A detailed study of the periodate oxidation of sialic acids in glycoproteins

Periodate oxidation of terminalN-acetyl- andN-glycoloylneuraminic acid residues in the mucins from edible bird nest substance and pig submandibular gland, respectively, can be carried out under conditions which exclusively give rise to the formation of the C-7 analogues of these sialic acids. In contrast, the C-8 compounds can be obtained in a maximum yield of about 40%. Under identical conditions,N-glycoloylneuraminic acid is oxidized about 1.5 times faster than theN-acetylated derivative. After release of the sialic acids by acid hydrolysis, the characterization of the oxidation products was carried out by TLC, by GLC and GLC-MS of the corresponding pertrimethylsilyl derivatives, and by 500-MHz¹H-NMR spectroscopy. In addition, molar response factors for GLC analysis and extinction coefficients in the orcinol/Fe³⁺/HCl assay were determined.     

Patterns of divergence during evolution of alpha 1-proteinase inhibitors in mammals

alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine proteinase inhibitor superfamily, has a primary role in controlling neutrophil elastase activity within the mammalian circulation. Several studies have indicated that the reactive center region of alpha 1-PI, the amino acid sequence of which is critical to recognition of and binding to target proteinases, is highly divergent within and among species. This appears to be a consequence of accelerated rates of evolution that may have been driven by positive Darwinian selection. In order to examine this and other features of alpha 1-PI evolution in more detail, we have isolated and sequenced cDNAs representing alpha 1- PI mRNAs of the mouse species Mus saxicola and Mus minutoides and have compared these with a number of other mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent of nonsynonymous substitution is generally high throughout the alpha 1-PI mRNA molecule, indicating greater overall rates of amino acid substitution. Within and among mouse species, the 5'-half of the mRNA, but not the 3'-half, has been homogenized by concerted evolution. Finally, the reactive center is under diversifying or positive Darwinian selection in murid rodents (rats, mice) and guinea pigs yet is under purifying selection in primates and artiodactyls. The significance of these findings to alpha 1-PI function and the possible selective forces driving evolution of serpins in general are discussed.      

Some effects of chromium toxicity on bush bean plants grown in soil

Summary Chromium applied to a noncalcareous soil at 50 ppm did not decrease yields of bush beans (Phaseolus vulgaris L. var Improved Tendergreen), but when EDTA (ethylenediamine tetraacetic acid) was added with it, it did. Very little Cr was present in leaves. In solution culture 10^-5 M Cr and higher were toxic. With solution culture the highest level of Cr in leaves was about 30 ppm and in general there was a decreasing gradient in Cr from roots to stems to leaves. EDTA had less effect in solution cultures on Cr toxicity because the Cr was already in solution. Chromium toxicity decreased cation levels in plants.     

A combined LDL receptor/LDL receptor adaptor protein 1 mutation as the cause for severe familial hypercholesterolemia

Familial hypercholesterolemia (FH) results from impaired catabolism of plasma low density lipoproteins (LDL), thus leading to high cholesterol, atherosclerosis, and a high risk of premature myocardial infarction. FH is commonly caused by defects of the LDL receptor or its main ligand apoB, together mediating cellular uptake and clearance of plasma LDL. In some cases FH is inherited by mutations in the genes of PCSK9 and LDLRAP1 (ARH) in a dominant or recessive trait. The encoded proteins are required for LDL receptor stability and internalization within the LDLR pathway. To detect the underlying genetic defect in a family of Turkish descent showing unregular inheritance of severe FH, we screened the four candidate genes by denaturing gradient gel electrophoresis (DGGE) mutation analysis. We identified different combinatory mixtures of LDLR- and LDLRAP1-gene defects as the cause for severe familial hypercholesterolemia in this family. We also show for the first time that a heterozygous LDLR mutation combined with a homozygous LDLRAP1 mutation produces a more severe hypercholesterolemia phenotype in the same family than a homozygous LDLR mutation alone.     

Magnetoelectric nanocomposite scaffold for high yield differentiation of mesenchymal stem cells to neural-like cells

While the differentiation factors have been widely used to differentiate mesenchymal stem cells (MSCs) into various cell types, they can cause harm at the same time. Therefore, it is beneficial to propose methods to differentiate MSCs without factors. Herein, magnetoelectric (ME) nanofibers were synthesized as the scaffold for the growth of MSCs and their differentiation into neural cells without factors. This nanocomposite takes the advantage of the synergies of the magnetostrictive filler, CoFe₂O ₄ nanoparticles (CFO), and piezoelectric polymer, polyvinylidene difluoride (PVDF). Graphene oxide nanosheets were decorated with CFO nanoparticles for a proper dispersion in the polymer through a hydrothermal process. After that, the piezoelectric PVDF polymer, which contained the magnetic nanoparticles, underwent the electrospun process to form ME nanofibers, the ME property of which has the potential to be used in areas such as tissue engineering, biosensors, and actuators.     

Ultrafine carbon particles induce apoptosis and proliferation in rat lung epithelial cells via specific signaling pathways both using EGF-R

Apoptosis and proliferation are important causes of adverse health effects induced by inhaled ultrafine particles. The molecular mechanisms of particle cell interactions mediating these end points are therefore a major topic of current particle toxicology and molecular preventive medicine. Initial studies revealed that ultrafine particles induce apoptosis and proliferation in parallel in rat lung epithelial cells, dependent on time and dosage. With these end points, two antagonistic reactions seem to be induced by the same extracellular stimulus. It was therefore investigated whether proliferation is induced directly by the particles or as a compensation of particle-caused cell death. Experimental conditions excluding compensatory proliferation demonstrated that both end points are induced independently by specific signaling pathways. Events eliciting signaling cascades leading to apoptosis and proliferation were studied with specific inhibitors of membrane receptors. Epidermal growth factor receptor (EGF-R) kinase activity was identified as essential for apoptosis as well as for proliferation. As ultrafine particle-induced proliferation alone was dependent on the activation of beta1-integrins, these membrane receptors are suggested to mediate the specificity of EGF-R signaling concerning the decision as to whether apoptosis or proliferation is triggered. Accordingly, MAP kinase signaling downstream of EGF-R showed comparable specificity with regard to receptor-dependent induction of apoptosis and proliferation. As key mediators of signaling cascades, the activation of extracellular signal-regulated kinases 1 and 2 proved to be specific for proliferation in a beta1-integrin-dependent manner, whereas phosphorylation of c-Jun NH2-terminal kinases 1 and 2 was correlated with the induction of apoptosis.     

Effect of short- and long-term strength exercise on cardiac oxidative stress and performance in rat

Increase in heart metabolism during severe exercise facilitates production of ROS and result in oxidative stress. Due to shortage of information, the effect of chronic strength exercise on oxidative stress and contractile function of the heart was assessed to explore the threshold for oxidative stress in this kind of exercise training. Male Wistar rats (80) were divided into two test groups exercised 1 and 3 months and two control groups without exercise. Strength exercise was carried by wearing a Canvas Jacket with weights and forced rats to lift the weights. Rats were exercised at 70% of maximum lifted weight 6 days/week, four times/day, and 12 repetitions each time. Finally, the hearts of ten rats/group were homogenized and MDA, SOD, GPX, and catalase (CAT) were determined by ELISA method. In other ten rats/group, left ventricle systolic and end diastolic pressures (LVSP and LVEDP) and contractility indices (LVDP and +dp/dt max) and relaxation velocity (−dp/dt max) were recorded. The coronary outflow was collected. Short- and long-term strength exercise increased heart weight and heart/BW ratio (P < 0.05). In the 3-month exercise group, basal heart rate decreased (P < 0.05). LVEDP did not change but LVDP, +dp/dt max, −dp/dt max, and coronary flow significantly increased in both exercise groups (P < 0.05). None of MDA or SOD, GPX, and CAT significantly changed. The results showed that sub-maximal chronic strength exercise improves heart efficiency without increase in oxidative stress index or decrease in antioxidant defense capacity. These imply that long-time strength exercise up to this intensity is safe for cardiac health.     

Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks

Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.     

NetPhosBac – A predictor for Ser/Thr phosphorylation sites in bacterial proteins

There is ample evidence for the involvement of protein phosphorylation on serine/threonine/tyrosine in bacterial signaling and regulation, but very few exact phosphorylation sites have been experimentally determined. Recently, gel‐free high accuracy MS studies reported over 150 phosphorylation sites in two bacterial model organisms Bacillus subtilis and Escherichia coli. Interestingly, the analysis of these phosphorylation sites revealed that most of them are not characteristic for eukaryotic‐type protein kinases, which explains the poor performance of eukaryotic data‐trained phosphorylation predictors on bacterial systems. We used these large bacterial datasets and neural network algorithms to create the first bacteria‐specific protein phosphorylation predictor: NetPhosBac. With respect to predicting bacterial phosphorylation sites, NetPhosBac significantly outperformed all benchmark predictors. Moreover, NetPhosBac predictions of phosphorylation sites in E. coli proteins were experimentally verified on protein and site‐specific levels. In conclusion, NetPhosBac clearly illustrates the advantage of taxa‐specific predictors and we hope it will provide a useful asset to the microbiological community.     

Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18‐1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho‐proteomics abolished the stimulatory effect of Munc18‐1 on SNARE complex formation (“SNARE‐templating”) and membrane fusion in vitro. Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18‐1‐null neurons expressing Munc18‐1_Y473D. Synaptic transmission was temporarily restored by high‐frequency stimulation, as well as by a Munc18‐1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non‐phosphorylatable Munc18‐1 supported normal synaptic transmission. We propose that SFK‐dependent Munc18‐1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post‐docking SNARE‐templating role of Munc18‐1, resulting in a largely abolished pool of releasable synaptic vesicles.