A novel oncogenic pathway by TLS-CHOP involving repression of MDA-7/IL-24 expression.

BACKGROUND: Translocated in liposarcoma-CCAAT/enhancer binding protein homologous protein (TLS-CHOP) (also known as FUS-DDIT3) chimeric oncoprotein is found in the majority of human myxoid liposarcoma (MLS), but its molecular function remains unclear. METHODS: We knockdowned TLS-CHOP expression in MLS-derived cell lines by a specific small interfering RNA, and analysed the gene expression profiles with microarray. RESULTS: TLS-CHOP knockdown inhibited growth of MLS cells, and induced an anticancer cytokine, melanoma differentiation-associated gene 7 (MDA-7)/interleukin-24 (IL-24) expression. However, double knockdown of TLS-CHOP and MDA-7/IL-24 did not inhibit MLS cell growth. CONCLUSION: Repression of MDA-7/IL-24 expression by TLS-CHOP is required for MLS tumour growth, and TLS-CHOP may become a promising therapeutic target for MLS treatment.

"Donut's shape" radiosurgical treatment planning for large cystic metastatic brain tumors.

BACKGROUND: Radiosurgical management of large cystic metastatic brain tumors represents a significant challenge. Nevertheless, modified dose planning has shown beneficial results in such cases. METHOD AND RESULTS: "Donut's shape" radiosurgical treatment planning is based on the chain-like application of multiple, small-sized isocenters for selective coverage of the contrast-enhancing tumor capsule and minimal irradiation of the central cystic area. Such an approach was used for the management of large cystic intracranial metastases, which were not accompanied by a significant mass effect and did not require immediate volume reduction. Treatment was done using Leksell Gamma Knife model C with automatic positioning system. The majority of treated lesions showed significant shrinkage after radiosurgery and no major complications were met. CONCLUSION: Large cystic metastatic brain tumors may be successfully treated with gamma knife radiosurgery alone using the proposed "donut's shape" dose planning with coverage of the contrast-enhancing tumor capsule by multiple small-sized isocenters.

Altered growth and branching patterns in synpolydactyly caused by mutations in HOXD13.

Hox genes regulate patterning during limb development. It is believed that they function in the determination of the timing and extent of local growth rates. Here, it is demonstrated that synpolydactyly, an inherited human abnormality of the hands and feet, is caused by expansions of a polyalanine stretch in the amino-terminal region of HOXD13. The homozygous phenotype includes the transformation of metacarpal and metatarsal bones to short carpal- and tarsal-like bones. The mutations identify the polyalanine stretch outside of the DNA binding domain of HOXD13 as a region necessary for proper protein function.

Development of an articulating ultrasonically activated device for laparoscopic surgery.

BACKGROUND: Ultrasonically activated devices (USADs) offer excellent coagulating dissection performance and are broadly used, particularly in endoscopic operations. Traditional USADs, however, have fixed linear shape and are thus limited in the directions from which organs can be approached. We have developed a small USAD transducer attached to the tip of an articulating device, offering a new kind of USAD in which the tip can bend as desired. We describe herein an evaluation of the coagulating dissection performance of this new articulating USAD and an in vivo confirmation of clinical usefulness. METHODS: To evaluate coagulating dissection performance, we compared coagulating shearing on porcine splenic arteries between the articulating USAD and a Harmonic Scalpel II (HSII), representing a traditional USAD. Changing the amplitude of vibration between 60 microm and 80 microm and grip force among 1, 2, and 3 N, we measured the time required for division and bursting pressure of coagulating dissection. An in vivo experiment in a pig was also used to confirm the usefulness of the articulating USAD in laparoscopic operations. RESULTS: Division time did not differ significantly between the articulating USAD and HSII with an 80-microm amplitude of vibration and a grip force of 2 or 3 N. Bursting pressure of blood vessels showed no significant difference between articulating USAD and HSII under all experimental conditions. In the in vivo experiment, the new bendable tip of the articulating USAD displayed coagulating dissection performance equivalent to that of the traditional USAD. CONCLUSIONS: We have developed a new articulating USAD that can broaden the range of methods and approaches available for USADs and improve usefulness and safety.

Identification of the pyramidal tract by neuronavigation based on intraoperative diffusion-weighted imaging combined with subcortical stimulation.

BACKGROUND/AIMS: To identify the pyramidal tract by neuronavigation based on intraoperative diffusion-weighted imaging (iDWI) combined with subcortical stimulation. METHODS: Seven patients with brain tumors near the deep white matter underwent resection surgery using neuronavigation based on iDWI to visualize white matter bundles. Subcortical electrical stimulation was performed and electromyography was measured at the extremities when surgical manipulation came near the position corresponding to the depicted bundle. We validated the bundle depicted on iDWI by considering the responses to subcortical stimulation and the distance between the stimulation site and the depicted bundle. RESULTS: Positive motor-evoked potentials were detected in 5 of 7 patients (8 stimulations) and the distance from the stimulation site to the depicted bundle was 0-4.7 mm (mean +/- SD, 1.4 +/- 2.1 mm). Negative (no) responses were obtained in all patients when the distance was more than 5 mm. The neuronavigation system had an average error of 0.79 +/- 0.25 mm and a maximum error of 2.0 mm (n = 16). CONCLUSION: Neuronavigation based on iDWI combined with subcortical stimulation allowed surgeons to identify the pyramidal tract and avoid inadvertent injury. Our findings demonstrate that the white matter bundles depicted by iDWI can contain the pyramidal tract.

Risk factors for regrowth of intracranial meningiomas after gamma knife radiosurgery: importance of the histopathological grade and MIB-1 index.

INTRODUCTION: The influence of histopathological grade and MIB-1 index of intracranial meningioma on the results of its radiosurgical management is not clear. The objective of the present retrospective study was to make an evaluation of these factors along with an analysis of other variables associated with progression-free survival after gamma knife radiosurgery (GKR). PATIENTS AND METHODS: Thirty-four intracranial meningiomas with known detailed histopathological diagnosis were analyzed. Tumors of WHO histopathological grades I, II, and III were diagnosed in 24, 3, and 7 cases, respectively. The median MIB-1 index was 1.3% (range: 0-31.9%). In 14 cases the MIB-1 index was 3.0% and more. In 26 cases the treatment was done at the time of tumor recurrence. Median volume of the neoplasm at the time of GKR was 4.1 mL (range: 0.4-43.1 mL). Median marginal dose was 12 Gy (range: 8-19 Gy). Median length of follow-up constituted 63 months (range: 19-132 months). RESULTS: Actuarial progression-free survival at 1, 3, 5, and 10 years constituted 100, 94, 83, and 58%, respectively. Histopathological grade II or III (p<0.0001), MIB-1 index 3% and more (p=0.0004), and non-skull base location (p=0.0026) of the tumor showed negative associations with progression-free survival in multivariate analyses. Actuarial progression-free survival at 5 years after GKR for benign and non-benign meningiomas constituted 100 and 45%, respectively (p<0.0001). CONCLUSION: Radiosurgery is a highly effective management option for benign intracranial meningiomas, but growth control of non-benign ones is significantly worse. It requires close neuroradiological follow-up and necessitates the search for modified treatment strategies.

Usefulness of intraoperative magnetic resonance imaging for glioma surgery.

BACKGROUND: Radical resection of gliomas can increase patient's survival. There is known concern, however, that aggressive tumour removal can result in neurological morbidity. The objective of the present study was to evaluate the usefulness of low magnetic field strength (0.3 Tesla) open intraoperative magnetic resonance imaging (iMRI) for complete resection of glioma with emphasis on functional outcome. METHODS: From 2000 to 2004, 96 patients with intracranial gliomas underwent tumour resection with the use of iMRI in Tokyo Women's Medical University. There were 50 men and 46 women; mean age was 39 years. Tumour volume varied from 1.2 ml to 198 ml (median: 36.5 mL). Resection rate and postoperative neurological status were compared between control group (46 cases, operated on during the initial period after installation of iMRI), and study group (50 most recent cases, in whom surgery was done using established treatment algorithm and improved image quality). FINDINGS: Overall, mean resection rate was 93%, and medial residual tumour volume was 0.17 ml. Total tumour removal was achieved in 44 cases (46%). Compared to control group, resection rate in the study group was significantly higher (91%, vs. 95%; P < 0.05), whereas residual tumour volume was significantly smaller (1.7 mL vs. 0.025 mL; P < 0.001). Nine patients in the control group (20%) and 24 in the study group (48%) experienced temporary postoperative neurological deterioration (P < 0.01), however, the rate of permanent morbidity evaluated 3 months after surgery did not differ significantly between the groups investigated (13% vs. 14%). CONCLUSIONS: Use of iMRI during surgery for intracranial gliomas permits to attain aggressive tumour resection with good functional outcome. Nevertheless, surgical experience with the iMRI system, establishment of treatment algorithm, and improvement of image quality are of paramount importance for optimal results.

Decreased type IV collagen expression by human decidual tissues in spontaneous abortion.

To provide some insight into the etiology of spontaneous abortion, the expression of type IV collagen was investigated in human decidual tissues obtained after spontaneous abortion (n = 17) and normal pregnancy (n = 22). Indirect immunofluorescent staining was performed for type I, III, and IV collagen as well as laminin, and Northern blot analysis was conducted to assess the expression of messenger ribonucleic acid for the alpha 1(IV) chain. Immunohistochemical analysis did not reveal any significant differences between normal pregnancy and spontaneous abortion with respect to interstitial collagens (type I and III collagen) and laminin in the decidual tissue. However, although pericellular immunostaining for type IV collagen was recognized around the decidual cells in normal pregnancy, very weak or no staining was observed in spontaneous abortion. Northern blot analysis revealed that the decidual expression of messenger ribonucleic acid for the alpha 1(IV) chain was significantly reduced in spontaneous abortion compared to that in normal pregnancy (P < 0.001). These results suggest that type IV collagen might play an important role in the maintenance of pregnancy and that decreased expression of this collagen could be associated with spontaneous abortion.

Decreased type III and V collagen expression in chorionic villi of hydatidiform mole.

To investigate the characteristic structure of hydatidiform mole, various types of collagen expression were determined in human villous tissues obtained from normal pregnancies (n = 17) and complete hydatidiform moles (n = 10). Indirect immunofluorescent staining was performed to detect type I, III, and VI collagen with specific monoclonal antibodies. Collagens were also extracted from the villous tissues obtained from normal pregnancy and hydatidiform mole by the salt precipitation method. Immunohistochemical staining for type I, III, and VI collagen revealed weak staining of the villous stroma in hydatidiform mole compared with that in normal pregnancy. Both the ratios of type III to type I collagen and the ratios of type V to type I collagen in the villous tissues were significantly decreased (P < 0.05) in molar pregnancy compared with those in normal pregnancy. These results suggest that alterations in the distribution and composition of collagen might play an important role in determining the pathophysiology and structure of hydatidiform mole.

Overexpression of type IV collagen in chorionic villi in hydatidiform mole.

To investigate the characteristic structure of hydatidiform mole, type IV collagen expression was determined in human villous tissues obtained from normal pregnancies (n = 17) and complete hydatidiform moles (n = 10). Indirect immunofluorescent staining was performed to detect type IV collagen with specific monoclonal antibody, and Northern blot analysis was performed to assess expression of messenger ribonucleic acid for the alpha1(IV) chain. In addition, serum levels of type I, III, and IV collagen were measured by RIA. Immunohistochemical staining for type IV collagen revealed stronger staining of the trophoblastic basement membrane in hydatidiform mole than in normal pregnancy. Northern blot analysis revealed that the villous expression of messenger ribonucleic acid for the alpha1(IV) chain was significantly increased in hydatidiform moles compared with normal pregnancy (P < 0.01). Although there were no differences in the serum type I and III collagen levels between hydatidiform mole and normal pregnancy, the type IV collagen level was significantly higher in patients with hydatidiform mole than in normal pregnancy (P < 0.05). These results suggest that type IV collagen might play an important role in determining the pathophysiology and structure of hydatidiform mole.

Assembly and sequencing of a cDNA covering the entire mouse alpha 1(IX) collagen chain.

Type IX collagen, a member of the FACIT family of collagenous proteins, contains heterotrimeric molecules of distinct alpha 1(IX), alpha 2(IX) and alpha 3(IX) chains. In this paper we describe the assembly and nucleotide sequence of a cDNA encoding the entire mouse alpha 1(IX) collagen chain. The nucleotide sequence provides for the first time the complete primary structure of the mouse chain. Knowledge of the complete structure of mouse alpha 1(IX) collagen will be useful for investigations of type IX collagen expression during normal mouse development and for the generation of transgenic mice with specific defects in this collagen.

Expression of trk receptors in the developing and adult human central and peripheral nervous system.

A family of tyrosine receptor kinases known collectively as trk receptors plays an essential role in signal transduction mediated by nerve growth factor and related neurotrophins. To localize the major trk receptors (trkA, B and C) in the developing and adult central (CNS) and peripheral (PNS) nervous system, we generated monoclonal antibodies (MAbs) to extracellular (MAbs E7, E13, E16, E21, E29) and intracellular (MAb I2) domains of human trkA fused to glutathione S-transferase. Several MAbs (E7, E13, E16) recognized glycosylated trkA (gp140trk and gp110trk) in Western blots, one MAb (E7) recognized non-glycosylated (p80trk) and glycosylated trkA in immunoprecipitation assays, and two MAbs (E13, E29) detected trkA on the cell surface of NIH3T3 cells transfected with a trkA cDNA. Although generated to trkA fusion proteins, this panel of MAbs also recognized trkB and trkC in flow cytometric studies of NIH3T3 cells transfected with trkB or trkC cDNAs. Thus, we used these pan-trk MAbs to probe selected regions of the CNS and PNS including the hippocampus, nucleus basalis of Meynert, cerebellum, spinal cord, and dorsal root ganglion (DRG) to localize trkA, B, and C receptors in the developing and adult human nervous system. These studies showed that trk receptors are expressed primarily by neurons and are detectable very early in the developing hippocampus, cerebellum, spinal cord, and DRG. Although the distribution and intensity of trk immunoreactivity changed with the progressive maturation of the CNS and PNS, immunoreactive trk receptors were detected in neurons of the adult human hippocampus, nucleus basalis of Meynert, cerebellum, spinal cord, and DRG. This first study of trk receptor proteins in the developing and adult human CNS and PNS documents the expression of these receptors in subsets of neurons throughout the developing and adult nervous system. Thus, although the expression of trk receptor proteins is developmentally regulated, the constitutive expression of these neurotrophin receptors by neurons in many regions of the adult human CNS and PNS implies that mature trk receptor-bearing neurons retain the ability to respond to neurotrophins long after terminal neuronal differentiation is complete.

Immunological properties of monoclonal antibodies to human and rat prolyl 4-hydroxylase.

Monoclonal antibodies to human (8 clones) and rat (12 clones) prolyl 4-hydroxylase [EC 1.14.11.2] were prepared and characterized as regards subclass, subunit specificity, inhibition and crossreactivity. Among the antibodies to the human enzyme, four clones showed the IgG1 subclass, two IgA, one IgG2b, and one IgM. Four clones reacted with the alpha subunit of the enzyme, while the others reacted with the beta subunit. The enzymatic activity was inhibited by four clones. Five clones crossreacted with the rat enzyme. One clone inhibited the rat enzyme. Among the antibodies to the rat enzyme, seven clones showed the IgG1 subclass, four IgG2a and one IgG2b. Seven clones reacted with the alpha subunit, and four with the beta subunit. One reacted with neither subunit. The enzymatic activity was inhibited by seven clones. Seven clones crossreacted with the human enzyme. Three clones inhibited the human enzyme.

Molecular cloning and sequencing of a cDNA encoding alanine-glyoxylate aminotransferase 2 from rat kidney.

Alanine-glyoxylate aminotransferase (AGT) 2 is a pyridoxal 5'-phosphate dependent, mitochondrial enzyme which, in the rat, is expressed at a high level in the kidney. The amino acid sequences of nine tryptic and seven CNBr peptides of the rat kidney AGT2 were determined. Three overlapping cDNAs encoding the AGT2 were cloned on the basis of its partial amino acid sequences by means of a polymerase chain reaction-based approach involving rat kidney poly(A)+ RNA. The complete cDNA sequence comprised 1,919 bases, and contained a 1,536-base open reading frame which encodes a polypeptide of 512 amino acid residues with a putative presequence consisting of 39 amino acid residues at the amino terminus, giving a precursor protein with a molecular mass of 57,150 Da. The sequence of AGT2 exhibits significant homology with neither peroxisomal AGT1 from human liver nor mitochondrial AGT1 from rat liver. However, the sequence of AGT2 exhibited 30.8, 29.2, and 27.1% identity with those of Escherichia coli 4-aminobutyrate aminotransferase, rat ornithine aminotransferase, and Pseudomonas cepacia 2,2-dialkylglycine decarboxylase, respectively. The active site sequences were also well conserved among these aminotransferases. AGT2, thus, is more similar to the other aminotransferases than to AGT1. The results suggest that the rat kidney AGT2 may play a biological role in amino acid metabolism distinct from that of AGT1.

Stretch-induced proliferation of cultured vascular smooth muscle cells and a possible involvement of local renin-angiotensin system and platelet-derived growth factor (PDGF).

This experiment was designed to investigate the possible involvement of angiotensin II (Ang II) and platelet-derived growth factor (PDGF) in the mechanism underlying stretch-induced proliferation of vascular smooth muscle cells (SMCs). SMCs from the rabbit aortic media were grown on polystyrene rubber-bottomed dishes coated with type I collagen. Cells were stretched cyclically by a vacuum-operated downward flexion of the culture dish bottom. A 1.4- to 1.6-fold increase in proliferation of SMCs was induced by cyclic stretching, as determined by [3H]-thymidine incorporation, in a stretch force-dependent manner in the range of 5% to 15% elongation, 30 cycles/min for 24 h. Expression of PDGF-B chain mRNA was up-regulated in a time-dependent manner in the range of 2 to 24 h, 10% elongation, and 30 cycles/min. Saralasin, a selective antagonist of Ang II, and captopril, an angiotensin I converting enzyme inhibitor, significantly suppressed the stretch-induced proliferation of SMCs. Blockade of angiotensinogen mRNA translation by antisense oligonucleotide inhibited proliferation under the mechanical strain. Stretch-induced proliferation was inhibited by 78% in the presence of anti-PDGF-AB neutralizing antibody. Increased expression of PDGF-B chain mRNA under the mechanical strain was inhibited by treatment with saralasin. Our results indicate that the stretch-induced proliferation of cultured SMCs is mediated at least in part via increased production of Ang II by the local renin-angiotensin system and a subsequent up-regulation of PDGF-B chain mRNA in an autocrine-paracrine manner.

Type VI collagen expression during growth of human ovarian follicles.

OBJECTIVE: To identify type VI collagen expression in human ovarian follicles during follicular growth. DESIGN: In vitro experiment. SETTING: Department of Obstetrics and Gynecology, Wakayama Medical College, Japan. PATIENT(S): Regularly cycling women who underwent adnexectomy. INTERVENTION(S): Immunohistochemistry and in situ hybridization for human type VI collagen. MAIN OUTCOME MEASURE(S): Expression of type VI collagen. RESULT(S): Expression of type VI collagen was observed in the theca cell layers during folliculogenesis, whereas no expression of type VI collagen was observed in the granulosa cell layers at the mRNA and protein levels. As the follicles grew, immunostaining for type VI collagen became intense in the theca cell layers, especially the theca externa. In preovulatory follicles, however, weak, fragmented, or discontinuous immunostaining of the theca cell layers was observed. This fragmented or discontinuous immunostaining was evident predominantly in the apical area of preovulatory follicles rather than in the basal area. CONCLUSION(S): Type VI collagen is present in the theca cell layers of follicles during folliculogenesis and plays an important role in interactions between the theca cells and extracellular matrix. These interactions may lead to changes in the shape, proliferation, migration, or differentiation of follicular cells during follicular development, maturation, and ovulation.

One step sandwich enzyme immunoassay for human type IV collagen using monoclonal antibodies.

Monoclonal antibodies were used in one step sandwich enzyme immunoassay (one step sandwich EIA) for human serum immunoreactive type IV collagen. The one step sandwich EIA using either polystyrene ball or microplate was characterized by carrying out two immunoreactions simultaneously, type IV collagen reacting with both a monoclonal antibody as a solid phase and a horseradish peroxidase-labeled monoclonal antibody (Fab') against human type IV collagen as a conjugate. Sensitivity of one step sandwich EIA system by using either polystyrene ball or microplate was 0.22 ng per tube or 0.04 ng per well for type IV collagen, and linearity was obtained between 0.22-40 ng/tube or 0.04-20 ng per well, respectively. Both methods gave reproducible quantitative analysis of immunoreactive type IV collagen levels in the sera of patients with hepatocellular carcinoma and patients with liver cirrhosis, which were apparently higher than the levels in the sera of healthy subjects. Protein immunoblotting shows that the immunoreactive type IV collagen trapped in our present one step sandwich EIA system was not the 7-S and NC1 domains of type IV collagen.

Concentration of serum laminin and type IV collagen in liver diseases assayed by a sandwich enzyme-immunoassay using monoclonal antibodies.

Serum laminin (P1 fragment) and type IV collagen levels were determined in patients with hepatic disorders. The method was based on a sandwich enzyme-immunoassay using two monoclonal antibodies that recognize different epitopes of either laminin or type IV collagen molecule. Laminin and type IV collagen levels in the serum of patients with chronic hepatic disorders were higher as compared with those in healthy control subjects, with the increment of serum type IV collagen being far greater than that of laminin. Since type IV collagen and laminin are major basement membrane components, it is suggested that the higher levels of these peptides may reflect a so-called capillarization of the perisinusoidal wall encountered in hepatic fibrogenesis. The assay system used in this experiment is simple and sensitive and can be applied to clinical evaluation of hepatic fibrosis.

A sandwich immunoassay for human prolyl 4-hydroxylase using monoclonal antibody.

Monoclonal antibody was used in a sandwich enzyme immunoassay and in a radioimmunoassay for human serum immunoreactive prolyl 4-hydroxylase. The enzyme immunoassay utilized a monoclonal antibody as a solid phase and horseradish peroxidase-labeled rabbit antibody (Fab') to human prolyl 4-hydroxylase as a conjugate. Sensitivity was 0.1 ng (0.4 fmol) of enzyme per tube. With a conjugate purified by an enzyme-bound affinity column, sensitivity was increased to 0.01 ng (0.04 fmol) per tube, and linearity was obtained between 0.01 to 30 ng (0.04-125 fmol) per tube. The radioimmunoassay used a 125I-labeled rabbit antibody (IgG) as the conjugate. Sensitivity of this technique was 0.4 ng of enzyme per tube. The enzyme immunoassay gave reproducible quantitation and evidenced a higher enzyme concentration in the serum of patients with liver disorders. Protein immunoblotting showed that the serum immunoreactive prolyl 4-hydroxylase trapped in the sandwich immunoassay was mainly the beta-subunit.