Is the failing heart out of fuel or a worn engine running rich? A study of mitochondria in old spontaneously hypertensive rats

Hypertension now affects about 600 million people worldwide and is a leading cause of death in the Western world. The spontaneously hypertensive rat (SHR), provides a useful model to investigate hypertensive heart failure (HF). The SHR model replicates the clinical progression of hypertension in humans, wherein early development of hypertension is followed by a long stable period of compensated cardiac hypertrophy that slowly progresses to HF. Although the hypertensive failing heart generally shows increased substrate preference towards glucose and impaired mitochondrial function, the cause-and-effect relationship between these characteristics is incompletely understood. To explore these pathogenic processes, we compared cardiac mitochondrial proteomes of 20-month-old SHR and Wistar-Kyoto controls by iTRAQ-labelling combined with multidimensional LC/MS/MS. Of 137 high-scoring proteins identified, 79 differed between groups. Changes were apparent in several metabolic pathways, chaperone and antioxidant systems, and multiple subunits of the oxidative phosphorylation complexes were increased (complexes I, III and IV) or decreased (complexes II and V) in SHR heart mitochondria. Respiration assays on skinned fibres and isolated mitochondria showed markedly lower respiratory capacity on succinate. Enzyme activity assays often also showed mismatches between increased protein expression and activities suggesting elevated protein expression may be compensatory in the face of pathological stress.     

Structure-activity relationship of leucyladenylate sulfamate analogues as leucyl-tRNA synthetase (LRS)-targeting inhibitors of Mammalian target of rapamycin complex 1 (mTORC1)

Leucyl-tRNA synthetase (LRS) plays an important role in amino acid-dependent mTORC1 signaling, which is known to be associated with cellular metabolism and proliferation. Therefore, LRS-targeting small molecules that can suppress mTORC1 activation may provide an alternative strategy to current anticancer therapy. In this work, we developed a library of leucyladenylate sulfate analogues by extensively modifying three different pharmacophoric regions comprising adenine, ribose and leucine. Several effective compounds were identified by cell-based mTORC1 activation assays and further tested for anticancer activity. The selected compounds mostly exhibited selective cytotoxicity toward five different cancer cell lines, supporting the hypothesis that the LRS-mediated mTORC1 pathway is a promising alternative target to current therapeutic approaches.     

Solution 1H NMR study of the active site molecular structure and magnetic properties of the cyanomet complex of the isolated alpha-chain from human hemoglobin A

The solution electronic and molecular structure for the heme pocket of the cyanomet complex of the isolated alpha-chain of human adult hemoglobin (HbA) has been investigated by homonuclear two-dimensional 1H NMR in order to establish an assignment protocol for the dimeric chain that will guide similar assignments in the intact, heterotetrameric HbA complex, and to compare the structures of the alpha-chain with its subunit in HbA. The target residues are those that exhibit significant (>0.2 ppm) dipolar shifts, as predicted by a "preliminary" set of magnetic axes determined from a small set of easily assigned active site residues. All 97 target residues (approximately 70% of total) were assigned by taking advantage of the temperature dependence predicted by the "preliminary" magnetic axes for the polypeptide backbone; they include all residues proposed to play a significant role in modulating the ligand affinity in the tetramer HbA. Left unassigned are the A-helix, the end of the G-helix and the beginning of the H-helix where dipolar shifts are less than 0.2 ppm. The complete assignments allow the determination of a robust set of orientation and anisotropies of the paramagnetic susceptibility tensor that leads to quantitative interpretation of the dipolar shifts of the alpha-chain in terms of the crystal coordinates of the alpha-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated alpha-chain relative to that in the intact HbA. The major magnetic axis, which is correlated with the tilt of the Fe-CN unit, is tilted approximately 10 degrees from the heme normal so that the Fe-CN unit is tilted toward the beta-meso-H in a fashion remarkably similar to the Fe-CO tilt in HbACO. It is concluded that a set of "preliminary" magnetic axes and the use of variable temperature two-dimensional NMR spectra are crucial to effective assignments in the cyanomet alpha-chain and that this approach should be similarly effective in HbA.     

Functional integrity of intravenous immunoglobulin following irradiation with a virucidal dose of gamma radiation.

Although intravenous immunoglobulins (IVIG) and other plasma therapeutics have had a relatively good safety record, improved methods for viral clearance are constantly being evaluated and incorporated into new manufacturing processes. Gamma irradiation has been used routinely to assure sterility of healthcare products and medical devices, but it has not been applied successfully as a viral inactivation method for biologics. We examine whether virucidal doses of gamma irradiation (50 kGy) can be delivered to a manufacturing intermediate form of IVIG, a fractionated plasma paste, with negligible effect on structural and functional integrity of purified IgG product. Immunoglobulins from paste were examined for radiation-induced damage by SDS-PAGE and ELISAs utilizing viral antigens specific for rubella, CMV and mumps. Fc domain integrity was assessed by immunoblotting, quantitatively comparing the binding of irradiated and non-irradiated materials to cell surface Fcgamma receptors, and by employing quantitative RT-PCR to study the kinetics of accumulation of mRNA for the immune modulatory cytokines IL-1alpha, IL-1beta, IL-4, IL-8, IFNgamma, and TNFalpha. The results demonstrate that Fab and Fc domains of IVIG remain essentially intact and functional after gamma irradiation to virucidal doses, suggesting that this method could be used to enhance the safety of IVIG products.     

Silencing of Vlaro2 for chorismate synthase revealed that the phytopathogen Verticillium longisporum induces the cross-pathway control in the xylem

The first leaky auxotrophic mutant for aromatic amino acids of the near-diploid fungal plant pathogen Verticillium longisporum (VL) has been generated. VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem.     

Identification of a Potent Endothelium-Derived Angiogenic Factor

The secretion of angiogenic factors by vascular endothelial cells is one of the key mechanisms of angiogenesis. Here we report on the isolation of a new potent angiogenic factor, diuridine tetraphosphate (Up₄U) from the secretome of human endothelial cells. The angiogenic effect of the endothelial secretome was partially reduced after incubation with alkaline phosphatase and abolished in the presence of suramin. In one fraction, purified to homogeneity by reversed phase and affinity chromatography, Up₄U was identified by MALDI-LIFT-fragment-mass-spectrometry, enzymatic cleavage analysis and retention-time comparison. Beside a strong angiogenic effect on the yolk sac membrane and the developing rat embryo itself, Up₄U increased the proliferation rate of endothelial cells and, in the presence of PDGF, of vascular smooth muscle cells. Up₄U stimulated the migration rate of endothelial cells via P2Y2-receptors, increased the ability of endothelial cells to form capillary-like tubes and acts as a potent inducer of sprouting angiogenesis originating from gel-embedded EC spheroids. Endothelial cells released Up₄U after stimulation with shear stress. Mean total plasma Up₄U concentrations of healthy subjects (N = 6) were sufficient to induce angiogenic and proliferative effects (1.34±0.26 nmol L^-1). In conclusion, Up₄U is a novel strong human endothelium-derived angiogenic factor.     

Protein tyrosine phosphatase 1B inhibitors isolated from Morus bombycis

Bioassay-guided fractionation of the chloroform-soluble fraction of Morus bombycis, using an in vitro PTP1B inhibitory assay led to the identification of three 2-arylbenzofurans, albafuran A (1), mulberrofuran W (2) and mulberrofuran D (6), along with three chalcone-derived Diels–Alder products, kuwanon J (3), kuwanon R (4), and kuwanon V (5). Compounds 1–6 showed remarkable inhibitory activity against PTP1B with IC₅₀ values ranging from 2.7 to 13.8 μM. Inhibition kinetics were analyzed by Lineweaver–Burk plots, which suggested that compounds 1–6 inhibited PTP1B in a mixed-type manner. The present results indicate that the respective lipophilic and hydroxyl groups of 2-arylbenzofurans and chalcone-derived Diels–Alder products play an important role in inhibition of PTP1B.     

Discovery of potent inhibitors of receptor protein tyrosine phosphatase sigma through the structure-based virtual screening

Receptor protein tyrosine phosphatase sigma (PTPσ) has proved to be a promising target for the development of therapeutics for the treatment of neurological diseases. Here, we report the first example for a successful application of the structure-based virtual screening to identify the novel small-molecule inhibitors of PTPσ. These inhibitors revealed high potencies with the associated IC₅₀ values ranging from 0.1 to 1.3 μM and were also screened for having desirable physicochemical properties as a drug candidate. Therefore, they deserve consideration for further development by structure–activity relationship studies to develop therapeutics for neurological diseases. Structural features relevant to the stabilization of the newly identified inhibitors in the active site of PTPσ are discussed in detail.