β-Glucan synthetase activity in Golgi vesicles ofPetunia hybrida

-Glucan synthetase activity has been demonstrated in a Golgi vesicle fraction isolated from pollen tubes ofPetunia hybrida. This-glucan synthetase activity differs from that of most other higher plants in its inability to incorporate [¹⁴C]glucose from GDP-[¹⁴C]glucose. UDP-[¹⁴C]glucose, however, is an appropriate glucose donor for this enzyme. The optimum conditions for this-glucan synthetase activity are: 1 mg Golgi vesicle protein/ml reaction mixture; pH=±8 and a temperature of 25°C. The newly synthesized alkali-insoluble glucan contains-1,3- as well as -1,4-glucosidic linkages.     

Single-base discrimination mediated by proofreading 3′ phosphorothioate-modified primers

It has been well known for decades that deoxyribonucleic acid (DNA) polymerases with proofreading function have a higher fidelity in primer extension as compared to those without 3′ exonuclease activities. However, polymerases with proofreading function have not been used in single nucleotide polymorphism (SNP) assays. Here, we describe a new method for single-base discrimination by proofreading the 3′ phosphorothioate-modified primers using a polymerase with proofreading function. Our data show that the combination of a polymerase with 3′ exonuclease activity and the 3′ phosphorothioate-modified primers work efficiently as a single-base mismatch-operated on/off switch. DNA polymerization only occurred from matched primers, whereas mismatched primers were not extended at the broad range of annealing temperature tested in our study. This novel single-base discrimination method has potential in SNP assays.     

Studies on the ɛ-Lysine Acylase

In order to study the enzymatic properties of the ?-lysine acylase in Achromobacter pestifer EA, experiments were carried out with the pure enzyme preparation. As a result of the investigation, it was found that this enzyme readily hydrolyzes ?-N-acyllysine and shows no α-amino acylase activity, and that the enzyme is not specifically activated by metal ions unlike the other acylases.     

Studies on the ɛ-Lysine Acylase

In succession to previous papers, screening experiments for ?-lysine acylase activity in microorganisms from soils have been carried out. As a result, the enzyme activity was observed in some soil bacteria. From the organisims showing the activity, the strain which showed the highest activity was isolated and the microorganism was confirmed to belong in Achromobacter pestifer. So, further screening experiments of the typical organisms of Achromobacter and Pseudomonas species have been carried out, but the found activity in several organisms was much lower than that in Achromobacter pestifer strain EA isolated by the authors. ?-Lysine acylase in this organism shows optical specificity and hydrolyses only L-isomer but not D-isomer of ?-acyllysine.     

Studies on the ɛ-Lysine Acylase

?-Lysine acylase of Achromobacter pestifer EA was purified by fractionations with ammonium sulfate and acetone, and by vertical zone electrophoresis. As a result, this bacterial ?-lysine acylase was obtained as an electrophoretically homogeneous protein, specific activity of which is the highest among ?-lysine acylases ever reported.     

Studies on the ɛ-Lysine Acylase

Since Achromobacter pestifer EA isolated from soils shows markedly high ?-Iysine acylase activity compared with those of the other microorganisms ever tested, cultural conditions for the production of this enzyme were investigated.

As a result, it was confirmed that simple medium containing 1% peptone, 5% glucose and some inorganic salts is most suitable for the enzyme production and that much more ?-Iysine acylase is produced by shaken culture or submerged culture in jar fermentor than by stationary culture. α-Amino acylase activity in this organism was also studied.     

Regulation of α-aminoadipyl-cysteinyl-valine,isopenicillin N synthetase,isopenicillin N isomerase and deacetoxycephalosporin C synthetase by nitrogen sources in Streptomyces lactamdurans

Summary Ammonium and asparagine produced a concentration-dependent reduction of cephamycin C biosynthesis by Streptomyces lactamdurans. Addition of ammonium salts at 1 mM concentration reduced cephamycin biosynthesis by resting cells of S. lactamdurans, whereas concentrations of asparagine above 10 mM were required to get the same effect. High ammonium concentrations decreased glutamine synthetase activity in cell extracts of S. lactamdurans in parallel to the reduction of antibiotic biosynthesis. Ammonium supplementation decreased the pool of glutamic acid and glutamine whereas the intracellular content of ammonium, alanine, and phosphoserine increased significantly. The pool of the tripeptide (l--aminoadipyl)-l-cysteinyl-d-valine, an intermediate in cephamycin biosynthesis, was greatly reduced in ammonium-supplemented cultures. Isopenicillin N synthetase, that converts the tripeptide (l--aminoadipyl)-l-cysteinyl-d-valine to isopenicillin N, isopenicillin N isomerase (that isomerises isopenicillin N to penicillin N) and deacetoxycephalosporin C synthetase (converting penicillin N into deacetoxycephalosporin C) were also reduced in ammonium-supplemented cultures. However, the activities of these enzymes were not inhibited in vitro by 40 mM ammonium, suggesting that the enzymes were repressed but not inhibited by ammonium in vivo.     

Recognition of tRNAGln by Helicobacter pylori GluRS2—a tRNAGln-specific glutamyl-tRNA synthetase

Accurate aminoacylation of tRNAs by the aminoacyl-tRNA synthetases (aaRSs) plays a critical role in protein translation. However, some of the aaRSs are missing in many microorganisms. Helicobacter pylori does not have a glutaminyl-tRNA synthetase (GlnRS) but has two divergent glutamyl-tRNA synthetases: GluRS1 and GluRS2. Like a canonical GluRS, GluRS1 aminoacylates tRNA^Glu1 and tRNA^Glu2. In contrast, GluRS2 only misacylates tRNA^Gln to form Glu-tRNA^Gln. It is not clear how GluRS2 achieves specific recognition of tRNA^Gln while rejecting the two H. pylori tRNA^Glu isoacceptors. Here, we show that GluRS2 recognizes major identity elements clustered in the tRNA^Gln acceptor stem. Mutations in the tRNA anticodon or at the discriminator base had little to no impact on enzyme specificity and activity.     

A dynamic checkpoint in oxidative lesion discrimination by formamidopyrimidine–DNA glycosylase

In contrast to proteins recognizing small-molecule ligands, DNA-dependent enzymes cannot rely solely on interactions in the substrate-binding centre to achieve their exquisite specificity. It is widely believed that substrate recognition by such enzymes involves a series of conformational changes in the enzyme–DNA complex with sequential gates favoring cognate DNA and rejecting nonsubstrates. However, direct evidence for such mechanism is limited to a few systems. We report that discrimination between the oxidative DNA lesion, 8-oxoguanine (oxoG) and its normal counterpart, guanine, by the repair enzyme, formamidopyrimidine-DNA glycosylase (Fpg), likely involves multiple gates. Fpg uses an aromatic wedge to open the Watson–Crick base pair and everts the lesion into its active site. We used molecular dynamics simulations to explore the eversion free energy landscapes of oxoG and G by Fpg, focusing on structural and energetic details of oxoG recognition. The resulting energy profiles, supported by biochemical analysis of site-directed mutants disturbing the interactions along the proposed path, show that Fpg selectively facilitates eversion of oxoG by stabilizing several intermediate states, helping the rapidly sliding enzyme avoid full extrusion of every encountered base for interrogation. Lesion recognition through multiple gating intermediates may be a common theme in DNA repair enzymes.     

Predators or prey? Spatio-temporal discrimination of human-derived risk by brown bears

Prey usually adjust anti-predator behavior to subtle variations in perceived risk. However, it is not clear whether adult large carnivores that are virtually free of natural predation adjust their behavior to subtle variations in human-derived risk, even when living in human-dominated landscapes. As a model, we studied resting-site selection by a large carnivore, the brown bear (Ursus arctos), under different spatial and temporal levels of human activity. We quantified horizontal and canopy cover at 440 bear beds and 439 random sites at different distances from human settlements, seasons, and times of the day. We hypothesized that beds would be more concealed than random sites and that beds would be more concealed in relation to human-derived risk. Although human densities in Scandinavia are the lowest within bear ranges in Western Europe, we found an effect of human activity; bears chose beds with higher horizontal and canopy cover during the day (0700?C1900?hours), especially when resting closer to human settlements, than at night (2200?C0600?hours). In summer/fall (the berry season), with more intensive and dispersed human activity, including hunting, bears rested further from human settlements during the day than in spring (pre-berry season). Additionally, day beds in the summer/fall were the most concealed. Large carnivores often avoid humans at a landscape scale, but total avoidance in human-dominated areas is not possible. Apparently, bears adjust their behavior to avoid human encounters, which resembles the way prey avoid their predators. Bears responded to fine-scale variations in human-derived risk, both on a seasonal and a daily basis.     

β-Glucan synthetase II activity upon callose formation in the flower ofArabidopsis thaliana

Callose formation was observed in the pollens during flower development and pollen tube grown in the pistil ofA. thaliana. The accumulation of callose occurred in the tetrad in the flower bud and pollen tube. Therefore, the activity of β-glucan synthetase II (GS II), which is responsible for synthesizing the callose, was measured in the flowers on the same developmental stages. The enzyme activity was increased by about 10% while the level of callose contents was increased by about 70% in tetrads. Then, callose accumulation was increased during pollen tube growth by about 30% higher than the other stages and enzyme activity was detected, 30% more too. These results suggest that callose plays an important role in the growth of pollen and pollen tube by increasing GS II activity.     

Increasing glutathione formation by functional expression of the γ-glutamylcysteine synthetase gene in Saccharomyces cerevisiae

A recombinant plasmid, pGMF, containing a gamma-glutamylcysteine synthetase gene (GSH-I) from Saccharomyces cerevisiae, was constructed with a copper-resistance gene as the selection marker and was introduced into S. cerevisiae YSF-31. The glutathione content of the recombinant strain was 1.5-fold (13.1 mg g dry cells(-1)) of that in the host strain.     

The tRNA synthetase paralog PoxA modifies elongation factor-P with (R)-β-lysine

The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with α-lysine at low efficiency. Cell-free extracts containing non-α-lysine substrates of PoxA modified EF-P with a change in mass consistent with addition of β-lysine, a substrate also predicted by genomic analyses. EF-P was efficiently functionally modified with (R)-β-lysine but not (S)-β-lysine or genetically encoded α-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases.     

Visual pattern discrimination by population retinal ganglion cells’ activities during natural movie stimulation

In the visual system, neurons often fire in synchrony, and it is believed that synchronous activities of group neurons are more efficient than single cell response in transmitting neural signals to down-stream neurons. However, whether dynamic natural stimuli are encoded by dynamic spatiotemporal firing patterns of synchronous group neurons still needs to be investigated. In this paper we recorded the activities of population ganglion cells in bullfrog retina in response to time-varying natural images (natural scene movie) using multi-electrode arrays. In response to some different brief section pairs of the movie, synchronous groups of retinal ganglion cells (RGCs) fired with similar but different spike events. We attempted to discriminate the movie sections based on temporal firing patterns of single cells and spatiotemporal firing patterns of the synchronous groups of RGCs characterized by a measurement of subsequence distribution discrepancy. The discrimination performance was assessed by a classification method based on Support Vector Machines. Our results show that different movie sections of the natural movie elicited reliable dynamic spatiotemporal activity patterns of the synchronous RGCs, which are more efficient in discriminating different movie sections than the temporal patterns of the single cells’ spike events. These results suggest that, during natural vision, the down-stream neurons may decode the visual information from the dynamic spatiotemporal patterns of the synchronous group of RGCs’ activities.     

Nα-Ipteroyltetra (γ-Glutamyl)]-Lysine as a Ligand for the Purification of Thymidylate Synthetase by Affinity Chromatography

N^α[Pteroyltetra (γ-glutamyl)]-lysine Sepharose was synthesized and shown to be a stable, high capacity affinity matrix capable of bringing about the purification of Lactobacillus casei thymidylate synthetase to maximum specific activity from crude extracts in high yield. Under conditions optimal for binding of thymidylate synthetase, dihydrofolate reductase was not bound.     

Does ornithine stimulate carbamoylphosphate synthetase?

The effect of ornithine on carbamoylphosphate formation of rat liver mitochondria treated with Triton X 100 was studied. The rate of carbamoylphosphate accumulation and citrulline formation depended on the ATP-, Pi-, N-acetylglutamate and protein concentration. At optimal conditions the rate of citrulline formation was at least 1.5-fold higher than the rate at which carbamoylphosphate accumulated (ornithine absent). A significant correlation between the amount of carbamoylphosphate formed and the citrulline/carbamoylphosphate ratio (ornithine effect) was found. In mitochondria the presence of a carbamoylphosphate degrading enzyme could be demonstrated. This enzyme may be one factor for the differences in the rate of carbamoylphosphate accumulation and the rate of citrulline synthesis.     

γ-glutamylcysteine synthetase from Plasmodium berghei

γ-glutamylcysteine synthetase (l-glutamate-l-cysteine ligase, γ-GCS, EC 6.3.2.2.), the rate limiting enzyme in glutathione biosynthetic pathway has been analysed in the asexual erythrocytic stages of rodent malaria parasite, Plasmodium berghei and its host erythrocytes. Cell-free parasite isolated by saponin lysis contained about 2 and 8 times higher activity of γ-GCS compared to P. berghei-infected and normal mice erythrocytes respectively. Subcellular fractionation revealed that the enzyme was mainly confined to the cytosolic part of the parasite. γ-GCS from P. berghei was purified employing ammonium sulphate precipitation, Sephadex G-200 gel filtration and anionic exchange chromatography on DEAE-cellulose. There was 51.6 fold purification of enzyme and its specific activity was 39.5 U/mg. SDS-PAGE showed P. berghei γ-GCS as a heterodimer dissociating into two non-identical sub-units of 66 kDa and 57 kDa. The enzyme was observed as white band of activity on native polyacrylamide gel stained for specific γ-GCS activity. Km values for l-Cys, ATP and l-Glu were 0.53 mM, 0.92 mM and 0.75 mM, respectively. The inhibition of γ-GCS activity by glutathione was found to be competitive with respect to glutamate (Ki = 1.53 mM) and non competitive to ATP and cysteine. Antimalarial drugs did not show any significant effect on parasite γ-GCS. Parasite enzyme induced humoral response in mice demonstrated by ELISA, IFA and immunoblotting and exhibited partial protection against P. berghei infection suggesting a significant role of P. berghei γ-GCS in malaria control.