that regulation via interdomain interactions is of broad relevance.