Dysregulated cyclin D1 expression early in head and neck tumorigenesis: in vivo evidence for an association with subsequent gene amplification

Cyclin D1 proto-oncogene is a key regulator of the mammalian cell-cycle acting at the restriction point in late G1. Amplification of the cyclin D1 locus, located on chromosome 11q13, as well as cyclin D1 protein overexpression have been reported in several human malignancies. The purpose of this study was to evaluate cyclin D1 gene copy status and protein expression during the multistep process of head and neck tumorigenesis, using a combination of fluorescence in situ hybridization and immunohistochemistry techniques. From 29 selected patients presenting with head and neck squamous carcinoma and whose tumor cytospins had been previously screened for presence (16 cases) or absence (13 cases) of amplification at the 11q13 band, we analysed 46 paraffin-embedded tissue specimens that demonstrated, besides the primary tumor, the presence of contiguous adjacent normal tissue and/or premalignant lesions. Of the 16 amplified cases, nine demonstrated a continuous progression from premalignant to invasive carcinoma and seven (77.7%) of these cases showed cyclin D1 gene amplification in premalignant lesions prior to development of invasive carcinoma. Increased cyclin D1 protein expression was observed in all 16 amplified tumors and five of the 13 (38.4%) non-amplified tumors. Interestingly, dysregulated cyclin D1 expression was also found in the premalignant lesions adjacent to all 16 amplified tumors, and it appeared to precede cyclin D1 gene amplification. In contrast no dysregulated expression was detected in the premalignant lesions of the non-amplified tumors. In conclusion, these findings provide strong evidence for early dysregulation of cyclin D1 expression during the tumorigenesis process and suggest that dysregulated increased expression precedes and possibly enables gene amplification.     

Effect of mild acid treatment on the survival, enteropathogenicity, and protein production in vibrio parahaemolyticus

Vibrio parahaemolyticus is an important food-borne enteropathogen that encounters various adverse conditions in its native environment or during infection. Effects of mild acid treatment on survival under stress conditions, enteropathogenicity, and protein production in this pathogen were investigated. Logarithmically grown cells, at pH 7.5 shifted to pH 5.0 for 30 min, were more resistant to subsequent acid challenge at pH 4.4. A two-phase adaptive procedure (pH 5.8 for 30 min; pH 5.0 for 30 min) was better than a single-phase procedure for enhancing the acid tolerance of this pathogen. The acid-adapted cells were cross-protected against the challenges of low salinity and thermal inactivation. One-dimensional polyacrylamide gel electrophoresis revealed that proteins with molecular masses of 6.4, 9.0, 13.6, 16.3, 18.9, 22.9, 24.4, 28.3, 33. 9, 36.9, 41.2, 47.6, 58.1, 65.6, 80.5, 88.2, and 96.9 kDa were induced or significantly enhanced, while proteins of 25.3, 30.1, 30. 7, and 91.7 kDa were significantly inhibited. Two-dimensional polyacrylamide gel electrophoresis revealed that 20 species of proteins were induced or significantly enhanced, while 26 species were inhibited. In assays conducted using the suckling mouse model, enteropathogenicity of the acid-adapted cells was significantly enhanced in terms of intestine/body weight ratio and in vivo recovery of infected cells.     

Mechanisms involved in the intrinsic isoniazid resistance of Mycobacterium avium

Isoniazid (INH), which acts by inhibiting mycolic acid biosynthesis, is very potent against the tuberculous mycobacteria. It is about 100-fold less effective against Mycobacterium avium. This difference has often been attributed to a decreased permeability of the cell wall. We measured the rate of conversion of radiolabelled INH to 4-pyridylmethanol by whole cells and cell-free extracts and estimated the permeability barrier imposed by the cell wall to INH influx in Mycobacterium tuberculosis and M. avium. There was no significant difference in the relative permeability to INH between these two species. However, the total conversion rate in M. tuberculosis was found to be four times greater. Examination of in vitro-generated mutants revealed that the major resistance mechanism for both species is loss of the catalase-peroxidase KatG. Analysis of lipid and protein biosynthetic profiles demonstrated that the molecular target of activated INH was identical for both species. M. avium, however, formed colonies at INH concentrations inhibitory for mycolic acid biosynthesis. These mycolate-deficient M. avium exhibited altered colony morphologies, modified cell wall ultrastructure and were 10-fold more sensitive to treatment with hydrophobic antibiotics, such as rifampin. These findings may significantly impact the design of new therapeutic regimens for the treatment of infections with atypical mycobacteria.     

Perioperative specific management of blood volume loss in craniosynostosis surgery

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.     

Age-related changes in gastric mucosal repair and proliferative activities in rats exposed acutely to aspirin

OBJECTIVES: (1) To determine whether and how outcome measurements in the ECASS trial are influenced by a shorter time window (0-3 vs. 3-6 h) between onset of symptoms and start of thrombolytic therapy using recombinant tissue plasminogen activator (rt-PA) in acute ischemic stroke. (2) To discuss the results of the ECASS 0- to 3-hour cohort with the results of the National Institute of Neurological Disorders and Stroke Study (NINDSS). DESIGN AND ANALYSIS: Analysis of the 0- to 3-hour and the 3- to 6-hour cohort in accordance with the ECASS protocol. Comparative analysis of the ECASS and NINDSS results following the NINDSS protocol using dichotomized endpoints. MAIN OUTCOME MEASURES: Primary endpoints: modified Rankin Scale, Barthel Index; secondary endpoints: combined Barthel/Rankin, long-term Scandinavian Stroke Scale, National Institutes of Health Stroke Scale, mortality at 30 and 90 days, occurrence of intracranial hemorrhage. NINDS trial endpoint: favorable outcome as defined in the NINDS trial. RESULTS: In ECASS, 87 patients were randomized within 3 h of stroke onset. Differences in favor of rt-PA treatment can be found for all primary and secondary outcome measures in the ECASS 0- to 3-hour cohort, except for mortality at day 30, which is somewhat higher in the rt-pA-treated group. However, due to the small sample size, the differences do not reach statistical significance. Early infarct signs (as defined by the ECASS protocol) are found as early as 2 h after stroke onset. Parenchymal hemorrhages are found significantly more often among rt-PA-treated patients. The results in the ECASS 0- to 3-hour cohort fit well with the results in NINDSS. CONCLUSION: Data from the 3-hour ECASS cohort support the efficacy of early thrombolytic therapy in acute hemispheric stroke patients. Comparing bleeding complications between the ECASS and NINDSS is difficult because of differences in the definition and occurrence of hemorrhagic events.     

Defect formation in epitaxial oxide dielectric layers due to substrate surface relief

Defects were characterized in epitaxial (001) CeO₂ films deposited and planarizedin situ on patterned (001) LaAlO₃ substrates by ion beam assisted deposition (IBAD). A hill and valley structure with steps running parallel to the [100] LaAlO₃ axis was produced on the surface of the substrate by photolithography and ion beam etching prior to film deposition. A conformai epitaxial CeO₂ layer of ∼ 100 nm thickness was deposited on the heated substrate by e-beam evaporation. Lattice-matching between the e-beam film and the substrate was of the type: (001) CeO₂∥(001) LaAlO₃ and [110] CeO₂∥[100] LaAlO₃. Evaporative deposition of additional film onto the conformai layer was accompanied by bombardment with a 500 eV argon/oxygen ion beam to promotein situ planarization. Extreme lattice misfit for the orientation (001) CeO₂∥(001)LaAlO₃ and [001] CeO₂∥[001] LaAlO₃ caused formation of dislocations in the e-beam CeO₂ film in the vicinity of individual ledges in the substrate surface. Coherence of the CeO₂ film was locally lost in the step regions of the hill and valley structure. The large patterned steps, which are composed of numerous adjacent ledges in the LaAlO₃ surface, caused nucleation of CeO₂ with a tilt misalignment of up to ∼5‡ about the substrate [100]. Nucleation and growth of nonepitaxial CeO₂ crystallites was observed along the step regions of the film during the IBAD portion of deposition. Defect formation in the e-beam ceria layer due to substrate surface relief indicates that “lattice engineering≓ of multilayer epitaxial structures may not be possible when nonplanar surfaces are created during device fabrication. The IBAD CeO₂ layer was more defective than the conformai layer deposited without the impinging ion beam, even in the portions of the film where epitaxy was maintained throughout both layers.     

Inhibition of NFkappaB activity through maintenance of IkappaBalpha levels contributes to dihydrotestosterone-mediated repression of the interleukin-6 promoter

Androgens repress expression of many genes, yet the mechanism of this activity has remained elusive. The cytokine, interleukin-6, is active in a variety of biological systems, and its expression is repressed by androgens. Accordingly we dissected the mechanism of androgen's ability to inhibit interleukin-6 expression at the molecular level. In a series of co-transfection assays, we found that 5alpha-dihydrotestosterone, through the androgen receptor, repressed activation of the interleukin-6 promoter, in part, by inhibiting NFkappaB activity. It did not appear that 5alpha-dihydrotestosterone inhibited NFkappaB by activating the androgen receptor to compete for the NFkappaB response element as we could not detect androgen receptor binding to the IL-6 promoter by DNase I footprinting assay. However, by electrophoretic mobility shift assay we found that 5alpha-dihydrotestosterone repressed formation of NFkappaB middle dotNFkappaB response element complex formation. In LNCaP prostate carcinoma cells, 5alpha-dihydrotestosterone achieved this effect through maintenance of IkappaBalpha protein levels in the face of phorbol ester, a stimulus that results in IkappaBalpha degradation. Finally, we confirmed that IkappaBalpha inhibits NFkappaB-mediated activation of the interleukin-6 promoter. These data suggest that maintenance of IkappaBalpha levels may represent the first identified mechanism for androgen-mediated repression of a natural androgen-regulated gene.     

Vertical bridgman techniques to homogenize zinc composition of CdZnTe substrates

In order to improve the Zn homogeneity along the axial direction of CdZnTe boule, we have employed a modified Bridgman technique using a (Cd, Zn) alloy source in communication with the melt, whose temperature has been gradually changed from 800 to 840°C during growth. Electron probe microanalysis (EPMA) measurements of Zn composition in the boule shows an excellent homogeneity of Zn along the axis of the CdZnTe boule compared with results in a boule grown by using a fixed source temperature. We have performed a numerical simulation to obtain the approximate temperatures of additional heating and cooling needed to improve the radial Zn homogeneity. CdZnTe boule has been grown by seeded vertical Bridgman furnace with two zones of heater and cooler. Ultraviolet/visible spectroscopic measurements of Zn composition over the length of the boule indicate that the radial distribution of Zn composition is very homogeneous in the body region of the boule, where the radial variation of Zn composition is ±0.0005.     

One-stage sternal stenting with homograft bone after cardiac operation in pediatric patients

Low cardiac output after open heart operations in neonates and infants carries a high mortality. Delayed sternal closure may be life-saving but may prolong hospital stay and increase costs. To circumvent these issues, we shaped homograft bone and interposed it between the sternal edges to allow primary wound closure in 2 pediatric patients. Midterm results are satisfactory.