Multipoint mapping studies of six loci on chromosome 11

The six loci, beta-globin (HBBC), parathyroid hormone (PTH), oncogene c-Ha-ras-1 (HRAS1), insulin (INS), calcitonin (CAL) and catalase (CAT) loci, have been mapped to 11p in the order: CAT-CAL-PTH-HBBC-(HRAS1-INS). The purpose of the current study was to examine the linkage relationships, especially the multipoint relationships of these loci in detail. In the 18 families studied, a significant sex difference in recombination was found for the HBBC: HRAS1 linkage with more recombination in the male parent than the female parent (22 vs. 2%). The results of the multipoint analyses provided further evidence for the order CAT-CAL-PTH-HBBC-(HRAS1-INS); however, the order of the last two tightly linked loci is still not clear.     

Design of Peptides Using α,β-Dehydro-residues: Synthesis,Crystal Structure and Molecular Conformation of N-Boc-L-Val-ΔPhe–ΔPhe-L-Ala-OCH3

To obtain general rules of peptide design using α,β-dehydro-residues, a sequence with two consecutive ΔPhe-residues, Boc-L -Val-ΔPhe–ΔPhe- L -Ala-OCH₃, was synthesized by azlactone method in solution phase. The peptide was crystallized from its solution in an acetone/water mixture (70:30) in space group P6₁ with a=b=14.912(3) Å, c= 25.548(5) Å, V=4912.0(6) ų. The structure was determined by direct methods and refined by a full matrix least-squares procedure to an R value of 0.079 for 2891 observed [I?3σ(I)] reflections. The backbone torsion angles ?₁=?54(1)°, ψ₁= 129(1)°, ω₁=?177(1)°, ?₂ =57(1)°, ψ₂=15(1)°, ω₂ =?170(1)°, ?₃=80(1)°, ψ₃ =7(2)°, ω₃=?177(1)°, ?₄ =?108(1)° and ψ^T₄=?34 (1)° suggest that the peptide adopts a folded conformation with two overlapping β-turns of types II and III′. These turns are stabilized by two intramolecular hydrogen bonds between the CO of the Boc group and the NH of ΔPhe³ and the CO of Val¹ and the NH of Ala⁴. The torsion angles of ΔPhe² and ΔPhe³ side chains are similar and indicate that the two ΔPhe residues are essentially planar. The folded molecules form head-to- tail intermolecular hydrogen bonds giving rise to continuous helical columns which run parallel to the c-axis. This structure established the formation of two β-turns of types II and III′ respectively for sequences containing two consecutive ΔPhe residues at (i+2) and (i+3) positions with a branched β-carbon residue at one end of the tetrapeptide.     

Regulation of endomembrane biogenesis in arabidopsis by phospatidic acid hydrolase

Coordination of membrane lipid biosynthesis is important for cell function during plant growth and development. Here we summarize our recent work on PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAH) which suggests that this enzyme is a key regulator of phosphaticylcholine (PC) biosynthesis in Arabidopsis thaliana. Disruption of PAH activity elevates phosphatidic acid (PA) levels and stimulates PC biosynthesis and biogenesis of the endoplasmic reticulum (ER). Furthermore, the activity of PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE (CCT), which is the key enzyme controlling the rate of PC biosynthesis, is directly stimulated by PA and expression of a constitutively active version of CCT replicates the effects of PAH disruption. Hence PAH activity can control the abundance of PA, which in turn can modulate CCT activity to govern the rate of PC biosynthesis. Crucially it is not yet clear how PAH activity is regulated in Arabidopsis but there is evidence that PAH1 and PAH2 are both phosphorylated and further work will be required to investigate whether this is functionally significant.     

Cytoprotective Effect of Recombinant Human Erythropoietin Produced in Transgenic Tobacco Plants

Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPO^M) by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT) genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC) promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPO^P) was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPO^P bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPO^P (20 U/ml) provides 2-fold better cytoprotection (44%) to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPO^M (21%). The cytoprotective effect of the asialo-rhuEPO^P was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2) and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.     

Understanding the binding interaction between phenyl boronic acid P1 and sugars: determination of association and dissociation constants using S–V plots,steady‐state spectroscopic methods and molecular docking

Continuous monitoring of glucose and sugar sensing plays a vital role in diabetes control. The drawbacks of the present enzyme‐based sugar sensors have encouraged the investigation into alternate approaches to design new sensors. The popularity of fluorescence sensors is due to their ability to bind reversibly to compounds containing diol. In this study we investigated the binding ability of phenyl boronic acid P1 for monosaccharides and disaccharides (sugars) in aqueous medium at physiological pH 7.4 using steady‐state fluorescence and absorbance. P1 fluorescence was quenched due to formation of esters with sugars. Absorbance and fluorescence measurements led to results that indicated that the sugars studied could be ordered in terms of their affinity to P1, as stated: sucrose > lactose > galactose > xylose > ribose > arabinose. In each case, the slope of modified Stern–Volmer plots was nearly 1, indicating the presence of only a single binding site in boronic acids for sugars. Docking studies were carried out using Schrodinger Maestro v.11.2 software. The binding affinity of phenyl boronic acid P1 with periplasmic protein (PDB ID 2IPM and 2IPL) was estimated using GlideScore.     

Temporal variations and pond age effect on plankton communities in semi-intensive freshwater marron (Cherax cainii,Austin and Ryan, 2002) earthen aquaculture ponds in Western Australia

The abundance and diversity of the plankton community represents the health of the aquatic ecosystem, and plays an important role in the growth of cultured animals under aquaculture conditions. The temporal variations of plankton abundance, taxonomic composition, diversity, evenness and species richness were studied in three old and three new semi-intensive marron (Cherax cainii, Austin and Ryan, 2002) ponds. Water parameters such as temperature, dissolved oxygen, pH, turbidity, TAN, nitrite, nitrate and reactive phosphate were recorded, and plankton samples were collected every two months, for one year of juvenile production cycle. A total of twenty-six phytoplankton and seven zooplankton genera were recorded. Chlorophyceae was the dominant class of phytoplankton throughout the year, followed by Trebouxiophyceae. Rotifera comprised 49.8% of the total zooplankton community (individuals L^?1), the largest proportion of any group. Temporal variations impacted the plankton abundance and community structure, and plankton abundance were more abundant during summer. The pond age did not influence the phytoplankton abundance, whereas zooplankton abundance was higher in older ponds.     

Enhanced Expression of Glia Maturation Factor Correlates with Glial Activation in the Brain of Triple Transgenic Alzheimer’s Disease Mice

We previously demonstrated that glia maturation factor (GMF), a brain specific protein, isolated, sequenced and cloned in our laboratory, induce expression of proinflammatory cytokines and chemokines in the central nervous system. We also reported that the up-regulation of GMF in astrocytes leads to the destruction of neurons suggesting a novel pathway of GMF-mediated cytotoxicity of brain cells, and implicated its involvement in the pathogenesis of inflammatory neurodegenerative diseases. In the present study, we examined the expressions of GMF in triple-transgenic Alzheimer’s disease (3xTg-AD) mice. Our results show a 13-fold up-regulation of GMF and 8–12-fold up-regulation of proinflammatory cytokines tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-1β, interferon gamma (IFN-γ), and chemokine (C–C motif) ligand 2 (CCL2) and C–X–C motif chemokine 10 (CXCL10/IP-10) mRNA as determined by quantitative real-time RT-PCR in the brain of 3xTg-AD mice as compared to non-transgenic (Non-Tg) mice. In conclusion, the increase in GMF and cytokine/chemokine expression was correlated with reactive glial fibrillary acidic protein positive astrocytes and ionized calcium binding adaptor molecule 1 (Iba-1)-positive microglia in 3xTg-AD mice.     

Molecular architecture of protein-RNA recognition sites

The molecular architecture of protein-RNA interfaces are analyzed using a non-redundant dataset of 152 protein-RNA complexes. We find that an average protein-RNA interface is smaller than an average protein-DNA interface but larger than an average protein–protein interface. Among the different classes of protein-RNA complexes, interfaces with tRNA are the largest, while the interfaces with the single-stranded RNA are the smallest. Significantly, RNA contributes more to the interface area than its partner protein. Moreover, unlike protein–protein interfaces where the side chain contributes less to the interface area compared to the main chain, the main chain and side chain contributions flipped in protein-RNA interfaces. We find that the protein surface in contact with the RNA in protein-RNA complexes is better packed than that in contact with the DNA in protein-DNA complexes, but loosely packed than that in contact with the protein in protein–protein complexes. Shape complementarity and electrostatic potential are the two major factors that determine the specificity of the protein-RNA interaction. We find that the H-bond density at the protein-RNA interfaces is similar with that of protein-DNA interfaces but higher than the protein–protein interfaces. Unlike protein-DNA interfaces where the deoxyribose has little role in intermolecular H-bonds, due to the presence of an oxygen atom at the 2′ position, the ribose in RNA plays significant role in protein-RNA H-bonds. We find that besides H-bonds, salt bridges and stacking interactions also play significant role in stabilizing protein-nucleic acids interfaces; however, their contribution at the protein–protein interfaces is insignificant.