NMR characterization of a pH-dependent equilibrium between two folded solution conformations of the pheromone-binding protein from Bombyx mori

NMR spectroscopic changes as a function of pH in solutions of the pheromone-binding protein of Bombyx mori (BmPBP) show that BmPBP undergoes a conformational transition between pH 4.9 and 6.0. At pH below 4.9 there is a single "acid form" (A), and a homogeneous "basic form" (B) exists at pH above 6.0. Between pH 5 and 6, BmPBP exists as a mixture of A and B in slow exchange on the NMR chemical shift time scale, with the transition midpoint at pH 5.4. The form B has a well-dispersed NMR spectrum, indicating that it represents a more structured, "closed" conformation than form A, which has a significantly narrower chemical shift dispersion. Conformational transitions of the kind observed here may explain heterogeneity reported for a variety of odorant-binding proteins, and it will be of interest to further investigate possible correlations with pH-dependent regulation of ligand binding and release in the biological function of this class of proteins.     

In vitro system to study realistic pulsatile flow and stretch signaling in cultured vascular cells

We developed a novel real-timeservo-controlled perfusion system that exposes endothelial cells grownin nondistensible or distensible tubes to realistic pulse pressures andphasic shears at physiological mean pressures. A rate-controlled flowpump and linear servo-motor are controlled by digitalproportional-integral-derivative feedback that employspreviously digitized aortic pressure waves as a command signal. Theresulting pressure mirrors the recorded waveform and can be digitallymodified to yield any desired mean and pulse pressure amplitude,typically 0-150 mmHg at shears of 0.5-15 dyn/cm².The system accurately reproduces the desired arterial pressure waveformand cogenerates physiological flow and shears by the interaction ofpressure with the tubing impedance. Rectangular glass capillary tubes[1-mm inside diameter (ID)] are used for real-time fluorescentimaging studies (i.e., pH_i, NO, Ca²⁺), whereassilicon distensible tubes (4-mm ID) are used for more chronic (i.e.,2-24 h) studies regarding signal transduction and geneexpression. The latter have an elastic modulus of12.4 · 10⁶ dyn/cm² similar to in vivovessels of this size and are studied with the use of a benchtop system.The new approach provides the first in vitro application of realisticmechanical pulsatile forces on vascular cells and should facilitatestudies of phasic shear and distension interaction and pulsatile signal transduction.

     

侧脑室注射一叶萩碱的心血管效应及作用机制

在麻醉大鼠侧脑室注射左旋一叶Ｑｉｕ碱（Ｌ－Ｓｅｃ）,记录动脉血压（ＡＰ）、心率（ＨＲ）及肾交感神经放电（ＲＳＮＤ）,观察前脑室周系统ＧＡＢＡ能紧张性活动改变引起的心血管效应。结果如下：（１）Ｌ－Ｓｅｃ可引起ＲＳＮＤ增加、ＡＰ升高和ＨＲ加快,并呈一定剂量－效应关系;但Ｌ－Ｓ盈余 于ｂｉｃｕｃｕｌｌｉｎｅ（Ｂｉｃ）。（２）Ｌ－Ｓｅｃ既能拮抗ｍｕｓｃｉｍｏｌ（Ｍｕｓ）,又能拮抗ｂａｃｌｏｆｅｎ（Ｂａｃ）     

Microsatellite diversity correlated with ecological-edaphic and genetic factors in three microsites of wild emmer wheat in North Israel

This study was conducted to test the effects of internal (genetic) and external factors on allelic diversity at 27 dinucleotide microsatellite (simple sequence repeat [SSR]) loci in three Israeli natural populations of Triticum dicoccoides from Ammiad, Tabigha, and Yehudiyya, north of the Sea of Galilee. The results demonstrated that SSR diversity is correlated with the interaction of ecological and genetic factors. Genetic factors, including genome (A vs. B), chromosome, motif, and locus, affected average repeat number (ARN), variance in repeat number (sigma), and number of alleles (NA) of SSRs, but the significance of some factors varied among populations. Genome effect on SSR variation may result from different motif types, particularly compound (or imperfect) versus perfect motifs, which may be related to different evolutionary histories of genomes A and B. Ecological factors significantly affected SSR variation. Soil-unique and soil-specific alleles were found in two edaphic groups dwelling on terra rossa and basalt soils across macro- and microgeographical scales. The largest contributions of genetic and ecological effects were found for diversity of ARN and NA, respectively. Multiple regression indicated that replication slippage and unequal crossing over could be important mutational mechanisms, but their significance varied among motifs. Edaphic stresses may affect the probability of replication errors and recombination intermediates and thus control diversity level and divergence of SSRs. The results may indicate that SSR diversity is adaptive, channeled by natural selection and influenced by both internal and external factors and their interactions.     

Acute effects of methoxamine on left ventricular-arterial coupling in streptozotocin-diabetic rats: a pressure-volume analysis

We determined the acute effects of methoxamine, a specific alpha1-selective adrenoceptor agonist, on the left ventricular-arterial coupling in streptozotocin (STZ)-diabetic rats, using the end-systolic pressure-stroke volume relationships. Rats given STZ 65 mg x kg(-1) iv (n = 8) were compared with untreated age-matched controls (n = 8). A high-fidelity pressure sensor and an electromagnetic flow probe measured left ventricular (LV) pressure and ascending aortic flow, respectively. Both LV end-systolic elastance E(LV,ES) and effective arterial elastance Ea were estimated from the pressure-ejected volume loop. The optimal afterload Q(load) determined by the ratio of Ea to E(LV,ES) was used to measure the optimality of energy transmission from the left ventricle to the arterial system. In comparison with controls, diabetic rats had decreased LV end-systolic elastance E(LV,ES), at 513 +/- 30 vs. 613 +/- 29 mmHg x mL(-1), decreased effective arterial elastance Ea, at 296 +/- 20 vs. 572 +/- 48 mmHg x mL(-1), and decreased optimal afterload Q(load), at 0.938 +/- 0.007 vs. 0.985 +/- 0.009. Methoxamine administration to STZ-diabetic rats significantly increased LV end-systolic elastance E(LV,ES), from 513 +/- 30 to 602 +/- 38 mmHg x mL(-1), and effective arterial elastance Ea, from 296 +/- 20 to 371 +/- 28 mmHg x mL(-1), but did not change optimal afterload Q(load). We conclude that diabetes worsens not only the contractile function of the left ventricle, but also the matching condition for the left ventricular-arterial coupling. In STZ-diabetic rats, administration of methoxamine improves the contractile status of the ventricle and arteries, but not the optimality of energy transmission from the left ventricle to the arterial system.     

Cytogenetic studies of three triazine herbicides. II. In vivo micronucleus studies in mouse bone marrow

Atrazine, simazine, and cyanazine are widely used preemergence and postemergence triazine herbicides that have made their way into the potable water supply of many agricultural communities. Although there are several contradictory genotoxicity studies in the literature, our previous in vitro studies with human lymphocytes showed that atrazine, simazine, and cyanazine did not induce sister chromatid exchanges (SCEs) or chromosome aberrations (CAs) up to the limits of solubility in aqueous medium using 0.5% dimethyl sulfoxide. To expand upon these results and to ensure that our in vitro findings could be replicated in an in vivo system, mice were treated with each triazine by two intraperitoneal injections, 24h apart. The animals were sacrificed and the bone marrow removed for micronucleus (MN) analysis, 24h after the last injection. Two to four independent trials were performed for MN analysis in polychromatic erythrocytes, and in some trials the spleen was removed, cultured, and analyzed for SCEs and CAs. None of the triazines investigated induced MN in the bone marrow, even at doses that caused significant bone marrow suppression and/or death. These results indicate that atrazine, simazine, and cyanazine are not genotoxic as measured by the bone marrow MN assay in mice following high dose exposures.     

An NMR and molecular modeling study of the site-specific binding of histamine by heparin, chemically modified heparin, and heparin-derived oligosaccharides

The diprotonated form of histamine binds site-specifically to heparin, a highly sulfated 1-->4 linked repeating copolymer comprised predominantly of 2-O-sulfo-alpha-L-iduronic acid (the I ring) and 2-deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl (the A ring). The binding is mediated by electrostatic interactions. The structural features of histamine and heparin, which are required for the site-specific binding, have been identified from the results of (1)H NMR studies of the binding of histamine by six heparin-derived oligosaccharides and four chemically modified heparins and molecular modeling studies. The results indicate that the imidazolium ring of diprotonated histamine is critical for directing site-specific binding, while the ammonium group increases the binding affinity. The imidazolium ring binds within a cleft, with the A ring of an IAI triad at the top of the cleft, and the I rings forming the two sides. The H3 proton of the A ring is in the shielding cone of the imidazolium ring. The carboxylate group of the I-ring at the reducing end of the IAI triad and possibly the sulfamido group of the A-ring are essential for site-specific binding, whereas the 2-O-sulfate group of the I ring and the 6-O-sulfate group of the A ring are not. The results indicate that histamine binds to the IAI triad with the I rings in the (1)C(4) conformation. Also, the configuration of the carboxylate group is critical, as indicated by the absence of site-specific binding of histamine by the related IAG sequence, where G is alpha-D-glucuronic acid. The molecular modeling results indicate that the N1H and N3H protons of the imidazolium ring of site-specifically bound histamine are hydrogen bonded to the carboxylates of the I rings at the nonreducing and reducing ends of the IAI trisaccharide sequence.     

Impaired vascular dynamics in normotensive diabetic rats induced by streptozotocin: tapered T-tube model analysis

This study is to explore the changes of arterial mechanical properties in streptozotocin (STZ)-diabetic rats, based on the exponentially tapered T-tube model. Rats given STZ 65 mg kg(-1)i.v. are compared with untreated weight- and age-matched controls. A high-fidelity pressure sensor and electromagnetic flow probe measured pulsatile pressure and flow waves in the ascending aorta, respectively. Diabetic rats exhibit isobaric vasodilatation that is characterized by an increase in cardiac output and no significant changes in aortic pressure. Total peripheral resistance of diabetic rats is lower than that of weight- and age-matched controls. Diabetic rats have higher total peripheral compliance (2.86+/-0.70 microl mm Hg(-1)) than do weight- (1.77+/-0.34 microl mm Hg(-1)) and age-matched (1.87+/-0.69 microl mm Hg(-1)) controls. Aortic characteristic impedance is reduced from 0.017+/-0.003 mm Hg min kg ml(-1)in weight- and 0.020+/-0.004 mm Hg min kg ml(-1)in age-matched controls to 0.010+/-0.004 mm Hg min kg ml(-1)in diabetic rats. Moreover, diabetic rats show shorter wave transit time in lower body circulation (17.86+/-1.91 ms) than do weight- (20.45+/-1.91) and age-matched (23.05+/-2.04 ms) controls. Under isobaric vasodilatation, the decreased resistance and increased compliance in peripheral circulation suggest that the contractile dysfunction of the smooth muscle cells may occur in resistance arterioles in diabetes. With unaltered aortic pressure, an impairment in aortic distensibility of STZ-diabetic rats is manifest on the reduced wave transit time rather than on the diminished aortic characteristic impedance.