The immunomodulatory benzodiazepine Bz-423 inhibits B-cell proliferation by targeting c-myc protein for rapid and specific degradation

Myc proteins regulate cell growth and are oncogenic in many cancers. Although these proteins are validated molecular anticancer targets, new therapies aimed at modulating myc have yet to emerge. A benzodiazepine (Bz-423) that was discovered in efforts to find new drugs for lupus was found recently to have antiproliferative effects on Burkitt's lymphoma cells. We now show that the basis for the antiproliferative effects of Bz-423 is the rapid and specific depletion of c-myc protein, which is coupled to growth-suppressing effects on key regulators of proliferation and cell cycle progression. c-Myc is depleted as a result of signals coupled to Bz-423 binding its molecular target, the oligomycin sensitivity-conferring protein subunit of the mitochondrial F(1)F(o)-ATPase. Bz-423 inhibits F(1)F(o)-ATPase activity, blocking respiratory chain function and generating superoxide, which at growth-inhibiting concentrations triggers proteasomal degradation of c-myc. Bz-423-induced c-myc degradation is independent of glycogen synthase kinase but is substantially blocked by mutation of the phosphosensitive residue threonine 58, which when phosphorylated targets c-myc for ubiquitination and subsequent proteasomal degradation. Collectively, this work describes a new lead compound, with drug-like properties, which regulates c-myc by a novel molecular mechanism that may be therapeutically useful.     

Reduction of the disintegrin and metalloprotease ADAM12 in preeclampsia

OBJECTIVES: The secreted form of ADAM12 is a metalloprotease that may be involved in placental and fetal growth. We examined whether the concentration of ADAM12 in first-trimester maternal serum could be used as a marker for preeclampsia. METHODS: We developed a semiautomated, time-resolved, immunofluorometric assay for the quantification of ADAM12 in serum. The assay detected ADAM12 in a range of 78-1248 microg/L. Serum samples derived from women in the first trimester of a normal pregnancy (n = 324) and from women who later developed preeclampsia during pregnancy (n = 160) were obtained from the First Trimester Copenhagen Study. ADAM12 levels were assayed in these serum samples. Serum levels of ADAM12 were converted to multiples of the median (MoM) after log-linear regression of concentration versus gestational age. RESULTS: Serum ADAM12 levels in women who developed preeclampsia during pregnancy had a mean log MoM of -0.066, which was significantly lower than the mean log MoM of -0.001 for ADAM12 levels observed in serum samples from women with normal pregnancy (P = .008). The mean log MoM was even lower in serum derived from preeclamptic women whose infant's weight at birth was less than 2,500 g (n = 27, mean log MoM of -0.120, P = .053). CONCLUSION: The maternal serum levels of ADAM12 are significantly lower during the first trimester in women who later develop preeclampsia during pregnancy when compared with levels in women with normal pregnancies. Because the secreted form of ADAM12 cleaves insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5, the IGF axis may play a role in preeclampsia. ADAM12 may be a useful early marker for preeclampsia. LEVEL OF EVIDENCE: II-2.     

The cholesterol hypothesis: time for the obituary?

The cholesterol hypothesis links cholesterol intake and blood levels to cardiovascular disease. It has had enormous impact on health care and society during decades, but has little or no scientific backing that is relevant for the human species. Apparently, the hypothesis is false and should be buried.     

Mesenchymal stem cells are susceptible to human herpesviruses, but viral DNA cannot be detected in the healthy seropositive individual

Allogeneic stem cell transplantation is often complicated by reactivation of herpesviruses. Mesenchymal stem cells (MSC) are immunomodulatory and may be used to treat graft-versus-host disease. We investigated if herpesviruses infect and can be transmitted by MSC, and if MSC suppress immune responses to various infectious agents. Mesenchymal stem cells from healthy seropositive donors were evaluated with polymerase chain reaction for the most common herpesviruses: cytomegalovirus (CMV), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2, Epstein-Barr virus (EBV) and varicella zoster virus. The cytopathological effect (CPE) was investigated and viral antigens analyzed by immunofluorescence after in vitro exposure to CMV, HSV-1 and EBV. We also studied MSC effect on lymphocyte stimulation induced by various infectious agents. No viral DNA could be detected in MSC isolated from healthy seropositive individuals. However, a CPE was noted and intracellular viral antigens detected after infection in vitro by CMV and HSV-1, but not by EBV. The CMV and HSV-1 infections were productive. Lymphocyte proliferation by herpesviruses, candida mannan and protein A from Staphylococcus aureus was suppressed by MSC. The data indicate that the risk of herpesvirus transmission by transplantation of MSC from healthy seropositive donors is low. However, MSC may be susceptible to infection if infused in a patient with CMV or HSV-1 viremia. MSC transplantation may compromise the host's defense against infectious agents.     

Identification of Y589 and Y599 in the juxtamembrane domain of Flt3 as ligand-induced autophosphorylation sites involved in binding of Src family kinases and the protein tyrosine phosphatase SHP2

Early signal relay steps upon ligand binding to the receptor tyrosine kinase Flt3 (ie, sites of Flt3 autophosphorylation and subsequent docking partners) are mainly unresolved. By immunoprecipitation of specific tryptic peptides contained in the juxtamembrane region of human Flt3 and subsequent radiosequencing, we identified the tyrosine residues 572, 589, 591, and 599 as in vivo autophosphorylation sites. Focusing on Y589 and Y599, we examined Flt3 ligand (FL)-mediated responses in wild-type-Flt3-(WT-Flt3-), Y589F-Flt3-, and Y599F-Flt3-expressing 32D cells. Compared with WT-Flt3-32D cells upon ligand stimulation, 32D-Y589F-Flt3 showed enhanced Erk activation and proliferation/survival, whereas 32D-Y599F-Flt3 cells hereby displayed substantially diminished responses. Both pY589 and pY599 were identified as association sites for signal relay molecules including Src family kinases and SHP2. Consistently, 32D-Y589F-Flt3 and 32D-Y599F-Flt3 showed decreased FL-triggered activation of Src family kinases. Interference with the Src-dependent negative regulation of Flt3 signaling may account for the enhanced mitogenic response of Y589F-Flt3. Y599 was additionally found to interact with the protein tyrosine phosphatase SHP2 in a phosphorylation-dependent manner. As Y599F-Flt3-32D was unable to associate with and to phosphorylate SHP2 and since silencing of SHP2 in WT-Flt3-expressing cells mimicked the Y599F-Flt3 phenotype, we hypothesize that recruitment of SHP2 to pY599 contributes to FL-mediated Erk activation and proliferation.     

Sex Difference in Formation of Propofol Metabolites: A Replication Study

Women recover faster from propofol anaesthesia and have been described to have a higher incidence of awareness during surgery, compared to men – an effect that may be inherent in sex differences in propofol metabolism. In an observational study, 98 ASA I‐II patients treated with continuous propofol infusion were recruited. The associations between sex and CYP2B6 and UGT1A9 polymorphisms with dose‐ and weight‐adjusted area under the total plasma level time curves (AUC) for propofol, and its metabolites propofol glucuronide (PG), 4‐hydroxypropofol (OHP) and hydroxyl glucuronide metabolites 4‐hydroxypropofol‐1‐O‐β‐D‐glucuronide (Q1G) and 4‐hydroxypropofol‐4‐O‐β‐D‐glucuronide (Q4G), were analysed. Significantly higher AUC of PG (1.3 times, p = 0.03), Q1G (2.9 times, p < 0.001), Q4G (2.4 times, p < 0.01) and OHP (4.6 times, p = 0.01) were found in women (n = 53) than in men (n = 45) after intravenous infusion of propofol using target‐controlled infusion system. There was, however, no significant impact of gene polymorphisms on propofol biotransformation. The results, which are supported by a previous pilot study using a propofol bolus dose, suggest that, compared to men, more rapid propofol metabolism may occur in women – a factor that may contribute to the mentioned differences in the efficacy of propofol anaesthesia between male and female patients.     

Sensitized Clotting Time as a Clinical Method

The so-called sensitized clotting time is compared with the prothrombin content of the blood as determined by Owren's method. The test was not reliable enough for control in anticoagulant therapy.     

Cataracts in transgenic mice caused by a human papillomavirus type 18 E7 oncogene driven by KRT1-14

Human papillomavirus type 18 (HPV18) is a common cause of cervical cancer. To create a mouse model for this common neoplastic disease, we used a human keratin 14 promoter to drive the HPV18 E7 oncogene to create transgenic mice. No mice up to a year of age developed cervical cancer. However, all transgenic mice and none of the controls developed progressive bilateral cortical cataracts. By 6 months of age, the cortex liquefied leaving the lens nucleus. Proliferation of lens epithelium formed multifocal nodules and free floating lens epithelial cells within the liquefied cortex. These cells were hyperplastic not neoplastic. Other HPV transgenic stocks develop cataracts suggesting this virus may have a broad cellular tropism.