Can the Quality of Pearls from the Japanese Pearl Oyster (<Emphasis Type="Italic">Pinctada fucata</Emphasis>) be Explained by the Gene Expression Patterns of the Major Shell Matrix Proteins in the Pearl Sac?

For pearl culture, the pearl oyster is forced open and a nucleus is implanted into the gonad with a mantle graft. The outer mantle epithelial cells of the implanted mantle graft elongate and surrounding the nucleus a pearl sac is formed. Shell matrix proteins secreted by the pearl sac play an important role in the regulation of pearl formation. Recently, seven shell matrix proteins were identified from the pearl oyster Pinctada fucata. However, there is a paucity of information on the function of these proteins and their gene expression patterns. Our study aims to elucidate the relationship between pearl type, quality, and gene expression patterns of six shell matrix proteins (msi60, n16, nacrein, msi31, prismalin-14, and aspein) in the pearl sac based on real-time PCR analysis. After culturing for about 2 months, the pearl sac tissues were collected from 22 individuals: 12 with high quality (HP), nine with low quality (LP), and one with organic (ORG) pearl formation. The surface of each of the 12 HP pearls was composed only of a nacreous layer; in contrast, that of the nine LP pearls was composed of nacreous and prismatic layers. The six target gene expressions were detected in all individuals. However, delta threshold cycle (ΔC _T) for msi31 was significantly higher in the HP than in the LP individuals (Mann–Whitney’s U test, p = 0.02). This means that the relative expression level of msi31, which constitutes the framework of the prismatic layer, was higher in the LP than in the HP individuals.     

Proteins binding to the 5''''-flanking regulatory elements of the human β-globin gene~1

The binding of nuclear proteins prepared from mouse erythroid tissue in different developmental stages to the 5'-flanking regulatory elements of human globin gene, two negative control regions(NCR1, -610 to -490 bp; NCR2,-338 to -233bp), was identified. Two stage specific protein factors corresponding to embryonic and fetal stages were found to be capable of binding to NCR2. These data provided evidence that the cis acting elements of the 5'-flanking region might be involved in the developmental control of globin gene and NCR2 might be responsible in part for the silence of globin gene in the embryonic and fetal stages.     

β-Galactosyl Yariv Reagent Binds to the β-1,3-Galactan of Arabinogalactan Proteins

Yariv phenylglycosides [1,3,5-tri(p-glycosyloxyphenylazo)-2,4,6-trihydroxybenzene] are a group of chemical compounds that selectively bind to arabinogalactan proteins (AGPs), a type of plant proteoglycan. Yariv phenylglycosides are widely used as cytochemical reagents to perturb the molecular functions of AGPs as well as for the detection, quantification, purification, and staining of AGPs. However, the target structure in AGPs to which Yariv phenylglycosides bind has not been determined. Here, we identify the structural element of AGPs required for the interaction with Yariv phenylglycosides by stepwise trimming of the arabinogalactan moieties using combinations of specific glycoside hydrolases. Whereas the precipitation with Yariv phenylglycosides (Yariv reactivity) of radish (Raphanus sativus) root AGP was not reduced after enzyme treatment to remove α-l-arabinofuranosyl and β-glucuronosyl residues and β-1,6-galactan side chains, it was completely lost after degradation of the β-1,3-galactan main chains. In addition, Yariv reactivity of gum arabic, a commercial product of acacia (Acacia senegal) AGPs, increased rather than decreased during the repeated degradation of β-1,6-galactan side chains by Smith degradation. Among various oligosaccharides corresponding to partial structures of AGPs, β-1,3-galactooligosaccharides longer than β-1,3-galactoheptaose exhibited significant precipitation with Yariv in a radial diffusion assay on agar. A pull-down assay using oligosaccharides cross linked to hydrazine beads detected an interaction of β-1,3-galactooligosaccharides longer than β-1,3-galactopentaose with Yariv phenylglycoside. To the contrary, no interaction with Yariv was detected for β-1,6-galactooligosaccharides of any length. Therefore, we conclude that Yariv phenylglycosides should be considered specific binding reagents for β-1,3-galactan chains longer than five residues, and seven residues are sufficient for cross linking, leading to precipitation of the Yariv phenylglycosides.Arabinogalactan proteins (AGPs) are a type of plant proteoglycans consisting of a Hyp-rich core protein and large arabinogalactan (AG) moieties (Fincher et al., 1983; Nothnagel, 1997). Although there are many molecular species of AGP differentiated by their core proteins, the AG moieties commonly comprise β-1,3-galactan main chains and β-1,6-galactan side chains, to which l-Ara and other auxiliary sugars, such as GlcA, 4-O-methyl-GlcA, l-Fuc, l-Rha, and Xyl, are attached (Fincher et al., 1983; Nothnagel, 1997; Seifert and Roberts, 2007). A commercial product of AGPs prepared from the acacia (Acacia senegal) tree is known as gum arabic and utilized as a food stabilizer. In the Japanese herbal remedy Juzen-Taiho-To, AGs from Astragalus membranaceus are the active ingredient (Majewska-Sawka and Nothnagel, 2000; Kiyohara et al., 2002). In intact plants, AGPs are implicated in various physiological events and serve as extracellular constituents and signaling molecules. For instance, an AGP from stylar transmitting tissue attracts pollen tubes and stimulates their elongation in tobacco (Nicotiana tabacum; Cheung et al., 1995).Yariv phenylglycosides [1,3,5-tri(p-glycosyloxyphenylazo)-2,4,6-trihydroxybenzene] are a group of chemical compounds that were initially developed as carbohydrate antigens for the purification of anti-glycoside antibody and sugar-binding protein (Yariv et al., 1962, 1967a). It then turned out that Yariv phenylglycosides specifically precipitate AGPs (Yariv et al., 1967b; Jermyn and Yeow, 1975). The specific interaction of AGPs with Yariv phenylglycosides forming brown-red precipitate is called Yariv reactivity and has been recognized as an important criterion in the definition of AGPs, even though a number of AGPs do not exhibit Yariv reactivity. Nevertheless, the structure involved in the interaction with Yariv phenylglycoside is presumed to be conserved in many AGPs. The interaction of Yariv phenylglycosides with AGP depends on the glycosyl residues attached to the phenylazotrihydroxybenzene core. In particular, β-glucosyl Yariv phenylglycoside (β-Glc-Yariv) and β-galactosyl Yariv phenylglycoside (β-Gal-Yariv) bind to AGPs, whereas α-glucosyl Yariv and α-galactosyl Yariv (α-Gal-Yariv) do not bind to AGPs (Jermyn and Yeow, 1975; Larkin, 1977, 1978; Nothnagel and Lyon, 1986). Because of the specific interaction with the β-glycosyl Yariv phenylglycosides (β-Yarivs), AGPs were formerly called “β-lectins” (Jermyn and Yeow, 1975; Gleeson and Jermyn, 1979; Nothnagel and Lyon, 1986).The β-Yarivs are useful tools for staining, detection, and quantification of AGPs. Using β-Glc-Yariv, β-lectins were shown to exist in angiosperm, gymnosperm, fern, moss, and liverwort, illustrating the wide distribution of AGPs in the plant kingdom (Jermyn and Yeow, 1975; Clarke et al., 1978). In addition, β-Yarivs are also used as chemical reagents in the purification of AGPs. A nonclassical AGP, xylogen, which is a signaling molecule inducing the differentiation to tracheary elements, has been purified from the culture medium of zinnia (Zinnia elegans) cells by precipitation with β-Glc-Yariv (Motose et al., 2004). As the treatment with β-Yarivs causes the perturbation of various physiological processes in plants, β-Yarivs are reliable cytochemical reagents to explore AGP functions. Application of β-Yarivs to cultured cells of Arabidopsis (Arabidopsis thaliana) induced programmed cell death, demonstrating the involvement of AGPs in the determination of cell fate (Gao and Showalter, 1999). In tobacco cultured cells, the treatment with β-Yarivs has indicated a possible role of AGPs in the orientation of cortical microtubules and the polymerization of F-actin (Sardar et al., 2006).Although Yariv phenylglycosides have been extensively utilized in studies of AGPs over 40 years, the identification of the target structures on AGPs required for β-Yariv reactivity remains elusive (Nothnagel, 1997; Seifert and Roberts, 2007). It has been proposed that β-Yarivs bind to the Hyp-rich core protein, based on the observation that deglycosylation treatment with hydrogen fluoride did not abolish the Yariv reactivity of gum arabic and a tobacco AGP (Akiyama et al., 1984). To the contrary, other reports have asserted the importance of the carbohydrate moieties for Yariv reactivity (Komalavilas et al., 1991). However, with regard to the specific carbohydrate structure required for interaction with β-Yarivs, the results were not always consistent: neither α-l-arabinofuranosyl residues nor β-1,6-galactan side chains were found to be involved in Yariv reactivity of AGPs from Gladiolus spp., radish (Raphanus sativus), and grape (Vitis vinifera; Gleeson and Clarke, 1979; Tsumuraya et al., 1987; Saulnier et al., 1992); partial acid hydrolysis to remove α-l-arabinofuranosyl residues diminished Yariv reactivity of a rose (Rosa spp.) AGP (Komalavilas et al., 1991); and mugwort (Artemisia vulgaris) pollen O-glycans consisting of a β-1,6-galactan core and branched α-l-arabinofuranosyl side chains precipitated with β-Glc-Yariv (Léonard et al., 2005). Accordingly, it has also been suggested that Yariv reactivity depends on the overall physical and chemical properties rather than a specific structural feature of AGPs.In this study, we demonstrate that the peptide component of AGPs is not required for Yariv reactivity. By sequentially trimming the AG moieties of AGPs with sets of specific glycoside hydrolases, we show that β-Gal-Yariv binds to the β-1,3-galactan main chains of radish root AGP. We confirm that β-1,6-galactan side chains are not necessary for Yariv reactivity, we identify β-1,3-galactopentaose (β-1,3-Gal₅) as the smallest carbohydrate structure to interact with β-Gal-Yariv, and we show that β-1,3-galactoheptaose (β-1,3-Gal₇) or longer β-1,3-galactosyl chains are required for the formation of insoluble precipitate with Yariv phenylglycoside. Based on computational modeling, a possible interaction mechanism between β-Gal-Yariv and β-1,3-galactan is suggested.     

Proteins binding to the 5‘—flanking regualtory elements of the human β—globin gene

The binding of nuclear proteins prepared from mouse erythroid tissue in different developmental stages to the 5‘-flanking regulatory elements of human β-globin gene,two negative control regions(NCR1,-610to-490 bp;NCR2,-338,to-233bp),was identified.Two stage specific protein factors corresponding to embryonic and fetal stages were found to be capable of binding to NCR2.These data provided evidence that the cis acting elements of the 5‘-flanking region might be involved in the developmental control of β-globin gene and NCR2 might be responsible in art for the silence of β-glolbin gene in the embryonic and fetal stages.     

Plumieribetin, a Fish Lectin Homologous to Mannose-binding B-type Lectins, Inhibits the Collagen-binding ��1��1 Integrin

Recently, a few fish proteins have been described with a high homology to B-type lectins of monocotyledonous plants. Because of their mannose binding activity, they have been ascribed a role in innate immunity. By screening various fish venoms for their integrin inhibitory activity, we isolated a homologous protein from the fin stings and skin mucus of the scorpionfish (Scorpaena plumieri). This protein inhibits α1β1 integrin binding to basement membrane collagen IV. By protein chemical and spectroscopic means, we demonstrated that this fish protein, called plumieribetin, is a homotetramer and contains a high content of anti-parallel β strands, similar to the mannose-binding monocot B-lectins. It lacks both N-linked glycoconjugates and common O-glycan motifs. Despite its B-lectin-like structure, plumieribetin binds to α1β1 integrin irrespective of N-glycosylation, suggesting a direct protein-protein interaction. This interaction is independent of divalent cations. On the cellular level, plumieribetin failed to completely detach hepatocarcinoma HepG2 cells and primary arterial smooth muscle cells from the collagen IV fragment CB3. However, plumieribetin weakened the cell-collagen contacts, reduced cell spreading, and altered the actin cytoskeleton, after the compensating α2β1 integrin was blocked. The integrin inhibiting effect of plumieribetin adds a new function to the B-lectin family, which is known for pathogen defense.     

Contribution of Specific Residues of the β-Solenoid Fold to HET-s Prion Function,Amyloid Structure and Stability

The [Het-s] prion of the fungus Podospora anserina represents a good model system for studying the structure-function relationship in amyloid proteins because a high resolution solid-state NMR structure of the amyloid prion form of the HET-s prion forming domain (PFD) is available. The HET-s PFD adopts a specific β-solenoid fold with two rungs of β-strands delimiting a triangular hydrophobic core. A C-terminal loop folds back onto the rigid core region and forms a more dynamic semi-hydrophobic pocket extending the hydrophobic core. Herein, an alanine scanning mutagenesis of the HET-s PFD was conducted. Different structural elements identified in the prion fold such as the triangular hydrophobic core, the salt bridges, the asparagines ladders and the C-terminal loop were altered and the effect of these mutations on prion function, fibril structure and stability was assayed. Prion activity and structure were found to be very robust; only a few key mutations were able to corrupt structure and function. While some mutations strongly destabilize the fold, many substitutions in fact increase stability of the fold. This increase in structural stability did not influence prion formation propensity in vivo. However, if an Ala replacement did alter the structure of the core or did influence the shape of the denaturation curve, the corresponding variant showed a decreased prion efficacy. It is also the finding that in addition to the structural elements of the rigid core region, the aromatic residues in the C-terminal semi-hydrophobic pocket are critical for prion propagation. Mutations in the latter region either positively or negatively affected prion formation. We thus identify a region that modulates prion formation although it is not part of the rigid cross-β core, an observation that might be relevant to other amyloid models.     

Analysis of Proteins Binding to the ITAM Motif of the β-Subunit of the High-Affinity Receptor for IgE (FcεRI)

Aggregation of the multichain (α β γ₂) high-affinity IgE receptor (Fcε RI) initiates a signaling cascade that results in the release of allergic mediators. The cytoplasmic tails of the Fcε RI-β and -γ subunits contain immunoreceptor tyrosine-based activation motifs (ITAMs). Phosphorylation of the γ ITAM mediates activation of Syk kinase and is sufficient for triggering the responses induced by Fcε RI crosslinking. Phosphorylation of the β ITAM is insufficient to mediate cell activation. The rat β ITAM contains three tyrosines (Tyr²¹⁸, Tyr²²⁴, and Tyr²²⁸) with an intermediate noncanonical tyrosine. Synthetic peptides based on the ITAM of the Fcε RI-β subunit were used to investigate the role of each phosphotyrosine in the binding of signaling proteins to this motif. Among the proteins that bind to phosphorylated β ITAM are Syk, Grb2, Shc, SHIP, and SHP-1, and binding does not depend on previous cell activation. Nonphosphorylated peptides do not bind these proteins. Syk binding to β -peptides is dependent on the number and position of phosphotyrosines in the ITAM. Phosphorylation of Tyr²¹⁸ seems to be most important for Syk binding. Recruitment of Syk and other signaling proteins to the β -subunit might be important for its amplifier role.     

Prediction of the β-Hairpins in Proteins Using Support Vector Machine

By using of the composite vector with increment of diversity and scoring function to express the information of sequence, a support vector machine (SVM) algorithm for predicting β-hairpin motifs is proposed. The prediction is done on a dataset of 3,088 non homologous proteins containing 6,027 β-hairpins. The overall accuracy of prediction and Matthew’s correlation coefficient are 79.9% and 0.59 for the independent testing dataset. In addition, a higher accuracy of 83.3% and Matthew’s correlation coefficient of 0.67 in the independent testing dataset are obtained on a dataset previously used by Kumar et al. (Nuclic Acid Res 33:154–159). The performance of the method is also evaluated by predicting the β-hairpins of in the CASP6 proteins, and the better results are obtained. Moreover, this method is used to predict four kinds of supersecondary structures. The overall accuracy of prediction is 64.5% for the independent testing dataset.     

Subunit Composition of the Zinc Proteins α- and β-Lipovitellin from Chicken

Chicken α- and β-lipovitellin are derived from parent vitellogenin proteins and contain four subunits (125, 80, 40, and 30 kDa) and two subunits (125 and 30 kDa), respectively. Metal analyses demonstrate both are zinc proteins containing 2.1 ± 0.2 mol of zinc/275 kDa per α-lipovitellin and 1.4 ± 0.2 mol of zinc/155 kDa per β-lipovitellin, respectively. The subunits of β-lipovitellin, Lv 1 (MW 125 kDa) and Lv 2 (MW 30 kDa), are separated by gel exclusion chromatography in the presence of zwittergent 3–16. Zinc elutes with Lv 1, suggesting that this subunit binds zinc in the absence of Lv 2. The subunits of α- and β-lipovitellin were separated by SDS-PAGE, digested with trypsin, and mapped by reverse-phase HPLC. The peptide maps of the 125-kDa subunits from α- and β-lipovitellin are essentially identical. Similar results are obtained for the 30-kDa subunits of both lipovitellins. The sequences of five and four peptides of the 125-kDa subunit of α- and β-Lv, respectively, and two peptides of the 30-kDa subunit of α- and β-lipovitellin were determined and match those predicted from the gene for vitellogenin II, Vtg II. Comparison of the amino acid composition of the 125- and 30-kDa subunits of α- and β-lipovitellin support the conclusion that they originate from the same gene. The sequences of peptides from the 80- and 40-kDa subunits of α-lipovitellin have not been found in the NCBI nonredundant data bank. The 27-amino acid N-terminal sequence of the 40-kDa protein is 56% similar to the last third of the Lv 1-coding region of the Vtg II gene, suggesting it may come from an analogous region of the Vtg I gene. We propose a scheme for the precursor—product relationship of Vtg I.     

Novel Peptide Inhibitors of β-Catenin Effectively Suppress the Tumorigenesis of Colorectal Cancer

Aberrant β-catenin activation promotes the proliferation and survival of several types of tumor cells, including colorectal cancer (CRC) cells. Synthetic peptides are drug candidates for treating various diseases; however, peptide inhibitors of β-catenin have been rarely reported. A series of peptide inhibitors for β-catenin (F15A_1–9k, F15A_2–9k, and F15A_3–9k) were designed and synthesized, and then used to treat human CRC cells (HT-29). Next, a series of in vitro assays, including cell counting, colony formation, flow cytometry, and Transwell assays, were performed to assess the biological effects of the peptides on CRC cells. Mouse xenograft models of HT-29 tumors were also used to evaluate the inhibitory effect of the peptide inhibitors on β-catenin expression in vivo. The inhibitory effect of the peptide inhibitors on β-catenin production was tested in a confocal laser scanning microscope study (CLSMS), and by H&E, TUNEL, and immunohistochemical (IHC) staining. The peptide inhibitors significantly reduced the viability of HT-29 cells in time- and concentration-dependent manners. Moreover, the peptide inhibitors for β-catenin significantly inhibited CRC tumorigenesis both in vitro and in vivo. Mechanistically, the peptide inhibitors for β-catenin inhibited the angiogenesis activity of HT-29 cells. When administered by itself, F15A_2–9k blocked cell division, induced apoptosis, and reduced the migration and invasion capabilities of HT-29 cells, while a combination of F15A_1–9k, F15A_2–9k, and F15A_3–9k showed even stronger inhibitory effects on HT-29 cells. In summary, the peptides designed to inhibit β-catenin demonstrated anti-tumor activity both in vitro and in vivo, suggesting their potential as therapeutic agents for treating CRC.

     

The Thalidomide-Binding Domain of Cereblon Defines the CULT Domain Family and Is a New Member of the β-Tent Fold

Despite having caused one of the greatest medical catastrophies of the last century through its teratogenic side-effects, thalidomide continues to be an important agent in the treatment of leprosy and cancer. The protein cereblon, which forms an E3 ubiquitin ligase compex together with damaged DNA-binding protein 1 (DDB1) and cullin 4A, has been recently indentified as a primary target of thalidomide and its C-terminal part as responsible for binding thalidomide within a domain carrying several invariant cysteine and tryptophan residues. This domain, which we name CULT (cereblon domain of unknown activity, binding cellular ligands and thalidomide), is also found in a family of secreted proteins from animals and in a family of bacterial proteins occurring primarily in δ-proteobacteria. Its nearest relatives are yippee, a highly conserved eukaryotic protein of unknown function, and Mis18, a protein involved in the priming of centromeres for recruitment of CENP-A. Searches for distant homologs point to an evolutionary relationship of CULT, yippee, and Mis18 to proteins sharing a common fold, which consists of two four-stranded β-meanders packing at a roughly right angle and coordinating a zinc ion at their apex. A β-hairpin inserted into the first β-meander extends across the bottom of the structure towards the C-terminal edge of the second β-meander, with which it forms a cradle-shaped binding site that is topologically conserved in all members of this fold. We name this the β-tent fold for the striking arrangement of its constituent β-sheets. The fold has internal pseudosymmetry, raising the possibility that it arose by duplication of a subdomain-sized fragment.     

Primordial linkage of β2-microglobulin to the MHC

β2-Microglobulin (β2M) is believed to have arisen in a basal jawed vertebrate (gnathostome) and is the essential L chain that associates with most MHC class I molecules. It contains a distinctive molecular structure called a constant-1 Ig superfamily domain, which is shared with other adaptive immune molecules including MHC class I and class II. Despite its structural similarity to class I and class II and its conserved function, β2M is encoded outside the MHC in all examined species from bony fish to mammals, but it is assumed to have translocated from its original location within the MHC early in gnathostome evolution. We screened a nurse shark bacterial artificial chromosome library and isolated clones containing β2M genes. A gene present in the MHC of all other vertebrates (ring3) was found in the bacterial artificial chromosome clone, and the close linkage of ring3 and β2M to MHC class I and class II genes was determined by single-strand conformational polymorphism and allele-specific PCR. This study satisfies the long-held conjecture that β2M was linked to the primordial MHC (Ur MHC); furthermore, the apparent stability of the shark genome may yield other genes predicted to have had a primordial association with the MHC specifically and with immunity in general.     

Contribution to the genetics of the serum β-lipoproteins in man

A study was made of the genetic behaviour of the factors Ag(x) and Ag(y) of the β-lipoproteins of human serum. It was found that these factors are controlled by a single pair of autosomal codominant genes with complete penetrance at birth. The gene frequencies were:

$$\begin{gathered} Milan . . . . Ag^x = 0,23 Ag^y = 0,77 \hfill \\ Berne . . . . Ag^x = 0,24 Ag^y = 0,76. \hfill \\ \end{gathered}$$     

Proteomic Analysis of Extracellular ATP-Regulated Proteins Identifies ATP Synthase β-Subunit as a Novel Plant Cell Death Regulator

Extracellular ATP is an important signal molecule required to cue plant growth and developmental programs, interactions with other organisms, and responses to environmental stimuli. The molecular targets mediating the physiological effects of extracellular ATP in plants have not yet been identified. We developed a well characterized experimental system that depletes Arabidopsis cell suspension culture extracellular ATP via treatment with the cell death-inducing mycotoxin fumonisin B1. This provided a platform for protein profile comparison between extracellular ATP-depleted cells and fumonisin B1-treated cells replenished with exogenous ATP, thus enabling the identification of proteins regulated by extracellular ATP signaling. Using two-dimensional difference in-gel electrophoresis and matrix-assisted laser desorption-time of flight MS analysis of microsomal membrane and total soluble protein fractions, we identified 26 distinct proteins whose gene expression is controlled by the level of extracellular ATP. An additional 48 proteins that responded to fumonisin B1 were unaffected by extracellular ATP levels, confirming that this mycotoxin has physiological effects on Arabidopsis that are independent of its ability to trigger extracellular ATP depletion. Molecular chaperones, cellular redox control enzymes, glycolytic enzymes, and components of the cellular protein degradation machinery were among the extracellular ATP-responsive proteins. A major category of proteins highly regulated by extracellular ATP were components of ATP metabolism enzymes. We selected one of these, the mitochondrial ATP synthase β-subunit, for further analysis using reverse genetics. Plants in which the gene for this protein was knocked out by insertion of a transfer-DNA sequence became resistant to fumonisin B1-induced cell death. Therefore, in addition to its function in mitochondrial oxidative phosphorylation, our study defines a new role for ATP synthase β-subunit as a pro-cell death protein. More significantly, this protein is a novel target for extracellular ATP in its function as a key negative regulator of plant cell death.ATP is a ubiquitous, energy-rich molecule of fundamental importance in living organisms. It is a key substrate and vital cofactor in many biochemical reactions and is thus conserved by all cells. However, in addition to its localization and functions inside cells, ATP is actively secreted to the extracellular matrix where it forms a halo around the external cell surface. The existence of this extracellular ATP (eATP)¹ has been reported in several organisms including bacteria (1), primitive eukaryotes (2), animals (3), and plants (4–6). This eATP is not wasted, but harnessed at the cell surface as a potent signaling molecule enabling cells to communicate with their neighbors and regulate crucial growth and developmental processes.In animals, eATP is a crucial signal molecule in several physiological processes such as neurotransmission (7, 8), regulation of blood pressure (9), enhanced production of reactive oxygen species (ROS) (10), protein translocation (11), and apoptosis (12). Extracellular ATP signal perception at the animal cell surface is mediated by P2X and P2Y receptors, which bind ATP extracellularly and recruit intracellular second messengers (13, 14). P2X receptors are ligand-gated ion channels that provide extracellular Ca²⁺ a corridor for cell entry after binding eATP, facilitating a surge in cytosolic [Ca²⁺] that is essential in activating down-stream signaling. P2Y receptors transduce the eATP signal by marshalling heteromeric G-proteins on the cytosolic face of the plasma membrane and activating appropriate downstream effectors.Although eATP exists in plants, homologous P2X/P2Y receptors for eATP signal perception have not yet been identified, even in plant species with fully sequenced genomes. Notwithstanding the obscurity of plant eATP signal sensors, some of the key downstream messengers recruited by eATP-mediated signaling are known. For example, eATP triggers a surge in cytosolic Ca²⁺ concentration (15–17) and a heightened production of nitric oxide (18–20) and reactive oxygen species (17, 21, 22). Altering eATP levels is attended by activation of plant gene expression (16, 21) and changes in protein abundance (5, 23), indicating that eATP-mediated signaling impacts on plant physiology. Indeed eATP has been demonstrated to regulate plant growth (20, 24–26), gravitropic responses (27), xenobiotic resistance (4), plant-symbiont interactions (28), and plant-pathogen interactions (23, 29). However, the mechanism by which eATP regulates these processes remains unclear, largely because the eATP signal sensors and downstream signal regulatory genes and proteins have not been identified.We previously reported that eATP plays a central regulatory role in plant cell death processes (5). Therefore, an understanding of the signaling components galvanized by eATP in cell death regulation might serve a useful purpose in providing mechanistic detail of how eATP signals in plant physiological processes. We found that eATP-mediated signaling negatively regulates cell death as its removal by application of ATP-degrading enzymes to the apoplast activates plant cell death (5). Remarkably, fumonisin B1 (FB1), a pathogen-derived molecule that activates defense gene expression in Arabidopsis (30), commandeers this eATP-regulated signaling to trigger programmed cell death (5). FB1 is a mycotoxin secreted by fungi in the genus Fusarium and initiates programmed cell death in both animal and plant cells (31, 32). In Arabidopsis, FB1 inaugurates cell death by inactivating eATP-mediated signaling via triggering a drastic collapse in the levels of eATP (5). FB1-induced Arabidopsis programmed cell death is dependent on the plant signaling hormone salicylic acid (33), which is a key regulator of eATP levels (29). Because concurrent application of FB1 and exogenous ATP to remedy the FB1-induced eATP deficit blocks death, FB1 and exogenous ATP treatments can therefore be used as probes to identify the key signal regulators downstream of eATP in cell death control. This is vital for achieving the global objective of elucidating the mechanism of eATP signaling in plant physiology.Gel-based proteomic analyses have been previously applied to successfully identify the novel role of eATP in the regulation of plant defense gene expression and disease resistance (23, 29). We have now employed FB1 and ATP treatments together with two-dimensional difference in-gel electrophoresis (DIGE) and matrix-assisted laser desorption-time of flight MS (MALDI-TOF MS) to identify the changes in Arabidopsis protein profiles associated with a shift from normal to cell death-inception metabolism. Additional reverse genetic analyses enabled us to definitively identify a putative ATP synthase β-subunit as a target for eATP-mediated signaling with an unexpected function in the regulation of plant programmed cell death.     

A ′Fragment Fitting Approach′ to Model Disulfide Loops by Utilizing Homologous Peptide Fragments from Unrelated Proteins of Known Structures: Application to the V3 Loop of the HIV-1 Envelope Glycoprotein gp120

The V3 loop of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 has gained considerable attention for developing subunit vaccines against HIV-1 and also as a target to develop anti-HIV-1 drugs. These endeavors would be significantly enhanced by understanding the structural aspects of this loop. The structure of the full-length V3 loop has not been defined yet. Therefore, a novel modeling technique, termed `Fragment Fitting Approach′ (FFA), was developed to model the V3 loop. This technique utilizes fragments (³ 6 residue long) with local sequence and secondary structure similarity from unrelated proteins with known x-ray crystallographic structure and concatenating the fragments to build the model. A systematic search method was devised to identify the fragments using the combined criteria of sequence and secondary structure identity and/or similarity, predicted by a combination of methods. FFA requires partial three-dimensional coordinates of the target sequence to be modelled to get the overall coordinate path correct. The method was validated with nine disulfide-bonded loops from the Protein data bank. The modelled structures conform well with the corresponding x-ray crystallographic structures. As the models were built using the x-ray coordinates with reasonable resolution (£ 3 Å), they are expected to have stereochemically correct structures. The modelled V3 loop structure might assist in structure-based drug design of anti-HIV-1 agents targeted to this loop.