Hzf Determines cell survival upon genotoxic stress by modulating p53 transactivation

A critical unresolved issue about the genotoxic stress response is how the resulting activation of the p53 tumor suppressor can lead either to cell-cycle arrest and DNA repair or to apoptosis. We show here that hematopoietic zinc finger (Hzf), a zinc-finger-containing p53 target gene, modulates p53 transactivation functions in an autoregulatory feedback loop. Hzf is induced by p53 and binds to its DNA-binding domain, resulting in preferential transactivation of proarrest p53 target genes over its proapoptotic target genes. Thus, p53 activation results in cell-cycle arrest in Hzf wild-type MEFs, while in Hzf(-/-) MEFs, apoptosis is induced. Exposure of Hzf null mice to ionizing radiation resulted in enhanced apoptosis in several organs, as compared to in wild-type mice. These findings provide novel insights into the regulation of p53 transactivation function and suggest that Hzf functions as a key player in regulating cell fate decisions in response to genotoxic stress.     

Crystal structures of the luciferase and green fluorescent protein from Renilla reniformis

Due to its ability to emit light, the luciferase from Renilla reniformis (RLuc) is widely employed in molecular biology as a reporter gene in cell culture experiments and small animal imaging. To accomplish this bioluminescence, the 37-kDa enzyme catalyzes the degradation of its substrate coelenterazine in the presence of molecular oxygen, resulting in the product coelenteramide, carbon dioxide, and the desired photon of light. We successfully crystallized a stabilized variant of this important protein (RLuc8) and herein present the first structures for any coelenterazine-using luciferase. These structures are based on high-resolution data measured to 1.4 Å and demonstrate a classic α/β-hydrolase fold. We also present data of a coelenteramide-bound luciferase and reason that this structure represents a secondary conformational form following shift of the product out of the primary active site. During the course of this work, the structure of the luciferase's accessory green fluorescent protein (RrGFP) was also determined and shown to be highly similar to that of Aequorea victoria GFP.     

Toxicity of fatty acid hydroperoxides towards Yarrowia lipolytica: Implication of their membrane fluidizing action

Linoleic acid hydroperoxide (HPOD), substrate of hydroperoxide lyase, an enzyme of the lipoxygenase pathway, can be transformed into many aromatic compounds, the so-called “green notes”. The presence of linoleic acid hydroperoxide in the culture medium of Yarrowia lipolytica, the yeast expressing the cloned hydroperoxide lyase of green bell pepper, undoubtedly exerted an inhibition on the growth and a toxic effect with 90% of yeast cells died after 120 min of exposition in 100 mM HPOD solution. The increase in cell membrane fluidity evaluated by measuring fluorescence generalized polarization with the increasing concentration of HPOD in the medium confirmed the fluidizing action of HPOD on yeast membrane. In addition, we determined by infrared spectroscopy measurement that this compound rapidly diffused into model phospholipids [1, 2-Dimyristoyl-D54-sn-Glycero-3-Phosphocholine (DMPC-D54)] bilayer, modifying their general physical state and their phase transition. In the presence of various concentrations of HPOD, the phase transition of DMPC-D54 occurred with an increase of both the corresponding wave number shift and the temperature range but the phase transition temperature was not modified. These results show that the toxic effects of HPOD on the yeast Yarrowia lipolytica may be initially linked to a strong interaction of this compound with the cell membrane phospholipids and components.     

Synthesis and characterization of pyrrolidine derivatives as potent agonists of the human melanocortin-4 receptor

A series of trans-4-phenylpyrrolidine-3-carboxamides were synthesized and characterized as potent ligands of the human melanocortin-4 receptor. Interestingly, a pair of diastereoisomers 20f-1 and 20f-2 displayed potent functional agonist and antagonist activity, respectively. Thus, the 3S,4R-compound 20f-1 possessed a K(i) of 11nM and an EC(50) of 24nM, while its 3R,4S-isomer 20f-2 exhibited a K(i) of 8.6 and an IC(50) of 65nM. Both compounds were highly selective over other melanocortin receptor subtypes. The MC4R agonist 20f-1 also demonstrated efficacy in diet-induced obese rats.     

Amino-caprolactam derivatives as gamma-secretase inhibitors

A series of amino-caprolactam sulfonamides were developed from a screening hit. Compounds with good in vitro and in vivo gamma-secretase activity are reported.     

Non-ischemic cardiomyopathy patients derive superior mortality benefit from cardiac resynchronization therapy

Background

Cardiac Resynchronization Therapy (CRT) is indicated for the treatment of advanced heart failure with severe systolic dysfunction and intraventricular conduction delay. Patient selection for this technology is vital, though it remains unclear which patients benefit most from CRT. We tested the hypothesis that patients with non-ischemic cardiomyopathy have a superior mortality benefit from CRT than ischemic cardiomyopathy patients.

Methods

We evaluated 95 CRT patients to determine which factors predict mortality.

Results

Patients with non-ischemic cardiomyopathy had a significantly better prognosis than patients with ischemic cardiomyopathy.

Conclusion

Larger prospective studies can substantiate this finding and better delineate which patients benefit most from CRT.     

Optimization of a solid phase extraction procedure for prostaglandin E2, F2 alpha and their tissue metabolites

The primary prostaglandins PGE(2) and PGF(2 alpha) are metabolized in tissues by a series of enzymatic and non-enzymatic reactions. To measure metabolic rates and individual reaction rates it is necessary to extract the parent prostaglandins and metabolites before the separation and quantification of each compound is achieved. Here we have established and optimized a solid phase extraction (SPE) procedure to recover PGE(2), PGF(2 alpha) and their six enzymatic and non-enzymatic tissue metabolites from aqueous solutions including urine, plasma and tissue homogenate. We have used octadecyl-bonded silica gel as the stationary phase and methanol-water mixtures as binary mobile phases. The volumes and concentrations of the washing and elution solutions were optimized individually for each PG. Recoveries of all PG standards were quantitative except for PGEM, which was recovered at 80% efficiency. Biological matrix components interfered with the extraction in a PG- and matrix-specific fashion. Inclusion of 1% formic acid in the loading mixture raised recoveries from urine, plasma and tissue homogenate to >or=90%. This SPE method is the first that has been optimized by systematic elution studies for PGE(2), PGF(2 alpha) and the complement of their tissue metabolites. The procedure is simple, robust and can serve as an effective pre-purification step before downstream separation and quantification of each tissue metabolite of PGE(2) and PGF(2 alpha) from complex biological matrices.     

Detecting tumor response to treatment using hyperpolarized 13C magnetic resonance imaging and spectroscopy

Measurements of early tumor responses to therapy have been shown, in some cases, to predict treatment outcome. We show in lymphoma-bearing mice injected intravenously with hyperpolarized [1-(13)C]pyruvate that the lactate dehydrogenase-catalyzed flux of (13)C label between the carboxyl groups of pyruvate and lactate in the tumor can be measured using (13)C magnetic resonance spectroscopy and spectroscopic imaging, and that this flux is inhibited within 24 h of chemotherapy. The reduction in the measured flux after drug treatment and the induction of tumor cell death can be explained by loss of the coenzyme NAD(H) and decreases in concentrations of lactate and enzyme in the tumors. The technique could provide a new way to assess tumor responses to treatment in the clinic.     

Development of a protein secretion system with the application of sec-dependent protein secretion components

In order to induce high levels of protein secretion, we have constructed a recombinant plasmid, designated pBP244, into which was incorporated key components of the type-II Sec-dependent secretion system, including LepB (signal peptidase), SecA (ATPase), and SecB (chaperone). The biological activities of the LepB, SecA, and SecB components expressed from genes harbored by pBP244 appeared to play their normal roles. In order to evaluate the protein secretion, a pspA (Streptococcus pneumoniae surface protein A) gene was cloned into pBP244, resulting in pBP438. S. typhimurium harboring pBP438 grown until the stationary phase, secreted a higher level of PspA into the culture supernatants than did the strain harboring pYA3494. The strain harboring pBP438 secreted a supernatant amount 1.71-fold, a periplasmic space amount 1.47-fold, and an outer membrane amount 1.49-fold higher than that of pYA3494. S. typhimurium chi8554 kept the Asd+ plasmid pBP244 and pBP438 for 60 generations in LB broth harboring DAP, thereby indicating that pBP244 and pBP438 were quite stable in the Salmonella strain.