Poly(β-hydroxybutyrate) production in oilseed leukoplasts of Brassica napus

Polyhydroxyalkanoates (PHAs) comprise a class of biodegradable polymers which offer an environmentally sustainable alternative to petroleum-based plastics. Production of PHAs in plants is attractive since current fermentation technology is prohibitively expensive. The PHA homopolymer poly(β-hydroxybutyrate) (PHB) has previously been produced in leaves of Arabidopsis thaliana (Nawrath et al., 1994, Proc Natl Acad Sci USA 91: 12760–12764). However, Brassica napus oilseed may provide a better system for PHB production because acetyl-CoA, the substrate required in the first step of PHB biosynthesis, is prevalent during fatty acid biosynthesis. Three enzymatic activities are needed to synthesize PHB: a β-ketothiolase, an acetoacetyl-CoA reductase and a PHB synthase. Genes from the bacterium Ralstonia eutropha encoding these enzymes were independently engineered behind the seed-specific Lesquerella fendleri oleate 12-hydroxylase promoter in a modular fashion. The gene cassettes were sequentially transferred into a single, multi-gene vector which was used to transform B. napus. Poly(β-hydroxybutyrate) accumulated in leukoplasts to levels as high as 7.7% fresh seed weight of mature seeds. Electron-microscopy analyses indicated that leukoplasts from these plants were distorted, yet intact, and appeared to expand in response to polymer accumulation. Received: 26 May 1999 / Accepted: 16 June 1999     

Misexpression of the catenin p120(ctn)1A perturbs Xenopus gastrulation but does not elicit Wnt-directed axis specification

Modulators of cadherin function are of great interest given that the cadherin complex actively contributes to the morphogenesis of virtually all tissues. The catenin p120(ctn) (formerly p120cas) was first identified as a src- and receptor-protein tyrosine kinase substrate and later shown to interact directly with cadherins. In common with beta-catenin and plakoglobin (gamma-catenin), p120(ctn) contains a central Armadillo repeat region by which it binds cadherin cytoplasmic domains. However, little is known about the function of p120(ctn) within the cadherin complex. We examined the role of p120(ctn)1A in early vertebrate development via its exogenous expression in Xenopus. Ventral overexpression of p120(ctn)1A, in contrast to beta-catenin, did not induce the formation of duplicate axial structures resulting from the activation of the Wnt signaling pathway, nor did p120(ctn) affect mesoderm induction. Rather, dorsal misexpression of p120(ctn) specifically perturbed gastrulation. Lineage tracing of cells expressing exogenous p120(ctn) indicated that cell movements were disrupted, while in vitro studies suggested that this may have been a consequence of reduced adhesion between blastomeres. Thus, while cadherin-binding proteins beta-catenin, plakoglobin, and p120(ctn) are members of the Armadillo protein family, it is clear that these proteins have distinct biological functions in early vertebrate development. This work indicates that p120(ctn) has a role in cadherin function and that heightened expression of p120(ctn) interferes with appropriate cell-cell interactions necessary for morphogenesis.     

Safe Oral Triiodo-L-Thyronine Therapy Protects from Post-Infarct Cardiac Dysfunction and Arrhythmias without Cardiovascular Adverse Effects

Background

A large body of evidence suggests that thyroid hormones (THs) are beneficial for the treatment of cardiovascular disorders. We have shown that 3 days of triiodo-L-thyronine (T3) treatment in myocardial infarction (MI) rats increased left ventricular (LV) contractility and decreased myocyte apoptosis. However, no clinically translatable protocol is established for T3 treatment of ischemic heart disease. We hypothesized that low-dose oral T3 will offer safe therapeutic benefits in MI.

Methods and Results

Adult female rats underwent left coronary artery ligation or sham surgeries. T3 (~6 μg/kg/day) was available in drinking water ad libitum immediately following MI and continuing for 2 month(s) (mo). Compared to vehicle-treated MI, the oral T3-treated MI group at 2 mo had markedly improved anesthetized Magnetic Resonance Imaging-based LV ejection fraction and volumes without significant negative changes in heart rate, serum TH levels or heart weight, indicating safe therapy. Remarkably, T3 decreased the incidence of inducible atrial tachyarrhythmias by 88% and improved remodeling. These were accompanied by restoration of gene expression involving several key pathways including thyroid, ion channels, fibrosis, sympathetic, mitochondria and autophagy.

Conclusions

Low-dose oral T3 dramatically improved post-MI cardiac performance, decreased atrial arrhythmias and cardiac remodeling, and reversed many adverse changes in gene expression with no observable negative effects. This study also provides a safe and effective treatment/monitoring protocol that should readily translate to humans.     

Biochemical and structural studies of yeast Vps4 oligomerization

The ESCRT (endosomal sorting complexes required for transport) pathway functions in vesicle formation at the multivesicular body, the budding of enveloped RNA viruses such as HIV-1, and the final abscission stage of cytokinesis. As the only known enzyme in the ESCRT pathway, the AAA ATPase (ATPase associated with diverse cellular activities) Vps4 provides the energy required for multiple rounds of vesicle formation. Like other Vps4 proteins, yeast Vps4 cycles through two states: a catalytically inactive disassembled state that we show here is a dimer and a catalytically active higher-order assembly that we have modeled as a dodecamer composed of two stacked hexameric rings. We also report crystal structures of yeast Vps4 proteins in the apo- and ATPγS [adenosine 5′-O-(3-thiotriphosphate)]-bound states. In both cases, Vps4 subunits assembled into continuous helices with 6-fold screw axes that are analogous to helices seen previously in other Vps4 crystal forms. The helices are stabilized by extensive interactions between the large and small AAA ATPase domains of adjacent Vps4 subunits, suggesting that these contact surfaces may be used to build both the catalytically active dodecamer and catalytically inactive dimer. Consistent with this model, we have identified interface mutants that specifically inhibit Vps4 dimerization, dodecamerization, or both. Thus, the Vps4 dimer and dodecamer likely form distinct but overlapping interfaces. Finally, our structural studies have allowed us to model the conformation of a conserved loop (pore loop 2) that is predicted to form an arginine-rich pore at the center of one of the Vps4 hexameric rings. Our mutational analyses demonstrate that pore loop 2 residues Arg241 and Arg251 are required for efficient HIV-1 budding, thereby supporting a role for this “arginine collar” in Vps4 function.     

Biosynthesis of a disialylated sequence in N-linked oligosaccharides: identification of an N-acetylglucosaminide (alpha 2----6)-sialyltransferase in Golgi apparatus from rat liver

Rat liver Golgi apparatus are shown to have a CMP-N-acetylneuraminate: N-acetylglucosaminide (alpha 2----6)-sialyltransferase which catalyzes the conversion of the human milk oligosaccharide LS-tetrasaccharide-a (NeuAc alpha 2----3Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----4Glc) to disialyllacto -N- tetraose containing the terminal sequence: (formula: see text) found in N-linked oligosaccharides of glycoproteins. The N-acetylglucosaminide (alpha 2----6)-sialyltransferase has a marked preference for the sequence NeuAc alpha 2----3-Gal beta 1---- 3GlcNAc as an acceptor substrate. Thus, the order of addition of the two sialic acids in the disialylated structure shown above is proposed to be first the terminal sialic acid in the NeuAc alpha 2----3Gal linkage followed by the internal sialic acid in the NeuAc alpha 2---- 6GlcNAc linkage. Sialylation in vitro of the type 1 branches (Gal beta 1---- 3GlcNAc -) of the N-linked oligosaccharides of asialo prothrombin to produce the same disialylated sequence is also demonstrated.     

Mechanisms of Human Erythrocytic Bioactivation of Nitrite

Nitrite signaling likely occurs through its reduction to nitric oxide (NO). Several reports support a role of erythrocytes and hemoglobin in nitrite reduction, but this remains controversial, and alternative reductive pathways have been proposed. In this work we determined whether the primary human erythrocytic nitrite reductase is hemoglobin as opposed to other erythrocytic proteins that have been suggested to be the major source of nitrite reduction. We employed several different assays to determine NO production from nitrite in erythrocytes including electron paramagnetic resonance detection of nitrosyl hemoglobin, chemiluminescent detection of NO, and inhibition of platelet activation and aggregation. Our studies show that NO is formed by red blood cells and inhibits platelet activation. Nitric oxide formation and signaling can be recapitulated with isolated deoxyhemoglobin. Importantly, there is limited NO production from erythrocytic xanthine oxidoreductase and nitric-oxide synthase. Under certain conditions we find dorzolamide (an inhibitor of carbonic anhydrase) results in diminished nitrite bioactivation, but the role of carbonic anhydrase is abrogated when physiological concentrations of CO₂ are present. Importantly, carbon monoxide, which inhibits hemoglobin function as a nitrite reductase, abolishes nitrite bioactivation. Overall our data suggest that deoxyhemoglobin is the primary erythrocytic nitrite reductase operating under physiological conditions and accounts for nitrite-mediated NO signaling in blood.     

Cell surface antigens on erythroid cells: A comparison of normal and anemic (WW) mice

Cell surface antigens of normal and anemic ($\text{W}\text{W}$) mouse erythroid cells have been examined in cytotoxicity assays with two rat antisera. When tested on fetal liver cells, a rat anti-erythroblast serum recognized antigen(s) present on erythroid cells early in development, while rat anti-adult red blood cell serum recognized antigen(s) present on mature erythroid cells. Each of these sera had different activity on normal (+/+ or $\text{W}\text{+}$) as compared to anemic ($\text{W}\text{W}$) erythroid cells.     

Screening for antiviral inhibitors of the HIV integrase-LEDGF/p75 interaction using the AlphaScreen luminescent proximity assay

Small-molecule inhibitors of HIV integrase (HIV IN) have emerged as a promising new class of antivirals for the treatment of HIV/AIDS. The compounds currently approved or in clinical development specifically target HIV DNA integration and were identified using strand-transfer assays targeting the HIV IN/viral DNA complex. The authors have developed a second biochemical assay for identification of HIV integrase inhibitors, targeting the interaction between HIV IN and the cellular cofactor LEDGF/p75. They developed a luminescent proximity assay (AlphaScreen) designed to measure the association of the 80-amino-acid integrase binding domain of LEDGF/p75 with the 163-amino-acid catalytic core domain of HIV IN. This assay proved to be quite robust (with a Z' factor of 0.84 in screening libraries arrayed as orthogonal mixtures) and successfully identified several compounds specific for this protein-protein interaction.