The promyelocytic leukemia protein stimulates SUMO conjugation in yeast.

The promyelocytic leukemia gene was first identified through its fusion to the gene encoding the retinoic acid receptor alpha (RARalpha) in acute promyelocytic leukemia (APL) patients. The promyelocytic leukemia gene product (PML) becomes conjugated in vivo to the small ubiquitin-like protein SUMO-1, altering its behavior and capacity to recruit other proteins to PML nuclear bodies (PML-NBs). In the NB4 cell line, which was derived from an APL patient and expresses PML:RARalpha, we observed a retinoic acid-dependent change in the modification of specific proteins by SUMO-1. To dissect the interaction of PML with the SUMO-1 modification pathway, we used the budding yeast Saccharomyces cerevisiae as a model system through expression of PML and human SUMO-1 (hSUMO-1). We found that PML stimulated hSUMO-1 modification in yeast, in a manner that was dependent upon PML's RING-finger domain. PML:RARalpha also stimulated hSUMO-1 conjugation in yeast. Interestingly, however, PML and PML:RARalpha differentially complemented yeast Smt3p conjugation pathway mutants. These findings point toward a potential function of PML and PML:RARalpha as SUMO E3 enzymes or E3 regulators, and suggest that fusion of RARalpha to PML may affect this activity.

Protease-activated receptor-1 (thrombin receptor) is expressed in mesenchymal portions of human hair follicle.

Protease nexin-1, a serine protease inhibitor, is expressed specifically in the dermal papilla (DP) of anagen hair follicles and is suggested to be one of the modulators of the cyclic growth of hair follicles. Accumulating evidence has shown that protease nexin-1 plays its biologic role by inhibiting thrombin action in various systems other than the hair follicle. Thrombin has various physiologic functions including blood coagulation cascade, mostly via activation of protease-activated receptors (PAR). In this study, we investigated the expression of PAR mRNA using RT-PCR in dissected human hair follicles. We showed that PAR-1 mRNA was expressed specifically in the mesenchymal portions, including DP and connective tissue sheath, of anagen hair follicles. Furthermore, immunoreactivity for PAR-1 was detected in the DP and lower portion of connective tissue sheath in the anagen and catagen phases and in the DP of telogen hair follicles. Because only a pharmacologic level (100 nM) of thrombin significantly stimulated cell proliferation and DNA synthesis of the cultured dermal papilla cells, thrombin does not seem to have a mitogenic effect on dermal papilla cells physiologically. These results raise the possibility that thrombin is involved in the cyclic hair growth through its receptor of PAR-1.

The stabilization mechanism of mutant-type p53 by impaired ubiquitination: the loss of wild-type p53 function and the hsp90 association.

Mutant-type p53 (mt p53) is largely accumulated in cancer cells due to its increased stability. To elucidate the mechanism of mt p53 stabilization, we analysed the turnover of p53 mutated at codon 248 whose alteration is most frequently found in human cancers. Proteasome inhibition induced the accumulation of ubiquitinated mt p53, indicating that the ubiquitinated forms were essentially unstable and degraded by the proteasome. The presence of a small amount of the ubiquitinated mt p53 relative to the abundant non-ubiquitinated form suggested that the mt p53 ubiquitination was a rate-limiting process in the slow turnover. Two phenomena destabilizing mt p53 via the ubiquitin-proteasome degradation were proved to be independent. First, the coexpression of wild-type p53 (wt p53) promoted mt p53 destabilization as feedback regulation. Second, geldanamycin also induced mt p53 destabilization through the dissociation of the protein from hsp90 but not through the restoration of wt p53 function. Neither the mutant-specific conformation nor the N-terminal phosphorylation seemed to contribute directly to the mt p53 stabilization. Further, a two-dimensional gel electrophoresis revealed that most of the post-translationally modified mt p53 was equally subjected to ubiquitination and subsequent proteasomal degradation. These findings are evidence that mt p53 stabilization depends on the impaired ubiquitination due to both the loss of wt p53 function and the hsp90 association.

Identification of Endogenous Gibberellins in the Leaves and Xylem Sap of Tea Plants.

Endogenous gibberellins (GAs) in the young leaves and xylem sap of tea plants (Camellia sinensis L.) were analyzed by GC-MS. The following GAs were identified by comparing their mass spectra and KRIs with those of authentic specimens: GA9 and GA20 in the leaves; GA9, GA12, GA15, GA20, GA44, GA51 and GA53 in the xylem sap.

Human ubiquitin-protein ligase Nedd4: expression, subcellular localization and selective interaction with ubiquitin-conjugating enzymes.

BACKGROUND: Nedd4 is a ubiquitin-protein ligase containing a calcium/lipid-binding domain, multiple WW domains and a C-terminal Hect domain, which is required for both the ubiquitin transfer and the association with E2 ubiquitin-conjugating enzymes. Nedd4 has been reported to be involved in the selective ubiquitination of some regulatory proteins in transcription and membrane transport. RESULTS: Three mRNA species for human Nedd4 were found to be 6.4-, 7.8- and 9.5-kb in size, and their expression patterns varied among normal tissues and cancer cell lines, indicating the tissue- and cell-specificities of Nedd4 expression. The Nedd4 protein, approximately 120 kDa in weight, was found in the cytoplasm, mainly in the perinuclear region and cytoplasmic periphery, of human cultured cells. Neural differentiation induced not only the down-regulation of Nedd4 but also the localization of the protein to both the cytoplasm and neurites. To identify the ubiquitination pathway that is linked to Nedd4, we demonstrated that specific E2 enzymes, including human Ubc4, UbcH5B, UbcH5C, UbcH6 and UbcH7, could transfer ubiquitin molecules to Nedd4 at the active cysteine residue, whereas E6AP accepted ubiquitins from Ubc4, UbcH5B, UbcH5C and UbcH7. Furthermore, nuclear localization of N-terminal deletion mutant Nedd4 enabled us to investigate the interaction between Nedd4 and E2 enzyme (Ubc4 or UbcH7) in the cell. The simultaneous expression of the full-length Nedd4 and E2 enzyme revealed the both proteins mostly colocalized in the cytoplasmic periphery, while the N-terminal deleted Nedd4 induced the nuclear and perinuclear colocalization with E2 enzyme. CONCLUSION: Our findings suggested that Nedd4 plays an important role in the cell regulation, including neural differentiation through cooperation with specific E2 ubiquitination pathways.

Identification of an ubiquitin-ligation system for the epidermal-growth-factor receptor--herbimycin A induces in vitro ubiquitination in rabbit-reticulocyte lysate.

Some receptor tyrosine kinases such as the receptors for epidermal-growth factor (EGF) and platelet-derived growth factor undergo polyubiquitination as a consequence of ligand binding. The EGF receptor is also ubiquitinated by treatment with herbimycin A, an ansamycin antibiotic widely used as a tyrosine kinase inhibitor. To investigate the mechanism of the receptor ubiquitination, we have established an assay system in which herbimycin-A-induced ubiquitination processes can be analyzed in vitro. We now show that herbimycin A treatment of the purified EGF receptor induces polyubiquitination of the receptor in rabbit-reticulocyte lysate. Both DEAE unadsorbed material (fraction I) and high salt eluate (fraction II) of the reticulocyte lysate are involved cooperatively in the ubiquitination process, where the ubiquitin-conjugating enzyme UBC4 can functionally substitute for fraction I. A ubiquitin-protein ligase-like activity, partially purified from fraction II by DEAE anion-exchange chromatography, also functions in concert with UBC4. The precise mechanism of herbimycin A-induced ubiquitination of the EGF receptor is not fully understood, however, our present findings suggest that direct interaction with herbimycin A results in some modification of the receptor which is recognized by the ubiquitin-conjugating system in rabbit-reticulocyte lysate.

Mouse/human sequence divergence in a region with a paternal-specific methylation imprint at the human H19 locus.

We have identified a region with characteristics of a paternal-specific methylation imprint at the human H19 locus. This region, extending from -2.0 kb upstream to the start of transcription, is heavily methylated in sperm and on the paternal allele in somatic cells. This methylation was preserved during pre-implantation. Structural analysis revealed the presence of CpG islands and a large direct repeat with a 400 bp sequence reiterated several times, but no significant sequence homology to the corresponding region of the mouse H19 gene. These findings could suggest a role for secondary DNA structure in genomic imprinting across the species, and they also present a puzzling aspect of the evolution of the H19 regulatory region in human and mouse.

Differentiation between aberrant ventricular conduction and ventricular ectopy in atrial fibrillation using RR interval scattergram.

BACKGROUND: Differentiation between aberrant ventricular conduction and ventricular ectopy during atrial fibrillation (AF) is of etiologic, prognostic, and therapeutic importance. We developed a noninvasive technique to diagnose aberrant ventricular conduction and ventricular ectopy in AF. METHODS AND RESULTS: We studied the Holter ECGs of 34 patients with paroxysmal AF and 62 patients with chronic AF. In all the patients, frequent wide QRS complexes were observed, and 32 patients were shown by electrophysiological examination to have ventricular ectopies or aberrant ventricular conductions. We obtained the RR interval scattergrams by plotting sequential pairs of RR intervals. Each point has the (n)th RR interval as its x value and the (n + 1)th RR interval as its y value. The irregularity of the RR intervals in AF resulted in widely scattered points delineated by the envelope along the axes. The y value of the envelope along the x axis indicates the shortest coupling interval to the preceding RR interval. Therefore, this curve defines the functional refractory period of atrioventricular conduction. The scattergram of the RR interval pairs immediately preceding the aberrant conduction (coupling points of aberrant conduction) specifically distributed along the envelope. In contrast, the coupling points of ventricular ectopies showed different distributions that had no relation to the envelope. That is, it included three typical patterns, ie, linear distribution below the envelope, linear distribution partially overlapped in the area of normal AF conduction, and chaotic distribution in the AF area. None of the scattergrams of ventricular ectopies showed curvilinear distribution along the envelope as aberrant conduction did. The specific distribution of the aberrant conduction on the RR interval scattergram suggested that aberrant conduction in AF could result from the difference of refractory periods between the AV node and bundle branch block. CONCLUSIONS: We conclude that the RR interval scattergram makes it possible to differentiate between aberrant ventricular conduction and ventricular ectopy in atrial fibrillation, and thus, it is a useful noninvasive clinical tool.

Arrhythmia analysis by successive RR plotting.

A successive RR interval plot was developed to analyze arrhythmia. The plot consisted of a set of points with the x-value of (N)th RR interval and the y-value of (N + 1)th RR interval. This method was applied in the arrhythmia analysis of Holter electrocardiograms obtained from 35 patients. In the analysis of ventricular premature contractions (VPCs) this method was useful not only in detecting VPCs but also in demonstrating coupling interval-dependent characteristics of VPCs. In the analysis of atrial fibrillation the successive RR plot enabled the authors to estimate the functional refractory period of the atrioventricular conduction. In conclusion, despite its simplicity, the successive RR plot was found to be powerful in analyzing arrhythmia. Specifically, the potential to analyze integrally the coupling interval-dependent properties of various types of arrhythmia makes it attractive as a clinical tool.

Neisseria gonorrhoeae dissemination and gonococcal meningitis.

Disseminated infection is a serious complication in approximately 2 percent of primary gonococcal infections. Meningeal infection is very rare; only 23 cases have been reported since 1922. We report a sexually active teenager with an acute febrile illness. From her cerebrospinal fluid cultures, Neisseria gonorrhoeae was identified. She recovered completely after treatment with ceftriaxone and penicillin. Possible explanations for gonococcal dissemination include unique strains of the organism as well as particular complement deficiencies of the host. Aggressive efforts by physicians to prevent, identify, and treat primary gonococcal diseases should continue because this will reduce the frequency of serious complications.

A Holter-tape analyzer employing circuits for calculation of correlation coefficients.

An analyzer for ventricular premature contraction (VPC) arrhythmias was developed. At 60 times real time, the analyzer processes the Holter tape in which the long-term ambulatory electrocardiogram was recorded. Template matching algorithm using Pearson product-moment correlation coefficient is employed. A microprocessor controls the analyzer. Circuits for calculation of correlation coefficients were developed to support insufficient computing speed of the microprocessor. Evaluation study shows that the sensitivity for detecting the normal QRS complexes and the VPCs were 98.9% and 99.4%, and that the specificity for these were 97.5% and 98.4%. Algorithm for high-speed calculation of correlation coefficients is also considered.

Prevalence of ventricular tachycardia in patients with different underlying heart diseases: a study by Holter ECG monitoring.

Twenty-four-hour Holter ECGs were recorded in 1089 patients. Ventricular tachycardia (VT) was observed in 184 tapes obtained from 81 patients (73 men and 8 women). Underlying heart diseases were present in 72 patients and no organic heart diseases were found in nine patients. The analysis of continuous 1-hour rhythm strips immediately before VT revealed that, in ischemic heart disease and hypertrophic cardiomyopathy, there was no correlation between the incidence of VT and the number or complexity of premature ventricular complexes (PVCs) within 1 hour before VT. In contrast, frequent or multiform PVCs were often observed during the pre-VT period in the patients with rheumatic heart disease or dilated cardiomyopathy. These findings suggest that the mechanism of VT may be different among the various underlying heart diseases. In addition, the mode of initiation of VT was evaluated. Only few episodes of VT occurred with the prematurity index value smaller than 1.0 or the vulnerability index value greater than 1.1. The correlation between the rate of VT and the preceding sinus rate was not significant, and the correlation between the rate of VT and the coupling interval of VT was weak. These facts suggest that the malignancy of VT, represented by the rate of VT, cannot be predicted by the preceding sinus rate or by the coupling interval of VT.

Clinical evidence suggesting vasospastic cause of myocardial infarction.

To examine the vasospastic cause of myocardial infarction (MI) we studied 1) the incidence of rest angina before MI, 2) clinical features of postinfarction angina and 3) the occurrence of MI in variant angina. 1) Of 178 patients with MI, 60 (34%) experienced rest angina for 1 day to 10 years before the onset of MI. The incidence of rest angina was significantly higher in patients having milder coronary stenosis of 75% or less (15/30, 50%) than in others having severe stenosis of 90% or more (45/148, 30%), p less than 0.05. 2) Postinfarction angina with ST elevation was observed in 16 patients (9%) and ST elevation developed in leads with pathological Q waves in all patients. The incidence of postinfarction angina was significantly higher in those having milder coronary stenosis than in others having severe stenosis, (27% versus 5%, p less than 0.005). Patients with postinfarction angina experienced rest angina before MI more frequently (81%) than others (29%, p less than 0.005). Sublingual nitroglycerin was effective in relieving postinfarction angina attacks and oral calcium antagonist prevented attacks in all patients. 3) MI developed in 9 of 97 patients with variant angina. Six patients had transmural and 3, non-transmural MI. Pathological Q waves and/or coronary T waves appeared in leads where ST elevation was observed during anginal attack. In 7 patients MI developed when antispastic agents were not used and in 2, when angina persisted even under treatment with calcium antagonist. These data strongly suggest that the coronary spasm can be a cause of MI in some patients.

Reflex heart rate and blood pressure changes during ST segment elevation in patients with variant angina.

Responses of heart rate and blood pressure to transient myocardial ischemia were analyzed in patients with variant angina. Heart rate changes during ST segment elevation were examined by means of a Holter ECG monitoring system. All 27 ST segment elevations from 10 patients with anterior ischemia were accompanied by an increase in heart rate by 12 +/- 2 bpm (mean +/- SEM, p less than 0.001) at peak ST segment elevation. With inferior ischemia in nine patients, heart rate decreased significantly by 4 +/- 1 bpm (n = 28, p less than 0.001). However, 9 of these 28 ST segment elevations showed a biphasic response of heart rate, that is, an initial increase and subsequent decrease. Such heart rate changes were not different between ST segment elevations with and without chest pain. With chest pain systolic blood pressure rose in anterior ischemia by 42 +/- 5 mm Hg (n = 10, p less than 0.001) but fell in inferior ischemia by 22 +/- 8 mm Hg (n = 7, p less than 0.05). We conclude that a different cardiovascular reflex occurs in response to inferior versus anterior ischemia and it is independent of chest pain.

Provocation of variant angina by alcohol ingestion.

The effect of alcohol on variant angina was studied in six patients who had a history of chest pain occurring with alcohol ingestion. On alcohol testing, Holter ECG monitoring was performed and a 12-lead ECG was recorded at the time of chest pain. In five, chest pain with ST elevation occurred 5.5 to 17.5 h after the ingestion of alcohol (100 to 150 ml as ethanol). These showed recurrent ST elevation on Holter ECG, most episodes being asymptomatic. Results of provocation testing were reproducible in all four patients in whom tests were repeated and ST elevation occurred in the same leads. No complications were observed. The Holter ECG revealed a higher heart rate after alcohol ingestion. The plasma level of alcohol was zero when angina occurred and plasma epinephrine, norepinephrine and serotonin were unchanged following alcohol ingestion. Alcohol ingestion may be a useful method of provoking variant angina, particularly in those who have a history of angina related to alcohol ingestion.