Nanoscale Dynamics of Protein Assembly Networks in Supersaturated Solutions.

Proteins in solution are conventionally considered macromolecules. Dynamic microscopic structures in supersaturated protein solutions have received increasing attention in the study of protein crystallisation and the formation of misfolded aggregates. Here, we present a method for observing rotational dynamic structures that can detect the interaction of nanoscale lysozyme protein networks via diffracted X-ray tracking (DXT). Our DXT analysis demonstrated that the rearrangement behaviours of lysozyme networks or clusters, which are driven by local density and concentration fluctuations, generate force fields on the femtonewton to attonewton (fN - aN) scale. This quantitative parameter was previously observed in our experiments on supersaturated inorganic solutions. This commonality provides a way to clarify the solution structures of a variety of supersaturated solutions as well as to control nucleation and crystallisation in supersaturated solutions.

A programmable DNA origami nanospring that reveals force-induced adjacent binding of myosin VI heads.

Mechanosensitive biological nanomachines such as motor proteins and ion channels regulate diverse cellular behaviour. Combined optical trapping with single-molecule fluorescence imaging provides a powerful methodology to clearly characterize the mechanoresponse, structural dynamics and stability of such nanomachines. However, this system requires complicated experimental geometry, preparation and optics, and is limited by low data-acquisition efficiency. Here we develop a programmable DNA origami nanospring that overcomes these issues. We apply our nanospring to human myosin VI, a mechanosensory motor protein, and demonstrate nanometre-precision single-molecule fluorescence imaging of the individual motor domains (heads) under force. We observe force-induced transitions of myosin VI heads from non-adjacent to adjacent binding, which correspond to adapted roles for low-load and high-load transport, respectively. Our technique extends single-molecule studies under force and clarifies the effect of force on biological processes.

Time-resolved X-ray Tracking of Expansion and Compression Dynamics in Supersaturating Ion-Networks.

Supersaturation of a solution system is a metastable state containing more solute than can be normally solubilized. Moreover, this condition is thermodynamically important for a system undergoing a phase transition. This state plays critical roles in deposition morphology in inorganic, organic, polymer and protein solution systems. In particular, microscopic solution states under supersaturated conditions have recently received much attention. In this report, we observed the dynamic motion of individual ion-network domains (INDs) in a supersaturated sodium acetate trihydrate solution (6.4 M) by using microsecond time-resolved and high accuracy (picometre scale) X-ray observations (diffracted X-ray tracking; DXT). We found that there are femto-Newton (fN) anisotropic force fields in INDs that correspond to an Angstrom-scale relaxation process (continuous expansion and compression) of the INDs at 25 µs time scale. The observed anisotropic force-field (femto-Newton) from DXT can lead to new explanations of how material crystallization is triggered. This discovery could also influence the interpretation of supercooling, bio-polymer and protein aggregation processes, and supersaturated systems of many other materials.

Photoconstructs of nerve guidance prosthesis using photoreactive gelatin as a scaffold.

We devised a novel nerve prosthesis composed of an elastomeric gelatinous tube and multifilament gelatinous fibers, both of which were prepared from styrene-derivatized gelatin, which allows in situ formation of a bioactive substance-incorporated gel. An in vitro study showed that the axonal regeneration potential of a photocured gelatin layer impregnated with laminin, fibronectin, and NGF was almost comparable with that of coated Matrigel. A nerve conduit and fibers prepared from photoreactive gelatin was subjected to visible-light irradiation with rotation in the presence of camphorquinone as a photoinitiator using a custom-designed apparatus. A sample of transparent gelatinous conduit with an inner diameter of 1.2 mm and a wall thickness of 0.6 mm and gelatin fibers ranging from 10 to 100 pm in diameter were produced. The photocured elastomeric gelatinous tube was flexible and had structural integrity that allowed mechanical handling without breaking. A novel nerve guidance prosthesis composed of tubes packed with fibers was assembled. This photofabrication technology may enable the design of a tailor-made shape and rapid morphogenesis and functional recovery of damaged nerve tissue.

Primary Ki-1 lymphoma in the central nervous system.

Large cell anaplastic malignant lymphoma with Ki-1 (CD30) antigen is a new entity among human non-Hodgkin's malignant lymphomas according Updated Kiel Classification and is also a very rare subtype in primary central nervous system (CNS) malignant lymphomas. The precise clinical characteristics and the significance of Ki-1 antigen have yet to be clarified. The authors herein report a case of Ki-1 positive primary T-cell CNS malignant lymphoma. A 49-year-old man presented with multiple mass lesions in the brain on MRI. Immunohistochemical investigations of biopsy specimens from the superior medullary velum revealed a large cell anaplastic T-cell lymphoma positive for Ki-1 antigen. After administering extensive chemo-radiotherapy, the patient has survived for more than 42 months after the onset of symptoms.

A lateral ventricular gliosarcoma arising in an ependymoma.

OBJECTIVE: We describe a 29-year-old man with gliosarcoma in the lateral ventricle. CASE: The patient presented with headache and impairment of consciousness. Computed tomography and magnetic resonance imaging localized the tumor to the right lateral ventricle and showed heterogeneous enhancement with administration of contrast agents. The tumor was partially removed via a transcallosal approach. Histologic examination disclosed gliosarcoma arising by malignant transformation of an ependymoma. POST-OPERATIVE COURSE: The patient died of tumor progression 78 days after admission, despite intensive radiotherapy and chemotherapy.

Vascular endothelial growth factor in chronic subdural haematomas.

OBJECTIVE: To elucidate molecular aspects of the mechanisms of expansion of chronic subdural haematomas (CSH), we examined the expression of two representative angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in CSH. METHODS: We quantified VEGF and bFGF in haematoma fluid and serum of 20 patients with CSH using an enzyme-linked immunosorbent assay. Mean concentrations of VEGF in the haematoma fluid (10277 pg/ml) and in serum, (355 pg/ml) were much greater than those of bFGF (haematoma, 3.04 pg/ml; serum, 4.74 pg/ml). Surgical specimens, including dura and the outer membrane of the CSH were analysed by in situ hybridisation to detect VEGF mRNA. Macrophages and vascular endothelial cells in the outer membrane over expressed VEGF mRNA. CONCLUSIONS: Enhanced production of VEGF by macrophages and vascular endothelial cells in the outer membrane is thought to be pathogenetically important in CSH.

Efficacy of single-bolus vs. frequent low-dose treatment with nitrosourea in experimental gliomas.

BACKGROUND: Malignant glioma remains a fatal disease. Continuous or frequent low-dose (FLD) chemotherapy with nitrosoureas reportedly causes fewer side-effects than single-bolus therapy without decreasing the antitumour effects. MATERIALS AND METHODS: To study the effect of FLD treatment with nimustine (ACNU) in rats with glioma, we intracerebrally inoculated with C6 glioma cells. We began the ACNU treatment 5 or 8 days later (total dose, 25 or 40 mg/kg) i.p. as either one bolus or smaller doses spread over 5 days week. RESULTS: At a total dose of 25 mg/kg beginning at day 8, survival duration did not differ between untreated controls and the FLD group, while the bolus significantly prolonged survival; the FLD group showed some improvement beyond control survival at 40 mg/kg (each p <0.001). Beginning treatment after 5 rather than 8 days prolonged survival somewhat further. CONCLUSION: FLD treatment with ACNU is less effective against experimental glioma in rats than bolus treatment.

[A case showing effective radiotherapy for a radiation-induced glioblastoma].

Radiation-induced glioblastoma is usually resistant to all treatments. We report a case with radiation-induced glioblastoma, in which radiotherapy was remarkably effective. A 14-year-old female with a history of acute lymphoblastic leukemia, at the age of 7, underwent 15 Gy of radiotherapy to the whole brain. She was admitted to our department due to the development of headache and nausea. Magnetic resonance imaging showed an irregularly enhanced mass in the left frontal lobe. Partial removal of the mass was performed and histological examination showed it to be glioblastoma with a high MIB-1 index. The patient underwent 40 Gy of local radiotherapy and chemotherapy with ACNU and Interferon-beta for 2 years. The residual tumor disappeared after the radiotherapy, and her status is still "complete remission", 29 months after the onset.

[Acute hepatitis B virus after chemotherapy for a case with germinoma].

A 28-year old man with HCG-producing germinoma had undergone chemotherapy and radiotherapy. On admission for the fifth session of maintenance chemotherapy, he was found to be positive for hepatitis B (HB)s antigen, but negative for HBs antibody. HBs antigen had been negative during previous admissions. Since liver function was normal, the patient underwent chemotherapy. During myelosuppression after chemotherapy, liver dysfunction developed and acute HB was diagnosed. He fortunately showed seroconversion 2 months after onset. Serum immunological examinations are required for patients receiving chemotherapy.

[Educational level of patients with germ cell tumor radiated in childhood].

In order to estimate the influence of radiotherapy on the intellectual development of children with brain tumor, we investigated the educational level of 21 patients with germ cell tumor who had undergone radiotherapy. They were divided into three groups in accordance with their age at the time of radiation; under school age group (under 6 years of age), elementary school age group (from 7 to 12 years of age), and junior high and high school age group (from 13 to 18 years of age). There were 2 cases in the under school age group, one of them graduated from high school and the other is presently a junior high school student. There were 5 cases in the elementary school age group. 3 of these graduated from university, 1 is presently a university student and 1 is a high school student. There were 14 cases in the junior high and high school age group. 2 of these are university students, 7 graduated from high school, 1 is presently a junior high school student, and 4 died because of tumor progression. The mean period of hospitalization of the patients who have been admitted to university was 63.0 days, and that of patients who have not been admitted university was 135 days. There is a statistical difference (p < 0.05). It could be concluded that the period of hospitalization rather than radiotherapy seemed to influence the educational status of children with brain tumor.

Fluid loading in rats increases serum brain natriuretic peptide concentration.

Hyponatremia after subarachnoid hemorrhage has been linked to high plasma concentration of atrial natriuretic peptide and brain natriuretic peptide. Volume expansion therapy to prevent symptomatic vasospasm, such as intensive hypertensive and hypervoremic therapy, may alter systemic concentration of these peptides. We therefore examine brain natriuretic peptide secretion in rats in response to acute volume expansion, infusing to 10 ml of saline over 1 h. In the 10 ml group, brain natriuretic peptide concentrations showed a significant increase from pre-infusion concentrations 1 h after initiation of infusion, but had begun to fall 1 h later. We suspect that high plasma concentration of brain natriuretic peptide after subarachnoid hemorrhage is partly caused by hypervoremic therapy.

Involvement of the multidrug resistance protein 3 in drug sensitivity and its expression in human glioma.

The multidrug resistance protein (MRP) family belongs to the ATP-binding cassette superfamily (ABC) of transporters, which are involved in ATP-dependent transport of hydrophobic compounds. One of the MRP family, MRP1, is partially associated with the multidrug resistance phenotype in brain tumors. In this study, we asked whether another MRP family gene, MRP3, could affect drug sensitivity to anticancer agents in human glioma cell lines and clinical glioma specimens. We first produced two antisense transfectants by introduction of antisense MRP3 cDNA into the glioma cell line NHG2, which endogenously expresses MRP3. The two MRP3 antisense transfectants showed 2- to 5-fold increases in drug sensitivity to etoposide and cisplatin compared with NHG2 cells, but their sensitivity to vincristine or nitrosourea was not changed. Two MRP3 cDNA sense transfectants of pig kidney cell lines showed 4- to 6-fold drug resistance to etoposide, but only 1.4- to 1.5-fold to cisplatin. We next compared the mRNA levels of four ABC transporters, multidrug resistance 1 (MDR1), MRP1, MRP2 and MRP3 in clinical samples, including 34 patients with gliomas, by quantitative RT-PCR analysis. In some of the clinical samples, increased expression of MRP1 and MRP3 was apparent in malignant gliomas. In situ hybridization revealed that glioma cells were stained with MRP3 probe. MRP3 may modulate drug sensitivity to certain anticancer agents in human gliomas.

Transcerebellomedullary fissure approach with special reference to methods of dissecting the fissure.

OBJECT: The purpose of the present study was to refine the transcerebellomedullary fissure approach to the fourth ventricle and to clarify the optimal method of dissecting the fissure to obtain an appropriate operative view without splitting the inferior vermis. METHODS: The authors studied the microsurgical anatomy by using formalin-fixed specimens to determine the most appropriate method of dissecting the cerebellomedullary fissure. While dissecting the spaces around the tonsils and making incisions in the ventricle roof, the procedures used to expose each ventricle wall were studied. Based on their findings, the authors adopted the best approach for use in 19 cases of fourth ventricle tumor. The fissure was further separated into two slit spaces on each side: namely the uvulotonsillar and medullotonsillar spaces. The floor of the fissure was composed of the tela choroidea, inferior medullary velum, and lateral recess, which form the ventricle roof. In this approach, the authors first dissected the spaces around the tonsils and then incised the taenia with or without the posterior margin of the lateral recess. These precise dissections allowed for easy retraction of the tonsil(s) and uvula and provided a sufficient view of the ventricle wall such that the deep aqueductal region and the lateral region around the lateral recess could be seen without splitting the vermis. The dissecting method could be divided into three different types, including extensive (aqueduct), lateral wall, and lateral recess, depending on the location of the ventricle wall and the extent of surgical exposure required. CONCLUSIONS: When the fissure is appropriately and completely opened, the approach provides a sufficient operative view without splitting the vermis. Two key principles of this opening method are sufficient dissection of the spaces around the tonsil(s) and an incision of the appropriate portions of the ventricle roof. The taenia(e) with or without the posterior margin of the lateral recess(es) should be incised.

Different responses of benign and atypical meningiomas to gamma-knife radiosurgery: report of two cases with immunohistochemical analysis.

Recent reports have shown that gamma-knife radiosurgery provides a safe and effective strategy for the management of brain tumors. To evaluate the role of stereotactic radiosurgery in the management of meningiomas, we investigated the histopathology of two patients. The patients, a 37-year-old man and a 54-year-old woman, presented with visual field disturbance or headache. Imaging studies demonstrated intracranial meningiomas--tentorial and sphenoid ridge, respectively. Each patient undewent subtotal surgical resection (more than 90% in both patients), followed by gamma-knife radiosurgery of the remnant tumor marginal doses of 15 Gy. Pathological examination of the original tumors revealed a meningothelial meningioma and an atypical meningioma, respectively. Enlargement of the remnant tumors 4 months after radiosurgery resulted in total surgical resection in both patients. Thirteen months later, the patient with the atypical meningioma underwent a third operation for early recurrence of the tumor. Histopathology was investigated, and MIB-1, p53, and bcl-2 labeling indexes (LI) were analyzed immunohistochemically. Histopathologically, the specimens showed necrosis and intratumoral vessel obliteration after radiosurgery in both cases. However, more remnant tumor cells survived in the atypical meningioma. Immunohistochemically, increased wild-type p53, decreased bcl-2 expression, and decreased MIB-1 LI were observed in the benign meningioma. In the atypical meningioma, on the contrary, MIB-1 LI was decreased and mutant-type p53 and bcl-2 expression were unchanged. The specimen from the third operation revealed an anaplastic meningioma, and MIB-1 LI was markedly increased. These findings suggest that the efficacy of radiosurgery may differ between benign and atypical meningiomas.

[Clinical trial of bradykinin-enhancing chemotherapy for a recurrent malignant glioma: a case report].

Patients with malignant glioma undergo a combined treatment with surgical resection, radiotherapy, and chemotherapy. Although those treatments usually show some restraining effects on the tumor, a relapse occurs in most of the patients within a few years. We have investigated the feasibility and safety of intra-arterial chemotherapy for malignant brain tumors by enhancing vascular permeability using intra-arterial bradykinin infusion. In 2001, The Committee of Ethics in Kyushu University approved our clinical trial of the bradykinin-enhancing chemotherapy for recurrent malignant gliomas. We here report the first case of our clinical trial. A 31-year-old man, who had undergone surgical resection followed by chemotherapy and irradiation for malignant progression of the left frontal astrocytoma over a period of 2 years, had a relapse of the tumor in the bilateral frontal lobes. After obtaining informed consent, bradykinin and carboplatin were infused through a microcatheter at the left A1 portion under general anesthesia. By dose escalation of bradykinin, the enhanced lesion in the bilateral frontal lobes diminished on magnetic resonance imaging after 3 trials with 3-week intervals, regardless of new lesions outside of the treated area. No neurological or physiological complication including myelosuppression was noted. Bradykinin-enhancing chemotherapy appeared to be effective and safe for malignant glioma. Because it was able to increase drug delivery to the tumor, it was possible to reduce the size of the dose of chemotherapeutic agent, which resulted in minimum complication.

Spinal intradural perimedullary arteriovenous fistula with varix in infant.

A rare occurrence of type IV spinal arteriovenous malformation (intradural perimedullary arteriovenous fistula) is described in an 18-month-old boy initially misdiagnosed with Guillain-Barré syndrome. An intramedullary mixed-intensity mass lesion at Th1 was demonstrated by magnetic resonance imaging together with flow voids over the dorsal aspect of the swollen spinal cord. Angiography demonstrated an intradural perimedullary arteriovenous fistula including an intraparenchymal vascular pocket. After partial embolisation of the posterior spinal arteries through the left intercostal-radicular artery, the arteriovenous fistula was removed completely together with an organised haematoma. The fistula directly opened into a vascular pocket, which was confirmed pathologically to be a varix. The postoperative course was uneventful, and the patient resumed ambulation within 4 months. The case, subclassifiable as a type IVb spinal perimedullary AVF, was unique given its location and the patient's age at presentation.

The specific expression of three novel splice variant forms of human metalloprotease-like disintegrin-like cysteine-rich protein 2 gene inBrain tissues and gliomas.

We have previously identified 67 exons on a yeast artificial chromosome contig spanning 1.5 Mb around the multidrug resistance 1 gene region of human chromosome 7q21.1. In this study, we identified three novel cytoplasmic variants (MDC2-gamma, MDC2-delta, and MDC2-epsilon) of the human metalloprotease-like disintegrin-like cysteine-rich protein 2 (MDC2) among these exons by screening a human brain cDNA library and also by using a reverse transcription polymerase chain reaction. Genomic sequence analysis strongly supported the idea that the variations in the cytoplasmic domain were generated by alternative splicing. The expression of MDC2 variant forms in human brain tissue and gliomas was examined by reverse transcription polymerase chain reaction and RNase protection assay. MDC2-epsilon was expressed only in the cortical and hippocampal regions in human brain, but not in gliomas. In contrast, MDC2-gamma was a major form expressed in human gliomas. Specific expression of these cytoplasmic variants of MDC2 in human brain and its malignancies is discussed.

Moyamoya disease.

Moyamoya disease is a specific chronic cerebrovascular occlusive disease first reported by Japanese surgeons in 1957. The disease is characterized by stenosis or occlusion of the terminal portions of the bilateral internal carotid arteries and abnormal vascular network in the vicinity of the arterial occlusion. It may cause ischemic attacks or cerebral infarction, which is more frequent in children than in adults. In adults, cerebral hemorrhage may occur. The disease is distributed in all age groups, but the highest peak is in childhood at less than 10 years of age. The characteristic histopathologic features of the steno-occlusive arteries are fibrocellular thickening of the intima containing proliferated smooth muscle cells and prominently tortuous and often duplicated internal elastic lamina. There is usually no atheromatous plaque in the arterial wall. Etiology of the disease is still unknown; however, multifactorial inheritance is considered possible because of a higher incidence of the disease in Japanese and Koreans and approximately 10% of familial occurrence among the Japanese. Recent genetic studies suggest some responsible genetic foci in chromosomes 3, 6 and 17.