Cloning, heterologous expression, and functional characterization of 5-epi-aristolochene-1,3-dihydroxylase from tobacco (Nicotiana tabacum).

Capsidiol is a bicyclic, dihydroxylated sesquiterpene produced by several solanaceous species in response to a variety of environmental stimuli. It is the primary antimicrobial compound produced by Nicotiana tabacum in response to fungal elicitation, and it is formed via the isoprenoid pathway from 5-epi-aristolochene. Much of the biosynthetic pathway for the formation of this compound has been elucidated, except for the enzyme(s) responsible for the conversion of 5-epi-aristolochene to its dihydroxylated form, capsidiol. Biochemical evidence from previous studies with N. tabacum (Whitehead, I. M., Threlfall, D. R., and Ewing, D. F., 1989, Phytochemistry 28, 775-779) and Capsicum annuum Hoshino, T., Yamaura, T., Imaishi, H., Chida, M., Yoshizawa, Y., Higashi, K., Ohkawa, H., Mizutani, J., 1995, Phytochemistry 38, 609-613. suggested that the oxidation of 5-epi-aristolochene to capsidiol was mediated by at least one elicitor-inducible cytochrome P450 hydroxylase. In extending these observations, we developed an in vivo assay for 5-epi-aristolochene hydroxylase activity and used it to demonstrate a dose-dependent inhibition of activity by ancymidol and ketoconazole, two well characterized inhibitors of cytochrome P450 enzymes. Using degenerate oligonucleotide primers designed to the well conserved domains found within most P450 enzymes, including the heme binding domain, cDNA fragments representing four distinct P450 families (CYP71, CYP73, CYP82, and CYP92) were amplified from a cDNA library prepared against mRNA from elicitor-treated cells using PCR. The PCR fragments were subsequently used to isolate full-length cDNAs (CYP71D20 and D21, CYP73A27 and A28, CYP82E1 and CYP92A5), and these in turn were used to demonstrate that the corresponding mRNAs were all induced in elicitor-treated cells, albeit with different induction patterns. Representative, full-length cDNAs for each of the P450s were engineered into a yeast expression system, and the recombinant yeast assessed for functional expression of P450 protein by measuring the CO difference spectra of the yeast microsomes. Only microsomal preparations from yeast expressing the CYP71D20 and CYP92A5 cDNAs exhibited significant CO difference absorbance spectra at 450 nm and were thus tested for their ability to hydroxylate 5-epi-aristolochene and 1-deoxycapsidiol, a putative mono-hydroxylated intermediate in capsidiol biosynthesis. Interestingly, the CYP71D20-encoded enzyme activity was capable of converting both 5-epi-aristolochene and 1-deoxycapsidiol to capsidiol in vitro, consistent with the notion that this P450 enzyme catalyzes both hydroxylations of its hydrocarbon substrate.

Differential induction of sesquiterpene metabolism in tobacco cell suspension cultures by methyl jasmonate and fungal elicitor.

Jasmonates are well documented for their ability to modulate the expression of plant genes and to influence specific aspects of disease/pest resistance traits. We and others have been studying the synthesis of sesquiterpene phytoalexins in elicitor/pathogen-challenged plants and have sought to determine if methyl jasmonate (MeJA) could substitute for fungal elicitors in the induction of capsidiol accumulation by tobacco cell cultures. The current results demonstrate that MeJA does in fact induce phytoalexin accumulation, but with a much more delayed induction time course than elicitor. While elicitor treatment induced strong but transient changes in key enzymes of sesquiterpene biosynthesis, sesquiterpene cyclase, and aristolochene/deoxy-capsidiol hydroxylase, MeJA did not. Instead, MeJA caused a protracted induction of cyclase activity and only a low level of hydroxylase activity. MeJA induced the expression of at least two sesquiterpene cyclase genes, including one that had not been observed previously in elicitor-induced mRNA populations. Only a small portion of the total sesquiterpene cyclase mRNA induced by MeJA was associated with polysomal RNA, suggesting that the MeJA treatment imposed both transcriptional and posttranscriptional regulation in tobacco cells. These results are not consistent with MeJA playing a role in orchestrating defense responses in elicitor-treated tobacco cells, but do provide evidence that MeJA induces a subset of genes coding for the biosynthesis of sesquiterpene phytoalexins.

Decreased NADH glutamate synthase activity in nodules and flowers of alfalfa (Medicago sativa L.) transformed with an antisense glutamate synthase transgene.

Legumes obtain a substantial portion of their nitrogen (N) from symbiotic N2 fixation in root nodules. The glutamine synthetase (GS, EC 6.3.1.2)/glutamate synthase (GOGAT) cycle is responsible for the initial N assimilation. This report describes the analysis of a transgenic alfalfa (Medicago sativa L.) line containing an antisense NADH-GOGAT (EC 1.4.1.14) under the control of the nodule-enhanced aspartate amino-transferase (AAT-2) promoter. In one transgenic line, NADH-GOGAT enzyme activity was reduced to approximately 50%, with a corresponding reduction in protein and mRNA. The transcript abundance for cytosolic GS, ferredoxin-dependent GOGAT (EC 1.4.7.1), AAT-2 (EC 2.6.1.1), asparagine synthase (EC 6.3.5.4), and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were unaffected, as were enzyme activities for AAT, PEPC and GS. Antisense NADH-GOGAT plants grown under symbiotic conditions were moderately chlorotic and reduced in growth and N content, even though symbiotic N2 fixation was not significantly reduced. The addition of nitrate relieved the chlorosis and restored growth and N content. Surprisingly, the antisense NADH-GOGAT plants were male sterile resulting from inviable pollen. A reduction in NADH-GOGAT enzyme activity and transcript abundance in the antisense plants was measured during the early stages of flower development. Inheritance of the transgene was stable and resulted in progeny with a range of NADH-GOGAT activity. These data indicate that NADH-GOGAT plays a critical role in the assimilation of symbiotically fixed N and during pollen development.

The alfalfa (Medicago sativa) TDY1 gene encodes a mitogen-activated protein kinase homolog.

Development of root nodules, specifically induction of cortical cell division for nodule initiation, requires expression of specific genes in the host and microsymbiont. A full-length cDNA clone and the corresponding genomic clone encoding a MAP (mitogen-activated protein) kinase homolog were isolated from alfalfa (Medicago sativa). The genomic clone, TDY1, encodes a 68.9-kDa protein with 47.7% identity to MMK4, a previously characterized MAP kinase homolog from alfalfa. TDY1 is unique among the known plant MAP kinases, primarily due to a 230 amino acid C-terminal domain. The putative activation motif, Thr-Asp-Tyr (TDY), also differs from the previously reported Thr-Glu-Tyr (TEY) motif in plant MAP kinases. TDY1 messages were found predominantly in root nodules, roots, and root tips. Transgenic alfalfa and Medicago truncatula containing a chimeric gene consisting of 1.8 kbp of 5' flanking sequence of the TDY1 gene fused to the beta-glucuronidase (GUS) coding sequence exhibited GUS expression primarily in the nodule parenchyma, meristem, and vascular bundles, root tips, and root vascular bundles. Stem internodes stained intensely in cortical parenchyma, cambial cells, and primary xylem. GUS activity was observed in leaf mesophyll surrounding areas of mechanical wounding and pathogen invasion. The promoter was also active in root tips and apical meristems of transgenic tobacco. Expression patterns suggest a possible role for TDY1 in initiation and development of nodules and roots, and in localized responses to wounding.

Long-term follow-up of medical versus surgical therapy for hypertrophic cardiomyopathy: a retrospective study.

In a retrospective analysis 139 patients with hypertrophic cardiomyopathy were followed up for 8.9 years (range 1 to 28 years). Patients were divided into two groups: Group 1 consisted of 60 patients with medical therapy and Group 2 of 79 patients with surgical therapy (septal myectomy). Groups 1 and 2 were subdivided according to the medical treatment. Group 1a received propranolol, 160 mg/day (n = 20); Group 1b verapamil, 360 mg/day (n = 18); and Group 1c, no therapy (n = 22). Group 2a received verapamil, 120 to 360 mg/day, after septal myectomy (n = 17) and Group 2b had no medical therapy after surgery (n = 62). In Group 1, 19 patients died (annual mortality rate 3.6%) and in Group 2, 17 patients died (mortality rate 2.4%, p = NS). Of the patients who died, approximately one half to two thirds in both Groups 1 and 2 died suddenly and the other one half to one third died because of congestive heart failure. The 10 year cumulative survival rate was 67% in Group 1, significantly smaller than that in Group 2 (84%, p less than 0.05). In the subgroups, the 10 year survival rate was 67% in Group 1a, 80% in 1b (p less than 0.05 versus 1a) and 65% in 1c (p less than 0.05 versus 1b). The 10 year survival rate was 100% in Group 2a (p less than 0.05 versus 1a, 1b, 1c) and 78% in Group 2b (p less than 0.05 versus 2a). It is concluded that cumulative survival rate is significantly better in surgically than in medically treated patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity-sensing rate responsive versus conventional fixed-rate pacing: a comparison of rate behavior and patient well-being during routine daily exercise.

Rate responsive single chamber pacing (VVIR) may be the pacemaker of choice in patients who are not suitable candidates for a dual chamber system. Several studies, most of them performed in an exercise laboratory, have shown a significantly higher exercise capacity demonstrating an improvement in cardiac output and anaerobic threshold compared to conventional fixed rate pacing (VVI). Expressing our idea that stress testing in an "artificial environment" on a bicycle or motor driven treadmill has its limitations and may be difficult to extend into patient's daily life, we designed an outdoor study imitating patient's daily activity. Twenty-one patients with an activity-sensing rate responsive pacemaker performed in a double blind fashion in VVI and VVIR mode the following test circuit: walking 170 meters on flat ground, 210 meters incline, climbing a flight of stairs, and the same circuit in reverse order, and therefore "downhill". Heart rate behavior was recorded by Holter monitoring and patients subjective feelings of well-being, i.e. fatigue and dyspnea were also evaluated. VVIR pacing responded promptly to exercise, i.e., walking on a flat ground, but no further significant increase in pacing rate was observed in relationship to the strength of physical activity while walking inclined or climbing stairs. While patients became exhausted, a nonphysiological decrease in heart rate sometimes occurred. Despite these limitations 6 of 12 patients who had a paced-only rhythm while exercising in both VVI and VVIR mode reported feeling significantly better in the VVIR mode, expressing less dyspnea and fatigue. In contrast, two of nine patients having only intermittently paced rhythm preferred the VVIR mode. Patients with lower ejection fraction (EF) were more likely to show subjectively a benefit while exercising in VVIR mode, compared to those with less reduced or normal EF. Despite the technical limitations of using a piezo crystal for rate adaptation, VVIR pacing is an important option in paced-only patients, but it seems less beneficial in patients with only intermittent paced rhythm.

[Long-term course of hypertrophic cardiomyopathy: drug versus surgical therapy]. / Langzeitverlauf der hypertrophen Kardiomyopathie: medikamentöse versus operative, Therapie.

139 patients with hypertrophic cardiomyopathy (HCM) have been followed up for 1-28 years (mean 8.9 years). Group 1 consisted of 60 patients (mean age 38 years) without indication for septal myectomy (SM) (no pressure gradient at rest in 8, pressure gradient less than 50 mm Hg in 52 cases); group 2 consisted of 79 patients (mean age 36 years) who had SM (pressure gradient at rest 70 mm Hg). Management in group 1 was the following: (1a) propranolol (n = 20) (160 mg/d), (1b) verapamil (n = 18) (360 mg/d) and (1c) no therapy (n = 22). 19 patients died in group 1 (mortality 3.6% year); 17 died in group 2 (mortality 2.4%/year). 10 year survival in group 1b was 80% and in groups 1a und 1c 67% and 65% respectively. Patients of group 1b had a higher survival rate (p less than 0.05) than the other subgroups. Surgery patients treated with verapamil (120-360 mg/d) (n = 17) had a 10-year survival rate of 100% compared to 78% for surgery patients (n = 34) without such treatment (p less than 0.05). In summary, it can be said that the overall survival rate after SM is better than that with medical treatment. Under verapamil, however, survival is not different from that after surgery. The most favorable outcome was observed in surgery patients under long-term therapy with verapamil, probably due to the reduction of systolic pressure overload (SM) and improvement in diastolic function (verapamil).

Abnormal segmental contraction velocity in coronary artery disease produced by isometric exercise and atrial pacing.

Since isometric exercise by sustained handgrip leads to a sizable increase in aortic pressure this maneuver was used in addition to atrial pacing to increase the imbalance between oxygen demand and supply in two groups of patients. Both groups were studied by left heart catheterization and cineangiography in the right anterior oblique projection, at rest, during atrial pacing and during combined pacing and handgrip exercise. Group 1, the control group, consisted of 10 patients without coronary artery disease having an ejection fraction of 0.61 to 0.82. Group 2 was composed of 10 patients with definite obstructive disease of one or more of the three main coronary arteries. At rest, ejection fraction was normal or nearly normal (range 0.54 to 0.78). Regional myocardial contraction performance was assessed by determining mean segmental shortening velocities at the basal (VSB), middle (VSM) and apical (VSA) short ventricular axes. Whereas at rest there was no significant difference between the two groups or any of the three velocities, during pacing, VSM and VSA were significantly smaller in Group 2 than in Group 1 (P smaller than 0.02). During pacing combined with handgrip exercise the difference between the two groups was clearly accentuated, all three velocities being highly significantly decreased in Group 2 (VSB, P smaller than 0.01; VSM and VSA, P smaller than 0.001). When evaluated individually the patients of Group 2 had in 9 segments during pacing values for VSB, VSM and VSA that were below the range of the normal subjects. During pacing combined with handgrip a newly abnormal shortening velocity was observed in 12 segments (VSB abnormal in 3 of 7, VSM in 4 of 7 and VSA in 5 of 7 instances). In conclusion, the combination of atrial pacing and handgrip exercise appears to be a useful stress maneuver to identify temporarily dysfunctioning segments in patients with coronary artery disease in whom atrial pacing alone is not sufficient to induce ischemic contraction disorders.