Functional Significance of Point Mutations in Stress Chaperone Mortalin and Their Relevance to Parkinson Disease

Mortalin/mtHsp70/Grp75 (mot-2), a heat shock protein 70 family member, is an essential chaperone, enriched in cancers, and has been shown to possess pro-proliferative and anti-apoptosis functions. An allelic form of mouse mortalin (mot-1) that differs by two amino acids, M618V and G624R, in the C terminus substrate-binding domain has been reported. Furthermore, genome sequencing of mortalin from Parkinson disease patients identified two missense mutants, R126W and P509S. In the present study, we investigated the significance of these mutations in survival, proliferation, and oxidative stress tolerance in human cells. Using mot-1 and mot-2 recombinant proteins and specific antibodies, we performed screening to find their binding proteins and then identified ribosomal protein L-7 (RPL-7) and elongation factor-1 α (EF-1α), which differentially bind to mot-1 and mot-2, respectively. We demonstrate that mot-1, R126W, or P509S mutant (i) lacks mot-2 functions involved in carcinogenesis, such as p53 inactivation and hTERT/hnRNP-K (heterogeneous nuclear ribonucleoprotein K) activation; (ii) causes increased level of endogenous oxidative stress; (iii) results in decreased tolerance of cells to exogenous oxidative stress; and (iv) shows differential binding and impact on the RPL-7 and EF-1α proteins. These factors may mediate the transformation of longevity/pro-proliferative function of mot-2 to the premature aging/anti-proliferative effect of mutants, and hence may have significance in cellular aging, Parkinson disease pathology, and prognosis.     

Efficient protocol for in vitro production of androgenic haploids of <Emphasis Type="Italic">Phlox drummondii</Emphasis>

Production of haploid plants has been restricted to only a few ornamental species. In this paper an efficient anther culture protocol has been devised for production of haploid plants of Phlox drummondii, a garden ornamental. Anthers with microspores at early- to late-uninucleate stages were inoculated on MS (Murashige and Skoog, Physiol Plant 15:473–479, 1962) basal medium containing 9% sucrose, 10 μM 2,4-D + 5 μM BA in the dark for callus induction. The callus (~2 mm) was transferred to MS medium containing 3% sucrose + 10 μM BA + 5 μM NAA under a 16 h photoperiod for multiplication. Anther-derived callus showed the greatest shoot differentiation (60% with greater than 3 shoots per culture) at 13 weeks after culture initiation when maintained on MS medium supplemented with 3% sucrose and cytokinin (7.5 μM BA). At 68 weeks, only 4.6% of cultures differentiated with less than one shoot per callus. Anther-derived shoots rooted readily on MS medium containing 7.5 μM IAA. Of 60 plants that regenerated from anther callus, 50% were haploid, 30% diploid, and 20% aneuploid. Developed protocol could be useful for the haploid induction of outcrossing ornamental plants for production of their homozygous double haploids.     

Endotracheal tube cuff pressure in three hospitals, and the volume required to produce an appropriate cuff pressure

BACKGROUND: Cuff pressure in endotracheal (ET) tubes should be in the range of 20-30 cm H2O. We tested the hypothesis that the tube cuff is inadequately inflated when manometers are not used. METHODS: With IRB approval, we studied 93 patients under general anesthesia with an ET tube in place in one teaching and two private hospitals. Anesthetists were blinded to study purpose. Cuff pressure in tube sizes 7.0 to 8.5 mm was evaluated 60 min after induction of general anesthesia using a manometer connected to the cuff pilot balloon. Nitrous oxide was disallowed. After deflating the cuff, we reinflated it in 0.5-ml increments until pressure was 20 cmH2O. RESULTS: Neither patient morphometrics, institution, experience of anesthesia provider, nor tube size influenced measured cuff pressure (35.3 +/- 21.6 cmH2O). Only 27% of pressures were within 20-30 cmH2O; 27% exceeded 40 cmH2O. Although it varied considerably, the amount of air required to achieve a cuff pressure of 20 cmH2O was similar with each tube size. CONCLUSION: We recommend that ET cuff pressure be set and monitored with a manometer.     

Linked for life: temporal and spatial coordination of late mitotic events

Establishing the temporal order of mitotic events is critical to ensure that each daughter cell receives a complete DNA complement. The spatial co-ordination of the cytokinetic ring site with the axis of chromosome segregation is likewise crucial. Recent studies in fungi indicate that regulators of chromosome segregation also participate in promoting mitotic exit and that the proteins that initiate mitotic exit, in turn, additionally regulate cytokinesis. These findings suggest that late mitotic events are coupled by employing one pathway to control multiple events. The regulatory mechanisms that ensure the spatial co-ordination of the mitotic spindle apparatus with the division site have also been elucidated recently in the asymmetrically dividing budding yeast. Interestingly, the spatial co-ordination of late mitotic events seems also to be important in higher eukaryotes.     

Molecular mechanisms of anti-angiogenic effect of curcumin

Modulation of pathological angiogenesis by curcumin (diferuloylmethane), the active principle of turmeric, seems to be an important possibility meriting mechanistic investigations. In this report, we have studied the effect of curcumin on the growth of Ehrlich ascites tumor cells and endothelial cells in vitro. Further, regulation of tumor angiogenesis by modulation of angiogenic ligands and their receptor gene expression in tumor and endothelial cells, respectively, by curcumin was investigated. Curcumin, when injected intraperitoneally (i.p) into mice, effectively decreased the formation of ascites fluid by 66% in EAT bearing mice in vivo. Reduction in the number of EAT cells and human umbelical vein endothelial cells (HUVECs) in vitro by curcumin, without being cytotoxic to these cells, is attributed to induction of apoptosis by curcumin, as is evident by an increase in cells with fractional DNA content seen in our results on FACS analysis. However, curcumin had no effect on the growth of NIH3T3 cells. Curcumin proved to be a potent angioinhibitory compound, as demonstrated by inhibition of angiogenesis in two in vivo angiogenesis assay systems, viz. peritoneal angiogenesis and chorioallantoic membrane assay. The angioinhibitory effect of curcumin in vivo was corroborated by the results on down-regulation of the expression of proangiogenic genes, in EAT, NIH3T3, and endothelial cells by curcumin. Our results on Northern blot analysis clearly indicated a time-dependent (0-24h) inhibition by curcumin of VEGF, angiopoietin 1 and 2 gene expression in EAT cells, VEGF and angiopoietin 1 gene expression in NIH3T3 cells, and KDR gene expression in HUVECs. Further, decreased VEGF levels in conditioned media from cells treated with various doses of curcumin (1 microM-1mM) for various time periods (0-24h) confirm its angioinhibitory action at the level of gene expression. Because of its non-toxic nature, curcumin could be further developed to treat chronic diseases that are associated with extensive neovascularization.     

Photolithographic patterning of C2C12 myotubes using vitronectin as growth substrate in serum-free medium

The C2C12 cell line is frequently used as a model of skeletal muscle differentiation. In our serum-free defined culture system, differentiation of C2C12 cells into myotubes required surface-bound signals such as substrate-adsorbed vitronectin or laminin. On the basis of this substrate requirement of myotube formation, we developed a photolithography-based method to pattern C2C12 myotubes, where myotubes formed exclusively on vitronectin surface patterns. We have determined that the optimal line width to form single myotubes is approximately 30 mum. To illustrate a possible application of this method, we patterned myotubes on the top of commercial substrate-embedded microelectrodes. In contrast to previous experiments where cell patterning was achieved by selective attachment of the cells to patterned surfaces in a medium that contained all of the factors necessary for differentiation, this study illustrates that surface patterning of a signaling molecule, which is essential for skeletal muscle differentiation in a defined system, can result in the formation of aligned myotubes on the patterns. This technique is being developed for applications in cell biology, tissue engineering, and robotics.     

Molecular and functional characterization of EhPAK3, a p21 activated kinase from Entamoeba histolytica

p21-activated kinases (PAKs) are a family of serine/threonine kinases whose activity is regulated by the binding of the small Rho family GTPases as well as by RhoGTPase independent mechanisms. PAKs have wide-ranging functions which include cytoskeletal organisation, cell motility, cell proliferation and survival. We have identified a PAK from Entamoeba histolytica - EhPAK3 that is distributed in the cytoplasm of unstimulated cells and localizes to the caps after induction of capping with Concanavalin A. EhPAK3 contains a GTPase interacting (CRIB) domain, an N-terminal pleckstrin homology (PH) domain and a C-terminal kinase domain. Among the PAKs of E. histolytica studied so far, EhPAK3 bears the maximum similarity to Dictyostelium discoideum PAKC (DdPAKC). Phylogenetic analysis showed that EhPAK3 was closely related to DdPAKC and forms a group with DdPAKA, Dd Myosin I heavy chain kinase (DdMIHCK), and a PAK reported earlier from E. histolytica EhPAK2. Recombinant full-length EhPAK3 undergoes auotophosphorylation and phosphorylates histone H1 in vitro in the absence of any small GTPase. This is the first comprehensive characterization of a PAK protein from E. histolytica, which has constitutive activity and has demonstrated a strong involvement in receptor capping.