General method for sequence-independent site-directed chimeragenesis

We have developed a simple and general method that allows for the facile recombination of distantly related (or unrelated) proteins at multiple discrete sites. To evaluate the sequence-independent site-directed chimeragenesis (SISDC) method, we have recombined beta-lactamases TEM-1 and PSE-4 at seven sites, examined the quality of the chimeric genes created, and screened the library of 2(8) (256) chimeras for functional enzymes. Probe hybridization and sequencing analyses revealed that SISDC generated a random library with little sequence bias and in which all targeted fragments were recombined in the desired order. Sequencing the genes from clones having functional lactamases identified 14 unique chimeras. These chimeras are characterized by a lower level of disruption, as calculated by the SCHEMA algorithm, than the library as a whole. These results illustrate the use of SISDC in creating designed chimeric protein libraries and further illustrate the ability of SCHEMA to identify chimeras whose folded structures are likely not to be disrupted by recombination.     

Nitric oxide synthase induction,cGMP elevation,and biopterin synthesis in vascular smooth muscle cells stimulated with interleukin-1beta in hypoxia

In cultured rat vascular smooth muscle cells (VSMC), inducible nitric oxide synthase (iNOS) expression evoked by interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha was greatly enhanced in hypoxia (2% O(2)), compared to in normoxia. In contrast, iNOS induction by interferon-gamma, lipopolysaccharide or their combination was barely influenced by hypoxia. These results indicate that iNOS induction is regulated by hypoxia in different manners, depending on the stimuli in VSMC. Nitric oxide (NO) production in response to stimulation with interferon-gamma plus lipopolysaccharide was significantly decreased in hypoxia, due to a decrease in the concentration of O(2) as a substrate. In contrast, the level of NO production in hypoxia was almost the same as that in normoxia when the cells were stimulated by IL-1beta. In addition, cGMP increased in response to IL-1beta in hypoxia to a level comparable to that in normoxia. Thus, it seems that the IL-1beta-induced expression of iNOS is up-regulated in hypoxia to compensate for a decrease in the enzyme activity due to the lower availability of O(2) as a substrate, and consequently a sufficient amount of NO is produced to elevate cGMP to an adequate level. In addition, the IL-1beta-induced synthesis of tetrahydrobiopterin, a cofactor for iNOS, was also greatly stimulated by hypoxia in VSMC.     

Butyric acid sensitizes Vero cells to ricin-induced apoptosis via accelerated activation of multiple signal transduction pathways

We found that the treatment with 1 mM butyric acid for 2 days renders Vero cells highly sensitive to ricin-induced apoptosis reflected by cytolysis concomitant with apoptotic cellular and nuclear morphological changes, DNA fragmentation, and increase in caspase-3 like activity, whereas butyric acid alone had no cytotoxic effect on Vero cells. During the treatment with butyric acid, gradual increase in alkaline phosphatase activity, an indicator for butyric acid-induced differentiation, was observed in Vero cells. Although the potency of ricin-mediated protein synthesis was increased in butyric acid-treated Vero cells as compared to untreated cells, the binding and internalization of ricin to the cells were not much affected. Furthermore, DNA fragmentation caused by other protein synthesis inhibitors such as diphtheria toxin and anisomysin were also highly potentiated in butyric acid-treated Vero cells, whereas the potencies of these toxins to inhibit the protein synthesis were not affected by butyric acid treatment. These results suggest that the apoptosis signaling pathway, which may be triggered by cytotoxic stress response caused by toxins, is sensitized in butyric acid-treated cells, while the pathways leading to the protein synthesis inhibition by these toxins are relatively unchanged. No significant differences in the expression levels of p21, p53, and Bcl-2 proteins were observed between butyric acid-treated and untreated Vero cells. The treatment with ricin resulted in the activation of p38 MAP kinase, and this activation occurred on an accelerated time schedule in butyric acid-treated Vero cells than in untreated cells. The specific inhibitor of p38 MAP kinase SB203580 showed a partial inhibitory effect on ricin-induced apoptosis in control Vero cells, but it was less effective in butyric acid-treated Vero cells. Taken together, our results suggest that butyric acid-treatment may result in sensitization of multiple intracellular signal transduction pathways including apoptotic signaling pathways and p38 MAP kinase pathway.     

Blood coagulation and fibrinolysis of rats fed fish oil: reduced coagulation factors especially involved in intrinsic pathway and increased activity of plasminogen activator inhibitor

Differences in the coagulation and fibrinolytic system of rats fed a fish oil based diet (fish oil diet) and fed a soybean oil based diet (control diet) were determined. Concentrations of plasma lipids were depressed in rats fed the fish oil diet. Prothrombin time (PT) and activated partial thromboplastin time (APTT) of rats fed the fish oil diet were longer than for the rats fed the control diet. Fish oil intake lowered the activities of most of the blood coagulation factors, and strongly depressed the factors involved in the intrinsic pathway. Fish oil also affected the fibrinolysis of rats. Plasminogen activator inhibitor (PAI) activity was elevated in rats fed the fish oil diet. In this study, both blood coagulation and fibrinolysis were down-regulated by feeding the fish oil diet.     

Artificial Extension of the Adenovirus Fiber Shaft Inhibits Infectivity in Coxsackievirus and Adenovirus Receptor-Positive Cell Lines

Recent studies demonstrate that virus-cellular receptor interactions are not the sole determinants of adenovirus (Ad) tropism. It has been shown that the fiber shaft length, which ranges from 6 to 23 beta-repeats in human Ads, also influences viral tropism. However, there is no report that investigates whether artificial extension of the shaft alters the infectivity profile of Ad. Therefore, we constructed Ad serotype 5 (Ad5) capsid-based longer-shafted Ad vectors by incorporating Ad2 shaft fragments of different lengths into the Ad5 shaft. We show that "longer-shafted" Ad vectors (up to 32 beta-repeats) could be rescued. We also show that longer-shafted Ad vectors had no impact on knob-CAR (coxsackievirus and Ad receptor) interaction compared to wild-type Ad. Nevertheless, gene transfer efficiencies of longer-shafted Ad vectors were lower in CAR-positive cell lines compared to wild-type Ad. We suggest that artificial extension of the shaft can inhibit infectivity in the context of CAR-positive cell lines without modification of knob-CAR interaction.     

Robustness of hen lysozyme monitored by random mutations

We investigated the robustness of hen lysozyme by using random mutant libraries. Six random mutant libraries containing 1, 1.5, 2, 3, 5 and 14 amino acid mutations per hen lysozyme were systematically constructed by varying the concentrations of Mg(2+) and Mn(2+) on polymerase chain reaction. The mutated genes from the six libraries were cloned to a yeast expression vector and a total of 4000 clones were screened on the basis of lysis activity and ELISA employing monoclonal antibody that recognized only lysozyme with native conformation. About 80% of the clones with an average of two amino acid mutations retained active structure. Almost all clones with an average of five mutations lost active structure. On the other hand, 80% of the clones with an average of two amino acid mutations retained both gross conformation and active structure and 24% of the clones with an average of 14 amino acid mutations retained gross conformation. These results show that gross conformation is robust against mutations and so is active structure to a lesser extent.     

Highly stable and efficient mRNA templates for mRNA-protein fusions and C-terminally labeled proteins

For high-throughput in vitro protein selection using genotype (mRNA)-phenotype (protein) fusion formation and C-terminal protein labeling as a post-selection analysis, it is important to improve the stability and efficiency of mRNA templates for both technologies. Here we describe an efficient single-strand ligation (90% of the input mRNAs) using a fluorescein-conjugated polyethylene glycol puromycin (Fluor-PEG Puro) spacer. This ligation provides a stable c-jun mRNA with a flexible Fluor-PEG Puro spacer for efficient fusion formation (70% of the input mRNA with the PEG spacer) in a cell-free wheat germ translation system. When using a 5' untranslated region including SP6 promoter and Omega29 enhancer (a part of tobacco mosaic virus Omega), an A(8) sequence (eight consecutive adenylate residues) at the 3' end is suitable for fusion formation, while an XA(8) sequence (XhoI and the A(8) sequence) is suitable for C-terminal protein labeling. Further, we report that Fluor-PEG N-t-butyloxycarbonylpuromycin [Puro(Boc)] spacer enhances the stability and efficiency of c-jun mRNA template for C-terminal protein labeling. These mRNA templates should be useful for puromycin-based technologies (fusion formation and C-terminal protein labeling) to facilitate high-throughput in vitro protein selection for not only evolutionary protein engineering, but also proteome exploration.