Polyelectrolyte complex micelles composed of c-raf antisense oligodeoxynucleotide-poly(ethylene glycol) conjugate and poly(ethylenimine): effect of systemic administration on tumor growth

An antisense oligodeoxynucleotide (ODN) delivery system based on polyelectrolyte complex (PEC) micelles composed of an ODN-poly(ethylene glycol) (PEG) conjugate and polyethylenimine (PEI) was demonstrated. The PEC micelles having a core/shell structure were spontaneously formed in an aqueous solution by ionic interactions between ODN part in the conjugate and PEI. The ODN/PEI polyelectrolyte complex formed an inner core while PEG chains surrounded it as a shell. The morphology of the micelles was visualized as a separate sphere by atomic force microscopy (AFM). When the micelles containing a c-raf antisense ODN were intravenously administered into tumor-bearing nude mice, significant antitumor activities against human lung cancer were observed. The intravenously injected micelles also showed significantly higher accumulation level in the solid tumor region compared to that of naked ODN.     

Purification, refolding, and characterization of recombinant Pseudomonas fluorescens lipase

Thermostable Pseudomonas fluorescens SIK W1 lipase (PFL), which is responsible for the spoilage of milk, was overexpressed as inclusion bodies in Escherichia coli. Renaturation of solubilized PFL was achieved by using size-exclusion protein refolding chromatography. The renatured enzyme was purified homogeneously using a combination of gel filtration and ion-exchange FPLC. Its specific activity was found to be enhanced in the presence of Ca2+. Secondary structural changes induced by Ca2+ were monitored by circular dichroism, which demonstrated that the activity increase of PFL in the presence of Ca2+ is strongly correlated with significant increases in alpha-helix and beta-sheet content. In the presence of Ca2+, the PFL structure was found resistant to denaturation by guanidine hydrochloride and to enzyme activity loss due to cosolvents like DMSO and trifluoroethanol, suggesting that Ca2+ plays an important role in inducing conformational changes and consequently in maintaining enzyme structural stability.     

Expression and purification of recombinant human angiopoietin-2 produced in Chinese hamster ovary cells

Angiopoietin-2 (Ang2) is a complex regulator of vascular remodeling that plays a role in both blood vessel sprouting and blood vessel regression through its receptor Tie2. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (20 microg/mL) of recombinant human Ang2 protein (rhAng2) with an amino-terminal FLAG-tag was constructed by transfecting the expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, and 0.32 microM. The rhAng2 secreted from rCHO cells was purified at a purification yield of 53.6% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng2 as a homodimeric glycoprotein form. Furthermore, rhAng2 binds to the Tie2 receptor and phosphorylates Tie2 in a concentration-dependent manner. Therefore, our rhAng2 could be useful for clarifying biological effect of exogenous Ang2 in the future.     

Molecular characterization of TEM-type beta-lactamases identified in cold-seep sediments of Edison Seamount (south of Lihir Island, Papua New Guinea)

To determine the prevalence and genotypes of beta-lactamases among clones of a metagenomic library from the cold-seep sediments of Edison seamount (10,000 years old), we performed pulse-field gel electrophoresis, antibiotic susceptibility testing, pI determination, and DNA sequencing analysis. Among the 8,823 clones of the library, thirty clones produced beta-lactamases and had high levels of genetic diversity. Consistent with minimum inhibitory concentration patterns, we found that five (16.7%) of thirty clones produced an extended-spectrum beta-lactamase. 837- and 259-bp fragments specific to blaTEM genes were amplified, as determined by banding patterns of PCR amplification with designed primers. TEM-1 was the most prevalent beta-lactamase and conferred resistance to ampicillin, piperacillin, and cephalothin. TEM-116 had a spectrum that was extended to ceftazidime, cefotaxime, and aztreonam. The resistance levels conferred by the pre-antibiotic era alleles of TEM-type beta-lactamases were essentially the same as the resistance levels conferred by the TEM-type alleles which had been isolated from clinically resistant strains of bacteria of the antibiotic era. Our first report on TEM-type beta-lactamases of the pre-antibiotic era indicates that TEM-type beta-lactamases paint a picture in which most of the diversity of the enzymes may not be the result of recent evolution, but that of ancient evolution.     

Purification and characterization of NADPH-dependent Cr(VI) reductase from Escherichia coli ATCC 33456

A soluble Cr(VI) reductase was purified from the cytoplasm of Escherichia coli ATCC 33456. The molecular mass was estimated to be 84 and 42 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, indicating a dimeric structure. The pI was 4.66, and optimal enzyme activity was obtained at pH 6.5 and 37 degrees C. The most stable condition existed at pH 7.0. The purified enzyme used both NADPH and NADH as electron donors for Cr(VI) reduction, while NADPH was the better, conferring 61%; higher activity than NADH. The Km values for NADPH and NADH were determined to be 47.5 and 17.2 micromol, and the Vmax values 322.2 and 130.7 micromol Cr(VI) min(-1)mg(-1) protein, respectively. The activity was strongly inhibited by N-ethylmalemide, Ag2+, Cd2+, Hg2+, and Zn2+. The antibody against the enzyme showed no immunological cross reaction with those of other Cr(VI) reducing strains.