DRG2 Depletion Promotes Endothelial Cell Senescence and Vascular Endothelial Dysfunction.

Endothelial cell senescence is involved in endothelial dysfunction and vascular diseases. However, the detailed mechanisms of endothelial senescence are not fully understood. Here, we demonstrated that deficiency of developmentally regulated GTP-binding protein 2 (DRG2) induces senescence and dysfunction of endothelial cells. DRG2 knockout (KO) mice displayed reduced cerebral blood flow in the brain and lung blood vessel density. We also determined, by Matrigel plug assay, aorta ring assay, and in vitro tubule formation of primary lung endothelial cells, that deficiency in DRG2 reduced the angiogenic capability of endothelial cells. Endothelial cells from DRG2 KO mice showed a senescence phenotype with decreased cell growth and enhanced levels of p21 and phosphorylated p53, γH2AX, senescence-associated ß-galactosidase (SA-ß-gal) activity, and senescence-associated secretory phenotype (SASP) cytokines. DRG2 deficiency in endothelial cells upregulated arginase 2 (Arg2) and generation of reactive oxygen species. Induction of SA-ß-gal activity was prevented by the antioxidant N-acetyl cysteine in endothelial cells from DRG2 KO mice. In conclusion, our results suggest that DRG2 is a key regulator of endothelial senescence, and its downregulation is probably involved in vascular dysfunction and diseases.

Hydrogen-bonding-driven enantioselective resolution against the Kazlauskas rule to afford γ-amino alcohols by Candida rugosa lipase.

Most lipases resolve secondary alcohols in accordance with the "Kazlauskas rule" to give the R enantiomers. In a similar manner to other lipases, Candida rugosa lipase (CRL) exhibits R enantioselectivity towards heptan-2-ol, although the enantiomeric ratio (E) is low (E=1.6). However, unexpected enantioselectivity (i.e., S enantioselectivity, E=58) of CRL towards 4-(tert-butoxycarbonylamino)butan-2-ol, which has a similar chain length to heptan-2-ol, has been observed. To develop a deeper understanding of the molecular basis for this unusual enantioselectivity, we have conducted a series of molecular modeling and substrate engineering experiments. The results of these computational and experimental analyses indicated that a hydrogen bond between the Ser450 residue and the nitrogen atom of the carbamate group is critical to stabilize the transition state of the S enantiomer.

Identification of a novel 11ß-HSD1 inhibitor from a high-throughput screen of natural product extracts.

Selective inhibitors of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) have considerable potential as a treatment for metabolic syndrome including type 2 diabetes mellitus and obesity. To identify 11ß-HSD1 inhibitors, we conducted high-throughput screening (HTS) of active natural product extracts from the Korea Chemical Bank, including Tanshinone I, Tanshinone IIA, and flavanone derivatives, and 2- and 3-phenyl-4H-chromen-4-one. Then Tanshinone IIA and its derivatives were targeted for the development of a lead compound according to the HTS results. However, the mechanism for anti-adipogenic effect through 11ß-HSD1 enzyme inhibition by Tanshinone IIA is not clear. Tanshinone IIA (2a) concentration-dependently inhibited 11ß-HSD1 activity in human and mouse 11ß-HSD1 overexpressed cells and 3T3-L1 adipocytes. Tanshinone IIA (2a) also inhibited 11ß-HSD1 enzyme activities in murine liver and fats. Furthermore, Tanshinone IIA (2a)-suppressed adipocyte differentiation of cortisone-induced adipogenesis in 3T3-L1 cells was associated with the suppression of the cortisone-induced adipogenesis-specific markers mRNA and protein expression. In 3T3-L1 preadipocytes, Tanshinone IIA (2a)-inhibited cortisone induced reactive oxygen species formation in a concentration-dependent manner. Thus, these results support the therapeutic potential of Tanshinone IIA (2a) as a 11ß-HSD1 inhibitor in metabolic syndrome patients.

Performance Comparison of Proton Exchange Membrane Water Electrolysis Cell Using Channel and PTL Flow Fields through Three-Dimensional Two-Phase Flow Simulation.

Water electrolysis technology is required to overcome the intermittency of renewable energy sources. Among various water electrolysis methods, the proton exchange membrane water electrolysis (PEMWE) cell has the advantages of a fast response and high current density. However, high capital costs have hindered the commercialization of PEMWE; therefore, it is important to lower the price of bipolar plates, which make PEMWE expensive. In addition, since the flow field inscribed in the bipolar plate significantly influences the performance, it is necessary to design the enhanced pattern. A three-dimensional two-phase flow model was used to analyze the two-phase flow and electrochemical reactions of the PEMWE anode. In order to compare the experimental results with the simulation, experiments were conducted according to the flow rate, and the results were in good agreement. First, as a result of comparing the performance of the channel and PTL (porous transport layer) flow fields, the channel flow field showed better performance than the PTL flow field. For the channel flow field, the higher the ratio of the channel width-to-rib width and the permeability of PTL, the performance got better. In the case of the PTL flow field, with the increased capillary pressure, the performance improved even if the PTL permeability decreased. Next, the direction of gravity affected the performance only when the channel flow field was used, and the X+ and Z+ directions were optimal for the performance. Finally, increasing the inlet flow rate could reduce the difference in performance between the channel and PTL flow fields, but the pressure drop gradually increased.

Transduction of the MPG-tagged fusion protein into mammalian cells and oocytes depends on amiloride-sensitive endocytic pathway.

BACKGROUND: MPG is a cell-permeable peptide with proven efficiency to deliver macromolecular cargoes into cells. In this work, we examined the efficacy of MPG as an N-terminal tag in a fusion protein to deliver a protein cargo and its mechanism of transduction. RESULTS: We examined transduction of MPG-EGFP fusion protein by live imaging, flow cytometry, along with combination of cell biological and pharmacological methods. We show that MPG-EGFP fusion proteins efficiently enter various mammalian cells within a few minutes and are co-localized with FM4-64, a general marker of endosomes. The transduction of MPG-EGFP occurs rapidly and is inhibited at a low temperature. The entry of MPG-EGFP is inhibited by amiloride, but cytochalasin D and methyl-beta-cyclodextrin did not inhibit the entry, suggesting that macropinocytosis is not involved in the transduction. Overexpression of a mutant form of dynamin partially reduced the transduction of MPG-EGFP. The partial blockade of MPG-EGFP transduction by a dynamin mutant is abolished by the treatment of amiloride. MPG-EGFP transduction is also observed in the mammalian oocytes. CONCLUSION: The results show that the transduction of MPG fusion protein utilizes endocytic pathway(s) which is amiloride-sensitive and partially dynamin-dependent. Collectively, the MPG fusion protein could be further developed as a novel tool of "protein therapeutics", with potentials to be used in various cell systems including mammalian oocytes.

Recyclable chaperone-conjugated magnetic beads for in vitro refolding of Burkholderia cepacia lipase.

Polymer-coated magnetic beads have become widely used in biological applications because of their facile recovery and easily modifiable surface. Herein, we report the application of magnetic beads to in vitro refolding of B. cepacia lipase. Magnetic particles (Fe3O4) prepared by co-precipitation of Fe2+ and Fe3+ ions under basic conditions were subsequently coated with carboxylic acid-containing polystyrene by emulsion polymerization. The polymer-coated magnetic beads were then conjugated with molecular chaperone proteins to assist with refolding. The chaperone-conjugated magnetic beads efficiently refolded B. cepacia lipase and were easily reused. The beads showed comparable refolding activity to the soluble chaperone, and retained more than 95% of their refolding activity after five cycles of refolding B. cepacia lipase.

Characterization of Organic Solvent-Tolerant Lipolytic Enzyme from Marinobacter lipolyticus Isolated from the Antarctic Ocean.

The Antarctic marine environment provides a good source of novel lipolytic enzymes that possess beneficial properties, i.e., resistance to extreme physical and chemical conditions. We found a lipolytic Escherichia coli colony that was transformed using genomic DNA from Marinobacter lipolyticus 27-A9 isolated from the Antarctic Ross Sea. DNA sequence analysis revealed an open reading frame of lipolytic enzyme gene. The gene translates a protein (LipA9) of 404 amino acids with molecular mass of 45,247 Da. Recombinant LipA9 was expressed in E. coli BL21 (DE3) cells and purified by anion exchange and gel filtration chromatography. The kcat/Km of LipA9 was 175 s-1 µM-1, and the optimum temperature and pH were 70 °C and pH 8.0, respectively. LipA9 had quite high organic solvent stability; it was stable toward several common organic solvents up to 50% concentration. Substrate specificity studies showed that LipA9 preferred a short acyl chain length of p-nitrophenyl ester and triglyceride. Sequence analysis showed that LipA9 contained catalytic Ser72 and Lys75 in S-x-x-K motif, like family VIII esterases. Homology modeling and site-directed mutagenesis studies revealed that Tyr141 and Tyr188 residues were located near the conserved motif and played an important role in catalytic activity.

Focusing mutations into the P. fluorescens esterase binding site increases enantioselectivity more effectively than distant mutations.

Rational design of enzymes with improved properties, such as enantioselectivity, usually focuses mutations within the substrate binding site. On the other hand, directed evolution of enzymes usually targets the entire protein and discovers beneficial mutations far from the substrate binding site. In this paper, we propose an explanation for this discrepancy and show that a combined approach--random mutagenesis within the substrate binding site--is better. To increase the enantioselectivity (E) of a Pseudomonas fluorescens esterase (PFE) toward methyl 3-bromo-2-methylpropionate, we focused mutagenesis into the substrate binding site at Trp28, Val121, Phe198, and Val225. Five of the catalytically active mutants (13%) showed better enantioselectivity than wild-type PFE. The increases in enantioselectivity were higher (up to 5-fold, reaching E = 61) than with mutants identified by random mutagenesis of the entire enzyme.

Biocatalysis in ionic liquids - advantages beyond green technology.

In recent years researchers have started to explore a particular class of organic solvents called room temperature ionic liquids - or simply ionic liquids - to identify their unique advantages for biocatalysis. Because they lack vapour pressure, ionic liquids hold potential as green solvents. Furthermore, unlike organic solvents of comparable polarity, they often do not inactivate enzymes, which simplifies reactions involving polar substrates such as sugars. Biocatalytic reactions in ionic liquids have also shown higher selectivity, faster rates and greater enzyme stability; however, these solvents present other challenges, among them difficulties in purifying ionic liquids and controlling water activity and pH, higher viscosity and problems with product isolation.

Development of Saccharomyces cerevisiae reductase YOL151W mutants suitable for chiral alcohol synthesis using an NADH cofactor regeneration system.

The aldo-keto reductases catalyze reduction reactions using various aliphatic and aromatic aldehydes/ketones. Most reductases require NADPH exclusively as their cofactors. However, NADPH is much more expensive and unstable than NADH. In this study, we attempted to change the five amino acid residues that interact with the 2'-phosphate group of the adenosine ribose of NADPH. These residues were selected based on a docking model of the YOL151W reductase and were substituted with other amino acids to develop NADH-utilizing enzymes. Ten mutants were constructed by site-directed mutagenesis and expressed in Escherichia coli. Among them, four mutants showed higher reductase activities than wild-type when using the NADH cofactor. Analysis of the kinetic parameters for the wild type and mutants indicated that the kcat/Km value of the Asn9Glu mutant toward NADH increased 3-fold. A docking model was used to show that the carboxyl group of Glu 9 of the mutant formed an additional hydrogen bond with the 2'-hydroxyl group of adenosine ribose. The Asn9Glu mutant was able to produce (R)-ethyl-4-chloro-3-hydroxyl butanoate rapidly when using the NADH regeneration system.

Bio-functionalization of metal-organic frameworks by covalent protein conjugation.

Bioconjugation of functional proteins onto metal-organic frameworks (MOFs) has been achieved using activation of pendent linking groups of the organic linkers on the surface of MOFs. Fluorescent microscopy revealed successful conjugation of an enhanced fluorescent protein onto MOFs. In addition, Candida-antarctica-lipase-B-conjugated MOFs showed no loss of enantioselectivity and activity in transesterification of (±)-1-phenylethanol.

Two-dimensional metal-organic frameworks with blue luminescence.

Three new isostructural two-dimensional metal-organic frameworks (MOFs), [M(bpydc)(H(2)O).H(2)O](n) where M = Zn (1); Co (2); Ni (3) and the bpydc is 2,2'-bipyridine-5,5'-dicarboxylate), were prepared by various methods such as hydrothermal, ultrasonic and microwave-assisted synthetic methods. Microcrystalline could be obtained by using ultrasonification or microwave irradiation in a short time. Their solid-state structures were revealed by X-ray crystallography. The coordination environment of the metal ions is distorted octahedral geometry. The metal ions are coordinated by two nitrogen atoms from the bipyridyl moiety, two oxygen atoms from one carboxylate in a bidentate manner, one oxygen atom from another carboxylate in a monodentate manner, and one oxygen atom from the aqua ligand. The multiple coordination modes of the bpydc ligand led to a novel topologically interesting two-dimensional sheet structure; a 6-connected uninodal net with Schläfli symbol of 3(3).4(4).5(5).6(2).7. Thermal and luminescence properties of the three MOFs were also investigated. The weight maintained constant in the range 290-342 degrees C for 1, 250-470 degrees C for 2, and 275-470 degrees C for 3 after the initial weight loss related to the dehydration steps. In particular, [Zn(bpydc)(H(2)O).H(2)O](n) (1) displays strong solid state blue luminescence.

Intracellular protein delivery by glucose-coated polymeric beads.

Glucose-coated polymeric beads have been prepared and applied to delivery of a model protein (enhanced green fluorescent protein) into mouse embryonic stem cells as well as Hela cells.

Improving the expression yield of Candida antarctica lipase B in Escherichia coli by mutagenesis.

Increasing the expression yield of active Candida antarctica lipase B (CAL-B) in Escherichia coli was achieved by using a codon-optimized synthetic gene and by mutagenesis to introduce hydrophilic residues on the surface of CAL-B. Five residues (four leucines and one isoleucine) on the surface of CAL-B were selected and changed with aspartate after codon optimization. While the codon-optimized synthetic gene of CAL-B did not increase the expression yield, the mutation increased the activity of the enzyme three-fold (3.3 mg/l of culture) compared to the wild type. The mutant enzyme had similar hydrolytic activity toward hydrolysis of p-nitrophenyl acetate or p-nitrophenyl butyrate and enantioselectivity toward hydrolysis of (R, S)-1-phenylethyl acetate compared to the wild-type enzyme.

Percutaneous balloon compression of trigeminal ganglion for the treatment of idiopathic trigeminal neuralgia : experience in 50 patients.

OBJECTIVE: We assessed the surgical results of percutaneous balloon compression in 50 patients with idiopathic trigeminal neuralgia. METHODS: Fifty patients with follow-up period of more than 12 months were retrospectively analyzed. The mean follow-up period was 42 months (range, 12-82). The mean age was 65.8 years (range, 27-83). Seventeen patients (34%) had other previous surgical procedures. The balloon was inflated by injecting radio-contrast media under brief general anesthesia according to Mullan's technique. The mean inflating time was 88 seconds (range, 60-120). The whole procedure took about 20 minutes. RESULTS: We reported excellent and good results in 70% of the cases, poor in 6% as annoying dysesthesia, recurrence in 16%, and 8% failure due to technical deficiencies. Forty-six patients (92%) were initially relieved of their pain. There were permanent motor weakness of the masseter muscle in 4% of patients and transitory diplopia in 8%. Neither anesthesia dolorosa nor keratitis occurred. Almost all patients (92%) were discharged postoperatively within two days. CONCLUSION: These results indicate that balloon compression would be an effective method with acceptable morbidity, technically, it can be performed rapidly and simply in the treatment of idiopathic trigeminal neuralgia.

Molecular basis for the enhanced lipase-catalyzed N-acylation of 1-phenylethanamine with methoxyacetate.

One of the commercial methods for preparing enantiopure amines is lipase-catalyzed kinetic resolution, although lipases catalyze aminolysis with only low activity. Interestingly, in 1997 Balkenhohl et al. used ethyl methoxyacetate instead of ethyl butyrate as an acylation reagent for the aminolysis of 1-phenylethanamine and increased the reaction rate more than a 100-fold. This method has been applied to other aminolysis reactions, but the molecular basis for the enhanced rate is not understood. A molecular-modeling study of the transition-state analogue for the aminolysis showed that an interaction between the beta-oxygen atom in methoxyacetate and the amine nitrogen atom might be a key factor in the rate enhancement. Other acylation reagents, such as methyl 3-methoxypropionate and methyl 4-methoxybutyrate, were chosen to test the influence of this interaction because these molecules can be spatially arranged to have similar interactions. The results were similar to that in the acylation with methoxyacetate. The initial aminolysis rates were improved (11-fold and sixfold, respectively) compared to that with butyrate. In contrast, alcoholysis with 1-phenylethanol afforded the same rate with all acyl donors.

Structure of an aryl esterase from Pseudomonas fluorescens.

The structure of PFE, an aryl esterase from Pseudomonas fluorescens, has been solved to a resolution of 1.8 A by X-ray diffraction and shows a characteristic alpha/beta-hydrolase fold. In addition to catalyzing the hydrolysis of esters in vitro, PFE also shows low bromoperoxidase activity. PFE shows highest structural similarity, including the active-site environment, to a family of non-heme bacterial haloperoxidases, with an r.m.s. deviation in 271 C(alpha) atoms between PFE and its five closest structural neighbors averaging 0.8 A. PFE has far less similarity (r.m.s. deviation in 218 C(alpha) atoms of 5.0 A) to P. fluorescens carboxyl esterase. PFE favors activated esters with small acyl groups, such as phenyl acetate. The X-ray structure of PFE reveals a significantly occluded active site. In addition, several residues, including Trp28 and Met95, limit the size of the acyl-binding pocket, explaining its preference for small acyl groups.