Structural and mechanistic insights into homocysteine degradation by a mutant of methionine γ‐lyase based on substrate‐assisted catalysis

Methionine γ‐lyse (MGL) catalyzes the α, γ‐elimination of l ‐methionine and its derivatives as well as the α, β‐elimination of l ‐cysteine and its derivatives to produce α‐keto acids, volatile thiols, and ammonia. The reaction mechanism of MGL has been characterized by enzymological studies using several site‐directed mutants. The Pseudomonas putida MGL C116H mutant showed drastically reduced degradation activity toward methionine while retaining activity toward homocysteine. To understand the underlying mechanism and to discern the subtle differences between these substrates, we analyzed the crystal structures of the reaction intermediates. The complex formed between the C116H mutant and methionine demonstrated that a loop structure (Ala51–Asn64) in the adjacent subunit of the catalytic dimer cannot approach the cofactor pyridoxal 5′‐phosphate (PLP) because His116 disrupts the interaction of Asp241 with Lys240, and the liberated side chain of Lys240 causes steric hindrance with this loop. Conversely, in the complex formed between C116H mutant and homocysteine, the thiol moiety of the substrate conjugated with PLP offsets the imidazole ring of His116 via a water molecule, disrupting the interaction of His116 and Asp241 and restoring the interaction of Asp241 with Lys240. These structural data suggest that the Cys116 to His mutation renders the enzyme inactive toward the original substrate, but activity is restored when the substrate is homocysteine due to substrate‐assisted catalysis.     

High‐Sensitivity C‐Reactive Protein in Japanese Patients with Type 2 Diabetes

Objective: To assess the relationship between high‐sensitivity (HS) C‐reactive protein (CRP) and metabolic syndrome (MetS) or atherosclerosis and to assess effects of strict metabolic control on the degree of inflammation and MetS in patients with type 2 diabetes. Research Methods and Procedures: Four hundred thirteen patients with diabetes were enrolled in the cross‐sectional study. Of these 413 patients, 161 patients were further admitted for 2.4 ± 0.4 weeks (mean ± SD) to investigate the change in HS‐CRP or other parameters under strict metabolic control. Results: Log‐transformed HS‐CRP value (log HS‐CRP) was strongly correlated with BMI (r = 0.448, p < 0.01). Log HS‐CRP was also correlated with the presence of MetS or each component of MetS. Furthermore, a positive significant trend in HS‐CRP levels was shown with an increasing number of MetS components (p < 0.05). Log HS‐CRP showed a significant positive correlation with carotid artery intima‐media thickness (IMT) (r = 0.152, p < 0.01). In multiple step‐wise regression analysis, BMI, hemoglobin A_1c, right IMT, duration of diabetes, and triglyceride were selected as explanatory variables for log HS‐CRP (R² = 0.412). Under strict metabolic control, HS‐CRP was significantly (p < 0.01) lower, together with lower levels of other markers for MetS. The change in HS‐CRP was significantly correlated with the change in BMI (r = 0.161, p = 0.04). Discussion: In subjects with type 2 diabetes, HS‐CRP levels are related to MetS and subclinical atherosclerosis. Strict weight management and metabolic control were associated with a reduction in HS‐CRP levels, and changes in HS‐CRP were related to changes in weight, supporting the hypothesis that lifestyle modification reduces inflammation and the risk of CHD.     

An Approach to Further Enhance the Cellular Productivity of Exogenous Protein Hyper-producing Chinese Hamster Ovary (CHO) Cells

The cell line D29, which was easily and rapidly established by the promoter-activated production and glutamine synthetase hybrid system, secreted recombinant human interleukin-6 (hIL-6) at a productivity rate of 39.5 μg 10⁻⁶ cells day⁻¹, one of the highest reported levels worldwide. The productivity rate was about 130-fold higher than that of the cell line A7, which was established without both promoter activation and gene amplification. Although D29 cells had a high copy number and high mRNA level of the hIL-6 gene as well as a high secretion rate of hIL-6, large amounts of intracellular hIL-6 protein accumulated in D29 cells compared to A7 cells. Northern blotting analysis showed no change in the GRP78/BiP expression level in D29 cells. In contrast, an electrophoresis mobility shift assay revealed strong activation of NF-κB in D29 cells. These results suggest that large amounts of hIL-6 translated from large amounts of hIL-6 mRNA cause excess accumulation of intact hIL-6 in the endoplasmic reticulum (ER), and that subsequent negative feedback signals via the ER overload response inhibit hIL-6 protein secretion. To enhance the hIL-6 productivity rate of D29 cells by releasing the negative feedback signals, the effect of pyrrolidinedithiocarbamate, an inhibitor of NF-κB activation, was examined. Suppression of NF-κB activation in D29 cells produced a 25% augmentation of the hIL-6 productivity rate. Therefore, in highly productive cells like D29 cells, the release of negative feedback signals could increase the total amount of recombinant protein secretion.     

Origins of mouse inbred strains deduced from whole-genome scanning by polymorphic microsatellite loci

Microsatellite loci are uniformly distributed at approximately 100-kbp intervals on all chromosomes except the chromosome Y, and genetic information about more than 9000 loci and high-throughput polymorphism analysis are now available. Taking advantage of these properties, we carried out whole-genome scanning using eight common inbred strains (CIS) of laboratory mice, including A/J, C57BL/6J, CBA/J, DBA/2J, SM/J, SWR/J, NC/Nga, and 129/SvJ, and eight wild-derived inbred strains (WIS), BGL2/Ms, CAST/Ei, JF1/Ms, MSM/Ms, NJL/Ms, PGN2/Ms, SK/CamEi, and SWN/Ms. We selected and located 1226 informative loci at 1.2-cM average intervals on all of the chromosomes of the 16 strains and compared the polymorphisms of the eight CIS with those from the eight WIS as subspecies representatives. More than 50% of the loci can be identified as WIS (therefore, subspecies-specific) alleles in the CIS genomes. We also discovered that the CIS chromosomes form a mosaic structure with an average ratio of domesticus to non-domesticus alleles of 3:1. Furthermore, the domesticus alleles were present much more frequently on the CIS chromosome X than on their autosomes, suggesting that successive backcrossing of non-domesticus stocks to domesticus stocks had been undergone at the beginning of CIS history.     

An acidic and thermostable carboxymethyl cellulase from the yeast Cryptococcus sp. S-2: purification, characterization and improvement of its recombinant enzyme production by high cell-density fermentation of Pichia pastoris

The extracellular carboxymethyl cellulase (CSCMCase) from the yeast, Cryptococcus sp. S-2, was produced when grown on cellobiose. It was purified to homogeneity from the supernatant by ultrafiltration, DEAE-5PW anion exchange column and TSK-Gel G3000SW gel filtration. The purified enzyme was monomeric protein with molecular mass of approximately 34 kDa. The optimum temperature and pH for the action of the enzyme were at 40–50 °C and 3.5, respectively. It was stable at pH range of 5.5–7.5 and retained approximately 50% of its maximum activity after incubating at 90 °C for 1 h. Moreover, it could able to hydrolyze carboxymethyl cellulose sodium salt higher than insoluble cellulose substrate such as Avicel, SIGMACELL^® and CM cellulose. Due to its action at acidic pH and moderately stable at high temperature, the gene encoding carboxymethyl cellulase (CSCMCase) was isolated and improved the enzyme yield by high cell-density fermentation of Pichia pastoris. The CSCMCase cDNA contains 1023 nucleotides and encodes a 341-amino acid. It was successfully expressed under the control of alcohol oxidase I promoter using methanol induction of P. pastoris fermentation in a 2L ABLE bioreactor. The production of the recombinant carboxymethyl cellulases was higher than that from Cryptococcus sp. S-2 of 657-fold (2.75 and 4.2 × 10⁻³ mg protein L⁻¹, respectively) indicating that the leader sequence of CSCMCase has been recognized and processed as efficiently by P. pastoris. Furthermore, the recombinant enzyme was purified in two-step of ultrafiltration and hydrophobic interaction chromatography which would be much more convenient for large-scale purification for successful industrial application.     

Muscle force per cross-sectional area is inversely related with pennation angle in strength trained athletes

The present study aimed to examine the effect of pennation angle on the force per cross-sectional area for elbow extensor muscles in strength-trained athletes. A total of 52 male bodybuilders (n = 32) and Olympic weightlifters (n = 20) did maximal isometric elbow extension on an isokinetic dynamometer. Muscle cross-sectional area (CSA) and muscle-fiber pennation angle (PA) of the triceps brachii muscles were measured by ultrasonography. Bodybuilders had significantly greater isometric elbow extension force (F), CSA and PA than weightlifters. The ratio of force to CSA (F/CSA) of bodybuilders was significantly lower than that of weightlifters. A significant positive correlation was observed between CSA and PA in both groups (r = 0.832, P < 0.001, and r = 0.682, P < 0.001, for bodybuilders and weightlifters, respectively). The F/CSA was negatively correlated to PA both for bodybuilders (r = -0.408, P < 0.05) and weightlifters (r = -0.465, P < 0.05). Thus present study indicates that the larger pennation angle is associated with the lower force relative to muscle CSA in strength-trained athletes.     

Periodic abdominal pumping supports leg development during metamorphosis in tenebrionid beetle Zophobas atratus

Rhythmic abdominal pumping movements in a pupa of giant mealworm beetle Zophobas atratus caused large hemolymph pressure pulses of approximately 20 mmHg. The abdominal pumping movements were completely blocked by transecting the ventral nerve cord (VNC) between the first and second abdominal ganglia. Transection of the VNC until 2 days after pupation caused a developmental defect of adult legs: morphogenesis of the tibial and tarsal segments was severely retarded, and the segments remained covered with a thick pupal cuticle. The developmental defect was rescued by artificially inducing rhythmic abdominal bending for 3 days after transection of VNC. Blocking of the abdominal pump did not increase the amount of water loss during the pupal period. The transplanted tibial segments lacking active tracheal ventilation could form a thick adult cuticle. The results suggest that abdominal pumping movements during the pupal period support the development of adult legs by facilitating hemolymph circulation.     

Kunitz-type protease inhibitors from acrorhagi of three species of sea anemones

Sea anemones are rich in biologically active polypeptides such as toxins and protease inhibitors. These polypeptides have so far been isolated from whole bodies, tentacles or secreted mucus. Recently, two novel peptide toxins with crab lethality have been isolated from acrorhagi (specialized aggressive organs elaborated by only certain species of sea anemones belonging to the family Actiniidae) of Actinia equina. This prompted us to survey biologically active polypeptides in the acrorhagi of two species of sea anemones, Anthopleura aff. xanthogrammica and Anthopleura fuscoviridis. No potent crab lethality was displayed by the acrorhagial extracts of both species. However, significantly high protease inhibitory activity was instead detected in the acrorhagial extracts of the two species and also in that of A. equina. From the acrorhagi of A. equina, A. aff. xanthogrammica and A. fuscoviridis, one (AEAPI), one (AXAPI) and two (AFAPI-I and AFAPI-III) protease inhibitors were isolated, respectively. The complete amino acid sequences of the four inhibitors were elucidated by N-terminal sequencing and sequencing of the C-terminal peptide fragment produced upon asparaginylendopeptidase digestion. The determined amino acid sequences revealed that all the four inhibitors are new members of the Kunitz-type protease inhibitor family.     

Isolation, purification, characterization and glycan-binding profile of a d-galactoside specific lectin from the marine sponge, Halichondria okadai

A lectin recognizing both Galbeta1-3GlcNAc and Galbeta1-4GlcNAc was purified from the demosponge Halichondria okadai by lactosyl-agarose affinity chromatography. The molecular mass of the lectin was determined to be 30 kDa by SDS-PAGE under reducing and non-reducing conditions and 60 kDa by gel permeation chromatography. The pI value of the lectin was 6.7. It was found to agglutinate trypsinized and glutaraldehyde-fixed rabbit and human erythrocytes in the presence and absence of divalent cations. The hemagglutinating activity by the lectin was inhibited by d-galactose, methyl-d-galactopyranoside, N-acetyl-d-galactosamine, methyl-N-acetyl-d-galactosaminide, lactose, melibiose, and asialofetuin. The K(d) of the lectin against p-nitrophenyl-beta-lactoside was determined to be 2.76x10(-5) M and its glycan-binding profile given by frontal affinity chromatography was shown to be similar to many other known galectins. Partial primary structure analysis of 7 peptides by cleavage with lysyl endopeptidase indicated that one of the peptides showed significant similarity with galectin purified from the sponge Geodia cydonium.     

Speciation of the Paradeontacylix spp. (Sanguinicolidae) of Seriola dumerili. Two new species of the genus Paradeontacylix from the Mediterranean

Two new species of teleost blood fluke belonging to the sanguinicolid genus Paradeontacylix are described from the greater amberjack, Seriola dumerili, i.e. Paradeontacylix ibericus n. sp. from the Iberian Peninsula and Paradeontacylix balearicus n. sp. from the Balearic Islands. P. ibericus n. sp. and P. balearicus n. sp. show morphological similarities with Paradeontacylix kampachi and Paradeontacylix grandispinus respectively, which occur in mixed infection in S. dumerili from Japan. Multivariate analysis of morphometrical data provided statistical evidence for the separation of four species. However, component by component analysis did not show statistically significant differences between P. balearicus and P. grandispinus. Molecular data based on rITS2 and mCO1 gene sequences also supported the separation into four species. Morphological and molecular data were used to examine phylogenetic relationships between Paradeontacylix species from S. dumerili and other species in the genus. The results coincided in revealing two main branches with P. kampachi+P. ibericus and (((P. grandispinus+P. balearicus) Paradeontacylix sanguinicoloides) Paradeontacylix godfreyi). Paradeontacylix odhneri, for which little data are available, was located basal in a separate branch. This is the only species of Paradeontacylix which parasitizes a non-carangid host which might probably explain the separation from the other species. Paired similarities between the Japanese and the Mediterranean species, despite the large geographic distance, could be explained by the speciation of parasite geminate lines before host separation by tectonic events. Consequently, geographic and historical isolation support the morphological and genetic differences leading to the evolution of the new species described here.