Identification and characterization of a cell cycle and apoptosis regulatory protein-1 as a novel mediator of apoptosis signaling by retinoid CD437

CD437, a novel retinoid, causes cell cycle arrest and apoptosis in a number of cancer cells including human breast carcinoma (HBC) by utilizing an undefined retinoic acid receptor/retinoid X receptor-independent mechanism. To delineate mediators of CD437 signaling, we utilized a random antisense-dependent functional knockout genetic approach. We identified a cDNA that encodes approximately 130-kDa HBC cell perinuclear protein (termed CARP-1). Treatments with CD437 or chemotherapeutic agent adriamycin, as well as serum deprivation of HBC cells, stimulate CARP-1 expression. Reduced levels of CARP-1 result in inhibition of apoptosis by CD437 or adriamycin, whereas increased expression of CARP-1 causes elevated levels of cyclin-dependent kinase inhibitor p21WAF1/CIP1 and apoptosis. CARP-1 interacts with 14-3-3 protein as well as causes reduced expression of cell cycle regulatory genes including c-Myc and cyclin B1. Loss of c-Myc sensitizes cells to apoptosis by CARP-1, whereas expression of c-Myc or 14-3-3 inhibits CARP-1-dependent apoptosis. Thus, apoptosis induction by CARP-1 involves sequestration of 14-3-3 and CARP-1-mediated altered expression of multiple cell cycle regulatory genes. Identification of CARP-1 as a key mediator of signaling by CD437 or adriamycin allows for delineation of pathways that, in turn, may prove beneficial for design and targeting of novel antitumor agents.     

Protein kinase C isoforms are translocated to microtubules in neurons

Activation of protein kinase C (PKC) increases microtubule (MT) growth lifetimes, resulting in extension of a nocodazole-sensitive population of MTs in Aplysia growth cones. We examined whether the two phorbol ester-activated PKCs in Aplysia, the Ca(2+)-activated PKC Apl I and the Ca(2+)-independent PKC Apl II, are associated with these MTs. Phorbol esters translocated PKC to the Triton X-100-insoluble fraction, and a significant portion of this translocated pool was sensitive to low concentrations of nocodazole. Low doses of nocodazole had no effect on the amount of PKC in the Triton X-100-insoluble fraction in the absence of phorbol esters, whereas higher doses of nocodazole reduced basal levels of PKC Apl II. The F-actin cytoskeletal disrupter, latrunculin A, removed both PKCs from the Triton X-100-insoluble fraction in both control and phorbol ester-treated nervous systems. PKC Apl II also directly interacted with purified MTs. In detergent-extracted cells, both PKCs immunolocalized predominantly with MTs. PKCs were associated with newly formed MTs invading the actin-rich peripheral growth cone domain after PKC activation. Our results are consistent with a central role for PKCs in regulating MT extension.     

Sildenafil (Viagra) induces powerful cardioprotective effect via opening of mitochondrial K(ATP) channels in rabbits

Sildenafil citrate (Viagra) is the pharmacological agent used to treat erectile dysfunction in men. Because this drug has a vasodilatory effect, we hypothesized that such an action may induce a preconditioning-like cardioprotective effect via opening of mitochondrial ATP-sensitive K (K(ATP)) channels. Rabbits were treated with sildenafil citrate (0.7 mg/kg iv) either 30 min (acute phase) or 24 h (delayed phase) before 30 min of ischemia and 3 h of reperfusion. Mitochondrial K(ATP) channel blocker 5-hydroxydecanoate (5-HD, 5 mg/kg iv) was given 10 min before ischemia-reperfusion. Infarct size was measured by tetrazolium staining. Sildenafil caused reduction in arterial blood pressure within 2 min of treatment, which returned to nearly baseline levels 3 min later. The infarct size (% risk area, means +/- SE) reduced from 33.8 +/- 1.7 in control rabbits to 10.8 +/- 0.9 during the acute phase (68% reduction, P < 0.05) and 19.9 +/- 2.0 during the delayed phase (41% reduction, P < 0.05). 5-HD abolished protection with an increase in infarct size to 35.6 +/- 0.4% and 36.8 +/- 1.6% during the acute and delayed phase, respectively (P < 0.05). Similar acute and delayed cardioprotective effects were observed when sildenafil was administered orally. Systemic hemodynamics also decreased after oral administration of the drug. However, these changes were mild and occurred slowly. For the first time, we demonstrate that sildenafil induces acute and delayed protective effects against ischemia-reperfusion injury, which are mediated by opening of mitochondrial K(ATP) channels.     

Transport of ions across peritoneal membrane

The electrical conductance of ions across the peritoneal membrane of young buffalo (approximately 18-24 months old) has been recorded. Aqueous solutions of NaF, NaNO3, NaCl, Na2SO4, KF, KNO3, KCl, K2SO4, MgCl2, CaCl2, CrCl3, MnCl2, FeCl3, CoCl2, and CuCl2 were used. The conductance values have been found to increase with increase in concentration as well as with temperature (15 to 35 degrees C) in these cases. The slope of plots of specific conductance, kappa, versus concentration exhibits a decrease in its values at relatively higher concentrations compared to those in extremely dilute solutions. Also, such slopes keep on increasing with increase in temperature. In addition, the conductance also attains a maximum limiting value at higher concentrations in the said cases. This may be attributed to a progressive accumulation of ionic species within the membrane. The kappa values of electrolytes follow the sequence for the anions: SO4(2-)>Cl->NO3->F- while that for the cations: K+>Na+>Ca2+>Mn2+>Co2+>Cu2+>Mg2+>Cr3+>Fe3+. In addition, the diffusion of ions depends upon the charge on the membrane and its porosity. The membrane porosity in relation to the size of the hydrated species diffusing through the membrane appears to determine the above sequence. As the diffusional paths in the membrane become more difficult in aqueous solutions, the mobility of large hydrated ions gets impeded by the membrane framework and the interaction with the fixed charge groups on the membrane matrix. Consequently, the membrane pores reduce the conductance of small ions, which are much hydrated. An increase in conductance with increase in temperature may be due to the state of hydration, which implies that the energy of activation for the ionic transport across the membrane follows the sequence of crystallographic radii of ions accordingly. The Eyring's equation, kappa=(RT/Nh)exp[-DeltaH*/RT]exp[DeltaS*/R], has been found suitable for explaining the temperature dependence of conductance in the said cases. This is apparent from the linear plots of log[kappaNh/RT] versus 1/T. The results indicate that the permeation of ions through the membrane giving negative values of DeltaS* suggest that there may be formation of either covalent linkage between the penetrating ions and the membrane material or else the permeation may not be the rate-determining step. On the one hand, a high DeltaS* value associated with the high value of energy of activation, Ea, for diffusion may suggest the existence of either a large zone of activation or loosening of more chain segments of the membrane. On the other hand, low value of DeltaS* implies that converse is true in such cases, i.e., either a small zone of activation or no loosening of the membrane structure upon permeation.     

On the occurrence of ternary complexes in agarose gels as studied by the contrast variation method

The contrast variation method by isotopic labelling as used in small-angle neutron scattering experiments is applied to the case of agarose systems (sol and gels) in order to cast some light on the occurrence of complexes (crystallosolvates). This approach proves to be helpful for giving further support to the existence of ternary complexes in agarose/water/DMSO gels and in agarose/DMSO sols. The results are discussed in the light of temperature-composition phase diagrams established previously.     

Genetic diversity analysis of North Africa’s barley using SSR markers

It was demonstrated that some North Africa barley accessions have diverse tolerance sources for abiotic stresses and a good nutritional quality, but the studies done were incomplete since they were realized separately in each country apart.To implement a more complete analysis, 31 barley accessions originated from North Africa (Algeria, Tunisia and Egypt) were analyzed using 11 SSR markers selected from the seven barley linkage groups for studying the genetic diversity among these chosen barley accessions.Over the 11 SSR markers, a total of 478 reproducible bands were scored with an average of 2.13 alleles/primer and the average polymorphism information content of 0.5.Genetic distance analysis of the 31 accessions showed a large genetic diversity and high number of different groups. The most accessions are clustered according to their eco-geographical origin, according to their pedigree and agronomic characters or according to the caryopsis character (hulled or naked caryopsis). This high number of obtained groups is an invaluable aid in crop improvement strategies and confirms the opinion suggesting that North Africa could be a secondary center of origin of barley. The various growing conditions and the multiple uses of barley in each country may be the cause of the large variability of the barley germplasm in each region.     

HDM2 antagonist MI-219 (spiro-oxindole), but not Nutlin-3 (cis-imidazoline), regulates p53 through enhanced HDM2 autoubiquitination and degradation in human malignant B-cell lymphomas

Background

Lymphomas frequently retain wild-type (wt) p53 function but overexpress HDM2, thereby compromising p53 activity. Therefore, lymphoma is a suitable model for studying the therapeutic value of disrupting the HDM2-p53 interaction by small-molecule inhibitors (SMIs). HDM2 have been developed and are under various stages of preclinical and clinical investigation. Previously, we examined the anti-lymphoma activity of MI-319, the laboratory grade of a new class of HDM2 SMI, the spiro-oxindole, in follicular lymphoma. Since then, MI-219, the clinical grade has become readily available. This study further examines the preclinical effects and mechanisms of MI-219 in a panel of human lymphoma cell lines as well as a cohort of patient-derived B-lymphcytes for its potential clinical use.

Results

Preclinical assessment of MI-219 was evaluated by means of an in vitro and ex vivo approach and compared to Nutlin-3, the gold standard. Characterization of p53 activity and stability were assessed by quantitative PCR, Western blot, and immunoprecipitation. Biological outcome was measured using Trypan blue exclusion assay, Annexin V/PI, PARP and caspase-3 cleavage. Surprisingly, the overall biological effects of Nutlin-3 were more delayed (48 h) while MI-219 triggered an earlier response (12-24 h), predominantly in the form of apoptotic cell death. Using a cell free autoubiquitination assay, neither agent interfered with HDM2 E3 ligase function. MI-219 was more effective in upregulating wt-p53 stabilization compared to Nutlin-3. MI-219, but not Nutlin-3, enhanced the autoubiquitination and degradation of HDM2.

Conclusions

Our data reveals unexpected differences between MI-219 and the well-studied Nutlin-3 in lymphoma cell lines and patient samples. We suggest a novel mechanism for MI-219 that alters the functional activity of HDM2 through enhanced autoubiquitination and degradation. Additionally, this mechanism appears to correspond to biological outcome. Our results provide evidence that different classes of HDM2 SMIs elicit molecular events that extend beyond HDM2-p53 dissociation which may be of biological and potentially therapeutic importance.     

Unique profile of ordered arrangements of repetitive elements in the C57BL/6J mouse genome implicating their functional roles

The entirety of all protein coding sequences is reported to represent a small fraction (~2%) of the mouse and human genomes; the vast majority of the rest of the genome is presumed to be repetitive elements (REs). In this study, the C57BL/6J mouse reference genome was subjected to an unbiased RE mining to establish a whole-genome profile of RE occurrence and arrangement. The C57BL/6J mouse genome was fragmented into an initial set of 5,321 units of 0.5 Mb, and surveyed for REs using unbiased self-alignment and dot-matrix protocols. The survey revealed that individual chromosomes had unique profiles of RE arrangement structures, named RE arrays. The RE populations in certain genomic regions were arranged into various forms of complexly organized structures using combinations of direct and/or inverse repeats. Some of these RE arrays spanned stretches of over 2 Mb, which may contribute to the structural configuration of the respective genomic regions. There were substantial differences in RE density among the 21 chromosomes, with chromosome Y being the most densely populated. In addition, the RE array population in the mouse chromosomes X and Y was substantially different from those of the reference human chromosomes. Conversion of the dot-matrix data pertaining to a tandem 13-repeat structure within the Ch7.032 genome unit into a line map of known REs revealed a repeat unit of ~11.3 Kb as a mosaic of six different RE types. The data obtained from this study allowed for a comprehensive RE profiling, including the establishment of a library of RE arrays, of the reference mouse genome. Some of these RE arrays may participate in a spectrum of normal and disease biology that are specific for mice.     

Effects of MASP-1 of the complement system on activation of coagulation factors and plasma clot formation

Background

Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis.

Methodology/Principal Findings

We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis.

Conclusions/Significance

We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation.