Identification of a Novel C-Terminal Truncated WT1 Isoform with Antagonistic Effects against Major WT1 Isoforms.

The Wilms' tumor gene WT1 consists of 10 exons and encodes a zinc finger transcription factor. There are four major WT1 isoforms resulting from alternative splicing at two sites, exon 5 (17AA) and exon 9 (KTS). All major WT1 isoforms are overexpressed in leukemia and solid tumors and play oncogenic roles such as inhibition of apoptosis, and promotion of cell proliferation, migration and invasion. In the present study, a novel alternatively spliced WT1 isoform that had an extended exon 4 (designated as exon 4a) with an additional 153 bp (designated as 4a sequence) at the 3' end was identified and designated as an Ex4a(+)WT1 isoform. The insertion of exon 4a resulted in the introduction of premature translational stop codons in the reading frame in exon 4a and production of C-terminal truncated WT1 proteins lacking zinc finger DNA-binding domain. Overexpression of the truncated Ex4a(+)WT1 isoform inhibited the major WT1-mediated transcriptional activation of anti-apoptotic Bcl-xL gene promoter and induced mitochondrial damage and apoptosis. Conversely, suppression of the Ex4a(+)WT1 isoform by Ex4a-specific siRNA attenuated apoptosis. These results indicated that the Ex4a(+)WT1 isoform exerted dominant negative effects on anti-apoptotic function of major WT1 isoforms. Ex4a(+)WT1 isoform was endogenously expressed as a minor isoform in myeloid leukemia and solid tumor cells and increased regardless of decrease in major WT1 isoforms during apoptosis, suggesting the dominant negative effects on anti-apoptotic function of major WT1 isoforms. These results indicated that Ex4a(+)WT1 isoform had an important physiological function that regulated oncogenic function of major WT1 isoforms

A Postoperative Intracranial Hemorrhage due to a vitamin K Deficiency in Treated Severe Subarachnoid Hemorrhage Patients : A Report of Two Cases

術後vitamin K(以下,VK)欠乏性頭蓋内出血をきたしたと考えられた破裂脳動脈瘤によるくも膜下出血(以下,SAH)の2症例を報告した.症例1は48歳女性.動脈瘤クリッピング術後10日目に急性硬膜外血腫,17日目には出血性梗塞を起こした.症例2は39歳男性.動脈瘤クリッピング術後14日目に急性硬膜外血腫を起こした.いずれも凝固系検査よりVK欠乏を疑い,VK_2静注を施行し,まもなく止血,以後再出血はなかった.症例2では急性硬膜外血腫手術中に血中 VKを測定し,VKの著しい低下を確認した.VK欠乏の原因としてSAHおよび手術侵襲によるVK消費亢進,抗生剤長期投与の結果としての腸内細菌叢の抑制によるVK産生の低下が考えられた.重症SAHの患者では汎発性血管内凝固症の準備状態にあり,加えてVK依存性凝固因子の産生低下がみられる.術後出血の原因としてVK欠乏症も念頭にいれておくべきと考えられる.The authors report 2 cases of a postoperative intracranial hemorrhage in treated severe subarachnoid hemorrhage (SAH) patients due to a vitamin K (VK) deficiency. These cases are described below. Case 1 : The patient, a 48-year-old woman, was admitted to our hospital because of an SAH, and a neck clipping was soon performed after admission. On the 10th postoperative day, an epidural hematoma was found, and on the 17th postoperative day, a hemorrhagic infarction occurred. Case 2 : The patient, a 39-year-old man, was admitted to our hospital because of an SAH, and a neck clipping was soon performed after admission. On the 14th postoperative day, an epidural hematoma was detected. The concentration of the serum VK during an evacuation of the hematoma was found to have markedly decreased, and a coagulation examination revealed that a VK deficiency had caused the postoperative hemorrhage. A subsequent adnlinistration of VK resolved the hemorrhagic complications in both patients. The VK deficiency in these cases may have been caused by an increased VK consumption due to the pre-DIC state after the SAH and the invasive surgery, and the supression of VK production by intestinal flora because of long-term antibiotic medication. It thus has been speculated that in severe SAH patients, a VK deficiency may be the cause of a postoperative hemorrhagic complication

The Combined Use of Composite Ceramic Granules and Fibrin Glue for Cranioplasties : Results of a Rat Model Study and Clinical Findings with Regard to Biocompatibility

水酸アパタイト・リン酸三カルシウム複合体顆粒(セラタイト^<(R)>)とフィブリン糊(ベリプラストP^<(R)>)を用いた頭蓋形成術を行った.動物実験では,ラット頭蓋骨を用い,本法の生体適合性を検討した.術後3ヶ月目の標本では,肉眼的には良好な頭蓋形成が得られたが,組織学的には新生骨は認められなかった.術後12カ月目の標本では,セラタイト^<(R)>を中心に新生骨が認められた.新生骨はセラタイト^<(R)>の表面に直接形成されており,また,一部にはセラタイト^<(R)>を取り囲むように新生骨が形成され,その顆粒の中にも新生骨が侵入しているのが観察された.以上の点から骨親和性および骨誘導能ともきわめて良好なことが示唆された.臨床的には開頭術後骨屑が少ない症例,再手術例への応用をはじめ,後頭蓋窩手術に伴う骨欠損の補嗔に応用した.術後の創感染はなく,良好な頭蓋形成が得られた.フィブリン湖を併用しているため髄液瘻の防止あるいは閉鎖にも有用であった.For cranioplasties, we have previously reported on the efficacy and safety of using a mixture of hydroxyapatite granules and the tricalciumphosphate composite (Ceratite^<(R)>) with fibrin glue (Beriplast P^<(R)>) . Continuing our research further, we have now investigated the biocompatibility of cranioplasty in rats. Two sets of Wistar rat (250-300 g) underwent a small craniectomy of the parietal bone, about 4 x 5 mm in size, without causing damage to the dura mater. The Ceratite^<(R)> in the form of granules, 0.3-0.6 mm in diameter, was then applied to the bone defect with Beriplast P^<(R)>. Postoperatively, at 3 and at 12 months, the results in each rat set, consisting of 6 rats each, were respectively evaluated. To accomplish this, 10 μm-thick sections from each set were prepared and stained with hematoxylin and eosin and examined under light microscopy. Results revealed that at 3 months postoperatively, there were signs that an excellent cranioplastic repair was in progress, but newly-formed bone had not yet to be seen among the Ceratite^<(R)> granules. However, at 12 months postoperatively, the repair appeared to be progressing well, for regenerated bone was observed amongst the Ceratite^<(R)> granules. Cranioplasties using Ceratite^<(R)> granules and Beriplast P^<(R)> have been performed for 22 neurosurgical patients, including one with a posterior fossa bone defect, and neither an infection nor local abnormal granulation were noted during the postoperative follow-up. These findings suggest that composite ceramics possess an excellent biocompatibility, and that the combined usage of composite granular ceramics and fibrin glue is both useful and safe for cranioplasties

A Giant Arteriovenous Malformation : Report of a Case that Showed Dramatic Neurological Improvement after Multi-staged Emboilization prior to the Surgical Removal of Malformation

本症例は右側頭頭頂後頭葉を占拠する巨大脳動静脈奇形で,著しい慢性脳虚血を伴い,頻回の痙攣発作と痴呆などの進行性神経脱落症候を呈した.全摘出はきわめて危険かつ困難であり,さらにCTにて大脳皮質下に広範な石灰化を認め,症状改善も困難と考えられていた.新開発のガイドワイヤーにより超選択的カテーテル挿入が可能となり,術前staged embolizationと術中embolizationの併用によりnormal perfusion pressure breakthroughの合併なく全摘出術に成功した.さらに術前embolizationの過程で,shunt flowの軽減に伴い神経脱落症候が劇的に改善したことも注目に値する.The authors describe a huge arteriovenous malformation (AVM) in the right temporo-parieto-occipital lobes that was successfully removed after a multi-staged embolization of the feeding arteries. The patient, a 54-year-old housewife, had presented uncontrolable epilepsy, progressive dementia, hemianopsia, hemiparesis, and hemisensory impairment of the left side. Angiography revealed a huge AVM mainly being nourished by the following sources : the parieto-occipital arteries of the posterior cerebra] artery, the temporal branches of the middle cerebral artery and dura] branches from the external carotid and vertebral arteries. Further, the cortical draining veins over the entire hemisphere were markedly, dilated, and the superior sagittal and straight sinuses visualized poorly, probably due to the congestion in the venous circulation caused by the arteriovenous shunt. CT scans also revealed multiple, club-like calcification in the parietal and frontal subcortical regions, indicating chronic brain ischemia caused by the remarkable arterial s_teal and/or venous hypertension. . Preoperative super-selective embolization was done in four sessions, using newly developed, low-friction, high -torque guide wires. and this resulted in a dramatic neurological improvement, especially with regard to the dementia. Then, following intraoperative embolization of the remaining feeding arteries, the AVM was success-fully removed. The patient tolerated these procedures well without hemodynamic complications, and after a few months of rehabilitation, she resumed her normal life

術後vitamin K 欠乏性頭蓋内出血をきたしたくも膜下出血の2例

術後vitamin K(以下,VK)欠乏性頭蓋内出血をきたしたと考えられた破裂脳動脈瘤によるくも膜下出血(以下,SAH)の2症例を報告した.症例1は48歳女性.動脈瘤クリッピング術後10日目に急性硬膜外血腫,17日目には出血性梗塞を起こした.症例2は39歳男性.動脈瘤クリッピング術後14日目に急性硬膜外血腫を起こした.いずれも凝固系検査よりVK欠乏を疑い,VK_2静注を施行し,まもなく止血,以後再出血はなかった.症例2では急性硬膜外血腫手術中に血中 VKを測定し,VKの著しい低下を確認した.VK欠乏の原因としてSAHおよび手術侵襲によるVK消費亢進,抗生剤長期投与の結果としての腸内細菌叢の抑制によるVK産生の低下が考えられた.重症SAHの患者では汎発性血管内凝固症の準備状態にあり,加えてVK依存性凝固因子の産生低下がみられる.術後出血の原因としてVK欠乏症も念頭にいれておくべきと考えられる.The authors report 2 cases of a postoperative intracranial hemorrhage in treated severe subarachnoid hemorrhage (SAH) patients due to a vitamin K (VK) deficiency. These cases are described below. Case 1 : The patient, a 48-year-old woman, was admitted to our hospital because of an SAH, and a neck clipping was soon performed after admission. On the 10th postoperative day, an epidural hematoma was found, and on the 17th postoperative day, a hemorrhagic infarction occurred. Case 2 : The patient, a 39-year-old man, was admitted to our hospital because of an SAH, and a neck clipping was soon performed after admission. On the 14th postoperative day, an epidural hematoma was detected. The concentration of the serum VK during an evacuation of the hematoma was found to have markedly decreased, and a coagulation examination revealed that a VK deficiency had caused the postoperative hemorrhage. A subsequent adnlinistration of VK resolved the hemorrhagic complications in both patients. The VK deficiency in these cases may have been caused by an increased VK consumption due to the pre-DIC state after the SAH and the invasive surgery, and the supression of VK production by intestinal flora because of long-term antibiotic medication. It thus has been speculated that in severe SAH patients, a VK deficiency may be the cause of a postoperative hemorrhagic complication

Apoptotic function of an Ex4a(+)WT1 isoform.

<p>(<b>A</b>) The role of Ex4a(+)WT1 in apoptosis. Ex4a(+) or Mock vector was transfected into WT1-expressing HT-1080 cells. Frequencies (%) of Annexin V-positive apoptotic cells and cells with loss of MMP were determined by flowcytometry after 24 h. Left, Frequencies (%) of Annexin V-positive apoptotic cells are shown. Right, Frequencies (%) of cells with mitochondrial membrane potential (MMP) loss are shown. Results are means and S.D. of three independent experiments. *, p<0.05. (<b>B</b>) Expression of Ex4a(+) and major WT1 isoforms during apoptosis. K562 cells were treated with the indicated concentrations of Dox for 12 h and analyzed for Annexin-V positive apoptotic cells and expression of Ex4a(+) and major WT1 isoforms by flowcytometry and RT-PCR, respectively. Upper, Frequencies (%) of Annexin V-positive apoptotic cells. Lower, RT-PCR using Ex4-F and Ex6-R primer pair that amplifies both Ex4a(+) and major WT1 isoforms. GAPDH is used as an internal control. Results are representative of three independent experiments. (<b>C</b>) Change of Ex4a(+)WT1 and major WT1 isoforms during apoptosis. K562 cells were treated with the indicated concentrations of Dox for 12 h and expression of Ex4a(+)WT1 and total WT1 isoforms including both Ex4a(+) and major WT1 isoforms were determined by quantitative real-time RT-PCR using Ex4a-F and Ex6-R primer pair and Ex6-F and Ex7-R primer pair, respectively. Actin is used as an internal control for normalization. Expression levels of Ex4a(+)WT1 and total WT1 in Dox-untreated cells are defined as 1.0. (<b>D</b>) Suppression of Ex4a(+)WT1 inhibits Dox-induced apoptosis. K562 cells were transfected with one μg of either of two WT1 Ex4a-specific siRNAs (si-4a-1 and si-4a-2) or a control siRNA (si-control) together with 2.0 μg of Ex4a(+)WT1 vector or 2.0 μg of empty vector (Mock), cultured for 24 h, treated with 4.0 μM Dox for 12 h, and then analyzed for Annexin-V positive apoptotic cells by flowcytometry. Frequencies (%) of Annexin V-positive apoptotic cells are shown. Results are mean and S.D. of three independent experiments. *, p<0.05.</p