Rapid Purification of Yeast Cytoplasmic Fumarate Reductase Affinity Chromatography on Blue Sepharose CL–6B

Abstract

The rapid and effective purification of soluble fumarate reductase from baker's yeast achieved by Blue Sepharose CL–6B chromatography. Cibacron Blue F3GA, the chromophore of Blue Sepharose, inhibited the activity of fumarate reductase. The enzyme bound to the column was selectively eluted by flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) or riboflavin. The purified enzyme was essentially homogeneous as indicated by polyacrylamide gel electrophoresis under non-denaturing conditions and under denaturing conditions in sodium dodecylsulfate. By this procedure, the enzyme could be rapidly purified with high yield from yeast cells.     

Studying the Morphology of Lyophilized Protein Solids Using X-ray Micro-CT: Effect of Post-freeze Annealing and Controlled Nucleation

The objective of this study was to determine how different techniques used during the freezing step of lyophilization affect morphology of the dried protein solids. Aqueous solutions containing recombinant human albumin, trehalose, and sodium phosphate buffer were dried after their freezing by shelf-ramp cooling, immersion in liquid nitrogen, or controlled ice nucleation. Some shelf-frozen solutions were heat treated (annealed) before the vacuum drying. We used three-dimensional (3D) X-ray micro-computed tomography (micro-CT) and scanning electron microscopy (SEM) to study the morphology of solids. The X-ray micro-CT images of the lyophilized microporous solids showed traces of varied size and structure ice crystals that were comparable to corresponding SEM images. A post-freeze heat treatment and a controlled nucleation both induced larger ice crystal ghosts in the solids. The variations in the structure of walls surrounding ice crystals, formed by the different freezing procedures, should affect the water vapor transition during the primary and secondary drying. Some solids also showed higher-density layer in the upper surface. Overall, the simple sample preparation procedures and the ample morphological information make the X-ray micro-CT appropriate for analyzing lyophilized pharmaceuticals.     

Inhibition of glycolysis induced by diethylstilbestrol in anaerobically grown yeast

In anaerobically grown yeast cells which lack functional mitochondria, the presence of diethylstilbestrol (DES) depressed glycolysis. The addition of the inhibitor markedly increased the cellular concentration of glycolytic intermediates which are formed prior to the pyruvate kinase step as well as to bring about an increase in the [ATP]/[ADP] ratio. Under these conditions an 18 fold decrease in the mass action ratio for pyruvate kinase [( pyruvate] [ATP]/[phosphoenolpyruvate] [ADP]) was noted, however, there was little if any effect on the other glycolytic enzymes. These results suggest that the depression of anaerobic glycolysis caused by DES results from a blockage at the level of the regulatory enzyme pyruvate kinase through a modification of its intracellular environment.     

Attentional Control and Interpretation of Facial Expression after Oxytocin Administration to Typically Developed Male Adults

Deficits in attentional-inhibitory control have been reported to correlate to anger, hostility, and aggressive behavior; therefore, inhibitory control appears to play an important role in prosocial behavior. Moreover, recent studies have demonstrated that oxytocin (OT) exerts a prosocial effect (e.g., decreasing negative behaviors, such as aggression) on humans. However, it is unknown whether the positively valenced effect of OT on sociality is associated with enhanced attentional-inhibitory control. In the present study, we hypothesized that OT enhances attentional-inhibitory control and that the positively valenced effect of OT on social cognition is associated with enhanced attentional-inhibitory control. In a single-blind, placebo-controlled crossover trial, we tested this hypothesis using 20 healthy male volunteers. We considered a decrease in the hostility detection ratio, which reflects the positively valenced interpretation of other individuals’ facial expressions, to be an index of the positively valenced effects of OT (we reused the results of our previously published study). As a measure of attentional-inhibitory control, we employed a modified version of the flanker task (i.e., a shorter conflict duration indicated higher inhibitory control). These results failed to demonstrate any significant behavioral effects of OT (i.e., neither a positively valenced effect on facial cognition nor an effect on attentional-inhibitory control). However, the enhancement of attentional-inhibitory control after OT administration significantly correlated to the positively valenced effects on the interpretation of uncertain facial cognition (i.e., neutral and ambiguous facial expressions).     

Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors

Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.     

Structural basis for DNA-cleaving activity of resveratrol in the presence of Cu(II)

Resveratrol (1, 3,5,4'-trihydroxy-trans-stilbene), a polyphenol found in grapes and other food products, is known as an antioxidant and cancer chemopreventive agent. However, 1 was shown to induce genotoxicity through a high frequency of micronucleus and sister chromatid exchange in vitro and DNA-cleaving activity in the presence of Cu(II). The present study was designed to explore the structure-activity relationship of 1 in DNA strand scission and to characterize the substrate specificity for Cu(II) and DNA binding. When pBR322DNA was incubated with 1 or its analogues differing in the number and positions of hydroxyl groups in the presence of Cu(II), the ability of 4-hydroxystilbene analogues to induce DNA strand scission is much stronger than that of 3-hydroxy analogues. The high binding affinity with both Cu(II) and DNA was also observed by 4-hydroxystilbene analogues. The reduction of Cu(II) which is essential for activation of molecular oxygen proceeded by addition of 1 to the solution of the Cu(II)-DNA complex, while such reduction was not observed with the addition of isoresveratrol, in which the 4-hydroxy group of 1 is changed to the 3-position. The results show that the 4-hydroxystilbene structure of 1 is a major determinant of generation of reactive oxygen species that was responsible for DNA strand scission.     

Enzymatic assay of allantoin in serum using allantoinase and allantoate amidohydrolase

A new enzymatic assay for specifically measuring allantoin concentration in serum has been developed. The currently used methods for allantoin analysis are time consuming and nonspecific or depend on the use of expensive equipment. In our method, allantoin is converted to allantoate by the action of allantoinase (EC 3.5.2.5). The allantoate produced is hydrolyzed to ureidoglycine and ammonia by the action of allantoate amidohydrolase (EC 3.5.3.9). Nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase (EC 1.4.1.4) subsequently acts on the ammonia produced, resulting in a change in absorbance at 340nm due to the consumption of reduced nicotinamide adenine dinucleotide phosphate. The amount of allantoin present is related to the change in the absorbance. The standard curve is linear up to at least 1mM allantoin. The procedure is simple, rapid, and accurate. The method has been used to measure serum allantoin levels after oral administration of purine nucleotides to experimental animals, including rats that have uricase catalyzing the conversion of urate to allantoin.     

Methylene blue protection against hypoxic injury in primary cultures of rat hepatocyte monolayers

Mitochondrial respiration is inhibited in cells exposed to hypoxia, and the oxidation of NADH to NAD(+) is blocked. As a result, oxidation reactions requiring NAD(+) are blocked, disrupting cellular metabolism. We studied the influence of methylene blue, which oxidizes NADH, on hypoxic damage to primary cultures of rat hepatocyte monolayers. During hypoxic treatment of hepatocytes, aspartate aminotransferase leaked out of the cells into the culture medium. However, addition of methylene blue to the medium repressed the hypoxic leakage of the enzyme. The exposure of hepatocytes to hypoxia decreased the acetoacetate/beta-hydroxybutyrate ratio which reflects the redox state of the cell. The level of the acetoacetate/beta-hydroxybutyrate ratio in hypoxic cells was increased by the addition of methylene blue. These results suggest that methylene blue protects against hypoxic injury due to its oxidation of NADH.     

Multiple signal transduction pathways mediated by 5-HT receptors

Among human serotonin (5-HT) receptor subtypes, each G protein-coupled receptor subtype is reported to have one G protein-signaling cascade. However, the signaling may not be as simple as previously thought to be. 5-HT5A receptors are probably the least well understood among the 5-HT receptors, but the authors found that 5-HT5A receptors couple to multiple signaling cascades. When the 5-HT5A receptors were expressed in undifferentiated C6 glioma cells, they modulated the level of second messengers. For example, activation of 5-HT5A receptors inhibited the adenylyl cyclase activity and subsequently reduced the cAMP level, as previously reported. In addition to this known signaling via Gi/Go, 5-HT5A receptors are coupled to the inhibition of ADP-ribosyl cyclase and cyclic ADP ribose formation. On the other hand, activation of 5-HT5A receptors transiently opened the K+ channels, presumably due to the increase in intracellular Ca2+ after formation of inositol (1,4,5) trisphosphate. The K+ currents were inhibited by both heparin and pretreatment with pertussis toxin, suggesting the cross-talk between Gi/Go protein and phopholipase C cascade. Thus, the authors results indicate that 5-HT5A receptors couple to multiple second messenger systems and may contribute to the complicated physiological and pathophysiological states. Although this multiple signaling has been reported only for 5-HT5A/5-HT1 receptors so far, it is possible that other 5-HT receptor subtypes bear similar complexity. As a result, in addition to the wide variety of expression patterns of each 5-HT receptor subtype, it is possible that multiple signal transduction systems may add complexity to the serotonergic system in brain function. The investigation of these serotonergic signaling and its impairment at cellular level may help to understand the symptoms of brain diseases.     

Detection of serum proteins by native polyacrylamide gel electrophoresis using Blue Sepharose CL-6B-containing stacking gels

Analysis of serum proteins by native polyacrylamide gel electrophoresis is difficult because albumin is abundant in serum and interferes with the resolution of other proteins, especially alpha-antitrypsin which has mobility that is very similar to that of albumin. We present here a method in which serum proteins are separated by polyacrylamide gel electrophoresis using stacking gels containing Blue Sepharose CL-6B, which has a high affinity for albumin, lipoproteins, kinases, and pyridine-nucleotide-dependent oxidoreductases. During electrophoresis, proteins that bind to Blue Sepharose CL-6B stay in the stacking gel and do not migrate into the separating gel. As a consequence, certain proteins, including alpha(1)-antitrypsin, can be detected as clear bands. This method overcomes the requirement for fractionation of serum samples prior to electrophoresis to remove albumin and allows the simultaneous analysis of many samples.