Functional study of the Nha1p C-terminus: involvement in cell response to changes in external osmolarity

Saccharomyces cerevisiae uses different mechanisms to adapt to changes in environmental osmolarity. Upon hyperosmotic shock, cells first mobilize a rapid rescue system that prevents excessive loss of ions and water; then in the adaptation period they accumulate a compatible solute (glycerol). When subjected to hypoosmotic shock, they rapidly release intracellular stocks of glycerol to reduce intracellular osmolarity and prevent bursting. The plasma membrane Nha1 alkali metal cation/H⁺ antiporter is not important in helping the cells to survive a sudden drop in external osmolarity, but is involved in the cell response to hyperosmotic shock. For this role, its long hydrophilic C-terminus is indispensable. The capacity of the Nha1 antiporter to transport potassium is regulated by Hog1 kinase. Upon sorbitol-mediated stress, the Nha1p potassium export activity decreases in order to maintain a higher intracellular concentration of solutes. The C-terminal-less Nha1 version is not inactivated and its potassium efflux activity renders cells very sensitive to hyperosmotic shock. Taken together, our results suggest an important role of Nha1p and its C-terminus in the immediate response to hyperosmotic shock as part of the rapid rescue mechanism.     

Expression of retinoblastoma protein and P16 proteins in classic Hodgkin lymphoma: relationship with expression of p53 and presence of Epstein-Barr virus in the regulation of cell growth and death

Deregulation of several genes involved in cell cycle control has been reported in classic Hodgkin lymphoma (cHL). This study aimed to investigate the expression of tumor suppressor proteins (P16(INK4A), retinoblastoma protein, and p53) in cHL in relation to the proliferation and apoptosis of Hodgkin/Reed-Sternberg (H/RS) cells, correlating with the status of Epstein-Barr virus (EBV). A total of 66 cHL cases and 10 nonneoplastic reactive lymphoid tissues were retrieved from the archives. Immunohistochemistry technique was used for the detection of protein expression. Presence of EBV infection was detected by EBV early RNA in situ hybridization. p16(INK4A) gene deletion status was assessed by fluorescence in situ hybridization technique. Expression of P16(INK4A) was observed in 49.2% of the cases, whereas positive retinoblastoma protein and p53 expressions in the H/RS cells were detected in 89.1% and 81.5% of the cases, respectively. Epstein-Barr virus positivity was detected in 53.0% of the cases. Proliferation marker, Ki-67 expression, was observed in 86.7% of the cases. There was no significant correlation between the expression of the various tumor suppressor proteins and Ki-67. Retinoblastoma protein and p53 were also not associated with the presence of EBV. An inverse relationship was observed between the expression of P16(INK4A) and the presence of EBV. There were no significant homozygous or hemizygous deletions of the p16(INK4A) gene. However, an aberrant copy number of chromosome 9 with the loss of one or more p16(INK4A) loci was detected in all cases assessable by fluorescence in situ hybridization. Loss of function of one or more tumor suppressor proteins may be involved in defective cell regulation of H/RS cells. Epstein-Barr virus may have a role in inhibiting P16(INK4A) expression, thus resulting in a perturbed p16(INK4A)-Rb cell cycle checkpoint.     

Mouse Models of Cognitive Disorders in Trisomy 21: A Review

Trisomy 21 (TRS21) is the most frequent genetic cause of mental retardation. Although the presence of an extra copy of HSA21 is known to be at the origin of the syndrome, we do not know which 225 HSA21 genes have an effect on cognitive processes. Mouse models of TRS21 have been developed using syntenies between HSA21 and MMU16, MMU10 and MMU17. Available mouse models carry extra fragments of MMU16 or of HSA21 that cover all of HSA21 (chimeric HSA21) or MMU16 (Ts16); some carry large parts of MMU16 (Ts65Dn, Ts1Cje, Ms1Cje), while others have reduced contiguous fragments covering the D21S17-ETS2 region or single transfected genes. This offers a nest design strategy for deciphering cognitive (learning, memory and exploration) and associated brain abnormalities involving each of these chromosomal regions. This review confirms the crucial but not exclusive contribution of the D21S17-ETS2 region encompassing 16 genes to cognitive disorders.     

Protective effect of glutathione against lipopolysaccharide-induced inflammation and mortality in rats

Objective To investigate protective effect of glutathione (GSH) against lipopolysaccharide (LPS)-induced inflammation and mortality in rats. Methods Male Sprague-Dawley rats were injected intraperitoneally (i. p.) with GSH (40 mg/kg) 30 min prior to LPS injection (20 mg/kg). Animal survival rate and the production of nitric oxide in serum were measured. Morphology of lung was observed using hematoxylin and eosin staining. Western blotting was used to assess the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and the phosphorylation of p38 in rat peritoneal macrophages. Results GSH significantly decreased LPS-induced mortality, inhibited serum NO production and eliminated lung histological injury. Furthermore, GSH inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins and the phosphorylation of p38 in LPS-stimulated rat peritoneal macrophages. Conclusions These results indicated that GSH suppressed LPS-induced systemic inflammatory response in rats and p38 MAPK signal pathway was involved in this process. Received 20 March 2006; returned for revision 21 May 2006; returned for final revision 11 June 2006; accepted by M. Katori 7 July 2006     

Prognostic value of immunohistochemical staining of p53, bcl-2, and Ki-67 in small cell lung cancer

Small cell lung cancer (SCLC) is one of the most fatal cancers in humans and many factors are known to be related to its poor prognosis. Immunohistochemical (IHC) stainings were done on SCLC specimens in order to investigate the prognostic value of the apoptosis-related gene expression and the tumor proliferative maker, and the relationships among these IHC results and patients clinical characteristics, chemoresponsiveness, and survival were analyzed. The medical records of 107 patients were reviewed retrospectively. IHC stainings for p53, bcl-2 and Ki-67 expressions were performed in the 66 paraffin-embedded biopsy samples. Sixty-six out of the 107 patients were evaluable for response rate and survival. The overall response rate was 75% (95% Confidence Interval=74-76%) and the median survival time was 14 months. The median survival time of limited stage was 16 months and that of extensive stage was 10 months. The prevalence of p53, bcl-2 and Ki-67 expression was 62%, 70%, and 49%, respectively. There were no correlations among the immunoreactivities of p53, bcl-2 and Ki-67 with clinical stage, chemoresponsiveness or overall survival. The clinical stage was the only prognostic factor influencing survival. The expression rates of p53, bcl-2, and Ki-67 were relatively high in SCLC without any prognostic significance. The exact clinical role of these markers should be defined through further investigations.     

The p75 neurotrophin receptor is widely expressed in conventional papillary thyroid carcinoma

Papillary thyroid carcinomas (PTCs) are associated with alterations in several proto-oncogenes related with nervous system development and function, such as TrkA and RET, which are commonly rearranged in these carcinomas. The other oncogenic event recently identified in PTC is the BRAF V600E mutation. Because the role of TrkA was not completely elucidated in thyroid cancer ethiopathogenesis, we decided to study the expression of active, phosphorylated TrkA and of its coreceptor p75 neurotrophin receptor (p75 NTR) in a series of 92 PTC (37 lesions of conventional PTC, 28 of follicular variant of PTC [FVPTC], and 27 of other variants of PTC) as well as in 21 samples of normal thyroid and nonneoplastic thyroid lesions used as a controls. We observed neoexpression of p75 NTR in PTC, particularly in conventional PTC and in other variants of PTC displaying a papillary growth pattern, rather than in FVPTC. No immunoexpression of p75 NTR was observed in normal thyroid nor in nonneoplastic thyroid lesions. The cellular localization of p75 NTR immunoexpression was also significantly associated with the growth pattern of PTC, being much more frequently detected in an apical localization in PTC with papillary architecture than in PTC with a follicular or solid growth pattern. This apical localization of p75 NTR was significantly associated with the presence of BRAF V600E. No significant differences were detected between normal thyroid, nonneoplastic lesions, and PTC (or any PTC variant) regarding expression/activation of TrkA, thus suggesting that by itself and in contrast to p75 NTR, TrkA is not altered during PTC development.     

Comparative study of the expression of cellular cycle proteins in cervical intraepithelial lesions

Interaction of human papilloma virus oncoproteins E6 and E7 with cell cycle proteins leads to disturbances of the cell cycle mechanism and subsequent alteration in the expression of some proteins, such as p16^INK4a, cyclin D1, p53 and KI67. In this study, we compared alterations in the expression of these proteins during several stages of intra-epitelial cervical carcinogenesis. Accordingly, an immunohistochemical study was performed on 50 cervical biopsies, including negative cases and intraepithelial neoplasias. The expression patterns of these markers were correlated with the histopathological diagnosis and infection with HPV. The p16^INK4a, followed by Ki67, showed better correlation with cancer progression than p53 and cyclin D1, which recommends their use in the evaluation of cervical carcinogenesis. These monoclonal antibodies can be applied to cervical biopsy specimens to identify lesions transformed by oncogenic HPV, separating CIN 1 (p16^INK4a positive) and identifying high-grade lesions by an increase in the cellular proliferation index (Ki67). In this way, we propose immunomarkers that can be applied in clinical practice to separate patients who need a conservative therapeutic approach from those who require a more aggressive treatment.     

幽门螺杆菌对SGC-7901细胞增殖凋亡及p27kipl表达的影响

Objective To explore the influence of Helicobacter pylori (Hp) on the proliferation, apoptosis and expression of p27kipl protein of gastric epithelial cells in vitro. Methods SC, C-7901 cell pro-liferation was examined by flow cytometry after incubation with different concentration of NCTC11637. The effect of NCTC11637 on cell apoptosis was also evaluated by flow cytometryo And the expression of p27kipl of gastric epithelial cells was detected by immunohistochemical analysis and in situ hybridization, respectively. Results Cell proliferation was enhanced when co-incubated with the Hp in low concentration (3.4 x 104 to 1.9 x 105 CFU/ml) but inhibited in higher concentration (≥4.8×106CFU/ml). However, cell apoptosis was increase when co-incubated with the Hp in high concentration (≥9.6 ×105 CFU/ml) showing concen-tration dependent picture. In addition, the co-incubation of SGC-7901 with Hp led to decrease of the expres-sion of p27kipl protein but not mRNA in a Hp concentration dependent way. Conclusion Hp could effect the gastric epithelial cells apoptosis and proliferation directly and influence the expression of p27kips protein which might facilitate gastric carcinoma.     

Correlation of cyclin D1 and E1 expression with bladder cancer presence, invasion, progression, and metastasis

The objective of this study was to determine the correlation of the expression of cyclin D1 and E1 with the expression of commonly altered cell cycle regulators and bladder cancer presence, staging, and clinical outcomes. We performed immunohistochemical staining for cyclin D1, cyclin E1, p53, p21, p27, and retinoblastoma protein (pRB) on serial cuts from normal urothelium from 9 controls, radical cystectomy specimens from 226 consecutive patients with advanced transitional cell carcinoma, and lymph nodes with metastasis from 50 of the 226 cystectomy patients. Cyclin D1 and E1 immunoreactivity were considered low when samples demonstrated less than 10% and 30% nuclear reactivity, respectively. Normal bladder urothelium from all 9 control patients showed uniformly intense expression of cyclin D1 and E1. Cyclin D1 expression was low in 99 (43.8%) of 226 cystectomy specimens and 25 (50.0%) of 50 metastatic lymph node specimens. Cyclin D1 immunoreactivity was not associated with any pathologic characteristics or clinical outcomes. Cyclin E1 expression was low in 125 (55.3%) of 226 cystectomy specimens and 22 (44.0%) of 50 metastatic lymph node specimens. Low cyclin E1 expression was significantly associated with advanced pathologic stage, lymphovascular invasion, and lymph node metastases. In multivariate analyses, low cyclin E1 expression was significantly associated with bladder cancer-specific mortality (P = .048), but not disease recurrence (P = .056). Low cyclin E1 expression was significantly associated with altered expression of pRB, p27, and cyclin D1. Low cyclin D1 expression was significantly associated with altered expression of pRB, p21, and cyclin E1. Cyclin E1 expression stratifies patients with bladder transitional cell carcinoma into those with more "indolent" behavior and those with features of biologically and clinically aggressive disease.