text:
This study presents a new method for collecting and handling saliva samples using an automated analytical microsyringe and microextraction by packed syringe (MEPS). The screening and determination of lidocaine in human saliva samples utilizing MEPS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were carried out. An exact volume of saliva could be collected. The MEPS C-8-cartridge could be used for 50 extractions before it was discarded. The extraction recovery was about 60%. The pharmacokinetic curve of lidocaine in saliva using MEPS-LC-MS/MS is reported.
In this study the use of micro-liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was investigated in routine bioanalysis application for separation and quantification of pro-drug AZD6319 (developed for aldezheimer treatment). Microextraction by packed sorbent (MEPS) was used as sample clean-up method. The focus of this study was put on the evaluation of the usability of smaller column diameters such as 1.0 and 0.3mm instead of 2.1mm in bioanalysis application to reduce solvent consumption and sample volumes. Solvent consumption was reduced by 80% when a 1.0mm column was used compared with 2.1mm column. Robustness of the micro-columns in terms of accuracy and precision was investigated. The application of LC-MS/MS for the quantitative analysis of AZD6319 in plasma samples showed good selectivity, accuracy and precision. The coefficients of determination (R-2) were >0.998 for all runs using plasma samples on the studied micro-columns. The inter-day accuracy values for quality control samples ranged from 99 to 103% and from 96 to 105% for 0.3x50mm and 1.0x50mm columns, respectively. The inter-day precision values ranged from 4.0 to 9.0% and from 4.0 to 8.0% for 0.3x50 and 1.0x50mm columns, respectively. In addition the sensitivity was increased by three times using a 1.0mm column compared with 2.1mm. Furthermore, robustness of the micro-columns from different manufacturers was investigated.
Environmental and predisposing genetic factors are known to play a crucial role in the development of systemic autoimmune diseases. With respect to the role of environmental factors, it is not known how and to what extent they contribute to the initiation and exacerbation of systemic autoimmunity. In the present study, I considered this issue and asked if environmental factors can induce autoimmunity in the absence of specific susceptible genes. The development of genetically controlled mercury- and silver-induced B cell activation and anti-nucleolar autoantibodies (ANolA) production in genetically heterozygous outbred Institute of Cancer Research (ICR), Naval Medical Research Institute (NMRI) and Black Swiss mouse stocks were analysed. Four weeks of treatment with both mercury and silver induced a strong B cell activation characterized by increased numbers of splenic antibody-secreting cells of at least one or more immunoglobulin (Ig) isotype(s) in all treated stocks. The three stocks also exhibited a marked increase in the serum IgE levels in response to mercury, but not silver. More importantly, in response to mercury a large numbers of ICR (88%), NMRI (96%) and Black Swiss (100%) mice produced different levels of IgG1 and IgG2a ANolA (a characteristic which is linked strictly to the H-2 genes). Similarly, but at lower magnitudes, treatment with silver also induced the production of IgG1 and IgG2a ANolA in 60% of ICR, 75% of NMRI and 100% of Black Swiss mice. Thus, the findings of this study suggest that long-term exposure to certain environmental factors can activate the immune system to produce autoimmunity per se, without requiring specific susceptible genes.
The amyloid beta (A beta) peptides are 39-42 residue-long peptides found in the senile plaques in the brains of Alzheimer's disease (AD) patients. These peptides self-aggregate in aqueous solution, going from soluble and mainly unstructured monomers to insoluble ordered fibrils. The aggregation process(es) are strongly influenced by environmental conditions. Several lines of evidence indicate that the neurotoxic species are the intermediate oligomeric states appearing along the aggregation pathways. This minireview summarizes recent findings, mainly based on solution and solid-state NMR experiments and electron microscopy, which investigate the molecular structures and characteristics of the A beta peptides at different stages along the aggregation pathways. We conclude that a hairpin-like conformation constitutes a common motif for the A beta peptides in most of the described structures. There are certain variations in different hairpin conformations, for example regarding H-bonding partners, which could be one reason for the molecular heterogeneity observed in the aggregated systems. Interacting hairpins are the building blocks of the insoluble fibrils, again with variations in how hairpins are organized in the cross-section of the fibril, perpendicular to the fibril axis. The secondary structure propensities can be seen already in peptide monomers in solution. Unfortunately, detailed structural information about the intermediate oligomeric states is presently not available. In the review, special attention is given to metal ion interactions, particularly the binding constants and ligand structures of A beta complexes with Cu(II) and Zn(II), since these ions affect the aggregation process(es) and are considered to be involved in the molecular mechanisms underlying AD pathology.
The link between many neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, and the aberrant folding and aggregation of proteins has prompted a comprehensive search for small organic molecules that have the potential to inhibit such processes. Although many compounds have been reported to affect the formation of amyloid fibrils and/or other types of protein aggregates, the mechanisms by which they act are not well understood. A large number of compounds appear to act in a nonspecific way affecting several different amyloidogenic proteins. We describe here a detailed study of the mechanism of action of one representative compound, lacmoid, in the context of the inhibition of the aggregation of the amyloid β-peptide (Aβ) associated with Alzheimer's disease. We show that lacmoid binds Aβ(1-40) in a surfactant-like manner and counteracts the formation of all types of Aβ(1-40) and Aβ(1-42) aggregates. On the basis of these and previous findings, we are able to rationalize the molecular mechanisms of action of nonspecific modulators of protein self-assembly in terms of hydrophobic attraction and the conformational preferences of the polypeptide.
Intermediate amyloidogenic states along the amyloid β peptide (Aβ) aggregation pathway have been shown to be linked to neurotoxicity. To shed more light on the different structures that may arise during Aβ aggregation, we here investigate surfactant-induced Aβ aggregation. This process leads to co-aggregates featuring a β-structure motif that is characteristic for mature amyloid-like structures. Surfactants induce secondary structure in Aβ in a concentration-dependent manner, from predominantly random coil at low surfactant concentration, via β-structure to the fully formed α-helical state at high surfactant concentration. The β-rich state is the most aggregation-prone as monitored by thioflavin T fluorescence. Small angle x-ray scattering reveals initial globular structures of surfactant-Aβ co-aggregated oligomers and formation of elongated fibrils during a slow aggregation process. Alongside this slow (minutes to hours time scale) fibrillation process, much faster dynamic exchange (k(ex) ∼1100 s(-1)) takes place between free and co-aggregate-bound peptide. The two hydrophobic segments of the peptide are directly involved in the chemical exchange and interact with the hydrophobic part of the co-aggregates. Our findings suggest a model for surfactant-induced aggregation where free peptide and surfactant initially co-aggregate to dynamic globular oligomers and eventually form elongated fibrils. When interacting with β-structure promoting substances, such as surfactants, Aβ is kinetically driven toward an aggregation-prone state.
Small organic molecules, like Congo red and lacmoid, have been shown to modulate the self-assembly of the amyloid beta peptide (A beta). Here, we show that A beta forms NMR invisible non-toxic co-aggregates together with lacmoid as well as Congo red. We find that the interaction involves two distinct kinetic processes and at every given time point only a small fraction of A beta is in the co-aggregate. These weak transient interactions kinetically redirect the aggregation prone A beta from self-assembling into amyloid fibrils. These findings suggest that even such weak binders might be effective as therapeutics against pathogenic protein aggregation.
Objectives: We quantified the effect of environmental temperature on mouse energy homeostasis and body temperature.Methods: The effect of environmental temperature (4e33 C) on body temperature, energy expenditure, physical activity, and food intake invarious mice (chow diet, high-fat diet, Brs3-/y, lipodystrophic) was measured using continuous monitoring.Results: Body temperature depended most on circadian phase and physical activity, but also on environmental temperature. The amounts ofenergy expenditure due to basal metabolic rate (calculated via a novel method), thermic effect of food, physical activity, and cold-inducedthermogenesis were determined as a function of environmental temperature. The measured resting defended body temperature matchedthat calculated from the energy expenditure using Fourier's law of heat conduction. Mice defended a higher body temperature during physicalactivity. The cost of the warmer body temperature during the active phase is 4e16% of total daily energy expenditure. Parameters measured indiet-induced obese and Brs3-/y mice were similar to controls. The high post-mortem heat conductance demonstrates that most insulation in miceis via physiological mechanisms.Conclusions: At 22 C, cold-induced thermogenesis isw120% of basal metabolic rate. The higher body temperature during physical activity isdue to a higher set point, not simply increased heat generation during exercise. Most insulation in mice is via physiological mechanisms, with littlefrom fur or fat. Our analysis suggests that the definition of the upper limit of the thermoneutral zone should be re-considered. Measuring bodytemperature informs interpretation of energy expenditure data and improves the predictiveness and utility of the mouse to model human energyhomeostasis.
Baseline signal output and communication between the periplasmic and cytoplasmic domains of the Escherichia colt aspartate chemoreceptor Tar(Ec) are both strongly influenced by residues at the C-terminus of transmembrane helix 2 (TM2). In particular, the cytoplasmic aromatic anchor, composed of residues Trp-209 and Tyr-210 in wild type Tar(Ec) is important for determining the CheA kinase-stimulating activity of the receptor and its ability to respond to chemoeffector-induced stimuli. Here, we have studied the effect on Tar(Ec) function of the six residue sequence at positions 207-212 Moving various combinations of aromatic residues among these positions generates substantial changes M receptor activity. Trp has the largest effect on function, both in maintaining normal activity and in altering activity when it is moved. Tyr has a weaker effect, and Phe has the weakest; however, all three aromatic residues can alter signal output when they are placed in novel positions. We also find that Gly-211 plays an important role in receptor function, perhaps because of the flexibility it introduces into the TM2-HAMP domain connector. The conservation of this Gly residue in the high-abundance chemoreceptors of E. coli and Salmonella enterica suggests that it may be important for the nuanced, bidirectional transmembrane signaling that occurs in these proteins.
The mammalian amyloid precursor protein (APP) protein family consists of the APP and the amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2). The neurotoxic amyloid beta-peptide (Abeta) originates from APP, which is the only member of this protein family implicated in Alzheimer disease. However, the three homologous proteins have been proposed to be processed in similar ways and to have essential and overlapping functions. Therefore, it is also important to take into account the effects on the processing and function of the APP-like proteins in the development of therapeutic drugs aimed at decreasing the production of Abeta. Insulin and insulin-like growth factor-1 (IGF-1) have been shown to regulate APP processing and the levels of Abeta in the brain. In the present study, we show that IGF-1 increases alpha-secretase processing of endogenous APP and also increases ectodomain shedding of APLP1 and APLP2 in human SH-SY5Y neuroblastoma cells. We also investigated the role of different IGF-1-induced signaling pathways, using specific inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK). Our results indicate that phosphatidylinositol 3-kinase is involved in ectodomain shedding of APP and APLP1, but not APLP2, and that MAPK is involved only in the ectodomain shedding of APLP1.
The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K-C-channel is a conserved glutamate in subunit III. However, the majority of the K-C-channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the K-C-channel, was found to depend on the conformation of Y241(Vc), located in subunit I at the interface with subunit III. Mutations of Y241(Vc) (to A/F/H/S) in the Vibrio cholerae cbb(3) eliminate catalytic activity, but also cause perturbations that propagate over a 28-angstrom distance to the active site heme b(3). The data suggest a linkage between residues lining the KC-channel and the active site of the enzyme, possibly mediated by transmembrane helix alpha 7, which contains both Y241(Vc) and the active site crosslinked Y255(Vc), as well as two Cu-B histidine ligands. Other mutations of residues within or near helix alpha 7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme.
Clustering sequences into families has long been an important step in characterization of genes and proteins. There are many algorithms developed for this purpose, most of which are based on either direct similarity between gene pairs or some sort of network structure, where weights on edges of constructed graphs are based on similarity. However, conserved synteny is an important signal that can help distinguish homology and it has not been utilized to its fullest potential.
Here, we present GenFamClust, a pipeline that combines the network properties of sequence similarity and synteny to assess homology relationship and merge known homologs into groups of gene families. GenFamClust identifies homologs in a more informed and accurate manner as compared to similarity based approaches. We tested our method against the Neighborhood Correlation method on two diverse datasets consisting of fully sequenced genomes of eukaryotes and synthetic data.
The results obtained from both datasets confirm that synteny helps determine homology and GenFamClust improves on Neighborhood Correlation method. The accuracy as well as the definition of synteny scores is the most valuable contribution of GenFamClust.
Several lines of evidence suggest mitochondrial dysfunction as a possible underlying mechanism of Alzheimer's disease (AD). Accumulation of the amyloid-beta peptide (Abeta), a neurotoxic peptide implicated in the pathogenesis of AD, has been detected in brain mitochondria of AD patients and AD transgenic mouse models. In vitro evidence suggests that the Abeta causes mitochondrial dysfunction e.g. oxidative stress, mitochondrial fragmentation and decreased activity of cytochrome c oxidase and TCA cycle enzymes. Here we review the link between mitochondrial dysfunctions and AD. In particular we focus on the mechanism for Abeta uptake by mitochondria and on the recently identified Abeta degrading protease in human brain mitochondria.
Mitochondrial presequences and other unstructured peptides are degraded inside mitochondria by presequence proteases (PrePs) identified in Arabidopsis thaliana (AtPreP), humans (hPreP), and yeast (Cym1/Mop112). The presequences of A. thaliana and human PreP are predicted to consist of 85 and 29 amino acids, respectively, whereas the Saccharomyces cerevisiae Cym1/Mop112 presequence contains only 7 residues. These differences may explain the reported targeting of homologous proteins to different mitochondrial subcompartments. Here we have investigated the targeting capacity of the PreP homologues' presequences. We have produced fusion constructs containing N-terminal portions of AtPreP(1-125), hPreP(1-69), and Cym1(1-40) coupled to green fluorescent protein (GFP) and studied their import into isolated plant, mammalian, and yeast mitochondria, followed by mitochondrial subfractionation. Whereas the AtPreP presequence has the capacity to target GFP into the mitochondrial matrix of all three species, the hPreP presequence only targets GFP to the matrix of mammalian and yeast mitochondria. The Cym1/Mop112 presequence has an overall much weaker targeting capacity and only ensures mitochondrial sorting in its host species yeast. Revisiting the submitochondrial localization of Cym1 revealed that endogenous Cym1/Mop112 is localized to the matrix space, as has been previously reported for the plant and human homologues. Moreover, complementation studies in yeast show that native AtPreP restores the growth phenotype of yeast cells lacking Cym1, demonstrating functional conservation.
Accumulation of amyloid-β peptide (Aβ), the neurotoxic peptide implicated in the pathogenesis of Alzheimer's disease (AD), has been shown in brain mitochondria of AD patients and of AD transgenic mouse models. The presence of Aβ in mitochondria leads to free radical generation and neuronal stress. Recently, we identified the presequence protease, PreP, localized in the mitochondrial matrix in mammalian mitochondria as the novel mitochondrial Aβ-degrading enzyme. In the present study, we examined PreP activity in the mitochondrial matrix of the human brain's temporal lobe, an area of the brain highly susceptible to Aβ accumulation and reactive oxygen species (ROS) production. We found significantly lower hPreP activity in AD brains compared with non-AD age-matched controls. By contrast, in the cerebellum, a brain region typically spared from Aβ accumulation, there was no significant difference in hPreP activity when comparing AD samples to non-AD controls. We also found significantly reduced PreP activity in the mitochondrial matrix of AD transgenic mouse brains (Tg mAβPP and Tg mAβPP/ABAD) when compared to non-transgenic aged-matched mice. Furthermore, mitochondrial fractions isolated from AD brains and Tg mAβPP mice had higher levels of 4-hydroxynonenal, an oxidative product, as compared with those from non-AD and nonTg mice. Accordingly, activity of cytochrome c oxidase was significantly reduced in the AD mitochondria. These findings suggest that decreased PreP proteolytic activity, possibly due to enhanced ROS production, contributes to Aβ accumulation in mitochondria leading to the mitochondrial toxicity and neuronal death that is exacerbated in AD. Clearance of mitochondrial Aβ by PreP may thus be of importance in the pathology of AD.
The world faces a global fishing crisis. Wild marine fisheries comprise nearly 15% of all animal protein in the human diet, but, according to the U.N. Food and Agriculture Organization, nearly 60% of all commercially important marine fish stocks are overexploited, recovering, or depleted (FAO 2012; Fig. 1). Some authors have suggested that the large population sizes of harvested marine fish make even collapsed populations resistant to the loss of genetic variation by genetic drift (e. g. Beverton 1990). In contrast, others have argued that the loss of alleles because of overfishing may actually be more dramatic in large populations than in small ones (Ryman et al. 1995). In this issue, Pinsky & Palumbi (2014) report that overfished populations have approximately 2% lower heterozygosity and 12% lower allelic richness than populations that are not overfished. They also performed simulations which suggest that their estimates likely underestimate the actual loss of rare alleles by a factor of three or four. This important paper shows that the harvesting of marine fish can have genetic effects that threaten the long-term sustainability of this valuable resource.
A key issue to understanding the transformations of terrestrial organic carbon in the ocean is to disentangle the latter from marine-produced organic matter. We applied a multiple stable isotope approach using 34S and 13C isotope signatures from estuarine dissolved organic matter (DOM), enabling us to constrain the contribution of terrestrial-derived DOM in an estuarine gradient of the northern Baltic Sea. The stable isotope signatures for dissolved organic sulfur (34SDOS) have twice the range between terrestrial and marine end members compared to the stable isotope signatures for dissolved organic carbon (13CDOC); hence, the share of terrestrial DOM in the total estuarine DOM can be calculated more precisely. DOM samples from the water column were collected using ultrafiltration on board the German RV Maria S Merian during a winter cruise, in the Bothnian Bay, Bothnian Sea, and Baltic proper. We calculated the terrestrial fraction of the estuarine DOC (DOCter) from both 13CDOC and 34SDOS signatures and applying fixed C: S ratios for riverine and marine end members to convert S isotope signatures into DOC concentrations. The 34SDOS signature of the riverine end member was +7.02‰, and the mean signatures from Bothnian Bay, Bothnian Sea, and Baltic proper were +10.27, +12.51, and +13.67‰, respectively, showing an increasing marine signal southwards (34SDOS marine end member 5 18.1‰). These signatures indicate that 87‰, 75‰, and 67‰, respectively, of the water column DOC is of terrestrial origin (DOCter) in these basins. Comparing the fractions of DOCter in each basin—that are still based on few winter values only—with the annual river input of DOC, it appears that the turnover time for DOCter in the Gulf of Bothnia is much shorter than the hydraulic turnover time, suggesting that high-latitude estuaries might be efficient sinks for DOCter.
Climate change is expected to have a strong effect on the Eastern Siberian Arctic Shelf (ESAS) region, which includes 40% of the Arctic shelves and comprises the Laptev and East Siberian seas. The largest organic carbon pool, the dissolved organic carbon (DOC), may change significantly due to changes in both riverine inputs and transformation rates; however, the present DOC inventories and transformation patterns are poorly understood. Using samples from the International Siberian Shelf Study 2008, this study examines for the first time DOC removal in Arctic shelf waters with residence times that range from months to years. Removals of up to 10%–20% were found in the Lena River estuary, consistent with earlier studies in this area, where surface waters were shown to have a residence time of approximately 2 months. In contrast, the DOC concentrations showed a strong nonconservative pattern in areas with freshwater residence times of several years. The average losses of DOC were estimated to be 30%–50% during mixing along the shelf, corresponding to a first-order removal rate constant of 0.3 yr−1. These data provide the first observational evidence for losses of DOC in the Arctic shelf seas, and the calculated DOC deficit reflects DOC losses that are higher than recent model estimates for the region. Overall, a large proportion of riverine DOC is removed from the surface waters across the Arctic shelves. Such significant losses must be included in models of the carbon cycle for the Arctic Ocean, especially since the breakdown of terrestrial DOC to CO2 in Arctic shelf seas may constitute a positive feedback mechanism for Arctic climate warming. These data also provide a baseline for considering the effects of future changes in carbon fluxes, as the vast northern carbon-rich permafrost areas draining into the Arctic are affected by global warming.
Both the ERCC1-XPF complex and the proteins involved in homoIogous recombination (HR) have critical roles in inter-strand cross-link (ICL) repair. Here, we report that mitomycin C-induced lesions inhibit replication fork elongation. Furthermore, mitomycin C-induced DNA double-strand breaks (DSBs) are the result of the collapse of ICL-stalled replication forks. These are not formed through replication run off, as we show that mitomycin C or cisplatin-induced DNA lesions are not incised by global genome nucleotide excision repair (GGR). We also suggest that ICL-lesion repair is initiated either by replication or transcription, as the GGR does not incise ICL-lesions. Furthermore, we report that RAD51 foci are induced by cisplatin or mitomycin C independently of ERCC1, but that mitomycin C-induced HR measured in a reporter construct is impaired in ERCC1-defective cells. These data suggest that ERCC1-XPF plays a role in completion of HR in ICL repair. We also find no additional sensitivity to cisplatin by siRNA co-depletion of XRCC3 and ERCC1, showing that the two proteins act on the same pathway to promote survival.
The mammalian ERCC1-XPF endonuclease has a suggested role in the repair of DNA double-strand breaks (DSB) by single-strand annealing (SSA). Here, we investigated the role of ERCC1 in homologous recombination in mammalian cells, and confirm a role of ERCC1 in SSA. Interestingly, we also report an unexpected role for ERCC1 in gene conversion. This provides support that gene conversion in mammalian somatic cells is carried out through synthesis-dependent strand annealing, rather than through a double Holliday Junction mechanism. Moreover, we find low frequencies of SSA and gene conversion in G1-arrested cells, suggesting that SSA is not a frequent DSB repair pathway in G1-arrested mammalian cells, even in the presence of perfect repeats. Furthermore, we find that SSA is not influenced by inhibition of CDK2 (using Roscovitine), ATM (using Caffeine and KU55933), Chk1 (using CEP-3891) or DNA-PK (using NU7026).
Proteomics is the global analysis of proteins that covers a broad range of technologies aimed at determining the identity and quantity of proteins expressed in the cell, their three-dimensional structure and interaction partners. In contrast to genome, proteome reflects more accurately on the dynamic state of the cell, tissue, or an organism. Therefore much is expected from proteomics to yield better disease markers for early diagnosis and therapy monitoring, as well as biomarkers that would indicate environmental exposure or provide prediction of biological age.
In this thesis, I have developed and applied robust and sensitive subproteomic approaches to study the effect of aging as well as and environmental pollution using different animal models.
In the first part, a high-throughput proteomic method based on liquid chromatography coupled to 2-dimensional gel electrophoresis (LC/2-DE) was developed. The usefulness of this method has been demonstrated by applying it to the assessment of marine pollution in a field experiment.
Next, I have utilized this subproteomic approach to study the effect of aging in mouse kidney of both genders. As a result, a protein expression signature of aging kidney was obtained, revealing gender-dependent alterations in proteome profiles of aging mouse kidney.
In order to further reduce the dynamic range of protein expression and increase the sensitivity of proteomic analysis, I have applied a shotgun mass spectrometry-based proteomic approach using isobaric tags for relative and absolute quantification (iTRAQ) coupled to liquid chromatography and tandem mass spectrometry (LC-MS/MS) to study age-related differences in peroxisome-enriched fractions from mouse liver. Only eight proteins showed statistically significant difference in expression (p<0.05) with moderate folds. This study indicates that age-depended changes in the liver proteome are minimal, suggesting that its proteome is efficiently maintained until certain age.
Finally, in the context of aging studies and the role of peroxisomes in aging, I have tested the utility of cell-penetrating peptides (CPPs) as agents for protein delivery into acatalasemic peroxisomes using yeast as a model. The results obtained suggest that CPPs may be suitable for the delivery of antioxidants to peroxisomes and in future could provide a tool for the protein therapy of age-related diseases.
Using a proteomic approach, we have developed a new method for the assessment of marine pollution that generates highly reproducible protein expression patterns and it is simple and scalable. The protocol is based on applying liquid chromatography (LC) coupled with two-dimensional electrophoresis (2-DE) to analyze changes in the protein expression pattern after exposure to marine pollution. The digestive gland of the sentinel "blue mussel" (Mytilus edulis) was batch-processed through a simple cell fractionation followed by ion-exchange chromatography and 2-DE. The selection of ligands, elution method, and small volume design was carefully considered to define a protocol that could be mainly robotized. A pilot study with samples collected from different Gothenburg harbor areas indicated that the clean area could be distinguished from the polluted ones based on a protein expression pattern (PES) composed of 13 proteins. Principal component analysis (PCA) and hierarchical clustering confirmed that the PES was sufficient to discriminate polluted and unpolluted areas and to provide a spatial gradient from the polluted source. Several proteins from the PES were identified by electrospray ionization tandem mass spectrometry (ESI−MS/MS), and they are involved in β-oxidation, amino acid metabolism, detoxification, protein degradation, organelle biogenesis, and protein folding. In the near future, this methodology could show potential advantages to assess marine pollution and could become a stable platform to elucidate ecotoxicological questions.
BACKGROUND: This study aims to analyze sex differences in mice aging kidney. We applied a proteomic technique based on subfractionation, and liquid chromatography coupled with 2-DE. Samples from male and female CD1-Swiss outbred mice from 28 weeks, 52 weeks, and 76 weeks were analysed by 2-DE, and selected proteins were identified by matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS).
RESULTS: This proteomic analysis detected age-related changes in protein expression in 55 protein-spots, corresponding to 22 spots in males and 33 spots in females. We found a protein expression signature (PES) of aging composed by 8 spots, common for both genders. The identified proteins indicated increases in oxidative and proteolytic proteins and decreases in glycolytic proteins, and antioxidant enzymes.
CONCLUSION: Our results provide insights into the gender differences associated to the decline of kidney function in aging. Thus, we show that proteomics can provide valuable information on age-related changes in expression levels of proteins and related modifications. This pilot study is still far from providing candidates for aging-biomarkers. However, we suggest that the analysis of these proteins could suggest mechanisms of cellular aging in kidney, and improve the kidney selection for transplantation.
Aging is a complex multifactorial phenomenon, which is believed to result from the accumulation of cellular damage to biological macromolecules. Peroxisomes recently emerged as another important source of reactive oxygen species (ROS) production in addition to mitochondria. However, the role of these organelles in the process of aging is still not clear. The aim of this study was to characterize the changes in protein expression profiles of young (10 weeks old) versus old (18 months old) mouse liver peroxisome-enriched fractions. We have applied shotgun proteomic approach based on liquid chromatography and tandem mass spectrometry (LC-MS/MS) combined with iTRAQ (isobaric tags for relative and absolute quantitation) labeling that allows comparative quantitative multiplex analysis. Our analysis led to identification and quantification of 150 proteins, 8 out of which were differentially expressed between two age groups at a statistically significant level (p < 0.05), with folds ranging from 1.2 to 4.1. These proteins involved in peroxisornal beta-oxidation, detoxification of xenobiotics and production of ROS. Noteworthy, differences in liver proteome have been observed between as well as within different age groups. In conclusion, our subproteomic quantitative study suggests that mouse liver proteome is sufficiently maintained until certain age.
The malarial parasite Plasmodium exports its own proteins to the cell surfaces of red blood cells (RBCs) during infection. Examples of exported proteins include members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family of proteins from Plasmodium falciparum. The presence of these parasite-derived proteins on surfaces of infected RBCs triggers the adhesion of infected cells to uninfected cells (rosetting) and to the vascular endothelium potentially obstructing blood flow. While there is a fair amount of information on the localization of these proteins on the cell surfaces of RBCs, less is known about how they can be exported to the membrane and the topologies they can adopt during the process. The first step of export is plausibly the cotranslational insertion of proteins into the endoplasmic reticulum (ER) of the parasite, and here, we investigate the insertion of three RIFIN and two STEVOR proteins into the ER membrane. We employ a well-established experimental system that uses N-linked glycosylation of sites within the protein as a measure to assess the extent of membrane insertion and the topology it assumes when inserted into the ER membrane. Our results indicate that for all the proteins tested, transmembranes (TMs) 1 and 3 integrate into the membrane, so that the protein assumes an overall topology of Ncyt-Ccyt. We also show that the segment predicted to be TM2 for each of the proteins likely does not reside in the membrane, but is translocated to the lumen.
The dynamics of DMPC in different isotropic bicelles have been investigated by NMR and EPR methods. The local dynamics were obtained by interpretation of 13C NMR relaxation measurements of DMPC in the bicelles, and these results were compared to EPR spectra of spin-labeled lipids. The overall size of the bicelles was investigated by PFG NMR translational diffusion measurements. The dynamics and relative sizes were compared among three different bicelles: [DMPC]/[DHPC] = 0.25, [DMPC]/[DHPC] = 0.5, and [DMPC]/[CHAPS] = 0.5. The local motion is found to depend much more strongly on the choice of the detergent, rather than the overall size of the bicelle. The results provide an explanation for differences in apparent dynamics for different peptides, which are bound to bicelles. This in turn determines under what conditions reasonable NMR spectra can be observed. A model is presented in which extensive local motion, in conjunction with the overall size, affects the spectral properties. An analytical expression for the size dependence of the bicelles, relating the radius of the bilayer region with physical properties of the detergent and the lipid, is also presented.
Mycobacterium tuberculosis R2-like ligand-binding oxidase (MtR2lox) belongs to a recently discovered group of proteins that are homologous to the ribonucleotide reductase R2 proteins. MtR2lox carries a heterodinuclear Mn/Fe cofactor and, unlike R2 proteins, a large ligand-binding cavity. A unique tyrosine-valine cross link is also found in the vicinity of the active site. To date, all known structures of R2 and R2lox proteins show a disordered C-terminal segment. Here, we present two new crystal forms of MtR2lox, revealing an ordered helical C-terminal. The ability of alternating between an ordered and disordered state agrees well with bioinformatic analysis of the protein sequence. Interestingly, ordering of the C-terminal helix shields a large positively charged patch on the protein surface, potentially used for interaction with other cellular components. We hypothesize that the dynamic C-terminal segment may be involved in control of protein function in vivo.
The Mycobacterium tuberculosis acid-induced operon MymA encodes the fatty acyl-CoA synthetase FadD13 and is essential for virulence and intracellular growth of the pathogen. Fatty acyl-CoA synthetases activate lipids before entering into the metabolic pathways and are also involved in transmembrane lipid transport. Unlike soluble fatty acyl-CoA synthetases, but like the mammalian integral-membrane very-long-chain acyl-CoA synthetases, FadD13 accepts lipid substrates up to the maximum length tested (C-26). Here, we show that FadD13 is a peripheral membrane protein. The structure and mutational studies reveal an arginine- and aromatic-rich surface patch as the site for membrane interaction. The protein accommodates a hydrophobic tunnel that extends from the active site toward the positive patch and is sealed by an arginine-rich lid-loop at the protein surface. Based on this and previous data, we propose a structural basis for accommodation of lipid substrates longer than the enzyme and transmembrane lipid transport by vectorial CoA-esterification.
Tuberculosis is a notorious disease responsible for the deaths of 1.4 million people worldwide. A third of the world's population is infected with Mycobacterium tuberculosis, the bacterium causing the disease. The increase of multi drug-resistant strains worsens the situation, and the World Health Organization has declared tuberculosis to be a global emergency. The bacterium envelopes itself with a unique set of very long-chain lipids that play an important role in virulence and drug resistance. Therefore enzymes involved in lipid metabolism are putative drug targets.
To allow entry into different metabolic pathways and transmembrane transport, fatty acids have to be activated. This is done primarily by fatty acyl-CoA synthetases (ACSs). We identified an ACS possibly involved in the bacterium's virulence and solved its structure. Structural interpretation combined with previously reported data gives us insights into the details of its function. This enzyme is known to harbor lipid substrates longer than the enzyme itself, and we now propose how this peripheral membrane protein accommodates its substrates.
Some of the most chemically challenging oxidations are performed by dinuclear metalloproteins belonging to the ferritin-like superfamily. We show that the ferritin-like protein, R2lox, from M. tuberculosis contains a new type of heterodinuclear Mn/Fe cofactor. This protein cofactor is capable of performing potent 2-electron oxidations as demonstrated by a novel tyrosine-valine crosslink observed in the protein.
Recently a new subclass of ribonucleotide reductase (RNR) R2 proteins, was identified in the intracellular pathogen Chlamydia trachomatis containing the same type of Mn/Fe cofactor mentioned above. The RNR R2 proteins use their metal site to generate a stable radical, essential for the reduction of ribonucleotides to their deoxy forms, the building blocks of DNA. With this work, we were able to characterize the architecture of this metal cofactor.
The membrane-bound detoxication enzyme microsomal glutathione transferase is localized predominantly in the liver of mammalian species, where it is present in high concentrations in the endoplasmic reticulum and the outer mitochondrial membrane. The enzyme conjugates a broad range of hydrophobic xenobiotics bearing reactive electrophilic centers to glutathione, which facilitates deactivation and excretion.
The microsomal glutathione transferase activity is increased several-fold upon the addition of sulfhydryl reagents, as well as by partial proteolysis. This unique property could reflect a mechanism of regulation of the enzyme in vivo. The microsomal glutathione transferase was purified from mouse and human liver and found to be similar to the rat enzyme in its structural properties, its activity profile and its ability to be activated by sulfhydryl reagents. The human enzyme displayed glutathione peroxidase/transferase activity towards products of lipid peroxidation, indicative of a functional role in protection against lipid peroxidation.
The histidine-specific reagent diethylpyrocarbonate (DEPC) rapidly inactivated the microsomal glutathione transferase and it was shown that 90 % of the activity was lost within the time period required for modification of the most reactive histidine. Inclusion of the substrate analogue S-hexylglutathione decreased the inactivation rate, indicating a functional importance for this histidine residue. Data is also presented which supports the involvement of arginine and lysine residue(s) in catalysis.
A random sequential catalytic mechanism could be defined for the microsomal glutathione transferase by the use of alternate substrate diagnosis. Chemical catalysis appears to rely on the lowering of the pKa of the thiol in glutathione to approximately 6.4. Activation of the enzyme was found to increase the catalytic efficiency towards a much broader range of substrates than was previously realized. This can be considered as a well-suited defence mechanism against toxic insult by electrophiles, when glutathione levels are usually decreased.
The thiol substrate specificity of microsomal glutathione transferase was investigated by the use of one glycyl- and eight y-glutamyl-modified glutathione analogues. The a-amino group of the glutamyl residue was demonstrated to be of preferential functional importance for the activity of the activated enzyme. The a-carboxyl of the glutamyl residue is obligatory for activity as demonstrated by the lack of activity with the descarboxy analogue 4-Abu-L-Cys-Gly. It was also noted that the activated microsomal glutathione transferase is more selective towards the thiol substrates than is the unactivated enzyme.
By comparing the tryptic cleavage products from intact and permeabilized microsomes, it was shown that the lysine-4 of the enzyme is located at the luminal side of the endoplasmic reticulum, whereas lysine-41 faces the cytosol. A hydrophobic stretch (positions 11-35) is the likely membranespanning region and additional membrane association is indicated. The active site, as well as the site of activation (cysteine-49), were demonstrated to face the cytosol.
Ribonucleotide reductase (RNR) catalyse the conversion of ribonucleotides to their corresponding deoxyribonucleotides in all organisms. The deoxyribonucleotides are the building blocks for DNA. Three different classes of RNR are found, class I, II and III. The class I RNRs operate under aerobic conditions, the class III RNRs operate under anaerobic conditions and the class II RNRs are indifferent to oxygen. All classes of RNR catalyse the reaction using a free radical mechanism. The free radical is generated to initiate the reaction mechanism but the generation differs between the classes.
I have worked with the anaerobic class III RNR from bacteriophage T4 and the work presented in this thesis involves several different aspects of the enzyme. The class III RNR from phage T4 can be used as a model for other class III RNRs.
From isotope labelling experiments, we show that a stable glycyl radical forms in the phage T4 class III RNR. I used site-directed mutagenesis to locate the glycyl radical to Gly580 in the NrdD protein of the T4 class III RNR. The glycyl radical is absolutely required for enzymatic activity.
Also using protein engineering, I show for the first time, the importance of cysteines in radical generation and the reaction mechanism of the class III RNRs. Four cysteines in the C-terminal of T4 NrdD are responsible for the last step in the generation of the glycyl radical at Gly580. Two cysteines in the active site of T4 NrdD, Cys79 and Cys290 are required for the reaction mechanism of the enzyme. A third residue within the active site, Asn311 is most likely also important for catalytic activity. A reaction mechanism that is different from the class I and II RNRs has been proposed.
The first crystal structure of a class III RNR, the class III RNR from phage T4 is presented. Structural relationships with the known class I RNR structure is discussed as well as similarities with another glycyl-radical enzyme.
Finally, the allosteric regulation of the class III RNR from phage T4 was characterized. Almost all RNRs are allosterically regulated to keep the deoxynucleotide pools balanced in the cell. Similarities to other RNRs as well as a unique feature of the class III RNR from phage T4 is discussed.
Di-iron carboxylate proteins perform a wide range of chemical reactions in the cell. These reactions often involve activation of dioxygen to generate highly oxidative species that can be used in catalysis. One member of the di-iron carboxylate family is the enzyme ribonucleotide reductase (RNR). This enzyme is essential for DNA synthesis since it is a key enzyme on the pathway for de-novo synthesis of deoxyribonucleotides, the building blocks of DNA. To perform its catalytic function RNR is dependent on a protein radical. In this thesis I have used x-ray crystallographic methods to investigate the mechanism of O2 activation and radical generation in the R2 subunit of RNR. These structural studies have lead to a proposal of a detailed mechanism, which could be common to most O2 activating di-iron carboxylate proteins. The alternative oxidase is a membrane protein that has been proposed to belong to the di-iron carboxylate family. This protein is a ubiquinol/oxygen oxidoreductase and can act as a terminal electron acceptor in the respiratory chain. I have used homology modelling to make a structural model of this enzyme, which provides new insights into its functions and its relationship to the other di-iron carboxylate proteins.
Given the strong oxidative power of the di-iron carboxylate proteins they would be very useful as oxidants in various industrial applications. Another part of my project has been aimed towards the design of a di-iron carboxylate enzyme tailored for industrial and environmental applications using a small and stable di-iron carboxylate protein as a starting framework, namely the bacterioferritin protein. This work has lead to the synthesis of a gene library with many potential enzymatic activities.
beta-N-Methylamino-l-alanine (BMAA) is an important non-protein amino acid linked to neurodegenerative diseases, specifically amyotrophic lateral sclerosis (ALS). Because it can be transferred and bioaccumulated higher up the food chain, it poses significant public health concerns; thus, improved detection methods are of prime importance for the identification and management of these toxins. Here, we report the successful use of N-hydroxysuccinimide ester of N-butylnicotinic acid (C-4-NA-NHS) for the efficient separation of BMAA from its isomers and higher sensitivity in detecting BMAA compared to the current method of choice using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization. Implementation of this efficient method allowed localization of BMAA in the non-visceral tissues of blue mussels, suggesting that more efficient depuration may be required to remove this toxin prior to consumption. This is a crucial method in establishing the absence or presence of the neurotoxic amino acid BMAA in food, environmental or biomedical samples.
The PepFect family of cell-penetrating peptides (CPPs) was designed to improve the delivery of nucleic acids across plasma membranes. We present here a comparative study of two members of the family, PepFect3 (PF3) and PepFect6 (PF6), together with their parental CPP transportan-10 (TP10), and their interactions with lipid membranes. We show that the addition of a stearyl moiety to TP10 increases the amphipathicity of these molecules and their ability to insert into a lipid monolayer composed of zwitterionic phospholipids. The addition of negatively charged phospholipids into the monolayer results in decreased binding and insertion of the stearylated peptides, indicating modification in the balance of hydrophobic versus electrostatic interactions of peptides with lipid bilayer, thus revealing some clues for the selective interaction of these CPPs with different lipids. The trifluoromethylquinoline moieties, in PF6 make no significant contribution to membrane binding and insertion. TP10 actively introduces pores into the bilayers of large and giant unilamellar vesicles, while PF3 and PF6 do so only at higher concentrations. This is consistent with the lower toxicity of PR and PF6 observed in previous studies.
Fourier transform infrared (FTIR) spectroscopy was used to study the structure of the recombinant antibodies 1E10, anti-CD20 and hR3, which are used as anti-cancer therapeutic drugs. We tested their sensitivity against different conditions and treatments such as pH, temperature, freeze-thaw cycles and drying, which are relevant for the practical usefulness of the drugs. All antibodies were stable against moderate temperature increases (up to 50 degrees C) and pH changes (range 5-9). 1E10 was sensitive to extreme pH values (pH 3 and 12), whereas hR3 was most sensitive to temperature (at and above 60 degrees C). We did not observe any significant changes upon freeze-thaw and drying treatments. The secondary structure content of all three antibodies was estimated to be similar to that of IgG with similar to 64% beta-sheet, 0% alpha-helix and similar to 36% other structure. (C) 2012 The International Alliance for Biological Standardization.
A sophisticated evolutionary conserved innate immune system has evolved in insects to fight pathogens and to restrict damage in harmful (danger) situations including cancer. A significant amount of knowledge about different infection models in Drosophila has been generated in past decades, which revealed functional resemblances and implications for vertebrate systems. However, how Drosophila responds towards multicellular parasitic nematodes and in danger situations is still little understood. Therefore, the aim of the thesis was to characterize multiple aspects of the host defense in the two important contexts mentioned above.
We analyzed the transcriptome profiles of nematode-infected Drosophila larvae with uninfected samples. For this we employed the entomopathogenic nematode Heterorhabditis bacteriophora with its symbiont Photorhabdus luminescence to infect Drosophila larvae. We found 642 genes were differentially regulated upon infection. Among them a significant portion belonged to immune categories. Further functional analysis identified a thioester containing protein TEP3, a recognition protein GNBP-like 3, the basement membrane component protein Glutactin and several other small peptides. Upon loss or reduced expression of these genes hosts showed mortality during nematode infections. This study uncovers a novel function for several of the genes in immunity.
Furthermore, we investigated the cellular response towards nematodes. When we eliminated hemocytes genetically (referred to as hml-apo) in Drosophila, we found hml-apo larvae are resistant to nematodes. Subsequent characterization of hml-apo larvae showed massive lamellocyte differentiation (another blood cell type which is rare in naïve larvae), emergence of melanotic masses, up- and down-regulation of Toll and Imd signaling respectively suggesting a pro-inflammatory response. Moreover, a striking defective leg phenotype in adult escapers from pupal lethality was observed. We identified nitric oxide (NO) as a key regulator of these processes. We also showed that imaginal disc growth factors 3 (IDGF3): (a) protects hosts against nematodes, (b) is a clotting component and (c) negatively regulates Wnt and JAK/STAT signaling. To follow larval behavior in the presence or absence of nematodes we monitored Drosophila larval locomotion behaviors using FIMtrack (a recently devised automated method) to elucidate evasive strategies of hosts. Finally, we characterized host defenses in three Drosophila leukemia models with and without nematode infection. Taken together, these studies shed light on host responses in two crucial circumstances, nematode infections and danger situations.
Human Transforming Growth Factor (TGF)-beta 1, one of three TGF-beta isoforms, is a pleotropic cytokine critical for many physiological and immunological processes. TGF-beta 1 is secreted in a latent form, linked to Latency Associated Protein (LAP). Analysis of Latent TGF-beta 1 by TGF-ELISA requires dissociation of TGF-beta 1 from LAP, e.g. by acidification of samples. The ELISA then measures total TGF-beta 1, equivalent to dissociated Latent TGF-beta 1 plus any free TGF-beta 1 present prior to acidification. Evolutionary conservation of TGF-beta 1 across mammals also renders TGF-beta 1 ELISAs reactive with TGF-beta 1 in bovine serum often used in human cell cultures. To enable a direct analysis of Latent TGF-beta 1, monoclonal antibodies were made against LAP from human latent TGF-beta 1 and used to develop a LAP ELISA detecting Latent TGF-beta 1. The ELISA did not react with LAP from human Latent TGF-beta 2 or 3, respectively, nor with Latent TGF-beta in bovine serum. EDTA-containing plasma from healthy subjects (n = 20) was analyzed by conventional TGF-beta 1 ELISA and LAP ELISA. By TGF-beta 1 ELISA, total TGF-beta 1 were detected in all samples (median 133 pM, range 34-348 pM); low levels of free TGF-beta 1 found in 8/20 non-addified samples showed that >98.5% of the total TGF-beta 1 derived from Latent TGF-beta 1. Latent TGF-beta 1 found in non-acidified samples by LAP ELISA (median 154 pM, range 48-403 pM) was comparable in molar levels to, and correlated with, total TGF-beta 1 (r(s) 0.96, p<0.0001). A similar agreement between the total TGF-beta 1 and the LAP ELISA was found with citrate- and heparin-containing plasma. The LAP ELISA facilitates analysis of Latent TGF-beta 1 without sample acidification and is not compromised by the presence of bovine serum in human cell supernatants.
The membrane protein monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii is responsible for the creation of intracellular membranes when overexpressed in Escherichia coli (E. coli). The present study investigates time dependent changes in composition and properties of E. coli membranes during 22 h of MGS induction. The lipid/protein ratio increased by 38% in MGS-expressing cells compared to control cells. Time-dependent screening of lipids during this period indicated differences in fatty acid modeling. (1) Unsaturation levels remained constant for MGS cells (~ 62%) but significantly decreased in control cells (from 61% to 36%). (2) Cyclopropanated fatty acid content was lower in MGS producing cells while control cells had an increased cyclopropanation activity. Among all lipids, phosphatidylethanolamine (PE) was detected to be the most affected species in terms of cyclopropanation. Higher levels of unsaturation, lowered cyclopropanation levels and decreased transcription of the gene for cyclopropane fatty acid synthase (CFA) all indicate the tendency of the MGS protein to force E. coli membranes to alter its usual fatty acid composition.
Certain membrane proteins involved in lipid synthesis can induce formation of new intracellular membranes in Escherichia coli, i.e., intracellular vesicles. Among those, the foreign monotopic glycosyltransferase MGS from Acholeplasma laidlawii triggers such massive lipid synthesis when overexpressed. To examine the mechanism behind the increased lipid synthesis, we investigated the lipid binding properties of MGS in vivo together with the correlation between lipid synthesis and MGS overexpression levels. A good correlation between produced lipid quantities and overexpressed MGS protein was observed when standard LB medium was supplemented with four different lipid precursors that have significant roles in the lipid biosynthesis pathway. Interestingly, this correlation was highest concerning anionic lipid production and at the same time dependent on the selective binding of anionic lipid molecules by MGS. A selective interaction with anionic lipids was also observed in vitro by P-31 NMR binding studies using bicelles prepared with E. coli lipids. The results clearly demonstrate that the discriminative withdrawal of anionic lipids, especially phosphatidylglycerol, from the membrane through MGS binding triggers an in vivo signal for cells to create a feed-forward stimulation of lipid synthesis in E. coil. By this mechanism, cells can produce more membrane surface in order to accommodate excessively produced MGS molecules, which results in an interdependent cycle of lipid and MGS protein synthesis.
In bioinformatics, machine learning methods have been used to predict features embedded in the sequences. In contrast to what is generally assumed, machine learning approaches can also provide new insights into the underlying biology. Here, we demonstrate this by presenting TargetP 2.0, a novel state-of-the-art method to identify N-terminal sorting signals, which direct proteins to the secretory pathway, mitochondria, and chloroplasts or other plastids. By examining the strongest signals from the attention layer in the network, we find that the second residue in the protein, that is, the one following the initial methionine, has a strong influence on the classification. We observe that two-thirds of chloroplast and thylakoid transit peptides have an alanine in position 2, compared with 20% in other plant proteins. We also note that in fungi and single-celled eukaryotes, less than 30% of the targeting peptides have an amino acid that allows the removal of the N-terminal methionine compared with 60% for the proteins without targeting peptide. The importance of this feature for predictions has not been highlighted before.
In the current work we characterize the uptake mechanism of two NickFect family members, NF51 and NF1, related to the biological activity of transfected plasmid DNA (pDNA). Both vectors condense pDNA into small negatively charged nanoparticles that transfect He La cells with equally high efficacy and the delivery is mediated by SCARA3 and SCARA.5 receptors. NF1 condenses DNA into less homogeneous and less stable nanoparticles than NF51. NF51/pDNA nanoparticles enter the cells via macropinocytosis, while NF1/pDNA complexes use clathrin- or caveolae-mediated endocytosis and macropinocytosis. Analysis of separated endosomal compartments uncovered lysomotropic properties of NF51 that was also proven by cotransfection with chloroquine. In summary we characterize how radical modifications in peptides, such as introducing a kink in the structure of NF51 or including extra negative charge by phospho-tyrosine substitution in NF1, resulted in equally high efficacy for gene delivery, although this efficacy is achieved by using differential transfection pathways.
Harnessing of a branched structure is a novel approach in the design of cell-penetrating peptides and it has provided highly efficient transfection reagents for intracellular delivery of nucleic acids. The new stearylated TP10 analogs, NickFects, condense plasmid DNA, splice correcting oligonucleotides and short interfering RNAs into stable nanoparticles with a size of 62-160 nm. Such nanoparticles have a negative surface charge (-11 to -18 mV) in serum containing medium and enable highly efficient gene expression, splice correction and gene silencing. One of the novel peptides, NickFect51 is capable of transfecting plasmid DNA into a large variety of cell lines, including refractory suspension and primary cells and in several cases exceeds the transfection level of commercially available reagent Lipofectamine (TM) 2000 without any cytotoxic side effects. Additionally we demonstrate the advantages of NickFect51 in a protein production system, QMCF technology, for expression and production of recombinant proteins in hardly transfectable suspension cells.
Microextraction by packed sorbent (MEPS) is a miniaturized, solid-phase extraction (SPE) technique that works online with gas chromatography (GC) and liquid chromatography (LC). Not only is the automation process with MEPS advantageous, but the much smaller volumes of the samples, solvents and dead space in the system also provide other significant advantages such as the speed and the simplicity of the sample preparation process. In this study MEPS has been evaluated for quantification of sensory neuron-specific receptors agonist (BAM8-22). Owing to the instability of BAMs, the focus was on fast extraction and determination of the peptide online using LC-MS/MS. Sorbents such as C2, C8 and ENV+ (hydroxylated polystyrenedivinylbenzene copolymer) were investigated in the present study. MEPS-C8 gave the best results compared with C2 and ENV and it was used for the method validation. The calibration curve was obtained within the concentration range of 20.03045nmol/L in plasma. The regression correlation coefficients for plasma samples were 0.99 for all runs (n=6). The between-batch accuracy and precision for BAM8-22 ranged from 13 to 2.0% and from 4.0 to 14%, respectively. Additionally, the accuracy and precision for BAM22-8 ranged from 13 to 7.0% and from 3.0 to 12%, respectively. The present method was used for pharmacokinetic studies for BAMs in plasma samples.
Chromatin is a highly dynamic, regulatory component in the process of transcription, repair, recombination and replication. The BRG1 and SNF2H proteins are ATP-dependent chromatin remodeling proteins that modulate chromatin structure to regulate DNA accessibility for DNA-binding proteins involved in these processes. The BRG1 protein is a central ATPase of the SWI/SNF complexes involved in chromatin remodeling associated with regulation of transcription. SWI/SNF complexes are biochemically hetero-geneous but little is known about the unique functional characteristics of the various forms. We have shown that SWI/SNF activity in SW13 cells affects actin filament organization dependent on the RhoA signaling pathway. We have further shown that the biochemical composition of SWI/SNF complexes qualitatively affects the remodeling activity and that the composition of biochemically purified SWI/SNF complexes does not reflect the patterns of chromatin binding of individual subunits. Chromatin binding assays (ChIP) reveal variations among subunits believed to be constitutive, suggesting that the plasticity in SWI/SNF complex composition is greater than suspected. We have also discovered an interaction between BRG1 and the splicing factor Prp8, linking SWI/SNF activity to mRNA processing. We propose a model whereby parts of the biochemical heterogeneity is a result of function and that the local chromatin environment to which the complex is recruited affect SWI/SNF composition.
We have also isolated the novel B-WICH complex that contains WSTF, SNF2H, the splicing factor SAP155, the RNA helicase II/Guα, the transcription factor Myb-binding protein 1a, the transcription factor/DNA repair protein CSB and the RNA processing factor DEK. The formation of this complex is dependent on active transcription and links chromatin remodeling by SNF2H to RNA processing.
By linking chromatin remodeling complexes with RNA processing proteins our work has begun to build a bridge between chromatin and RNA, suggesting that factors in chromatin associated assemblies translocate onto the growing nascent RNA.
The exact role of high density lipoprotein in atheroprotection is not well understood yet due to its complex nature; it comprises more than ten subclasses that vary in size, composition, and function. Isolation and characterization of these subclasses is an important step for further studies addressing their functions in health and disease. In this work, we present a novel method that is relatively simple and efficient for isolation of high density lipoprotein subclasses. The method depends on fractional filtration of the subclasses through a preformed gel membrane system under the effect of an electric field, where the stepwise isolation of the subclasses depends on differences in their rates of migration in polyacrylamide gel. Using this design, we were able to isolate seven high density lipoprotein subclasses with relative molecular masses of 42,000-50,000; 71,000; 103,000; 124,000; 150,000; 182,000; and 219,000. All the subclasses contained apolipoprotein A-I, phosphatidylcholine, sphingomyelin, free cholesterol, esterified cholesterol, and triacylglycerols. Some fractions of some samples contained the apolipoproteins A-II, C-I, C-II, C-III, and E. A subclass of molecular mass of 106,000 was identified and isolated from a healthy young subject that contained albumin and apoA-I with some free and esterified cholesterol, but with no triacylglycerols. This electrofiltration technique offers a novel tool for isolating pure native high density lipoprotein subclasses in a concentrated form that can be used directly for detailed studies of their physicochemical and physiological properties.
Novel injectable thermosensitive PNIPAM/hyaluronic acid hydrogels containing various amounts of chitosan-g-acrylic acid coated PLGA (ACH-PLGA) micro/nanoparticles were synthesized and designed to facilitate the regeneration of cartilage tissue. The ACH-PLGA particles were used in the hydrogels to play a triple role: first, the allyl groups on the chitosan-g-acrylic acid shell act as crosslinkers for PNIPAM and improved the mechanical properties of the hydrogel to mimic the natural cartilage tissue. Second, PLGA core acts as a carrier for the controlled release of chondrogenic small molecule melatonin. Third, they could reduce the syneresis of the thermosensitive hydrogel during gelation. The optimum hydrogel with the minimum syneresis and the maximum compression modulus was chosen for further evaluations. This hydrogel showed a great integration with the natural cartilage during the adhesion test, and also, presented an interconnected porous structure in scanning electron microscopy images. Eventually, to evaluate the cytotoxicity, mesenchymal stem cells were encapsulated inside the hydrogel. MTT and Live/Dead assay showed that the hydrogel improved the cells growth and proliferation as compared to the tissue culture polystyrene. Histological study of glycosaminoglycan (GAG) showed that melatonin treatment has the ability to increase the GAG synthesis. Overall, due to the improved mechanical properties, low syneresis, the ability of sustained drug release and also high bioactivity, this injectable hydrogel is a promising material system for cartilage tissue engineering.
This thesis is aimed at summarizing some of the alternative in vitro methods and models that have been used to study both adult and developmental neurotoxicity (DNT), and also to pinpoint some of the important aspects of using alternative in vitro methods. The aim of the papers included in this thesis was to challenge the hypothesis that neurotoxicity and DNT of chemicals can be studied using robust endpoints for proliferation and neural differentiation, such as neurite outgrowth, mRNA expression and protein expression, in two different cell lines. The aim was also to characterize the two cell lines and identify marker genes important for differentiation and to evaluate if these markers could be used as indicators for DNT. The hypothesis being that any chemical that change the expression of important genes for the developmental process could possibly result in DNT for the cells. The current developmental neurotoxicity testing guidelines, using animal models, are time consuming, expensive, ethically questionable and have relatively low sensitivity. Because of this, there has been a paradigm shift towards developing and using alternative methods capable of testing and screening large number of substances. The next generation of developmental neurotoxicity testing is predicted to consist of both in silico and in vitro testing that have to be used in a combined fashion so that it will generate a more rapid and efficient toxicity testing. The idea is to use a battery of refined endpoint studies that identify the specific toxicity of a compound, discriminate between different neural subpopulations and the different stages of neural differentiation. The use of transcriptomic approaches has been suggested as an example of such an endpoint. In this thesis we have evaluated the human neuroblastoma cell line SH-SY5Y and the murine neural progenitor cell line C17.2 in their ability to detect neurotoxic and developmental neurotoxic compounds. We have evaluated this by using functional endpoints, such as neurite outgrowth, cell membrane potential and phenotype ratios. We have also studied the effect of selected chemicals on the levels of mRNA markers specific for different neural cell populations or for neural differentiation in general. We have performed whole genome gene expression on the two cell lines during differentiation and identified and selected a limited number of genes that have been evaluated for their ability to detect developmental neurotoxicity. Both cell lines showed that they have the capability to identify neurotoxic and developmental neurotoxic compounds and could possibly serve as an addition to the testing battery of neurotoxicity in the future. Some of the focus of this thesis has been directed towards the neurodevelopmental effects of the neurotoxic compound acrylamide. Most people get exposed to acrylamide through food consumption and from environmental pollution. Since acrylamide crosses the placental barrier, it creates a risk for developmental consequences. We found that acrylamide affected both cell proliferation and differentiation in both cell lines. Acrylamide affected both neuronal and the glial phenotypes in the C17.2 cell line. We also revealed that acrylamide attenuated neural differentiation at concentrations that were seven orders of magnitude lower than the estimated plasma concentration of free acrylamide in the fetus. Low concentrations of acrylamide altered the gene expression of several genes involved in the retinoic acid signaling as well as the CREB signaling pathways during retinoic acid driven differentiation in the SH-SY5Y cells. Since sub-micromolar concentrations seem to inhibit the differentiation process in both cell lines, developmental neurotoxicity induced by daily intake of acrylamide is a matter of concern. We found that the C17.2 cell line could function as a good model for detecting acute neurotoxicity by evaluating the cell membrane potential of the cells in combination with gene expression of neural and stress marker genes.


text:
I received this late last night and it is clear that we are probably on the door on the 28-29.my intel that came from someone in the HB market was called back to Hong Kong for Tuesday.
This info lines up with other info on OM (Open Mic) as well as lining up with many other intel providers scenarios.
How many times I have said good-bye only to have to hang in there again.
At this time, I can honestly say I firmly believe that the football game is over and China has won and US has lost but China has won for Humanity.
I think markets will open still in quiet mode. I will say that Iraq has been ready for so long it is sad that they were used as a pawn in a vicious chess game
We should be very thankful that Abadi knew how to play the US and went with the BRICS over a year ago. Martha

subset: commoncrawl


text:
Happy Friday, everyone! We had a great week at Collaborate 13, thank you to everyone who stopped by the booth. We also blogged about "Oracle VM and Oracle Linux pre-validated, pre-tested with FlexPod", released version 4.2.12 of Oracle VM VirtualBox and version 4.63 of Oracle Secure Global Desktop. Lots of great stuff going on!
Speaking of Oracle Secure Global Desktop, our Friday Tip this week is on that very product. In this video tip, Mohan Prabhala covers how to disable or enable copy functionality in applications to increase security:
We'll see you next week!


text:
By Joel Shangle
Heading into the 2016 Bassmaster Classic on Grand Lake in Oklahoma, Simms Pro James Elam was tabbed as a potential favorite. Pretty heady expectations for a young Elite Series pro fishing in his first Classic. No surprise, though, to the many northern Oklahoma anglers who Elam had regularly beaten on Grand Lake since he was a teenager.
Fast forward to the final week before the 2017 Classic on Lake Conroe in Texas, and you'll find a slightly different James Elam. Less pressure, fewer preconceived notions about the fishery, and a more open mind about what might happen on this San Jacinto River impoundment come March 24-26. Read more:
SIMMS: Last year's Classic on Grand Lake had to have felt a little weird to you. Awesome, but weird. How different does it feel to not be competing on your home lake this year?
Elam: I feel like I'm fishing more of a normal tournament, mostly. I can just go to a lake I've never fished before and fish it like I would any other day. Last year at this time, I had the opportunity to practice a whole lot, and to fish the lakes around Grand Lake so I could have a better understanding of how the fish might act. The pressure was definitely on me at that event – I was supposed to do well there because I've done well there in the past.
SIMMS: It sounds like you prefer being able to approach a fishery "fresh", for lack of a better term.
Elam: I do like that. Conroe is a blank, clean-slate lake for me, and I've always done really well in situations like that. I can show up for the first day without any preconceived notions, and just work off of the clues I find when I'm on the water.
SIMMS: That's a pretty accurate analysis of your whole Elite Series career, isn't it? You've had a lot of those "clean-slate lakes" to decipher.
Elam: Until I qualified for the Elite Series and started fishing it in 2013, I'd really only fished in Arkansas, Oklahoma, Texas and Missouri, and all of those lakes were non-grass lakes. I'd never fished a tidal fishery or a grass lake. I'd never fished for northern smallmouth, or for Coosa River, Alabama spotted bass. There were just a lot things I hadn't seen yet. I realize that they're all bass and they're really not that different, but I still had some learning to do. A lake like Toledo Bend, for example, there are certain things you look for, and I had no idea what those things were until I'd fished there a couple of times.
SIMMS: It's worked out pretty well for you, though. Are you aware that since the Delaware River tournament at the end of the 2014 season, your average Elite Series and Open finish is 40 to 50 places higher?
Elam: Wow, no, I wasn't aware of that. I guess that's just due to the learning curve of traveling around the country and fishing all of these different lakes. I think it's less of a guessing game now; or rather, I'm making more educated guesses. I just didn't have enough of that cross-country experience for it to click, but I do feel a lot more calm and prepared than I did when I was first starting out on the Elites. And it's not just the fishing part of it. It's knowing how to eat better, being in better physical shape, just getting everything set up better when I'm competing. Nothing matches tournament experience, though. That's where the mental part of the game really develops.
SIMMS: Apply that statement to the upcoming Classic. It's a lot busier than a regular tournament: it's only a three-day format, and you know you'll have major travel times between the lake and the stadium in Houston.
Elam: The non-fishing part, I'll have to be in shape to only go with six hours of sleep a night. I'll have to be sure I'm eating right, so I don't get run down. But the fishing part, I'll maybe take a few more chances, maybe risk a little bit more going for bigger bags. We're not playing for points at the Classic. Say I go out the first day and catch 15 pounds, am sort of in the middle of the pack: I'll fish Day 2 differently than I would a regular Elite event, where I might be okay with 15 more pounds and some good Angler of the Year points. A lake like Conroe, you know those big ones are in there, I think it might be worth it to risk a little more.
SIMMS: Talk a little more about that Conroe fishery. Most of the world knows it as the lake where Keith Combs obliterated the three-day tournament record with 110 pounds, so it's not out of the question for somebody to make up a lot of ground with a monster bag on Day 2 or Day 3
Elam: It's really kind of a treat when we go to a lake like this because there are so many big fish. It keeps your attention when you know you could be having a so-so day, and at 2 p.m., you get an 8- to 10-pound bite that changes the whole tournament. You never feel like you're really out of it on a lake like that. You're definitely not going to run around throwing 8-pound line – it's more meat-and-potatoes baits and heavy equipment.
SIMMS: How do you expect it to fish during that three-day Classic window? Combs' giant bags came in the summer, when fish were offshore, but it won't fish like that in late March.
Elam: I expect to catch some fish flippin' in shallow water with a big rod and 25-pound line. I think that's my strongest skill set when it comes to Conroe. I think we also have a good chance of catching post-spawn fish on a Carolina-rig and a crankbait, and I feel like I do fairly well with both of those tactics. I'd expect it to be a shallow, junk-fishing deal where a lot of the fish will be in 4 to 8 feet. The bluegill will probably have a big influence, maybe there'll be a shad spawn, and there are a lot of docks. I'm looking forward to it, I think it's going to fish well.


text:
Take a walk in these Samba trainers from adidas Originals. In a black colourway these quality trainers first came to prominence in the early 1960s initially as a football trainer, this statement shoe quickly became a firm favourite in the terraces thanks to its unique look. With a distinctive upper featuring spice yellow branding and trimmings, there is a textured gum sole plus padded ankle collar so you can assure not just style but comfort. Finished off with the iconic adidas Originals branding on the tongue and heel providing one final quality stamp of approval.
View the embedded image gallery online at:


text:
Passage of the Princess
- Discontinued
- Published By: Devine Destinies
- Author: Shannon Rouchelle
- Page: 144
- Word Count: 44190
- Publication Date: April 8, 2012
- Heat Level:
- Available Formats: Adobe Acrobat, Epub, Hiebook, Microsoft Reader, Mobipocket, Mobipocket (.mobi), Palm DOC/iSolo, Rocket, Sony PDF
- Categories: Dark, Romance
The Princess of Peace.
Princess Cassandra Wellington is destined to fulfill a quest and lead her people to freedom and prosperity. With the sacred Ruby Idol in her procession, she travels across dangerous lands with the evil Delmirths on her trail.
When her adventure takes a bad turn of events, Cassandra is rescued from death by a handsome warrior. Keidon Roke soon steals her heart and the two continue on the journey to return the statue to the Sunken Islands. But love and lust threaten the completion of the quest. Can they successfully enter the sacred land pure and untouched, or will passion destroy them?
To read excerpts, you need to have an account with us. Please log in first.


text:
An idea of "ultimate responsibility" in Alexander Technique fascinates me. It strikes me that this idea of how Alexander regards responsibility makes his work unique. Wondering about this assumption is interesting, because most assumptions are the act of intentionally setting up a given characteristic. Assumptions work like axioms; they branch off and lead down a very specific pathways of action, pre-empting all others.
It seems Alexander jumped to an assumption involving a person's ability to direct their own actions. This assumption states the person is ultimately responsible for what he does, no matter how it feels internally after routines have been adopted and feel as natural as breathing. In Alexander's world, there is no subconscious, only divided consciousness or undivided wholeness. Sometimes I wonder if this is one of F.M. Alexander's mistakes.
We do not really know all of the sophisticated responses that are put into motion in a single order we have given ourselves. Because humans are adaptable, we forget the routines we have installed or been conditioned to repeat as the "standing orders" from our past experience. Are we meant to know the ways they are carried out? Alexander thought we should know, that we need to bring our choices into awareness, but I am not so sure.
To undo the order, it seems more efficient to trace it back to its origin and change it there, rather than to piecemeal the change directly by messing with the whole system. In some ways, it's tempting to mess with the system as you become capable of doing so. But all these adjustments are merely compensations, …until you find the originating order and the subsequent directives. You would usually only want to bother because these directives have not worked so well to solve the problem, or the circumstances have changed. Once you find these directives, then you may update them and choose different ways of responding, …and the habit is completely and totally transformed; no looking back. This is where an ideal of "insight" or even "enlightenment" comes from – which I believe is deserved.
I wonder why Alexander did not put together that we are always falling and always uprighting our bodies; I guess he did, but he didn't spell this one out very specifically either. I imagine by the time someone was able to gain this secret for themselves, their coordination was so completely re-sensitized that it was a moot point.
For this reason, I think this assumption of "ultimate responsibility" is very much worth exploring.
How are we responsible for the ways we respond to a desire? How does knowing and responding to a desire work in particular, in me?


text:
Devon Energy Hobbs, NM, USA
Location: Hobbs, NM, US Job ID: 17611 Qualified applicants are considered without regard to race, color, religion, sex, sexual orientation, gender identity, national origin, age, disability, status as a protected veteran or other characteristics protected by law. For more information on Equal Employment Opportunity, please follow these links: EEO is the Law EEO is the Law Supplement Pay Transparency Provision BASIC ACCOUNTABILITY The ESP Optimization Technician is responsible for providing technical assistance to the production foreman, including but not limited to production review and trouble shooting of wells and analysis of mechanical failures. Be familiar with a variety of production applications. Must have computer skills etc. EDUCATION High School diploma or equivalent EXPERIENCE Requires a minimum of two years of Field Service experience with Electrical Submersible Pumps (ESP) JOB RESPONSIBILITIES Monitors, trouble shoots and tests downhole ESP equipment including Downhole Monitoring Tools in order to maximize well productivity and ESP run life. Assist with the set up and start of Centrilift Motor Controller's and VSD's Shoot fluid levels, weigh and analyze data from all types of artificial lift systems (i.e. rod pump, plunger lift, & ESP wells) Strong commitment to safety and environment Work closely with Production Foreman, EHS and Lease Operators Maintain compliance, in conjunction with and through close coordination with the Foreman and EHS Dept. for all regulatory, environmental and safety issues Handle other special projects as assigned Supports and maintains all company policies May be required to work long hours, on holidays, be called out after shift hours and on weekends Work outside in all types of weather conditions Capable of climbing stairs, ladders, work around various equipment safely OTHER REQUIREMENTS Oil and Gas Industry Experience Required Practice safe work habits at all times Demonstrate commitment to Devon Attributes Strong commitment to safety and environment Intermediate computer skills Strong verbal and written communication skills Able to work within a team environment Creative individual, capable of analyzing and solving mechanical problems Position requires operation of Company vehicles. Incumbent must meet requirements of Company Policy Must be able to safely and effectively wear Self Contained Breathing equipment and work in an H2S environment Capable of safely climbing stairs, ladders, fixed to various equipment during production and drilling operations Devon Energy is committed to ensuring equal employment opportunity, including providing reasonable accommodations to individuals with a disability. Applicants with a physical or mental disability who require a reasonable accommodation for any part of the application or hiring process may contact the Devon Talent Acquisition Team at firstname.lastname@example.com .


text:
Review: Désirée Zamorano. The Amado Women. El Paso TX: Cinco Puntos Press, 2014.
Michael Sedano
Love is complicated, not the many-splendored thing of songs of yore. Take the four Amado women, for instance. In their own way, each is loved, or finds love, or thinks she does, in Désirée Zamorano's first novel for Cinco Puntos Press, The Amado Women. But there's a wide gap between perceived love, and happiness. Zamorano is not kind to her characters in ways that will unnerve some readers, but at the end of the book, the reader is going to want more.
Men are less so. Men are shits. There's a medical doctor who won't listen to a child's complaints yet ignoring them could be lethal. There's Amado père, a philandering jerk who turns mawkish when Mercy dumps him. There's Nataly's married lover who wants sex, not love nor companionship. She doesn't see that. There's one decent guy, Celeste's friend, whose passion she refuses to allow. Then there's Jack Levine. All you need to know about this brutal asshole is in the novel's opening pages. There's a lot more to Jack and his own love life, but to share details would be a spoiler.
There are two more Amado women, the Levine girls. Is their future to replicate the other Amado women's? And like mom, the children are in danger. Daddy doesn't like Becky, the youngest. Becky's body hurts but her bruises don't show, either. Like mother, like daughter? The first-born, Miriam, perhaps is daddy's girl and doesn't get beaten. When Sylvia does something "wrong", the first words out of Miriam's mouth when daddy walks in the door are to snitch on mom and Becky. Of all these people, Miriam may be in the greatest danger, danger of growing up to be a survivor through connivance. Again, to reveal details will ruin the twists that Zamorano skillfully crafts for her readers.
As one reads the stories of these four women and two girls, a reader begins to question if Désirée Zamorano is being ironic with that surname. Are these women loved? Do they know love, or is their experience the detritus of love? Might the only genuine, lasting love be that between mother and child?
As Zamorano's novel unfolds, the latter seems the most accurate assessment of these women's lives. Sisterly love is just as complicated as women with men. Nataly and Celeste are at each other's throats. Nataly, the youngest and her mother's favorite, resents Celeste's decision to leave their home town of Orange for school in Arcata. Celeste went off to Humboldt State and never came back. For her part, Celeste sneers at her sister's art career and privately gloats that Nataly works as a waitress while her art career goes nowhere. Sylvia keeps secrets from her family, instead shares the private details of her hellish life with a confidante.
Désirée Zamorano's focus is these women's experience, electing to zoom in on the drama and skimming over what the women look like. Are they brown? Except for a smattering of Spanish phrases and a child who chokes to death on a tortilla de harina, we don't really see them as ethnic, nor as others see them.
Women aren't their looks so Zamorano gives little attention to superficial matters, describing women in general terms and tokens like purses. Nataly is beautiful. Celeste has short spiky hair. Mercy touches up her canas. Sylvia has bruises that don't show, and money that does.
Zamorano has chosen to downplay physical appearance, so vital to a woman's place in contemporary culture. Not until the action is spinning down does the author let the reader "see" what some of the characters look like, the New York artist, the confidante. These moments of recognition at the end of the book enhance the story, adding dimension to minor, but key, players, and a measure of ineluctable satisfaction, even if looks don't matter.
With her characters reeling through yearning, loneliness, illness, discord, tragedy, unfolding crisis that brings them together, Zamorano allows the superficial issues of appearance to provide momentary respite from some really terrible events happening to people whom we care about, even if they don't care as much as the reader.
With so much in this novel that a reviewer cannot talk about, what, then are we to make of the Amado women? When the reader steps back from the page inductively and looks for connections between these particular women and women generally, the fog begins clearing. The Amado Women is a novel about consequences and accountability.
Bad decisions lead to messy outcomes. Indecision leads to a damaged spirit and worse—Sylvia. But so does hasty decision--Nataly. Doing the right thing for oneself is something that requires risk—Celeste and Mercy. Then outside forces beyond one's influence or control happen, and knock one for a loop. The woman who is reluctant to take a reasoned risk is unprepared for life's vagaries and will live one of those lives of quiet desperation that demand a second novel to resolve all that unfinished business.
I hope that's Désirée Zamorano's plan because I want to know what happened after the final page.
Visit Cinco Puntos Press to order a copy, or ask your local indie bookseller to get your copies through Consortium Book Sales and Distribution.
Altadena Event Brings Delightful Surprise
It was a difficult weekend in that far too many wonderful events were scheduled for the same time across the Los Angeles basin. In my neck of the woods, Luis J. Rodriguez was reading at the Pasadena main library and later that day part of a Poet Laureate Address to the Nation.
My camera wanted to go Pasadena, but my heart took me instead to Altadena, where Daniel Castro was leading a discussion of a PBS documentary, Latino Americans: 500 Years of History. Episode 5, Prejudice and Pride 1965-1980.
That was a grand decision on my part, not only for the entertaining documentary but for the delightful surprise that cropped up. Sancho has a book!
Southern Californians enjoyed years of "The Sancho Show" on Pasadena City College's FM radio station, KPCC. Sancho had consejos for young listeners: go to school, study, groove to good sounds, stay out of trouble. Then KPCC went private and the station went to the dogs. The programmers dumped Sancho, the European Hour, and a midnight jazz program, under the guise that, as their star deejay whined, "we want to play our music" emphasis on the "our."
As I took my seat in Altadena Library's community room, I noticed poet Mario Angel Escobar seated near the wall. I threw him a head nod and turned attention to the film and discussion. That was when the delightful surprise hit me.
Escobar is a publisher. Izote Press' book, Dear Chavela, was authored by Sancho, who in his non-radio persona is Dr. Daniel Castro, retired now but formerly president of LA Trade-Technical College, where he met Mario. Escobar related the story, and it's as wonderfully heart-warming as Dear Chavela, and reflects the raison d'être of Dear Chavela and Castro's mission as an educator and as Sancho.
Escobar was being arrested on the campus of LA Trade Tech, after a fist fight. Castro intervened, directing the campus cop to release the plumbing student into the president's custody. Escobar explained his frustration at flunking his plumbing classes. Castro told the kid to explore something different, entonces.
Mario enrolled in philosophy and literature classes and excelled. He got his first degree and kept going until he attained a doctorate and has made a career in teaching and writing. Can I get an ¡Ajua! for that?
Izote Press' Dear Chavela is the richest 52 pages you will read this year, if you order copies. You'll read it the first time in an hour, then re-read favorites over and over.
A pastiche on advice columns, Chavela is the advice columnist for The Chicano Times, headquartered at Marrano Beach, California. Chavela is the world's foremost authority on cosas Chicana, hence the letters.
In his introduction, Castro promises "My goal was to make you, my friends, smile, think, and dream a little. You may laugh, ponder, get angry agree or disagree, maybe even cry—but the goal is to get you thinking." Promises made, promises kept.
Castro captures the colloquial voice of young people like the ones you grew up with. I heard my own voice, that of my primas and primos, tias and tios, in the epistolary style.
The kid who doesn't speak Spanish when "everyone" expects that and gets to wondering, "what am I?"
The kid whose name is mispronounced, or denied by anglo teachers in favor of a nickname. The kid asks, "who am I?"
There's a laugh-out-loud piece on nicknames and their origins.
There's a tear-jerker piece on the assimilated kid named Brandon Swartz who gets confronted by cultura when his unknown-to-him grandfather dies in Morenci, where his mother comes from. At home, she calls him "son" but in Arizona he's "mi'jo." Like the kid with the Mexican names, Brandon asks, "who am I? What am I?"
Each of the thirty-two pieces in the volume runs a few paragraphs, a page or two at most. Many of Castro's voices are young--elementary and junior high—making Dear Chavela totally appropriate for kids that age. Any reader coming out of the bicultural experience will find a lot to enjoy in this book.
All proceeds from sales go to Castro's memorial scholarship named for Castro's son, Quetzalcoatl, who died in an automobile accident.
Click here for a video and information on The Sancho Show and CDs.
Click here for Izote Press and to order copies of Dear Chavela.
Fire, Friends and Fun on Olympic Blvd
I lost track of time and when my wife called me away from writing, I grabbed nothing and headed to the car. We had seats at Cal Tech for the Escher Quartet with guitarist Jason Vieaux. A fabulous concert indeed, yet I had time to remember my camera, but did not. Chamber Music listeners will enjoy The Coleman Concert series at Cal Tech. It's the best—without exception—music bargain in California. The Eroica Trio wraps the season in April, followed by the $15,000 competition for chamber performers.
There was a host of artists attending. Margaret Garcia, who is learning glass work, was there with LA Art News publisher Kathi Milligan. Polaroid Image Transer artist Mario Trillo and cartoonist Sergio Hernandez joined writer Angel Guerrero in the house. Poet Gloria Enedina Alvarez and artist José Lozano, whose book Little Chanclas is earning him lots of attention, also joined the audience.
The heart of the studio is Jaime's glass blowing studio. After a successful funding drive, Jaime bought the equipment housed in the loading dock of the industrial building. Guerrero will train young people in the generally inaccessible technology, in addition to Jaime's own stupendous work.
Guests stationed themselves along the rail above the work floor. Guerrero's students worked with skilled confidence shaping and blowing a glob of molten glass into a sphere. Jaime's key apprentice, Tyler Straight, guided the younger apprentices through the process. Suddenly a clinking noise told the sad tale; the piece had gone out of control and broke.
Tyler, Jaime announced, has earned an internship at the Corning Glass Works in New York state. That achievement is a clear indicator of the quality instruction and guidance Jaime Guerrero provides, and Tyler's talent. La Bloga congratulates Tyler on this outstanding first step in a major career in art and technology of glass.
I missed Araceli Silva's jewelry studio. I remembered Silva's beadwork displayed at Artemio Rodriguez' studio years ago and had lost track of her. I still have. A visit to her webpage illustrates new styles, semi-precious mineral work, but no beading. Drat.
Watch La Bloga for news of future events at 3030.


text:
Program Details
The online Bachelor of Science in Healthcare Administration is designed for students who wish to earn the skills and knowledge to enter and thrive within the healthcare industry. The curriculum provides a strong basis to aid students entering the job market or going on to further their educations while supplementing prior knowledge in order to strengthen individuals as a whole. Focusing on ethics, leadership, sustainability, and communication, this program readies its graduates for success in areas of the public and private sector, such as health insurance providers, consulting firms, and pharmaceutical companies.
- A minimum of a 2.2 college GPA on a 4.0 scale and the equivalent of 45 semester hours or an associate degree*
- All transcript(s)
*This is a bachelor completion program. Students who do not have 45 or more semester hours may be eligible for enrollment in the online Associate of Arts program as a pathway to a bachelors degree.
School Accreditation Statement
Southern Association of Colleges and Schools, Commission on Colleges