Inverted Linear Halbach Array for Separation of Magnetic Nanoparticles.

A linear array of Nd-Fe-B magnets has been designed and constructed in an inverted Halbach configuration for use in separating magnetic nanoparticles. The array provides a large region of relatively low magnetic field, yet high magnetic field gradient in agreement with finite element modeling calculations. The magnet assembly has been combined with a flow channel for magnetic nanoparticle suspensions, such that for an appropriate distance away from the assembly, nanoparticles of higher moment aggregate and accumulate against the channel wall, with lower moment nanoparticles flowing unaffected. The device is demonstrated for iron oxide nanoparticles with diameters of ~ 5 and 20 nm. In comparison to other approaches, the inverted Halbach array is more amenable to modeling and to scaling up to preparative quantities of particles.

A case of acral lentginous melanoma: the correlation between CD95L expression on melanoma cells and apoptosis of tumor infiltrating lymphocytes.

There is an increasing amount of evidence that melanoma cells express the ligand for CD95 (CD95L), a potent inducer of apoptosis which contributes to creating the immune privileged circumstances of tumor sites. However, it still remains to be demonstrated whether the capacity of melanoma cells to express CD95L is acquired during the progression. We addressed this question with a case of acral lentiginous melanoma by employing immunostaining using an antibody directed against CD95L as well as by in situ TUNEL staining. H&E-staining of tumor specimens revealed that there were two different growth patterns. The central part of the tumor showed a deeper invasion into the dermis (Breslow thickness >4 -mm). The horizontally growing edge of the tumor proliferated more superficially (Breslow thickness<3-mm). Relatively fewer lymphocytes were observed around the melanoma nests in central areas, which expressed detectable amounts of CD95L. In contrast, more lymphocytes were observed among the melanoma cells in the peripheral lesion, where CD95L was not detected. To evaluate the relevance of the CD95L expression, in situ TUNEL staining was performed. This indicated a significant correlation of lymphocyte apoptosis with CD95L expression on melanoma cells. Together the data suggest that expression of CD95L is turned on depending on the level of melanoma, and that it may tribute to creating immune privileged circumstances by initiating apoptosis of tumor filrating lymphocytes.

Sp3, but not Sp1, mediates the transcriptional activation of the p21/WAF1/Cip1 gene promoter by histone deacetylase inhibitor.

We previously reported that both sodium butyrate and trichostatin A (TSA), both of which are known as inhibitors of histone deacetylase, arrest human tumor cells at G1 and G2-M and activate the cyclin-dependent kinase inhibitor, the p21/WAF1/Cip1 gene promoter, through the Sp1 sites. In this study, we identified Sp1 and Sp3 as major factors binding to the Sp1 sites of the p21/WAF1/Cip1 promoter in MG63 cells through electrophoretic mobility shift assays and showed that TSA treatment did not change their binding activities. However, GAL4-Sp3 but not GAL4-Sp1 fusion protein supported the TSA-mediated gene induction from a luciferase reporter plasmid driven by five GAL4 DNA-binding sites. Moreover, the ectopic expression of dominant negative Sp3 repressed the enhancement by TSA of the p21/WAF1/Cip1 promoter and Sp1 site-driven promoter. Taken together, these results suggest that histone deacetylase inhibitor up-regulates p21/WAF1/Cip1 transcription by Sp3 but not by Sp1.

Histone deacetylase inhibitor activates the p21/WAF1/Cip1 gene promoter through the Sp1 sites.

Trichostatin A (TSA), a specific histone deacetylase inhibitor, induces histone hyperacetylation and modulates the expression of some genes. We examined the effects of TSA on MG63 cells. TSA induced growth arrest and expression of the p21/WAF1/Cip1 protein. A close correlation between the level of histone acetylation and induction of the p21/WAF1/Cip1 protein was detected. Using several mutant p21/WAF1/Cip1 promoter fragments, mutation of either of two Sp1 sites at -82 or -69 of the p21/WAF1/Cip1 promoter reduced the responsiveness to TSA. This finding indicates that TSA activates the p21/WAF1/Cip1 promoter through the Sp1 sites in a p53-independent manner.

Identification by differential display of eight known genes induced during in vivo intimal hyperplasia.

To achieve a better understanding of the mechanism of intimal thickening, we used a rabbit model in which aorta was denuded mechanically by a balloon catheter. Total RNA was prepared from each aorta 1, 2, 7, 14, 23, or 30 days after denudation, and from intact aorta of non-denuded control rabbits. Subsequently, using the differential display method, we identified eight genes that were expressed differently during the time course after injury. One of them, RESP18 (encoding regulated endocrine secretory protein 18), was suppressed during the acute reaction. The other seven showed increase in expression during the acute phase: the genes for hTAFII68 (human TATA-binding protein associated factor), NPAT (nuclear protein mapped to the AT locus), OSF2 (osteoblast-specific factor 2), Pyst1, casein kinase 1 alpha, integrin alpha 1, and XP-C complementing protein. Although hTAFII68, NPAT, OSF2, and Pyst1 are thought to be related to transcription, not all four are positive regulators. Considering that none of these genes had previously been reported as being implicated in intimal hyperplasia, we conclude that many known or unknown genes play roles in this process. We believe that differential display is an effective method for screening genes whose variations in expression can provide clues toward understanding the molecular mechanism of intimal hyperplasia.

Thrombolytic therapy by pro-Urokinase combined with tissue plasminogen activator in an acute internal carotid occlusion.

Acute occlusion of the internal carotid artery by a cardiac embolus was quickly recanalized by the new thrombolytic agent, pro-Urokinase, combined with tissue plasminogen activator, without any side-effects. This is the first reported case of thrombolytic therapy of a cerebral artery using these agents in combination.

P2 aneurysm approached via the temporal horn: technical case report.

OBJECTIVE AND IMPORTANCE: We report the use of a transcortical transventricular approach to a P2 aneurysm, which was located near the choroidal fissure, protruded into the temporal horn, and was considered to be too difficult to approach by the conventional subtemporal route. CLINICAL PRESENTATION: An 81-year-old woman suddenly developed severe headache with vomiting and subsequently lost consciousness. Computed tomographic scans revealed a diffuse intraventricular hemorrhage and subarachnoid hemorrhage. Cerebral angiography disclosed a saccular aneurysm in the right P2 segment. INTERVENTION: On the 16th day after admission, successful neck clipping was easily performed through the temporal horn via the inferior temporal gyrus. The postoperative course was uneventful. CONCLUSION: This special approach may be preferable in such cases, because it protects the brain from the detrimental effects of strong temporal retraction and provides a wider working space.

Binding of NF-Y transcription factor to one of the cis-elements in the myeloperoxidase gene promoter that responds to granulocyte colony-stimulating factor.

The expression of the myeloperoxidase (MPO) gene is restricted to cells of the myeloid cell lineage and is induced by granulocyte colony-stimulating factor (G-CSF). In this study, a series of deletion mutations was introduced in the promoter of the human MPO gene, which was then fused to the chloramphenicol acetyltransferase gene. The G-CSF-induced promoter activity was examined in mouse myeloid precursor FDC-P1 transformants that constitutively express the G-CSF receptor. A G-CSF-responsive element (GRE) in the MPO gene was found approximately 800 base pairs upstream from the transcription initiation site. When the 5'-flanking region of the human MPO gene contained this element, it yielded promoter activity in cells cultured with G-CSF but not in cells cultured with interleukin 3. Gel shift assays with the element showed that a specific nuclear factor(s) (NF/G-CSF) binds to the element. The NF/G-CSF was purified by affinity chromatography using an oligonucleotide of GRE. Protein sequence analysis of the purified NF/G-CSF indicated that NF/G-CSF is a ubiquitous transcription factor, NF-Y, which is composed of three subunits. The recombinant NF-Y was then shown to bind to GRE in a combination of the three subunits.

Butyrate activates the WAF1/Cip1 gene promoter through Sp1 sites in a p53-negative human colon cancer cell line.

Butyrate is a well known colonic luminal short chain fatty acid, which arrests cell growth and induces differentiation in various cell types. We examined the effect of butyrate on the expression of WAF1/Cip1, a potent inhibitor of cyclin-dependent kinases, and its relation to growth arrest in a p53-mutated human colon cancer cell line WiDr. Five millimolar butyrate completely inhibited the growth of WiDr and caused G1-phase arrest. WAF1/Cip1 mRNA was rapidly induced within 3 h by treatment with 5.0 mM butyrate, and drastic WAF1/Cip1 protein induction was detected. Using several mutant WAF1/Cip1 promoter fragments, we found that the butyrate-responsive elements are two Sp1 sites at -82 and -69 relative to the transcription start site. We also found that a TATA element at -46 and two overlapping consensus Sp1 sites at -60 and -55 are essential for the basal promoter activity of WAF1/Cip1. These findings suggest that butyrate arrests the growth of WiDr by activating the WAF1/Cip1 promoter through specific Sp1 sites in a p53-independent fashion.

Quantitative visualization of flow through a centrifugal blood pump: effect of washout holes.

To clarify the effect of washout holes on the flow in a centrifugal blood pump to prevent blood stagnation, a quantitative flow visualization technique was applied to compare flows in models with and without washout holes. A scaled-up model of a prototype pump and a high speed video camera were used for the flow visualization, and images were processed by particle tracking velocimetry. Particular attention was paid to the flow through the gaps behind and in front of the impeller. The results showed that in the gap behind the impeller, washout holes caused not only an inward flow, but also an increase in the tangential velocities. In the gap in front of the impeller, washout holes caused an outward flow and a decrease in the tangential velocities. Head flow characteristics were little affected by the washout holes in this initial design for which the flow through the washout holes was set to be approximately 10% of the flow in the external circuit. These results suggest that the flow through washout holes is significant in the prevention of blood stagnation in 2 ways. First, the inward radial velocity behind the impeller and outward velocity in front of the impeller result in fluid exchange, and second, a tangential velocity increase reduces fluid stagnation behind the impeller.

[Natural course of non-operative cases of acute subdural hematoma: sequential computed tomographic study in the acute and subacute stages].

We have studied 20 non-operative cases of traumatic acute subdural hematoma in the acute and subacute stages by sequential computed tomography (CT). 20 patients were divided into three groups as is shown below; 8 patients with rapid complete resolution within 24 hours (rapid resolution group), 10 patients with slow resolution beyond 24 hours, mainly in the subacute stage (slow resolution group), and 2 patients worsening clinically due to the increase of subdural fluid collection in the subacute stage (subacute worsening group, what is called, "subacute subdural hematoma"). In the rapid resolution group, CT showed mixed density thin subdural hematoma in 6 patients; delayed subdural effusion in 3 patients; cerebral contusion in 2 patients; and diffuse brain swelling in 2 patients. We reviewed 8 of our cases and 13 reported cases. As a result, we consider that the main pathological mechanisms of rapid resolution types were, in the elderly, the washout of the hematoma by the leakage of cerebrospinal fluid (CSF) and, in the young, the compression of the hematoma by brain swelling. In the subacute worsening group, CT showed, in the acute stage, mixed density thick subdural hematoma with brain atrophy and no intraaxial lesions and, in the subacute stage, the increase of low density subdural fluid collection with marked mass effect. We reviewed 2 of our cases and 19 reported cases. As a result, we related the increase of subdural fluid collection in the subacute stage with the CSF leakage into the subdural space due to the tearing of arachnoid membrane. However, massive CSF leakage into the subdural space, producing marked mass effect, may be joined by other factors such as osmotic pressure gradient or oozing from the outer membrane of the hematoma.

Percutaneous transluminal angioplasty of the internal carotid artery with a continuous-perfusion dilatation catheter.

A patient who lost consciousness and had a convulsion caused by brain ischemia during balloon inflation in connection with percutaneous transluminal angioplasty was successfully treated with a continuous-perfusion dilatation catheter without complications.

[A case report of anterior mediastinal cystic lymphangioma].

Fourty seven years old woman came to our hospital for further examination of incidentally found abnormal chest shadow. Chest US examination revealed lobulated cystic mass. The cyst wall was thin and smooth. Chest computed tomography showed water density cystic mass. Preoperative diagnosis was pericardial cyst. Operation was done. Lobulated cyst was attached to pericardium and diaphragma. Though adhesion to the pericardium was loose, adhesion to the diaphragma was tight. To achieve complete resection of the cyst, partial resection of the diaphragma was needed. Postoperative course was uneventful. Cystic lymphangioma is benign but complications such as infection or bleeding were reported. Then complete resection of the cyst should be done.

Histone deacetylase inhibitor activates the WAF1/Cip1 gene promoter through the Sp1 sites.

Treatment of cultured cells with trichostatin A (TSA), a specific histone deacetylase inhibitor, induces the histone hyperacetylation and modulates expression of some mammalian genes. We examined the effects of TSA on cell growth arrest, and its relation to expression of the WAF1/Cip1 gene, a potent inhibitor of cyclin-dependent kinases, in a p53-mutated human osteosarcoma cell line MG63. TSA at 500 ng/ml induced growth arrest at both G1 and G2/M phases, and the expressions of the WAF1/Cip1 mRNA and protein. We also examined the changes of acetylated isoforms of histone H4. Dose-response and kinetic analysis suggest a close correlation between the level of histone acetylation and the induction of the WAF1/Cip1 expressions. Using several mutant WAF1/Cip1 promoter fragments, we found that the TSA responsive elements are two Sp1 sites at -82 and -69 relative to the transcription start site. These findings indicate that TSA induces the WAF1/Cip1 promoter through the typical Sp1 sites, in a p53-independent fashion. Furthermore, the Sp1-luc plasmid, containing SV40 promoter-derived three consensus Sp1 binding sites, was markedly activated by TSA, compared to the mutant Sp1-luc plasmid. These results demonstrate that transcriptional activation through the Sp1 sites of the WAF1/Cip1 promoter by TSA coincides with induced hyperacetylation of histone H4.

Transient hydrocephalus due to movement of a clot plugging the aqueduct.

A rare case of transient hydrocephalus is reported. A 64-year-old woman presented with headache. Computerized tomography (CT) scan revealed hydrocephalus with tiny blood clots in the left foramen of Monro and in the aqueduct. Six hours after the onset, the signs and symptoms disappeared spontaneously. The second CT showed improvement of the hydrocephalus with migration of the clot into the i.v. ventricle. Aqueductal trapping and releasing of the clot formed by bleeding from the choroid plexus located in the left foramen of Monro was suspected for the origin of the transient hydrocephalus.

Role of the intrachain disulfide bond of ovalbumin during conversion into S-ovalbumin.

Disulfide-reduced and carboxymethylated ovalbumin was treated at pH 9.9 and 55 degrees C for 24 h as a specific condition for preparation of S-ovalbumin. The stability and conformation of the product were investigated. Such alkaline treatment converted native protein to S-ovalbumin, but this modified ovalbumin was not stabilized, according to results of calorimetric analysis. Instead, it had lost its native like conformation; the magnitude of CD spectra decreased. The conformation after alkaline treatment was not clear, but the possibility of aggregation was excluded by electrophoretic analysis. These observations showed that the transformation of native ovalbumin into S-ovalbumin requires the presence of the disulfide bond.

CT findings of extravasation of contrast medium from a ruptured aneurysm during cerebral angiography--a case report and six others from the literature.

We present a case of ruptured aneurysm in which extravasation of contrast medium was suspected during cerebral angiography and confirmed by computed tomography. In cases of ruptured aneurysm, post-angiographic computed tomography before operation (measurement of the Hounsfield unit numbers and grading by them) is necessary for establishing the diagnosis of extravasation of contrast medium and for grasping its degree and extent.

Comparative study of the effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on generation and mobilization of neutrophils in cyclophosphamide-treated neutropenic mice.

To examine the differential in vivo effects of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the recovery from neutropenia caused by chemotherapy, we directly compared the actions of these CSFs on neutrophil count in the bone marrow, spleen, and peripheral blood of mice treated with an anticancer drug, cyclophosphamide (CPA). Both G-CSF and GM-CSF stimulated the generation of neutrophils in bone marrow, but GM-CSF is only about 1/20 as effective as G-CSF. Both CSFs also stimulated the release of the generated neutrophils therefrom. G-CSF mobilized the neutrophils into both the circulation pool and the marginal pool, and these neutrophils were able to migrate into the inflammatory site when stimulated by casein. On the other hand, GM-CSF mobilize the neutrophils into the marginal pool but not the circulation pool, and they were not able to migrate into the inflammatory site. These findings indicate that G-CSF physiologically plays an important role as a factor which stimulates neutrophil mobilization from bone marrow into circulation as well as neutrophil generation in the bone marrow.

A quantitative visualization study of flow in a scaled-up model of a centrifugal blood pump.

A quantitative flow visualization study of a scaled-up model of a centrifugal blood pump was performed. Since the size of the scaled-up model was three times as large as the original pump under development, and the kinematic viscosity of the saline solution used as the working fluid was approximately one-third that of the blood, we obtained a similar flow at one twenty-seventh the angular velocity of the original pump. The flow was visualized by seeding the saline solution with neutrally buoyant particles and by illuminating the model with a laser light sheet. Since the gap flow behind the impeller is important for thrombus formation, it was recorded by a high-speed video camera, and the velocity field was evaluated automatically by particle tracking velocimetry. It was shown that in the gap behind the impeller there existed a region where the velocity profile was almost flat which can be called a core region. The results indicated the effectiveness of the present visualization technique for centrifugal blood pumps.

Serum levels of anti-Staphylococcus aureus-specific IgE in patients with atopic dermatitis.

We investigated the role of Staphylococcus aureus-specific IgE in patients with atopic dermatitis (AD). The titer of serum S. aureus-specific IgE was measured using the RAST method in 67 patients with AD and correlated with serum LDH, eosinophil count and total IgE. The titer of S. aureus-specific IgE was elevated in 41 patients but was not detected in 26 patients. The mean serum level of total IgE was higher in the positive group than in the negative group, but the eosinophil count and LDH levels were not different between the two groups. S. aureus was detected and cultured from the skin of 33/41 (80%) patients in the positive group, but only from the skin of 5/26 (19%) patients of the negative group. Our results suggest that S. aureus-specific antibody is present in patients with moderate-to-severe atopic dermatitis and may be involved in the pathogenesis of AD.