Actinorhodin biosynthesis: structural requirements for post-PKS tailoring intermediates revealed by functional analysis of ActVI-ORF1 reductase

Actinorhodin (ACT) produced by Streptomyces coelicolor A3(2) is an aromatic polyketide antibiotic, whose basic carbon skeleton is derived from type II polyketide synthase (PKS). Although an acyl carrier protein (ACP) serves as an anchor of nascent intermediates during chain elongation in the type II PKS complex, it generally remains unknown when an ACP-free intermediate is released from the complex to post-PKS modification ("tailoring") steps. In ACT biosynthesis, a stereospecific ketoreductase (RED1) encoded by actVI-ORF1 reduces the 3beta-keto group of a proposed bicyclic intermediate to an (S) secondary alcohol. The bicyclic intermediate is formed from the steps of PKS and its closely associated enzymes and lies at the interface toward ACT-tailoring steps. To clarify whether RED1 recognizes the ACP-bound bicyclic intermediate or the ACP-free bicyclic intermediate, recombinant RED1 was purified for enzymatic characterization. RED1 was heterologously expressed in Escherichia coli and purified using Ni-chelate and gel filtration column chromatographies to homogeneity in soluble form. Enzymatic studies in vitro on RED1 with synthetic analogues, in place of an unstable bicyclic intermediate, showed that RED1 recognizes 3-oxo-4-naphthylbutyric acid (ONBA) as a preferred substrate and not its N-acetylcysteamine thioester. This strongly suggests that RED1 recognizes ACP-free bicyclic beta-keto acid as the first committed intermediate of tailoring steps. Kinetic studies of RED1 showed high affinity with ONBA, consistent with the requirement for an efficient reduction of a labile beta-keto carboxylic acid. Interestingly, the methyl ester of ONBA acted as a competitive inhibitor of RED1, indicating the presence of strict substrate recognition toward the terminal acid functionality.     

Muscle metaboreflex attenuates spontaneous heart rate baroreflex sensitivity during dynamic exercise

Hypoperfusion of active skeletal muscle elicits a reflex pressor response termed the muscle metaboreflex. Dynamic exercise attenuates spontaneous baroreflex sensitivity (SBRS) in the control of heart rate (HR) during rapid, spontaneous changes in blood pressure (BP). Our objective was to determine whether muscle metaboreflex activation (MRA) further diminishes SBRS. Conscious dogs were chronically instrumented for measurement of HR, cardiac output, mean arterial pressure, and left ventricular systolic pressure (LVSP) at rest and during mild (3.2 km/h) or moderate (6.4 km/h at 10% grade) dynamic exercise before and after MRA (via partial reduction of hindlimb blood flow). SBRS was evaluated as the slopes of the linear relations (LRs) between HR and LVSP during spontaneous sequences of at least three consecutive beats when HR changed inversely vs. pressure (expressed as beats x min(-1) x mmHg(-1)). During mild exercise, these LRs shifted upward, with a significant decrease in SBRS (-3.0 +/- 0.4 vs. -5.2 +/- 0.4, P<0.05 vs. rest). MRA shifted LRs upward and rightward and decreased SBRS (-2.1 +/- 0.1, P<0.05 vs. mild exercise). Moderate exercise shifted LRs upward and rightward and significantly decreased SBRS (-1.2 +/- 0.1, P<0.05 vs. rest). MRA elicited further upward and rightward shifts of the LRs and reductions in SBRS (-0.9 +/- 0.1, P<0.05 vs. moderate exercise). We conclude that dynamic exercise resets the arterial baroreflex to higher BP and HR as exercise intensity increases. In addition, increases in exercise intensity, as well as MRA, attenuate SBRS.     

Spontaneous baroreflex control of heart rate during exercise and muscle metaboreflex activation in heart failure

In heart failure (HF), there is a reduced baroreflex sensitivity at rest, and during dynamic exercise there is enhanced muscle metaboreflex activation (MRA). However, how the arterial baroreflex modulates HR during exercise is unknown. We tested the hypothesis that spontaneous baroreflex sensitivity (SBRS) is attenuated during exercise in HF and that MRA further depresses SBRS. In seven conscious dogs we measured heart rate (HR), cardiac output, and left ventricular systolic pressure at rest and during mild and moderate dynamic exercise, before and during MRA (via imposed reductions of hindlimb blood flow), and before and after induction of HF (by rapid ventricular pacing). SBRS was assessed by the sequences method. In control, SBRS was reduced from rest with a progressive resetting of the baroreflex stimulus-response relationship in proportion to exercise intensity and magnitude of MRA. In HF, SBRS was significantly depressed in all settings; however, the changes with exercise and MRA occurred with a pattern similar to the control state. As in control, the baroreflex stimulus-response relationship showed an intensity- and muscle metaboreflex (MMR)-dependent rightward and upward shift. The results of this study indicate that HF induces an impairment in baroreflex control of HR at rest and during exercise, although the effects of exercise and MRA on SBRS occur with a similar pattern as in control, indicating the persistence of some vagal activity.     

Biological evaluation of de-O-sulfonated analogs of salacinol, the role of sulfate anion in the side chain on the alpha-glucosidase inhibitory activity

De-O-sulfonated analogs (10a, Y(-)=CH(3)OSO(3) and 10b, Y(-)=Cl) of salacinol, a naturally occurring glycosidase inhibitor, and its diastereomer (12a, Y(-)=CH(3)OSO(3)) with L-thiosugar moiety (1,4-dideoxy-1,4-epithio-L-arabinitol) were prepared. Their inhibitory activities against intestinal maltase and sucrase were examined and compared with those of the parent alpha-glycosidase inhibitor, salacinol (1a). Compounds 10a and 10b showed a potent inhibitory activity equal to that of 1a against both enzymes, although 12a was a weak inhibitor against sucrase and maltase. These results indicated that the O-sulfonate anion moiety of 1a is not essential for the inhibitory activity.     

Effects of posture on peripheral vascular responses to lower body positive pressure

We tested the hypothesis that peripheral vascular responses (in the lower and upper limbs) to application of lower body positive pressure (LBPP) are dependent on the posture of the subjects. We measured heart rate, stroke volume, mean arterial pressure, leg and forearm blood flow (using the Doppler ultrasound technique), and leg (LVC) and forearm (FVC) vascular conductance in 11 subjects (9 men, 2 women) without and with LBPP (25 and 50 mmHg) in supine and upright postures. Mean arterial pressure increased in proportion to increases in LBPP and was greater in supine than in upright subjects. Heart rate was unchanged when LBPP was applied to supine subjects but was reduced in upright ones. Leg blood flow and LVC were both reduced by LBPP in supine subjects [LVC: 4.8 (SD 4.0), 3.6 (SD 3.5), and 1.4 (SD 1.8) ml.min(-1).mmHg(-1) before LBPP and during 25 and 50 mmHg LBPP, respectively; P < 0.05] but were increased in upright ones [LVC: 2.0 (SD 1.2), 3.4 (SD 3.4), and 3.0 (SD 2.0) ml.min(-1).mmHg(-1), respectively; P < 0.05]. Forearm blood flow and FVC both declined when LBPP was applied to supine subjects [FVC: 1.3 (SD 0.6), 1.0 (SD 0.4), and 0.9 (SD 0.6) ml. min(-1).mmHg(-1), respectively; P < 0.05] but remained unchanged in upright ones [FVC: 0.7 (SD 0.4), 0.7 (SD 0.4), and 0.6 (SD 0.5) ml.min(-1).mmHg(-1), respectively]. Together, these findings indicate that the leg vascular response to application of LBPP is posture dependent and that the response differs in the lower and upper limbs when subjects assume an upright posture.     

Schistosoma mekongi and Schistosoma japonicum: Differences in the distribution of eggs in the viscera of mice

The difference in the distribution of Schistosoma eggs in the viscera has not been clearly elucidated in the two closely related species Schistosoma japonicum and Schistosoma mekongi. In this study, we quantitatively compared the distribution of eggs in mice infected with the two species. In S. mekongi-infected mice, 56.6% to 69.4% of total eggs were found in the distal small intestine 9 to 15 weeks after infection, while in S. japonicum-infected mice, 48.8% to 71.8% of eggs were found in the proximal small intestine during the same period. There were significantly more eggs in the liver in mice infected with S. japonicum than in those infected with S. mekongi. The number of adult worms recovered did not differ between the two species during the study period. The total number of eggs laid in the tissues also did not differ between the two species at 12 to 15 weeks postinfection, but in the earlier period the total number of eggs was significantly fewer in S. mekongi-infected than in S. japonicum-infected mice, suggesting the delayed maturation of the former compared with the latter. These results clearly show that S. japonicum and S. mekongi exhibit different oviposition behavior in their hosts.     

Degeneration of hrpZ gene in Pseudomonas syringae pv. tabaci to evade tobacco defence: an arms race between tobacco and its bacterial pathogen

The HrpZ harpin of Pseudomonas syringae is known to induce a hypersensitive response (HR) in some plants. In P. syringae pv. tabaci (Pta), the harpin gene hrpZ has been spontaneously disrupted by an internal deletion in its open reading frame and a frame shift. The loss of the ability of the recombinant harpin polypeptide of Pta to induce HR despite the high sensitivity of tobacco plants to harpin led us to investigate the meaning of the disrupted hrpZ gene in the virulence of Pta 6605. The hrpZ gene from P. syringae pv. pisi was introduced into wild-type (WT) Pta. The hrpZ-complemented Pta secreted harpin into the culture medium, but failed to cause disease symptoms by both infiltration and spray inoculation. Inoculation with the hrpZ-complemented Pta induced defence responses in tobacco plants, whereas the defence responses of tobacco plants were not prominent on inoculation with WT Pta. These results indicate that an ancestor of Pta might have disrupted hrpZ by an internal deletion to evade plant defences and confer the ability to cause disease in tobacco plants.     

Components of essential oils extracted from leaves and shoots of abies species in Japan

The essential oils extracted from the leaves and the shoots of five Abies species (Pinaceae) growing in Japan, i.e., A. firma, A. homolepis, A. veitchii, A. mariesii, and A. sachalinensis, were characterized by GC-FID and GC/MS analyses. The yields of the essential oils extracted from A. sachalinensis were the highest among them. A significant amount of α-pinene was contained in the essential oils of all the Abies species examined. In A. homolepis and A. veitchii, significant differences in the content of the essential oils were found depending on whether these were extracted from the leaves or from the shoots. Regarding the enantiomeric ratio of α-pinene, the (+)-enantiomer was predominant in the oil extracted from the leaves of A. firma, while (-)-α-pinene was present in higher amounts in the oils of A. veitchii (leaves and shoots), A. mariesii (leaves and shoots), and A. sachalinensis (shoots). The fact that there may be a quantitative and qualitative difference in the components of the essential oils extracted from the different parts of a plant was investigated by cluster analysis.