X-ray structures of the <Emphasis Type="Italic">Pseudomonas cichorii</Emphasis> D-tagatose 3-epimerase mutant form C66S recognizing deoxy sugars as substrates

Pseudomonas cichorii D-tagatose 3-epimerase (PcDTE), which has a broad substrate specificity, efficiently catalyzes the epimerization of not only D-tagatose to D-sorbose but also D-fructose to D-psicose (D-allulose) and also recognizes the deoxy sugars as substrates. In an attempt to elucidate the substrate recognition and catalytic reaction mechanisms of PcDTE for deoxy sugars, the X-ray structures of the PcDTE mutant form with the replacement of Cys66 by Ser (PcDTE_C66S) in complexes with deoxy sugars were determined. These X-ray structures showed that substrate recognition by the enzyme at the 1-, 2-, and 3-positions is responsible for enzymatic activity and that substrate-enzyme interactions at the 4-, 5-, and 6-positions are not essential for the catalytic reaction of the enzyme leading to the broad substrate specificity of PcDTE. They also showed that the epimerization site of 1-deoxy 3-keto D-galactitol is shifted from C3 to C4 and that 1-deoxy sugars may bind to the catalytic site in the inhibitor-binding mode. The hydrophobic groove that acts as an accessible surface for substrate binding is formed through the dimerization of PcDTE. In PcDTE_C66S/deoxy sugar complex structures, bound ligand molecules in both the linear and ring forms were detected in the hydrophobic groove, while bound ligand molecules in the catalytic site were in the linear form. This result suggests that the sugar-ring opening of a substrate may occur in the hydrophobic groove and also that the narrow channel of the passageway to the catalytic site allows a substrate in the linear form to pass through.     

Membrane marker movement on sympathetic axons in tissue culture

In cultures of dissociated chick sympathetic ganglia, retrograde (somatopetal) movement of concanavalin A receptors in the axolemma can be observed directly. Such movement was visualized by using concanavalin A coated red blood cells (ConA-RBCs) as membrane markers. Forty-eight percent of all ConA-RBCs which bound to sympathetic nerve fibers moved somatopetally at rates ranging from 11-84 μ/hr with a mean and standard error of 49 ± 6 μ/hr (n = 18). On nongrowing nerve fibers, the ConA- RBCs within 60 μ of the cell body showed retrograde movement, while on elongating neurites only those markers within 30 μ showed such movement. The rate of retrograde ConA-RBC movement appeared to increase with distance from the cell soma. The binding of ConA-RBCs to sympathetic neurites was specific for concanavalin A receptors since pretreatment with either concanavalin A or α-methylglucopyranoside prevented this binding. Untreated polystyrene beads (1.1 μ) which bound nonspecifically to the neurite membrane also showed retrograde movement. These beads moved somatopetally at rates similar to those of the ConA-RBCs but did so regardless of their initial distance from the soma. These data suggest that retrograde movement of surface elements might be a general property of cultured sympathetic nerve fibers.     

Possible Involvement of Jasmonic Acid in Tuberization of Yam Plants

Jasmonic acid (JA) was isolated from yam leaves (Dioscorea batatas),with the guidance of assay for tuber-inducing activity whichwas carried out by cultures of single-node segments of potatostems in vitro, and identified by HPLC and mass spectrometry.The level of endogenous JA in the leaves of the plants, whichbeing 5 ? 10^–7 M at the first harvest, increased continuouslywith the growth of the plants. The effect of exogenous JA onthe tuberization of yam plants was examined in vitro, and itwas found to have strong tuber-inducing activity. The thresholdconcentration of JA for induction of yam tuberization was ca.10^–7M. These results suggest that tuberization of yamplants is controlled by JA. (Received February 25, 1991; Accepted April 15, 1991)     

The effects of thromboxane A2 inhibitors (OKY-046 and ONO-3708) and leukotriene inhibitors (AA-861 and LY-171883) on CCl4-induced chronic liver injury in mice

The effects of OKY-046, a selective thromboxane A2 (TxA2) synthetase inhibitor, ONO-3708, a novel TxA2 receptor antagonist, AA-861, a selective 5-lipoxygenase inhibitor and LY-171883, a peptide leukotrienes (p-LTs) receptor antagonist on the chronic liver injury were investigated in mice. The chronic liver injury was induced by the injection of carbon tetrachloride (CCl4) two times a week for twelve weeks in mice. In chronic liver injury models, significant histopathological changes in the liver and extensive elevation of glutamate transaminase (GOT and GPT) activity were observed. Administration of OKY-046, ONO-3708, AA-861 and LY-171883 for 12 weeks suppressed the elevation of serum GOT and GPT levels and histopathological changes in CCl4-induced chronic liver injury. These results suggest that TxA2 and LTs inhibitors are effective for the onset and development of chronic liver injury in mice.     

Evaluation of four phosphopeptide enrichment strategies for mass spectrometry-based proteomic analysis

Information about phosphorylation status can be used to prioritize and characterize biological processes in the cell. Various analytical strategies have been proposed to address the complexity of phosphorylation status and comprehensively identify phosphopeptides. In this study, we evaluated four strategies for phosphopeptide enrichment, using titanium dioxide (TiO₂) and Phos-tag ligand particles from in-gel or in-solution digests prior to mass spectrometry-based analysis. Using TiO₂ and Phos-tag magnetic beads, it was possible to enrich phosphopeptides from in-gel digests of phosphorylated ovalbumin separated by Phos-tag SDS-PAGE or in-solution serum digests, while minimizing non-specific adsorption. The tip-column strategy with TiO₂ particles enabled enrichment of phosphopeptides from in-solution digests of whole-cell lysates with high efficiency and selectivity. However, the tip-column strategy with Phos-tag agarose beads yielded the greatest number of identified phosphopeptides. The strategies using both types of tip columns had a high degree of overlap, although there were differences in selectivity between the identified phosphopeptides. Together, our results indicate that multi-enrichment strategies using TiO₂ particles and Phos-tag agarose beads are useful for comprehensive phosphoproteomic analysis.     

Haptoglobin gene promoter polymorphism and haplotypes are unique in different populations

We investigated the distribution of haptoglobin (HP) alleles and haplotypes among Africans (Ghanaians), Europeans (from South Africa), and Chinese. HP*1F was present only in Africans and Europeans, whereas HP*del was unique to Chinese. Six base substitutions at the promoter region were population specific. Only 3 out of 18 haplotypes were shared among the populations. A probable application of HP in human population genetics appears legitimate.