Radiotherapy modulates expression of EGFR, ERCC1 and p53 in cervical cancer

Cervical cancer is a public health problem and the molecular mechanisms underlying radioresistance are still poorly understood. Here, we evaluated the modulation of key molecules involved in cell proliferation, cell cycle and DNA repair in cervical cancer cell lines (CASKI and C33A) and in malignant tissues biopsied from 10 patients before and after radiotherapy. The expression patterns of epidermal growth factor receptor (EGFR), excision repair cross-complementation group 1 (ERCC1) and p53 were evaluated in cancer cell lines by quantitative PCR and western blotting, and in human malignant tissues by immunohistochemistry. The mutation status of TP53 gene was evaluated by direct sequencing. Among cell lines, absent or weak modulations of EGFR, ERCC1 and p53 were observed after exposure to 1.8 Gy. Conversely, increased expressions of p53 (5/10 patients; P=0.0239), ERCC1 (5/10 patients; P=0.0294) and EGFR (4/10 patients; P=0.1773) were observed in malignant tissues after radiotherapy with the same radiation dose. TP53 mutations were found only in one patient. Here we show that a single dose of radiotherapy induced EGFR, ERCC1 and p53 expression in malignant tissues from cervical cancer patients but not in cancer cell lines, highlighting the gap between in vitro and in vivo experimental models. Studies on larger patient cohorts are needed to allow an interpretation that an upregulation of p53, EGFR and ERCC1 may be part of a radioresistance mechanism.

Radiotherapy modulates expression of EGFR, ERCC1 and p53 in cervical cancer.

Cervical cancer is a public health problem and the molecular mechanisms underlying radioresistance are still poorly understood. Here, we evaluated the modulation of key molecules involved in cell proliferation, cell cycle and DNA repair in cervical cancer cell lines (CASKI and C33A) and in malignant tissues biopsied from 10 patients before and after radiotherapy. The expression patterns of epidermal growth factor receptor (EGFR), excision repair cross-complementation group 1 (ERCC1) and p53 were evaluated in cancer cell lines by quantitative PCR and western blotting, and in human malignant tissues by immunohistochemistry. The mutation status of TP53 gene was evaluated by direct sequencing. Among cell lines, absent or weak modulations of EGFR, ERCC1 and p53 were observed after exposure to 1.8 Gy. Conversely, increased expressions of p53 (5/10 patients; P=0.0239), ERCC1 (5/10 patients; P=0.0294) and EGFR (4/10 patients; P=0.1773) were observed in malignant tissues after radiotherapy with the same radiation dose. TP53 mutations were found only in one patient. Here we show that a single dose of radiotherapy induced EGFR, ERCC1 and p53 expression in malignant tissues from cervical cancer patients but not in cancer cell lines, highlighting the gap between in vitro and in vivo experimental models. Studies on larger patient cohorts are needed to allow an interpretation that an upregulation of p53, EGFR and ERCC1 may be part of a radioresistance mechanism.

Surface determination through atomically resolved secondary-electron imaging.

Unique determination of the atomic structure of technologically relevant surfaces is often limited by both a need for homogeneous crystals and ambiguity of registration between the surface and bulk. Atomically resolved secondary-electron imaging is extremely sensitive to this registration and is compatible with faceted nanomaterials, but has not been previously utilized for surface structure determination. Here we report a detailed experimental atomic-resolution secondary-electron microscopy analysis of the c(6 × 2) reconstruction on strontium titanate (001) coupled with careful simulation of secondary-electron images, density functional theory calculations and surface monolayer-sensitive aberration-corrected plan-view high-resolution transmission electron microscopy. Our work reveals several unexpected findings, including an amended registry of the surface on the bulk and strontium atoms with unusual seven-fold coordination within a typically high surface coverage of square pyramidal TiO5 units. Dielectric screening is found to play a critical role in attenuating secondary-electron generation processes from valence orbitals.

A case of pancreatic ductal adenocarcinoma with marked infiltration with IgG4-positive cells.

A 75-year-old man was diagnosed as having pancreatic ductal carcinoma containing remarkable lymphocytic and plasma cell infiltration, as revealed by the cytological examination of endoscopic ultrasound guided fine-needle aspiration (EUS-FNA) specimen. The EUS-FNA specimen showed small amounts of atypical epithelium with noticeable lymphocytes and plasma cells. A pancreatic resection was performed, and the histopathological features showed an invasive pancreatic ductal carcinoma with autoimmune pancreatitis (AIP) lymphoplasmacytic sclerosing pancreatitis (LPSP)-like lesions. Most of the plasma cells were immunoreactive to anti-IgG4 antibody. EUS-FNA may be necessary for the differential diagnosis of AIP and pancreatic cancer, and close attention should be given to the presence of marked lymphoplasmacytic cells in EUS-FNA specimens while making the diagnosis.

Imaging and elemental mapping of biological specimens with a dual-EDS dedicated scanning transmission electron microscope.

A dedicated analytical scanning transmission electron microscope (STEM) with dual energy dispersive spectroscopy (EDS) detectors has been designed for complementary high performance imaging as well as high sensitivity elemental analysis and mapping of biological structures. The performance of this new design, based on a Hitachi HD-2300A model, was evaluated using a variety of biological specimens. With three imaging detectors, both the surface and internal structure of cells can be examined simultaneously. The whole-cell elemental mapping, especially of heavier metal species that have low cross-section for electron energy loss spectroscopy (EELS), can be faithfully obtained. Optimization of STEM imaging conditions is applied to thick sections as well as thin sections of biological cells under low-dose conditions at room and cryogenic temperatures. Such multimodal capabilities applied to soft/biological structures usher a new era for analytical studies in biological systems.

Risk factors to cause tooth formation anomalies in chemotherapy of paediatric cancers.

This study aimed to investigate the risk factors of tooth formation anomalies in anti-cancer chemotherapies. Long-term survivors treated by conventional chemotherapy (n = 26), conventional chemotherapy with high-dose chemotherapy (HDC) (n = 14), and HDC with total body irradiation (TBI) (n = 6) were analysed for the incidence of tooth agenesis, microdonts, and short-rooted teeth. The tooth agenesis and/or microdonts were found in second premolars and second molars, but not in first molars or central incisors. The ratio of subjects with tooth agenesis and/or microdonts was 66.7% and 18.2% in subjects administered conventional chemotherapy at <4 years and ≥ 4 years of age, respectively, while it was 100% and 25% in subjects administered HDC at <4 years and ≥ 4 years of age. The incidence of tooth formation anomalies did not related with the duration of conventional chemotherapy but increased by HDC. The incidence of tooth formation anomalies did not show significantly differences between the HDC with and without TBI groups, and was higher in busulfan-administered subjects than in subjects given cyclophosphamide. It may be concluded that the high-risk group of tooth agenesis is the subjects with HDC under 4 years of age. However, protocols of conventional chemotherapy are not an important risk factor to cause the tooth formation anomalies.

Atomic imaging using secondary electrons in a scanning transmission electron microscope: experimental observations and possible mechanisms.

We report detailed investigation of high-resolution imaging using secondary electrons (SE) with a sub-nanometer probe in an aberration-corrected transmission electron microscope, Hitachi HD2700C. This instrument also allows us to acquire the corresponding annular dark-field (ADF) images both simultaneously and separately. We demonstrate that atomic SE imaging is achievable for a wide range of elements, from uranium to carbon. Using the ADF images as a reference, we studied the SE image intensity and contrast as functions of applied bias, atomic number, crystal tilt, and thickness to shed light on the origin of the unexpected ultrahigh resolution in SE imaging. We have also demonstrated that the SE signal is sensitive to the terminating species at a crystal surface. A possible mechanism for atomic-scale SE imaging is proposed. The ability to image both the surface and bulk of a sample at atomic-scale is unprecedented, and can have important applications in the field of electron microscopy and materials characterization.

Endocannabinoids contribute to metabotropic glutamate receptor-mediated inhibition of GABA release onto hippocampal CA3 pyramidal neurons in an isolated neuron/bouton preparation.

Retrograde synaptic signaling by endogenous cannabinoids (endocannabinoids) is a recently discovered form of neuromodulation in various brain regions. In hippocampus, it is well known that endocannabinoids suppress presynaptic inhibitory neurotransmitter release in CA1 region. However, endocannabinoid signaling in CA3 region remains to be examined. Here we investigated whether presynaptic inhibition can be caused by activation of postsynaptic group I metabotropic glutamate receptors (mGluRs) and following presynaptic cannabinoid receptor type 1 (CB1 receptor) using mechanically dissociated rat hippocampal CA3 pyramidal neurons with adherent functional synaptic boutons. Application of group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) reversibly suppressed spontaneous inhibitory postsynaptic currents (IPSCs). In the presence of tetrodotoxin (TTX), frequency of miniature IPSCs was significantly reduced by DHPG, while there were no significant changes in minimum quantal size and sensitivity of postsynaptic GABA(A) receptors to the GABA(A) receptor agonist muscimol, indicating that this suppression was caused by a decrease in GABA release from presynaptic nerve terminals. Application of CB1 synthetic agonist WIN55212-2 (mesylate(R)-(+)-[2,3-dihydro-5-methyl-3-[4-morpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone) or endocannabinoid 2-arachidonoylglycerol also suppressed the spontaneous IPSC. The inhibitory effect of DHPG on spontaneous IPSCs was abolished by SR-141716 (5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide), a CB1 receptor antagonist. Furthermore, postsynaptic application of GDP-betaS blocked the DHPG-induced inhibition of spontaneous IPSCs, indicating the involvement of endcannabinoid-mediated retrograde synaptic signaling. These results provide solid evidence for retrograde signaling from postsynaptic group I mGluRs to presynaptic CB1 receptors, which induces presynaptic inhibition of GABA release in rat hippocampal CA3 region.

Imaging single atoms using secondary electrons with an aberration-corrected electron microscope.

Aberration correction has embarked on a new frontier in electron microscopy by overcoming the limitations of conventional round lenses, providing sub-angstrom-sized probes. However, improvement of spatial resolution using aberration correction so far has been limited to the use of transmitted electrons both in scanning and stationary mode, with an improvement of 20-40% (refs 3-8). In contrast, advances in the spatial resolution of scanning electron microscopes (SEMs), which are by far the most widely used instrument for surface imaging at the micrometre-nanometre scale, have been stagnant, despite several recent efforts. Here, we report a new SEM, with aberration correction, able to image single atoms by detecting electrons emerging from its surface as a result of interaction with the small probe. The spatial resolution achieved represents a fourfold improvement over the best-reported resolution in any SEM (refs 10-12). Furthermore, we can simultaneously probe the sample through its entire thickness with transmitted electrons. This ability is significant because it permits the selective visualization of bulk atoms and surface ones, beyond a traditional two-dimensional projection in transmission electron microscopy. It has the potential to revolutionize the field of microscopy and imaging, thereby opening the door to a wide range of applications, especially when combined with simultaneous nanoprobe spectroscopy.

Inhibition of the transient receptor potential cation channel TRPM2 by 2-aminoethoxydiphenyl borate (2-APB).

BACKGROUND AND PURPOSE: Transient receptor potential melastatin 2 (TRPM2) is a non-selective Ca(2+)-permeable cation channel and is known to be activated by adenosine 5'-diphosphoribose (ADP-ribose) and hydrogen peroxide. TRPM2 current responses are reported to be drastically potentiated by the combination of each of these ligands with heat. Furthermore, the combination of cyclic ADP-ribose with heat also activates TRPM2. Although flufenamic acid, antifungal agents (miconazole and clotrimazole), and a phospholipase A(2) inhibitor (N-(p-amylcinnamoyl)anthranilic acid) inhibit TRPM2, their inhibition was either gradual or irreversible. EXPERIMENTAL APPROACH: To facilitate future research on TRPM2, we screened several compounds to investigate their potential to activate or inhibit the TRPM2 channels using the patch-clamp technique in HEK293 cells, transfected with human TRPM2. KEY RESULTS: 2-aminoethoxydiphenyl borate (2-APB) exhibited a rapid and reversible inhibition of TRPM2 channels that had been activated by its ADP-ribose or cADP-ribose and heat in a dose-dependent manner (IC(50) about 1 microM). 2-APB also inhibited heat-evoked insulin release from pancreatic islets, isolated from rats. CONCLUSIONS AND IMPLICATIONS: 2-APB proved to be a powerful and effective tool for studying the function of TRPM2.

Proteomic analysis of stage I primary lung adenocarcinoma aimed at individualisation of postoperative therapy.

Although postoperative adjuvant chemotherapy (PAC) with uracil-tegafur significantly improves the prognosis of patients with stage I lung adenocarcinoma, subset analysis has revealed that only 11.5% of patients with stage IB derive actual benefit from such therapy. Therefore, it is extremely important to identify patients for whom adjuvant chemotherapy will be beneficial. We performed comprehensive protein analysis of 24 surgically resected specimens of stage I adenocarcinoma using liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by bioinformatical investigations to identify protein molecules. Furthermore, we carried out immunohistochemical studies of 90 adenocarcinoma specimens to validate the results of LC-MS/MS. We detected two kinds of protein molecules (myosin IIA and vimentin) by LC-MS/MS. We confirmed their immunohistochemical expression and distribution, and evaluated the relationship between the expression of these proteins and prognosis after adjuvant chemotherapy. Patients with no expression of either myosin IIA or vimentin showed a significantly better outcome regardless of PAC using uracil-tegafur. However, we were unable to select responders to uracil-tegafur using these proteins. Cases of adenocarcinoma lacking expression of either myosin IIA or vimentin show a good outcome without PAC, and therefore do not require such treatment.

Hypercalcemia in childhood acute lymphoblastic leukemia: frequent implication of parathyroid hormone-related peptide and E2A-HLF from translocation 17;19.

Hypercalcemia is relatively rare but clinically important complication in childhood leukemic patients. To clarify the clinical characteristics, mechanisms of hypercalcemia, response to management for hypercalcemia, incidence of t(17;19) and final outcome of childhood acute lymphoblastic leukemia (ALL) accompanied by hypercalcemia, clinical data of 22 cases of childhood ALL accompanied by hypercalcemia (>12 mg/dl) reported in Japan from 1990 to 2005 were retrospectively analyzed. Eleven patients were 10 years and older. Twenty patients had low white blood cell count (<20 x 10(9)/l), 15 showed hemoglobin> or =8 g/dl and 14 showed platelet count > or =100 x 10(9)/l. Parathyroid hormone-related peptide (PTHrP)-mediated hypercalcemia was confirmed in 11 of the 16 patients in whom elevated-serum level or positive immunohistochemistry of PTHrP was observed. Hypercalcemia and accompanying renal insufficiency resolved quickly, particularly in patients treated with bisphosphonate. t(17;19) or add(19)(p13) was detected in five patients among 17 patients in whom karyotypic data were available, and the presence of E2A-HLF was confirmed in these five patients. All five patients with t(17;19)-ALL relapsed very early. Excluding the t(17;19)-ALL patients, the final outcome of ALL accompanied by hypercalcemia was similar to that of all childhood ALL patients, indicating that the development of hypercalcemia itself is not a poor prognostic factor.

Deficiency of tenascin-C attenuates liver fibrosis in immune-mediated chronic hepatitis in mice.

Tenascin-C (TNC), an extracellular matrix glycoprotein, is upregulated in chronic liver disease. Here, we investigated the contribution of TNC to liver fibrogenesis by comparing immune-mediated hepatitis in wild-type (WT) and TNC-null (TNKO) mice. Eight-week-old BALB/c mice received weekly intravenous injections of concanavalin A to induce hepatitis, and were sacrificed one week after the 3rd, 6th, 9th, and 12th injections. In WT livers, immunohistochemical staining revealed a gradual increase in TNC deposition. TNC mRNA levels also increased sequentially and peaked after the 9th injection. Collagen deposition, stained with picrosirius red, was significantly less intense in TNKO mice than in WT mice, and procollagen I and III transcripts were significantly upregulated in WT mice compared with TNKO mice. Inflammatory infiltrates were most prominent after the 3rd-6th injections in both groups and were less intense in TNKO mice than in WT mice. Interferon-gamma, tumour necrosis factor-alpha, and interleukin-4 mRNA levels were significantly higher in WT mice than in TNKO mice, while activated hepatic stellate cells (HSCs) and myofibroblasts, a cellular source of TNC and procollagens, were more common in WT livers. Transforming growth factor (TGF)-beta1 mRNA expression was significantly upregulated in WT mice, but not in TNKO mice. In conclusion, TNC can promote liver fibrogenesis through enhancement of inflammatory response with cytokine upregulation, HSC recruitment, and TGF-beta expression during progression of hepatitis to fibrosis.

Delirium during intravenous sedation with midazolam alone and with propofol in dental treatment.

A 62-year-old man visited our clinic for dental implantation under intravenous sedation. He demonstrated increased psychomotor activity and incomprehensible verbal contact during intravenous sedation. Although delirium caused by midazolam or propofol in different patients has been reported, the present case represents a delirium that developed from both drugs in the same patient, possibly because of the patient's smaller tolerance to midazolam and propofol.

Effect of ZnO addition on bioactive CaO-SiO2-P2O5-CaF2 glass-ceramics containing apatite and wollastonite.

Some ceramics show bone-bonding ability, i.e. bioactivity. Apatite formation on ceramics is an essential condition to bring about direct bonding to living bone when implanted into bony defects. A controlled surface reaction of the ceramic is an important factor governing the bioactivity and biodegradation of the implanted ceramic. Among bioactive ceramics, glass-ceramic A-W containing apatite and wollastonite shows high bioactivity, as well as high mechanical strength. In this study, glass-ceramics containing zinc oxide were prepared by modification of the composition of the glass-ceramic A-W. Zinc oxide was selected to control the reactivity of the glass-ceramics since zinc is a trace element that shows stimulatory effects on bone formation. Glass-ceramics were prepared by heat treatment of glasses with the general composition: xZnOx(57.0-x)CaOx35.4SiO(2)x7.2P(2)O(5)x0.4CaF(2) (where x=0-14.2mol.%). Addition of ZnO increased the chemical durability of the glass-ceramics, resulting in a decrease in the rate of apatite formation in a simulated body fluid. On the other hand, the release of zinc from the glass-ceramics increased with increasing ZnO content. Addition of ZnO may provide bioactive CaO-SiO(2)-P(2)O(5)-CaF(2) glass-ceramics with the capacity for appropriate biodegradation, as well as enhancement of bone formation.

Therapy-related myelodysplastic syndrome in childhood: a retrospective study of 36 patients in Japan.

We report here a retrospective analysis of 36 children with therapy-related myelodysplastic syndrome (t-MDS) diagnosed between 1990 and 1999 in Japan. Their median age was 7.7 years and the median latency period for the development of t-MDS was 38.5 months. The primary tumors were hematologic in 15 of the cases and nonhematologic in 21. Chromosomal abnormalities were detected in 32/34(94%) patients: abnormalities of chromosomes 5and/or 7 in 41% and notably, 11q23 abnormalities in 31%. The prognosis of children with t-MDS was very poor as compared to children with primary MDS (5 year survival: 16% versus 54%, p<0.0001).

Celiac plexus block: evaluation of injectate spread by three-dimensional computed tomography.

We describe a case of pancreatic cancer in which the spread pattern of injectate during neurolytic celiac plexus block was evaluated by three-dimensional helical computed tomography. Three-dimensional images provide excellent visualization of the spread patterns of injectate in a target site, which appears to provide patients with effective relief from intractable pain.

Positive association of CYP11B2 gene polymorphism with genetic predisposition to essential hypertension.

Predispositions to essential hypertension and cardiovascular diseases are possibly associated with gene polymorphisms of the renin-angiotensin system. Gene polymorphisms of angiotensinogen and angiotensin-converting enzyme genes have been suggested to be risk factors for hypertension and myocardial infarction. Concerning the polymorphism of aldosterone synthase (CYP11B2) gene, earlier studies have shown inconsistent results in terms of its relation to hypertension. In the present case-control study, we investigated the association of -344T/C polymorphism in the promoter region of human CYP11B2 gene with genetic predisposition to hypertension. The genotype of -344T/C polymorphism was determined in essential hypertension subjects (n=250) and normotensive subjects (n=221). The distributions of three genotypes (TT, TC, and CC) were significantly different between the hypertensive and the normotensive groups (chi(2)=9.61, P=0.008). Namely, the frequency of C allele was higher in the hypertensive patients than in the normotensive subjects (34.2 vs 26.5%, P=0.010). Our data suggest that the -344C allele of CYP11B2 gene polymorphism is associated with the genetic predisposition to develop essential hypertension.

Clinical implication of multiple risk factor control in the management of diabetic macrovasucular complications.

Using cross-sectional and prospective analyses, the risk factors for macroangiopathy (MA) in nonobese Type 2 diabetic patients were evaluated. In the cross-sectional study, we determined a cutoff point for each variable at which changes in the prevalence of total MA reached statistically significant levels. In the prospective study, those who met more than four out of seven control criteria as set forth in the Multiclinical Study for Diabetic Macroangiopathy (MSDM) had less risk of MA in Type 2 diabetes initially diagnosed without MA compared with those who fulfilled less than three factors. These results suggest that multiple risk factor control is the most effective and reasonable way to lower the incidence of MA in Type 2 diabetes.

Comparison of the effects of amlodipine and losartan on 24-hour ambulatory blood pressure in hypertensive patients.

Effects of amlodipine (AML), a long-acting calcium antagonist, and losartan (LOS), an angiotensin II receptor antagonist, on 24-hr blood pressure profile were compared in 15 patients with essential hypertension. After 4 weeks of placebo period, the patients were treated with AML or LOS in a random crossover design for 12-16 weeks each. Either drug was given once daily at 0800 and the doses were titrated so that the office blood pressure was reduced lower than 140/90mmHg. At the end of each period, 24-hr blood pressure was monitored. Average office blood pressure was lowered from 158 +/- 2/ 98 +/- 2 mmHg to 134 +/- 1/87 +/- 1 mmHg by AML and 134 +/- 2/88 +/- 1 mmHg by LOS. Average 24-hr blood pressure was also reduced from 144 +/- 3/ 92 +/- 2 mmHg to 131 +/- 2/84 +/- 2 mmHg by AML and 135 +/- 3/85 +/- 2 mmHg by LOS. The averaged 24-hr systolic blood pressure was significantly lower in AML than in LOS (p < 0.05). Then, the 24-hr blood pressure was analyzed for four segments; morning (0530-0900 h), daytime (0930-1800 h), evening (1830-2300 h) and night (2330-0500 h). Although the daytime blood pressure was comparable between AML and LOS, systolic blood pressure in the evening and morning hours were lower in AML than in LOS (133 +/- 2 vs. 138 +/- 3mmHg,p<0.01; 129 +/- 3 vs. 134 +/- 4,p<0.05). Troughtopeakratio of antihypertensive effect on systolic blood pressure was significantly greater in AML than in LOS (62 +/- 5% vs. 55 +/- 4%, p < 0.05). Either drug did not cause reflective increase in pulse rate over 24 hours. These results suggest that both AML and LOS are equally effective in lowering daytime blood pressure without eliciting reflex tachycardia, however, the antihypertensive effect of AML lasts longer than that of LOS. Such information seems important to achieve 24-hr blood pressure control using these drugs.