Synthesis and evaluation of new endomorphin analogues modified at the Pro2 residue

Six new endomorphin analogues, incorporating constrained amino acids in place of native proline have been synthesized. Residues of (S)-azetidine-2-carboxylic acid (Aze), 3,4-dehydro-(S)-proline (Δ³Pro), azetidine-3-carboxylic acid (3Aze) and dehydro-alanine (ΔAla) have been used to prepare [Δ³Pro²]EM-2 (1), [Aze²]EM-1 (2), [Aze²]EM-2 (3), [3Aze²]EM-1 (4), [3Aze²]EM-2 (5) and [ΔAla²]EM-2 (6). Binding assays and functional bioactivities for μ- and δ-receptors are reported. The highest affinity, bioactivity and selectivity are shown by peptides 2 and 3 containing the Aze residue.     

Optimized DNA extraction methods for encysted embryos of the endangered fairy shrimp, Branchinecta sandiegonensis

The San Diego fairy shrimp Branchinecta sandiegonensis is a federally endangered species endemic to vernal pools in southern California, USA. Filling events in these habitats are highly variable, with some pools failing to hold water long enough for reproduction over many successive years. Studies of this species are thus hindered by the relatively rare appearance of aquatically active life history phases. Because diapausing cysts are abundant and present at all times, they provide an underutilized opportunity for both species identification and genetic studies. However, methods for extracting DNA from cysts are technically challenging because of their structure and size. Here we present a protocol for extracting DNA from B. sandiegonensis cysts in sufficient quantities for “DNA Barcoding”, microsatellite analysis and other genotyping and sequencing applications. The technique will aid in population genetic studies and species identification (since taxonomic keys only distinguish among adults), and will be applicable to other crustaceans with similar diapausing cysts.     

Change in conformation with reduction of alpha-helix content causes loss of neutrophil binding activity in fully cytotoxic Shiga toxin 1

Shiga toxins (Stx) play an important role in the pathogenesis of hemolytic uremic syndrome, a life-threatening renal sequela of human intestinal infection caused by specific Escherichia coli strains. Stx target a restricted subset of human endothelial cells that possess the globotriaosylceramide receptor, like that in renal glomeruli. The toxins, composed of five B chains and a single enzymatic A chain, by removing adenines from ribosomes and DNA, trigger apoptosis and the production of pro-inflammatory cytokines in target cells. Because bacteria are confined to the gut, the toxins move to the kidney through the circulation. Polymorphonuclear leukocytes (PMN) have been indicated as the carriers that "piggyback" shuttle toxins to the kidney. However, there is no consensus on this topic, because not all laboratories have been able to reproduce the Stx/PMN interaction. Here, we demonstrate that conformational changes of Shiga toxin 1, with reduction of α-helix content and exposition to solvent of hydrophobic tryptophan residues, cause a loss of PMN binding activity. The partially unfolded toxin was found to express both enzymatic and globotriaosylceramide binding activities being fully active in intoxicating human endothelial cells; this suggests the presence of a distinct PMN-binding domain. By reviewing functional and structural data, we suggest that A chain moieties close to Trp-203 are recognized by PMN. Our findings could help explain the conflicting results regarding Stx/PMN interactions, especially as the groups reporting positive results obtained Stx by single-step affinity chromatography, which could have preserved the correct folding of Stx with respect to more complicated multi-step purification methods.     

Oxidation and reduction of sulfite contribute to susceptibility and detoxification of SO2 in Populus × canescens leaves

Key Message

The critical level for SO ₂ susceptibility of Populus × canescens is approximately 1.2 μL L ^?1 SO ₂ . Both sulfite oxidation and sulfite reduction and assimilation contribute to SO ₂ detoxification.

Abstract

In the present study, uptake, susceptibility and metabolism of SO₂ were analyzed in the deciduous tree species poplar (Populus × canescens). A particular focus was on the significance of sulfite oxidase (SO) for sulfite detoxification, as SO has been characterized as a safety valve for SO₂ detoxification in herbaceous plants. For this purpose, poplar plants were exposed to different levels of SO₂ (0.65, 0.8, 1.0, 1.2 μL L^?1) and were characterized by visible injuries and at the physiological level. Gas exchange parameters (stomatal conductance for water vapor, CO₂ assimilation, SO₂ uptake) of the shoots were compared with metabolite levels (sulfate, thiols) and enzyme activities [SO, adenosine 5′-phosphosulfate reductase (APR)] in expanding leaves (80–90 % expanded). The critical dosage of SO₂ that confers injury to the leaves was 1.2 μL L^?1 SO₂. The observed increase in sulfur containing compounds (sulfate and thiols) in the expanding leaves strongly correlated with total SO₂ uptake of the plant shoot, whereas SO₂ uptake rate was strongly correlated with stomatal conductance for water vapor. Furthermore, exposure to high concentration of SO₂ revealed channeling of sulfite through assimilatory sulfate reduction that contributes in addition to SO-mediated sulfite oxidation to sulfite detoxification in expanding leaves of this woody plant species.     

Peroxisomes contribute to biosynthesis of extracellular glycolipids in fungi

Many microorganisms secrete surface‐active glycolipids. The basidiomycetous fungus Ustilago maydis produces two different classes of glycolipids, mannosylerythritol lipids (MEL) and ustilagic acids (UAs). Here we report that biosynthesis of MELs is partially localized in peroxisomes and coupled to peroxisomal fatty acid degradation. The acyltransferases, Mac1 and Mac2, which acylate mannosylerythritol with fatty acids of different length, contain a type 1 peroxisomal targeting signal (PTS1). We demonstrate that Mac1 and Mac2 are targeted to peroxisomes, while other enzymes involved in MEL production reside in different compartments. Mis‐targeting of Mac1 and Mac2 to the cytosol did not block MEL synthesis but promoted production of MEL species with altered acylation pattern. This is in contrast to peroxisome deficient mutants that produced MELs similar to the wild type. We could show that cytosolic targeting of Mac1 and Mac2 reduces the amount of UA presumably due to competition for overlapping substrates. Interestingly, hydroxylated fatty acids characteristic for UAs appear in MELs corroborating cross‐talk between both biosynthesis pathways. Therefore, peroxisomal localization of MEL biosynthesis is not only prerequisite for generation of the natural spectrum of MELs, but also facilitates simultaneous assembly of different glycolipids in a single cell.     

Activation of the EPOR-β common receptor complex by cibinetide ameliorates impaired wound healing in mice with genetic diabetes

Diabetes is characterized by poor wound healing which currently lacks an efficacious treatment. The innate repair receptor (IRR) is a master regulator of tissue protection and repair which is expressed as a response injury or metabolic stress, including in diabetes. Activation of the IRR might provide benefit for diabetic wound healing. A specific IRR agonist cibinetide was administered in an incisional wound healing model performed mice with genetic diabetes (db⁺/db⁺) and compared to the normal wild-type. Animals were treated daily with cibinetide (30 μg/kg/s.c.) or vehicle and euthanized 3, 7, and 14 days after the injury to quantitate vascular endothelial growth factor (VEGF), malondialdehyde (MAL), phospho-Akt (pAkt), phospho e-NOS (p-eNOS), and nitrite/nitrate content within the wound. Additional evaluations included quantification of skin histological change, angiogenesis, scar strength, and time to complete wound closure. Throughout the wound healing process diabetic animals treated with vehicle exhibited increased wound MAL with reduced VEGF, pAkt, peNOS and nitrite/nitrate, all associated with poor re-epitheliziation, angiogenesis, and wound breaking strength. Cibenitide administration significantly improved these abnormalities. The results suggest that cibinetide-mediated IRR activation may represent an interesting strategy to treat diabetes-associated wound healing.     

Cofactor requirement of ribosome-inactivating proteins from plants

A large number of type 1 ribosome-inactivating proteins (RIPs)from plants (families of Caryophyllaceae, Cucurbitaceae, Euphorbiaceae,Phytolaccaceae, and Poaceae) were examined for their requirementfor ATP and supernatant factors for full activity. A markedrequirement was observed with agrostin among Caryophyllaceae,gelonin among Euphorbiaceae, and with both barley RIP and tritin-Samong Poaceae. The distribution of cofactor requirement in Phytolaccaceaediscriminates leaf forms (cofactor-independent) from seed androot forms (cofactor-dependent). The results are discussed onthe basis of the present knowledge on the tissue localizationof RIPs and on the sensitivity of ribosomes to conspecific RIPs. Key words: Cofactors, ribosome-inactivating proteins, RNA-N-glycosidase, up-regulation     

News & Notes: Correlation of Resistance to the Alkaloid Lycorine with the Degree of Suppressiveness in Petite Mutants of Saccharomyces cerevisiae

In previous papers (Del Giudice et al. Curr Genet 8:493–497, 1984; Massardo et al. Curr Genet 17:455–457, 1985) we have shown that strains of Saccharomyces cerevisiae that are devoid of mitochondrial DNA (rho ^o) are resistant to the alkaloid lycorine isolated from Amaryllis plants, whereas strains containing mitochondrial DNA (rho ⁻ , mit ⁻ , or rho ⁺ ) are sensitive to this drug. In addition, we were able to show that the so-called hypersuppressive petites, whose mitochondrial genomes consist of short regions of DNA containing an ori sequence, show intermediate resistance. In this paper, we demonstrate that the degree of suppressiveness of a rho ⁻ mutant correlates with the degree of resistance to lycorine. Received: 20 September 1996 / Accepted: 10 January 1997