Automated affinity chromatography measurements of compound mixtures using a lab-on-valve apparatus coupled to electrospray ionization mass spectrometry

We report a fully automated affinity chromatography system using a lab-on-valve (LOV) apparatus coupled to an electrospray ionization ion-trap mass spectrometer (ESI-MS). The system allows simultaneous measurements of multiple ligand affinities to proteins immobilized on beads. Bead regeneration, column repacking, and repetitive measurements are achieved on the time scale of several minutes. In this study, the system was used to screen the binding of a peptide mixture to human and Trypanosoma brucei (T. brucei) truncated Pex5 (tPex5) proteins. Equilibrium dissociation constants (K(d)) were measured for T. brucei tPex5 and compared to the values obtained by a fluorescence-based competition assay. The three peptides that showed affinity toward tPex5 had K(d) values that were comparable in magnitude (within a factor of 5) and showed the same ranking order as those from manual fluorescence measurements. With 12 min of sample infusion, the entire sample-to-sample cycle takes about 15 min and can be repeated without any preparation between runs. For T. brucei tPex5 affinity measurements, 1 mg of protein was sufficient for 35 repetitive analyses in the automated LOV-ESI-MS apparatus. The system allows rapid determination of K(d) in the range of 10(-5)-10(-7) M for sample mixtures and is suitable for screening a large number of compounds against multiple proteins.     

Glutamate regulation of non-quantal release of acetylcholine in the rat neuromuscular junction

Glutamate, previously demonstrated to participate in regulation of the resting membrane potential in skeletal muscles, also regulates non-quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non-quantal ACh secretion was estimated by the amplitude of endplate hyperpolarization (H-effect) following blockade of skeletal muscle post-synaptic nicotinic receptors by (+)-tubocurarine and cholinesterase by armin (diethoxy-p-nitrophenyl phosphate). Glutamate was shown to inhibit non-quantal release but not spontaneous and evoked quantal secretion of ACh. Glutamate-induced decrease of the H-effect was enhanced by glycine. Glycine alone also lowered the H-effect, probably due to potentiation of the effect of endogenous glutamate present in the synaptic cleft. Inhibition of N-methyl-d-aspartate (NMDA) receptors with (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine (MK801), dl-2-amino-5-phosphopentanoic acid (AP5) and 7-chlorokynurenic acid or the elimination of Ca2+ from the bathing solution prevented the glutamate-induced decrease of the H-effect with or without glycine. Inhibition of muscle nitric oxide synthase by NG-nitro-l-arginine methyl ester (l-NAME), soluble guanylyl cyclase by 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and binding and inactivation of extracellular nitric oxide (NO) by haemoglobin removed the action of glutamate and glycine on the H-effect. The results suggest that glutamate, acting on post-synaptic NMDA receptors to induce sarcoplasmic synthesis and release of NO, selectively inhibits non-quantal secretion of ACh from motor nerve terminals. Non-quantal ACh is known to modulate the resting membrane potential of muscle membrane via control of activity of chloride transport and a decrease in secretion of non-quantal transmitter following muscle denervation triggers the early post-denervation depolarization of muscle fibres.     

Distribution of PER protein,pigment-dispersing hormone,prothoracicotropic hormone,and eclosion hormone in the cephalic nervous system of insects

Investigations performed on adult insects revealed that putative components of the central pacemaker, the protein Period (PER) and the pigment-dispersing hormone (PDH), are immunocytochemically detectable in discrete sets of brain neurons throughout the class of Insecta, represented by a bristletail, mayfly, damselfly, 2 locust species, stonefly, 2 bug species, goldsmith beetle, caddisfly, honeybee, and 2 blowfly species. The PER-positive cells are localized in the frontal protocerebrum and in most species also in the optic lobes, which are their only location in damselfly and goldsmith beetle. Additional PER-positive cells occur in a few species either in the deuto- and tritocerebrum or in the suboesophageal ganglion. The PER staining was always confined to the cytoplasm. The PDH immunoreactivity consistently occurs in a cluster of perikarya located frontoventrally at the proximal edge of the medulla. The mayfly and both locust species possess additional PDH neurons in 2 posterior cell clusters at the proximal edge of the medulla, and mayfly, waterstrider, and 1 of the blowfly species in the central brain. PDH-positive fibers form a fanlike arrangement over the frontal side of the medulla. Two or just 1 bundle of PDH-positive fibers run from the optic lobe to the protocerebrum, with collaterals passing over to the contralateral optic lobe. Antisera to the prothoracicotropic (PTTH) and the eclosion (EH) hormones, which in some insects regulate the molting and ecdysis rhythms, respectively, typically react with a few neurons in the frontal protocerebrum. However, the PTTH-positive neurons of the mayfly and the damselfly and the EH-positive neurons of the caddisfly are located in the suboesophageal ganglion. No PTTH-like antigen was detected in locusts, and no EH-like antigens were detected in the damselfly, stonefly, locusts, and the honeybee. There are no signs of co-localization of the PER-, PDH-, PTTH-, and EH-like antigens in identical neurons.     

Exogenous administration of gangliosides inhibits Fc epsilon RI-mediated mast cell degranulation by decreasing the activity of phospholipase C gamma

Gangliosides released from tumor cells, as well as administered exogenously, suppress the immune responses by largely unknown mechanisms. We show here that a pretreatment of rat basophilic leukemia cells with isolated brain gangliosides inhibited the release of preformed secretory mediators from cells activated via FcepsilonRI but not Thy-1 glycoprotein. Exogenously administered gangliosides also affected the cell-substrate adhesion and the levels of polymeric filamentous actin in Ag-activated cells. Although the production of phosphoinositides was also decreased, enzymatic activity of phosphatidylinositol 3-kinase was not inhibited. Gangliosides had no or only marginal effect on the association of aggregated FcepsilonRI with glycosphingolipid-enriched membranes and on tyrosine phosphorylation of FcepsilonRI and the linker for activation of T cells. Though pretreatment with gangliosides did not inhibit the association of linker for activation of T cells with phospholipase C (PLC)gamma1 and PLCgamma2, tyrosine phosphorylation of these enzymes, as well as their enzymatic activities and association with detergent-insoluble signaling assemblies were reduced. This resulted in a decreased production of inositol 1,4,5-trisphosphate and an inhibition of Ca(2+) mobilization. The combined data support the concept that exogenously administered gangliosides interfere with those properties of glycosphingolipid-enriched membranes that are important for the formation of plasma membrane-associated signaling assemblies containing PLCgamma but not for initial tyrosine phosphorylation of FcepsilonRI subunits.     

The cycling and distribution of PER-like antigen in relation to neurons recognized by the antisera to PTTH and EH in Thermobia domestica

The cephalic nervous system of the firebrat contains antigens recognized by antisera to the clock protein period (PER), the prothoracicotropic hormone (PTTH) and the eclosion hormone (EH). The content of the 115 kDa PER-like antigen visualized on the western blots fluctuates in diurnal rhythm with a maximum in the night. The oscillations entrained in a 12:12 h light/dark (LD) cycle persist in the darkness and disappear in continuous light. They are detected by immunostaining in 14 pairs of the protocerebral neurons and are extreme in four suboesophageal neurons and two cells in each corpus cardiacum that contain PER only during the night phase. No circadian fluctuations occur in three lightly stained perikarya of the optic lobe. Five cell bodies located in each brain hemisphere between the deuto-and the tritocerebrum retain weak immunoreactivity under constant illumination. In all cells, the staining is confined to the cytoplasm and never occurs in the cell nuclei. The cells containing PER-like material do not react with the anti-PTTH and anti-EH antisera, which recognize antigens of about 50 and 20 kDa, respectively. The anti-PTTH antiserum stains in each brain hemisphere seven neurons in the protocerebrum, eight in the optic lobe, and 3–5 in the posterior region of the deutocerebrum. The antiserum to EH reacts in each hemisphere with just two cells located medially to the mushroom bodies. No cycling of the PTTH-like and EH-like antigens was detected.     

Unique molecular architecture of silk fibroin in the waxmoth, Galleria mellonella

Proteins of silk fibers are characterized by reiterations of amino acid repeats. Physical properties of the fiber are determined by the amino acid composition, the complexity of repetitive units, and arrangement of these units into higher order arrays. Except for very short motifs of 6-10 residues, the length of repetitive units and the number of these units concatenated in higher order assemblies vary in all spider and lepidopteran silks analyzed so far. This paper describes an exceptional silk protein represented by the 500-kDa heavy chain fibroin (H-fibroin) of the waxmoth, Galleria mellonella. Its non-repetitive N-terminal (175 residues) and C-terminal (60 residues) parts, the overall gene organization, and the nucleotide sequence around the TATA box show that it is homologous to the H-fibroins of other Lepidoptera. However, over 95% of the protein consists of highly ordered repetitive structures that are unmatched in other species. The repetitive region includes 11 assemblies AB(1)AB(1)AB(1)AB(2)(AB(2))AB(2) of remarkably conserved polypeptide repeats A (63 amino acid residues), B(1) (43 residues), and B(2) (18 residues). The repeats contain a high proportion of Gly (31.6%), Ala (23.8%), Ser (18.1%), and of residues with long hydrophobic side chains (16% for Leu, Ile, and Val combined). The presence of the GLGGLG and SSAASAA(AA) motifs suggests formation of pleated beta-sheets and their stacking into crystallites. Conspicuous conservation of the apolar sequence VIVI followed by DD or ED is interpreted as indicating the importance of hydrophobicity and electrostatic charge in H-fibroin cross-linking. The environment of G. mellonella larvae within bee cultures requires continuous production of silk that must be both strong and elastic. The spectacular arrangement of the repetitive H-fibroin region apparently evolved to meet these requirements.     

Lignin degrading system of white-rot fungi and its exploitation for dye decolorization

With global attention and research now focused on looking for the abatement of pollution, white-rot fungi is one of the hopes of the future. The lignin-degrading ability of these fungi have been the focus of attention for many years and have been exploited for a wide array of human benefits. This review highlights the various enzymes produced by white-rot fungi for lignin degradation, namely laccases, peroxidases, aryl alcohol oxidase, glyoxal oxidase, and pyranose oxidase. Also discussed are the various radicals and low molecular weight compounds that are being produced by white-rot fungi and its role in lignin degradation. A brief summary on the developments in research of decolorization of dyes using white-rot fungi has been made.     

Alterations in Ca2+-channels during the development of diabetic cardiomyopathy

In order to examine the status of Ca²⁺ channels in heart sarcolemma during the development of diabetes, rats were injected intravenously with 65 mg/kg streptozotocin and hearts were removed 1, 3 and 8 weeks later. Crude membranes from the ventricular muscle were prepared and the specific binding of ³H-nitrendipine was studied by employing different concentrations of this Ca ²⁺-antagonist. A significant decrease in both dissociation constant and maximal number of 3H-nitrendipine binding was observed in 3 and 8 weeks diabetic preparations. No such alterations were evident in diabetic brain membranes. Treatment of diabetic animals with insulin prevented the occurrence of these changes in the myocardium. The altered ³H-nitrendipine binding characteristics in diabetic heart membranes may not be due to the high levels of circulating catecholamines in this experimental model because no such changes were seen upon injecting a high dose (40 mg/kg) of isoproterenol in rats for 24 hr. The reduced number of ³H-nitrendipine binding sites may decrease Ca²⁺-influx through voltage sensitive Ca²⁺ channels and partly explain the depressed cardiac contractile force development in chronic diabetes whereas the increased affinity of Ca²⁺ channels may partly explain the increased sensitivity of diabetic heart to Ca ²⁺.     

Survival factor-like activity of small peptides in hybridoma and CHO cells cultures

Synthetic peptides containing three to six amino acid residues were previously shown to improve key parameters of monoclonal antibody-producing mouse hybridoma cultures. The aim of the current work was to investigate whether small peptides also exert analogous beneficial impact on a CHO-K1-derived cell line (XMK-111-10) engineered for production of the human model glycoprotein SEAP (secreted alkaline phosphatase). Similar to hybridoma cultures, growth and SEAP production profiles of CHO XMK-111-10 were modulated by peptides. Both viable cell density and SEAP production were increased by tetraalanine or by a fraction of wheat gluten hydrolysate. Whereas tetraglycine increased the peak viable cell density, the growth-suppressing tripeptide Gly-Lys-Gly significantly boosted SEAP production. All peptide-supplemented cultures showed slight improvement of culture viability during the decline phase of the batch cultures, suggesting a survival factor-like activity of the peptides.