A novel chloride channel in Vicia faba guard cell vacuoles activated by the serine/threonine kinase, CDPK

Calcium-Dependent Protein Kinases (CDPKs) in higher plants contain a C-terminal calmodulin-like regulatory domain. Little is known regarding physiological CDPK targets. Both kinase activity and multiple Ca2+-dependent signaling pathways have been implicated in the control of stomatal guard cell movements. To determine whether CDPK or other protein kinases could have a role in guard cell signaling, purified and recombinant kinases were applied to Vicia faba guard cell vacuoles during patch-clamp experiments. CDPK activated novel vacuolar chloride (VCL) and malate conductances in guard cells. Activation was dependent on both Ca2+ and ATP. Furthermore, VCL activation occurred in the absence of Ca2+ using a Ca2+-independent, constitutively active, CDPK* mutant. Protein kinase A showed weaker activation (22% as compared with CDPK). Current reversals in whole vacuole recordings shifted with the Nernst potential for Cl-and vanished in glutamate. Single channel recordings showed a CDPK-activated 34 +/- 5 pS Cl- channel. VCL channels were activated at physiological potentials enabling Cl- uptake into vacuoles. VCL channels may provide a previously unidentified, but necessary, pathway for anion uptake into vacuoles required for stomatal opening. CDPK-activated VCL currents were also observed in red beet vacuoles suggesting that these channels may provide a more general mechanism for kinase-dependent anion uptake.     

Determination of cell fate by c-Abl activation in the response to DNA damage

The cellular response to DNA damage includes growth arrest and activation of DNA repair. Certain insights into how DNA damage is converted into intracellular signals that control the genotoxic stress response have been derived from the finding that the c-Abl protein tyrosine kinase is activated by ionizing radiation and other DNA-damaging agents. c-Abl associates with the DNA-dependent protein kinase (DNA-PK) and is activated by DNA-PK-dependent phosphorylation. The ataxia telangiectasia mutated (ATM) gene product also contributes to c-Abl activation. The demonstration that c-Abl binds to p53, induces the transactivation function of p53 and activates p21 expression has supported involvement of c-Abl in regulation of the p53-dependent G1 arrest response. Interaction between c-Abl and the Rad51 protein has also provided support for involvement of c-Abl in recombinational repair of DNA strand breaks. Defects in G1 arrest and repair predispose to replication of damaged templates and, in the event of irreparable DNA lesions, induction of apoptosis. The available evidence indicates that c-Abl effects a proapoptotic function by a mechanism largely independent of p53. c-Abl also functions as an upstream effector of the proapoptotic JNK/SAPK and p38 MAPK pathways. In addition, c-Abl-dependent inhibition of PI 3-kinase contributes to the induction of apoptosis. The findings thus suggest that, in response to genotoxic stress, c-Abl functions in determining cell fate, that is growth arrest and repair or induction of apoptosis. The physiologic function of c-Abl may reside in control of the cellular response to DNA strand breaks that occur during DNA replication, genetic recombination and gene rearrangements.     

Adenovirus-mediated expression of PML suppresses growth and tumorigenicity of prostate cancer cells

Our previous studies demonstrated that the promyelocytic leukemia gene, PML, encodes a growth and transformation suppressor. Overexpression of PML inhibits cancer cell growth in vitro and in vivo. In this study, we further explored the possibility of applying PML as a potential agent for developing prostate cancer gene therapy using an adenovirus delivery system. We have constructed and produced the recombinant PML-adenovirus, Ad-PML, in which the full-length PML cDNA is driven by the strong cytomegalovirus promoter. In LNCaP, DU145, and PC-3 prostate cancer cell lines, an infection efficiency of 90% can be achieved at a concentration of 2, 10, and 100 multiplicity of infection (MOI), respectively. Western blotting and immunofluorescence staining demonstrated that the AD-PML-infected cells expressed a high level of PML protein. The protein expression peaked at days 3-4 postinfection, and a detectable level of PML was found at day 18 after viral infection. To test the effect of Ad-PML on the growth of prostate cancer cells, the DU145 and LNCaP cells were infected with 10 and 2 MOI of Ad-PML. We found that the growth rate of the Ad-PML-infected DU145 and LNCaP cells were significantly inhibited. A tumorigenicity test in nude mice showed that the Ad-PML-treated DU145 cells failed to form tumors. Most importantly, direct injection of Ad-PML into DU145-induced tumors was able to repress tumor growth in nude mice by 64%. Taken together, these data indicate that PML is a tumor growth suppressor in prostate cancer and that Ad-PML may be a potential candidate for human prostate cancer therapy.     

Regeneration of sudomotor and sensory nerve fibres after digital replantation and microneurovascular toe-to-hand transfer

We analyzed the informations concerning the late postoperative evolution for 405 (60%) of 760 patients with colon cancer operated between 1968-1996 in the Surgical Clinic from Tg.-Mure? and we found a global 5-year survival rate of 35.57%. The conclusion after this study was that the most important worsening prognostic factors were the advanced stage evolution of the tumor and the occlusion. The females had a better prognosis than the males. The rural patients had a better prognosis than the urban. In our study the younger age isn't a worsening factor for survival. The patients with a longer history of the symptoms had an unexpected better survival rate than the others. Localization of the tumors at the site of the descending colon and flexures is an important negative prognostic factor. The penetrating feature of the tumor is more important than the lymphatic invasion for prognosis. Subtotal colectomy performed for stenotic tumors of the left colon, even in emergency for occlusions gave us one of the best 5-year survival rate (75%). The histopathologic type of undifferentiated carcinoma and the mucinous adenocarcinoma are associated with the poorest survival rate. The lymphoma has a better survival rate than the carcinoma. The perioperative blood transfusion, even though is associated with a low survival rate it is not an important prognostic factor.     

Recombinant PML adenovirus suppresses growth and tumorigenicity of human breast cancer cells by inducing G1 cell cycle arrest and apoptosis

Our previous studies demonstrated that the promyelocytic leukemia gene, PML which involved in the 15;17 translocation in acute promyelocytic leukemia (APL) is a growth and transformation suppressor. In this study, recombinant PML adenovirus, Ad-PML was constructed and used to infect human breast cancer cells in vitro and in vivo, the anti-oncogenic function of PML and its mechanism of growth suppressing effect in breast cancer cells were examined. We showed that Ad-PML effectively infected the MCF-7 and SK-BR-3 cells. A high level of PML protein was expressed within 24 h post-infection and a detectable level remained at day 16. Ad-PML significantly suppressed the growth rate, clonogenicity, and tumorigenicity of breast cancer cells. Intratumoral injections of MCF-7-induced tumors by high titer Ad-PML suppressed tumor growth in nude mice by about 80%. The injection sites expressed high level of PML and associated with a massive apoptotic cell death. To elucidate the molecular mechanism of PML's growth suppressing function, we examined the effect of Ad-PML on cell cycle distribution in MCF-7 and SK-BR-3 cells. We found that Ad-PML infection caused a cell cycle arrest at the G1 phase. We further showed that G1 arrest of MCF-7 cells is associated with a significant decrease in cyclin D1 and CDK2. An increased expression of p53, p21 and cyclin E was found. The Rb protein became predominantly hypophosphorylated 48 h post-infection. These findings indicate that PML exerts its growth suppressing effects by modulating several key G1 regulatory proteins. Our study provides important insight into the mechanism of tumor suppressing function of PML and suggests a potential application of Ad-PML in human cancer gene therapy.     

A transient outward-rectifying K+ channel current down-regulated by cytosolic Ca2+ in Arabidopsis thaliana guard cells

Sustained (noninactivating) outward-rectifying K+ channel currents have been identified in a variety of plant cell types and species. Here, in Arabidopsis thaliana guard cells, in addition to these sustained K+ currents, an inactivating outward-rectifying K+ current was characterized (plant A-type current: IAP). IAP activated rapidly with a time constant of 165 ms and inactivated slowly with a time constant of 7.2 sec at +40 mV. IAP was enhanced by increasing the duration (from 0 to 20 sec) and degree (from +20 to -100 mV) of prepulse hyperpolarization. Ionic substitution and relaxation (tail) current recordings showed that outward IAP was mainly carried by K+ ions. In contrast to the sustained outward-rectifying K+ currents, cytosolic alkaline pH was found to inhibit IAP and extracellular K+ was required for IAP activity. Furthermore, increasing cytosolic free Ca2+ in the physiological range strongly inhibited IAP activity with a half inhibitory concentration of approximately 94 nM. We present a detailed characterization of an inactivating K+ current in a higher plant cell. Regulation of IAP by diverse factors including membrane potential, cytosolic Ca2+ and pH, and extracellular K+ and Ca2+ implies that the inactivating IAP described here may have important functions during transient depolarizations found in guard cells, and in integrated signal transduction processes during stomatal movements.     

Three tRNA binding sites in rabbit liver ribosomes and role of the intrinsic ATPase in 80S ribosomes from higher eukaryotes

Three tRNA binding sites have been found in organisms of all domains (former kingdoms) with only one exception: Four binding sites have been reported for cytoplasmic 80S ribosomes from rabbit liver. Therefore, the issue was reconsidered, and the data revealed that rabbit liver ribosomes contain three tRNA binding sites, underlining the universal character of this ribosomal feature. Furthermore, a first analysis of the role of the ribosome intrinsic ATPase was performed. This ATPase is found in ribosomes of higher eukarya but not in lower eukarya such as yeast or ribosomes of the domains archea and bacteria. The results suggest that the intrinsic ATPase fulfills the same function as the essential third elongation factor EF-3, an ATPase in higher fungi (yeast etc.), that facilitates the release of the deacylated tRNA from the E site.     

Ultrastructure of rat kidney after long-term dehydration at different altitudes

Calcific deposits in tendinous tissue are common to middle-aged patients. The condition usually resolves spontaneously, but refractory cases can be expedited by needle barbotage or surgery.     

The human myoepithelial cell is a natural tumor suppressor

Myoepithelial cells, which surround ducts and acini of glandular organs, form a natural border separating proliferating epithelial cells from basement membrane and underlying stroma. Myoepithelial cells in situ and in vitro constitutively express high amounts of proteinase inhibitors that include tissue inhibitor of metalloproteinase 1, protease nexin-II, alpha-1 antitrypsin, and maspin. Human myoepithelial xenografts (HMS-X, HMS-3X, and HMS-4X), which our laboratory has established, accumulate an abundant extracellular matrix containing sequestered proteinase inhibitors. Humatrix, a gel that we have derived from HMS-X, inhibits tumor cell invasion (down to 25% +/- 10% of Matrigel control; P < 0.01), and our recently established human myoepithelial cell lines, HMS-1, HMS-3, and HMS-4, inhibit tumor cell invasion in cellular invasion (down to 42% +/- 7% of control; P < 0.05) and in conditioned media assays (down to 30% +/- 8% of control; P < 0.01). The anti-invasive effects of HMS-1, HMS-3, and HMS-4 can be enhanced by phorbol 12-myristate 13-acetate (down to 2% +/- 1% of control) by a maspin-dependent mechanism and abolished by dexamethasone (up to 95% +/- 5% of control) by a maspin-independent mechanism (P < 0.01). HMS-X, HMS-3X, HMS-4X, and Humatrix inhibit tumor invasion and metastasis in severe combined immunodeficient mice (P < 0.001). The cumulative data suggest that myoepithelial cells are natural paracrine suppressors of invasion and metastasis and may specifically inhibit the progression of precancerous disease states to invasive cancer in vivo.