Decreased diffusion in central pontine myelinolysis.

Two patients with central pontine myelinolysis (CPM) were studied with diffusion-weighted MR imaging 1 week after onset of tetraplegia. In both patients, affected white matter showed hyperintensity on diffusion-weighted images associated with a decrease in apparent diffusion coefficient (ADC) values. In one patient studied serially, ADC values normalized by 3 weeks after tetraplegia. Early in the clinical course, diagnosis of CPM can sometimes be difficult. Hyperintensity on diffusion-weighted images may therefore have diagnostic utility. Decreased lesional ADC values support the notion that CPM is a consequence of relative intracellular hypotonicity.

14-3-3 is involved in p75 neurotrophin receptor-mediated signal transduction.

The low affinity neurotrophin receptor (p75NTR) has been shown to mediate the apoptosis signaling to neural cells. However, the specific mechanisms of intracellular signal transduction of this process are largely unknown. To understand p75NTR-mediated signal transduction, we previously identified a protein that interacts with the intracellular domain of p75NTR, and we named it p75NTR-associated cell death executor (NADE). To elucidate further the signaling mechanisms utilized by p75NTR and NADE, we screened for NADE-binding protein(s) with the yeast two-hybrid method, and we identified 14-3-3epsilon as a NADE-binding protein in vivo. To examine whether 14-3-3epsilon affects the induction of p75NTR-mediated apoptosis, wild type or various deletion mutant forms of 14-3-3epsilon were co-expressed in HEK293, PC12nnr5, and oligodendrocytes. Interestingly, transient expression of the mutant form of 14-3-3epsilon lacking the 208-255 amino acid region blocked nerve growth factor-dependent p75NTR/NADE-mediated apoptosis, although this mutant form of 14-3-3epsilon continued to associate with NADE. These results suggest that 14-3-3epsilon plays an important role in the modulation of nerve growth factor-dependent p75NTR/NADE-mediated apoptosis.

Expression of p75NTR and its associated protein NADE in the rat cochlea.

OBJECTIVES/HYPOTHESIS: To investigate the expression of the low-affinity neurotrophin receptor p75 (p75NTR) and its associated protein NADE in the cochlea of the developing and the adult rat. Studies such as this one will help to predict the functional role of p75NTR and NADE in cochlear development. STUDY DESIGN: Histochemical evaluation of p75NTR and NADE in the rat cochlea was performed. METHODS: Immunohistochemical analysis was used to localize p75NTR and NADE in the rat cochlea at postnatal (PN) days PN0, PN2, PN4, PN6, PN8, PN10, and PN13 and in the adult. Confocal laser scanning microscopy was used to analyze whole-mount specimens. RESULTS: Immunoreactivity of both p75NTR and NADE was observed in pillar cells. However, these proteins displayed reciprocal expression patterns. Expression of p75NTR was detected at PN0 and PN2, but disappeared after PN4. In contrast, NADE expression was initially detected at PN2 and persisted into adulthood. CONCLUSIONS: The neurotrophin receptor p75NTR and NADE have distinct and independent roles in developing and mature cochlea.

Negative regulation of Fas-mediated apoptosis by FAP-1 in human cancer cells.

FAP-1 (Fas-associated phosphatase-1) was previously identified as a protein that associates with a negative regulatory domain (C-terminal 15 amino acids) of Fas using the yeast 2-hybrid system. Functional analysis indicated that FAP-1 expression correlates with resistance to Fas-induced apoptosis in human cancer cells. We first generated anti-FAP-1 polyclonal antibody and confirmed the interaction of FAP-1 and Fas in vivo. FAP-1 interacted with wild-type, but not mutant, Fas (tPLV) in 293T cells after transfecting FAP-1 and Fas or its mutant. To investigate the functional role of FAP-1 in Fas-mediated signal transduction, we established stable transfectants of FAP-1 in 3 human cancer cell lines. Apoptosis assays demonstrated that cancer cells over-expressing FAP-1 increased the resistance to Fas-induced apoptosis by the anti-Fas antibody CH-11 in contrast with the wild types or the vector-transfected cells. In addition, FAP-1 regulated the activity of both caspases 3 and 8. Our data indicate a functional role for FAP-1 as a negative regulator of Fas-mediated apoptosis in human cancer cells and suggest that an additional signal-transducing molecule may be required for complete suppression of Fas-mediated apoptosis.

NADE, a p75NTR-associated cell death executor, is involved in signal transduction mediated by the common neurotrophin receptor p75NTR.

The low affinity neurotrophin receptor p75NTR can mediate cell survival as well as cell death of neural cells by NGF and other neurotrophins. To elucidate p75NTR-mediated signal transduction, we screened p75NTR-associated proteins by a yeast two-hybrid system. We identified one positive clone and named NADE (p75NTR-associated cell death executor). Mouse NADE has marked homology to the human HGR74 protein. NADE specifically binds to the cell-death domain of p75NTR. Co-expression of NADE and p75NTR induced caspase-2 and caspase-3 activities and the fragmentation of nuclear DNA in 293T cells. However, in the absence of p75NTR, NADE failed to induce apoptosis, suggesting that NADE expression is necessary but insufficient for p75NTR-mediated apoptosis. Furthermore, p75NTR/NADE-induced cell death was dependent on NGF but not BDNF, NT-3, or NT-4/5, and the recruitment of NADE to p75NTR (intracellular domain) was dose-dependent. We obtained similar results from PC12 cells, nnr5 cells, and oligodendrocytes. Taken together, NADE is the first signaling adaptor molecule identified in the involvement of p75NTR-mediated apoptosis induced by NGF, and it may play an important role in the pathogenesis of neurogenetic diseases.

A novel mammalian nuclear protein similar to Schizosaccharomyces pombe Prp1p/Zer1p and Saccharomyces cerevisiae Prp6p pre-mRNA splicing factors.

We have cloned a novel 100-kDa mammalian protein, which was recognized by an anti-peptide antibody against an epitope-containing nuclear localization signal of NF-kappaB p65 subunit. Predicted amino acid sequence of the protein is similar to those of yeast splicing factors, Prp1p/Zer1p of Schizosaccharomyces pombe and Prp6p of Saccharomyces cerevisiae. Among these proteins, tetratrico peptide repeat (TPR) motif, which mediates protein-protein interactions, is conserved, whereas leucine zipper motif is found only in the 100-kDa protein. Indirect immunofluorescent staining showed that the 100-kDa protein localized in the nucleus in HeLa cells.

Functional interaction of Fas-associated phosphatase-1 (FAP-1) with p75(NTR) and their effect on NF-kappaB activation.

The common neurotrophin receptor p75(NTR), a member of the tumor necrosis factor (TNF) receptor superfamily, plays an important role in several cellular signaling cascades, including that leading to apoptosis. FAP-1 (Fas-associated phosphatase-1), which binds to the cytoplasmic tail of Fas, was originally identified as a negative regulator of Fas-mediated apoptosis. Here we have shown by co-immunoprecipitation that FAP-1 also binds to the p75(NTR) cytoplasmic domain in vivo through the interaction between the third PDZ domain of FAP-1 and C-terminal Ser-Pro-Val residues of p75(NTR). Furthermore, cells expressing a FAP-1/green fluorescent protein showed intracellular co-localization of FAP-1 and p75(NTR) at the plasma membrane. To elucidate the functional role of this physical interaction, we examined TRAF6 (TNF receptor-associated factor 6)-mediated NF-kappaB activation and tamoxifen-induced apoptosis in 293T cells expressing p75(NTR). The results revealed that TRAF6-mediated NF-kappaB activation was suppressed by p75(NTR) and that the p75(NTR)-mediated NF-kappaB suppression was reduced by FAP-1 expression. Interestingly, a mutant of the p75(NTR) intracellular domain with a single substitution of a Met for Val in its C-terminus, which cannot interact with FAP-1, displayed enhanced pro-apoptotic activity in 293T transfected cells. Thus, similar to Fas, FAP-1 may be involved in suppressing p75(NTR)-mediated pro-apoptotic signaling through its interaction with three C-terminal amino acids (tSPV). Thus, FAP-1 may regulate p75(NTR)-mediated signal transduction by physiological interaction through its third PDZ domain.

Nuclear translocation of nuclear factor kappa B in early 1-cell mouse embryos.

Nuclear factor kappa B (NF-kappaB) is a transcription factor that controls the expression of a number of genes under cellular redox potential. It has recently been found that NF-kappaB plays a pivotal role in morphogenesis and embryonic development, e.g., in formation of Drosophila malanogaster ventral structures and chicken limb buds. However, the role of NF-kappaB in preimplantation development in mammals is not yet understood. In this study, we show that RelA, one of the subunits of NF-kappaB, is expressed in mouse eggs and embryos from the metaphase II (MII) oocyte to the blastocyst stage. Therefore, it is thought that RelA is maternally expressed and that it continues to be expressed during preimplantation development. Immunofluorescence analysis showed that RelA protein was mainly distributed in the cytoplasm of embryos, whereas nuclear translocation of RelA, evidence for NF-kappaB activation, was observed only at the early 1-cell stage. Finally we studied the effects of NF-kappaB inhibitors, pyrrolidine dithiocarbamate and N-acetyl-L-cysteine, on the preimplantation development of mouse embryos. When these inhibitors were added to the culture medium from the early 1-cell stage, subsequent development through the 2-cell stage was inhibited. However, little, if any, influence on the progression through the 2-cell stage was observed when the inhibitors were added at the late 1-cell or the 2-cell stage. Taken together, the results suggest that the activation of NF-kappaB at the early 1-cell stage is required for the development of mouse embryos beyond the 2-cell stage.

[Clinical evaluation of irinotecan combined with cisplatin by divided administration in patients with untreated primary non-small cell lung cancer].

The efficacy and safety of irinotecan (CPT-11) combined with cisplatin (CDDP) were assessed in 24 previously untreated patients with primary non-small cell lung cancer. CPT-11 (60 mg/m2) and CDDP (30 mg/m2) were administered in combination at weekly intervals, on days 1.8, and 15 of the treatment course. During treatment, the patients were evaluated for adverse drug reactions and response. The response rate was 58.3% for all patients and 60.9% for the patients who completed the full treatment course. The median survival time was 13.0 months. The major adverse reactions were myelosuppression and gastro-intestinal disorders, but no treatment-related deaths were observed. Myelosuppressions included grade 3 or 4 leukopenia (25.0%) and anemia (33.3%). Grade 3 and higher gastro-intestinal reactions included nausea and vomiting (8.3%), diarrhea (12.5%), and anorexia (16.7%). These results suggest that combined weekly CPT-11 and CDDP therapy is capable of achieving a favorable tumor response with less toxicity, and thus worth consideration as a treatment option. Given that only 33.3% of the patients finished the full treatment course, further study should be devoted to the subject of CDDP and/or CPT-11 dosages.

Size separation of sodium dodecyl sulfate complexes of human plasma proteins by capillary electrophoresis employing linear polyacrylamide as a sieving polymer.

Electrophoretic conditions to separate sodium dodecyl sulfate (SDS) complexes of human plasma proteins according to their size differences, by capillary electrophoresis employing linear polyacrylamide as a sieving matrix (LPA-CE), have been examined. Using the optimized separation conditions, SDS complexes of human plasma proteins not treated with reducing agents were separated into about 40 peaks and shoulders within 60 min. The molecular mass values of major peaks in a separation pattern were estimated from a plot of molecular mass and migration time for standard proteins and some of the major plasma proteins have been identified on the pattern. The electrophoretic conditions were successfully applied for the analysis of proteins in immunoglobulin G (IgG) myeloma sera.

QM is a novel zinc-binding transcription regulatory protein: its binding to c-Jun is regulated by zinc ions and phosphorylation by protein kinase C.

A novel method was developed for cloning of zinc-binding proteins. We used 65Zn2+ as a probe to screen a human lung cDNA library, and isolated QM using this approach. QM appears to be a negative regulator of c-Jun that acts by binding to the leucine zipper region of c-Jun. We demonstrated that QM bound zinc ions and that such binding was necessary for the interaction of QM with c-Jun. We also showed that protein kinase C introduced about 1 mol of phosphate into 1 mol of QM. The binding of QM to c-Jun was decreased by 60% when QM had been phosphorylated. These results suggest that QM is a novel zinc-binding transcription regulatory protein and that interaction between QM and c-Jun is regulated by zinc ions and phosphorylation.

Separation and characterization of a novel isoenzyme of cyclic nucleotide phosphodiesterase from rat cerebrum.

Anion-exchange chromatography on a Mono-Q column of the supernatant fraction, after ultracentrifugation, from a homogenate of rat cerebrum, prepared under isotonic conditions in the presence of protease inhibitors, yielded a novel isoenzyme of cyclic nucleotide phosphodiesterase (PDE) with properties unlike those of known PDEs. The isoenzyme was insensitive to stimulation by Ca2+/calmodulin and cyclic GMP, and it hydrolyzed both cyclic AMP and cyclic GMP with KM values of 0.109 +/- 0.008 microM and 1.78 +/- 0.04 microM, respectively. The ratio of Vmax of hydrolysis of cyclic GMP to that of cyclic AMP was 1.90 +/- 0.07. Nicardipine (PDE I inhibitor), SK&F 94120 (PDE III inhibitor), rolipram (PDE IV inhibitor) and zaprinast (PDE V inhibitor) had very weak inhibitory effects on the PDE activity of the isoenzyme. These results suggest that the isoenzyme is a novel and previously unreported species of PDE, which we tentatively designate PDE VIII.

The role of the accessory pathway in the onset of atrial fibrillation in Wolff-Parkinson-White syndrome--electrophysiological examination before and after surgical ablation.

To determine the role of the accessory pathway in the pathogenesis of atrial fibrillation, we compared electrophysiological findings in 17 patients (44.7 +/- 10.2 years) with a history of atrial fibrillation before and after surgical ablation of the accessory pathway. The PA interval was shortened, and the atrial refractory periods and the potential minimal wavelength of an atrial impulse (FRPA/PA) were significantly increased, after surgery. Fragmented atrial activity (an increase of 150% or more in the duration of the high right atrial electrogram) was observed in 80% of the patients before surgery and in 25% after surgery. Its zone was significantly decreased after surgery. Repetitive atrial firing was defined as the occurrence of 3 or more successive atrial electrograms induced by a premature stimulation. This was observed in 60% of the patients before surgery, but in none after surgery. Atrial fibrillation was induced in 16 patients during the preoperative study, but in only 1 patient postoperatively. In conclusion, these results suggest that accessory pathways affect atrial vulnerability and play an important role in the onset of atrial fibrillation in WPW syndrome.

Differential effect of pharmacological autonomic blockade on some electrophysiological properties of the human ventricle and atrium.

OBJECTIVE: This study investigated the dominance of each limb of the autonomic nervous system and tested sympathetic-vagal interactions in the human ventricle and atrium after administration of propranolol and atropine. PATIENTS AND METHODS: The 90% monophasic action potential duration (MAPD90) and the effective refractory period (ERP) at the right ventricular apex (RV) and the right lateral atrium (RA) were measured in 14 patients. The MAPD90 was measured during constant RV and RA pacing (cycle length 600 ms) and the ERP was measured at a driven cycle length of 600 ms. Electrophysiological variables were measured during a control period, after propranolol (0.15 mg/kg loading dose followed by 0.1 mg/min infusion), and after autonomic blockade (atropine 0.04 mg/kg). RESULTS: Both RV MAPD90 and RV ERP increased after propranolol (RV MAPD90 from 268 (26) ms to 275 (26) ms, p < 0.005; RV ERP from 252 (25) ms to 258 (26) ms, p < 0.0005) and then decreased to below the control values after autonomic blockade (RV MAPD90 256 (24) ms; RV ERP 239 (25) ms, p < 0.0005 v propranolol, p < 0.0005 v control). In contrast, both RA MAPD90 and RA ERP increased after propranolol (RA MAPD90 from 242 (19) ms to 260 (19) ms; RA ERP from 216 (21) ms to 230 (18) ms, p < 0.0005), and then increased slightly more after autonomic blockade (RA MAPD90 265 (16) ms, p = 0.09; RA ERP 235 (16) ms, p = 0.07), thus remaining above control values (p < 0.0005). CONCLUSIONS: The results indicate (a) that in the human ventricle vagal stimulation and sympathetic beta stimulation are antagonistic and that direct vagal stimulation predominates over beta stimulation, with sympathetic-vagal interaction being minimal and (b) that in the human atrium vagal stimulation and beta stimulation are synergistic and beta stimulation predominates over vagal stimulation, with direct vagal stimulation having a minimal effect.

Long-term results of catheter ablation for idiopathic ventricular tachycardia originated from the right ventricular outflow.

We performed catheter ablation in 10 consecutive patients with idiopathic monomorphic right ventricular tachycardia and studied the long-term outcome. All ventricular tachycardias had a left bundle branch block configuration with an inferior axis, and originated from right ventricular outflow. Antiarrhythmic drugs (3-6 drugs) had been ineffective in controlling ventricular tachycardia. The 2 patients who underwent direct-current ablation (2 shocks of 150 J) had no recurrence of ventricular tachycardia and did not require antiarrhythmic drugs during a follow-up of 56 and 51 months, respectively. Of the 8 patients who underwent radiofrequency ablation (30-40 watt, 20-40 sec, 2-15 application, using Inter Nova RA 50, 13.56 MHz), 1 patient had no recurrence of ventricular tachycardias and did not require antiarrhythmic drugs, 4 patients had no recurrence of ventricular tachycardias but did require anti-arrhythmic drugs, and 3 patients experienced recurrence of non-sustained ventricular tachycardia despite the use of antiarrhythmic drugs during a follow-up of 15-40 months. There were no complications except for cardiac perforation which occurred immediately after direct-current ablation in 1 patient. In conclusion, long-term success in preventing ventricular tachycardia was achievable with direct-current ablation, but this success was associated with serious risks, such as cardiac perforation. Radiofrequency ablation was safer than direct-current ablation, but had a lower long-term success rate.

Early afterdepolarizations in a patient with idiopathic monomorphic right ventricular tachycardia.

To identify the role of afterdepolarizations in the induction of idiopathic monomorphic right ventricular tachycardia (VT), monophasic action potentials (MAPs) were recorded in a patient with this type of VT. The VT had a left bundle branch block configuration and inferior axis, and originated in the right ventricular outflow tract (RVOT). MAPs were recorded with a contact electrode at the origin of the VT, as well as other ventricular sites. The VT was induced by the intravenous administration of isoproterenol and/or rapid ventricular pacing and was preceded by short-long-short sequences of RR intervals. Early afterdepolarizations (EADs) in MAPs were recorded at the origin of VT (RVOT), but not recorded at other ventricular sites. These data suggest that catecholamine sensitive triggered activity seems to be the mechanism of idiopathic monomorphic right VT and EADs can be recorded in association with the occurrence of this type of VT.

Effect of alternating combination chemotherapy consisting of cyclophosphamide, doxorubicin, vincristine, cisplatin, and etoposide for small cell lung cancer on hematopoietic progenitors in the peripheral blood.

The effects of a combination chemotherapy (CAV-PVP) consisting of cyclophosphamide, doxorubicin, hydrochloride (dox) and vincristine (CAV) alternating with cisplatin and etoposide (PVP) on peripheral blood hematopoietic progenitor cells (PBHPs) were studied in five patients with small cell lung cancer (SCLC). The kinetics of the CFU-GM levels were different during the CAV and PVP phases. None of the five patients displayed a rebound increase in the level of peripheral blood CFU-GM during the CAV phase. In contrast, all five patients displayed a rebound increase in peripheral blood CFU-GM levels during the PVP phase of the alternative combination chemotherapy (3-5 weeks after the initiation of PVP regimen). These findings indicate the optimal timing for leukapheresis to obtain PBHPs in SCLC patients which have been treated with an alternating combination chemotherapy consisting of CAV-PVP.

[The effects of pharmacological autonomic blockade on the conduction system in patients with and without sinus node dysfunction].

Electrophysiological studies were performed in 26 patients with sick sinus syndrome (SSS) and 12 controls before and after pharmacologic autonomic blockade (PAB) with propranolol (0.15 mg/kg) and atropine (0.04 mg/kg). Sinus cycle length (SCL) shortened significantly after PAB in both groups. PA and HV intervals did not change after PAB. AH interval shortened and Wenckebach period increased after PAB in SSS group. Maximum corrected sinus node recovery time shortened in the control group but did not change in the SSS group after PAB. Calculated sinoatrial conduction time shortened after PAB in both groups. Refractory periods of the atrioventricular (AV) node and the ventricle shortened significantly, but those of the atrium did not change after PAB. Linear relations existed between the change of SCL and the change of AH interval and of the Wenckebach period. These results suggested that the autonomic nervous system had the same effects on the sinus and the AV nodes in patients with SSS, and that parasympathetic tone was predominant on the sinus node, the perinodal tissue and the AV node under resting conditions.