Substitution F659G in the Irr1p/Scc3p cohesin influences the cell wall of Saccharomyces cerevisiae

The sister chromatid cohesion complex of Saccharomyces cerevisiae is composed of proteins termed cohesins. The complex forms a ring structure that entraps sister DNAs, probably following replication. The mechanism of cohesion is universal and the proteins participating in this process are evolutionarily highly conserved. We investigated the Irr1p/Scc3p cohesin subunit, an under-studied protein. We show that the presence of a mutated copy of IRR1 gene, encoding the F658G substitution in Irr1p, changes the sensitivity of the heterozygous irr1-1/IRR1 diploid to cell wall-affecting compounds. Microscopic images indicate that chitin distribution in the mutant cell wall is affected, although the biochemical composition of the cell wall is not drastically changed. This observation suggests that irr1-1 mutation in heterozygous state may influence the cell wall integrity and indicates a possible link between mechanisms regulating the cell wall biosynthesis, nuclear migration and chromosome segregation.     

Radiation response and cell cycle regulation of p53 rescued malignant keratinocytes

Mutations in the tumor suppressor gene p53 were found in more than 90% of all human squamous cell carcinomas (SCC). To study the function of p53 in a keratinocyte background, a tetracycline-controlled p53 transgene was introduced into a human SCC cell line (SCC15), lacking endogenous p53. Conditional expression of wild-type p53 protein upon withdrawal of tetracycline was accompanied with increased expression of p21(WAF1/Cip1) resulting in reduced cell proliferation. Flow-cytometric analysis revealed that these cells were transiently arrested in the G1/S phase of the cell cycle. However, when SCC15 cells expressing p53 were exposed to ionizing radiation (IR), a clear shift from a G1/S to a G2/M cell cycle arrest was observed. This effect was greatly depending on the presence of wild-type p53, as it was not observed to the same extent in SCC15 cells lacking p53. Unexpectedly, the p53- and IR-dependent G2/M cell cycle arrest in the keratinocyte background was not depending on increased expression or stabilization of 14-3-3sigma, a p53-regulated effector of G2/M progression in colorectal cancer cells. In keratinocytes, 14-3-3sigma (stratifin) is involved in terminal differentiation and its cell cycle function in this cell type might diverge from the one it fulfills in other cellular backgrounds.     

Inhibition of p38 MAPK Signaling Augments Skin Tumorigenesis via NOX2 Driven ROS Generation

p38 mitogen-activated protein kinases (MAPKs) respond to a wide range of extracellular stimuli. While the inhibition of p38 signaling is implicated in the impaired capacity to repair ultraviolet (UV)-induced DNA damage—a primary risk factor for human skin cancers—its mechanism of action in skin carcinogenesis remains unclear, as both anti-proliferative and survival functions have been previously described. In this study, we utilized cultured keratinocytes, murine tumorigenesis models, and human cutaneous squamous cell carcinoma (SCC) specimens to assess the effect of p38 in this regard. UV irradiation of normal human keratinocytes increased the expression of all four p38 isoforms (α/β/γ/δ); whereas irradiation of p53-deficient A431 keratinocytes derived from a human SCC selectively decreased p38α, without affecting other isoforms. p38α levels are decreased in the majority of human cutaneous SCCs assessed by tissue microarray, suggesting a tumor-suppressive effect of p38α in SCC pathogenesis. Genetic and pharmacological inhibition of p38α and in A431 cells increased cell proliferation, which was in turn associated with increases in NAPDH oxidase (NOX2) activity as well as intracellular reactive oxygen species (ROS). These changes led to enhanced invasiveness of A431 cells as assessed by the matrigel invasion assay. Chronic treatment of p53^-/-/SKH-1 mice with the p38 inhibitor SB203580 accelerated UV-induced SCC carcinogenesis and increased the expression of NOX2. NOX2 knockdown suppressed the augmented growth of A431 xenografts treated with SB203580. These findings indicate that in the absence of p53, p38α deficiency drives SCC growth and progression that is associated with enhanced NOX2 expression and ROS formation.     

The basis of asymmetry in IS2 transposition

In the first step of IS2 transposition, the formation of an IS2 minicircle, the roles of the two IS ends differ. Terminal cleavage initiates exclusively at the right inverted repeat (IRR) - the donor end - whereas IRL is always the target. At the resulting minicircle junction, the two abutted ends are separated by a spacer of 1 or 2 basepairs. In this study, we have identified the determinants of donor and target function. The inability of IRL to act as a donor results largely from two sequence differences between IRL and IRR - an extra basepair between the conserved transposase binding sequences and the end of the element, and a change of the terminal dinucleotide from CA-3' to TA-3'. These two changes also impose a characteristic size on the minicircle junction spacer. The only sequences required for the efficient target function of IRL appear to be contained within the segment from position 11-42. Although IRR can function as a target, its shorter length and additional contacts with transposase (positions 1-7) result in minicircles with longer, and inappropriate, spacers. We propose a model for the synaptic complex in which the terminus of IRL makes different contacts with the transposase for the initial and final strand transfer steps. The sequence differences between IRR and IRL, and the behavioural characteristics of IRL that result from them, have probably been selected because they optimize expression of transposase from the minicircle junction promoter, Pjunc.     

Expression of p53, bcl-2 and Ki-67 in cervical intraepithelial neoplasia and invasive squamous cell carcinoma of the uterine cervix

OBJECTIVE: To assess the expression of p53, bcl-2 and Ki-67 in the progression of cervical neoplasia. STUDY DESIGN: A total of 131 cervical specimens, consisting of normal cervical epithelium (n = 43), cervical intraepithelial neoplasia (CIN) lesions (n =40) and cervical squamous cell carcinomas (SCCs) (n = 48) were examined immunohistochemically in paraffin sections for expression of p53, bcl-2 and Ki-67. RESULTS: Immunoreactivity of p53 was found in 27% of SCC cases, but it had no significant relationship with SCC staging (p = 0.791). Immunoreactivity of bcl-2 was observed in 33% of CIN 3 cases. We found a significant relationship (chi2 test: p = 0.009) between the expression of bcl-2 and CIN grading. Ki-67 index was higher in high grade CIN (HGCIN: CIN 2 and 3) and SCC lesions compared to normal cervices. Ki-67 index showed a correlation with bcl-2 protein expression (p = 0.030), but not with p53 protein expression (p = 0.239). CONCLUSION: HGCIN is an early stage to demonstrate the alteration of bcl-2 and Ki-67 expressions. Progression of neoplasia in the uterine cervix is accompanied by an increase of antiapoptotic protein, bcl-2 as well as cellular proliferation.     

Analysis of p16 expression and allelic imbalance / loss of heterozygosity of 9p21 in cutaneous squamous cell carcinomas

Deletions of the short arm of chromosome 9 have been reported in different types of malignancies. This chromosomal region contains a number of known tumour suppressor genes, including the p16INK4A (CDKN2A), p15INK4B and MTAP tumour suppressor genes located at 9p21. In this study twenty-two paraffin embedded invasive cutaneous SCC were examined for allelic imbalance/ loss of heterozygosity (AI/LOH) of the 9p region (in particular 9p21), and for p16 protein expression. DNA was isolated from microdissected sections of normal and tumour cells and analysed for AI/LOH by using six fluorescently labelled microsatellite markers that map to the 9p region. P16 protein expression was examined by immunohistochemistry. At each of the six microsatellite markers the majority of SCC analysed showed AI/LOH. Overall both AI/LOH within the CDKN2A locus and absence of p16 protein expression were frequent among the cutaneous SCC analysed, suggesting that p16 inactivation may play a role in cutaneous SCC development. The majority of the SCC analysed also had AI/LOH of the marker within the MTAP gene, and at markers flanking the CDKN2A gene; thus further investigation as to a possible role for these genes in the development of cutaneous SCC is warranted.     

Inhibition of NK cell activity through TGF-beta 1 by down-regulation of NKG2D in a murine model of head and neck cancer

In an orthotopic murine model of head and neck squamous cell carcinoma (SCC VII/SF) we studied NK cell-mediated immunity following vaccination with a recombinant vaccinia virus expressing IL-2 (rvv-IL-2). SCC VII/SF tumor cells were injected into the oral cavity of C3H/HeJ mice on day 0. Mice were vaccinated on days 7, 10, and 14 with rvv-IL-2 and control vaccines. Phenotypes, numbers, and biological activities of NK cells were determined following vaccination. Levels of expression of NK-activating receptor NKG2D and CD16 on NK cell surface were assayed in the vaccinated mice. Expression of NKG2D ligands, Rae1, and H60 on SCC VII/SF cells was also examined. Vaccination with rvv-IL-2 resulted in expansion of NK cells. NK cells isolated from rvv-IL-2-vaccinated mice had significantly higher biological activities compared with mice treated with control vaccines. NK cells from tumor-bearing mice expressed significantly lower levels of NKG2D and CD16 compared with rvv-IL-2 vaccinated mice. SCC VII/SF tumors expressed NKG2D ligand Rae 1, although H60 was not present. SCC VII/SF tumors expressed high levels of TGF-beta1, which were down-modulated by vaccination with rvv-IL-2. Incubation of NK cells with tumor homogenate or cultured supernatant of SCC VII/SF cells reduced the expression of NKG2D and CD16. This inhibition appeared to be mediated by TGF-beta1. SCC VII/SF tumors in the oral cavity of the mice secrete high quantities of TGF-beta1, which reduce the expression of NK cell receptor NKG2D as well as CD16 and inhibits biological functions of NK cells.     

Heat shock protein 27 and p16 immunohistochemistry in cervical intraepithelial neoplasia and squamous cell carcinoma

Heat shock protein 27 (hsp27) is expressed by squamous cell carcinoma of the uterine cervix. Results from an earlier study by our group indicted that hsp27 may be a diagnostic marker for cervical intraepithelial neoplasia (CIN) and carcinoma. p16 expression is known to be elevated in intraepithelial uterine cervical cancer and grades 2 and 3 lesions (CIN2, CIN3), but has also been reported to be negative in 5-20% of cervical cancer and CIN lesions. The aim of our study was to confirm immunohistochemically the expression of hsp27 and p16 in cervical lesions. Formalin-fixed, paraffin-embedded cervical tissue specimens obtained between 2002 and 2010 were investigated for hsp27 and p16 expression. Positive staining was detected for hsp27 in 63% of normal cervical tissues, 47% of CIN1 lesions, 75% of CIN2 lesions, 92% of CIN3 lesions, and 100% of squamous cell carcinomas (SCC); the corresponding rates for p16 positivity were 29, 47, 67, 92, and 75%, respectively. Positive staining for both hsp27 and p16 was observed in 6% of normal cervical tissues and in 19% of CIN1, 18% of CIN2, 85% of CIN3, and 75% of SCC specimens. Hsp27 or p16 positivity had a sensitivity of 95.6 or 84.7% and a specificity of 37.2 or 70.5%, respectively, for the identification of CIN3 or SCC lesions; when both hsp27 and p16 were assessed, both the sensitivity and specificity were improved. In conclusion, both hsp27 and p16 immunohistochemistry is a useful tool for the diagnosis of CIN3 lesions or cervical SCC.     

Preserved Pancreatic β-Cell Development and Function in Mice Lacking the Insulin Receptor-Related Receptor

Receptors of the insulin/insulinlike growth factor (IGF) family have been implicated in the regulation of pancreatic beta-cell growth and insulin secretion. The insulin receptor-related receptor (IRR) is an orphan receptor of the insulin receptor gene (Ir) subfamily. It is expressed at considerably higher levels in beta cells than either insulin or IGF-1 receptors, and it has been shown to engage in heterodimer formation with insulin or IGF-1 receptors. To address whether IRR plays a physiologic role in beta-cell development and regulation of insulin secretion, we have characterized mice lacking IRR and generated a combined knockout of Ir and Irr. We report that islet morphology, beta-cell mass, and secretory function are not affected in IRR-deficient mice. Moreover, lack of IRR does not impair compensatory beta-cell hyperplasia in insulin-resistant Ir(+/-) mice, nor does it affect beta-cell development and function in Ir(-/-) mice. We conclude that glucose-stimulated insulin secretion and embryonic beta-cell development occur normally in mice lacking Irr.     

Asthma,Type 1 and Type 2 Diabetes Mellitus,and Inflammatory Bowel Disease amongst South Asian Immigrants to Canada and Their Children: A Population-Based Cohort Study

BACKGROUND

There is a high and rising rate of immune-mediated diseases in the Western world. Immigrants from South Asia have been reported to be at higher risk upon arrival to the West. We determined the risk of immune-mediated diseases in South Asian and other immigrants to Ontario, Canada, and their Ontario-born children.

METHODS

Population-based cohorts of patients with asthma, type 1 diabetes (T1DM), type 2 diabetes (T2DM), and inflammatory bowel disease (IBD) were derived from health administrative data. We determined the standardized incidence, and the adjusted risk of these diseases in immigrants from South Asia, immigrants from other regions, compared with non-immigrant residents of Ontario. The risk of these diseases in the Ontario-born children of immigrants were compared to the children of non-immigrants.

RESULTS

Compared to non-immigrants, adults from South Asia had higher risk of asthma (IRR 1.56, 95%CI 1.51-1.61) and T2DM (IRR 2.59, 95%CI 2.53-2.65). Adults from South Asia had lower incidence of IBD than non-immigrants (IRR 0.32, 95%CI 0.22-0.49), as did immigrants from other regions (IRR 0.29, 95%CI 0.20-0.42). Compared to non-immigrant children, the incidence of asthma (IRR 0.66, 95%CI 0.62-0.71) and IBD (IRR 0.47, 95%CI 0.33-0.67) was low amongst immigrant children from South Asia. However, the risk in Ontario-born children of South Asian immigrants relative to the children of non-immigrants was higher for asthma (IRR 1.75, 95%CI 1.69-1.81) and less attenuated for IBD (IRR 0.90, 95%CI 0.65-1.22).

CONCLUSION

Early-life environmental exposures may trigger a genetic predisposition to the development of asthma and IBD in South Asian immigrants and their Canada-born children.     

Interaction of the autonomic nervous system with intrinsic cardiac rate regulation in the guinea-pig, Cavia porcellus

In the denervated mammalian heart a change in right atrial pressure will still alter heart rate (intrinsic rate response, IRR). We have examined the IRR in isolated right atria of the guinea-pig maintained in oxygenated Krebs–Henseleit solution at 37°C, to compare with and extend studies in other species, and to determine whether the guinea-pig is a suitable model for electrophysiological studies of the IRR. Baseline diastolic transmural pressure was set at 2 mmHg. A 6-mmHg increase in right atrial pressure (RAP) caused an increase in atrial rate that reached a steady value of 15 min⁻¹ after 1–2 min. This response was enhanced by carbamylcholine and attenuated by isoprenaline. The influence of RAP on the rate response to vagal stimulation was examined. With RAP set at 8 mmHg, the reduction in atrial rate following vagal stimulation was 72±5% of that at 2 mmHg (n=6, mean±S.E., P<0.005). Continuous vagal stimulation produced a sustained bradycardia, and the effect of this bradycardia on the IRR was examined. When atrial rate was reduced 6% by vagal stimulation, the IRR was augmented to 202±21% of the control (n=6, P<0.005). This augmentation was larger (P<0.05) than that seen when atrial rate was reduced 8% by carbamylcholine (130±8% of control; n=7, P<0.05). Overall, the IRR in the guinea-pig is similar to that in the rabbit, and shows similar interactions with the autonomic nervous system.     

ATR signaling cooperates with ATM in the mechanism of low dose hypersensitivity induced by carbon ion beam

Little work has been done on the mechanism of low dose hyper-radiosensitivity (HRS) and later appeared radioresistance (termed induced radioresistance (IRR)) after irradiation with medium and high linear energy transfer (LET) particles. The aim of this study was to find out whether ATR pathway is involved in the mechanism of HRS induced by high LET radiation. GM0639 cells and two ATM deficient/mutant cells, AT5BIVA and AT2KY were irradiated by carbon ion beam. Thymidine block technique was developed to enrich the G2-phase population. Radiation induced early G2/M checkpoint was quantitatively assess with dual-parameter flow cytometry by detecting the cells positive for phospho-histone H3. The involvement of ATR pathway in HRS/IRR response was detected with pretreatment of specific inhibitors prior to carbon ion beam. The link between the early G2/M checkpoint and HRS/IRR under carbon ion beam was first confirmed in GM0639 cells, through the enrichment of cell population in G2-phase or with Aurora kinase inhibitor that attenuates the transition from G2 to M phase. Interestingly, the early G2/M arrest could still be observed in ATM deficient/mutant cells with an effect of ATR signaling, which was discovered to function in an LET-dependent manner, even as low as 0.2 Gy for carbon ion radiation. The involvement of ATR pathway in heavy particles induced HRS/IRR was determined with the specific ATR inhibitor in GM0639 cells, which affected the HRS/IRR occurrence similarly as ATM inhibitor. These data demonstrate that ATR pathway may cooperate with ATM in the mechanism of low dose hypersensitivity induced by carbon ion beam.     

Effect of alpha-2 adrenergic receptor stimulation on short-circuit current across isolated skin of the toad Bufo arenarum

1. The effect of the alpha-2 adrenergic agonist clonidine on short-circuit current (SCC) across isolated skins of Bufo arenarum toads was investigated. 2. Clonidine inhibited basal SCC in a dose-dependent manner. 3. Blockade of the effect of clonidine on basal SCC by the selective alpha-2 antagonist yohimbine supports the hypothesis that the inhibitory effect is mediated by the stimulation of alpha-2 adrenergic receptors. 4. The fact that the inhibitory effect of clonidine is higher in skins with spontaneous positive SCC than in the negative ones, and that the alpha-2 agonist was unable to alter amiloride-induced negative SCC suggests that the inhibitory effect of clonidine may probably be mediated by inhibition of sodium transport.     

Development and inter-rater reliability of the Liverpool adverse drug reaction causality assessment tool

Aim

To develop and test a new adverse drug reaction (ADR) causality assessment tool (CAT).

Methods

A comparison between seven assessors of a new CAT, formulated by an expert focus group, compared with the Naranjo CAT in 80 cases from a prospective observational study and 37 published ADR case reports (819 causality assessments in total).

Main Outcome Measures

Utilisation of causality categories, measure of disagreements, inter-rater reliability (IRR).

Results

The Liverpool ADR CAT, using 40 cases from an observational study, showed causality categories of 1 unlikely, 62 possible, 92 probable and 125 definite (1, 62, 92, 125) and ‘moderate’ IRR (kappa 0.48), compared to Naranjo (0, 100, 172, 8) with ‘moderate’ IRR (kappa 0.45). In a further 40 cases, the Liverpool tool (0, 66, 81, 133) showed ‘good’ IRR (kappa 0.6) while Naranjo (1, 90, 185, 4) remained ‘moderate’.

Conclusion

The Liverpool tool assigns the full range of causality categories and shows good IRR. Further assessment by different investigators in different settings is needed to fully assess the utility of this tool.     

Downregulation of RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1, and HYAL2 in non-small cell lung cancer

Chromosomal and genome abnormalities of 3p are frequent in many epithelial tumors, including lung cancer. Several critical regions with a high frequency of hemi-and homozygous deletions in tumors are known for 3p, and more than 20 cancer-related genes occur in 3p21.3. Quantitative real-time PCR was used to measure the mRNA level for tumor-suppressor and candidate genes of 3p21.3 (RBSP3/CTDSPL, NPRL2/G21, RASSF1A, ITGA9, HYAL1, and HYAL2) in major types of non-small cell lung cancer (NSCLC): squamous cell lung cancer (SCC) and lung adenocarcinoma (AC). A significant (2-to 100-fold) and frequent (44–100%) decrease in mRNA levels was observed in NSCLC. The mRNA level decrease and its frequency depended on the histological type of NSCLC for all genes. The downregulation of RASSF1A and ITGA9 was significantly associated with AC progression; the same tendency was observed for RBSP3/CTDSPL, NPRL2/G21, HYAL1, and HYAL2. In SCC, the downregulation of all genes was not associated with the clinical stage, tumor cells differentiation, and metastasis in lymph nodes. The RBSP3/CTDSPL, NPRL2/G21, ITGA9, HYAL1, and HYAL2 mRNA levels significantly (5-to 13-fold on average) decreased at a high frequency (83–100%) as early as SCC stage I. Simultaneous downregulation of all six genes was observed in some tumor samples and was independent of the gene position in 3p21.3 and the functions of the protein products. The Spearman correlation coefficient r _s was 0.63–0.91, p < 0.001. The highest r _s values were obtained for gene pairs ITGA9-HYAL2 and HYAL1-HYAL2, whose products mediate cell-cell adhesion and cell-matrix interactions; coregulation of the genes was assumed on this basis. Both genetic and epigenetic mechanisms proved to be important for downregulation of RBSP3/CTDSPL and ITGA9. This finding supported the hypothesis that the cluster of cancerrelated genes in the extended 3p21.3 locus is simultaneously inactivated during the development and progression of lung cancer and other epithelial tumors. A significant and frequent decrease in the mRNA level of the six genes in SCC could be important for developing specific biomarker sets for early SCC diagnosis and new approaches to gene therapy of NSCLC.