Number Of Tyrosine Research Articles

Identification of a specific α-synuclein peptide (α-Syn 29-40) capable of eliciting microglial superoxide production to damage dopaminergic neurons

BackgroundMisfolded α-synuclein (α-Syn) aggregates participate in the pathogenesis of synucleinopathies, such as Parkinson’s disease. Whereas much is known about how the various domains within full-length α-Syn (FL-α-Syn) contribute to the formation of α-Syn aggregates and therefore to their neurotoxicity, little is known about whether the individual peptides that can be generated from α-syn, possibly as intermediate metabolites during degradation of misfolded α-Syn aggregates, are neurotoxic themselves.MethodsA series of synthesized α-Syn peptides, corresponding to the locus in FL-α-Syn containing alanine 30, substitution of which with a proline causes a familial form of Parkinson’s disease, were examined for their capacity of inducing release of microglial superoxide. The neurotoxicity of these peptides was measured according to their influence on the ability of neuroglial cultures deficient in gp91phox, the catalytic unit of NADPH oxidase (Nox2), or wild-type cultures to take up 3H-labeled dopamine and on the number of tyrosine hydroxylase-staining-positive neurons. Western blots and confocal images were utilized to analyze membrane translocation of p47phox and p67phox, phosphorylation of p47phox and Erk1/2 kinase, and binding of α-Syn peptides to gp91phox. Activation of brain microglia in mice injected with α-Syn peptides was demonstrated by immunostaining for major histocompatibility complex (MHC)-II along with qPCR for Iba-1 and MHC-II.ResultsWe report α-Syn (29-40) as a specific peptide capable of activating microglial Nox2 to produce superoxide and cause dopaminergic neuronal damage. Administered to mice, this peptide also activated brain microglia to increase expression of MHC-II and Iba-1 and stimulated oxidation reaction. Exploring the underlying mechanisms showed that α-Syn (29-40) peptide triggered Nox2 to generate extracellular superoxide and its metabolite H2O2 by binding to the catalytic unit gp91phox of Nox2; diffusing into cytosol, H2O2 activated Erk1/2 kinase to phosphorylate p47phox and p67phox and further activated Nox2, establishing a positive feedback loop to amplify the Nox2-mediated response.ConclusionsCollectively, our study suggests novel information regarding how α-Syn causes neuronal injury, possibly including mechanisms involving abnormal metabolites of α-Syn aggregates.

Treadmill step training promotes spinal cord neural plasticity after incomplete spinal cord injury.

A large body of evidence shows that spinal circuits are significantly affected by training, and that intrinsic circuits that drive locomotor tasks are located in lumbosacral spinal segments in rats with complete spinal cord transection. However, after incomplete lesions, the effect of treadmill training has been debated, which is likely because of the difficulty of separating spontaneous stepping from specific training-induced effects. In this study, rats with moderate spinal cord contusion were jected to either step training on a treadmill or used in the model (control) group. The treadmill training began at day 7 post-injury and lasted 20 ± 10 minutes per day, 5 days per week for 10 weeks. The speed of the treadmill was set to 3 m/min and was increased on a daily basis according to the tolerance of each rat. After 3 weeks of step training, the step training group exhibited a sig-nificantly greater improvement in the Basso, Beattie and Bresnahan score than the model group. The expression of growth-associated protein-43 in the spinal cord lesion site and the number of tyrosine hydroxylase-positive ventral neurons in the second lumbar spinal segment were greater in the step training group than in the model group at 11 weeks post-injury, while the levels of brain-derived neurotrophic factor protein in the spinal cord lesion site showed no difference between the two groups. These results suggest that treadmill training significantly improves functional re-covery and neural plasticity after incomplete spinal cord injury.

Abstract 2884: Preclinical studies on tumor retention, antitumor efficacy and toxicity of thermally responsive polypeptide-based radionuclide intratumoral depot in nude mice

Abstract Despite clear advantages of brachytherapy over external beam radiotherapy, current implementations of this approach have several limitations. These include the need for general anesthesia or intravenous sedation, complicated placement procedures and the need for post -treatment re-excision for device removal. An alternative approach that circumvents these constraints would be clearly attractive. In a previous study, we demonstrated that a thermally sensitive elastin-like polypeptide (ELP) displayed increased tumor retention through a thermally triggered in-situ coacervation and delayed tumor growth through the therapeutic action of a conjugated radionuclide, 131I, over a non-thermally sensitive ELP following intratumoral (i.t.) administration. However, this “smart” radionuclide carrier did not lead to complete tumor regression and had a small effect on survival. To improve the antitumor efficacy of this thermally sensitive ELP-radionuclide conjugate (ELP-depot), we investigated the effects of three orthogonal parameters upon radionuclide retention in tumor following i.t administration: the ELP concentration, molecular weight and tyrosine content (hydrophobicity). Furthermore, using the ELP-depot optimized in the above studies, we examined the antitumor efficacy and toxicity in two human tumors (human head and neck squamous carcinoma (FaDu) and human prostate (PC-3) tumor) xenografts in nude mice. The results demonstrate that the injected concentration, the transition temperature (Tt), and the hydrophobicity are critical design parameters with significant influence over radionuclide tumor retention. A 16-fold increase in concentration from 62.5 μM to 1000 μM resulted in a 5-fold increase in retention (16% ID versus 85% ID) 7 days post-injection (p < 0.01, ANOVA). Decreasing the Tt (a value dependent upon the molecular weight) from 30 °C to 24 °C resulted in a 14-fold increase in tumor retention over the same time period (p < 0.01, ANOVA). Tyrosine content-induced the changes of ELP hydrophobicity have significantly affected the behavior of radionuclide in tumor, presenting that the tumor retention, radioactivity stability and antitumor efficacy were enhanced with the increase in the tyrosine number from 1,4 to 7. This optimized ELP depot achieved a 100% of tumor response to this radiotherapy in both tumor xenografts and no significant toxicity was found in either of tumor models. Interestingly, complete tumor regression (tumor size turned out to be zero at the end of the experiment) rate was 67% in FaDu and 77% in PC-3. Together, all our data indicates that the ELP radionuclide depot is an ideal intratumoral radionuclide carrier, because it is injectable, biocompatible, has long tumor retention and good antitumor efficacy for local radiotherapy of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2884. doi:1538-7445.AM2012-2884

Acute Myeloid Leukemia as a Model for Cancer Therapy

Acute Myeloid Leukemia as a Model for Cancer Therapy

L-Tryptophan Production by Auxotrophic and Analogue Resistant Mutants of Aureobacterium flavescens

A number of tyrosine plus phenylalanine double auxotrophic mutants were isolated by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) treatment of a locally isolated strain of Aureobacterium flavescens of which 11A39 and 11A17 were selected on the basis of their tryptophan production in a mineral salt medium over other isolated mutant strains. The mutational block in the aromatic amino acid biosynthetic pathway of the selected double auxotrophs were determined. By controlling pH of the production medium to near neutrality, the active growth period could be extended up to 72 h and more tryptophan was accumulated compared to pH unregulated culture where the active growth ceased after 48 h. Further improvement of the tryptophan production has been achieved by stepwise isolation of a mutant strain resistant to the tryptophan analogues p-fluorotryptophan (FT) and 5-methyl tryptophan (MT) from the 11A39. Demand for L-tryptophan as food additive and therapeutic agent is increasing day by day throughout the World, particularly in the underdeveloped and developing countries like India. Still to date India depends on other countries for L-tryptophan. The aim of this work is to develop a potent high yielding, feed back insensitive mutant strain and optimization of its medium pH for maximum production of tryptophan.

Syk and Lyn mediate distinct Syk phosphorylation events in FcɛRI-signal transduction: Implications for regulation of IgE-mediated degranulation

Spleen tyrosine kinase (Syk) is a key regulatory factor in the IgE-mediated allergic signal transduction pathway in mast cells and basophils. Syk is phosphorylated on a number of tyrosines following the binding of IgE/allergen complexes to FcɛRI receptors leading to initiation of inflammatory signaling via downstream enzymes and scaffolding proteins. We examined the kinases responsible for the phosphorylation of key Syk tyrosines in rat RBL-2H3 basophilic cells and bone marrow-derived mast cells (BMMCs). The phosphorylation of Syk tyrosine 346 was completely blocked by the novel Src family kinase inhibitor BIRA766, suggesting this tyrosine is a pure substrate for Src family kinases. This was supported by the findings that kinase-dead (KD) Syk was efficiently phosphorylated on this tyrosine and that a specific Syk inhibitor BAY61-3606 was without effect. The phosphorylation of other Syk tyrosines 317, 342, 519 and 520 was reduced by Syk and Src family inhibitors, suggesting a role for auto- and trans-phosphorylation. Lyn was the predominant Src family kinase expressed and activated in RBL-2H3 cells, meanwhile Lyn knockdown with a specific siRNA interfered with the phosphorylation of all Syk tyrosines and the Syk substrates SLP-76 and LAT. Pharmacological inhibition of Syk completely blocked the degranulation of RBL-2H3 and BMMCs. However, Lyn knockdown sensitized RBL-2H3 cells to FcɛRI-induced degranulation. We showed that whilst interference with Lyn expression disrupts FcɛRI proximal signaling via Syk and its direct substrates including SLP-76 and LAT, distal activation of downstream proteins including Erk is enhanced. This study identifies the responsible kinases for the phosphorylation of key Syk tyrosines and the propagation of FcɛRI receptor mediated signal transduction in allergic responses.

Exposure to intermittent hypoxia (IH) for 7 days reduces the number of tyrosine hydroxylase‐immunoreactive (TH‐ir) neurons in the caudal NTS

In neonates, hypoxia‐ischemia results in cell loss within the NTS A2 cell group. To determine if exposure to IH in adult rats resulted in a loss within the A2 cell group, we counted the number of TH‐ir neurons in the NTS in normoxic rats (n=11) and rats exposed to IH (between 8am and 4pm alternate 21% and 10% O2 every 3 min for 7 days, n=7). TH‐ir was revealed by treatment of sections with a monoclonal TH antibody (Chemicon) followed by a secondary labeled with Alexa Fluor 594. Photomicrographs of NTS sections were counted by at least 3 investigators blind as to the origin of the sections. In normoxic controls, sections of caudal NTS (Bregma ‐14.3) had 21±2 TH‐ir neurons/section compared to 11±3 TH‐ir neurons/section in IH exposed rats (p=.008). Sections of intermediate NTS (Bregma ‐13.6) had 27±3 TH‐ir neurons/section in normoxic rats compared to 14±5 TH‐ir neurons/section in IH exposed rats (p=.04). Sections of rostral NTS (Bregma ‐12.9) had 16±6 TH‐ir neurons/section in normoxic rats compared to 13+6 TH‐ir neurons/section in IH exposed rats (p=.17). These results indicate that exposure to IH reduces the number of TH‐ir neurons in caudal regions of the NTS. A reduction in the number of catecholaminergic neurons might contribute to the pathophysiology associated with exposure to IH.

Role of acetylcholine receptors and dopamine transporter in regulation of extracellular dopamine in rat carotid body cultures grown in chronic hypoxia or nicotine.

Using dissociated rat carotid body (CB) cultures, we compared levels of extracellular dopamine (DA) around oxygen-sensitive glomus cells grown for approximately 12 days in normoxia (Nox; 20% O2), chronic hypoxia (CHox; 6% O2), or chronic nicotine (CNic; 10 microM nicotine, 20% O2), with or without acetylcholine (ACh) receptor (AChR) agonists/antagonists and blockers of DA uptake. In Nox cultures, extracellular DA, determined by HPLC and normalized to the number of tyrosine hydroxylase-positive glomus cells present, was augmented by acute (approximately 15-min) exposure to hypoxia (5% O2; approximately 6x basal), high extracellular K+ (30 mM; approximately 10x basal), nomifensine (1 microM; a selective DA uptake inhibitor; approximately 3x basal), and nicotine (100 microM; approximately 5x basal), but not methylcholine (300 microM; a specific muscarinic agonist). In contrast, in CHox cultures where basal DA release is markedly elevated (approximately 9x control), the stimulatory effect of high K+ (3-4x basal) and acute hypoxia (approximately 2x basal) on DA release persisted, but nicotine and nomifensine were no longer effective and methylcholine had a partial inhibitory effect. In CNic cultures, basal DA levels were also elevated (approximately 9x control), similar to that in CHox cultures; however, although acute hypoxia had a stimulatory effect on DA release (approximately 2x basal), nicotine, nomifensine, and high K+ were ineffective. The elevated basal DA in both CHox and CNic cultures was attenuated by acute or chronic treatment with mecamylamine (100 microM), a nicotinic AChR (nAChR) antagonist. In addition, long-term (16-h), but not acute (15-min), treatment with the muscarinic antagonist atropine (1 microM) produced an additional enhancement of basal DA levels in CHox cultures. Thus, after chronic hypoxia or nicotine in vitro, extracellular DA levels around CB chemoreceptor cell clusters appear to be set by a variety of factors including released ACh, positive and negative feedback regulation via nAChRs and muscarinic AChRs, respectively, and modulation of DA transporters. These results provide insight into roles of endogenous transmitters in the adaptation of CB chemoreceptors to chronic hypoxia and suggest pathways by which neuroactive drugs, e.g., nicotine, can interfere with the protective chemoreflex response against hypoxia.

Expression of Mixed Lineage Kinase-1 in Pancreatic β-Cell Lines at Different Stages of Maturation and during Embryonic Pancreas Development

Events controlling differentiation to insulin-secreting beta-cells in the pancreas are not well understood, although beta-cells are thought to arise from pluripotent ductal precursor cells. To search for signaling proteins that might be involved in beta-cell maturation, we analyzed protein kinase expression in two developmentally and functionally distinct pancreatic beta-cell lines, RIN-5AH and RIN-A12, by reverse transcriptase polymerase chain reaction. A number of tyrosine and serine/threonine kinases were identified in both lines. One protein kinase, mixed lineage kinase-1 (MLK-1), was expressed at both the RNA and protein levels in RIN-5AH cells, which display an immature beta-cell phenotype, but was not detected in the more mature RIN-A12 cells. Furthermore, levels of MLK-1 mRNA and protein were increased after brief stimulation of RIN-5AH cells with either the differentiation inducer, sodium butyrate, or with serum after serum starvation. These increases in expression were independent of phenotypic markers such as insulin secretion or surface expression of major histocompatibility class I- and A2B5-reactive ganglioside. In addition, increases in MLK-1 expression in the stimulated RIN-5AH cells were accompanied by phosphorylation of MLK-1 on serine but not tyrosine. Antisense oligonucleotides to two distinct regions of MLK-1 caused RIN-5AH cells, but not RIN-A12 cells, to adopt a highly undifferentiated morphology, with a reduction in DNA synthesis and MLK-1 protein levels and elevated glucagon mRNA levels, but with no effect on insulin mRNA. In an immunohistochemical survey of embryonic mouse tissues, we found that temporal expression of MLK-1 was regulated in a tissue-specific manner. In the embryonic pancreas, MLK-1 expression was evident in ductal cells from day 13 to 16 but was not detected in late stage gestation or neonatal pancreas. These data suggest that MLK-1 is regulated in immature pancreatic beta-cells and their ductal precursors at the level of functional maturity and may therefore play a role in beta-cell development.

Isolation and Characterization of Cajanus cajan Lectin

Cajanus cajan lectin was isolated by ammonium sulfate fractionation and affinity chromatography on an IgM-Sepharose 6B column. Gel filtration and SDS-PAGE showed size homogeneity of the lectin. The lectin with Mr 18,000 on SDS-PAGE had gel filtration behavior which was consistent with a molecular weight of 39 kDa and a Stokes radius of 2.74 nm. The results showed that the lectin is a dimer composed of identical subunits with N- and C-terminal residues of threonine and alanine, respectively. The glycoprotein lectin contained 3% concanavalin A-reactive neutral carbohydrates. Its amino acid composition is characterized by high contents of acidic amino acids. The number of tyrosine and tryptophan residues per mole of the lectin was determined to be 14 and 4, respectively, by spectrophotometry. Results on the effects of large numbers of saccharides on lectin-mediated hemagglutination and lectin-IgM precipitation showed that the C. cajan lectin was specific for mannose and glucose. A comparative study of the properties of C. cajan lectin and concanavalin also presented.

Solvent perturbation spectra of yellow mealworm α-amylase and of wheat protein inhibitors

1. 1. The number of tyrosine and tryptophan residue-equivalents on the surfaces of the α-amylase and of two of its protein inhibitors has been determined. 2. 2. The solvent-exposed tyrosine and tryptophan residue-equivalents in the dimeric inhibitor are respectively four and two times as much as those ones of the monomeric inhibitor. 3. 3. On the basis of the homology among their polypeptide chains, it is suggested that the outer tryptophan residues in either inhibitors are located at the positions 4 and 51 of the primary structure. 4. 4. On the interaction of the monomeric inhibitor with the amylase, two tryptophan residue-equivalents became no more accessible to the solvent.

Regulatory properties of 3‐deoxy‐D‐arabino‐heptulosonate‐7‐phosphate synthase isozymes from Candida maltosa

AbstractThe first step in the biosynthesis of aromatic amino acids by Candida maltosa was catalysed by two isozymes of 3‐deoxy‐d‐arabino‐heptulosonate 7‐phosphate (DAHP) synthase. The formation of which was constitutive. The phenylalanine‐sensitive and the tyrosine‐sensitive DAHP synthases were partially purified by hydroxylapatite chromatography and several properties of the separated isozymes were studied. Both enzymes showed a molecular weight of 90,000. The Km‐values of the tyrosine‐sensitive DAHP synthase were 0.20 mm for erythrose‐4‐phosphate (E‐4‐P) and 0.50 mm for phosphoenolpyruvate (PEP). Inhibition of this isozyme reaction by l‐tyrosine was competitive with respect to E‐4‐P (Ki = 0.028 mm) and non‐competitive with respect to PEP. The Km‐values of the phenylalanine‐sensitive DAHP synthase were estimated to be 0.65 mm for E‐4‐P and 0.63 mm for PEP; a Ki of 0.097 mm was obtained for l‐phenylalanine. A number of tyrosine and phenylalanine analogues and biosynthetic intermediates also inhibited the two isozyme reactions. For example, we observed a strong inhibitory effect of p‐hydroxyphenylpyruvate on the tyrosine‐sensitive DAHP synthase and of phenylpyruvate on the phenylalanine‐sensitive isozyme. Prephenate and arogenate had a less pronounced effect on the phenylalanine‐sensitive DAHP synthase.

Conformational properties of human immunoglobulin G subclasses analysis by temperature-perturbation and solvent-perturbation spectroscopy

Conformational properties of human myeloma immunoglobulins G belonging to four subclasses (IgG1 Van, IgG2 Kom, IgG3 Pla, IgG4 Ang), and also Fab, Fc and pFc′ fragments derived from IgG1 Van, IgG2 Kom and IgG3 Pla have been studied by temperature-perturbation and solvent-perturbation spectroscopy. It has been shown that the immunoglobulins studied practically do not differ in the number of tyrosine and tryptophan residues exposed to different solvent perturbants (saccharose, glycerol, dimethylsulfoxide). The same regularity is observed for isolated Fab and Fc fragments. At the same time, the immunoglobulins compared and their proteolytic fragments significantly differ in the number of aromatic chromophores perturbed by temperature. These data indicate that immunoglobulins of different subclasses and their subunits have a different rigidity of structure in relation to thermal perturbation. The Fc subunits of IgG1 are characterized by the lowest rigidity of structure of internal hydrophobic cores of domains (characterized by the rigidity of the microenvironment of tryptophan residues), as compared with the Fc subunits of IgG2 and IgG3. In the case of IgG1 and IgG2, these differences seem to be brought about by a different rigidity of structure of C H2 domains, since thermal-perturbation spectra of the pFc′ fragments of these subclasses practically coincide. The total number of chromophores exposed to different solvent perturbants in the isolated Fab and Fc fragments practically coincides with the number of exposed chromophores in intact immunoglobulins. Similar coincidence is observed for the tryptophan residues perturbed by temperature. These data indicate that neither the conformation of surface sites nor the conformation of internal hydrophobic cores of domains significantly changes on isolation of Fab and Fc fragments. At the same time, many more tyrosine residues are perturbed by temperature in the intact immunoglobulin G1 Van than in the corresponding sum of isolated Fab and Fc fragments, while for IgG2 Kom, which has the same length of hinge region, these values practically coincide. This fact can be explained by the greater temperature dependence of motions of subunits in IgG1 Van as compared with IgG2 Kom, and as a result of this by the higher mutual temperature-dependent influence of subunits on their internal structure (on interdomain interactions).

Separation and properties of isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthetase from Saccharomyces cerevisiae.

The phenylalanine-sensitive and the tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthetases in yeast were separated by affinity chromatography and several properties of the separated isozymes were studied. The nature of the chromatographic process was also investigated. Both DAHP synthetases were inactivated by EDTA. The inactivation was reversed by Co2+ or Mn2+ and to a lesser extent by Zn2+. Phosphoenolpyruvate protected both enzymes from inactivation by EDTA or by heat treatment. Both enzymes showed a broad pH optimal range between 6.5 and 8.0 and a molecular weight of 65 000. The concentration of effector for 50% inhibition of each isozyme was approximately 5 × 10−5 M phenylalanine and 2 × 10−4 M tyrosine. A number of tyrosine and phenylalanine analogues also inhibited the enzyme reaction. Kinetic data were obtained for each of the separated isozymes both in the presence and in the absence of inhibitors. Considerable departure from Michaelis–Menten kinetics was observed in several instances. Our kinetic results are significantly different from those previously reported for the ammonium sulfate fractionated isozymes. These differences may be due to structural changes in the enzymes caused by the ammonium sulfate procedure.

Studies on the properties of chemically modified actin III. Carbethoxylation

Diethylpyrocarbonate was used for the specific carbethoxylation of the histidyl residues of G- and F-actins. The characteristic properties of carbethoxylated actins were studied. The extent of carbethoxylation depended on the actin to reagent ratio, and equal numbers of histidyl residues were carbethoxylated in G- and F-actins. The number of tyrosine and SH groups did not change on diethylpyrocarbonate treatment. 30% of the bound nucleotide of G-actin was removed, whereas a high degree of carbethoxylation did not change the bound nucleotide of F-actin. The characteristic properties of F-actin (viscosity, repolymerizability, actomyosin formation, activation of myosin ATPase) were not altered by the treatment even on carbethoxylation of 70% of the histidyl residues. The characteristic properties of G-actin (polymerizability, actomyosin formation and activation of myosin ATPase) were almost lost on carbethoxylation of about half the histidyl residues. The treatment did not change the helix content of G-actin (the 233-mμ Cotton effect). These results lead to the assumption that the histidyl residues, which have some role in determining the characteristic properties of actin, are buried in F-actin.