Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon alpha-2a for the treatment of hepatitis C

A potent, long-lasting form of interferon alpha-2a mono-pegylated with a 40 kilodalton branched poly(ethylene glycol) was designed, synthesized, and characterized. Mono-pegylated interferon alpha-2a was comprised of four major positional isomers involving Lys31, Lys121, Lys131, and Lys134 of interferon. The in vitro anti-viral activity of pegylated interferon alpha-2a was found to be only 7% of the original activity. In contrast, the in vivo antitumor activity was severalfold enhanced compared to interferon alpha-2a. Pegylated interferon alpha-2a showed no immunogenicity in mice. After subcutaneous injection of pegylated interferon alpha-2a, a 70-fold increase in serum half-life and a 50-fold increase in mean plasma residence time concomitant with sustained serum concentrations were observed relative to interferon alpha-2a. These preclinical results suggest a significantly enhanced human pharmacological profile for pegylated interferon alpha-2a. Results of Phase II/III hepatitis C clinical trials in humans confirmed the superior efficacy of pegylated interferon alpha-2a compared to unmodified interferon alpha-2a.     

High-level expression and purification of immunogenic recombinant SAG1 (P30) of Toxoplasma gondii in Escherichia coli

Surface antigen 1 (SAG1) of Toxoplasma gondii is a good candidate for diagnosis and vaccine development, but recombinant SAG1 produced in Escheichia coli often loses its specific immunogenicity due to the incorrect folding. In the present study, a truncated SAG1 was highly expressed in E. coli as a fusion protein, about 30% of the total protein of the cell lysate. After a simple purification and refolding procedure, purified rSAG1 can be recognized by human Toxoplasma-infective serum, and ELISA kits constructed by rSAG1 can sensitively and specifically detect toxoplasma infection.     

Protein-protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET)

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM-only protein that contains four tandemly repeated LIM motifs with an N-terminal single zinc finger (half LIM motif). FHL3 expresses predominantly in human skeletal muscle. In this report, FHL3 was shown to be a novel interacting partner of FHL2 using the yeast two-hybrid assay. Furthermore, site-directed mutagenesis of FHL3 indicated that the LIM2 of FHL3 is the essential LIM domain for interaction with FHL2. Green fluorescent protein (GFP) was used to tag FHL3 in order to study its distribution during myogenesis. Our result shows that FHL3 was localized in the focal adhesions and nucleus of the cells. FHL3 mainly stayed in the focal adhesion during myogenesis. Moreover, using site-directed mutagenesis, the LIM1 of FHL3 was identified as an essential LIM domain for its subcellular localization. Mutants of GFP have given rise to a novel technique, two-fusion fluorescence resonance energy transfer (FRET), in the determination of protein-protein interaction at particular subcellular locations of eukaryotic cells. To determine whether FHL2 and FHL3 can interact with one another and to locate the site of this interaction in a single intact mammalian cell, we fused FHL2 and FHL3 to different mutants of GFP and studied their interactions using FRET. BFP/GFP fusion constructs were cotransfected into muscle myoblast C2C12 to verify the colocalization and subcellular localization of FRET. We found that FHL2 and FHL3 were colocalized in the mitochondria of the C2C12 cells and FRET was observed by using an epi-fluorescent microscope equipped with an FRET specific filter set.     

Molecular cloning and characterization of a RING-H2 finger protein, ANAPC11, the human homolog of yeast Apc11p

Yeast Apc11p together with Rbx1 and Roc2/SAG define a new class of RING-H2 fingers in a superfamily of E3 ubiquitin ligases. The human homolog of Apc11p, ANAPC11 was identified during a large-scale partial sequencing of a human liver cancer cDNA library and partial characterization was performed. This 514 bp full-length cDNA has a predicted open reading frame (ORF) encoding 84 amino acids. The ORF codes for ANAPC11, the human anaphase promoting complex subunit 11 (yeast APC11 homolog), which possesses a RING-H2 finger motif and exhibits sequence similarity to subunits of E3 ubiquitin ligase complexes. In Northern blot hybridization with poly(A) RNA of various human tissues using radio-labelled ANAPC11 cDNA probe, we found strong signals detected in skeletal muscle and heart; moderate signals detected in brain, kidney, and liver; and detectable but low signals in colon, thymus, spleen, small intestine, placenta, lung, and peripheral blood leukocyte. The ANAPC11 gene is located at the human chromosome 17q25. ANAPC11 is distributed diffusely in the cytoplasm and nucleus with discrete accumulation in granular structures in all the cell lines (AML 12, HepG2, and C2C12) transfected. Expression level of ANAPC11 is found higher in certain types of cancer determined in the RNA dot blot experiment.     

Elfin is expressed during early heart development

Elfin (previously named CLIM1) is a protein that possesses an N-terminal PDZ domain and a C-terminal LIM domain. It belongs to the family of Enigma proteins. Enigma proteins are a family of cytoplasmic proteins that contain an N-terminal PDZ domain and a series of C-terminal LIM domains. By virtue of these two protein interacting domains, Enigma proteins are capable of protein-protein interactions. It has been proposed that Enigma proteins may act as adapters between kinases and the cytoskeleton. We have previously shown that Elfin is most abundantly expressed in the heart and it colocalizes with alpha-actinin 2 at the Z-disks of the myocardium. In this report, Elfin was shown to localize at the actin stress fibers of myoblasts, as revealed by green fluorescent protein (GFP) tagging. In situ hybridization and immunostaining showed that Elfin expression begins at an early stage in mouse development and is present throughout the developing heart. Taken together, our experimental results suggest that Elfin may play an important role in myofibrillogenesis and heart development.     

Comparative induction of CYP1A1 expression by pyridine and its metabolites

We compared pyridine and five of its metabolites in terms of (i) in vivo induction of CYP1A1 expression in the lung, kidney, and liver in the rat and (ii) in vitro binding to, and activation of, the aryl hydrocarbon receptor (AhR) in cytosol from rat liver or Hepa1c1c7 cells. Following a single 2.5 mmol/kg ip dose of either pyridine, 2-hydroxpyridine, 3-hydroxypyridine, 4-hydroxypyridine, N-methylpyridinium, or pyridine N-oxide, CYP1A1 activity (ethoxyresorufin O-deethylase), protein level (as determined by Western blotting), and mRNA level (as determined by Northern blotting) were induced by pyridine, N-methylpyridinium, and pyridine N-oxide in the lung, kidney, and liver. The induction by N-methylpyridinium or pyridine N-oxide was comparable to or greater than that by pyridine in some tissues. 2-Hydroxypyridine and 3-hydroxypyridine caused tissue-specific induction or repression of CYP1A1, whereas 4-hydroxypyridine had no effect on the expression of the enzyme. Pyridine and its metabolites elicited weak activation of the aryl hydrocarbon receptor in a gel retardation assay in cytosol from rat liver but not Hepa 1c1c7 cells. However, the receptor activation did not parallel the in vivo CYP1A1 induction by the pyridine compounds, none of which inhibited binding of ?(3)H2,3,7, 8-tetrachlorodibenzo-p-dioxin to AhR in a competitive assay in rat liver cytosol. The findings are consistent with a role of pyridine metabolites in CYP1A1 induction by pyridine but do not clearly identify the role of aryl hydrocarbon receptor in the induction mechanism.     

Attenuated nitric oxide synthase activity and protein expression accompany intestinal ischemia/reperfusion injury in rats

Intestinal ischemia/reperfusion (I/R) leads to bowel impairment via the release of reactive oxygen species (ROS) and neutrophil infiltration. In addition to modulating intestinal integrity, nitric oxide (NO(*)) inhibits neutrophil activation and scavenges ROS. Attenuated endogenous NO(*) formation may result in the accrual of these deleterious stimuli. Therefore, we determined nitric oxide synthase (NOS) activity in anesthetized rats subjected to 1 h of superior mesenteric ischemia or ischemia followed by reflow. NOS activity was measured in intestinal tissue homogenates as the conversion rate of (3)H-L-arginine to (3)H-L-citrulline. Our results demonstrate that intestinal ischemia leads to a decrease in NOS activity indicating lower NO(*) formation in the animal model. The attenuation in NOS activity was not reversed following 4 h of reperfusion. Western blot analysis revealed that the decline in enzyme activity was accompanied by reduced intestinal NOS III (endothelial constitutive NOS) expression. These findings provide biochemical evidence for impaired NO(*) formation machinery in intestinal I/R injury.     

Redesigning the substrate specificity of an enzyme: isocitrate dehydrogenase

Despite the structural similarities between isocitrate and isopropylmalate, isocitrate dehydrogenase (IDH) exhibits a strong preference for its natural substrate. Using a combination of rational and random mutagenesis, we have engineered IDH to use isopropylmalate as a substrate. Rationally designed mutations were based on comparison of IDH to a similar enzyme, isopropylmalate dehydrogenase (IPMDH). A chimeric enzyme that replaced an active site loop-helix motif with IPMDH sequences exhibited no activity toward isopropylmalate, and site-directed mutants that replaced IDH residues with their IPMDH equivalents only showed small improvements in k(cat). Random mutants targeted the IDH active site at positions 113 (substituted with glutamate), 115, and 116 (both randomized) and were screened for activity toward isopropylmalate. Six mutants were identified that exhibited up to an 8-fold improvement in k(cat) and increased the apparent binding affinity by as much as a factor of 80. In addition to the S113E mutation, five other mutants contained substitutions at positions 115 and/or 116. Most small hydrophobic substitutions at position 116 improved activity, possibly by generating space to accommodate the isopropyl group of isopropylmalate; however, substitution with serine yielded the most improvement in k(cat). Only two substitutions were identified at position 115, which suggests a more specific role for the wild-type asparagine residue in the utilization of isopropylmalate. Since interactions between neighboring residues in this region greatly influenced the effects of each other in unexpected ways, structural solutions were best identified in combinations, as allowed by random mutagenesis.     

Protein-protein interaction of FHL2, a LIM domain protein preferentially expressed in human heart, with hCDC47

In the yeast two-hybrid library screening, the heart-specific FHL2 protein was found to interact with hCDC47. In vitro interaction study between FHL2 protein and hCDC47 was demonstrated. From the results of domain studies by the yeast two-hybrid assay, the second and third LIM domains in conjunction with the first half LIM domain of FHL2 were identified to be important in binding with hCDC47. Besides, in Northern blot hybridization of human cancer cell lines, the highest FHL2 mRNA expression was detected in colorectal adenocarcinoma SW480 and HeLa cell S3. Our results imply that FHL2 protein may associate with cancer development and may act as a molecular adapter to form a multicomplex with hCDC47 in the nucleus, thus it plays an important role in the specification or maintenance of the terminal differentiated phenotype of heart muscle cells.