Purification and characterization of porcine skeletal muscle aminopeptidase T, a novel metallopeptidase homologous to leukotriene A4 hydrolase

A novel aminopeptidase, Aminopeptidase T (APase T), was purified from porcine skeletal muscle following successive column chromatography: twice on DEAE-cellulose, hydroxyapatite, and Sephacryl S-200 HR using Leu-β-naphthylamide (LeuNap) as a substrate. The molecular mass of the enzyme was 69 kDa on SDS-PAGE. The optimum pH towards LeuNap of the enzyme was about 7. The enzyme activity was strongly inhibited by bestatin and was negatively affected by ethylenediaminetetraacetic acid (EDTA). Chlorine-activated APase T liberated Leu, Ala, Met, Pro, and Arg from Nap derivatives. The APase T gene consisted of an ORF of 1,836 bp encoding a protein of 611 amino acid residues. The APase T was highly homologous to bovine, human, and mouse Leukotriene A(4) hydrolase (LTA(4)H), a bifunctional enzyme which exhibits APase and epoxide hydrolase activity.     

Venous thromboembolism after total joint arthroplasty: results from a Japanese multicenter cohort study

Introduction

Real-world evidence of the effectiveness of pharmacological thromboprophylaxis for venous thromboembolism (VTE) is limited. Our objective was to assess the effectiveness and safety of thromboprophylactic regimens in Japanese patients undergoing joint replacement in a real-world setting.

Method

Overall, 1,294 patients (1,073 females and 221 males) who underwent total knee arthroplasty (TKA) and 868 patients (740 females and 128 males) who underwent total hip arthroplasty (THA) in 34 Japanese national hospital organization (NHO) hospitals were enrolled. The primary efficacy outcome was the incidence of deep vein thrombosis (DVT) detected by mandatory bilateral ultrasonography up to post-operative day (POD) 10 and pulmonary embolism (PE) up to POD28. The main safety outcomes were bleeding (major or minor) and death from any cause up to POD28.

Results

Patients undergoing TKA (n = 1,294) received fondaparinux (n = 360), enoxaparin (n = 223), unfractionated heparin (n = 72), anti-platelet agents (n = 45), or no medication (n = 594). Patients undergoing THA (n = 868) received fondaparinux (n = 261), enoxaparin (n = 148), unfractionated heparin (n = 32), anti-platelet agents (n = 44), or no medication (n = 383). The incidence rates of sonographically diagnosed DVTs up to POD10 were 24.3% in patients undergoing TKA and 12.6% in patients undergoing THA, and the incidence rates of major bleeding up to POD28 were 1.2% and 2.3%, respectively. Neither fatal bleeding nor fatal pulmonary embolism occurred. Significant risk factors for postoperative VTE identified by multivariate analysis included gender (female) in both TKA and THA groups and use of a foot pump in the TKA group. Only prophylaxis with fondaparinux reduced the occurrence of VTE significantly in both groups. Propensity score matching analysis (fondaparinux versus enoxaparin) showed that the incidence of DVT was lower (relative risk 0.70, 95% confidence interval (CI) 0.58 to 0.85, P = 0.002 in TKA and relative risk 0.73, 95% CI 0.53 to 0.99, P = 0.134 in THA) but that the incidence of major bleeding was higher in the fondaparinux than in the enoxaparin group (3.4% versus 0.5%, P = 0.062 in TKA and 4.9% versus 0%, P = 0.022 in THA).

Conclusions

These findings indicate that prophylaxis with fondaparinux, not enoxaparin, reduces the risk of DVT but increases bleeding tendency in patients undergoing TKA and THA.

Trial registration

University Hospital Medical Information Network Clinical Trials Registry: UMIN000001366. Registered 11 September 2008.     

Variation of the oxidation state of verdoheme in the heme oxygenase reaction

Heme oxygenase (HO) converts hemin to biliverdin, CO, and iron applying molecular oxygen and electrons. During successive HO reactions, two intermediates, α-hydroxyhemin and verdoheme, have been generated. Here, oxidation state of the verdoheme-HO complexes is controversial. To clarify this, the heme conversion by soybean and rat HO isoform-1 (GmHO-1 and rHO-1, respectively) was compared both under physiological conditions, with oxygen and NADPH coupled with ferredoxin reductase/ferredoxin for GmHO-1 or with cytochrome P450 reductase for rHO-1, and under a non-physiological condition with hydrogen peroxide. EPR measurements on the hemin-GmHO-1 reaction with oxygen detected a low-spin ferric intermediate, which was undetectable in the rHO-1 reaction, suggesting the verdoheme in the six-coordinate ferric state in GmHO-1. Optical absorption measurements on this reaction indicated that the heme degradation was extremely retarded at verdoheme though this reaction was not inhibited under high-CO concentrations, unlike the rHO-1 reaction. On the contrary, the Gm and rHO-1 reactions with hydrogen peroxide both provided ferric low-spin intermediates though their yields were different. The optical absorption spectra suggested that the ferric and ferrous verdoheme coexisted in reaction mixtures and were slowly converted to the ferric biliverdin complex. Consequently, in the physiological oxygen reactions, the verdoheme is found to be stabilized in the ferric state in GmHO-1 probably guided by protein distal residues and in the ferrous state in rHO-1, whereas in the hydrogen peroxide reactions, hydrogen peroxide or hydroxide coordination stabilizes the ferric state of verdoheme in both HOs.     

On the role of the first transmembrane domain in cation permeability and flux of the ATP-gated P2X2 receptor

P2X receptors are a family of seven ligand-gated ion channels (P2X1-P2X7) that open in the presence of ATP. We used alanine-scanning mutagenesis and patch clamp photometry to study the role of the first transmembrane domain of the rat P2X2 receptor in cation permeability and flux. Three alanine-substituted mutants did not respond to ATP, and 19 of the 22 functional receptors resembled the wild-type receptor with regard to the fraction of the total ATP-gated current carried by calcium or the permeability of calcium relative to cesium. The remaining three mutants showed modest changes in calcium dynamics. Two of these occurred at sites (Gly30 and Phe44) that are unlikely to interact with permeating cations in a meaningful way. The third was a conserved tyrosine (Tyr43) that may form an inter-pore binding site for calcium. The data suggest that, with the possible exception of Tyr43, the first transmembrane domain contributes little to the permeation properties of the P2X2 receptor.     

Exercise therapy for prevention of falls in people with Parkinson's disease: A protocol for a randomised controlled trial and economic evaluation

Background

People with Parkinson's disease are twice as likely to be recurrent fallers compared to other older people. As these falls have devastating consequences, there is an urgent need to identify and test innovative interventions with the potential to reduce falls in people with Parkinson's disease. The main objective of this randomised controlled trial is to determine whether fall rates can be reduced in people with Parkinson's disease using exercise targeting three potentially remediable risk factors for falls (reduced balance, reduced leg muscle strength and freezing of gait). In addition we will establish the cost effectiveness of the exercise program from the health provider's perspective.

Methods/Design

230 community-dwelling participants with idiopathic Parkinson's disease will be recruited. Eligible participants will also have a history of falls or be identified as being at risk of falls on assessment. Participants will be randomly allocated to a usual-care control group or an intervention group which will undertake weight-bearing balance and strengthening exercises and use cueing strategies to address freezing of gait. The intervention group will choose between the home-based or support group-based mode of the program. Participants in both groups will receive standardized falls prevention advice. The primary outcome measure will be fall rates. Participants will record falls and medical interventions in a diary for the duration of the 6-month intervention period. Secondary measures include the Parkinson's Disease Falls Risk Score, maximal leg muscle strength, standing balance, the Short Physical Performance Battery, freezing of gait, health and well being, habitual physical activity and positive and negative affect schedule.

Discussion

No adequately powered studies have investigated exercise interventions aimed at reducing falls in people with Parkinson's disease. This trial will determine the effectiveness of the exercise intervention in reducing falls and its cost effectiveness. This pragmatic program, if found to be effective, has the potential to be implemented within existing community services.

Trial registration

The protocol for this study is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12608000303347).     

Cytochemical investigation of genic male-sterility in Chinese cabbage

A genic male sterile Chinese cabbage, Brassica campestris L. ssp. chinensis Makino, was examined using cytological and cytochemical methods to characterize the process of pollen abortion in this plant. Thick sections of both fertile and sterile anthers at different developmental stages were stained using Toluidine Blue O, Periodic Acid-Schiff’s (PAS) reaction and Sudan Black B to detect cytochemical changes that may occur in the distribution of insoluble polysaccharide and lipid storage bodies. Pollen abortion in sterile anthers occurs at an early stage of microspore development. During early microspore development, reductions in the number of starch grains in the connective tissue of fertile anthers coincide with the accumulation of starch grains in cells of the anther wall. In the late microspore stage, a large vacuole forms in the microspore, and tapetal cells synthesize and accumulate lipid droplets. The cellular organization of tapetal cells in sterile anthers appears similar to that in fertile anthers, except for the absence of lipid droplets in cells of sterile anthers and diffusely labeled tapetal polysaccharides, suggesting defects in nutrient storage. Supported by National Natural Science Foundation of CHINA (30170060)     

AMP and IMP dissociate actomyosin into actin and myosin

We investigated to determine why heating of squid muscle at 60 degrees C induced the liberation of actin from myofibrils. When a mixture of a myofibrillar fraction and a low-molecular sarcoplasmic fraction prepared from squid muscle was heated at 60 degrees C, actin liberation occurred. When a myofibrillar fraction was heated with ATP, AMP, or IMP, actin liberation occurred. Hence, AMP is perhaps one of the factors causing actin liberation in postmortem squid muscle. It was found that AMP and IMP reversibly dissociated actomyosin of chicken, bovine, and porcine skeletal muscles into actin and myosin on incubation at 0 degrees C at pH 7.2 in 0.2 M KCl. These results led us to conclude that AMP and IMP were the most responsible factors causing actin liberation from myofibrils in the heated muscle and causing reversible dissociation of actomyosin on storage of skeletal muscle at a low temperature. Hence, AMP and IMP are possible factors causing the resolution of rigor mortis in muscles.