CT angiography for surgical planning in face transplantation candidates.

SUMMARY: Facial allotransplantation replaces missing facial structures with anatomically identical tissues, providing desired functional, esthetic, and psychosocial benefits far superior to those of conventional methods. On the basis of very encouraging initial results, it is likely that more procedures will be performed in the near future. Typical candidates have extremely complex vascular anatomy due to severe injury and/or multiple prior reconstructive attempts; thus, each procedure is uniquely determined by the defects and vascular anatomy of the candidate. We detail CT angiography vascular mapping, noting the clinical relevance of the imaging, the angiosome concept and noninvasive delineation of the key vessels, and current controversies related to the vascular anastomoses.

Anti-tumor activity against multiple myeloma by combination of KW-2478, an Hsp90 inhibitor, with bortezomib.

Heat shock protein 90 (Hsp90) is a promising target for anti-tumor therapy. We previously reported the anti-tumor activity of a novel Hsp90 inhibitor, KW-2478, in multiple myeloma (MM) as a single agent. In this study, we examined the combinational effect of KW-2478 and bortezomib, a proteasome inhibitor, in vitro and in vivo. In vitro, KW-2478 enhanced bortezomib-induced cell growth inhibition, both in MM cell lines and primary patient MM cells. The combination of KW-2478 and bortezomib also induced caspase activation in MM cell lines. Interestingly, the combination synergistically enhanced the expression of Hsp70B, a homolog of Hsp70, in human MM cells and peripheral blood mononuclear cells, indicating Hsp70B could be a surrogate biomarker for the combination of Hsp90 and proteasome inhibitors. In vivo, the combination of KW-2478 with bortezomib showed synergistic anti-tumor activity without significant body weight loss in a subcutaneously inoculated human myeloma model. Furthermore, the combination also showed synergistic reduction of tumor burden in bone marrow in an orthotopic myeloma model. Our results strongly suggest that combination of KW-2478 with bortezomib could exhibit enhanced anti-tumor activity against human myeloma.

Sensitivity of epidermal growth factor receptor and ErbB2 exon 20 insertion mutants to Hsp90 inhibition.

The mature epidermal growth factor receptor (EGFR) neither associates with nor requires the molecular chaperone heat-shock protein 90 (Hsp90). Mutations in EGFR exons 18, 19, and 21 confer Hsp90 chaperone dependence. In non-small cell lung cancer (NSCLC), these mutations are associated with enhanced sensitivity to EGFR inhibitors in vitro and with clinical response in vivo. Although less prevalent, insertions in EGFR exon 20 have also been described in NSCLC. These mutations, however, confer resistance to EGFR inhibitors. In NSCLC, exon 20 insertions have also been identified in the EGFR family member ErbB2. Here, we examined the sensitivity of exon 20 insertion mutants to an Hsp90 inhibitor currently in the clinic. Our data demonstrate that both EGFR and ErbB2 exon 20 insertion mutants retain dependence on Hsp90 for stability and downstream-signalling capability, and remain highly sensitive to Hsp90 inhibition. Use of Hsp90 inhibitors should be considered in NSCLC harbouring exon 20 insertions in either EGFR or ErbB2.

A radicicol derivative, KF58333, inhibits expression of hypoxia-inducible factor-1alpha and vascular endothelial growth factor, angiogenesis and growth of human breast cancer xenografts.

A novel oxime derivative of radicicol, KF58333, binds to the heat shock protein 90 (Hsp90) and destabilizes its associated signaling molecules. These effects play a critical role in the growth inhibition of tumor cells. To further investigate the effects of this agent, it was administered to two human breast cancer cell lines, KPL-1 and KPL-4, both in vitro and in vivo. KF58333 dose-dependently inhibited the growth and vascular endothelial growth factor (VEGF) secretion, concomitantly with a decrease in VEGF mRNA expression, in each cell line. This agent also suppressed the increase of VEGF secretion and expression induced by hypoxia (1% O(2)). Intravenous injections of this agent into nude mice bearing either KPL-1 or KPL-4 xenografts significantly inhibited the tumor growth associated with a decrease in the Ki67 labeling index and microvascular area and an increase in apoptosis and the necrotic area. These findings indicate that the antitumor activity of this radicicol derivative may be partly mediated by decreasing VEGF secretion from tumor cells and inhibiting tumor angiogenesis. To explore the action mechanisms of the anti-angiogenic effect, the expression level of hypoxia-inducible factor (HIF)-1alpha was investigated. KF58333 provided a significant decrease in the HIF-1alpha protein expression under both normoxic and hypoxic conditions. In contrast, the mRNA expression of HIF-1alpha was not decreased by this agent. It is suggested that the post-transcriptional down-regulation of HIF-1alpha expression by this agent may result in a decrease of VEGF expression and tumor angiogenesis.

Development of interferon-alpha resistant subline from human chronic myelogenous leukemia cell line KT-1.

OBJECTIVE: Interferon-alpha (IFN-alpha) is one of the most effective therapeutic agents for a number of hematological malignancies, including chronic myelogenous leukemia (CML). Nevertheless, its efficacy is limited because of the development of resistance to IFN-alpha therapy. Previously, we established the novel human CML cell line KT-1, which is sensitive to the antiproliferative effects of IFN-alpha. Here, we report the establishment of an IFN-alpha-resistant subline, KT-1/A3R alpha 1000, by culturing KT-1/A3 cells (IFN-alpha-sensitive subline of KT-1) with increasing concentrations of IFN-alpha, in order to analyze the mechanism of acquisition of IFN-alpha resistance in CML cells after IFN-alpha therapy. SUBJECTS AND METHODS: We developed an IFN-alpha-resistant tumor cell variant, KT-1/A3R alpha 1000, from the KT-1/A3 cell line by culturing cells with increasing concentrations of IFN-alpha. This subline was examined for its ability to proliferate and its resistance to apoptosis in high concentrations of IFN-alpha. The induction of the ISGF3 complex in response to IFN-alpha alpha in KT-1/A3R alpha 1000 was compared with that in the parental cell. RESULTS: The levels of interferon-stimulated gene factor 3 components (STAT1, STAT2, and p48) proteins and STAT2 tyrosine phosphorylation induced after IFN-alpha treatment were unchanged, but formation of the ISGF3 complex was remarkably reduced in KT-1/A3R alpha 1000 cells compared to parental cells. CONCLUSION: The KT-1/A3R alpha 1000 subline is a useful model for studying the mechanism of IFN-alpha resistance after IFN-alpha therapy.

Stereospecific antitumor activity of radicicol oxime derivatives.

PURPOSE: Radicicol is a novel hsp90 antagonist, distinct from the chemically unrelated benzoquinone ansamycin compounds, geldanamycin and herbimycin. Both geldanamycin and radicicol bind in the aminoterminal nucleotide-binding pocket of hsp90, destabilizing the hsp90 client proteins, many of which are essential for tumor cell growth. We describe here antitumor activity of a novel oxime derivative of radicicol, KF58333. We also investigated the mechanism of antitumor activity of KF58333 in comparison with its oxime isomer KF58332. METHODS: Antiproliferative activities were determined in a panel of breast cancer cell lines in vitro. We also examined inhibition of hsp90 function and apoptosis induction in erbB2-overexpressing human breast carcinoma KPL-4 cells in vitro. Direct binding activity to hsp90 was assessed by hsp90-binding assays using geldanamycin or radicicol beads. In animal studies, we investigated plasma concentrations of these compounds after i.v. injection in BALB/c mice and antitumor activity against KPL-4 cells transplanted into nude mice. Inhibition of hsp90 function and induction of apoptosis in vivo were investigated using tumor specimens from drug-treated animals. RESULTS: KF58333 showed potent antiproliferative activity against all breast cancer cell lines tested in vitro, and was more potent than its stereoisomer KF58332. These results are consistent with the ability of KF58333 to deplete hsp90 client proteins and the induction of apoptosis in KPL-4 cells in vitro. Interestingly, KF58333, but not KF58332, showed significant in vivo antitumor activity accompanied by induction of apoptosis in KPL-4 human breast cancer xenografts. Although the plasma concentrations of these compounds were equivalent, KF58333, but not KF58332, depleted hsp90 client proteins such as erbB2, raf-1 and Akt in the tumor specimen recovered from nude mice. CONCLUSIONS: These results suggest that inhibition of hsp90 function, which causes depletion of hsp90 client proteins in tumor, contributes to the antitumor activity of KF58333, and that the stereochemistry of the oxime moiety is important for the biological activity of radicicol oxime derivatives.

Effects of hostility and lifestyle on coronary heart disease among middle-aged urban Japanese.

OBJECTIVE: To explore the association between multi-dimensional aspects of hostility and coronary heart disease among middle-aged urban Japanese. SUBJECTS AND METHODS: We conducted a case-control study. Cases were consecutive patients with acute coronary syndrome admitted to a hospital in Japan. Fifty-three patients (45 men and 8 women) aged 35 to 65 were enrolled. For each case, two sex and age (+/-2 years) matched controls were recruited from among participants in a health check-up program at a health promotion center located in the same area as the hospital. Two questionnaires, both with four components, were used to measure hostility and coping with anger: the one was for anger, hostility, physical aggression and verbal aggression, and the other for aggression, social inhibition, guilt, and controlled affect. RESULTS: The scores of all components from two questionnaires were higher for cases than controls, but the differences were not significant. Multivariate analysis showed that anger, fat intake, alcohol consumption and house size were significantly associated with the etiology of acute coronary syndrome. CONCLUSION: Anger, lifestyle, and socioeconomic status play important roles for the etiology of coronary heart disease in middle-aged urban Japanese.

Sensitivity to interferon-alpha and interferon-stimulated gene factor 3 binding activity in human chronic myelogenous leukemia cell line KT-1.

The mechanism of responsiveness of chronic myelogenous leukemia (CML) cells to interferon (IFN)-alpha was examined by using two subclones of CML cell line KT-1 which exhibited significantly different sensitivities to the antiproliferative and apoptosis-inducing effects of IFN-alpha. IFN-stimulated gene factor 3 (ISGF3) formation by IFN-alpha was reduced in the IFN-alpha-resistant subclone compared to the IFN-alpha-sensitive subclone. We conclude that the level of ISGF3 formation is responsible for the difference in IFN-alpha responses between these subclones.

Novel oxime derivatives of radicicol induce erythroid differentiation associated with preferential G(1) phase accumulation against chronic myelogenous leukemia cells through destabilization of Bcr-Abl with Hsp90 complex.

Chronic myelogenous leukemia (CML) is a clonal disorder of a pluripotent hematopoietic stem cells characterized by a chimeric bcr-abl gene giving rise to a p210(Bcr-Abl) protein with dysregulated tyrosine kinase activity. Radicicol, a macrocyclic antifungal antibiotic, binds to the N-terminal of heat shock protein 90 (Hsp90) and destabilizes Hsp90-associated proteins such as Raf-1. This study investigated the effect of radicicol, novel oxime derivatives of radicicol (KF25706 and KF58333), and herbimycin A (HA), a benzoquinoid ansamycin antibiotic, on the growth and differentiation of human K562 CML cells. Although KF25706 and KF58333 induced the expression of glycophorin A in K562 cells, radicicol and HA caused erythroid differentiation transiently. Cell cycle analysis showed that G(1) phase accumulation was observed in K562 cells treated with KF58333. KF58333 treatment depleted p210(Bcr-Abl), Raf-1, and cellular tyrosine phosphorylated proteins in K562 cells, whereas radicicol and HA showed transient depletion of these proteins. KF58333 also down-regulated the level of cell cycle-dependent kinases 4 and 6 and up-regulated cell cycle-dependent kinase inhibitor p27(Kip1) protein without an effect on the level of Erk and Hsp90 proteins. Immunoprecipitation analysis showed that p210(Bcr-Abl) formed multiple complexes with Hsp90, some containing p23 and others Hsp70; KF58333 treatment dissociated p210(Bcr-Abl) from Hsp90/p23 chaperone complexes. Furthermore, KF58333 induced apoptosis in K562 cells and administration of KF58333 prolonged the survival time of SCID mice inoculated with K562 cells. These results suggest that KF58333 may have therapeutic potential for the treatment of CML that involves abnormal cellular proliferation induced by p210(Bcr-Abl).

Visualization of mitochondrial oxygen fixation in brain slices by gas-tissue autoradiography.

We have developed a novel autoradiographic method of visualizing oxygen fixation with sufficient delivery of [(15)O]O(2)/O(2). Brain slices (400 microm) were preincubated in Krebs-Ringer phosphate buffer and exposed to [(15)O]O(2) in a chamber. Fixation of [(15)O]O(2) correlated with the polarographically measured oxygen consumption among tissue slices from various organs (r=0.84). The fixation of [(15)O]O(2) by brain slices was significantly reduced (7. 2% of the control) by heat-treatment or dose dependently by NaCN (18. 2% of the control on 50 mM NaCN pretreatment). The (15)O radioactivity in the brain slices prepared from rotenone injected rats was also reduced compared to the control (56.8% of the control side). In an autoradiographic study, (15)O radioactivity showed a heterogeneous distribution both in coronal and sagittal sections. Autoradiography of young and senescent rat brain sections showed reduction of oxygen uptake with aging in the cerebrum, the senescent being 77.4% of the young. This method provides information regarding basic oxygen consumption of tissue slices under condition of sufficient O(2) delivery, which reflects mitochondrial electron transport.

KF25706, a novel oxime derivative of radicicol, exhibits in vivo antitumor activity via selective depletion of Hsp90 binding signaling molecules.

Radicicol, a macrocyclic antifungal antibiotic, has been shown to bind to the heat shock protein 90 (Hsp90) chaperone, interfering with its function. Hsp90 family chaperones have been shown to associate with several signaling molecules and play an essential role in signal transduction, which is important for tumor cell growth. Because radicicol lacks antitumor activity in vivo in experimental animal models, we examined the antitumor activity of a novel radicicol oxime derivative, radicicol 6-oxime (KF25706), on human tumor cell growth both in vitro and in vivo. KF25706 showed potent antiproliferative activities against various human tumor cell lines in vitro and inhibited v-src- and K-ras-activated signaling as well as radicicol. In addition, Hsp90 family chaperone-associated proteins, such as p185erbB2, Raf-1, cyclin-dependent kinase 4, and mutant p53, were depleted by KF25706 at a dose comparable to that required for antiproliferative activity. KF25706 was also shown to compete with geldanamycin for binding to Hsp90. KF29163, which is an inactive derivative of radicicol, was less potent both in p185erbB2 depletion and Hsp90 binding. More importantly, KF25706 showed significant growth-inhibitory activity against human breast carcinoma MX-1 cells transplanted into nude mice at a dose of 100 mg/kg twice daily for five consecutive i.v. injections. KF25706 was also shown to possess antitumor activity against human breast carcinoma MCF-7, colon carcinoma DLD-1, and vulval carcinoma A431 cell lines in vivo in an animal model. Finally, we confirmed the depletion of Hsp90-associated signaling molecules (Raf-1 and cyclin-dependent kinase 4) with ex vivo Western blotting analysis using MX-1 xenografts. In agreement with in vivo antitumor activity, KF25706 depleted Hsp90-associated molecules in vivo, whereas KF29163 and radicicol did not show this activity in vivo. Taken together, these results suggest that antitumor activity of KF25706 may be mediated, at least in part, by binding to Hsp90 family proteins and destabilization of Hsp90-associated signaling molecules.

[A study on standardization of the Five-Factor Personality Inventory for Children (FFPC)].

The purpose of this study is to construct and standardize the Five Factor Personality Inventory for Children (FFPC). Fifty items were chosen through two preliminary surveys. Then the 50-item questionnaire was administered to 3,386 primary school children. Based on the results of principal component analysis, 40 items were selected for the FFPC final version. The mean scores, standard deviations, and score distributions of the five-factor scales were computed. One sample of 215 children was tested twice, the interval being 9 weeks. The test-retest correlations ranged between .778 and .854. Scale scores correlated with teacher nominations of the various kinds of personality. The FFPC five-factor scales were shown to have high internal consistency and test-retest reliability. The factorial validity, construct validity, and concurrent validity were also confirmed.

[Development of the Japanese version of the Buss-Perry Aggression Questionnaire (BAQ)].

The Aggression Questionnaire (Buss & Perry, 1992) has been used to investigate links between personality factors and health outcomes. We developed the Japanese version of the Buss-Perry Aggression Questionnaire (BAQ) and assessed validity and reliability of the scale. Study I (N = 1,125 college students) used a 45-item rating questionnaire measuring each of four components of aggression: Anger, Hostility, Physical Aggression, and Verbal Aggression. Four aggression subscales emerged clearly from exploratory factor analysis. Study II (N = 611 college students) used a 24-item questionnaire and replicated factor structure and factor loadings of Study I. The scales were shown to be highly internally consistent, and stable at appropriate levels over 4-month time period. Normative data, factorial validity, and external evidence of construct, convergent, and discriminant validity for the scales were also presented.

Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with geldanamycin.

The molecular chaperone Hsp90 plays an essential role in the folding and function of important cellular proteins including steroid hormone receptors, protein kinases and proteins controlling the cell cycle and apoptosis. A 15 A deep pocket region in the N-terminal domain of Hsp90 serves as an ATP/ADP-binding site and has also been shown to bind geldanamycin, the only specific inhibitor of Hsp90 function described to date. We now show that radicicol, a macrocyclic antifungal structurally unrelated to geldanamycin, also specifically binds to Hsp90. Moreover, radicicol competes with geldanamycin for binding to the N-terminal domain of the chaperone, expressed either by in vitro translation or as a purified protein, suggesting that radicicol shares the geldanamycin binding site. Radicicol, as does geldanamycin, also inhibits the binding of the accessory protein p23 to Hsp90, and interferes with assembly of the mature progesterone receptor complex. Radicicol does not deplete cells of Hsp90, but rather increases synthesis as well as the steady-state level of this protein, similar to a stress response. Finally, radicicol depletes SKBR3 cells of p185erbB2, Raf-1 and mutant p53, similar to geldanamycin. Radicicol thus represents a structurally unique antibiotic, and the first non-benzoquinone ansamycin, capable of binding to Hsp90 and interfering with its function.

Radicicol leads to selective depletion of Raf kinase and disrupts K-Ras-activated aberrant signaling pathway.

Activation of Ras leads to the constitutive activation of a downstream phosphorylation cascade comprised of Raf-1, mitogen-activated protein kinase (MAPK) kinase, and MAPK. We have developed a yeast-based assay in which the Saccharomyces cerevisiae mating pheromone-induced MAPK pathway relied on co-expression of K-Ras and Raf-1. Radicicol, an antifungal antibiotic, was found to inhibit the K-ras signaling pathway reconstituted in yeast. In K-ras-transformed, rat epithelial, and K-ras-activated, human pancreatic carcinoma cell lines, radicicol inhibited K-Ras-induced hyperphosphorylation of Erk2. In addition, the level of Raf kinase was significantly decreased in radicicol-treated cells, whereas the levels of K-Ras and MAPK remained unchanged. These results suggest that radicicol disrupts the K-Ras-activated signaling pathway by selectively depleting Raf kinase and raises the possibility that pharmacological destabilization of Raf kinase could be a new and powerful approach for the treatment of K-ras-activated human cancers.

[A case of tsutsugamushi disease which occurred in south western Shikoku].

We report a case of tsutsugamushi disease found in south western Shikoku. A 64-year-old male who lived in Towa Village in Kochi, developed a fever and headache on April 6, 1994, and was admitted to Uwajima City Hospital on April 15, with a ten-day history of illness. He had an eschar on the right anterior side of the breast and an enlargement of the right axillary lymph node, without a rash. Laboratory data showed mild liver injury and atypical lymphocytes with 6% in peripheral blood. After his blood was drawn for rickettsial isolation, the minocycline was administered. His symptoms improved rapidly and was discharged in good condition. We successfully isolated the causative agent, Rickettsia tsutugamushi, and designated it as the Shiba strain. High antibody titer against the Kato, Karp and Gilliam strains was detected in serum on admission and increased during the course of the disease. In Shikoku, tsutsugamushi disease is rare and only 13 cases were reported during last ten years. Especially in south western district of Shikoku, there have been no case reported since 1960. This case is important epidemiologically and suggests that we should pay attention to this disease.

Ultrastructural histochemistry of carbonic anhydrase in rat osteoclasts.

Carbonic anhydrase (CA) was localized in osteoclasts of rat tibial metaphysis by light and electron microscopy using the histochemical method of Hansson. Ultrastructurally, positive staining was seen in the cytosol, mitochondrial cristae, Golgi apparatus, some nuclei and lysosomelike vesicles, and on the bone surface beneath the osteoclast. The presence of the enzyme within the Golgi ++apparatus, lysosomelike vesicles, and on the bone surface beneath the osteoclast suggests that some CA may be secreted into the resorbing zone. According to literature, the above localization of CA suggests that there may be three isozymes of CA in the rat osteoclast. These are ubiquitous cytosolic CA (CA-II), mitochondrial CA (CA-V) and secretory CA (CA-VI).

Mutational replacements of conserved amino acid residues in the alpha subunit change the catalytic properties of Escherichia coli F1-ATPase.

Four Escherichia coli mutants with defects in the alpha subunit of H+-ATPase (F0F1) (strain KF154, Pro-281----Leu; KF101 and KF131, Ala-285----Val; KF114, Arg-376----Cys) were isolated, and the kinetic properties of their F1-ATPases were studied. All the mutations so far identified are clustered in the two defined regions of the alpha subunit. With F1 of strain KF114, as with F1 of uncA401 (Ser-373----Phe; T. Noumi, M. Futai, and H. Kanazawa (1984) J. Biol. Chem. 259, 10076-10079), the rate of multisite hydrolysis of ATP was 4 X 10(-3)-fold lower than that with wild-type F1, suggesting that residues Ser-373 and Arg-376 or the regions in their vicinities are essential for positive catalytic cooperativity. With F1 from strain KF101, multisite hydrolysis was higher (about 40% of that of the wild type), but the F1 was unstable and showed defective interaction with the membrane sector (F0). The F1 from KF154 had lower multisite hydrolysis (about 10% of that of the wild type) but could support slow growth by oxidative phosphorylation.