Lipase-catalyzed enantioselective esterification of (S)-naproxen hydroxyalkyl ester in organic media

A lipase-catalyzed, enantioselective esterification process in organic solvents was developed for the synthesis of (S)-naproxen hydroxyalkyl ester. With the selection of lipase (Candida rugosa lipase) and reaction medium (isooctane and cyclohexane), a high enantiomeric ratio of <100 for the enzyme was obtained. 1,4-Butanediol was the best acyl acceptor. The carbon chain length of the alcohol had a major effect on the enzyme activity and enantioselectivity of lipase-catalyzed esterification.     

Improved immunomatrix methods to detect protein:protein interactions

Immunoprecipitation (IP) and coimmunoprecipitation (co-IP) are key techniques for studying protein-protein interactions. These methods utilize immobilized Protein A or Protein G to isolate antibody-bound target antigens. The main disadvantage of traditional IP and co-IP is that the conditions used to elute the precipitated antigen also release the antibody thus contaminating the antigen and destroying the antibody support. To overcome these problems, we describe two methods to generate a reusable antibody support by cross-linking the antibody to immobilized Protein A or Protein G, or by coupling it directly to the resin (see Scheme 1). Antibody cross-linking can be done in 1 h while antibody coupling requires 4 h. IP or co-IP is accomplished by incubating the antibody resin with the protein sample. Washes and elutions are carried out in a spin column to reduce resin loss and decrease assay time. Target proteins are eluted with 0.1 M glycine (pH 2.8) and the resin-bound antibody is re-equilibrated in phosphate-buffered saline (PBS) for reuse. Our studies have demonstrated that the immobilization efficiency for the antibody coupling method was similar for several species of antibody. Furthermore, we illustrate that using both methods of antibody immobilization yield IP and co-IP results similar to traditional protocols but eliminate the antibody heavy and light chain contamination.     

Total synthesis of human and rat coupling factor-6 amide and pressor effects in the rat

Mitochondrial coupling factor-6 (CF-6) is a component of the ATP synthase complex essential for energy transduction. CF-6, which is localized to the surface of endothelial cells (ECs) and released by shear stress, has been implicated as an endogenous vasoconstrictor. Previous methods of obtaining CF-6 through purification and recombinant methods were laborious and inefficient. Here, we describe the chemical synthesis of human CF-6, (33-108)-NH(2), its C-terminal fragment (55-108)-NH(2), which is termed pCF-6; the rat CF-6, (33-108)-NH(2), its C-terminal fragment pCF-6, (55-108)-NH(2); and two N-terminal fragments of the rat pro-coupling factor-6, (24-52)-NH(2) and (33-52)-NH(2). Biological activities of each peptide were initially screened with bioassays and verified by in vivo studies. Accordingly, intravenous administration of CF-6, pCF-6, rat CF-6, and rat pCF-6 produced a modest but statistically significant increase in blood pressure and heart rate in urethane anesthetized rats, whereas the N-terminal rat pro-coupling factor-6, (24-52)-NH(2) and (33-52)-NH(2) caused no significant pressor response. Thus, the biologically active site probably resides at the C-terminal portion of CF-6 peptides.     

Chlorophyll reduction in the seed of Brassica napus with a glutamate 1-semialdehyde aminotransferase antisense gene*

Chlorophyll reduction in the seed of Brassica can be achieved by downregulating its synthesis. To reduce chlorophyll synthesis, we have used a cDNA clone of Brassica napus encoding glutamate 1-semialdehyde aminotransferase (GSA-AT) to make an antisense construct for gene manipulation. Antisense glutamate 1-semialdehyde aminotransferase gene (Gsa) expression, directed by a Brassica napin promoter, was targeted specifically to the embryo of the developing seed. Transformants expressing antisense Gsa showed varying degrees of inhibition resulting in a range of chlorophyll reduction in the seeds. Seed growth and development were not affected by reduction of chlorophyll. Seeds from selfed transgenic plants germinated with high efficiency and growth of seedlings was vigorous. Seedlings from T₂ transgenic lines segregated into three distinctive phenotypes: dark green, light green and yellow, indicating the dominant inheritance of Gsa antisense gene. These transgenic lines have provided useful materials for the development of a low chlorophyll seed variety of B. napus.     

Plant regeneration through shoot formation from callus of Areca catechu L.

In order to establish and optimize an in vitro micropropagation protocol of Venus fly trap (Dionaea muscipula Ellis), a carnivorous plant, the effects of medium type, MS medium concentration, pH, and cytokinin and auxin types on shoot proliferation and root formation were investigated using 3-month-old shoots. The shoot proliferation was most effective in 2.3 M kinetin-supplemented 1/3MS medium at pH 5.5. The best conditions for rooting were 1/3MS medium supplemented with 0.5 M IBA. All subcultured shoots produced extensive root systems after 5–6 weeks culture. When plantlets after rooting were planted in plastic pots filled with 1:1 peat moss and sand, the survival rate of plantlets was almost 100%, exhibiting normal development. With subculture every 8 weeks, hundreds of the plants were propagated from a single plant within a year.     

Updated classification of hemangiomas and other vascular anomalies

Vascular anomalies comprise a widely heterogenous group of tumors and malformations. Great confusion has arisen because of the term hemangioma has been and is continued to be used to represent a multitude of vascular entities. This review presents the updated classification of vascular anomalies with the goal of clarifying the term hemangioma. In addition, newer clinical concepts in hemangiomas and other vascular tumors is presented. Hemangioma subtypes and hemangioma variants are also discussed, and a brief review of pyogenic granuloma and Kaposiform hemangioendothelioma is provided. Finally, the immunohistochemical marker GLUT1 is reviewed, a marker that heralds a new era in vascular anomalies research.     

Purification of human plasma haptoglobin by hemoglobin-affinity column chromatography

Haptoglobin (Hp) is an acute-phase protein; its plasma levels increase consistently in response to infection and inflammation. The concentration of human plasma Hp is ranged between 1 and 1.5 mg/ml. Similar to blood type, individual human Hp is classified as Hp 1-1, 2-1, or 2-2. The structural and functional analysis of the Hp, however, has not been studied in detail due to its difficult isolation procedure. Previously, we reported a single step for the purification of porcine Hp. In this study, we established a purification method using a high capacity hemoglobin-affinity column. Briefly, DEAE-purified human hemoglobin was first coupled to Sepharose 4B to prepare an affinity column in a 15-ml bed volume. Following a flow through of human plasma and an extensive wash, the bound material was eluted with a solution of 0.15 M NaCl, pH 11 (adjusted by ammonium), to remove low-affinity bound proteins. The high-affinity bound Hp was then eluted with 0.15 M NaCl containing 5 M urea, pH 11, and collected in tubes containing 100 microl of 1 M Tris buffer, pH 7.0. The biological activity of dialyzed Hp was retained as it formed a complex with hemoglobin on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using this procedure, approximately 10 mg of Hp 1-1, with homogeneity greater than 96%, was obtained from 15 ml of human plasma. Affinity purified Hp 2-1 or 2-2, however, contained trace amounts of apoA-I with the similar approach. The Hp could be further purified by HPLC using a Superose 12 gel-permeation chromatography, if desired, to achieve 100% purity. All the phenotypes of purified Hp consisted of alpha and beta chains on SDS-PAGE in the presence of a reducing reagent, further confirmed by a Western blot analysis. We conclude that human hemoglobin-affinity column was most suitable for the isolation of Hp 1-1 in large quantities. Whereas, one additional step using a gel-permeation was necessary for that of Hp 2-1 and 2-2.     

Hydrogen production with immobilized sewage sludge in three-phase fluidized-bed bioreactors

Municipal sewage sludge was immobilized with a modified alginate gel entrapment method, and the immobilized cells were used to produce hydrogen gas in a three-phase fluidized bed. The hydrogen-producing fluidized beds were operated at different liquid velocity (U(0)) and hydraulic retention time (HRT). The results show that in response to operating liquid velocities, the fluidized-bed system had three flow regimes, namely, plug flow, slug flow, and free bubbling. Pressure fluctuation analysis was used to analyze the hydrodynamic properties in this three-phase fluidized bed when it was under a steady-state production of biogas. With a steady-state biogas production rate (U(g)) of 0.196 mL/s/L, a transition state occurred at a liquid velocity (U(0)) of 0.85 cm/s. As U(0) < 0.85 cm/s, the system was basically a nonhomogeneous fluidized bed, whereas the bed became homogeneous when U(0) was higher than 0.85 cm/s. The fluidized bed can be stably carried out at high loading rates (HRT as low as 2 h). Hydrogen fermentation results show that the maximal hydrogen production rate was 0.93 L/h/L and the best yield (Y(H)2(/sucrose)) was 2.67 mol H(2)/mol sucrose.     

Enhancement and prolongation of baculovirus-mediated expression in mammalian cells: focuses on strategic infection and feeding

The baculovirus/insect cell system has been widely used for recombinant protein production. Since the finding that baculovirus was able to infect hepatocytes in 1995, various attempts to utilize baculovirus as a gene delivery vehicle into mammalian cells have been reported. In this study, we intended to explore the possibility of utilizing a baculovirus/mammalian cell system as a nonlytic, continuous protein production system. A recombinant baculovirus vector carrying enhanced green fluorescent protein (EGFP) under the control of cytomegalovirus immediate-early (CMV-IE) promoter was constructed. This virus was used to infect four common mammalian cell lines, and HeLa was found to yield the highest expression level. Additions of butyrate and valproic acid both enhanced the expression level, but butyrate exhibited a more profound effect. More importantly, HeLa cells were found to be superinfected by baculovirus, a result not observed in the conventional baculovirus/insect cell system. The effects of multiplicity of infection (MOI) and infection timing were also compared. High MOI up to 800 increased the expression in the short term (4 days), but the relatively higher cell death and lower cell density compromised the overall protein yield thereafter. The highest overall expression for a long term was obtained at MOI = 200 when the cells were initially infected at the mid-exponential phase and superinfected with additional baculovirus (MOI = 200) together with a one-time supplement of butyrate. In summary, the strategic infection and feeding enhanced the expression level 9-fold (compared with unsuperinfected culture) and prolonged the duration of expression to 16 days. This study reveals that this baculovirus/mammalian cell system has great potential to become a novel continuous, nonlytic expression system.