Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate:ATP/ADP isopentenyltransferases.

It has been believed that the key step in cytokinin biosynthesis is the addition of a 5-carbon chain to the N(6) of AMP. To identify cytokinin biosynthesis enzymes that catalyze the formation of the isopentenyl side chain of cytokinins, the Arabidopsis genomic sequence was searched for genes that could code for isopentenyltransferases. This resulted in the identification of nine putative genes for isopentenyltransferases. One of these, AtIPT4, was subjected to detailed analysis. Overexpression of AtIPT4 caused cytokinin-independent shoot formation on calli. As shoot formation on calli normally occurs only when cytokinins are applied, it suggested that this gene product catalyzed cytokinin biosynthesis in plants. Recombinant AtIPT4 catalyzed the transfer of an isopentenyl group from dimethylallyl diphosphate to the N(6) of ATP and ADP, but not to that of AMP. AtIPT4 did not exhibit the DMAPP:tRNA isopentenyltransferase activity. These results indicate that cytokinins are, at least in part, synthesized from ATP and ADP in plants.

Identification of CRE1 as a cytokinin receptor from Arabidopsis.

Cytokinins are a class of plant hormones that are central to the regulation of cell division and differentiation in plants. It has been proposed that they are detected by a two-component system, because overexpression of the histidine kinase gene CKI1 induces typical cytokinin responses and genes for a set of response regulators of two-component systems can be induced by cytokinins. Two-component systems use a histidine kinase as an environmental sensor and rely on a phosphorelay for signal transduction. They are common in microorganisms, and are also emerging as important signal detection routes in plants. Here we report the identification of a cytokinin receptor. We identified Arabidopsis cre1 (cytokinin response 1) mutants, which exhibited reduced responses to cytokinins. The mutated gene CRE1 encodes a histidine kinase. CRE1 expression conferred a cytokinin-dependent growth phenotype on a yeast mutant that lacked the endogenous histidine kinase SLN1 (ref. 10), providing direct evidence that CRE1 is a cytokinin receptor. We also provide evidence that cytokinins can activate CRE1 to initiate phosphorelay signalling.

The Cytokinin-hypersensitive genes of Arabidopsis negatively regulate the cytokinin-signaling pathway for cell division and chloroplast development.

We isolated Arabidopsis thaliana mutants that respond more sensitively than the wild type to cytokinins. The calli produced from the mutants exhibit typical cytokinin responses, including rapid proliferation and chloroplast development in response to lower levels of cytokinins than in the wild type. The mutations are recessive and belong to two complementation groups designated ckh1 and ckh2 for cytokinin-hypersensitive. CKH1 and CKH2 were mapped to the top of chromosome I and the middle of chromosome II, respectively. The cytokinin levels in these mutants were not increased. We speculate that the CKH1 and CKH2 gene products negatively regulate the signaling pathway leading from cytokinin perception to cell proliferation and chloroplast development.

Biochemical characterization of a putative cytokinin-responsive His-kinase, CKI1, from Arabidopsis thaliana.

His-Asp phosphorelays are evolutionary-conserved powerful biological tactics for intracellular signal transduction. Such a phosphorelay is generally made up of "sensor histidine (His)-kinases", "response regulators", and "histidine-containing (HPt) phosphotransmitters". Results from recent intensive studies suggested that in the higher plant Arabidopsis thaliana, His-Asp phosphorelays may be widely used for propagating environmental stimuli, such as phytohormones (e.g., ethylene and cytokinin). In this study, we characterized, in vitro, the putative cytokinin-responsive CKI1 His-kinase, in terms of His-Asp phosphorelays. It was demonstrated for the first time that the receiver domain in this sensor exhibits a strong phosphohistidine phosphatase activity toward some Arabidopsis HPt phosphotransmitters (AHP1 and AHP2), suggesting the functional importance of the receiver domain for a resumed interaction of the sensor His-kinase with other His-Asp phosphorelay components.

Cytokinin signaling.

Although cytokinin plays a central role in plant development, our knowledge of the biosynthesis, distribution, perception and signal transduction of cytokinin is limited. Recent molecular-genetic studies have, however, implicated involvement of a two-component system in cytokinin signal transduction. Furthermore, new mutants with altered cytokinin responses and genes involved in cytokinin signaling have been identified.

Gibberellin A3 causes a decrease in the accumulation of mRNA for ACC oxidase and in the activity of the enzyme in azuki bean (Vigna angularis) epicotyls.

Differential screening, aimed at the isolation of cDNA clones of mRNAs whose accumulation is influenced by GA3, resulted in the isolation of a cDNA clone of an mRNA whose level was decreased by GA3 in segments of epicotyls of Vigna angularis. The putative protein encoded by this cDNA resembled the 1-aminocyclopropane-1-carboxylate oxidases (ACC oxidases) identified in other plant species (about 80% homology at the amino acid level). Thus, the corresponding gene was designated AB-ACO1 (azuki bean ACC oxidase). GA3 also decreased the activity of ACC oxidase in azuki bean epicotyls, but it did not decrease the rate of ethylene evolution. In fact, GA3 increased the rate of ethylene evolution and the level of ACC. Thus, GA3 seemed to increase the production of ethylene by promoting the synthesis of ACC.

CKI1, a histidine kinase homolog implicated in cytokinin signal transduction.

Although cytokinin plays a central role in plant development, little is known about cytokinin signal transduction. Five Arabidopsis thaliana mutants that exhibit typical cytokinin responses, including rapid cell division and shoot formation in tissue culture in the absence of exogenous cytokinin, were isolated by activation transferred DNA tagging. A gene, CKI1, which was tagged in four of the five mutants and induced typical cytokinin responses after introduction and overexpression in plants, was cloned. CKI1 encodes a protein similar to the two-component regulators. These results suggest that CKI1 is involved in cytokinin signal transduction, possibly as a cytokinin receptor.

Exercise performance after PTMC in mitral stenosis: the relation with hemodynamics, ventilatory response and skeletal muscle function.

We examined the contribution of hemodynamics, ventilatory response and skeletal muscle function to the exercise performance after percutaneous transvenous mitral commissurotomy (PTMC). Nine patients with mitral stenosis (MS) underwent symptom-limited maximal ergometer exercise before, at 1 week and 3 months after PTMC. At 3 months after PTMC four patients (group 1) revealed an improved exercise performance (peak oxygen uptake (VO2) 747 +/- 145 to 1,039 +/- 175 ml/min; p < 0.01), and five patients (group 2) showed an unchanged exercise performance. Exercise performance in group 1 already increased at 1 week after PTMC. Peak cardiac output (Q) in group 1 increased (4.9 +/- 0.7 to 6.9 +/- 1.0 L/min, p < 0.05) at 3 months after PTMC. The slope of the minute ventilation (VE)-carbon dioxide production (VCO2) relation during exercise decreased at 1 week after PTMC (40.2 +/- 4.2 to 30.5 +/- 3.8, p < 0.05), and decreased slope of VE-VCO2 remained at 3 months after PTMC. However in group 2 these parameters did not change. There was a significant correlation between percent change of peak VO2 and percent change of peak Q from before to 3 months after PTMC (r = 0.84, p < 0.01). However there were no differences in reduction of mean pulmonary artery pressure, pulmonary vascular resistance and maximum isometric force (MIF) of left quadriceps between two groups. These findings suggest that the increased Q and improved excessive ventilation were important for increase in exercise performance in patients with MS after PTMC.

Interleukin-6 receptor expression in the peripheral B cells of patients with multicentric Castleman's disease.

Interleukin-6 (IL-6) is an important regulator of terminal B-cell differentiation. Inappropriate oversynthesis of IL-6 may play primary role in the pathogenesis of multicentric Castleman's disease (MCD). We investigated the expression of the IL-6 receptor (IL-6R) in peripheral B cells from three patients with MCD, as well as the responsiveness of these cells to IL-6. Flow-cytometric analysis showed that IL-6R was significantly expressed on the peripheral B cells of two of three patients. The B cells expressing IL-6R spontaneously produced increased levels of immunoglobulin G (IgG). IL-6R-expressing B cells from one patient showed hyper-responsiveness to IL-6.

The presence of CD28-negative T cells in a patient with multicentric Castleman's disease.

We found increased numbers of CD28-negative T cells in a patient with multicentric Castleman's disease (MCD), who also had significantly decreased interleukin-2 (IL-2) production and impaired T-cell proliferation. The presence of CD28-negative T cells may be indicative of a functional T-cell defect in MCD.

Changes in peripheral plasma luteinizing hormone and testosterone concentrations and semen quality in normal and cryptorchid dogs during sexual maturation.

Changes in peripheral plasma luteinizing hormone (LH) and testosterone (T) concentrations and semen quality were investigated in four unilaterally cryptorchid (UC), three bilaterally cryptorchid (BC), and five normal beagles, from 4 to 48 weeks of age. Blood samples were collected at 2- or 4-week intervals, and the LH and T concentrations were determined by radioimmunoassay. Semen samples were collected weekly by digital manipulation. The mean LH and T concentrations in the normal dogs increased after 20 and 22 weeks of age and reached approximately 4 ng/ml and 3 ng/ml respectively at 36 weeks of age. Semen volume and total spermatozoa count increased apparently at 34 and 35 weeks of age respectively. Spermatozoa motility and percentage of abnormal spermatozoa varied less after 38 weeks of age. Plasma LH and T concentrations in the UC and BC dogs had a tendency to remain lower than values in the normal dogs after 26 weeks of age. The semen volume and spermatozoa count in the UC dogs did not increase markedly. The spermatozoa count was less than half that in normal dogs, and the percentage of abnormal spermatozoa was higher throughout the experimental period. All BC dogs had azoospermia. Thus it was evident that development of spermatogenic function during sexual maturation in the UC and BC dogs was insufficient compared with that in normal dogs.

Bioactive glass promoted formation of nodules in periodontal-ligament fibroblasts in vitro.

The effects of bioactive glass and vitamin D3 on cultured fibroblasts derived from periodontal-ligament, with respect to their proliferation and alkaline-phosphatase activity were studied. The cells were cultured with or without the bioactive glass and/or vitamin D3, then the number and alkaline-phosphatase activity of the cells were measured periodically until the 33rd day. Formation of mineralized deposits was assessed by staining with alizarin red and von Kossa staining techniques. Fewer fibroblasts grew when they were cultured in the presence of bioactive glass and/or vitamin D3 as compared to those cultures without them. Alkaline-phosphatase activity was greater in the fibroblasts cultured with bioactive glass and vitamin D3 than in the cells grown without them. Mineralized deposits assessed by alizarin red and von Kossa staining techniques were observed microscopically around the fibroblasts cultured with bioactive glass and/or vitamin D3. A nodule visible after drying was evident only when both bioactive glass and vitamin D3 were present in culture. The results showed that although the bioactive glass and vitamin D3 decreased cell proliferation, they increased the alkaline-phosphatase activity of the fibroblasts which formed a nodule, suggesting an effect which might be useful for implant materials.

Is ST/HR slope a predictor of coronary artery lesion in effort angina pectoris? A comparison of exercise hemodynamic variables.

This study evaluated the clinical value of the ST/HR slope and exercise hemodynamic variables for the severity of coronary artery lesions. Twenty-nine patients with effort angina pectoris underwent ergometer exercise during right side heart catheterization. The ST/HR slope failed to differentiate three groups stratified by the number of involved coronary vessels. The ST/HR slope was uncorrelated with the Gensini score (r = 0.24). By contrast, pulmonary capillary pressure (PCP), cardiac output index (CI) and CI/PCP at peak exercise correlated significantly with the Gensini score (r = 0.73, 0.53 and 0.69, respectively). We conclude that the ST/HR slope is not a useful predictor of the severity of coronary artery lesions, while exercise hemodynamic variables measured by right side heart catheter are useful.

[Applicability to exercise test of thermodilution technique for right ventricular ejection fraction].

To assess the reliability of right ventricular ejection fraction (RVEF) during exercise measured by thermodilution technique using a modified Swan-Gantz catheter with a fast-response thermister, we measured RVEF under several conditions in 19 patients with cardiac disease. Measurements were repeated 5 times in each condition, and average RVEF and coefficient of variation (CV) were evaluated. 1) Injectate volume did not affect RVEF and CV. 2) A reduction in RVEF occurred with the thermister moved from proximal portion to distal portion within the pulmonary artery. 3) There were no differences in measurements of RVEF and CV between those during spontaneous breathing and those during apnea. 4) Postural change from supine to sitting decreased RVEF (38 +/- 8 to 35 +/- 9%; p < 0.05) and increased CV (7 +/- 2 to 13 +/- 5%). 5) Exercise increased RVEF (35 +/- 9 to 37 +/- 10%; p < 0.05) but did not change CV (13 +/- 5 vs 13 +/- 5%) compared with rest in the sitting position. 6) Cardiac rhythm (sinus vs atrial fibrillation) did not affect CV. 7) Average value of RVEF and CV during exercise were not different among 3, 4, 5 times repeated measurements. We considered that thermodilution technique for RVEF was applicable to exercise test, and 3 measurements were enough to determine the average value of RVEF during exercise.

Instability of F-actin in the absence of ATP: a small amount of myosin destabilizes F-actin.

The effects of the neutral salt concentration, pH, and coexistence of myosin on the denaturation of F-actin without ATP at low temperature were studied using the DNase I inhibition assay. The percent denaturation of F-actin gradually increased with a decrease in pH from 8.0 to 5.2, on incubation for 2 weeks in the presence of 50 mM KCl at 0 degrees C. This change was much faster in 0.5 M KCl and more than 75% of the F-actin became denatured on incubation for 1 week at pH 5.2. The buffer composition was found to exert a strong influence on the denaturation of F-actin. That is, there was a tendency for the denaturation of F-actin at pH 6.0 to be faster in MES[2-(N-morpholino)ethanesulfonic acid]-NaOH buffer than in sodium phosphate buffer, the critical concentrations of actin in 0.5 M KCl being 0.31 mg/ml for MES-NaOH buffer and 0.15 mg/ml for sodium phosphate buffer. A sigmoidal relationship was found between the percent denaturation of F-actin and the KCl concentration added, the greatest change occurring at KCl concentrations between 0.25 and 0.75 M. The time courses of the denaturation of F-actin showed that the percent denaturation rose at first and that in time the rate of the increase decreased. In the case of pH 8.0 and 0.5 M KCl, it took about 1 week for the denaturation rate to begin to drop. The pH of 6.0 further promoted the instability of F-actin exposed to high KCl concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of the blood flow during exercise in chronic heart failure--compensatory mechanism to the decreased cardiac output.

To evaluate the blood flow distribution during exercise, 51 patients with chronic heart failure underwent ergometer exercise testing measuring cardiac output and leg blood flow. At the given workrate (10 watts and 25 watts) cardiac index (L/min/m2) was significantly lower in NYHA class III patients than class I patients (at 10 watts, 4.08 +/- 1.05 in class I, 4.01 +/- 1.29 in class II and 3.00 +/- 0.89 in class III, p less than 0.05; I vs III), while leg blood flow (L/min/m2) was similar among 3 groups (at 10 watts, 1.19 +/- 0.32, 1.29 +/- 0.25 and 1.16 +/- 0.29, ns). Consequently, residual blood flow (L/min/m2) was significantly lower in class III than class I (at 10 watts, 2.89 +/- .92 and 2.78 +/- 1.27 and 1.84 +/- 0.71, p less than 0.05: I vs III). The results at 25 watts were similar. Serum noradrenaline was significantly higher in class III patients than class I patients at both 10 and 25 watts. We concluded that in severe heart failure, agreater blood flow is distributed to the working leg muscle as compared with less severe heart failure. And such an increased distribution of blood flow to working leg plays a role to compensate an insufficient cardiac output response in patients with severe heart failure.

Determination of diltiazem hydrochloride enantiomers in dog plasma using chiral stationary-phase liquid chromatography.

The separation and determination of d- and l-diltiazem hydrochloride in dog plasma by a two-column high-performance liquid chromatographic technique are described. Diltiazem hydrochloride and its metabolites were extracted from dog plasma and analyzed on a conventional column (Nucleosil 5C18) with a volatile buffer system. The column effluent of diltiazem hydrochloride was collected and evaporated. The enantiomeric ratio of the collected diltiazem was determined using a chiral column (Chiralcel OC). The method was accurate and sensitive.

Separation of beta-lactam antibiotics by micellar electrokinetic chromatography.

The retention behaviour of beta-lactam antibiotics in micellar electrokinetic chromatography (EKC) was investigated. Sodium dodecyl sulphate (SDS) and sodium N-lauroyl-N-methyltaurate were used an anionic surfactants at concentrations of 0.05-0.3 M. It was found that the retention of ionic substances in micellar EKC is determined by the following three factors: the electrophoretic migration of the ionic substances, the interaction between the ionic substances and ionic surfactants and solubilization of the solute by the micellar phase. A difference in the retention behaviours of cationic substances was observed between the two anionic surfactants, which have different groups neighbouring the charge-bearing groups. The effect of an ion-pairing reagent was also investigated to make the effect of the micelle clearer. All test solutes were successfully separated by micellar EKC at SDS concentrations above 0.1 M, with theoretical plate numbers ranging from 70,000 to 260,000.