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Notes: Abstract: cDNA clones were isolated from cultured fibro-blasts of a patient previously reported as having GM2-gangliosidosis due to defective processing of the precursor β-hexosaminidase α chain. Sequence analysis of a clone containing the entire protein coding sequence showed a single nucleotide substitution, from G to A, at nucleotide residue no. 1444, which resulted in a change in amino acid residue no. 482, from the normal glutamic acid to lysine. This transversion was confirmed in two other cDNAs from the same unamplified library. The results collectively indicate that the change from the strongly negative to strongly positive charge at amino acid residue no. 482 is responsible for the defective processing of the enzyme in this patient.

Notes: Abstract: By using a sensitive method, we assayed lysocom-pounds of gangliosides and asialogangliosides in tissues from four patients with GM2 gangliosidosis (one with Sand-hoff disease and three with Tay-Sachs disease) and from three patients with GM1 gangliosidosis [one with infantile type (fetus), one with late-infantile, and one with adult type]. In the brain and spinal cord of all the patients except for an adult GM 1 gangliosidosis patient, abnormal accumulation of the lipids was observed, though the concentration in the fetal tissue was low. In GM2 gangliosidosis, the amounts of lyso GM2 ganglioside accumulated in the brain were similar among the patient with Sandhoff disease and the patients with Tay-Sachs disease, whereas the concentration of asialo lyso GM2 ganglioside in the brain was higher in the former patient than in the latter patients. By comparing the sphingoid bases of neutral sphingolipids, gangliosides, and lysosphingolipids, it was suggested that lysosphingolipids in the diseased tissue are synthesized by sequential glycosylation from free sphingoid bases, but not by deacyla-tion of the sphingolipids. Because lysosphingolipids are known to be cytotoxic, the abnormally accumulated lysosphingolipids may well be the pathogenetic agent for the neuronal degeneration in gangliosidoses.

Notes: MATSUOKA, K., et al.: Electrophysiological Features of Atrial Tachycardia Arising from the Atrioven-tricular Annulus. Atrial tachycardia (AT) arises from various sites in the atrium and the mechanisms are nonuniform. McGuire et al. reported that the cells around the atrioventricular annuli resembled nodal cells in their cellular electrophysiology. The purpose of this study was to delineate the electrophysiological features of AT arising from the atrioventricular (AV) annulus (AVAT). The study included five patients with six AVATs that were abolished by the radiofrequency energy delivery. The location of the AV annuli was defined by using the AV ratio of the local electrograms and the amplitude of the ventricular electrograms, in addition to the anatomic findings under fluoroscopic guidance. The tachycardia cycle lengths were 403 ± 117 ms. An AV ratio of the electrograms at the successful ablation sites was 0.4 ± 0.4 at the tricuspid annulus and 1.5 ± 0.3 at the mitral annulus. Small doses (mean 3.2 ± 1.8 mg) of adenosine triphosphate could terminate all the tachycardia episodes for five of the ATs without the development of AV nodal conduction block. The successful ablation sites were located at the right mid-septum in 1 AT, right posteroseptum in 2 ATs, right posterolateral region in 1 AT, and left anteroseptum in 2 ATs. These findings suggest that the cells with nodal-type action potentials around both annuli might play an important role in the genesis of AVAT.

Notes: Radiofrequency (RF) catheter ablation of supraventricular tachycardias causes local parasympathetic denervation. This study used heart rate variability (HRV) to evaluate the effects of ablation of atrial tachycardia (AT) arising from the atrioventricular annulus (AVAT) on autonomic function. Ten patients with AVAT were referred for ablation (group AT) and compared with 8 patients with paroxysmal atrial fibrillation who underwent PV isolation (group Paf), and 13 patients with idiopathic ventricular tachycardia successfully treated by ablation (group VT). Time and frequency domain analysis of HRV on 24-hour ambulatory ECG recordings was performed before and after ablation. Root mean square of differences of consecutive N-N intervals (rMSSD), percentage of difference between consecutive N-N intervals 〉50 ms (pNN50), and high frequency (HF) component were measured to examine the effects on parasympathetic nerve activity. In group AT, rMSSD, pNN50, and HF decreased significantly after ablation, while they remained unchanged in group Paf and group VT. These observations suggest that parasympathetic denervation after ablation was limited to group AT, and depended on the site of energy delivery along the tricuspid or mitral valve as opposed to atrial or ventricular muscle.

Notes: The purpose of this study was to compare the electrophysiological characteristics of posterior and anterior atrioventricular junctional reentrant tachycardia (AVJRT) during radiofrequency (RF) catheter ablation of a slow pathway. Twenty-four patients with common A VJRT, including 4 posterior (P) and 20 anterior AVJRT (A) were studied. We analyzed the retrograde atrial activation sequence of junctional rhythm and the presence of transient HA block during slow pathway ablation. When HA block developed, the AH interval before ablation and immediately after the end of energy delivery was measured. Successful ablation sites were divided into three groups; high (H), middle (M), and low (L) from the His bundle to the floor of the coronary sinus orifice. The results were: (1) the number of successful ablation sites were H 0, M 1, L 3 in P and H 1, M 8, L 11 in A; (2) the HA interval during AVJRT in P was longer than that in A (109 ± 48 ms vs 43 ± 6 ms, P 〈 0.01); (3) the retrograde atrial activation sequence during Junctional rhythm was strictly concordant with that during AVJRT in both groups, but HA block developed during slow pathway ablation more often in P than in A (100% vs 30%, P 〈 0.01); and (4) The AH interval did not lengthen after HA block developed in P. These data suggest that another pathway does exist from the A V node to the atrium in addition to anterograde fast pathway and slow pathway, and that this pathway is used as the retrograde limb of P.

Notes: Radiofrequency (RF) catheter ablation of supraventricular tachycardias (SVT) has been shown to result in local parasympathetic denervation. The purpose of this study was to estimate the correlation between RF cumulative energy and parasympathetic denervation at three different ablation sites. Methods: 45 patients who underwent RF ablation of 36 AV reentrant tachycardias and 9 AV nodal reentrant tachycardias were studied. Twenty patients had left free-wall accessory pathways (group L), 8 patients right free-wall accessory pathways (group R), and 17 patients septal accessory path ways (n = 8) or slow pathways (n -9)(groupS). Time and frequency domain analysis of heart rate variability on 24-hour ambulatory ECG recordings was performed before and after RF ablation, pNN50 and the high frequency (0.15 to 0.40 Hz, HF) component were measured to examine the effects on parasympathetic nerve activity. The values of Δ pNN50 and Δ HF were expressed as the percent change of pNN50 and HF that occurred after versus before RF ablation. Results: Both pNN50 and HF significantly decreased after RF ablation in all three groups. In group S, there was a significant correlation between RF cumulative energy and Δ pNN50r = 0.66, P 〈 0.01) or Δ HF (r = 0.58, P 〈 0,05). In contrast, there was no correlation between RF cumulative energy and Δ pNNSO or Δ HF in either group L or group R. Conclusion: These data suggest that RF ablation produces parasympathetic denervation at all three sites along the mitral or tricuspid annulus and that parasympathetic fibers may be located predominantly in the septal area.

Notes: CND41, a DNA binding protein of chloroplast nucleoids, may function as a negative regulator of chloroplast gene expression. The reduction of CND41 in an antisense transformant accelerated plastid development in shoot apex cells and early young leaves, and caused a dwarf phenotype and altered leaf morphology. Plant height and leaf shape could be restored almost to those of the wild type by application of gibberellins (GAs), clearly indicating that a reduction in GA content was a prime cause of the dwarf phenotype in CND41 antisense transformants. The transformants had reduced endogenous levels of active gibberellin (GA1), a biologically active GA, compared to those of wild-type plants. Possible relationships between chloroplast development affected by CND41 function and GA biosynthesis are discussed.

Notes: [Auszug] Fas is the death receptor, transducing cell death signaling upon stimulation by Fas ligand. During Fas-initiated cell death signaling, the formation of Fas-death inducing signaling complex (Fas-DISC) is the first step. Here we have identified a new component of Fas-DISC which we call ...

Notes: Abstract The present study was done to identify and characterize the isoenzymes of cyclic nucleotide phosphodiesterase (PDE) and to determine their intracellular distribution in human kidney and heart. The in vitro effects of new cardiotonic agents, namely, NSP-805 (4,5-dihydro-5-methyl-6-[4-[(2-methyl-3-oxo-1-cyclopentenyl)amino] phenyl]-3(2H)-pyridazinone), TZC-5665 (6-[4-[2-[3-(5-chloro-2-cyanophenoxy)-2-hydroxypropylamino]-2-methylpropylamino]phenyl]-5-methyl-4,5-dihydro-3(2 H)-pyridazinone) and its metabolites, OPC-18790 ((±)-6-[3-(3,4-dimethoxybenzylamino)-2-hydroxypropoxy]-2-(1H)-quinolinone), MS-857 (4-acetyl-l-methyl-7-(4-pyridyl)-5,6,7,8-tetrahydro-3(2H)-isoquinolinone) and E-1020 (1,2-dihydro-6-methyl-2-oxo-5-(imidazo[1,2-a]pyridin-6yl)-3-pyridine carbonitrile hydrochloride monohydrate), on these human PDE isoenzymes were also investigated. PDE isoenzymes were separated from cytosolic and particulate fractions of homogenates of human kidney and heart by DEAE-Sepharose chromatography. PDE isoenzymes were identified by their elution characteristics, substrate specificities, sensitivities to regulation by effectors and by the use of isoenzyme-specific inhibitors. In a cytosolic fraction from kidney, Ca2+/calmodulin-dependent PDE (CaM-PDE), cyclic GMP-stimulated PDE (cGS-PDE), cyclic GMP-inhibited PDE (cGI-PDE) and two forms of cyclic AMP-specific PDE (cAMP-PDE) were resolved. One form of cAMP-PDE (cAMP-PDEα), which was eluted at a lower ionic strength than cGI-PDE during DEAE-Sepharose chromatography, was newly recognized in human tissues, though the other form (cAMP-PDEβ), which eluted later than cGI-PDE, had been previously isolated. Both of these cAMP-specific PDEs had similar properties in that they predominantly hydrolyzed cAMP with similar K m values for cAMP and were inhibited to almost equal extents by 3-isobutyl-l-methylxanthine (IBMX) but were hardly inhibited by cGMP. However, cAMP-PDEα was inhibited about 10 times more weakly (on the basis of IC50 values) by rolipram (4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone) and Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone than was cAMP-PDEβ. In a cytosolic fraction from heart ventricle, four distinct PDE isoenzymes, CaM-PDE, cGS-PDE, cGI-PDE and cAMP-PDEβ, were recognized. cAMP-PDEβ was the major component of the cAMP-hydrolyzing activity in the cytosolic fraction from human kidney, while CaM-PDE and cGI-PDE represented more than 90% of the total cAMP phosphodiesterase activity in the cytosolic fraction from human heart. In the particulate fractions from human kidney and heart, three activities, those of cGI-PDE and of two forms of cAMP-PDE, were identified. In kidney, cAMP/PDEα was the main cAMP-hydrolyzing PDE, while in heart cGI-PDE accounted for most of the activity. Furthermore, we evaluated the inhibitory effects on these human PDE isoenzymes of newly synthesized compounds with inotropic effects, namely, NSP-805, metabolites of TZC-5665 referred to as M-1 (6-(4-aminophenyl)-5-methyl-4,5-dihydro-3(2H)-pyridazinone) and M-2 (6-(4-acetyl-aminophenyl)-5-methyl-4,5-dihydro-3(2H)-pyridazinone), OPC-18790, MS-857 and E-1020. These drugs potently inhibited the activity of cGI-PDE and very weakly inhibited the activities of CaM-PDE and cGS-PDE. With respect to inhibitory effects on cardiac cAMP-PDEβ, there were some differences between the pyridazinone derivatives, for example NSP-805 and M-2, and the nonpyridazinone derivatives OPC-18790, MS-857 and E-1020. From the IC50 values, it was clear that the pyridazinone derivatives inhibited the activity of cGI-PDE at concentrations that were two to four orders of magnitude lower than that required for the inhibition of the activity of cAMP-PDEβ, while for the nonpyridazinone derivatives the difference was about one order of magnitude. The inhibition of the activity of human cardiac cGI-PDE by NSP-805, M2, OPC-18790, MS-857 and E-1020 was competitive with respect to cAMP with K i values of 0.012, 0.32, 0.42, 1.3 and 0.15 μmol/l, respectively. These results suggest that there may be two isoforms of cAMP-PDE, which exist not only in the particulate fraction but also in the cytosolic fraction of human tissues, and that PDE inhibitors, which exert their cardiotonic effects by inhibiting cGI-PDE, have different selectivities with respect to the inhibition of the other human PDE isoenzymes.

Notes: Abstract. The present study was done to identify and characterize the isoenzymes of cyclic nucleotide phosphodiesterase (PDE) and to determine their intracellular distribution in human kidney and heart. The in vitro effects of new cardiotonic agents, namely, NSP-805 (4,5-dihydro-5-methyl-6-[4-[(2-methyl-3-oxo-1-cyclopentenyl)amino]phenyl]-3(2H)-pyridazinone), TZC-5665 (6-[4-[2-[3-(5-chloro-2-cyanophenoxy)-2-hydroxypropylamino]-2-methylpropylamino]phenyl]-5-methyl-4,5-dihydro-3(2H)-pyridazinone) and its metabolites, OPC-18790 ((±)-6-[3-(3,4-dimethoxybenzylamino)-2-hydroxypropoxy]-2-(1H)- quinolinone), MS-857 (4-acetyl-1-methyl-7-(4-pyridyl)-5,6,7,8-tetrahydro-3(2H)-isoquinolinone) and E-1020 (1,2-dihydro-6-methyl-2-oxo-5-(imidazo[1,2-a]pyridin-6-yl)-3-pyridine carbonitrile hydrochloride monohydrate), on these human PDE isoenzymes were also investigated.   PDE isoenzymes were separated from cytosolic and particulate fractions of homogenates of human kidney and heart by DEAE-Sepharose chromatography. PDE isoenzymes were identified by their elution characteristics, substrate specificities, sensitivities to regulation by effectors and by the use of isoenzyme-specific inhibitors.   In a cytosolic fraction from kidney, Ca2+/calmodulin-dependent PDE (CaM-PDE), cyclic GMP-stimulated PDE (cGS-PDE), cyclic GMP-inhibited PDE (cGI-PDE) and two forms of cyclic AMP-specific PDE (cAMP-PDE) were resolved. One form of cAMP-PDE (cAMP-PDEα), which was eluted at a lower ionic strength than cGI-PDE during DEAE-Sepharose chromatography, was newly recognized in human tissues, though the other form (cAMP-PDEβ), which eluted later than cGI-PDE, had been previously isolated. Both of these cAMP-specific PDEs had similar properties in that they predominantly hydrolyzed cAMP with similar K m values for cAMP and were inhibited to almost equal extents by 3-isobutyl-1-methylxanthine (IBMX) but were hardly inhibited by cGMP. However, cAMP-PDEα was inhibited about 10 times more weakly (on the basis of IC50 values) by rolipram (4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone) and Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone than was cAMP-PDEβ. In a cytosolic fraction from heart ventricle, four distinct PDE isoenzymes, CaM-PDE, cGS-PDE, cGI-PDE and cAMP-PDEβ, were recognized. cAMP-PDEβ was the major component of the cAMP-hydrolyzing activity in the cytosolic fraction from human kidney, while CaM-PDE and cGI-PDE represented more than 90% of the total cAMP phosphodiesterase activity in the cytosolic fraction from human heart. In the particulate fractions from human kidney and heart, three activities, those of cGI-PDE and of two forms of cAMP-PDE, were identified. In kidney, cAMP-PDEα was the main cAMP-hydrolyzing PDE, while in heart cGI-PDE accounted for most of the activity. Furthermore, we evaluated the inhibitory effects on these human PDE isoenzymes of newly synthesized compounds with inotropic effects, namely, NSP-805, metabolites of TZC-5665 referred to as M-1 (6-(4-aminophenyl)-5-methyl-4,5-dihydro-3(2H)-pyridazinone) and M-2 (6-(4-acetyl-aminophenyl)-5-methyl-4,5-dihydro-3(2H)-pyridazinone), OPC-18790, MS-857 and E-1020. These drugs potently inhibited the activity of cGI-PDE and very weakly inhibited the activities of CaM-PDE and cGS-PDE. With respect to inhibitory effects on cardiac cAMP-PDEβ, there were some differences between the pyridazinone derivatives, for example NSP-805 and M-2, and the nonpyridazinone derivatives OPC-18790, MS-857 and E-1020. From the IC50 values, it was clear that the pyridazinone derivatives inhibited the activity of cGI-PDE at concentrations that were two to four orders of magnitude lower than that required for the inhibition of the activity of cAMP-PDEβ, while for the nonpyridazinone derivatives the difference was about one order of magnitude. The inhibition of the activity of human cardiac cGI-PDE by NSP-805, M2, OPC-18790, MS-857 and E-1020 was competitive with respect to cAMP with K i values of 0.012, 0.32, 0.42, 1.3 and 0.15 μmol/l, respectively.   These results suggest that there may be two isoforms of cAMP-PDE, which exist not only in the particulate fraction but also in the cytosolic fraction of human tissues, and that PDE inhibitors, which exert their cardiotonic effects by inhibiting cGI-PDE, have different selectivities with respect to the inhibition of the other human PDE isoenzymes.