Molecular cloning, structural analysis and mass spectrometric identification of native dioscorins of various yam species.

BACKGROUND: Dioscorins are the major storage proteins of yam tubers. However, the molecular nature of their heterogeneity in tubers has not been fully elucidated. In this study the authors isolated the dioscorin gene families of Dioscorea japonica and Dioscorea pseudojaponica, performed matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and elucidated which dioscorin isoforms are the major constituents in tubers. RESULTS: The dioscorin gene families of D. japonica (Dj-dioA1-Dj-dioA4, Dj-dioB1 and Dj-dioB2) and D. pseudojaponica (Dp-dioA1-Dp-dioA5 and Dp-dioB1) were cloned from cDNA libraries of yam tubers. The dioscorins isolated from Dioscorea alata (Da-dioscorins), D. japonica (Dj-dioscorins) and D. pseudojaponica (Dp-dioscorins) were mainly monomers, with a few dimers. The monomers contained one intramolecular disulfide bond (Cys(28)-Cys(187)) and belonged to Class A dioscorins with two cysteine residues. The dimers consisted of Class B dioscorins with one intermolecular disulfide bond (Cys(40)-Cys(40)). Results of MALDI-TOF-MS revealed that the Da-dioscorins were mainly encoded by Da-dioA2, Da-dioA3 and Da-dioA4. The majority of the Dj-dioscorins were encoded by Dj-dioA1, Dj-dioA2, Dj-dioA3 and Dj-dioB2. The Dp-dioscorins mainly comprised proteins encoded by Dp-dioA1, Dp-dioA3, Dp-dioA4, Dp-dioB1 and Dp-dioB2. CONCLUSION: Determination of the constituents of dioscorin isoforms in yam tubers provides a basis for future studies of their physiological and biomedical functions.

Different modulating effects of adenosine on neonatal and adult polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNs) are the major leukocytes in the circulation and play an important role in host defense. Intact PMN functions include adhesion, migration, phagocytosis, and reactive oxygen species (ROS) release. It has been known for a long time that adenosine can function as a modulator of adult PMN functions. Neonatal plasma has a higher adenosine level than that of adults; however, little is known about the modulating effects of adenosine on neonatal PMNs. The aim of this study was to investigate the effects of adenosine on neonatal PMN functions. We found that neonatal PMNs had impaired adhesion, chemotaxis, and ROS production abilities, but not phagocytosis compared to adult PMNs. As with adult PMNs, adenosine could suppress the CD11b expressions of neonatal PMNs, but had no significant suppressive effect on phagocytosis. In contrast to adult PMNs, adenosine did not significantly suppress chemotaxis and ROS production of neonatal PMNs. This may be due to impaired phagocyte reactions and a poor neonatal PMN response to adenosine. Adenosine may not be a good strategy for the treatment of neonatal sepsis because of impaired phagocyte reactions and poor response.

Recombinant dioscorins of the yam storage protein expressed in Escherichia coli exhibit antioxidant and immunomodulatory activities.

Dioscorins, the major storage proteins in yam tubers, exhibit biochemical and immunomodulatroy activities. To investigate the potential application of dioscorins in biomedical research, we expressed the dioscorin genes Dj-dioA3 and Dp-dioA2 from Dioscorea japonica and Dioscorea pseudojaponica, respectively, in E. coli and routinely obtained approximately 15 mg proteins per liter Escherichia coli culture (mg/L) to 30 mg/L of rDj-dioscorinA3 and 4 to 8 mg/L of rDp-dioscorinA2. Western blot analyses revealed that both recombinant dioscorins contained epitopes with similar antigenicities to those of the native dioscorins. Results from dithiothreitol (DTT) treatment followed by monobromobimane (mBBr) staining showed that both recombinant dioscorins, like the native dioscorins, contain an intramolecular disulfide bond between Cys(28) and Cys(187) residues. Circular dichroism spectroscopy findings indicated that the secondary structural contents of the recombinant dioscorins showed high similarity to those of their corresponding native dioscorins. Both recombinant dioscorins, like the native dioscorins, exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and Toll-like receptor 4 signaling activities, and stimulated the phagocytosis of E. coli by macrophage. Overall, our results indicated that substantial amounts of recombinant dioscorins can be purified easily from E. coli and that these recombinant dioscorins are appropriate for application in future investigations of the biomedical functions of dioscorins.

Identification of immunodeficient molecules in neonatal mononuclear cells by proteomic differential displays.

Human newborns are known to be susceptible to microbial infection. This susceptibility is generally attributed to immaturity of the newborn immune system. However, the mechanisms for impaired immunity in newborns are still incompletely defined. In this study, we sought to elucidate the protein differential display between adult and neonatal mononuclear cells (MNC) using a proteomic approach. MNC samples from cord blood and adult peripheral blood were subjected to 2-D PAGE analysis. Differential protein displays between cord blood and adult MNC were determined and validated. There were 34 differentially expressed proteins between cord blood and adult MNC identified by 2-D PAGE. The differentially displayed proteins were clustered into two major signal pathways, cellular processing and purine metabolism. After validation by Western blot, we found more abundant arginase-1 (ARG1) and Rho GDP-dissociation inhibitor 2 (RhoGDI2), while less adenosine deaminase (ADA) and ß-actin in cord blood MNC. In functional validation, we found that lower ADA was proven to enhance the TNF-α production by cord blood monocytes. The results from this study discovered the proteomic displays for altered immunity between adult and neonatal MNC that support a understanding of the correction of impaired immune response in newborns.