Merlin inhibits Wnt/ß-catenin signaling by blocking LRP6 phosphorylation.

Merlin, encoded by the NF2 gene, is a tumor suppressor that acts by inhibiting mitogenic signaling and is mutated in Neurofibromatosis type II (NF2) disease, although its molecular mechanism is not fully understood. Here, we observed that Merlin inhibited Wnt/ß-catenin signaling by blocking phosphorylation of LRP6, which is necessary for Wnt signal transduction, whereas mutated Merlin in NF2 patients did not. Treatment with Wnt3a enhanced phosphorylation of Ser518 in Merlin via activation of PAK1 in a PIP2-dependent manner. Phosphorylated Merlin dissociated from LRP6, allowing for phosphorylation of LRP6. Tissues from NF2 patients exhibited higher levels of ß-catenin, and proliferation of RT4-D6P2T rat schwannoma cells was significantly reduced by treatment with chemical inhibitors of Wnt/ß-catenin signaling. Taken together, our findings suggest that sustained activation of Wnt/ß-catenin signaling due to abrogation of Merlin-mediated inhibition of LRP6 phosphorylation may be a cause of NF2 disease.

Phosphorylation of purified recombinant hepatitis B virus-X protein by mitogen-activated protein kinase and protein kinase C in vitro.

The recombinant human hepatitis B virus-X protein (rhHBx) has been expressed as inclusion bodies in Escherichia coli and purified. By sequential dialysis of urea, rhHBx was folded into the native structure, which was demonstrated by both the efficacy of its transcriptional activation of the adenovirus major late promoter, fluorescence and circular dichroism (CD) analysis. The increase in CD values at 220 nm and a corresponding blue shift of the intrinsic fluorescence emission confirmed the ability of HBx to refold in lower concentrations of urea to produce the active protein. After purification and renaturation, the rhHBx protein was found to be phosphorylated by protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). In vivo phosphorylation of HBx was also demonstrated. Although PKC and MAPK enhance the HBx phosphorylation in vitro, neither protein kinase A nor caseine kinase II (CKII) phosphorylate HBx protein, though there are possible substrate residues of both kinases in HBx protein. Phosphoamino acid analysis of the total acid hydrolyzed HBx showed that serine residues can be phosphorylated by PKC or MAPK.

Rapid and simple purification of human immunodeficiency virus 1 epitope gp41.

In order to develop a reliable and inexpensive serodiagnostic method, part of the transmembrane glycoprotein gene of HIV-1, gp41', (HIV-env 548-646) was cloned into an expression vector, pCT10 with a sequence encoding a hydroxylamine cleavage site and with a part of Lac Z gene (Lac 2": 834 base pairs) as a fusion partner. Overexpression of Lac Z"-gp41' was induced in E. coli and the gp41' fusion protein was purified to homogeneity by centrifugation, hydroxylamine cleavage and an ion-exchange chromatography. Western blot analysis and enzyme-linked immunosorbant assay (ELISA) using the purified gp41 fragment showed high sensitivity and specificity of gp41 as an antigen to detect anti HIV-1 antibodies in testing human sera. These results suggest that this simple and rapid purification method is reliable for obtaining a large quantity of purified gp41'.

Overexpression and simple purification of human immunodeficiency virus-1 gag epitope derived from a recombinant antigen in E. coli and its use in ELISA.

To develop a test for diagnosis of human immunodeficiency virus-1 (HIV-1) exposure sensitivity, a part of the gag gene was cloned and expressed in Escherichia coli, using expression vectors containing a trp promoter. The immunoreactivity of recombinant protein was determined using HIV-1 specific antibodies in a Western blot analysis. The recombinant plasmid, pYHCgag3, gag gene was fused to the trpE' gene linked to the hydroxylamine (HA) cleavage recognition sequence which was induced to overexpress a core antigen (gag a.a. 121-398 from plasmid BH10) as fusion protein in the form of insoluble inclusion body. Recombinant gag was purified by a simple single step purification procedure. After partial purification of inclusion bodies and subject to the HA-cleavage treatment, gag protein was further purified to homogeneity using DEAE-Sepharose chromatography. The purified core antigen offered reliable results with high sensitivity and specificity for identification of HIV-1 antibodies when tested in the enzyme-linked immunosorbent assay (ELISA). These results suggest that mass production of recombinant core antigen will provide a valuable resource to HIV-1 serodiagnostics for the screening of large groups of blood donors to prevent HIV-1 infection.

Overexpression and purification of human immunodeficiency virus type 1 env derived epitopes in Escherichia coli.

In order to develop a reliable and inexpensive serodiagnostic method, a part of envelope gene of HIV-1, gp120' and gp41' (HIV-1 env a.a. 295-474 and a.a. 556-647) was cloned into a T7 expression vector (pET3d). The fusion protein (gp120'-gp41') was overexpressed in Escherichia coli, then purified to homogeneity by a simple gel filtration chromatography. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using the purified fusion protein showed a high sensitivity and a specificity for the detection of anti HIV-1 antibodies in testing human plasma. These results suggest that the expression scheme employing a direct expression vector and the rapid purification method are reliable and applicable for obtaining a large quantity of HIV-1 env protein for diagnoses of HIV-1 infections.

Effects of timing of coil embolization after aneurysmal subarachnoid hemorrhage on procedural morbidity and outcomes.

OBJECTIVE: To elucidate the effect of treatment timing on procedural clinical outcomes after aneurysmal subarachnoid hemorrhage (SAH) for patients treated by endosaccular coil embolization. METHODS: A group of 327 patients who were consecutively treated, during a 46-month period, for ruptured intracranial aneurysms by coil embolization within 30 days after SAH were evaluated. Outcomes were assessed by comparing immediate pretreatment World Federation of Neurological Surgeons (WFNS) grades, 72-hour posttreatment WFNS grades, and modified Glasgow Outcome Scale scores at 6 months for patients treated within 48 hours (Group 1), 3 to 10 days (Group 2), or 11 to 30 days (Group 3) after SAH. RESULTS: The three interval-to-treatment groups included 33, 38, and 29% of the patients, respectively. Before treatment, 70% of the patients in Group 1, 78% of those in Group 2, and 83% of those in Group 3 were in good clinical grades (i.e., WFNS Grade 1 or 2). After coil embolization, the WFNS grades were either unchanged or improved for 93.5% of the patients in Group 1, 89.5% of those in Group 2, and 91.5% of those in Group 3. After 6 months, 81.3% of the patients in Group 1 experienced good outcomes (modified Glasgow Outcome Scale scores of 1 or 2), as did 84% of those in Group 2 and 80% of those in Group 3. No statistical difference was demonstrated between the three groups when they were compared for these two variables. CONCLUSION: The interval between endovascular treatment and SAH did not affect periprocedural morbidity rates or 6-month outcomes. Coil embolization should therefore be performed as early as possible after aneurysmal SAH, to prevent aneurysmal rerupture.

Five-year experience in using coil embolization for ruptured intracranial aneurysms: outcomes and incidence of late rebleeding.

OBJECT: During a 5-year period 317 patients presenting with aneurysmal subarachnoid hemorrhage were successfully treated by coil embolization within 30 days of hemorrhage. The authors followed patients to assess the stability of aneurysm occlusion and its longer-term efficacy in protecting patients against rebleeding. METHODS: Patients were followed for 6 to 65 months (median 22.3 months) by clinical review, angiography performed at 6 months posttreatment, and annual questionnaires. Stable angiographic occlusion was evident in 86.4% of small and 85.2% of large aneurysms with recurrent filling in 38 (14.7%) of 259 aneurysms. Rebleeding was caused by aneurysm recurrence in four patients (between 11 and 35 months posttreatment) and by rupture of a coincidental untreated aneurysm in one patient. Annual rebleeding rates were 0.8% in the 1st year, 0.6% in the 2nd year, and 2.4% in the 3rd year after aneurysm embolization, with no rebleeding in subsequent years. Rebleeding occurred in three (7.9%) of 38 recurrent aneurysms and in one (0.4%) of 221 aneurysms that appeared stable on angiography. CONCLUSIONS: Periodic follow-up angiography after coil embolization is recommended to identify aneurysm recurrence and those patients at a high risk of late rebleeding.

Anterior cervical corpectomy and fusion using miniplate and screws in a 7-year-old child with eosinophilic granuloma of the cervical spine.

STUDY DESIGN: This is a case report of a 7-year-old child with eosinophilic granuloma in the cervical spine, which underwent anterior cervical corpectomy and fusion by using Miniplate and screws. OBJECTIVES: To describe the use of Miniplate and screws for pediatric cervical anterior fusion. SUMMARY OF BACKGROUND DATA: Eosinophilic granuloma is a rare disease causing destructive bony lesions of the cervical spine in children. A complete resection and fusion were considered to be the preferable treatment in our case. However, cervical spinal fusion and instrumentation in children may be technically difficult because of the size of the vertebral body and the iliac bone. In addition, a proper device for an internal fixation in pediatric patients is not yet available. METHODS: A case of eosinophilic granuloma in pediatric spine was presented. RESULTS: We confirmed successful bony fusion and the restoration of the normal cervical curvature without recurrence of the tumor 2 years after the procedure. CONCLUSIONS: For proper internal fixation and prevention of dislodgement of the grafted bone, we used the Miniplate and screws as internal fixator after intralesional resection of the tumor mass.

Usefulness of MR imaging for diseases of the small intestine: comparison with CT.

OBJECTIVE: To evaluate the usefulness of MR imaging for diseases of the small intestine, emphasizing a comparison with CT. MATERIALS AND METHODS: Thirty-four patients who underwent both CT and MR imaging using FLASH 2D and HASTE sequences were analyzed. All patients had various small bowel diseases with variable association of peritoneal lesions. We compared the detectabilities of CT and MR imaging using different MR pulse sequences. The capability for analyzing the characteristics of small intestinal disease was also compared. RESULTS: MR imaging was nearly equal to CT for detecting intraluminal or peritoneal masses, lesions in the bowel and mesentery, and small bowel obstruction, but was definitely inferior for detecting omental lesions. The most successful MR imaging sequence was HASTE for demonstrating bowel wall thickening, coronal FLASH 2D for mesenteric lesions, and axial FLASH 2D for omental lesions. MR imaging yielded greater information than CT in six of 12 inflammatory bowel diseases, while it was equal to CT in six of seven neoplasms and inferior in five of seven mesenteric ischemia. In determining the primary causes of 15 intestinal obstructions, MR imaging was correct in 11 (73%) and CT in nine (60%) patients. CONCLUSION: MR imaging can serve as an alternative diagnostic tool for patients with suspected inflammatory bowel disease, small intestinal neoplasm or obstruction.

Radiosurgery for metastatic spinal tumors: follow-up MR findings.

BACKGROUND AND PURPOSE: MR imaging is the primary tool for evaluation and monitoring of spinal tumors. We retrospectively analyzed the MR imaging findings before and after SRS for metastatic spinal tumors. MATERIALS AND METHODS: We reviewed MR imaging findings on 79 metastatic spinal tumor lesions in 44 patients (29 male and 15 female)who had undergone radiosurgery between November 2003 and April 2008. Posttreatment MR imaging was evaluated retrospectively for 3 aspects: 1) changes in tumor volume; 2) changes in T2 signal intensity;and 3) changes in contrast enhancement patterns. RESULTS: With regard to tumor volume on MR images, 32 lesions(40.5%) decreased in volume (group 1), 39 (49.4%) showed no change (group 2), and 8 (10.1%) increased in volume (group 3). T2 signal intensities were unchanged in 4 lesions (type 1), homogeneously increased in 3 (type 2), and changed to a homogeneously dark signal in 4 (type 4). The T2 signal intensity was increased and inter mixed with dark signal intensity (type 3) in 68 lesions. A decrease in contrast enhancement with or without non-enhancing foci was seen in 73 lesions. A persistent homogeneous enhancement pattern was seen in all 4 of the type 1 lesions, in 1 of the 3 type 2 lesions, and in 1 of the 68 type 3 lesions. CONCLUSIONS: Main MR imaging features of locally controlled metastatic spinal tumors included no increase in tumor volume, increased T2 signal intensity with intermixed T2 dark signal intensity,and decreased contrast enhancement. Follow-up MR imaging also provided several patterns of tumor recurrence [corrected].

Cloning and functional analysis of the GSH1/MET1 gene complementing cysteine and glutathione auxotrophy of the methylotrophic yeast Hansenula polymorpha.

The Hansenula polymorpha GSH1/MET1 gene was cloned by complementation of glutathione-dependent growth of H. polymorpha gsh1 mutant isolated previously as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resistant and cadmium ion sensitive clone. The H. polymorpha GSH1 gene was capable of restoring cadmium ion resistance, MNNG sensitivity, normal glutathione level and cell proliferation on minimal media without addition of cysteine or glutathione, when introduced into the gsh1 mutant cells. It was shown that the H. polymorpha GSH1 gene has homology to the Saccharomyces cerevisiae MET1 gene encoding S-adenosyl-L-methionine uroporphyrinogen III transmethylase, responsible for the biosynthesis of sulfite reductase cofactor, sirohaem. The H. polymorpha GSH1/MET1 gene deletion cassette (Hpgsh1/met1::ScLEU2) was constructed and corresponding null mutants were isolated. Crossing data of the point gsh1 and null gsh1/met1 mutants demonstrated that both alleles were located to the same gene. The null gsh1/met1 mutant showed total growth restoration on minimal media supplemented with cysteine or glutathione as a sole sulfur source, but not with inorganic (sulfate, sulfite) or organic (methionine, S-adenosylmethionine) sources of sulfur. Moreover, both the point gsh1 and null gsh1/met1 mutants displayed increased sensitivity to the toxic carbon substrate methanol, formaldehyde, organic peroxide and cadmium ions.

A computerised relational database for auditing endovascular treatment of patients with intracranial aneurysms.

We describe the development and design of a database for auditing patients with intracranial aneurysms and their endovascular treatment. The database has been used since 1992. Our department's version now contains records of over 800 patients and well over 1,000 aneurysms. The advantages of a relational database for this type of audit are discussed. Copies of the software can be obtained free of charge from the authors.

Spinal clear cell meningioma presented with progressive paraparesis in infancy.

Clear cell meningioma, about 20 cases of which have been reported in the literature, is a morphological variant of meningioma. The authors report a case of spinal clear cell meningioma that occurred in a child. A 14-month-old girl showed gradually progressive paraparesis 1 month after she started to walk. Magnetic resonance image showed an intradural extramedullary mass compressing the conus medullaris and cauda equina. Complete excision of the tumor was done, and the patient gradually recovered from motor weakness and neurogenic bladder. Histological examinations along with immunohistochemical and ultrastructural investigations allowed a diagnosis of clear cell meningioma. During the follow-up period, a recurrent mass lesion was detected on the 8-month follow-up MR image in the same region. Because clear cell meningioma might be biologically aggressive, postoperative adjuvant therapy and close follow-up investigation should be considered.

Naturally acquired antibody responses to the C-terminal region of merozoite surface protein 1 of Plasmodium vivax in Korea.

We expressed a protein in Saccharomyces cerevisiae in order to evaluate the humoral immune responses to the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax. This protein (Pv200(18)) had a molecular mass of 18 kDa and was reactive with the sera of individuals with patent vivax malaria on immunoblotting analysis. The levels of immunoglobulin M (IgM) and IgG antibodies against Pv200(18) were measured in 421 patients with vivax malaria (patient group), 528 healthy individuals from areas of nonendemicity (control group 1), and 470 healthy individuals from areas of endemicity (control group 2), using the indirect enzyme-linked immunosorbent assay (ELISA) method. To study the longevity of the antibodies, 20 subjects from the patient group were also tested for the antibody levels once a month for 1 year. When the cutoff values for seropositivity were determined as the mean + 3 x standard deviation of the antibody levels in control group 1, both IgG and IgM antibody levels were negative in 98.5% (465 of 472) of control group 2. The IgG and IgM antibodies were positive in 88.1% (371 of 421) and 94.5% (398 of 421) of the patient group, respectively. The IgM antibody became negative 2 to 4 months after the onset of symptoms, whereas the IgG antibody usually remained positive for more than 5 months. In conclusion, indirect ELISA using Pv200(18) expressed in S. cerevisiae may be a useful diagnostic method for vivax malaria.