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A simple procedure for the fabrication of sub-10 nm scale Si nanopillars in a 2-D array using reactive ion etching with 8 nm Co nanoparticles as etch masks is demonstrated. The obtained Si nanopillars are single crystalline tapered pillar structures of 5 nm (top) x 8 nm (bottom) with a density of approximately 4 x 10(10) pillars cm(-2) on the substrate, similar to the density of Co nanoparticles distributed before the ion etching process. The uniform spatial distribution of the Si nanopillars can also be patterned into desired positions. Our fabrication method is straightforward and requires mild process conditions, which can be extended to patterned 2-D arrays of various Si nanostructures.

We have previously shown that stable overexpression of the thrombospondin-2 (TSP-2) gene inhibited the tumor growth and angiogenesis of human squamous cell carcinoma xenotransplants. To investigate the potential antitumoral efficacy of systemic TSP-2 therapy, we expressed a recombinant 80 kDa fragment of human TSP-2 (TSP-2/NTF), encompassing the N-terminal globular region through the three type 1 repeats, in human kidney 293 EBNA cells, using a modified pCEP4 expression vector. Daily intraperitoneal injections of TSP-2/NTF resulted in a significant inhibition of the growth of human A431 squamous cell carcinomas in vivo and in reduced tumor vascularization. To further investigate possible mechanisms of the antiangiogenic activity of TSP-2/NTF, several in vitro angiogenesis assays were performed in human dermal microvascular endothelial cells. TSP-2/NTF inhibited vascular endothelial growth factor induced migration of human dermal microvascular endothelial cells and inhibited tube formation on Matrigel in vitro. TSP-2/NTF also inhibited vascular endothelial growth factor induced angiogenesis in an in vivo Matrigel assay. Moreover, TSP-2/NTF potently induced human dermal microvascular endothelial cell apoptosis in vitro but did not affect A431 tumor cell proliferation or apoptosis. These findings identify TSP-2/NTF as a potent systemic inhibitor of tumor growth and angiogenesis, acting by direct inhibition of several endothelial cell functions involved in neovascularization.

In contrast to the extensive molecular and functional characterization of blood vascular endothelium, little is known about the mechanisms that control the formation and lineage-specific differentiation and function of lymphatic vessels. The homeobox gene Prox1, the vertebrate homologue of the Drosophila prospero gene, has been recently identified to be required for the induction of lymphatic vascular development from preexisting embryonic veins, and studies in Prox1-deficient mice have confirmed Florence Sabin's original hypothesis about the origin of the lymphatic vascular system from embryonic veins. The recent establishment of cell culture models for the selective propagation of blood vascular and lymphatic endothelial cells, together with the findings that these cells maintain their lineage-specific differentiation in vitro, has led to the discovery that Prox1 expression is sufficient to induce a lymphatic phenotype in blood vascular endothelium. Ectopic expression of Prox1 downregulated blood vascular-associated genes and also upregulated some of the known lymphatic endothelial cell markers. Together, these studies suggest that the blood vascular phenotype represents the default endothelial differentiation and they identify an essential role of Prox1 in the program specifying lymphatic endothelial cell fate.

Previous studies have demonstrated that the specificity of Src homology 2 (SH2) and phosphotyrosine-binding domain interactions are mediated by phosphorylated tyrosines and their neighboring amino acids. Two of the first phosphotyrosine-based binding sites were found on middle T antigen of polyoma virus. Tyr-250 acts as a binding site for ShcA, whereas Tyr-315 forms a binding site for the SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase. However, genetic analysis of a given phosphotyrosine's role in signaling can be complicated when it serves as a binding site for multiple proteins. The situation is particularly difficult when the phosphotyrosine serves as a secondary binding site for a protein with primary binding determinates elsewhere. Mutation of a tyrosine residue to phenylalanine blocks association of all bound proteins. Here we show that the mutation of the amino acids following the phosphorylated tyrosine to alanine can reveal phosphotyrosine function as a secondary binding site, while abrogating the phosphotyrosine motif's role as a primary binding site for SH2 domains. We tested this methodology by using middle T antigen. Our results suggest that Tyr-250 is a secondary binding site for phosphatidylinositol 3-kinase, whereas Tyr-315 is a secondary binding site for a yet-to-be-identified protein, which is critical for transformation.

Within the vascular system, the mucin-type transmembrane glycoprotein T1alpha/podoplanin is predominantly expressed by lymphatic endothelium, and recent studies have shown that it is regulated by the lymphatic-specific homeobox gene Prox1. In this study, we examined the role of T1alpha/podoplanin in vascular development and the effects of gene disruption in mice. T1alpha/podoplanin is first expressed at around E11.0 in Prox1-positive lymphatic progenitor cells, with predominant localization in the luminal plasma membrane of lymphatic endothelial cells during later development. T1alpha/podoplanin(-/-) mice die at birth due to respiratory failure and have defects in lymphatic, but not blood vessel pattern formation. These defects are associated with diminished lymphatic transport, congenital lymphedema and dilation of lymphatic vessels. T1alpha/podoplanin is also expressed in the basal epidermis of newborn wild-type mice, but gene disruption did not alter epidermal differentiation. Studies in cultured endothelial cells indicate that T1alpha/podoplanin promotes cell adhesion, migration and tube formation, whereas small interfering RNA-mediated inhibition of T1alpha/podoplanin expression decreased lymphatic endothelial cell adhesion. These data identify T1alpha/podoplanin as a novel critical player that regulates different key aspects of lymphatic vasculature formation.

Tissue-type plasminogen activator (tPA) is a multidomain serine protease that converts the zymogen plasminogen to plasmin. tPA contains two kringle domains which display considerable sequence identity with those of angiostatin, an angiogenesis inhibitor. TK1-2, a recombinant kringle domain composed of t-PA kringles 1 and 2 (Ala(90)-Thr(263)), was produced by both bacterial and yeast expression systems. In vitro, TK1-2 inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor, and epidermal growth factor. It did not inhibit proliferation of non-endothelial cells. TK1-2 also inhibited in vivo angiogenesis in the chick embryo chorioallantoic membrane model. These results suggest that the recombinant kringle domain of t-PA is a selective inhibitor of endothelial cell growth and identifies this molecule as a novel anti-angiogenic agent.

We have developed an electromagnetic (EM) wave propagation theory through a single layer and multiple layers in the near-field and far-field regions, and have constructed a matrix formalism in terms of the boundary conditions of the EM waves. From the shielding efficiency (SE) against EM radiation in the near-field region calculated by using the matrix formalism, we propose that the effect of multiple layers yields enhanced shielding capability compared to a single layer with the same total thickness in conducting layers as the multiple layers. We compare the intensities of an EM wave propagating through glass coated with conducting indium tin oxide (ITO) on one side and on both sides, applying it to the electromagnetic interference (EMI) shielding filter in a flat panel display such as a plasma display panel (PDP). From the measured intensities of EMI noise generated by a PDP loaded with ITO coated glass samples, the two-side coated glass shows a lower intensity of EMI noise compared to the one-side coated glass. The result confirms the enhancement of the SE due to the effect of multiple layers, as expected in the matrix formalism of EM wave propagation in the near-field region. In the far-field region, the two-side coated glass with ITO in multiple layers has a higher SE than the one-side coated glass with ITO, when the total thickness of ITO in both cases is the same.

Urokinase plasminogen activator (uPA) belongs to a family of proteins that contains kringle domain and plays an important role in inflammation, tissue remodeling, angiogenesis, and tumor metastasis by pericellular plasminogen activation. Kringle domains of plasminogen have been shown to demonstrate anti-angiogenic and anti-tumor activities. Here, we report our investigation of the kringle domain of uPA for anti-angiogenic activity and a possible cellular mechanism of action. The recombinant kringle domain of uPA (Asp(45)-Lys(135)) (UK1) inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor (VEGF), or epidermal growth factor. It also inhibited migration of endothelial cells induced by VEGF or uPA, and in vivo angiogenesis on the chick chorioallantoic membrane. It did not block plasminogen activation by activated uPA in clot lysis and chromogenic substrate assays. Neither binding of UK1 to immobilized uPA receptor nor competitive inhibition of uPA binding were confirmed by real-time interaction analysis. However, internalization of UK1 followed by translocation from cytosol to nucleus was determined to be specific to endothelial cells. It also elicited a transient increase of Ca(2+) flux of more than 2-fold within 2 min of exposure in an endothelial cell-specific manner. These results suggest that the kringle domain of uPA exhibits anti-angiogenic activity and that its anti-angiogenic activity may occur through a different mechanism from inhibition of uPA-uPA receptor interaction or uPA proteolytic activity and may be associated with endothelial-cell specific internalization not mediated by the uPA receptor.

In mammals, the lymphatic vascular system develops by budding of lymphatic progenitor endothelial cells from embryonic veins to form a distinct network of draining vessels with important functions in the immune response and in cancer metastasis. However, the lineage-specific molecular characteristics of blood vascular versus lymphatic endothelium have remained poorly defined. We isolated lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BVECs) by immunomagnetic isolation directly from human skin. Cultured LECs but not BVECs expressed the lymphatic markers Prox1 and LYVE-1 and formed LYVE-1-positive vascular tubes after implantation in vivo. Transcriptional profiling studies revealed increased expression of several extracellular matrix and adhesion molecules in BVECs, including versican, collagens, laminin, and N-cadherin, and of the growth factor receptors endoglin and vascular endothelial growth factor receptor-1/Flt-1. Differential immunostains of human skin confirmed the blood vessel-specific expression of these genes. During embryonic development, endoglin expression was gradually down-regulated on lymphatic endothelium whereas vascular endothelial growth factor receptor-1 was absent from lymphatics. We also identified several genes with specific expression in LECs. These results demonstrate that some lineage-specific genes are only expressed during distinct developmental stages and they identify new molecular markers for blood vascular and lymphatic endothelium with important implications for future studies of vascular development and function.

Signal transducer and activator of transcription (STAT) proteins are both tyrosine- and serine-phosphorylated, mediating signal transduction and gene regulation. Following gene regulation, STAT activity in the nucleus is then terminated by a nuclear protein phosphatase(s), which remains unidentified. Using novel antibody arrays to screen the Stat1-specific protein phosphatase(s), we identified a SHP-2-Stat1 interaction in the A431 cell nucleus. SHP-2 and Stat1 nuclear localization and their association in response to either epidermal growth factor or interferon-gamma (IFNgamma) were confirmed by immunofluorescent staining and affinity precipitation assays. The SHP-2 C-terminal region containing protein-tyrosine phosphatase activity interacted with the C-terminal SH2 transcriptional activation domain of Stat1. In SHP-2-/- mouse fibroblast cells, Stat1 phosphorylation at both the tyrosine residue Tyr(701) and the serine residue Ser(727) by IFNgamma was enhanced and prolonged. Consistently, purified GST-SHP-2 dephosphorylated Stat1 at both tyrosine and serine residues when immunoprecipitated phospho-Stat1 or a peptide corresponding to the sequence surrounding Tyr(P)(701) or Ser(P)(727) of Stat1 was used as the substrate. Overexpression of SHP-2 in 293T cells inhibited IFNgamma-dependent Stat1 phosphorylation and suppressed Stat1-dependent induction of luciferase activity. Our findings demonstrate that SHP-2 is a dual-specificity protein phosphatase involved in Stat1 dephosphorylation at both tyrosine and serine residues and plays an important role in modulating STAT function in gene regulation.

Early during development, one of the first indications that lymphangiogenesis has begun is the polarized expression of the homeobox gene Prox1 in a subpopulation of venous endothelial cells. It has been shown previously that Prox1 expression in the cardinal vein promotes and maintains the budding of endothelial cells that will form the lymphatic vascular system. Prox1-deficient mice are devoid of lymphatic vasculature, and in these animals endothelial cells fail to acquire the lymphatic phenotype; instead, they remain as blood vascular endothelium. To investigate whether Prox1 is sufficient to induce a lymphatic fate in blood vascular endothelium, Prox1 cDNA was ectopically expressed by adenoviral gene transfer in primary human blood vascular endothelial cells and by transient plasmid cDNA transfection in immortalized microvascular endothelial cells. Transcriptional profiling combined with quantitative real-time reverse transcription-polymerase chain reaction and Western blotting analyses revealed that Prox1 expression up-regulated the lymphatic endothelial cell markers podoplanin and vascular endothelial growth factor receptor-3. Conversely, genes such as laminin, vascular endothelial growth factor-C, neuropilin-1, and intercellular adhesion molecule-1, whose expression has been associated with the blood vascular endothelial cell phenotype, were down-regulated. These results were confirmed by the use of specific antibodies against some of these markers in sections of embryonic and adult tissues. These findings validate our previous proposal that Prox1 is a key player in the molecular pathway leading to the formation of lymphatic vasculature and identify Prox1 as a master switch in the program specifying lymphatic endothelial cell fate. That a single gene product was sufficient to re-program the blood vascular endothelium toward a lymphatic phenotype corroborates the close relationship between these two vascular systems and also suggests that during evolution, the lymphatic vasculature originated from the blood vasculature by the additional expression of only a few gene products such as Prox1., (Copyright 2002 Wiley-Liss, Inc.)

The unfolding of bovine thyroglobulin (Tg) in guanidine hydrochloride (GuHCl) solution was studied by following the fluorescence and circular dichroism. With increasing GuHCl concentrations, the emission maximum of the intrinsic fluorescence clearly red-shifted in two stages. At concentrations of GuHCl less than 1.2 M or more than 1.6 M, the red shift showed a cooperative manner. At concentrations of GuHCl between 1.2 and 1.6 M, an unfolding intermediate was observed. It was further characterized by the increased binding of the fluorescence probe 1-anilinonaphthalene-8-sulfonic acid (ANS). No significant changes of the secondary structure were indicated by CD spectra at the concentrations of GuHCl between 1.2 and 1.6 M. The conformation of this state has properties similar to those of a molten globule state which may exist in the folding pathway of the protein. Further changes in fluorescence properties occurred at concentrations of denaturant higher than 1.6 M with a significant red shift of the emission maximum from 340 to 347 nm and a marked decrease in ANS binding. This in vitro study gave a clue to understand the biochemical mechanism for the occurrence of aggregation and molecular chaperone binding during Tg maturation in vivo.

The distinct roles of angiopoietin (Ang)-1 and Ang2, counteracting ligands for the endothelium-specific Tie2 receptor, in tumor development and progression have remained poorly understood. We investigated the expression of Ang1 and Ang2 during multistep mouse skin carcinogenesis and in human squamous cell carcinoma (SCC) xenografts. Expression of Ang2, but not of Ang1, was up-regulated in angiogenic tumor vessels already in early stages of skin carcinogenesis and was also strongly increased in SCCs. Stable overexpression of Ang1 in human A431 SCCs resulted in a more than 70% inhibition of tumor growth, associated with enhanced Tie2 phosphorylation levels, as compared with low levels in control transfected tumors. No major changes in the vascular density, vascular endothelial growth factor mRNA and protein expression, and vascular endothelial growth factor receptor-2 phosphorylation levels were observed in Ang1-expressing tumors. However, the fraction of tumor blood vessels with coverage by alpha-smooth muscle actin-positive periendothelial cells was significantly increased, indicative of an increased vascular maturation status. These findings identify an inhibitory role of Ang1/Tie2 receptor-mediated vessel maturation in SCC growth and suggest that up-regulation of its antagonist, Ang2, during early-stage epithelial tumorigenesis contributes to the angiogenic switch by counteracting specific vessel-stabilizing effects of Ang1.

We investigated the combined effects of p53 gene transfer and irradiation and its still unclear interaction mechanism in human gliomas. Four human glioma cell lines expressing mutant type p53 (U373 and A172) and wild-type p53 (D54MG and EFC-2) were transfected by adenoviral vectors bearing p53 gene at 50 multiplicity of infection. Two days after transfection, cells were irradiated (3, 6, and 9 Gy). The cytotoxicity was evaluated by clonogenic assay. The quantitative analysis of apoptosis and cell cycle analysis were performed using flow cytometry. Irradiation combined with adenoviral p53 transfection significantly increased cytotoxicity, which was additive in cell lines with wild-type p53 and more than additive in cell lines with mutant p53. The combination of two modalities increased the apoptotic population by 14% in A172 cells and 20% in D54 MG cells, which were the sum of apoptosis from each modality. Adenoviral p53 transfection increased the G1 phase fraction and concomitant decrease of radioresistant S phase fraction in A172 and D54MG cells. Our study demonstrated that p53 gene transfer combined with irradiation increased absolute cytotoxicity in human glioma cells used in this experiment. The interaction mechanism for increased cytotoxicity involved, in part, increased apoptosis and change of cell cycle profile.

The dissociation and unfolding behavior of the GCN4 leucine zipper has been studied using SDS titration. Circular dichroism (CD) spectra showed that the alpha-helix content of the leucine zipper (20 microM) decreased during the sodium dodecyl sulfate (SDS) titration. However, the alpha-helix content of the leucine zipper still remained significant in the presence of 1 mM SDS, with little change detected when the SDS concentration further increased to 2 mM. The dimer dissociation of the leucine zipper is also a co-operative process during SDS titration; with no dimer remaining when SDS concentration reached 1 mM, as shown by electrophoresis and the the theta(222)/theta(208) ratio. Our results indicate that SDS efficiently induces leucine zipper dimer dissociation with the monomers still partially folded. The experimental results provide important evidence for the previous model that partial helix formation precedes dimerization in coiled coil folding.

Anatomical lesions of hypothalamic area associated with hypodipsic hypernatremia have been reported only rarely. We report here a case of hypodipsic hypernatremia induced by a hypothalamic lesion. A 25-yr-old man, who had been treated with radiation for hypothalamic tumor 5-yr before, was admitted for evaluation of hypernatremia and hypokalemia. He never felt thirst despite the elevated plasma osmolality and usually refused to drink intentionally. Plasma arginine vasopressin (AVP) level was normal despite the severe hypernatremic hyperosmolar state and urine was not properly concentrated, while AVP secretion was rapidly induced by water deprivation and urine osmolality also progressively increased to the near maximum concentration range. All of these findings were consistent with an isolated defect in osmoregulation of thirst, which was considered as the cause of chronic hypernatremia in the patient without an absolute deficiency in AVP secretion. Hypokalemia could be induced by activation of the renin-angiotensin-aldosterone system as a result of volume depletion. However, inappropriately low values of plasma aldosterone levels despite high plasma renin activity could not induce symptomatic hypokalemia and metabolic alkalosis. The relatively low serum aldosterone levels compared with high plasma renin activity might result from hypernatremia. Hypernatremia and hypokalemia were gradually corrected by intentional water intake only.

The noncoding RNA Xist has been shown to be essential for X-chromosome inactivation and to coat the inactive X-chromosome (Xi). Thus, an important question in understanding the formation of Xi is whether the binding reaction of Xist is necessary for X-chromosome inactivation. In this article, we demonstrate the failure of X-chromosome silencing if the association of Xist with the X-chromosome is inhibited. The chromatin-binding region was functionally mapped and evaluated by using an approach for studying noncoding RNA function in living cells that we call peptide nucleic acid (PNA) interference mapping. In the reported experiments, a single 19-bp antisense cell-permeating PNA targeted against a particular region of Xist RNA caused the disruption of the Xi. The association of the Xi with macro-histone H2A is also disturbed by PNA interference mapping.

The angiogenic switch during tumorigenesis is thought to be induced by a change in the balance of pro- angiogenic and anti-angiogenic factors. To elucidate the biological role of the endogenous angiogenesis inhibitor thrombospondin-2 (TSP-2) during multistep carcinogenesis, we subjected TSP-2-deficient and wild-type mice to a chemical skin carcinogenesis regimen. Surprisingly, TSP-2 expression was strongly upregulated in the mesenchymal stroma of wild-type mice throughout the consecutive stages of tumorigenesis whereas the angiogenesis factor, vascular endothelial growth factor, was induced predominantly in tumor cells. TSP-2 deficiency dramatically enhanced susceptibility to skin carcinogenesis and resulted in accelerated and increased tumor formation. The angiogenic switch occurred in early stages of pre-malignant tumor formation, and tumor angiogenesis was significantly enhanced in TSP-2-deficient mice. While TSP-2 deficiency did not affect tumor differentiation or proliferation, tumor cell apoptosis was significantly reduced. These results reveal upregulation of an endogenous angiogenesis inhibitor during multi step tumorigenesis and identify enhanced stromal TSP-2 expression as a novel host anti-tumor defense mechanism.

Recent increase in the incidence of lung cancer often makes it difficult to differentiate between lung cancer and tuberculosis (TB), due to their radiologic similarities. Fine needle aspiration biopsy (FNAB) has been widely employed for the diagnosis of lung cancer and TB, but the diagnostic accuracy of TB is not high enough. As a rapid screening test for tuberculosis, we evaluated serological tests using Mycobacterium tuberculosis PPD and lipoarabinomannan (LAM) antigens. A total of 95 patients with indication of FNAB cytology from initial CT findings were enrolled. 25 patients had TB, 76 thoracic malignancy, and six (7.9%) of the lung cancer patients also had TB, indicating much higher prevalence of TB in thoracic tumor patients. Antibodies to PPD were elevated in 18 (72.0%) of 25 TB patients and in 22 (31.4%) of 70 patients with thoracic malignancy. In contrast, only 3 (4.7%) of 64 healthy controls aged 40 or above were seropositive to PPD antigen. The prevalence of anti-PPD antibodies in thoracic tumor patients was therefore significantly greater than that amongst the healthy controls (p<0.001, chi-square test). However, no significant difference in the prevalence of anti-LAM antibodies was found between study subjects and controls. This study demonstrates that thoracic tumor patients have significantly elevated antibodies to PPD; therefore, high anti-PPD seroreactivity in thoracic tumor patients should be cautiously interpreted. A longitudinal investigation on seropositive thoracic tumor patients is required to determine the role of the serological test for TB in lung cancer patients.

Increased expression of VEGF in several types of tumours has been shown to correlate with poor prognosis. We used a replication-deficient adenoviral vector containing antisense VEGF cDNA (Ad5CMV-alphaVEGF) to down-regulate VEGF expression and increase the efficiency of delivery of the antisense sequence in the human breast cancer cell line MDA231-MB. Transfection of these cells with Ad5CMV-alphaVEGF in vitro reduced secreted levels of VEGF protein without affecting cell growth. Moreover, injection of the Ad5CMV-alphaVEGF vector into intramammary xenografts of these cells established in nude mice inhibited tumour growth and reduced the amount of VEGF protein and the density of microvessels in those tumours relative to tumours treated with the control vector Ad5(dl312). Our results showed that antisense VEGF(165)cDNA was efficiently delivered in vivo via an adenoviral vector and that this treatment significantly inhibited the growth of established experimental breast tumours. The Ad5CMV-alphaVEGF vector may be useful in targeting the tumour vasculature in the treatment of breast cancer., (Copyright 2001 Cancer Research Campaign http://www.bjcancer.com Copyright 2001 Cancer Research Campaign.)

Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells., (Copyright 2001 Academic Press.)

Murine Xist is an essential transcript for X chromosome inactivation (X inactivation). According to recently revised structure, Xist is at least 17.8 kb long. It consists of seven exons and there are two major transcripts in female somatic cells. In this study we further defined the molecular structures of the two isoforms, namely short (S) and long (L) forms by northern blot and RNAse protection assay (RPA). The following lines of evidences suggest that mouse Xist depends on differential polyadenylation, not alternative splicing, to generate the two RNA isoforms: (1) only one band was detectable with the northern probes spanning the 3' end of Xist. (2) RPA showed the 3' termini of both S and L forms, and there are putative polyadenylation signals and hairpin structures close to these ends. (3) Analyses by splice site prediction program did not show any evidence of splice motifs in the sequence of L form. (4) Alignments between Xist 3' end (ESTs) and genomic sequence support the absence of splicing event in the region. The newly revised structure of Xist isoforms may have different stability and roles in the process of X inactivation.

The five Saccharomyces cerevisiae Not proteins are associated with the Ccr4 and Caf1 proteins in 1.2 MDa and 2 MDa complexes. The Not proteins have been proposed to repress transcription of promoters that do not contain a canonical TATA sequence, while the Ccr4 and Caf1 proteins are required for non-fermentative gene expression. The mechanism of transcriptional regulation by the Ccr4-Not complex is unknown and the role of its different components is unclear. Only Not1p is essential for yeast viability.Here, we show that most strains carrying combinations of two null alleles of the non-essential CCR4-NOT genes are non-viable. This would suggest that the Ccr4-Not complex is essential. We find that Not1p consists of at least two domains, a C-terminal domain that is essential for yeast viability, and a N-terminal domain that is dispensable but required for yeast wild-type growth. The essential C-terminal domain of Not1p can associate with Not5p, and both proteins are present in 1.2 and 2 MDa complexes in the absence of the N-terminal Not1p domain. In contrast, in the absence of the N-terminal domain of Not1p, Ccr4p does not efficiently associate in large complexes nor with the C-terminal domain of Not1p. Healthy growth is observed when both domains of Not1p are expressed in trans, and is correlated with their physical association, together with Ccr4p, in large complexes. These results are consistent with the essential function of Not1p lying within the Ccr4-Not complex., (Copyright 2000 Academic Press.)

Purpose: The authors' goal was to assess the diagnostic accuracy and clinical effect of MRI compared with echocardiography and catheterization in the evaluation of cardiac defects with situs ambiguous., Method: Twenty-two patients with visceral heterotaxy syndrome were included., Results: Because situs determined by the relation between the pulmonary artery and bronchi showed most predominantly a tendency toward lateralization, this was regarded as the standard reference of situs determination. For the purpose of this study, patients were classified as having right isomerism (n = 13) or left isomerism groups (n = 9). MRI has several advantages compared with echocardiography or cardiac angiography for examining patients with situs ambiguous. (1) The bronchial, pulmonary arterial, and splenic situs can be readily determined, and discrepancies (n = 2) can be assessed easily. (2) Venoatrial connections are adequately imaged. In particular, all types of total and partial anomalous pulmonary venous return are delineated, regardless of whether restrictions of pulmonary blood flow or pulmonary venous obstructions are involved (n = 4). The courses of vertical veins were easily identified, and the prearterial position was revealed in only one of seven right isomerisms with total anomalous pulmonary venous return. The drain pattern of the hepatic vein can be visualized using three-dimensional spatial information and is useful for total cavopulmonary connection design. (3) Associated complicated cardiac anomalies, particularly the size or peripheral stenosis of the pulmonary arteries, may be evaluated, and this information is useful for palliative shunt operations., Conclusion: Because of its wide field of view and imaging, which is not restricted by associated anomalies, a thorough understanding of the cardiovascular anatomy of the situs ambiguous can be achieved using MRI, which is of considerable value in the surgical correction of this complicated anomaly. MRI can obviate or facilitate catheterization in these critically ill patients.

Malignant gliomas are highly angiogenic and aggressive tumors. IFN-beta has been used for the treatment of patients with malignant glioma; however, its antitumor mechanism in vivo remains unclear. To understand the in vivo antitumor effect and mechanism of recombinant human IFN-beta (rhIFN-beta) depending on the stages of tumor development or progression, we used orthotopic xenograft brain tumors generated by stereotactic intracerebral implantation of U-87 human glioma cells in nude mice. Mice bearing tumors 7 days (group 1) and 21 days (group 2) postimplant were treated with 2 x 10(5) IU/day of rhIFN-beta or saline i.p. for 15 days, respectively. Tumor growth was suppressed by 69.6% in group 1 and 10.8% in group 2 compared with tumors of each control group treated with saline. rhIFN-beta-treated group 1 animals showed 38% reduction in vascularization along with a 2.5-fold increase of the apoptotic index and no change in the proliferative index as compared with untreated tumors. The expression level of vascular endothelial cell growth factor and basic fibroblast growth factor was not affected by rhIFN-beta treatment. rhIFN-beta showed inhibitory activity on proliferation of U-87 cells, human umbilical vein endothelial cells, and PAM 212 murine keratinocytes in vitro. Our results indicate that the in vivo antitumor effect of rhIFN-beta on malignant gliomas may be mediated, at least in part, via angiogenesis inhibition rather than antiproliferative activity and that rhIFN-beta may be more effective for the treatment of malignant glioma patients at an early stage with minimal or microscopic tumor burdens rather than at an advanced stage of tumor development.

We investigated the antineoplastic potentials of recombinant adenovirus containing wild-type p53 cDNA (Ad5CMV-p53) for malignant gliomas. In four human glioma cell lines (U-251 and LG expressing endogenous mutant p53, and U-87 and EFC-2 expressing wild-type p53) and two rat glioma cell lines (9L and C6, each expressing mutant and wild-type p53), gene transfer efficiency determined by X-gal staining and Western blotting was varied (10-99% at 10-500 multiplicity of infection, MOI). Growth inhibitory effect was drastic (>90% at 100 MOI) in U-251 cells and only moderate or minimal in other cell lines harboring wild-type p53 or low gene transfer efficiency. Ex vivo transduction of U-251 cells with Ad5CMV-p53 suppressed the in vivo tumorigenicity of the cells. Histopathologic examination for Ad5CMV-p53 toxicity to rat brains showed inflammatory reactions in half of the tested brains at 10(8) MOI. U-251 cells were inoculated intracerebrally in nude mice and injected Ad5CMV-p53 into the tumor, in which neither the tumor suppression nor the survival benefit was observed. In conclusion, heterogeneity of the cellular subpopulations of malignant glioma in p53 status, variable and insufficient gene delivery to tumor, and adenoviral toxicity to brain at higher doses may be limiting factors to be solved in developing adenovirus-p53 gene therapy for malignant gliomas.

High-resolution computed tomography (HRCT) was carried out in 36 patients with congenital left-to-right shunt disease and 10 normal control subjects to assess the feasibility of CT in the evaluation of pulmonary hemodynamics. The patients had a left-to-right or a bidirectional shunt and the hemodynamic data obtained by cardiac catheterization in these patients were compared to the information obtained by CT imaging. The pulmonary/systemic blood flow (Q(p)/Q(s)) ratio and pulmonic/systemic resistance (R(p)/R(s)) ratio had a significant correlation with the pulmonary artery/bronchus (PA/Br) ratio (r = 0.54 and r = -0.37, respectively) and pulmonary vein/bronchus (PV/Br) ratio (r = 0.66 and r = -0.66, respectively), and the R(p)/R(s) and mean PA pressure also showed a significant correlation with the PA/PV ratio (r = 0.53 and r = -0.61, respectively) in the mid-lung field when accompanying bronchi were 4. 0-5.9 mm in diameter. There was no correlation between the hemodynamic data and the size of the central and hilar PA or with the rate of PA tapering. With HRCT, it is possible to evaluate pulmonary hemodynamics in patients with congenital heart disease with a left-to-right or bidirectional shunt, particularly R(p)/R(s) and mean PA pressure, which have been very difficult to obtain noninvasively. The small-sized pulmonary vessel/Br ratio or the small-sized PA/PV ratio could offer very useful information, but the dimension of the central PA provided the least useful information.

Kringle 1-3 of human plasminogen is a potent inhibitor of endothelial cell proliferation. To understand a possible role for the unique cystine bridge between kringle 2 and kringle 3, we disrupted the interkringle disulfide bond by mutating Cys(169) and Cys(297) to serine residues. The yield of the mutant during the refolding process was decreased significantly. Anti-endothelial cell proliferative activity of the mutant was similar to that of the wild type. There was no significant difference in in vivo antiangiogenic activity between the wild type and the mutant in chorioallantoic membrane assay. However, in the mutant, the weak lysine binding capability of kringle 2 was not detected and its mobility in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis is different from that of the wild type. These results support the notion that the overall antiangiogenic function of angiostatin is mediated by individual kringles, and suggest that the lysine binding capability of kringle 2 is likely not important for the antiangiogenic activity of kringle 1-3., (Copyright 2000 Academic Press.)

The XIST gene plays an essential role in X Chromosome (Chr) inactivation during the early development of female humans. It is believed that the XIST gene, not encoding a protein, functions as an RNA. The XIST cDNA is unusually long, as its full length is reported to be 16.5 kilobase pairs (kb). Here, comparison of sequences from the genomic interval downstream to the 3' end of the human XIST gene against the human EST database brought to light a number of human EST sequences that are mapped to the region. Furthermore, PCR amplification of human cDNA libraries and RNA fluorescence in situ hybridization (RNA-FISH) demonstrate that the human XIST gene has additional 2.8 kb downstream sequences which have not been documented as a part of the gene. These data show that the full-length XIST cDNA is, in fact, 19.3 kb, not 16.5 kb as previously reported. The newly defined region contains an intron that may be alternatively spliced and seven polyadenylation signal sequences. Sequences in the newly defined region show overall sequence similarity with the 3' terminal region of mouse Xist, and three subregions exhibit quite high sequence conservation. Interestingly, the new intron spans the first two sub-regions that are absent in one of the two isoforms of mouse Xist. Taken together, we revise the structure of human XIST cDNA and compare cDNA structures between human and mouse XIST/Xist. al. 1992). This gene, called XIST/Xist (X inactive specific transcript), shows several interesting features. First, both human and mouse XIST/Xist cDNA are unusually long, reportedly 16.5 kb and 17.8 kb, respectively (Brown et al. 1992; Hong et al. 1999). Second, the transcript does not seem to encode a protein, on the basis of the lack of a significant open reading frame, absence of the Xist RNA from polysomes, and localization of the transcript in the nucleus (Brockdorff et al. 1992; Brown et al. 1992). Third, the XIST/Xist RNA physically associates with, or 'coats,' the inactive X Chr (Brown et al. 1992; Clemson et al. 1996). Fourth, XIST/Xist transcripts can be observed as early as the four-cell stage, and upon the initiation of X-inactivation, the steady-state level of the transcript rises dramatically, apparently by stabilization of the RNA (Panning et al. 1997; Sheardown et al. 1997). Although the function of XIST/Xist is not known, deletion of the gene leads to failure of X-inactivation, and knock-out mice die around the gastrulation stage (Marahrens et al. 1997; Penny et al. 1996). In this report, we revise the structure of the human XIST cDNA and discuss structural features of the newly defined region.

Endostatin, a carboxyl-terminal fragment of collagen XVIII is known as an anti-angiogenic agent, that specifically inhibits the proliferation of endothelial cell and the growth of several primary tumor. We report here the purification and characterization of the recombinant murine endostatin (rmEndostatin) which was expressed in a prokaryotic expression system. This rmEndostatin has similar physiochemical properties of yeast-produced recombinant endostatin, and it also specifically inhibits the proliferation and migration of bovine capillary endothelial cells stimulated by basic fibroblast growth factor. The biological activity of rmEndostatin was also shown by its anti-angiogenic ability on the chorioallantoic membrane of chick embryo in vivo. In this article, we demonstrate the refolding and purification of rmEndostatin, expressed using E. coli system, to a biologically active and soluble form. In addition, these results confirm the activity of endostatin as a potent anti-angiogenic agent.

Purpose: The purpose of our study was to determine specific CT findings of tuberculous pneumonia presenting as segmental or lobar consolidation along with a pathologic review of specimens with similar radiographic patterns., Method: CT findings of 45 cases of proven tuberculous pneumonia and 21 proven nontuberculous pneumonia were compared. Pathologic findings of five surgically resected tuberculous pneumonia cases were also investigated. The presence of fluid bronchogram (linear, branching shadow of fluid attenuation) and inner low attenuation/cavitation in the area of consolidation, luminal dilatation, and wall thickening of proximal bronchi were the main points sought on CT scan. In addition, the presence of bronchogenic dissemination, lymph node enlargement, and pleural lesions was also checked for in the unaffected area of both lungs., Results: The following bronchial changes were seen in the tuberculous pneumonia and nontuberculous pneumonia groups, respectively: fluid bronchogram in 68.9 and 23.8% (p < 0.05), bronchial luminal dilatation in 60.0 and 23.8% (p < 0.05), and bronchial wall thickening of the proximal airway leading to the area of consolidation in 52.8 and 7% (p < 0.05). Bronchogenic dissemination outside the consolidation appeared in 88.9 and 52.4% (p < 0.05), respectively. In the tuberculous pneumonia group, lymph node enlargement and pleural reaction were seen in 55.6 and 35.6%, respectively, but in 42.9 and 57.1% in the nontuberculous pneumonia group (p > 0.05). Histologically, tuberculous pneumonia showed either bronchioles containing inflammatory exudates and submucosal granuloma or alveoli containing aggregates of alveolar macrophages or cellular debris., Conclusion: Fluid bronchogram in the area of homogeneous consolidation, bronchial luminal dilatation, and bronchial wall thickening of the proximal airway were the bronchial changes more significantly prominent in the tuberculous pneumonia group. We suspect that these findings may represent tuberculous bronchitis in small airways.

Human malignant gliomas are highly vascularized and aggressive tumors. Angiogenesis inhibitors have been shown to induce regression of a variety of primary and metastatic tumors in vivo. However, their usefulness in treating brain tumors is not well understood. Angiostatin, a multiple kringle (1-4 of 5)-containing fragment of plasminogen, is one of the highly effective natural cryptic angiogenesis inhibitors. In our study, the therapeutic efficacy of non-glycosylated and small molecular size recombinant kringles 1-3 (rPK1-3) was examined in the treatment of brain tumors generated by stereotactic intracerebral implantation of U-87 human glioma cells in nude mice. Mice bearing tumors 7 days post-implant were treated daily with rPK1-3 (100 mg/kg) s.c. for 21 days. Treated animals showed suppressed brain tumor growth by greater than 71.2% along with a 3-fold increase of apoptotic index and suppressed vascularization by 78.9%, without any observable signs of toxicity. Analysis of bFGF and VEGF expression in the tumors of treated animals using immuno-histochemical methods showed near complete absence of growth factors. Our results indicate that the non-glycosylated, small molecular size rPK1-3 is an efficient tumoristatic agent for the treatment of intracranial human glioma xenografts in mice and might provide new strategies for the treatment of brain tumors., (Copyright 1999 Wiley-Liss, Inc.)

In this report, we present structural data for the murine Xist gene. The data presented in this paper demonstrate that the murine Xist transcript is at least 17.4 kb, not 14.3 kb as previously reported. The new structure of the murine Xist gene described herein has seven exons, not six. Exon VII encodes an additional 3.1 kb of information at the 3' end. Exon VII contains seven possible sites for polyadenylation; four of these sites are located in the newly discovered 3' end. Consequently, it is possible that several distinct transcripts may be produced through differential polyadenylation of a primary transcript. Alternative use of polyadenylation signals could result in size changes for exon VII. Two major species of Xist are detectable by Northern analysis, consistent with differential polyadenylation. In this paper, we propose a model for the role of the Xist 3' end in the process of X-chromosome counting and choice during embryonic development.

MMAC/PTEN, a tumor suppressor gene located on chromosome 10q, has recently been shown to act as a phosphatidylinositol 3,4,5-triphosphate phosphatase and to modulate cell growth and apoptosis. Somatic mutations of MMAC/PTEN have been reported in a number of human cancers, especially in glioblastoma multiforme (GBM), although the number of identified mutations (approximately 10-35%) is significantly lower than the frequency of LOH affecting the MMAC/PTEN locus in the specimens (approximately 75-95%). To further investigate the possible alterations that may affect MMAC/PTEN, we examined the expression of the gene by reverse transcription-PCR in a series of gliomas. A significant difference (P < 0.001) was observed between the expression of MMAC/PTEN in GBMs versus lower grades of gliomas, thus mimicking the difference in allelic deletion associated with the locus in these tumors. Furthermore, Kaplan-Meier survival plots, adjusted for age and tumor grade, showed a significantly better prognosis for patients whose tumors expressed high levels of MMAC/PTEN. Additionally, immunostaining of GBMs revealed little or no MMAC/PTEN expression in about two-thirds of the tumors, whereas the other approximately one-third of tumors had significantly higher levels of expression. However, in about two-thirds of the high-expressing specimens, a heterogeneous pattern of expression was observed, indicating that certain cells within the tumor failed to express MMAC/PTEN. The combination of these results suggest that, in addition to molecular alterations affecting the gene, altered expression of MMAC/PTEN may play a significant role in the progression of GBM and patient outcome.

The MET protooncogene encodes a transmembrane tyrosine kinase identified as the receptor of a polypeptide known as hepatocyte growth factor/scatter factor. We performed PCR-based single-strand conformational polymorphism and sequencing analysis of the tyrosine kinase domain of the MET gene (exon 15-19) in 75 primary liver cancers. Three missense mutations were detected exclusively in 10 childhood hepatocellular carcinomas (HCCs), while no mutations were detected in 16 adult HCCs, 21 cholangiocarcinomas, or 28 hepatoblastomas. The extremely short incubation period from hepatitis B virus infection to the genesis of childhood HCC as compared with the adult HCC suggests that there may be an additional mechanism that accelerates the carcinogenesis of childhood HCC. Our results indicate that mutations of the tyrosine kinase domain of the MET gene may be involved in the acceleration of the carcinogenesis in childhood HCC.

The satellite repeat structure of the mammalian centromere contains the CENP-B protein binding site. Using the peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH), we show by direct PNA-DNA binding that all detectable CENP-B sites in a mammalian genome might have the same sequence. Two species-specific PNA 17-mers, pMm and pMc, were identified from CENP-B binding sites of Mus musculus and M. caroli, respectively. Fluorescence in situ hybridization confirmed that pMc hybridized to M. caroli centromeres only; however, pMm cross-hybridized to M. musculus and human centromeres. By using a series of CENP-B PNA 17-mers containing 1, 2, 3, 5, and 7 base-pair mismatches to their DNA counterparts, we further demonstrate that PNA-FISH can discriminate between two CENP-B DNA sequences that differ by a single base-pair in mouse and human centromeres, suggesting the degree of conservation of CENP-B sequences throughout the genome. In comparison with DNA oligonucleotides, PNA oligomers demonstrate the higher sequence specificity, improved stability, reproducibility, and lower background. Therefore, PNA oligomers have significant advantages over DNA oligonucleotide probes in analyzing microsatellites in a genome.

Abnormalities in the p16INK4a tumor suppressor gene are found in many lung cancer cell lines and primary lung cancer tissue. To examine its tumor suppressor function and potential adequacy in cancer gene replacement therapy, wild-type p16INK4a gene was inserted in an adenovirus derived gene delivery system and introduced into lung cancer cell lines (NCI-H441 and NCI-H157) that did not express p16INK4a. Western blot assay and immunocytochemistry demonstrated production of wild-type p16 protein in these cell lines. The biological function of exogenous p16 protein was confirmed by the inhibition of pRB phosphorylation. The expression of exogenous p16 protein via recombinant adenovirus significantly inhibited cancer cell growth and colony formation in vitro of NSCLC that can not express endogenous p16. The flow cytometric analysis showed these results correlated with G1 cell cycle arrest. These observations suggest the value of adenovirally-mediated p16INK4a gene replacement therapy for lung cancer.

Accurate assessment and replacement of blood loss and fluid-electrolyte deficit during craniosynostosis repair is difficult owing to patient size and the diversity of surgical technique. Forty-three patients undergoing primary craniosynostosis repair over a 10-year period were studied retrospectively to determine blood loss and fluid deficit and to assess blood transfusion practices during both intraoperative and postoperative periods. Blood loss was calculated on the basis of estimated red cell mass (ERCM) and fluid-electrolyte imbalance was investigated with blood samplings. Blood transfusion was considered appropriate if the postoperative or posttransfusion ERCM was within 12% of the preoperative value. Estimated fluid requirement (EFR) was used in 4 ml kg(-1) h(-1) except for neonates. Intraoperatively, 80% of all patients were appropriately managed with respect to blood transfusion and EFR. Postoperatively only 20% of the patients receiving transfusions were transfused appropriately. In 23.3% of these patients (10/43) unexpected respiratory distress developed immediately after their recovery from the anesthesia. With the measurement of estimated blood volume and allowable blood loss, appropriate transfusion could be achieved for the successful treatment of the primary craniosynostosis.

The loss of large segments or an entire copy of chromosome 10 is the most common genetic alteration in human glioblastomas. To address the biological and molecular consequences of this chromosomal alteration, we transferred a human chromosome 10 into a glioma cell clone devoid of an intact copy. The hybrid cells exhibited an altered cellular morphology, a decreased saturation density, and a suppression of both anchorage-independent growth and tumor formation in nude mice. The hybrids also expressed the recently identified candidate tumor suppressor gene MMAC1/PTEN. To further identify gene products that may be involved in glioma progression, a subtractive hybridization was performed between the human glioblastoma cells and the phenotypically suppressed hybrid cells to identify differentially expressed gene products. Sixty-one clones were identified, with nine clones being preferentially expressed in the hybrid cells. Four cDNA clones represented markers of differentiation in glial cells. Two cDNA clones shared homology with platelet derived growth factor-alpha and the insulin receptor, respectively, both genes previously implicated in glioma progression. A novel gene product that was expressed predominantly in the brain, but which did not map to chromosome 10, was also identified. This clone contained an element that was also present in three additional clones, two of which also exhibited differential expression. Consequently, the presence of a functional copy of chromosome 10 in the glioma cells results in differential expression of a number of gene products, including novel genes as well as those associated with glial cell differentiation.

The results are reported of a retrospective review of the presentation and outcome of 43 pineal region tumors treated from 1982 to 1996, including 20 identified tumors: 5 germinomas, 8 teratomas, 2 embryonal carcinomas, 1 endodermal sinus tumor, 2 pineocytomas and 2 pineoblastomas. Of the 43 tumors reviewed, 36 were located in the pineal region, 5 in the suprasellar, and 2 in both the pineal and suprasellar regions. Twenty patients underwent surgical resection: total in 6 and partial in 10, while only a biopsy was taken in 4 cases. Fifteen patients were managed on the basis of serum CSF tumor markers and radiation response. Twenty-three patients with germinomas received radiotherapy (RT) and had a 5-year survival rate of 87%. Fifteen patients with non-germinomatous germ cell tumors received RT and chemotherapy following direct surgery, and 5 died (mortality rate of 33.3%). The overall survival rate of the 43 patients with pineal tumors was 79.1% (34/43) and the death rate was 20.9% (9/43). It is now recognized that the wide variety of tumor types found in the pineal region necessitates different modes of treatment, and improved microsurgical and stereotactic surgical techniques have made mortality and morbidity rates acceptably low. Because the radiation response and CSF cytology are not enough to determine optimum treatment, a tissue diagnosis should be obtained in all patients.

The high-resolution computed tomography (HRCT) studies for bronchial asthma (BA) have revealed abnormal radiologic findings such as bronchial wall thickening, bronchiectasis, emphysema and mosaic pattern of lung attenuation. But the clinical significance of these findings are not yet clarified. In this study, we quantified the bronchial wall thickness and evaluated HRCT features in 57 BA subjects (338 bronchi) who had precipitating factors of irreversible airway remodeling, 19 COPD subjects (70 bronchi) and 10 healthy subjects (23 bronchi). Then we correlated HRCT findings with the clinical features, pulmonary functions and methacholine PC20 (PC20M) and studied their clinical significance. The bronchial wall for BA was about 1.48 mm thicker than that for COPD and about 2.34 mm thicker than for healthy controls (p < 0.0001, respectively). But the individual mean ratio of bronchial wall thickness to luminal diameter (BWT/LD) in asthmatics did not correlate with the clinical features, lung functions and PC20M. Abnormal HRCT findings, such as bronchiectasis (17.5%), emphysema (5.3%) and mosaic pattern of lung attenuation (17.5%) were found in BA. These findings were more common in BA with moderate to severe airflow limitation (FEV1 < 80%, p < 0.05) and patients with these changes had a more prolonged history of asthma (p < 0.05). PC20M was higher in BA with these abnormal changes (p < 0.001) but these patients' FEV1 (p < 0.05), FEF25-75 (p < 0.05) and specific airway conductance (p < 0.05) were lower than those having BA without such findings. In this study we showed that the bronchial wall was more significantly thickened in BA but that it did not correlate well with the clinical features, lung functions and PC20M. Additionally, patients having BA with abnormal airway and air space HRCT findings had a prolonged history of asthmatic symptoms, loss of lung functions and decreased bronchial hyperresponsiveness. These results suggested the possibility that HRCT can be used for the differentiation of BA from COPD or healthy controls. Furthermore, patients having BA with abnormal HRCT changes demonstrate poor lung function and less hyperreactive bronchi than those without. We concluded that HRCT may be useful for the prognosis and treatment of bronchial asthma cases who have the precipitating factors of irreversible airway remodelling.

Potentiometric responses of polyurethane (PU)-based membranes containing valinomycin and varying amounts of plasticizer (DOA) and/or lipophilic additive (KTpClPB) were examined as a function of soft segment [poly(tetramethylene ether glycol)] contents in aromatic diisocyanate-based PU matrices. Upon increasing the weight percentages (w(soft)) of soft segments, which in part behave like a built-in plasticizer, providing the matrices with rubbery structure (glass transition temperature below -58 °C), the amounts of DOA and/or KTpClPB necessary to result in near-Nerntian response (e.g., slope > 50 mV/decade) to potassium were substantially lowered. The apparent effect of adding plasticizer to PU-based membranes was comparable to that resulting from an increase of free carrier concentration in normal PVC-based membranes. Owing to the chemical interaction between mobile anionic sites and urethane chains, plasticizer-free PU membranes could be prepared with the PU matrices with high soft segment contents (w(soft) ≥ 60 wt %). PUs composed of 60 ≤ w(soft) < 80 wt % were recommended as the matrix for fabricating ISE membranes with no or low plasticizer content.

A radioisotope first pass study was done on patients over a period of 1 to 15 years (average 4.6 years) after repair for ventricular septal defect or arterial septal defect with a left to right shunt. The age of the patients ranged from 6 to 32 years (average 14.2 year) at the time of the study. The total work of exercise and the right and left ventricular ejection fraction(EF) were evaluated at rest and after exercise. The results were compared with the preoperative hemodynamic findings and with the age of patient at the time of the operation. 1) When the total work of exercise was divided with the maximal exercise capacity of the normal individual corresponding to the patients' height and body surface area (the percentage of total work), it were very low with the average of 40% of normal. There was no sexual difference, but the percentage of total work of exercise had significant correlation with the patients' age at the time of operation (r = -0.52, p < 0.01) and post-exercise left ventricular ejection fraction (LVEF)(r = -0.39, p < 0.05). 2) LVEF at rest had some correlation with the preoperative mean pulmonary arterial pressure (r = -0.29, p = 0.05), but showed no relationship with Qp/Qs or Rp/Rs ratios. The right ventricular ejection fraction (RVEF) at rest had no relations with the preoperative hemodynamic findings with maximal workload. 3) The post-exercise RVEF showed linear correlation with the preoperative Rp/Rs ratio (r = -0.49, p < 0.005), and mean pulmonary arterial pressure (r = -0.37, p < 0.05). The post-exercise LVEF had no significant correlation with any preoperative hemodynamic factors. 4) When greater than 5% increase in ventricular EF after exercise is considered normal, the group with the normal right and left ventricular responses (n = 11) showed normal preoperative Rp/Rs ratio (7.6 +/- 4.1). In the group with normal left, but abnormal right ventricular response (n = 9) and the group with abnormal biventricular response (n = 11), both demonstrated incremental increase in Rp/Rs ratio (20.1 +/- 11.3, 26.3 +/- 19.8 respectively). Normal right, but abnormal left ventricular reaction (n = 2) was noted in patients with residual aortic valvular insufficiency and residual ventricular septal defect. In conclusion, post-operative ventricular response was much more sensitive and informative than that of ventricular function at rest and to detect subclinical cardiac dysfunction. Post-exercise RVEF was closely correlated with preoperative pulmonary vascular hemodynamics, while post-exercise LVEF seemed to be a major determinant of working capacity after repair.

Whereas most ocular complications following cerebral angiography are benign and transient, central retinal artery occlusion following cerebral angiography produces severe permanent visual deficits in nearly 40% of patients. Physicians ordering and performing cerebral angiography should be aware of this complication, since immediate attention to patients with visual disturbance from central retinal artery occlusion may save useful vision. All previously reported cases of central retinal artery occlusion following cerebral angiography have occurred after direct percutaneous carotid angiography. We report a case of central retinal artery occlusion following transfemoral cerebral angiography using a #5 French catheter and meglumine iothalamate (Conray-60) contrast.