Polyoxygenated Cyclohexenes and Other Constituents of Cleistochlamys kirkii Leaves.

Thirteen new metabolites, including the polyoxygenated cyclohexene derivatives cleistodiendiol (1), cleistodienol B (3), cleistenechlorohydrins A (4) and B (5), cleistenediols A-F (6-11), cleistenonal (12), and the butenolide cleistanolate (13), 2,5-dihydroxybenzyl benzoate (cleistophenolide, 14), and eight known compounds (2, 15-21) were isolated from a MeOH extract of the leaves of Cleistochlamys kirkii. The purified metabolites were identified by NMR spectroscopic and mass spectrometric analyses, whereas the absolute configurations of compounds 1, 17, and 19 were established by single-crystal X-ray diffraction. The configuration of the exocyclic double bond of compound 2 was revised based on comparison of its NMR spectroscopic features and optical rotation to those of 1, for which the configuration was determined by X-ray diffraction. Observation of the co-occurrence of cyclohexenoids and heptenolides in C. kirkii is of biogenetic and chemotaxonomic significance. Some of the isolated compounds showed activity against Plasmodium falciparum (3D7, Dd2), with IC50 values of 0.2-40 µM, and against HEK293 mammalian cells (IC50 2.7-3.6 µM). While the crude extract was inactive at 100 µg/mL against the MDA-MB-231 triple-negative breast cancer cell line, some of its isolated constituents demonstrated cytotoxic activity with IC50 values ranging from 0.03-8.2 µM. Compound 1 showed the most potent antiplasmodial (IC50 0.2 µM) and cytotoxic (IC50 0.03 µM, MDA-MB-231 cell line) activities. None of the compounds investigated exhibited translational inhibitory activity in vitro at 20 µM.

A novel mutation in GATA6 causes pancreatic agenesis.

Heterozygous mutations in GATA6 have been linked to pancreatic agenesis and cardiac malformations. The aim of this study was to describe a new mutation in GATA6 in an infant with pancreatic agenesis, associated with truncus arteriosus and absent gallbladder. Clinical data were obtained from chart review. Gene sequencing was performed on genomic DNA. The patient was a female infant diagnosed shortly after birth with a severe cardiac malformation, absent gallbladder, anomalous hepatic blood flow, unilateral hydronephrosis and hydroureter, neonatal diabetes, and pancreatic exocrine insufficiency. Despite prolonged intensive management care, she died at 3 months of age because of cardiac complications. Analysis of her genomic DNA revealed a novel missense mutation of GATA6. The novel mutation described in this case extends the list of GATA6 mutations causing pancreatic agenesis and cardiac malformations.

Iron-regulated surface determinant B (IsdB) promotes Staphylococcus aureus adherence to and internalization by non-phagocytic human cells.

Staphylococcus aureus is a human pathogen that causes invasive and recurring infections. The ability to internalize into and persist within host cells is thought to contribute to infection. Here we report a novel role for the well-characterized iron-regulated surface determinant B (IsdB) protein which we have shown can promote adhesion of 293T, HeLa cells and platelets to immobilized bacteria independently of its ability to bind haemoglobin. IsdB bound to the active form of the platelet integrin αIIb ß3 , both on platelets and when the integrin was expressed ectopically in CHO cells. IsdB also promoted bacterial invasion into human cells. This was clearly demonstrated with bacteria lacking fibronectin-binding proteins (FnBPs), which are known to promote invasion in the presence of fibronectin. However, IsdB also contributed significantly to invasion by cells expressing FnBPs in the presence of serum. Thus IsdB appears to be able to interact with the broader family of integrins that bind ligands with the RGD motif and to act as a back up mechanism to promote interactions with mammalian cells.

PRDM14 inhibits 293T cell proliferation by influencing the G1/S phase transition.

PRDM14 (PRDI-BF1 and RIZ domain-containing 14), a transcription factor, plays important roles in primordial germ cell specification and embryonic stem cell pluripotency, and supports the maintenance of self-renewal by promoting the expression of stem cell markers while also repressing the expression of differentiation factors. As a proto-oncogene, the ectopic expression of PRDM14 can enhance breast cell growth and reduce breast cell sensitivity to chemotherapeutic drugs. Conversely, knockdown of PRDM14 expression induces apoptosis in breast cancer cells and restores their sensitivity to chemotherapeutic drugs. Here, we sought to identify the role of PRDM14 in 293T cells. PRDM14-infected 293T cells exhibited an abnormal morphology, and we found that ectopic expression of PRDM14 inhibits colony formation, cell proliferation and metastasis. In addition, our data indicated that PRDM14 influences the G1/S phase transition of 293T cells by inducing the expression of cell cycle regulators. In conclusion, these results showed that PRDM14 inhibits 293T cell proliferation by influencing the G1/S phase transition and impacts cell migration by regulating the level of MMP/TIMP expression, thus mediating extracellular matrix degradation.

Non-ventricular, Clinical, and Functional Features of the RyR2(R420Q) Mutation Causing Catecholaminergic Polymorphic Ventricular Tachycardia.

INTRODUCTION AND OBJECTIVES: Catecholaminergic polymorphic ventricular tachycardia is a malignant disease, due to mutations in proteins controlling Ca(2+) homeostasis. While the phenotype is characterized by polymorphic ventricular arrhythmias under stress, supraventricular arrhythmias may occur and are not fully characterized. METHODS: Twenty-five relatives from a Spanish family with several sudden deaths were evaluated with electrocardiogram, exercise testing, and optional epinephrine challenge. Selective RyR2 sequencing in an affected individual and cascade screening in the rest of the family was offered. The RyR2(R420Q) mutation was generated in HEK-293 cells using site-directed mutagenesis to conduct in vitro functional studies. RESULTS: The exercise testing unmasked catecholaminergic polymorphic ventricular tachycardia in 8 relatives (sensitivity = 89%; positive predictive value = 100%; negative predictive value = 93%), all of them carrying the heterozygous RyR2(R420Q) mutation, which was also present in the proband and a young girl without exercise testing, a 91% penetrance at the end of the follow-up. Remarkably, sinus bradycardia, atrial and junctional arrhythmias, and/or giant post-effort U-waves were identified in patients. Upon permeabilization and in intact cells, the RyR2(R420Q) expressing cells showed a smaller peak of Ca(2+) release than RyR2 wild-type cells. However, at physiologic intracellular Ca(2+) concentration, equivalent to the diastolic cytosolic concentration, the RyR2(R420Q) released more Ca(2+) and oscillated faster than RyR2 wild-type cells. CONCLUSIONS: The missense RyR2(R420Q) mutation was identified in the N-terminus of the RyR2 gene in this highly symptomatic family. Remarkably, this mutation is associated with sinus bradycardia, atrial and junctional arrhythmias, and giant U-waves. Collectively, functional heterologous expression studies suggest that the RyR2(R420Q) behaves as an aberrant channel, as a loss- or gain-of-function mutation depending on cytosolic intracellular Ca(2+) concentration.

Mutations in the Corneal Endothelial Dystrophy-Associated Gene SLC4A11 Render the Cells More Vulnerable to Oxidative Insults.

PURPOSE: To investigate the effect of mutations in SLC4A11 on cellular localization of the protein, mitochondrial function, and apoptosis due to oxidative stress. Mutations in SLC4A11 have been associated with 2 different forms of corneal endothelial dystrophy that lead to degeneration of the corneal endothelium, causing opacity of the cornea and gradual vision loss. METHODS: HEK 293 cells were transfected with wild-type SLC4A11 or mutants, Ser213Leu, Arg233Cys, Gly418Asp, and Thr584Lys, and exposed to oxidative stress. Cellular localization of the proteins was detected by confocal microscopy, whereas mitochondrial dysfunction, reactive oxygen species (ROS) generation, and apoptosis were analyzed by flow cytometry and a colorimetric assay. Expressions of antioxidant genes were quantitated by real-time polymerase chain reaction. RESULTS: Although wild-type SLC4A11 was localized on the cell membrane, mutant proteins were found diffused in the cytoplasm. Mutations in SLC4A11 caused an increase in generation of ROS and mitochondrial dysfunction due to oxidative stress. NRF2, HO-1, and NQO expression decreased significantly, and a higher rate of apoptosis was detected in cells with mutant proteins under oxidative stress. CONCLUSIONS: Our data suggest that mutations in SLC4A11 cause retention of the protein in the cytoplasm and generate increased reactive oxygen species. We found that cells containing mutant SLC4A11 are more vulnerable to oxidative and mitochondrial damage, less able to overcome oxidative stress through the expression of sufficient levels of antioxidant genes, and are more prone to apoptotic death.