Competitive Inhibition of Abscisic Acid-Regulated Gene Expression by Stereoisomeric Acetylenic Analogs of Abscisic Acid

The properties of two enantiomeric synthetic acetylenic abscisic acid (ABA) analogs (PBI-51 and PBI-63) in relation to ABA-sensitive gene expression are reported. Using microspore-derived embryos of Brassica napus as the biological material and their responsiveness to ABA in the expression of genes encoding storage proteins as a quantitative bioassay, we measured the biological activity of PBI-51 and PBI-63. Assays to evaluate agonistic activity of either compound applied individually showed a dose-dependent increase in napin gene expression on application of PBI-63. Maximal activity of about 40 [mu]M indicated that PBI-63 was an agonist, although somewhat weaker than ABA. PBI-63 has a similar stereochemistry to natural ABA at the junction of the ring and side chain. In contrast, PBI-51 showed no agonistic effects until applied at 40 to 50 [mu]M. Even then, the response was fairly weak. PBI-51 has the opposite stereochemistry to natural ABA at the junction of the ring and side chain. When applied concurrently with ABA, PBI-63 and PBI-51 had distinctly different properties. PBI-63 (40 [mu]M) and ABA (5 [mu]M) combined gave results similar to the application of either compound separately with high levels of induction of napin expression. PBI-51 displayed a reversible antagonistic effect with ABA, shifting the typical ABA dose-response curve by a factor of 4 to 5. This antagonism was noted for the expression of two ABA-sensitive genes, napin and oleosin. To test whether this antagonism was at the level of ABA recognition or uptake, ABA uptake was monitored in the presence of PBI-51 or PBI-63. Neither compound decreased ABA uptake. Treatments with either PBI-51 or PBI-63 showed an effect on endogenous ABA pools by permitting increases of 5- to 7-fold. It is hypothesized that this increase occurs because of competition for ABA catabolic enzymes by both compounds. The fact that ABA pools did not decrease in the presence of PBI-51 suggests that PBI-51 must exert its antagonistic properties through direct competition with ABA at a hormone-recognition site.     

A 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus enhances acyl exchange between acyl-CoA and phosphatidylcholine

The gene encoding a 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus was over-expressed in developing seeds of Arabidopsis thaliana . Biochemical analysis of T₂ and T₃ A. thaliana seeds revealed a significant increase in polyunsaturated fatty acids (FAs) (18:2 ^{cis Δ9,12} and 18:3 ^{cis Δ9,12,15}) at the expense of very long monounsaturated FA (20:1 ^{cis Δ11}) and saturated FAs. In vitro assays demonstrated that recombinant B. napus ACBP (rBnACBP) strongly increases the formation of phosphatidylcholine (PC) in the absence of added lysophosphatidylcholine in microsomes from ΔYOR175c yeast expressing A. thaliana lysophosphatidylcholine acyltransferase ( AthLPCAT ) cDNA or in microsomes from microspore-derived cell suspension cultures of B. napus L. cv. Jet Neuf. rBnACBP or bovine serum albumin (BSA) were also shown to be crucial for AthLPCAT to catalyse the transfer of acyl group from PC into acyl-CoA in vitro . These data suggest that the cytosolic 10-kDa ACBP has an effect on the equilibrium between metabolically active acyl pools (acyl-CoA and phospholipid pools) involved in FA modifications and triacylglycerol bioassembly in plants. Over-expression of ACBP during seed development may represent a useful biotechnological approach for altering the FA composition of seed oil.     

Identification of an alpha3-fucosyltransferase and a novel alpha2- fucosyltransferase activity in cercariae of the schistosome Trichobilharzia ocellata: biosynthesis of the Fucalpha1-->2Fucalpha1-- >3[Gal(NAc)beta1-->4]GlcNAc sequence

Fucose is a major constituent of the protein- and lipid-linked glycans of the various life-cycle stages of schistosomes. These fucosylated glycans are highly antigenic and seem to play a role in the pathology of schistosomiasis. In this article we describe the identification and characterization of two fucosyltransferases (FucTs) in cercariae of the avian schistosome Trichobilharzia ocellata, a GDP-Fuc:[Galbeta1-- >4]GlcNAcbeta-R alpha1-->3-FucT and a novel GDP-Fuc:Fucalpha-R alpha1-- >2-FucT. Triton X-100 extracts of cercariae were assayed for FucT activity using a variety of acceptor substrates. Type 1 chain (Galbeta1- ->3GlcNAc) based compounds were poor acceptors, whereas those based on a type 2 chain (Galbeta1-->4GlcNAc), whether alpha2'-fucosylated, alpha3'-sialylated, or unsubstituted, and whether present as oligosaccharide or contained in a glycopeptide or glycoprotein, all served as acceptor substrates. In this respect the schistosomal alpha3- FucT resembles human FucT V and VI rather than other known FucTs. N- ethylmaleimide, an inhibitor of several human FucTs, had no effect on the activity of the schistosomal alpha3-FucT, whereas GDP-beta-S was strongly inhibitory. Large scale incubations were carried out with Galbeta1-->4GlcNAc, GalNAcbeta1-->4GlcNAcbeta-O -(CH2)8COOCH3 and Fucalpha1-->3GlcNAcbeta1-->2Man as acceptor substrates and the products of the incubations were isolated using a sequence of chromatographic techniques. By methylation analysis and 2D-TOCSY and ROESY1H-NMR spectroscopy the products formed were shown to be Galbeta1-- >4[Fucalpha1-->2Fucalpha1-->3]GlcNAc, GalNAcbeta1-->4[Fucalpha1-- >2Fucalpha1-->3]GlcNAcbe ta-O-(CH2)8COOCH3, and Fucalpha1-->2Fucalpha1-- >3GlcNAcbeta1-->2Man, respectively. It is concluded that the alpha2- FucT and alpha3-FucT are involved in the biosynthesis of the (oligomeric) Lewisx sequences and the Fucalpha1-->2Fucalpha1-->3GlcNAc structural element that have been described on schistosomal glycoconjugates.      

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p - 2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)- 1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.      

Role of Far Infra-Red Therapy in Dialysis Arterio-Venous Fistula Maturation and Survival: Systematic Review and Meta-Analysis

Introduction

A well-functioning arteriovenous fistula (AVF) is the best modality for vascular access in patients with end-stage renal disease (ESRD) requiring haemodialysis (HD). However, AVFs’ main disadvantage is the high rate of maturation failure, with approximately one third (20%–50%) not maturing into useful access. This review examine the use of Far-Infra Red therapy in an attempt to enhance both primary (unassisted) and secondary (assisted) patency rates for AVF in dialysis and pre-dialysis patients.

Method

We performed an online search for observational studies and randomised controlled trials (RCTs) that evaluated FIR in patients with AVF. Eligible studies compared FIR with control treatment and reported at least one outcome measure relating to access survival. Primary patency and secondary patency rates were the main outcomes of interest.

Results

Four RCTs (666 patients) were included. Unassisted patency assessed in 610 patients, and was significantly better among those who received FIR (228/311) compared to (185/299) controls (pooled risk ratio of 1.23 [1.12–1.35], p = 0.00001). In addition, the two studies which reported secondary patency rates showed significant difference in favour of FIR therapy- 160/168 patients - compared to 140/163 controls (pooled risk ratio of 1.11 [1.04–1.19], p = 0.003).

Conclusion

FIR therapy may positively influence the complex process of AVF maturation improving both primary and secondary patency rates. However blinded RCTs performed by investigators with no commercial ties to FIR therapy technologies are needed.     

Ontogenetic variations in the venom proteome of the Amazonian snake Bothrops atrox

Background  

Bothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Previous studies have demonstrated that the biological and pharmacological activities of B. atrox venom alter with the age of the animal. Here, we present a comparative proteome analysis of B. atrox venom collected from specimens of three different stages of maturation: juveniles, sub-adults and adults.     

Recombinant NY-ESO-1 cancer antigen: production and purification under cGMP conditions

The cancer-testis antigen, NY-ESO-1, has been engineered into a bacterial expression plasmid which incorporates a His6-tag. The plasmid was transfected into E. coli strain BL21 and Master and Working cell banks generated from this expression system. Three 15-litre fermentations were performed under cGMP (code of Good Manufacturing Practice) conditions and the crude NY-ESO-1 tagged protein isolated as solubilised inclusion bodies. A three-step cGMP chromatography process (immobilised metal affinity, anion exchange, and hydrophobic interaction) was utilised to purify the protein. The purified NY-ESO-1 is being used in early stage human cancer vaccine trials in Australia and the U.S.A.     

Acid growth effects in maize roots: Evidence for a link between auxin-economy and proton extrusion in the control of root growth

The role of proton excretion in the growth of apical segments of maize roots has been examined. Growth is stimulated by acidic buffers and inhibited by neutral buffers. Organic buffers such as 2[N-morpholino] ethane sulphonic acid (MES) — 2-amino-2-(hydroxymethyl)propane-1,3 diol (Tris) are more effective than phosphate buffers in inhibiting growth. Fusicoccin(FC)-induced growth is also inhibited by neutral buffers. The antiauxins 4-chlorophenoxyisobutyric acid (PCIB) and 2-(naphthylmethylthio) propionic acid (NMSP) promote growth and H⁺-excretion over short time periods; this growth is also inhibited by neutral buffers. We conclude that growth of maize roots requires proton extrusion and that regulation of root growth by indol-3yl-acetic acid (IAA) may be mediated by control of this proton extrusion.Abbreviations IAA indol-3yl-acetic acid - ABA abscisic acid - FC fusicoccin - PCIB 4-chlorophenoxy-isobutyric acid - MES 2(N-morpholino)ethane sulphonic acid - Tris 2-amino-2-(hydroxymethyl) propane-1,3-diol - NMSP 2-(naphthylmethylthio)propionic acid     

Increased T lymphocytes and IgMEA-receptor lymphocytes in Hodgkin's disease spleens.

Splenic lymphocytes from 11 patients with Hodgkin's disease were compared to lymphocytes of six spleens from patients with nonlymphoproliferative diseases. T lymphocytes were increased in patients with histological involvement by Hodgkin's disease. Likewise, lymphocytes from spleens with histological involvement showed increased rosette formation with immunologlobulin M-coated sheep red blood cells (IgMEA). A similar increase in T lymphocytes and in IgMEA rosette formation was not observed with normal peripheral blood lymphocytes, control spleens, or with Hodgkin's disease spleens without evidence of histological involvement.