Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner

In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ataxia telangiectasia mutated (ATM) gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, we generated gene-targeted Chk2-deficient mice. Unlike ATM(-/-) and p53(-/-) mice, Chk2(-/-) mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2(-/-) mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G(1)/S arrest. Quantitative comparison of the G(1)/S checkpoint, apoptosis, and expression of p53 proteins in Chk2(-/-) versus ATM(-/-) thymocytes suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis was restored in Chk2(-/-) thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3(+) related (ATR) were mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis.     

Hypergonadotropic ovarian failure associated with an inherited mutation of human bone morphogenetic protein-15 (BMP15) gene

Hypergonadotropic ovarian failure is a common cause of female infertility. It is a heterogeneous disorder that, in the most severe forms, is a result of ovarian dysgenesis (OD). Most OD cases are associated with major X-chromosome abnormalities, but the pathogenesis of this disorder is still largely undefined in patients with a normal karyotype. Animal models showed the important role in female reproduction played by the product of a gene located at Xp11.2 in humans (BMP15). BMP15 is an oocyte-specific growth/differentiation factor that stimulates folliculogenesis and granulosa cell (GC) growth. We report two sisters with a normal karyotype who are affected with hypergonadotropic ovarian failure due to OD. The familial presentation suggested a genetic origin, and candidate genes were screened for mutations. A heterozygous nonconservative substitution in the pro region of BMP15 (Y235C) was identified in both sisters but not in 210 control alleles. This mutation was inherited from the father. Mutant BMP15 appears to be processed abnormally, is associated with reduced GC growth, and antagonizes the stimulatory activity of wild-type protein on GC proliferation. In conclusion, the first natural mutation in human BMP15 is associated with familial OD, indicating that the action of BMP15 is required for the progression of human folliculogenesis. This condition represents an exceptional example of X-linked human disease exclusively affecting heterozygous females who inherited the genetic alteration from the unaffected father. BMP15 defects are involved in the pathogenesis of hypergonadotropic ovarian failure in humans.     

p53 Represses the Mevalonate Pathway to Mediate Tumor Suppression

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Cross Talk among the Glycoproteins Involved in Herpes Simplex Virus Entry and Fusion: the Interaction between gB and gH/gL Does Not Necessarily Require gD

The gD, gB, and gH/gL glycoprotein quartet constitutes the basic apparatus for herpes simplex virus (HSV) entry into the cell and fusion. gD serves as a receptor binding glycoprotein and trigger of fusion. The conserved gB and gH/gL execute fusion. Central to understanding HSV entry/fusion has become the dissection of how the four glycoproteins engage in cross talk. While the independent interactions of gD with gB and gD with gH/gL have been documented, less is known of the interaction of gB with gH/gL. So far, this interaction has been detected only in the presence of gD by means of a split green fluorescent protein complementation assay. Here, we show that gB interacts with gH/gL in the absence of gD. The gB-gH/gL complex was best detected with a form of gB in which the endocytosis and phosphorylation motif have been deleted; this form of gB persists in the membranes of the exocytic pathway and is not endocytosed. The gB-gH/gL interaction was detected both in whole transfected cells by means of a split yellow fluorescent protein complementation assay and, biochemically, by a pull-down assay. Results with a panel of chimeric forms of gB, in which portions of the glycoprotein bracketed by consecutive cysteines were replaced with the corresponding portions from human herpesvirus 8 gB, favor the view that gB carries multiple sites for interaction with gH/gL, and one of these sites is located in the pleckstrin-like domain 1 carrying the bipartite fusion loop.Entry of herpes simplex virus (HSV) into the cell requires a multipartite apparatus made of a quartet of viral glycoproteins, gD, gB, and the heterodimer gH/gL, and a multistep process that culminates in the fusion of the virion envelope with cell membranes (5, 6, 10, 25, 36, 41). gD serves as the receptor-binding glycoprotein, able to interact with alternative receptors, nectin1, herpesvirus entry mediator (HVEM) and, in some cells, modified heparan sulfate (9, 13, 30, 39). It can also be engineered to accept heterologous ligands able to interact with selected receptors present on tumor cells and thus represents a tool to redirect HSV tropism (21, 28, 29, 42). The heterodimer gH/gL and gB execute fusion and constitute the conserved fusion apparatus across the Herpesviridae family. gB structure in the postfusion conformation shows a trimer with a central coiled coil (19). gH shows elements typical of type 1 fusion glycoproteins, in particular, helices able to interact with membranes, and two heptad repeats potentially able to form a coiled coil (12, 15-18). The discovery that a soluble form of gD enables entry of gD-null virions revealed that gD serves the additional function of triggering fusion and led to the view that the major roles of gD are to sense that virus has reached a receptor-positive cell and to signal to gB and gH/gL that fusion is to be executed (8). Biochemical and structural analyses showed that the C-terminal region of the gD ectodomain, containing the profusion domain required for fusion but not for receptor binding, can undergo major conformational changes (11, 24). Specifically, it binds the gD core and masks or hinders the receptor binding sites, conferring upon the molecule a closed, auto-inhibited conformation (24). Alternatively, it may unfold, conferring upon gD an open conformation. It was proposed that the C terminus of gD unfolds from gD core at receptor binding and recruits gH/gL and gB to a quaternary complex. A key feature of the model was that complexes among the glycoprotein quartet were not preformed, but, rather, they would assemble at the onset of or at fusion execution.Central to understanding HSV entry/fusion has become the dissection of the interactions that occur among the members of the glycoprotein quartet and their significance to the process. A first evidence of a gD-gH/gL interaction was provided in coimmunoprecipitation studies (35). Interactions between gD and gH/gL and between gD and gB were subsequently detected by split green fluorescence protein (GFP) complementation assays, implying that gD can recruit gB and gH/gL independently of one another, a result that argues against a stepwise recruitment of the glycoproteins to gD. In agreement with the proposed model, the interaction between gH/gL and gB was detected in the presence of transfected or soluble gD (1, 2). However, further studies highlighted levels of complexity not foreseen in the initial model. Thus, pull-down analyses showed that the interaction sites in gD with gB and with gH/gL lie in part outside the C-terminal portion of the gD ectodomain, that resting virions contain small amounts of gD in complex with gB and with gH/gL prior to encountering cells, and that de novo gD-gB complexes were not detected at virus entry into the cell (14).A major objective of current studies was to analyze the interaction of gB with gH/gL. We documented the interaction by two independent assays, i.e., by a complementation assay of split yellow fluorescent protein Venus (herein indicated as YFP) (31) in whole cells and, biochemically, by a pull-down assay. The latter was applied recently in our laboratory and is based on the ability of One-Strep-tagged proteins (e.g., gH) to specifically absorb to Strep-Tactin resin and thus retain any protein in complex (14). To preliminarily search for gB regions critical for the interaction with gH/gL, we engineered chimeric forms of HSV-1 and human herpesvirus 8 (HHV-8) gB in which the cysteines were preserved. While none of the chimeras was completely defective in the interaction, the interactions in the chimeras carrying substitutions in the pleckstrin-like domain 1—the domain that carries the bipartite fusion loops—were hampered. Altogether, the results underscore the ability of gB to interact with gH/gL in the absence of gD and favor the view that sites in gB for interaction with gH/gL involve multiple contacts, one of which is located in the domain that carries the fusion loops.     

Galvanic microparticles increase migration of human dermal fibroblasts in a wound-healing model via reactive oxygen species pathway

Electrical signals have been implied in many biological mechanisms, including wound healing, which has been associated with transient electrical currents not present in intact skin. One method to generate electrical signals similar to those naturally occurring in wounds is by supplementation of galvanic particles dispersed in a cream or gel. We constructed a three-layered model of skin consisting of human dermal fibroblasts in hydrogel (mimic of dermis), a hydrogel barrier layer (mimic of epidermis) and galvanic microparticles in hydrogel (mimic of a cream containing galvanic particles applied to skin). Using this model, we investigated the effects of the properties and amounts of Cu/Zn galvanic particles on adult human dermal fibroblasts in terms of the speed of wound closing and gene expression. The collected data suggest that the effects on wound closing are due to the ROS-mediated enhancement of fibroblast migration, which is in turn mediated by the BMP/SMAD signaling pathway. These results imply that topical low-grade electric currents via microparticles could enhance wound healing.     

New HPLC–MS method for the simultaneous quantification of the antileukemia drugs imatinib,dasatinib, and nilotinib in human plasma

A new method using high performance liquid chromatography coupled with electrospray mass spectrometry is described for the quantification of plasma concentration of tyrosine kinase inhibitors imatinib, dasatinib and nilotinib. A simple protein precipitation extraction procedure was applied on 250 μl of plasma aliquots. Chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a gradient (acetonitrile and water + formic acid 0.05%) on a C18 reverse phase analytical column with 20 min of analytical run, at flow rate of 1 ml/min. Mean intra-day and inter-day precision for all compounds were 4.3 and 11.4%; mean accuracy was 1.5%; extraction recovery ranged within 95 and 114%. Calibration curves ranged from 10,000 to 62.5 ng/ml. The limit of quantification was set at 78.1 ng/ml for imatinib and at 62.5 ng/ml for dasatinib and nilotinib. This novel developed methodology allows a specific, sensitive and reliable simultaneous determination of the three tyrosine kinase inhibitors imatinib, dasatinib and nilotinib in a single chromatographic run, useful for drugs estimation in plasma of patients affected by chronic myeloid leukemia.     

The K(ATP)+ channel is involved in a low-amplitude permeability transition in plant mitochondria

Pea (Pisum sativum) stem mitochondria, energized by NADH, succinate or malate plus glutamate, underwent a spontaneous low-amplitude permeability transition (PT), which could be monitored by dissipation of the electrical potential (deltapsi) or swelling. The occurrence of the latter effects was dependent on O2 availability, because O2 shortage anticipated the manifestation of both deltapsi dissipation and swelling. Spontaneous deltapsi collapse was also monitored in sucrose-resuspended mitochondria and again O2 deprivation caused an anticipation of the phenomenon. However, in this case deltapsi dissipation was not accompanied by a parallel mitochondrial swelling. The latter effect was, indeed, evident only if mitochondria were resuspended in KCl (as osmoticum), or other cations with a molecular mass up to 100 Da (choline+). PT was also induced by protonophores (carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or free fatty acids) or valinomycin (only in KCl). The FCCP-induced dissipation of deltapsi and swelling were inhibited by ATP and stimulated (anticipated) by cyclosporin A or O2 shortage. The FCCP-induced PT was accompanied by the release of pyridine nucleotides from the matrix and of cytochrome c from the intermembrane space of KCl-resuspended mitochondria. The spontaneous and FCCP-induced low-amplitude PT of plant mitochondria are interpreted as due to the activity of a recently identified K(ATP)+ channel whose open/closed state is dependent on polarization of the inner membrane and on the oxidoreductive state of some sulfhydryl groups.