Band 16, Heft 3

Post-mitotic neurons are typically terminally differentiated and in a quiescent status. However, in Alzheimer disease (AD), many neurons display ectopic re-expression of cell cycle-related proteins. Cyclin-dependent kinase 11 (CDK11) mRNA produces a 110-kDa protein (CDK11p110) throughout the cell cycle, a 58-kDa protein (CDK11p58) that is specifically translated from an internal ribosome entry site and expressed only in the G2/M phase of the cell cycle, and a 46-kDa protein (CDK11p46) that is considered to be apoptosis specific. CDK11 is required for sister chromatid cohesion and the completion of mitosis. In this study, we found that the expression patterns of CDK11 vary such that cytoplasmic CDK11 is increased in AD cellular processes, compared to a pronounced nuclear expression pattern in most controls. We also investigated the effect of amyloid precursor protein (APP) on CDK11 expression in vitro by using M17 cells overexpressing wild-type APP and APP Swedish mutant phenotype and found increased CDK11 expression compared to empty vector. In addition, amyloid-β25–35 resulted in increased CDK11 in M17 cells. These data suggest that CDK11 may play a vital role in cell cycle re-entry in AD neurons in an APP-dependent manner, thus presenting an intriguing novel function of the APP signaling pathway in AD.

Pharmacological inhibition of protein kinases that are responsible for the phosphorylation of the carboxy-terminal domain (CTD) of RNA Pol II during transcription by 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) leads to severe inhibition of mRNA synthesis and activates p53. Transcription of the p53 effectors that are induced under these conditions, such as p21 or PUMA, must bypass the requirement for CTD phosphorylation by the positive elongation factor P-TEFb. Here, we have downregulated SNW1/SKIP, a splicing factor and a transcriptional co-regulator, which was found to interact with P-TEFb and synergistically affect Tat-dependent transcription elongation of HIV 1. Using the colon cancer derived cell line HCT116, we have found that both doxorubicin- and DRB-induced expression of p21 or PUMA is insensitive to SNW1 downregulation by siRNA. This suggests that transcription of stress response genes, unlike, e.g., the SNW1-sensitive mitosis-specific genes, can proceed uncoupled from regulators that normally function under physiological conditions.

The tight junction protein claudin-4 is frequently overexpressed in pancreatic cancer, and is also a receptor for Clostridium perfringens enterotoxin (CPE). The cytotoxic effects of CPE are thought to be useful as a novel therapeutic tool for pancreatic cancer. However, the responses to CPE via claudin-4 remain unknown in normal human pancreatic duct epithelial (HPDE) cells. We introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture as a model of normal HPDE cells in vitro. hTERT-HPDE cells treated with or without 10% FBS and pancreatic cancer cell lines PANC-1, BXPC3, HPAF-II and HPAC were treated with CPE. In Western blotting, the expression of claudin-4 protein in hTERT-HPDE cells treated with 10% FBS was as high as it was in all of the pancreatic cancer cell lines. In hTERT-HPDE cells with or without 10% FBS, cytotoxicity was not observed at any concentration of CPE, whereas in all pancreatic cancer cell lines, CPE had a dose-dependent cytotoxic effect. In hTERT-HPDE cells with 10% FBS, claudin-4 was localized in the apical-most regions, where there are tight junction areas, in which in all pancreatic cancer cell lines claudin-4 was found not only in the apical-most regions but also at basolateral membranes. In hTERT-HPDE cells with 10% FBS after treatment with CPE, downregulation of barrier function and claudin-4 expression at the membranes was observed. In HPAC cells, the sensitivity to CPE was significantly decreased by knockdown of claudin-4 expression using siRNA compared to the control. These findings suggest that, in normal HPDE cells, the lack of toxicity of CPE was probably due to the localization of claudin-4, which is different from that of pancreatic cancer cells. hTERT-HPDE cells in this culture system may be a useful model of normal HPDE cells not only for physiological regulation of claudin-4 expression but also for developing safer and more effective therapeutic methods targeting claudin-4 in pancreatic cancer.

We used a continuum model based on the Helfrich free energy to investigate the binding dynamics of a lipid bilayer to a BAR domain surface of a crescent-like shape of positive (e.g. I-BAR shape) or negative (e.g. F-BAR shape) intrinsic curvature. According to structural data, it has been suggested that negatively charged membrane lipids are bound to positively charged amino acids at the binding interface of BAR proteins, contributing a negative binding energy to the system free energy. In addition, the cone-like shape of negatively charged lipids on the inner side of a cell membrane might contribute a positive intrinsic curvature, facilitating the initial bending towards the crescent-like shape of the BAR domain. In the present study, we hypothesize that in the limit of a rigid BAR domain shape, the negative binding energy and the coupling between the intrinsic curvature of negatively charged lipids and the membrane curvature drive the bending of the membrane. To estimate the binding energy, the electric potential at the charged surface of a BAR domain was calculated using the Langevin-Bikerman equation. Results of numerical simulations reveal that the binding energy is important for the initial instability (i.e. bending of a membrane), while the coupling between the intrinsic shapes of lipids and membrane curvature could be crucial for the curvature-dependent aggregation of negatively charged lipids near the surface of the BAR domain. In the discussion, we suggest novel experiments using patch clamp techniques to analyze the binding dynamics of BAR proteins, as well as the possible role of BAR proteins in the fusion pore stability of exovesicles.

Published data concerning the effects of hypertonicity on cell motility have often been controversial. The interpretation of results often rests on the premise that cell responses result from cell dehydration, i.e. osmotic effects. The results of induced hypertonicity on cell movement of Dictyostelium discoideum amoebae and human melanoma HTB-140 cells reported here show that: i) hypertonic solutions of identical osmolarity will either inhibit or stimulate cell movement depending on specific solutes (Na+ or K+, sorbitol or saccharose); ii) inhibition of cell motility by hypertonic solutions containing Na+ ions or carbohydrates can be reversed by the addition of calcium ions; iii) various cell types react differently to the same solutions, and iv) cells can adapt to hypertonic solutions. Various hypertonic solutions are now broadly used in medicine and to study modulation of gene expression. The observations reported suggest the need to examine whether the other responses of cells to hypertonicity can also be based on the solute-dependent cell responses besides cell dehydration due to the osmotic effects.

The ADAM (A Disintegrin and Metalloprotease) family of transmembrane proteins plays important roles in embryogenesis and tissue formation based on their multiple functional domains. In the present study, for the first time, the expression patterns of the premature and the active forms of six members of the ADAM proteins — ADAM9, ADAM10, ADAM12, ADAM17, ADAM22 and ADAM23 — in distinct parts of the developing chicken brain were investigated by quantitative Western blot analysis from embryonic incubation day (E) 10 to E20. The results show that the premature and the active forms of various ADAM proteins are spatiotemporally regulated in different parts of the brain during development, suggesting that the ADAMs play a very important role during embryonic development.

Yeast two-hybrid (Y2H) and isothermal titration calorimetry (ITC) methods were used to further study the mutational effect of non-erythroid alpha spectrin (αII) at position 22 in tetramer formation with beta spectrin (βII). Four mutants, αII-V22D, V22F, V22M and V22W, were studied. For the Y2H system, we used plasmids pGBKT7, consisting of the cDNA of the first 359 residues at the N-terminal region of αII, and pGADT7, consisting of the cDNA of residues 1697–2145 at the C-terminal region of βII. Strain AH109 yeast cells were used for colony growth assays and strain Y187 was used for β-galactosidase activity assays. Y2H results showed that the C-terminal region of βII interacts with the N-terminal region of αII, either the wild type, or those with V22F, V22M or V22W mutations. The V22D mutant did not interact with βII. For ITC studies, we used recombinant proteins of the αII N-terminal fragment and of the erythroid beta spectrin (βI) C-terminal fragment; results showed that the Kd values for V22F were similar to those for the wild-type (about 7 nM), whereas the Kd values were about 35 nM for V22M and about 90 nM for V22W. We were not able to detect any binding for V22D with ITC methods. This study clearly demonstrates that the single mutation at position 22 of αII, a region critical to the function of nonerythroid α spectrin, may lead to a reduced level of spectrin tetramers and abnormal spectrin-based membrane skeleton. These abnormalities could cause abnormal neural activities in cells.

Severe acute pancreatitis (SAP) is a common disease with a poor prognosis. Heart failure is one cause of SAP patient death. Intermedin (IMD) is a potent endogenous cardio-protective substance. Administration of exogenous IMD showed beneficial effects in cardiovascular diseases. The aim of this study was to investigate the myocardial damage in SAP and to determine the therapeutic potential of IMD for SAP. Using an SAP rat model, we examined endogenous IMD expression following SAP induction, and determined the effect of IMD on myocardial function, histological morphology, apoptosis-related gene expression, and prognosis. Our results indicated that the cardiac function and histological structure were significantly disrupted in SAP rats. Infusion of exogenous IMD significantly preserved cardiac function and ameliorated myocardial damage. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) revealed that myocardial apoptosis was extensively present in SAP rats, and IMD infusion led to increased expression of the prosurvival factor Bcl-2, but decreased pro-apoptotic factors Bax and caspase-3. In addition, IMD infusion also reversed the change of IMD receptor systems in SAP rat heart tissue. Furthermore, we found that IMD infusion greatly decreased mortality of SAP rats. In conclusion, administration of SAP produced therapeutic effects in SAP through modulating apoptotic and pro-survival gene expression, inhibiting myocardial apoptosis, preserving cardiac function, and a useful therapeutic agent for SAP, and provides us an insight for a clinical trial of IMD for treating human severe acute pancreatitis.

The use of tissue-specific promoter elements in the treatment of cervical cancer has been explored in this paper. The P105 promoter of human papillomavirus 18 (HPV18) was utilised to direct tissue-specific expression in a number of cell types. Expression was examined in three cervical carcinoma cell lines: HeLa (HPV18 positive), SiHa (HPV16 positive), and C33A cells (HPV negative); the epithelial cell line, H1299; and the foetal fibroblast cell line, MRC5, utilising a luciferase expression vector. Expression was highest in the cervical cell lines by a factor of at least 80. The effect of a number of mutations in the P105 promoter on expression levels was examined. Three deletion constructs of the long control region (LCR) were investigated: an 800 bp fragment (LCR800), a 400 bp fragment (LCR400), and a 200 bp fragment (LCR200), as well as the full length product LCR of HPV18 (LCR1000). The LCR800 construct of the HPV18 P105 promoter had the highest level of expression in the cervical cell lines and was also highest in the HPV18-positive HeLa cell line. Site-directed mutagenesis was then employed on the LCR800 construct to create four further constructs that each had inactivating mutations in one of the four E2 binding sites (E2BSs). Overall, this study indicated that the LCR800 construct of the HPV18 P105 promoter could be utilised as a tissuerestricted promoter in cervical cancer cells.

Recently, stem cell biology has become an interesting topic, especially in the context of treating diseases and injuries using transplantation therapy. Several varieties of human stem cells have been isolated and identified in vivo and in vitro. Ideally, stem cells for regenerative medical application should be found in abundant quantities, harvestable in a minimally invasive procedure, then safely and effectively transplanted to either an autologous or allogenic host. The two main groups of stem cells, embryonic stem cells and adult stem cells, have been expanded to include perinatal stem cells. Mesenchymal stem cells from perinatal tissue may be particularly useful in the clinic for autologous transplantation for fetuses and newborns, and after banking in later stages of life, as well as for in utero transplantation in case of genetic disorders. This review highlights the characteristics and therapeutic potential of three human mesenchymal stem cell types obtained from perinatal sources: Wharton’s jelly, the amnion, and the chorion.