Crystallographic dissection of the thermal motion of protein-sugar complex

The crystal structure of turkey egg lysozyme (TEL) complexed with di-N-acetylchitobiose (NAG2) was refined at 1.19 A resolution by the full-matrix least-squares method with anisotropic temperature factors, and its thermal motion was evaluated by the TLS method. The average ESDs of atomic parameters of nonhydrogen atoms were 0.030 A for coordinates and 0.025 A(2) for anisotropic temperature factors. The active site cleft of TEL binds the alpha-anomer of NAG2 in a nonproductive binding mode with its pyranose rings parallel to a beta-sheet. The TEL structure was compared with the re-refined 1.12 A structure of native TEL. The RMS difference for equivalent Calpha atoms was 0.103 A and a relatively large difference was observed in the region of residues 104-125 rather than in the beta-sheet region where NAG2 was bound. In contrast, the temperature factor of the beta-sheet region was significantly decreased by the NAG2 binding. The TLS model that describes the rigid body motion in translation, libration, and screw motion was adopted for the evaluation of the molecular motion of TEL and NAG2, and the TLS parameters were determined by the least-squares fit to U(ij). The contribution of the external motion of TEL was estimated to be 55.8% of the observed temperature factor for the native structure and 45.9% for the NAG2 complex. The internal motion of TEL represented with atomic thermal ellipsoids was very similar between the native and complex structures except the NAG2 binding region. In the structure of NAG2, the rigid body motion dominates the thermal motion. The center of rotation of NAG2, 4.45A far from the center of gravity, is on the nitrogen atom of the acetylamino group that is hydrogen bonded to the main-chain peptide groups of Asn49 and Ala107. The rigid body motion of NAG2 indicates that the acetylamino group is most strongly bound to the active site, and the recognition of this group is a crucial step of the substrate binding.     

Class VI myosin moves processively along actin filaments backward with large steps.

Among a superfamily of myosin, class VI myosin moves actin filaments backwards. Here we show that myosin VI moves processively on actin filaments backwards with large ( approximately 36 nm) steps, nevertheless it has an extremely short neck domain. Myosin V also moves processively with large ( approximately 36 nm) steps and it is believed that myosin V strides along the actin helical repeat with its elongated neck domain that is critical for its processive movement with large steps. Myosin VI having a short neck cannot take this scenario. We found by electron microscopy that myosin VI cooperatively binds to an actin filament at approximately 36 nm intervals in the presence of ATP, raising a hypothesis that the binding of myosin VI evokes "hot spots" on actin filaments that attract myosin heads. Myosin VI may step on these "hot spots" on actin filaments in every helical pitch, thus producing processive movement with 36 nm steps.     

Dietary Zn deficiency does not influence systemic blood pressure and vascular nitric oxide signaling in normotensive rats

Because zinc (Zn) is an important component for cell protection against certain oxygen species, it has been suggested that Zn deficiency impairs the potent oxidant defense capacity, which is constitutively provided in the vascular system. However, the influence of dietary Zn deficiency on systemic blood pressure and vascular system is controversial and unclear. We therefore examine the effect of dietary Zn deficiency on systemic blood pressure, a potent superoxide scavenger, aortic Cu/Zn superoxide dismutase (SOD) activity, a most representative synthase of the endothelium-derived relaxing factor, and aortic endothelial nitric oxide synthase (eNOS) expression. Furthermore, the direct effects of intravenous administration of NOS inhibitor, N ^ω-nitro-l-arginine methyl ester (l-NAME), and a SOD mimetic compound, tempol, in normotensives were tested in Wistar-Kyoto (WKY) rats. A Zn-deficient diet (4 wk) contributed to growth retardation, the decrease in thymus weight, and the lower levels of serum Zn compared with the standard diet group. However, no significant difference in conscious systolic and diastolic blood pressure was found in the Zn-deficiency group. The administration of l-NAME caused an increase in the mean arterial pressure (MAP) levels in the two groups of rats and the involvement of the vasodilator nitric oxide (NO) in the regulation of systemic BP in the normotensive state. On the other hand, administration of the superoxide scavenger, tempol, led to a decrease in MAP levels in the two groups of rats, indicating the participation of the oxygen free radical, superoxide, in the maintenance of the systemic BP in a normotensive state. There were no significant differences between the Zn-deficient diet group and the standard diet group in the normotensive state. eNOS expression and Cu/Zn SOD activity in the aorta were also intact in Zn-deficient normotensive rats. These findings suggest that the 4 wk of Zn deficiency was inadequate to alter systemic blood pressure and focal NO signaling in the normotensive state. Long-term Zn deficiency affects the neuronal, immune, and hematopoietic systems, which contribute to systemic and/or local circulation. However, Zn deficiency alone does not cause hypertension and local vascular dysfunction in the normotensive state.     

Existence of ghrelin-immunopositive and -expressing cells in the proventriculus of the hatching and adult chicken

Ghrelin was isolated from the rat stomach as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) and has been found in the gastrointestinal tract of many vertebrates. Although the sequence and structure of chicken ghrelin has recently been determined, morphological characteristics of ghrelin cells in the chicken gastrointestinal tract are still obscure. In this study, we investigated ghrelin expression and distribution of ghrelin-producing cells in the hatching and adult chicken gastrointestinal tract by RT-PCR, immunohistochemistry and in situ hybridization. Ghrelin mRNA expression was observed mainly in the proventriculus in the hatching chicken and in the proventriculus, pylorus and duodenum of the adult chicken by RT-PCR. Ghrelin-immunopositive (ghrelin-ip) cells in the proventriculus were located at the mucosal layer but not in the myenteric plexus or smooth muscle layer. The number of ghrelin-ip cells in the adult chicken was greater than that in the hatching chicken. Interestingly, in the adult chicken, the number of ghrelin-ip cells were almost the same as that of ghrelin mRNA-expressing (ghrelin-ex) cells; however, in the hatching chicken, the number of ghrelin-ex cells was greater than that of ghrelin-ip cells. These results clearly demonstrate that ghrelin-producing cells exist in the chicken gastrointestinal tract, especially in the proventriculus, from hatching to adult stages of development, as well as in mammals.     

Simultaneous nitrification and denitrification by controlling vertical and horizontal microenvironment in a membrane-aerated biofilm reactor

Nitrogen and carbon components in domestic modified wastewater were completely removed by simultaneous nitrification and denitrification using a membrane-aerated biofilm reactor where biofilm was fixed on a hollow-fiber membrane. To measure the spatial distribution of pH, ammonium and nitrate ions and to observe microbes inside the biofilm fixed on the membrane, microelectrodes and the fluorescence in situ hybridization (FISH) method were applied. Due to plug flow in the vertical direction (from the bottom to the top of the reactor), ammonium nitrogen was gradually removed and negligible nitrate nitrogen was detected throughout the reactor. FISH revealed that ammonia-oxidizing bacteria were mainly distributed inside the biofilm and other bacteria, which included denitrifying bacteria, were mainly distributed outside the biofilm and over the suspended sludge. In order to characterize bacterial activity in the vertical direction of the reactor, nitrification rates at lower, central and upper points were calculated using microelectrode data. The nitrification rate at the lower point was 7 and 125 times higher than those at the central and upper points, respectively. These results show that the removal of carbon and nitrogen compounds was accomplished efficiently by using various kinds of bacteria distributed vertically and horizontally in a single reactor.     

Does enhanced expression of the Na+-CA2+ exchanger increase myocardial vulnerability to ischemia/reperfusion injury in rabbit hearts?

The present study focused on examining the efficacy of feeding a rutin-glucose derivative (G-rutin) to inhibit glycation reactions that can occur in muscle, kidney and plasma proteins of diabetic rats. Both thiobarbituric acid-reactive substance levels and protein carbonyl contents in muscle and kidney were significantly (p < 0.05) reduced in streptozotocin-induced diabetic rats fed G-rutin supplemented diet, compared to diabetic rats fed control diet. The N -fructoselysine content in muscle and kidney, a biomarker of early glycation reaction, was markedly (p < 0.05) increased by diabetes, but significantly (p < 0.05) reduced in diabetic rats fed G-rutin. Advanced glycation end-products (AGEs) in serum and kidney protein were measured by immunoblot using anti-AGE antibody, and were also reduced in diabetic rats fed dietary G-rutin. Feeding G-rutin also slightly inhibited aldose reductase activity in these animals. These results demonstrate for the first time that dietary G-rutin consumption can provide potential health benefits that are related to the inhibition of tissue glycation reactions common to diabetes.     

Interruption of signal transduction between G protein and PKC-ε underlies the impaired myocardial response to ischemic preconditioning in postinfarct remodeled hearts

We have recently shown that the protective mechanism of ischemic preconditioning (PC) is impaired in the myocardium that survived infarction and underwent postinfarct ventricular remodeling. In this study, we examined the hypothesis that failure of PC to activate PKC- underlies the refractoriness of the remodeling heart to PC. Circumflex coronary arteries were ligated in rabbits to induce infarction and subsequent ventricular remodeling, and only sham operations were performed in controls. Hearts were isolated before (i.e. 4 days later) or after (i.e. 2 weeks later) remodeling of the left ventricle and used for isolated buffer-perfused heart experiments. Myocardial infarction was induced in isolated hearts by 30 min global ischemia/2 h reperfusion, and its size was measured by tetrazolium staining. Using separate groups of hearts, tissue biopsies were taken before and after PC, and PKC translocation was assessed by Western blotting. Areas infarcted in vivo by coronary ligation (CL) were excluded from subsequent infarct size/PKC analyses. In the hearts 4 days after CL, PC with 2 cycles of 5 min ischemia/5 min reperfusion induced PKC- translocation from cytosol to particulate fractions and limited infarct size to 40% of control value. In the hearts remodeled 2 weeks after CL, PC failed to induce PKC- translocation and infarct size limitation. In this group, PKC activity and hemodynamic responses to adenosine were similar to those in sham-operated controls. When remodeling after CL was prevented by valsartan infusion (10 mg/kg/day), an angiotensin II type 1 (AT₁) receptor blocker, PC could induce both infarct limitation and PKC- translocation. The present results suggest that persistent activation of AT₁ receptors during remodeling disturbed the PC signaling between G proteins and PKC-, which underlies the refractoriness of the remodeled myocardium to PC.     

New bulky chiral Lewis acid catalyst: 3,3'-di(2-mesitylethynyl)binaphthol-titanium(IV) complex

A new chiral Lewis acid catalyst 9 was prepared in situ from a 1:2 molar mixture of (R)-3,3'-di(2-mesitylethynyl)binaphthol (6) and titanium(IV) isopropoxide at ambient temperature. The 3- and 3'-substituents on 6 were effective for preventing undesired aggregation between Ti(IV) complexes and increasing the enantioselectivity (up to 82% ee) in the Diels-Alder reaction of methacrolein with cyclopentadiene.