Overexpression and characterization of thermostable serine protease in Escherichia coli encoded by the ORF TTE0824 from Thermoanaerobacter tengcongensis.

A novel extracellular serine protease derived from Thermoanaerobacter tengcongensis, designated tengconlysin, was successfully overexpressed in Escherichia coli as a soluble protein by recombination of an N-terminal Pel B leader sequence instead of the original presequence and C-terminal 6x histidine tags. The purified protein was activated by 0.1% sodium dodecyl sulfate (SDS) treatment but not by thermal treatment. The molecular weight of tengconlysin estimated by SDS-polyacrylamide gel electrophoresis analysis and gel filtration chromatography was 37.9 and 36.2 kDa, respectively, suggesting that the enzyme is monomeric. The N-terminal sequence of mature tengconlysin was LDTAT, suggesting that it is a preproprotein containing a 29 amino acid presequence (predicted from the SigP program) and a 117 amino acid prosequence in the N-terminus. The C-terminal putative propeptide (position 469-540 in the preproprotein) did not inhibit the protease activity. The optimum temperature for tengconlysin activity was 90 degrees C in the presence of 1 mM calcium ions and the optimum pH ranged from 6.5 to 7.0. Activity inhibition studies suggest that the protease is a serine protease. The protease was stable in 0.1% SDS and 1-4 M urea at 70 degrees C in the presence of calcium ions and was activated by the denaturing agents.

The expression and activation of matrix metalloproteinase-1 after subarachnoid haemorrhage in rats.

BACKGROUND: Collagen lattice contraction has been reported as another aspect of the pathogenesis of cerebral vasospasm after subarachnoid haemorrhage (SAH). Recently, some authors have suggested that matrix metalloproteinase-1 (MMP-1) plays an important role in collagen lattice contraction. Therefore, this study aimed to clarify a role of MMP-1 during cerebral vasospasm in a rat SAH model. METHOD: We used a single-SAH model in rats and assessed the basilar arteries (BAs) at 30 minutes and on 2 days after SAH by cross-sectional area measurement and other histological parameters. Immunohistochemistry and Western blot analysis were used to quantify MMP-1 expression and activation. RESULTS: BAs in rats significantly exhibited severe cerebral vasospasm at 30 minutes after SAH and moderate vasospasm on Day 2. The immunohistochemistry and Western blotting performed in BAs of rats demonstrated that both expression and activation in MMP-1 peaked at 30 minutes after SAH and then declined to the control level. CONCLUSIONS: MMP-1 is expressed and activated in a parallel time course to the development of cerebral vasospasm in an experimental model of SAH.

Training in Neurovascular Intervention Usefulness of in-Vitro Model and Clinical Practice.

SUMMARY: We introduce our training tools and system of neurovascular intervention. An in vitro cerebral vascular model was used for the young residents to understand the basic interventional techniques and devices. The model included several vascular lesions such as cerebral aneurysm, dural arterio-venous fistula, or carotid artery stenosis. Endovascular procedures in the model were performed under fluoroscopic or direct visual control, and consecutive haemodynamic changes were visualized by using digital subtraction angiography and direct observation. Thus, traineess could have an easy understanding of clinical conditions. New medical devices, such as platinum coils, were successfully implanted in the model under stable conditions. After the initial training using vascular model, the residents had started clinical experiences under the control of senior surgeons. Although it is difficult to describe usefulness of our clinical training, we believe that we provide enough good quality and quantity of clinical cases to the residents. Because our endovascular team has recently had150-200 interventional procedures every year, one resident can have experienced more than 100 cases per year. The qualification of a Board Certified Specialist of the Japanese Society of Intravascular Neurosurgery (JSIN) requires that the applicant must have experienced more than 100 cases for four years. So our residents can have enough case materials to qualify the board examination.

Accumulation of cell cycle regulatory proteins, p21 and p27, induced after hyperthermia in human glioma cells.

It was investigated whether there was a relationship between p53 p21 and p27 induction pathways in the cellular response of glioma cells to hyperthermia. Two glioma cell lines were employed. A-172 cells had the wild-type of p53, and U251 cells had the mutant-type of p53. An adenovirus harbouring wild-type p53 was also used for the overexpression. The protein induction by hyperthermia was monitored by Western blot analysis. In U251 cells, the expression of wild-type p53 and hyperthermia had an additional cytotoxic effect, but did not affect A-172 cells. Significant p21 accumulation by hyperthermia was recognized in A-172 cells, and was also recognized in p53-transduced U251 cells. On the other hand, the accumulation of p27 by hyperthermia was not seen in A-172 or U251 cells, and the exogenous expression of p53 did not affect the accumulation of p27 by hyperthermia in U251 cells. These findings suggest that the p53-p21 pathway is involved in the signal transduction after hyperthermia, rather than the p27 pathway.

[Usefulness of a new mechanical detachable coil for neurovascular intervention].

The electrolytically detachable platinum coil (Guglielmi Detachable Coil: GDC) is a safe and efficient endovascular tool for treatment of cerebral aneurysms. However, the GDC still has some problems, including a prolonged detaching time and high cost. The Detach Coil System (DCS) is a newly developed platinum detachable coil for the treatment of neurovascular diseases. This has a mechanical "screw" detachment system, which can be detached faster than the GDC. The platinum coil is mounted to the tip of the delivery wire by the "screw" system. For detaching the coil, 20-25 times anti-clockwise rotation of the delivery wire using a "detach locking device" is required. We report our preliminary clinical experience of using the DCS in 11 patients. This series included 5 sacral aneurysms, 3 dissecting aneurysms, and 3 dural arteriovenous fistulas. Seventy-five coils were used in total, of which 5 coils were retrieved and 70 coils were implanted. The detaching time of each DCS was 15-20 seconds, which was much faster than that of the GDC. All lesions were successfully treated without symptomatic complications. In the limited number of cases, our result suggest that the DCS allowed safe and fast endovascular treatment of neurovascular disease at a lower cost.

Down-regulation of integrin alpha(v)beta(3) expression and integrin-mediated signaling in glioma cells by adenovirus-mediated transfer of antisense urokinase-type plasminogen activator receptor (uPAR) and sense p16 genes.

Interaction between the extracellular matrix and integrin receptors on cell surfaces leads not only to cell adhesion but also to intracellular signaling events that affect cell migration, proliferation, and survival. The vitronectin receptor alpha(v)beta(3) integrin is of key importance in glioma cell biology. The expression of urokinase-type plasminogen activator receptor (uPAR) was recently shown to co-regulate with the expression of alpha(v)beta(3) integrin. Moreover, restoration of the p16 protein in glioma cells inhibits the alpha(v)beta(3) integrin-mediated spreading of those cells on vitronectin. Thus we hypothesized that adenovirus-mediated down-regulation of uPAR and overexpression of p16 might down-regulate the expression of alpha(v)beta(3) integrin and the integrin-mediated signaling in glioma cells, thereby defeating the malignant phenotype. In this study, we used replication-deficient adenovirus vectors that contain either a uPAR antisense expression cassette (Ad-uPAR) or wild-type p16 cDNA (Ad-p16) and a bicistronic adenovirus construct in which both the uPAR antisense and p16 sense expression cassettes (Ad-uPAR/p16) are inserted in the E1-deleted region of the vector. Infecting the malignant glioma cell line SNB19 with Ad-uPAR, Ad-p16, or Ad-uPAR/p16 in the presence of vitronectin resulted in decreased alpha(v)beta(3) integrin expression and integrin-mediated biological effects, including adhesion, migration, proliferation, and survival Our results support the therapeutic potential of simultaneously targeting uPAR and p16 in the treatment of gliomas.

Grafting of encapsulated genetically modified cells secreting GDNF into the striatum of parkinsonian model rats.

In order to deliver glial cell line-derived neurotrophic factor (GDNF) into the brain, we have established a cell line that produces GDNF in a continuous fashion by genetic engineering. These cells were encapsulated and grafted into parkinsonian model rats that had received unilateral intrastriatal injection of 6-hydroxydopamine 2 weeks earlier. Neurochemical analysis showed that GDNF has been produced from the capsule for 6 months after grafting and histological analysis revealed good survival of GDNF-producing cells in the capsule 6 months after grafting. The density of nigrostriatal dopaminergic fibers in the striatum as well as the number of dopaminergic cell bodies in the substantia nigra recovered significantly after GDNF-producing cell grafting. These results suggest the possible application of GDNF-producing cell grafting for the treatment of Parkinson's disease.

Neurotrophic factor-secreting cell grafting for cerebral ischemia: preliminary report.

In this experiment, we examined a possible protective effect of encapsulated neurotrophic factor-secreting cell grafting for ischemic injury. We established a basic fibroblast growth factor (bFGF)-secreting cell line by genetic manipulation. We enveloped these cells into polymer capsules, which consist of a semipermeable membrane, and implanted them into the right striatum of rats. At 6 days after implantation, these rats received right middle cerebral artery occlusion (MCAO) using interluminal suture technique. At 24 h after MCAO, rats were sacrificed and their cerebral infarction volume was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining and image analysis. We found approximately 30% reduction in infarct volume in the encapsulated bFGF-secreting cell grafting groups vs. the encapsulated naive BHK cell grafting group or the without implantation group. We measured bFGF secretion from encapsulated bFGF-secreting cells using enzyme-linked immunosorbent assay (ELISA). The retrieved capsules continued to secrete bFGF. There was no significant difference of bFGF secretion between the capsules before and after transplantation. A large number of viable BHK-bFGF cells was observed within the full length of the retrieved capsule. These results indicate that encapsulated bFGF-secreting cell grafting exerts a protective effect on ischemic injury.

Primary Ewing's sarcoma/peripheral primitive neuroectodermal tumor at the vertex of the skull with elevated serum carcinoembryonic antigen: case report.

A primary Ewing's sarcoma arising in the skull is relatively rare. Although a small number of case reports noted elevated carcinoembryonic antigen (CEA) in patients with primary central nervous system (CNS) neoplasms, there is no report of Ewing's sarcoma/peripheral primitive neuroectodermal tumor (PNET) with elevated serum levels of CEA. A 7-year-old boy who had episodes of headache and vomiting had noticed a solid mass in the vertex of the head. Imaging studies revealed a large intra- and extracranial tumor at the vertex of the skull. Hematological examination demonstrated high serum levels of CEA: 91.09 ng/ml. The patient initially underwent an embolization of the bilateral middle meningeal arteries with Gelfoam particles. One week later, the patient was operated on and a subtotal resection of the tumor was performed. On histopathological and molecular genetic examination, the tumor was diagnosed as a Ewing's sarcoma/peripheral PNET. Immunohistochemical study showed strongly positive staining for CEA in the tumor cells. The serum level of CEA was normalized at 0.83 ng/ml after the tumor was removed and the boy underwent radiotherapy and 3 courses of chemotherapy. This is the first reported case of a primary Ewing's sarcoma/peripheral PNET at the vertex of the skull with elevated serum CEA.

[Use of the GDC for embolization of a tumor fed by a cavernous branch of the internal carotid artery].

Currently, embolization of small branches of the internal carotid artery (ICA) can be embolized through superselective microcatheterization, followed by the injection of liquid or particulate embolic materials. Often, however, a microcatheter cannot be placed in a stable enough position to allow an endovascular surgeon to perform a safe embolization, and the reflux of embolic agents into the main trunk of the ICA is a major concern. Meticulous technique and a detailed knowledge of the vascular anatomy of the cavernous sinus region are necessary to maximize devascularization of the lesion and to minimize the risk of complications. This report describes the case of a patient with a hypervascular tumor whose feeding vessel from the cavernous ICA was successfully occluded with polyvinyl alcohol (PVA) combined with a regular Guglielmi detachable coil (GDC). A 62-year-old woman had a left-sided petroclival meningioma, which was diagnosed based on computed tomography and magnetic resonance studies. Transfemoral angiographic studies demonstrated that the tumor was fed by intracavernous branches of the left ICA. We believed that another embolic agent would have presented a risk of reflux into the ICA, with possible unwanted occlusion of normal intracranial arteries. A single GDC was sufficient to occlude the feeding artery, and the patient underwent successful surgery 3 days after the endovascular procedure. The GDC can eliminate the ICA supply to hypervascular tumors safely when liquid or particle embolic materials would present a risk of reflux into normal arteries. This device can be positioned and repositioned and can be detached without mechanical force. It may also decrease the risk of unwanted embolization of normal intracranial arteries.

High cyclin E/low p27Kip1 expression is associated with poor prognosis in astrocytomas.

Cyclin E and p27Kip1 are co-regulators of the G1- to S-phase transition and closely related to tumor behavior. The purpose of this study was to examine expression of cyclin E and p27Kip1 in astrocytomas and to evaluate the relationships between expression of these cell-cycle regulators and prognosis of patients with astrocytoma. Tissue samples from 130 astrocytomas (WHO grade 1 n = 5, grade 2 n = 23, grade 3 n = 64, grade 4 n = 38) were examined immunohistochemically for cyclin E and p27Kip1 expression. Patient charts were reviewed for clinical presentation, and survival was followed. The cyclin E labeling index (LI) tended to increase with tumor grade (Kruskal-Wallis, P = 0.0104). For patients with primary astrocytomas, the 50% survival times for the low cyclin E LI (< 5%) group and the high cyclin E LI (> or = 5%) group were 53.7 months and 19.8 months. In combined analysis of cyclin E and p27Kip1 expression, the low cyclin E/high p27Kip1 LI (> or = 50%) group had the best survival (50% survival time: 103.2 months), the low cyclin E/low p27Kip1 LI (> or = 50%) and the high cyclin E/high p27Kip1 LI groups moderate survival (24.1 and 27.5 months), and the high cyclin E/low p27Kip1 LI group the worst survival (13.1 months). Multivariate analysis identified the combined factor, high cyclin E/low p27Kip1, as a novel independent prognostic factor for survival time (P = 0.0037, relative risk = 2.4). This study suggested that combined analysis of cyclin E and p27Kip1 expression was considered to be potentially useful in predicting the prognosis of patients with astrocytoma.

Combined use of sphenoidal electrodes and the dipole localization method for the identification of the mesial temporal focus.

UNLABELLED: We attempted to sub-classify four cases who show temporal spikes on standard scalp electroencephalogram (EEG), using sphenoidal electrodes and the dipole localization METHOD: In a case with mesial temporal epilepsy, spikes showed phase reversal in a sphenoidal electrode, and the spike dipoles were estimated to be in the mesial temporal lobe. In a case with lateral temporal epilepsy, spikes showed no phase reversal in a sphenoidal electrode, and the spike dipoles were estimated to be in the lateral temporal lobe. In two cases out of four, spikes showed phase reversal in sphenoidal electrodes, whilst the dipoles were estimated to be in the frontal lobe. Clinical features also suggested a diagnosis of frontal lobe epilepsy. In one of the two cases in which frontal lobe epilepsy was suspected, ictal dipoles as well as interictal spike dipoles indicated participation of the frontal lobe in the genesis of seizures. Nevertheless, only mesial temporal lobectomy was performed based on results obtained by invasive subdural electrodes. As a result, seizures were not controlled. Although sphenoidal electrodes were useful for differentiating between mesial and lateral temporal lobe foci, it is advisable to use them in combination with the dipole localization method to identify frontal lobe foci.

Effects of aging on cerebral vasospasm after subarachnoid hemorrhage in rabbits.

BACKGROUND AND PURPOSE: The effects of aging on cerebral vasospasm after subarachnoid hemorrhage (SAH) remain to be elucidated. The aim of this study was to clarify age-related differences of vasospasm and of papaverine reactivity in the responses of basilar arteries after SAH in rabbits. METHODS: Rabbits receiving a single injection of arterial blood into the cisterna magna were divided into 3 groups: young (2 to 3 months old), adult (6 to 9 months old), and old (20 to 40 months old). Vertebrobasilar angiograms were obtained before SAH and 1, 2, 4, and 7 days after SAH. Papaverine was administrated selectively via the vertebral artery on day 2, and serial angiography was performed for up to 2 hours. Vessel structures were assessed with light microscopy on days 1, 2, 4, and 7 after SAH and at 10, 30, and 60 minutes after papaverine infusion. RESULTS: Mortality from SAH in old rabbits was 40%, whereas that of young and adult rabbits was 0%. Angiograms revealed that SAH induced maximal constriction of the basilar arteries on day 2 in all age groups, and the constrictions were significantly increased with age at all time points investigated. The degree of dilatation of spastic basilar arteries after intra-arterial papaverine administration significantly decreased with age. Duration of the efficacy of papaverine became significantly shorter with age. Vessel diameter returned to the preinfusion value approximately 120, 60, and 30 minutes after infusion in young, adult, and old rabbits, respectively. Light microscopy in old rabbits showed luminal narrowing and corrugation of the internal elastic lamina not only in the basilar arteries but also in small arteries and intraparenchymal arterioles. CONCLUSIONS: This study suggests that aging increases the degree of vasospasm in rabbits. The impaired reactivity to papaverine with aging might imply the early transition of the aged vessel to the papaverine-resistant chronic stage.

Coil embolization of intradural pseudoaneurysms caused by arterial injury during surgery: report of two cases.

Intradural pseudoaneurysms arose in two patients as a result of arterial injury incurred during surgery. In the first patient, the pseudoaneurysm developed in the middle cerebral artery, at the site of vessel perforation during aneurysmal surgery. In the second patient, the pseudoaneurysm developed in the anterior communicating artery after removal of a tuberculum sellae meningioma. These aneurysms had small ostia and were successfully embolized with electrolytically detachable coils. The clinical features and the treatment of intracranial pseudoaneurysms are discussed.

Inhibition of poly(ADP-ribose) polymerase attenuates cerebral vasospasm after subarachnoid hemorrhage in rabbits.

BACKGROUND AND PURPOSE: Poly(ADP-ribose) polymerase (PARP) is important in modulating inflammation, which has been implicated in cerebral vasospasm after subarachnoid hemorrhage (SAH). We investigated the role of PARP in vasospasm using 3-aminobenzamide (3-AB), a PARP inhibitor, in a rabbit model. METHODS: Twenty-four New Zealand White rabbits were divided into 4 groups: (1) no treatment (control group, n=6); (2) blood injection without pretreatment (SAH-only group, n=6); (3) blood injection with pretreatment by vehicle (SAH+vehicle group, n=6); and (4) blood injection with pretreatment by 3-AB (SAH+3-AB group, n=6). We used the single-hemorrhage model of SAH, injecting autologous arterial blood into the cisterna magna. Angiography was performed before (baseline) and after (day 2) SAH, and the diameter of the basilar artery (BA) was measured. Animals were euthanatized after the second angiogram. After perfusion and fixation, the brains were cut into sections for hematoxylin and eosin and immunohistochemical staining for poly(ADP-ribosyl)ation. RESULTS: In the control group, there were no differences in the BA lumen caliber between baseline and day 2 (96.8+/-10.4%). Cerebral vasospasm in the SAH+3-AB group (88.2+/-6. 2%) was remarkably attenuated in comparison with that in the SAH-only group (64.9+/-8.0%) and the SAH+vehicle group (65.6+/-10. 8%). The BA in the SAH+3-AB group showed less corrugation of the tunica elastica interna than that in the SAH-only and SAH+vehicle groups. Staining for poly(ADP-ribosyl)ation was markedly inhibited in smooth muscle and adventitial cells of the BA in the SAH+3-AB group compared with other groups. CONCLUSIONS: Inhibiting ADP-ribosylation attenuates cerebral vasospasm after SAH in rabbits, and PARP activation may play an important role in the development of cerebral vasospasm.

Expression of Bcl-2, Bcl-x, and Bax proteins in astrocytomas in relation to patient survival.

The Bcl-2 family is composed of a group of related proteins that either prevent or promote apoptosis. This study was undertaken to assess the prognostic value of Bcl-2, Bcl-x, and Bax in patients with astrocytomas. Tissue samples from 104 astrocytomas (WHO grade 2, 21 cases: grade 3, 49 cases; grade 4, 34 cases), including 68 primary and 36 recurrent tumors, were examined immunohistochemically for Bcl-2, Bcl-x, and Bax expression. Patient charts were reviewed for clinical presentation, and survival was followed. The mean values of the Bcl-2, Bcl-x, and Bax labeling indexes (LI) were 15.9 +/- 13.1%, 53.2 +/- 35.3%, and 25.9 +/- 23.2%, respectively. Statistical analysis showed that the Bcl-x LI of high-grade (grade 3 or 4) astrocytomas was higher than that of low-grade (grade 2) tumors (P = 0.0064). There were no significant differences in patient survival between the high- and low-LI groups of Bcl-2, Bcl-x, and Bax. Since the mechanism and regulation of apoptosis are still unclear, it seems difficult to use the Bcl-2 family as a biological marker in predicting the prognosis of patients with astrocytomas.

The importance of venous hypertension in the formation of dural arteriovenous fistulas: a case report of multiple fistulas remote from sinus thrombosis.

Various hypotheses have been reported concerning the pathogenesis of dural arteriovenous fistulas (DAVFs). However, it is still controversial whether sinus thrombosis or venous hypertension has a greater influence on the formation of DAVFs. We present a rare case of multiple DAVFs that developed after sinus thrombosis. Chronic venous hypertension secondary to sinus thrombosis in the left transverse-sigmoid sinus induced the multiple DAVFs, including one in the right cavernous sinus, which was remote from the occluded sinus. This case indicates the importance of venous hypertension in the formation of DAVFs.

Peritumoral brain edema in intracranial meningiomas: effects of radiological and histological factors.

OBJECTIVE: We examined the radiological and histological features influencing the development of peritumoral brain edema (PTBE) among patients with meningiomas. METHODS: Factors causing PTBE were retrospectively analyzed for 125 patients with primary intracranial meningiomas. These factors included tumor size, tumor location, brain-tumor interface, signal intensity on T2-weighted scans, contrast enhancement, and cyst formation (as observed on magnetic resonance imaging scans), as well as tumor vascularity and blood supply (as observed in digital subtraction angiography studies). We defined the edema/tumor volume ratio as the edema index, and we used this index to evaluate PTBE. RESULTS: A relationship between the tumor size and the volume of PTBE was observed. Convexity and middle fossa meningiomas demonstrated the greatest increases in mean edema indices. Meningothelial, anaplastic, microcystic, and angiomatous subtypes exhibited higher edema indices than did other types. Multivariate analysis demonstrated two significant radiological factors: cortical penetration (as defined by the disappearance of the arachnoid layer on magnetic resonance imaging scans) (relative risk, 2.067; P = 0.0148) and vascular supply from the pial-cortical arteries (as observed on angiograms) (relative risk, 2.087; P = 0.0082). CONCLUSION: Tumor infiltration into adjacent brain parenchyma and a pial-cortical blood supply are critical factors for the development of PTBE among patients with meningiomas.