Occurrence of Potential Brassinosteroid Precursor Steroids in Seeds of Wheat and Foxtail Millet

Triticum aestivum L.) and foxtail millet (Setaria italica Beauv.) were found by GC-MS to contain, in addition to bulk sterols, 4-en-3-one steroids including 24-ethylcholesta-4,24(28)Z- dien-3-one (a new steroid), 24-methylcholest-4-en-3-one, 24-ethylcholesta-4,22E-dien-3-one and 24-ethylcholest-4-en-3-one, as well as 5α-steroidal 3-one compounds including 24-methyl-5α-cholestan-3-one, 24-ethyl-5α-cholestan-3-one and 24-ethyl 5α-cholest-22E-en-3-one (in S. italica only). Analysis of free sterol and steryl ester fractions indicated that campestanol and sitostanol were present at high levels in both seeds. These results suggest that the seeds of T. aestivum and S. italica synthesize campestanol from campesterol via 24-methylcholest-4-en-3-one and 24-methyl-5α-cholestan-3-one as has already been demonstrated in Arabidopsis thaliana L., and also produce sitostanol from sitosterol via 24-ethylcholest-4-en-3-one and 24-ethyl-5α-chotestan-3-one. Biosynthetic relationships of campestanol and sitostanol with C₂₈ and C₂₉ brassinosteroids are discussed. Received 4 September 1998/ Accepted in revised form 26 November 1998     

Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding

Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to d-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2′-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.     

Solution structure of agelenin, an insecticidal peptide isolated from the spider Agelena opulenta, and its structural similarities to insect-specific calcium channel inhibitors

Agelenin, isolated from the Agelenidae spider Agelena opulenta, is a peptide composed of 35 amino acids. We determined the three-dimensional structure of agelenin using two-dimensional NMR spectroscopy. The structure is composed of a short antiparallel beta-sheet and four beta-turns, which are stabilized by three disulfide bonds. Agelenin has characteristic residues, Phe9, Ser28 and Arg33, which are arranged similarly to the pharmacophore of the insect channel inhibitor, omega-atracotoxin-Hv1a. These observations suggest that agelenin and omega-atracotoxin-Hv1a bind to insect calcium channels in a similar manner. We also suggest that another mode of action may operate in the channel inhibition by omega-agatoxin-IVA and omega-atracotoxin-Hv2a.     

Static interaction between muscle mechanoreflex and arterial baroreflex in determining efferent sympathetic nerve activity

Elucidation of the interaction between the muscle mechanoreflex and the arterial baroreflex is essential for better understanding of sympathetic regulation during exercise. We characterized the effects of these two reflexes on sympathetic nerve activity (SNA) in anesthetized rabbits (n = 7). Under open-loop baroreflex conditions, we recorded renal SNA at carotid sinus pressure (CSP) of 40, 80, 120, or 160 mmHg while passively stretching the hindlimb muscle at muscle tension (MT) of 0, 2, 4, or 6 kg. The MT-SNA relationship at CSP of 40 mmHg approximated a straight line. Increase in CSP from 40 to 120 and 160 mmHg shifted the MT-SNA relationship downward and reduced the response range (the difference between maximum and minimum SNA) to 43 +/- 10% and 19 +/- 6%, respectively (P < 0.01). The CSP-SNA relationship at MT of 0 kg approximated a sigmoid curve. Increase in MT from 0 to 2, 4, and 6 kg shifted the CSP-SNA relationship upward and extended the response range to 133 +/- 8%, 156 +/- 14%, and 178 +/- 15%, respectively (P < 0.01). A model of algebraic summation, i.e., parallel shift, with a threshold of SNA functionally reproduced the interaction of the two reflexes (y = 1.00x - 0.01; r(2) = 0.991, root mean square = 2.6% between estimated and measured SNA). In conclusion, the response ranges of SNA to baroreceptor and muscle mechanoreceptor input changed in a manner that could be explained by a parallel shift with threshold.     

Dialysate dihydroxyphenylglycol as a window for in situ axoplasmic norepinephrine disposition

To examine basal axoplasmic norepinephrine (NE) kinetics at the in situ cardiac sympathetic nerve ending, we applied a dialysis technique to the heart of anesthetized cats and performed the dialysate sampling with local administration of a pharmacological tool through a dialysis probe. The dialysis probe was implanted in the left ventricular wall, and dihydroxyphenylglycol (DHPG, an index of axoplasmic NE) levels were measured by liquid chromatogram-electrochemical detection. Control dialysate DHPG levels were 161+/-19 pg/ml. Pargyline (monoamine oxidase inhibitor, 1 mM) decreased the dialysate DHPG levels to 38+/-10 pg/ml. Further alpha-methyl-para-tyrosine, omega-conotoxin GVIA, desipramine (NE synthesis, release and uptake blockers) decreased the dialysate DHPG levels to 64+/-19, 106+/-15, 110+/-22 pg/ml, respectively. In contrast, reserpine (vesicle NE transport inhibitor, 10 microM) increased the dialysate DHPG levels to 690+/-42 pg/ml. Thus, NE synthesis, metabolism and recycling (release, uptake and vesicle transport) affected basal intraneuronal NE disposition at the nerve endings. Measurement of DHPG levels through a dialysis probe provides information about basal intraneuronal NE disposition at the cardiac sympathetic nerve endings. Yohimbine (alpha(2)-adrenoreceptor blocker, 10 microM) and U-521 (catechol-O-methyltransferase blocker, 100 microM) did not alter the dialysate DHPG levels. Furthermore, there were no significant differences in the reserpine induced DHPG increment between the presence and absence of desipramine (10 microM) or alpha-methyl-para-tyrosine (100 mg/kg i.p.). These results may be explained by the presence of two axoplasmic pools of NE, filled by NE taken up and synthesized, and by NE overflow from vesicle. The latter pool of NE may be closed to the monoamine oxidase system in the axoplasma.     

Comprehensive analysis of mycolic acid subclass and molecular species composition of Mycobacterium bovis BCG Tokyo 172 cell wall skeleton (SMP-105)

The mycobacterial cell envelope consists of a characteristic cell wall skeleton (CWS), a mycoloyl arabinogalactan peptidoglycan complex, and related hydrophobic components that contribute to the cell surface properties. Since mycolic acids have recently been reported to play crucial roles in host immune response, detailed molecular characterization of mycolic acid subclasses and sub-subclasses of CWS from Mycobacterium bovis BCG Tokyo 172 (SMP-105) was performed. Mycolic acids were liberated by alkali hydrolysis from SMP-105, and their methyl esters were separated by silica gel TLC into three subclasses: alpha-, methoxy-, and keto-mycolates. Each mycolate subclass was further separated by silver nitrate (AgNO(3))-coated silica gel TLC into sub-subclasses. Molecular weights of individual mycolic acid were determined by MALDI-TOF mass spectrometry. alpha-Mycolates were sub-grouped into cis, cis-dicyclopropanoic (alpha1), and cis-monocyclopropanoic-cis-monoenoic (alpha2) series; methoxy-mycolates were sub-grouped into cis-monocyclopropanoic (m1), trans-monocyclopropanoic (m2), trans-monoenoic (m3), cis-monocyclopropanoic-trans-monoenoic (m4), cis-monoenoic (m5), and cis-monocyclopropanoic-cis-monoenoic (m6) series; and keto-mycolates were sub-grouped into cis-monocyclopropanoic (k1), trans-monocyclopropanoic (k2), trans-monoenoic (k3), cis-monoenoic (k4), and cis-monocyclopropanoic-cis-monoenoic (k5) series. The position of each functional group, including cyclopropane rings and methoxy and keto groups, was determined by analysis of the meromycolates with fast atom bombardment (FAB) mass spectrometry and FAB mass-mass spectrometry, and the cis/trans ratio of cyclopropane rings and double bonds were determined by NMR analysis of methyl mycolates. Mycolic acid subclass and molecular species composition of SMP-105 showed characteristic features including newly-identified cis-monocyclopropanoic-trans-monoenoic mycolic acid (m4).