Galectin-3 in apoptosis,a novel therapeutic target

During the past decade, extensive progress has been made toward understanding the molecular basis for the regulation of apoptosis. In mammalian cells undergoing apoptosis, two distinct mechanisms or pathways are operated and are triggered by cell death stimuli from intra- or extra-cellular environments, namely the intrinsic or extrinsic pathways, resulting in mitochondrial membrane depolarization. Several lines of evidence from our laboratories and others have indicated that galectin-3 plays an important role in these pathways by binding to various ligands. Here the authors provide a brief discussion on the role of endogenous or extra-cellular galectin-3 in the regulation of apoptosis and how it could be used as a therapeutic target using natural plant products as its functional inhibitors.     

Electroporation of embryogenic protoplasts of sweet orange (<Emphasis Type="Italic">Citrus sinensis</Emphasis> (L.) Osbeck) and regeneration of transformed plants

Summary Electroporation conditions were optimized for the transfection of protoplasts isolated from an embryogenic cell line of sweet organe [Citrus sinensis (L.) Osbeck ev. Hamlin]. Electric field strength (375–450 V cm⁻¹) vector DNA concentration (100 μgml⁻¹), carrier DNA concentration (100 μgml⁻¹), electroporation buffer (pH 8), and preelectroporation heat shock of protoplasts (5 min at 45°C) were optimized. The plasmid vector pBI221 containing the β-glucuronidase (GUS) coding sequence under the control of the CaMV 35S promoter was used and GUS activity was measured 24h after electroporation. All variables significantly affected transfection efficiency and when optimal conditions for each were combined. GUS activity was 7714 pmol 4-methylumbelliferone (MU) mg⁻¹ (protein) min⁻¹. Protoplasts were then electroporated in the presence of green fluorescent protein (GFP) expression vectors pARS101 or pARS108. Green fluorescent embryos were selected, plants regenerated, and integration of the transgene was confirmed by Southern blot analysis. Both plasmids were constructed using EGFP, a GFP variant 35 times brighter than wtGFP, having a single, red-shifted excitation peak, and optimized for human codon-usage. pARS101 was constructed by placing EGFP under the control of a 35S–35S promoter containing 33 bp of the untranslated leader sequence from alfalfa mosaic virus. pARS108 was constructed similarly except sequences were added for transport and retention of EGFP in the lumen of the endoplasmic reticulum. Mention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the US Department of Agriculture and does not imply its approval to the exclusion of other products or veudors that may also be suitable.     

Variation Within and Between Phytophthora Species from Rubber and Citrus Trees in China, Determined by Polymerase Chain Reaction Using RAPDs

Variation among 39 isolates of Phytophthora of six morphological species (P. citrophthora. P. parasitka, P. capsici, P. palmivora and P. meadii. from rubber and citrus trees, and P. colocasiae from taro) was studied using random amplified polymorphic DNA (RAPD) analysis. Ten randomly-chosen 10-mer primers were used. Generally, the banding patterns were similar within species and different between species, but no one primer was able to distinguish all six species from one another. Cluster analysis on pooled data from all the primers gave six groups of isolates corresponding to the six morphological species. The group corresponding to P. citrophthora was divided further into subgroups that were related to host species and geographical location. This work confirmed the existing morphological classification of Phytophthora isolates from rubber and citrus trees in tropical China and showed the validity of using RAPDs to study the taxonomy of Phytophthora.     

Isolation and characterization of two entomopathogenic fungi attackingDiaphorina citri (Homoptera, Psylloidea) in Indonesia

In an attempt to suppress the propagation of citrus greening disease in Indonesia, we explored pathogens ofDiaphorina citri which vectors the disease. At two orange orchards, manyD. citri adults were found to be dead and covered with fungal mycelia. Two fungi,Paecilomyces fumosoroseus andHirsutella citriformis, were consistently isolated from the infected insects. Molecular phylogeny of their 18S rDNA sequences showed that they belong to the ascomycetous clade of the Clavicipitales/Hypocreales, which embraces many entomopathogenic fungi. When healthy adults ofD. citri were inoculated with conidia of theP. fumosoroseus, the insects died within 6 d.     

Season‐long mating disruption of citrus leafminer,Phyllocnistis citrella Stainton,with an emulsified wax formulation of pheromone

The citrus leafminer, Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae), is a major worldwide pest of citrus. Larval feeding by this insect facilitates proliferation of citrus bacterial canker, Xanthomonas axonopodis pv. citri. Herein, we describe a season‐long disruption trial of P. citrella with a newly developed, emulsified wax dispenser of pheromone (SPLAT‐CLM^TM). A formulation containing a 3 : 1 blend of (Z,Z,E)‐7,11,13‐hexadecatrienal:(Z,Z)‐7,11‐hexadecadienal at a 0.2% loading rate of active ingredient by weight and deployed twice per season (24 weeks total) at 490 g of formulation/ha caused season‐long disruption of male moth catch in pheromone traps as well as reduced leaf infestation. Analysis of pheromone release from dispensers by gas chromatography revealed that effective disruption of P. citrella occurred at a deployment rate of 126 μg of (Z,Z,E)‐7,11,13‐hexadecatrienal/ha/h. Direct observation of moth behaviour in the field suggested that disruption by this formulation occurred by a non‐competitive mechanism. A formulation of the 3 : 1 attractive blend at a 0.02% pheromone loading rate caused only 2–6 weeks of disruption per deployment and did not reduce leaf infestation during mid and end of the season evaluations. A formulation containing 0.2% of (Z,Z)‐7,11‐hexadecadienal alone and deployed at 490 g/ha caused 6–7 weeks of moth disruption to pheromone traps and did not prevent leaf infestation, while an identical formulation loaded with 0.02% (w/w) of (Z,Z)‐7,11‐hexadecadienal alone had no effect on P. citrella orientation to pheromone traps. The SPLAT formulation evaluated herein appears to be an excellent release device for (Z,Z,E)‐7,11,13‐hexadecatrienal given that approximately 100 days of steady release occurred following an initial brief (ca. 7 days) burst of higher release. The advantages of SPLAT as a formulation for P. citrella disruption include low cost of manufacturing, biodegradable and weather resistant characteristics, and flowability allowing machine application. Mating disruption should be an effective alternative to insecticides for management of P. citrella and may reduce the incidence of citrus canker.     

Characterization of Citrus Rootstock Responses to Tylenchulus semipenetrans (Cobb)

Citrus rootstocks which significantly limited the reproduction of Tylenchulus semipenetrans (Cobb) "Citrus" and "Poncirus" biotypes responded to infection by producing a hypersensitive-type response in the root hypodermis, wound periderm and/or cavities in the root cortex, and/or abnormal vacuoles in nurse cell cytoplasm. Rootstocks which limited nematode reproduction also had significantly fewer nematodes in the rhizoplane within 8 d of inoculation than did rootstocks which did not limit reproduction. Germplasm sources of the cellular responses which limited citrus nematode reproduction were identified.     

Tylenchulus graminis n. sp. and T. palustris n. sp. (Tylenchulidae), from Native Flora of Florida,with Notes on T. semipenetrans and T. furcus

Tylenchulus graminis n. sp. and T. palustris n. sp. are described and illustrated from broomsedge (Andropogon virginicus L.) and pop ash (Fraxinus caroliniana Mill.), respectively. T. graminis resembles T. furcus in having a distinct anus, but T. graminis second-stage juveniles (J2) do not have a bifid tail. T. semipenetrans does not have a perceptible anus. The mature female of T. graminis has a mucronate pointed terminus while T. semipenetrans has a smooth and round terminus. T. graminis males have wider stylet knobs and basal bulb and a longer tail than T. semipenetrans males. T. graminis J2 have a longer posterior body portion (without large fat globules) than T. semipenetrans J2. T. palustris resembles T. semipenetrans in having an undetectable anus but differs by the short and conoid mature female postvulval section. The male of T. palustris has larger stylet knobs and basal bulb than those of T. semipenetrans and a bluntly rounded tail terminus, which is tapered in T. semipenetrans. T. palustris differs from T. furcus and T. graminis in having an undetectable anus, by the conoid postvulval section of mature females, by the shorter and rounded tail of males, and the shorter J2 posterior body section without large fat globules. T. graminis and T. palustris are parasites of indigenous flora of Florida.     

Host discrimination by the endoparasitoid Psyllaephagus pistaciae (Hymenoptera: Encyrtidae): A case of time-dependent ability

Host-discrimination behavior by the koinobiont parasitoid, Psyllaephagus pistaciae Ferrière (Hymenoptera: Encyrtidae), the major biocontrol agent of the common pistachio psylla, Agonoscena pistaciae Burckhardt & Lauterer (Hemiptera: Psyllidae), in Iran, was investigated in the laboratory. The results demonstrated that P. pistaciae quickly detected and avoided freshly parasitized hosts, either after antennating or by probing with their ovipositor. However, this discriminatory ability declined with time, probably influenced by both external and internal markers left by the previous ovipositing female. The results also confirmed that female P. pistaciae responded to changes in host quality associated with the parasitoid's larval development 4 days after the initial parasitization, clearly indicating that the second female could detect the presence of the larvae and adjust her host-selection decision. In addition, psyllid nymphs treated topically with a solution of Dufour's gland were rejected by the parasitoid, showing that Dufour's gland secretion had a significant effect as a host marking chemical. The current study also showed that superparasitism increased the host-mortality and that the rate of encapsulation decreased, suggesting that, when two eggs are laid in the same psyllid nymph, only one parasitoid develops to an adult.     

Symptoms induced by transgenic expression of p23 from Citrus tristeza virus in phloem‐associated cells of Mexican lime mimic virus infection without the aberrations accompanying constitutive expression

Citrus tristeza virus (CTV) is phloem restricted in natural citrus hosts. The 23‐kDa protein (p23) encoded by the virus is an RNA silencing suppressor and a pathogenicity determinant. The expression of p23, or its N‐terminal 157‐amino‐acid fragment comprising the zinc finger and flanking basic motifs, driven by the constitutive 35S promoter of cauliflower mosaic virus, induces CTV‐like symptoms and other aberrations in transgenic citrus. To better define the role of p23 in CTV pathogenesis, we compared the phenotypes of Mexican lime transformed with p23‐derived transgenes from the severe T36 and mild T317 CTV isolates under the control of the phloem‐specific promoter from Commelina yellow mottle virus (CoYMV) or the 35S promoter. Expression of the constructs restricted to the phloem induced a phenotype resembling CTV‐specific symptoms (vein clearing and necrosis, and stem pitting), but not the non‐specific aberrations (such as mature leaf epinasty and yellow pinpoints, growth cessation and apical necrosis) observed when p23 was ectopically expressed. Furthermore, vein necrosis and stem pitting in Mexican lime appeared to be specifically associated with p23 from T36. Phloem‐specific accumulation of the p23Δ158–209(T36) fragment was sufficient to induce the same anomalies, indicating that the region comprising the N‐terminal 157 amino acids of p23 is responsible (at least in part) for the vein clearing, stem pitting and, possibly, vein corking in this host.