Crystal structure of nitrile hydratase from a thermophilic Bacillus smithii

The crystal structure of the nitrile hydratase (NHase) from Bacillus smithii SC-J05-1 was determined. Our analysis of the structure shows that some residues that seem to be responsible for substrate recognition are different from those of other NHases. In particular, the Phe52 in the beta subunit of NHase from B. smithii covers the metal center partially like a small lid and narrows the active site cleft. It is well known that the NHase from B. smithii especially prefers aliphatic nitriles for its substrate rather than aromatic ones, and we can now infer that the Phe52 residue may play a key role in the substrate specificity for this enzyme. This finding leads us to suggest that substitution of these residues may alter the substrate specificity of the enzyme.     

Electrocardiograms Corresponding to the Development of Myocardial Infarction in Anesthetized WHHLMI Rabbits (Oryctolagus cuniculus), an Animal Model for Familial Hypercholesterolemia

The aim of this study was to determine whether features indicative of myocardial ischemia occur in the electrocardiograms (ECG) in myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits, an animal model for human familial hypercholesterolemia. ECG were recorded in 110 anesthetized WHHLMI rabbits (age, 10 to 39 mo) by using unipolar and bipolar limb leads with or without chest leads. We noted the following electrocardiographic changes: T wave inversion (37.4%), ST segment depression (31.8%), deep Q wave (16.3%), reduced R wave amplitude (7.3%), ST segment elevation (2.7%), and high T wave (1.8%). These ECG changes resembled those in human patients with coronary heart disease. Histopathologic examination revealed that the left ventricular wall showed acute myocardial lesions, including loss of cross-striations, vacuolar degeneration, coagulation necrosis of cardiac myocytes, and edema between myofibrils, in addition to chronic myocardial lesions such as myocardial fibrosis. The coronary arteries that caused these ECG changes were severely stenosed due to atherosclerotic lesions. Ischemic ECG changes corresponded to the locations of the myocardial lesions. Normal ECG waveforms were similar between WHHLMI rabbits and humans, in contrast to the large differences between rabbits and mice or rats. In conclusion, ischemic ECG changes in WHHLMI rabbits reflect the location of myocardial lesions, making this model useful for studying coronary heart disease.Abbreviations: CHD, coronary heart disease; ECG, electrocardiogram; WHHL, Watanabe heritable hyperlipidemic; WHHLMI, myocardial infarction-prone Watanabe heritable hyperlipidemicCoronary heart disease (CHD) is prevalent in developed countries, including the United States.^16,24 Although potent compounds (for example, statins to inhibit cholesterol synthesis) have been developed to reduce the public health burden of this disease, CHD remains a leading cause of death, and further efforts are needed to reduce associated morbidity and mortality.²⁵ In evaluating the therapeutic effects of CHD interventions, the electrocardiogram (ECG) is an essential tool for examining myocardial function.³⁹In humans, various ischemic ECG changes occur in association with myocardial ischemia and infarction, such as high T wave, ST segment elevation, emergence of the deep Q wave, reduction of R wave amplitude, resolution of ST segment elevation, and T wave inversion.^21,39 In addition, ST segment depression is a typical change observed with subendocardial ischemia.^2,7In the study of myocardial ischemia, animal models that show ECG waveforms comparable to those of human patients with CHD play an important role. This similarity is important not only for assessing the effects of agents for the treatment of CHD but also for assessing adverse effects of newly developed agents on cardiac function. Although ECG have been used to study myocardial ischemia in several species including pigs, dogs, rabbits, rats, and mice,^{3,9,10,14,18,23} most of these studies used coronary ligation models. These models do not fully reflect the events that occur during myocardial ischemia caused by atherosclerotic coronary stenosis, which is seen typically in patients with CHD.The Watanabe heritable hyperlipidemic (WHHL) rabbit⁴⁰ and the myocardial infarction-prone WHHL (WHHLMI) rabbit³³ are animal models for the study of human myocardial ischemia. WHHLMI rabbits spontaneously develop hypercholesterolemia due to a deficiency of receptors for low-density lipoproteins and manifest severe coronary atherosclerosis and myocardial infarction. Importantly, lipoprotein metabolism in WHHL and WHHLMI rabbits resembles that in humans.^28,30 Using these advantages of the WHHL and WHHLMI models, we and others have been studying the effects of hypocholesterolemic and antiatherosclerotic agents on coronary atherosclerosis.^29,32,34 However, ECG were not examined in these studies. Because the rabbit heart is electrophysiologically similar to the human heart,^27,38 using ECG to monitor myocardial function in the WHHLMI rabbit may be valuable.In the present study, we examined whether ECG changes observed in WHHLMI rabbits reflect myocardial ischemia and whether those changes correspond to ECG features in human patients with CHD.     

Two c-type cytochromes, NirM and NirC, encoded in the nir gene cluster of Pseudomonas aeruginosa act as electron donors for nitrite reductase.

Three c-type cytochromes, NirM, NirC, and NirN, are encoded in the nirSMCFDLGHJEN gene cluster for cytochrome cd(1)-type nitrite reductase (NIR) of Pseudomonas aeruginosa. nirS is the structural gene for NIR. NirM (cytochrome c(551)) is reported to be a physiological electron donor for nitrite reductase. The respective functions of NirC and NirN have remained unclear. In this study, we produced recombinant NirC and NirN in P. aeruginosa, and purified them from the periplasmic fraction. N-terminal amino acid sequences of the purified proteins showed that the N-terminal 31 and 18 residues of NirC and NirN precursors were cleaved, respectively, indicating that cleaved peptides act as signals for membrane translocation. In addition, the ability of NirC for electron donation to nitrite reductase was investigated. NirC, as well as NirM, was able to mediate the electron donation from the membrane electron pathway to NIR, suggesting that the structural gene for NIR is followed by the genes for two electron donors for NIR.     

A novel family of hemicellulolytic α-glucuronidase

Investigation of the xylanolytic enzyme system of the xylose-fermenting yeast Pichia stipitis resulted in the discovery of an extracellular α-glucuronidase efficiently debranching hardwood glucuronoxylan. This activity is not exhibited by more extensively investigated α-glucuronidases of glycoside hydrolase (GH) family 67, operating on substrates in which the uronic acid is linked to the non-reducing xylopyranosyl residues of main chain fragments. The N-terminus of the purified enzyme corresponded exactly to the P. stipitis gene ABN67901 coding for a protein of unknown function. BLAST search revealed the presence of similar genes in genomes of other microorganisms. These results lead to the emergence of a new family of α-glucuronidases.     

Degradation of carbohydrate moieties of arabinogalactan-proteins by glycoside hydrolases from Neurospora crassa

Arabinogalactan-proteins (AGPs) are a family of plant proteoglycans having large carbohydrate moieties attached to core-proteins. The carbohydrate moieties of AGPs commonly have β-(1→3)(1→6)-galactan as the backbone, to which other auxiliary sugars such as l-Ara and GlcA are attached. For the present study, an α-l-arabinofuranosidase belonging to glycoside hydrolase family (GHF) 54, NcAraf1, and an endo-β-(1→6)-galactanase of GHF 5, Nc6GAL, were identified in Neurospora crassa. Recombinant NcAraf1 (rNcAraf1) expressed in Pichia pastoris hydrolyzed radish AGPs as well as arabinan and arabinoxylan, showing relatively broad substrate specificity toward polysaccharides containing α-l-arabinofuranosyl residues. Recombinant Nc6GAL (rNc6GAL) expressed in P. pastoris specifically acted on β-(1→6)-galactosyl residues. Whereas AGP from radish roots was hardly hydrolyzed by rNc6GAL alone, β-(1→6)-galactan side chains were reduced to one or two galactan residues by a combination of rNcAraf1 and rNc6GAL. These results suggest that the carbohydrate moieties of AGPs are degraded by the concerted action of NcAraf1 and Nc6GAL secreted from N. crassa.     

gammaepsilon Sub-complex of thermophilic ATP synthase has the ability to bind ATP

The isolated epsilon subunit of F(1)-ATPase from thermophilic Bacillus PS3 (TF(1)) binds ATP [Y. Kato-Yamada, M. Yoshida, J. Biol. Chem. 278 (2003) 36013]. The obvious question is whether the ATP binding concern with the regulation of ATP synthase activity or not. If so, the epsilon subunit even in the ATP synthase complex should have the ability to bind ATP. To check if the ATP binding to the epsilon subunit within the ATP synthase complex may occur, the gammaepsilon sub-complex of TF(1) was prepared and ATP binding was examined. The results clearly showed that the gammaepsilon sub-complex can bind ATP.     

Correction to: Herding mechanisms to maintain the cohesion of a harem group: two interaction phases during herding

Journal of Ethology - The article Herding mechanisms to maintain the cohesion of a harem group.     

Mulberry leaf powder prevents atherosclerosis in apolipoprotein E-deficient mice

Mulberry is commonly used to feed silkworms. Here we examined whether a dietary intake of mulberry leaf (ML) could affect atherogenesis in vivo and in vitro. Apolipoprotein E-deficient mice were fed either normal chow (control group) or a diet containing 1% ML powder (ML group) from 6 weeks of age. The mice were sacrificed after 12 weeks. The susceptibility of plasma lipoprotein to oxidation was assessed using diene formation. A significant increase in the lag time of lipoprotein oxidation was detected in the ML group compared with the control group. Furthermore, the ML group showed a 40% reduction in atherosclerotic lesion size in the aortae compared with the control. We also examined the direct anti-oxidative activity of ML in vitro. Aqueous extract of ML had a strong scavenging effect on 1,1-diphenyl-2-picrylhydrazyl and inhibited lipoprotein oxidation. These results confirm that ML contains anti-oxidative substances that might help prevent atherosclerosis.