A simple method using 31P-NMR spectroscopy for the study of protein phosphorylation

Nonradioactive 31P-NMR spectroscopy has previously been used for the study of protein phosphorylations. However, the procedures does not seem to be easy for non-experts of this field, hence, this approach has not been widely used. We introduce here a simple protocol with 31P-NMR spectroscopy to study in vitro phosphorylation in receptor proteins. The effectiveness of this method was verified using synthetic peptides and recombinant proteins of the C-terminus of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor, whose phosphorylations are considered to have important roles in synaptic plasticity. We show that a decrease in the pH of the sample solution after the phosphorylation reaction is critical for the separation of the phosphorylation signals. In the analysis of the C-terminal portion of the GluR2 AMPA receptor, the phosphorylation sites of which had not hitherto been well clarified, we found the presence of at least three protein kinase C (PKC) phosphorylation sites. Furthermore, this method allows prediction of the origins of each of the phosphorylation peaks. Thus, the techniques we described here is useful for examination of protein phosphorylation and permits us to safely conduct repetitive experiments.     

Effect of graft-versus-host disease on the outcome of bone marrow transplantation from an HLA-identical sibling donor using GVHD prophylaxis with cyclosporin A and methotrexate.

The effect of graft-versus-host disease (GVHD) on relapse incidence and survival has been analyzed in several studies, but previous studies included heterogeneous patients. Therefore, we analyzed the data of 2114 patients who received unmanipulated bone marrow graft from an HLA-identical sibling donor with a GVHD prophylaxis using cyclosporin A and methotrexate. Among the 1843 patients who survived without relapse at 60 days after transplantation, 435 (24%) developed grade II-IV acute GVHD. Among the 1566 patients who survived without relapse at 150 days after transplantation, 705 (47%) developed chronic GVHD. The incidence of relapse was significantly lower in patients who developed acute or chronic GVHD, but disease-free survival (DFS) was significantly inferior in patients who developed acute GVHD. A benefit of 'mild' GVHD was only seen in high-risk patients who developed grade I acute GVHD. The strongest association between GVHD and a decreased incidence of relapse was observed in patients with standard-risk acute myelogenous leukemia/myelodysplastic syndrome. In conclusion, the therapeutic window between decreased relapse and increased transplant-related mortality due to the development of GVHD appeared to be very narrow.     

Radiation tolerance of the cochlear nerve at the γ-knife in rabbits

Since the first treatment of acoustic neurinoma using the γ-knife by Leksell, a series of cases have been reported with good control rates. However, the most frequent complication is delayed hearing loss which occurs in more than 50% of patients. The purpose of this study was to define a safe dose by analyzing the radiosurgical dose-response relationship and histological effects on the normal cochlear nerve in rabbit. The rabbits had computed tomography (CT)-guided stereotactic radiosurgery on their cochlear nerves in the internal auditory canal with a 4 mm collimator focusing of a γ-unit. Maximum doses of 10, 20, 30, 40, 60, 80, 100, 200 and 500 Gy were administered. After the radiosurgery, auditory brain stem responses (ABR) and the behavior of the rabbits were evaluated periodically. At the conclusion, histological investigations were performed. No physiological or histological findings were observed from doses of 30 Gy or below during the 12 month period after the radiosurgery. A dose of 100 Gy caused a severe ABR threshold elevation, vestibular dysfunction and facial palsy. Necrosis and demyelination of nerves were observed pathologically. In this study, we determined that the safe dose to the normal cochlear nerve during radiosurgery was under 40 Gy in rabbits, and complications seemed to vary due to individual differences in radiation tolerance.     

Superiority of hepatic arterial infusion in preventing catabolism of 5-FU compared with portal vein infusion revealed by an in vivo 19F NMR study

Purpose: The aim of this study was to identify the route of administration of 5-FU with the greatest pharmacological advantage in a rat model using noninvasive in vivo ¹⁹F nuclear magnetic resonance (NMR) spectroscopy. Methods: 5-FU (50 mg/kg) was administered to anesthetized Wistar rats cannulated into the hepatic artery, portal vein or tail vein and 11 NMR spectra were acquired from the liver region to 60.5 min every 5.5 min. Results: With systemic i.v. (tail vein) infusion, the ¹⁹F-NMR signal for 5-FU from the liver region peaked in the first spectrum (0–5.5 min), and then gradually decreased. The signal for the 5-FU catabolite α-fluoro-β-alanine (FBAL) gradually increased to the sixth spectrum (0–33.0 min) and then plateaued. Following portal vein infusion the intensity of the first 5-FU spectrum was twice as high as that following i.v. infusion, but the intensity decreased and the FBAL signal increased gradually in the sixth spectrum as systemic i.v. infusion. In contrast, the intensity of the 5-FU signal following hepatic artery infusion was the same as that following portal vein infusion in the first spectrum, and maintained a strong intensity to the final spectrum (60.5 min). The FBAL signal was detected from the second spectrum following hepatic artery infusion, but its intensity was significantly weaker than that following i.v. or portal vein infusion. Conclusions: Hepatic arterial infusion resulted in the active form of 5-FU being present for a longer time and its degradation in the liver being suppressed compared with the results following portal vein infusion. This catabolic advantage of hepatic areterial infusion could lead to a more potent anti-tumor activity against liver metastases, but could also lead to significant host toxicity including biliary toxicity. We recommend that the dose/schedule of 5-FU administered via the hepatic artery should be adjusted carefully. Received: 4 August 1997 / Accepted: 16 January 1998     

Mutation Analysis of the WT1 Gene in Myelodysplastic Syndromes

The WT1 tumor suppressor gene was examined for mutations in a panel of 44 patients with myelo-dysplastic syndromes (MDS) including acute myelogenous leukemias (AML) secondary to MDS, using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis and sequencing analysis. A WT1 mutation was detected in one out of 17 cases of AML secondary to MDS. This mutation exists upstream of the zinc finger region and is predicted to produce a truncated WT1 protein lacking the zinc finger region. No mutations were detected in 27 MDS patients who had not progressed to AML. This is the first report of analysis for WT1 mutations in a large number of MDS patients, suggesting that WT1 mutations are uncommon in MDS. Abnormalities in this gene may, however, contribute to a small proportion of cases showing progression from MDS into AML.     

Evaluation of syringomyelia with three-dimensional constructive interference in a steady state (CISS) sequence

The purpose of this study was to evaluate a three-dimensional (3D) constructive interference in steady state (CISS) sequence in the assessment of syringomyelia. Eleven patients with syringomyelia were prospectively studied with magnetic resonance imaging. All patients underwent sagittal imaging with T1- and T2-weighted spin-echo (SE), and 3D-CISS sequences. The SE and 3D-CISS images, as well as multiplanar reconstruction (MPR) images of the 3D-CISS sequence, were analyzed with regard to image quality, degree of artifacts, visualization of the extent and internal structure of the syringomyelia, and contrast-to-noise ratio (CNR) of the fluid within the syringomyelia. Contrast between the spinal cord and cerebrospinal fluid (CSF), as well as delineation was significantly poorer for the T1-weighted SE sequence than for the 3D-CISS sequence (P < 0.01), while there was no significant difference between the T2-weighted SE sequence and the 3D-CISS sequence. Artifacts induced by CSF flow were significantly more for the T2-weighted SE sequence than for the 3D-CISS sequence (P < 0.01). Although the extent of syringomyelia was delineated equally among the three sequences in 9 of 11 patients, it was better for the 3D-CISS sequence than for the SE sequences in the remaining two. Septation and communication between the cavities were best detected by the 3D-CISS MPR images. The CNR of the 3D-CISS sequence was significantly higher than that of the SE sequence (P < 0.01). The 3D-CISS sequence demonstrates the extent and internal structures of syringomyelia better than conventional SE sequences and should be added to SE sequences in the evaluation of syringomyelia.     

Postoperative interstitial pneumonia: 2 cases after lobectomy

Two patients with postoperative interstitial pneumonia are reported. Preoperative diagnosis was primary lung cancer without idiopathic interstitial pneumonia (IIP). Within one week after operation acute interstitial pneumonia (AIP) occurred on the nonoperated side and developed. Steroid therapy was performed but one was dead. AIP is a fatal complication after pulmonary resection and steroid therapy may be useful in some cases of postoperative AIP.     

Nationwide survey on complementary and alternative medicine in cancer patients in Japan.

PURPOSE: To determine the prevalence of use of complementary and alternative medicine (CAM) by patients with cancer in Japan, and to compare the characteristics of CAM users and CAM nonusers. PATIENTS AND METHODS: A questionnaire on cancer CAM and the Hospital Anxiety and Depression Scale were delivered to 6,607 patients who were treated in 16 cancer centers and 40 palliative care units. RESULTS: There were 3,461 available replies for a response rate of 52.4%. The prevalence of CAM use was 44.6% (1,382 of 3,100) in cancer patients and 25.5% (92 of 361) in noncancer patients with benign tumors. Multiple logistic regression analysis determined that history of chemotherapy, institute (palliative care units), higher education, an altered outlook on life after cancer diagnosis, primary cancer site, and younger age were strongly associated with CAM use in cancer patients. Most of the CAM users with cancer (96.2%) used products such as mushrooms, herbs, and shark cartilage. The motivation for most CAM use was recommendation from family members or friends (77.7%) rather than personal choice (23.3%). Positive effects were experienced by 24.3% of CAM users with cancer, although all of them received conventional cancer therapy concurrently. Adverse reactions were reported by 5.3% of cancer patients. CAM products were used without sufficient information by 57.3% of users with cancer and without a consultation with a doctor by 60.7% of users. CONCLUSION: This survey revealed a high prevalence of CAM use among cancer patients, without sufficient information or consultation with their physicians. Oncologists should not ignore the CAM products used by their patients because of a lack of proven efficacy and safety.     

Identification of Ki23819, a highly potent inhibitor of kinase activity of mutant FLT3 receptor tyrosine kinase.

Constitutively active internal tandem duplication (ITD) in the juxtamembrane domain of Fms-like tyrosine kinase 3 (FLT3), a type III receptor tyrosine kinase, is the most common molecular defect associated with acute myeloid leukemia. Its presence confers a poor outcome in patients with acute myeloid leukemia who receive conventional chemotherapy. FLT3-ITD has therefore been considered to be an attractive molecular target for a novel therapeutic modality. We describe here the identification and characterization of Ki23819 as a novel FLT3 inhibitor. Ki23819 suppressed proliferation and induced apoptosis of FLT3-ITD-expressing human leukemia cell lines. The growth-inhibitory effect of Ki23819 on MV4-11 cells was superior to that of SU11248, another FLT3 inhibitor (IC(50)<1 vs 3-10 nM). Ki23819 inhibited the autophosphorylation of FLT3-ITD more efficiently than that of wild-type FLT3. FLT3-ITD-dependent activation of the downstream signaling proteins ERK and STAT5 was also inhibited within similar concentration ranges. Thus, Ki23819 is a potent in vitro inhibitor of FLT3.