Characterization of horizontally transferred ß-fructofuranosidase (ScrB) genes in Agrilus planipennis.

The emerald ash borer (Agrilus planipennis) is an important invasive insect pest of Fraxinus spp. that feeds on host tissues containing high levels of sucrose. However, little is known about how it digests sucrose. Here, using larval midgut transcriptome data and preliminary genome sequence efforts, two ß-fructofuranosidase-encoding ScrB genes, AplaScrB-1 and AplaScrB-2, were identified, and proved to reside within the A. planipennis genome. Homology and phylogenetic analysis revealed that they were acquired by A. planipennis via horizontal gene transfer (HGT) from bacteria, possibly an event independent from that reported in bark beetles (eg ScrB genes). Microsynteny between A. planipennis DNA scaffold #2042940, which hosts AplaScrB-1, and a region in the Tribolium castaneum chromosome LG4 suggested that A. planipennis gained this gene after the separation of Buprestidae and Tenebrionidae. Although both of the putative AplaScrB proteins have conserved ß-fructofuranosidase motifs, only AplaScrB-2 was predicted to be a secretory protein. Expression of AplaScrB-1 seemed constitutive during development and in all tissues examined, whereas AplaScrB-2 showed a peak expression in adults and in the midgut. We propose that acquisition of these genes by A. planipennis from bacteria is adaptive, and specifically AplaScrB-2 is involved in breaking down dietary sucrose to obtain energy for development.

Cloning and characterization of a Gasp homolog from the spruce budworm, Choristoneura fumiferana, and its putative role in cuticle formation.

Proteins that are capable of binding chitin play essential roles in the synthesis and structural integrity of the insect cuticle and peritrophic matrix. In the course of developing expressed sequence tag (EST) libraries for the eastern spruce budworm, Choristoneura fumiferana, we identified an abundant cDNA encoding a homolog of the Drosophila "gasp" gene (Gene Analogous to Small Peritrophins). For the present work, we undertook the characterization of this new homolog, CfGasp, in an effort to identify its role during larval development. As shown for DmGasp, the C. fumiferana homolog was found to contain three type-2 chitin-binding domains (CBDs), which were also found in Gasp orthologs retrieved from GenBank. In a phylogenetic analysis, these Gasp proteins formed a tight cluster, distinct from the midgut-specific peritrophins with which they share the cysteine-containing CBDs so far considered absent from cuticular proteins. However, unlike what has been shown for peritrophins, CfGasp transcript levels were low in larval midguts and most abundant in epidermis, while they were low in trachea and ovaries. Transcript levels increased during larval molts in a pattern similar to that observed for exocuticular proteins in other insects. In addition, the recombinant protein was shown to be capable of binding chitin. Altogether, these results suggest a structural role for CfGasp in exocuticle formation.

Molecular and serological characterization of hepatitis B virus genotype A and D infected blood donors in Poland.

Hepatitis B virus (HBV) genotypes have distinct geographical distributions and influence severity of clinical outcome and response to antiviral therapies. HBV polymorphism in HBV surface antigen (HBsAg) positive first time blood donors from Poland was examined. HBV serological markers and HBV DNA were tested in 170 samples. Whole genome (n = 53) or specific region sequences: pre-S/S and basic core promoter/precore (BCP/PC) region (91 and 154 samples, respectively) were phylogenetically analyzed. The median age of infected donors was 21 years. Anti-HBs, anti-HBe and hepatitis B e antigen were detected in 5%, 92.4% and 10.5% of tested donors, respectively. The HBV DNA load ranged between unquantifiable and 3.1 x 10(10) IU/mL (median: 4.10 x 10(3) IU/mL). Genotypes A2 (81.2%) and D (18.8%) co-circulated. Phylogenetic analyses revealed differences between the genotypes. Viral load and level of HBsAg tended to be lower in genotype D. The median HBsAg/HBV DNA ratio expressed in IU/mL was one for both genotypes, but very low or very high ratios appeared more frequent in genotype D infections. Higher amino acid variability in the surface proteins (median: 4%vs 1.5%; P = 0.01) and in the major hydrophilic region was observed in genotype D (P = 0.01). BCP/PC region analysis revealed the double mutation 1762T/1764A in 49/125 (39.2%) genotype A2 and 6/29 (20.7%) genotype D strains (P = 0.08). Mutations in PC and BCP regions correlated neither with HBsAg nor HBV DNA levels. HBV genotype A2 is dominant in HBsAg positive donors in Poland. Minority genotype D strains are significantly more substituted than genotype A2 strains potentially affecting the course of infection.

Cloning, expression and characterization of four serpin-1 cDNA variants from the spruce budworm, Choristoneura fumiferana.

Four cDNAs (Cfserpin-1a, Cfserpin-1b, Cfserpin-1c and Cfserpin-1d) of the Choristoneura fumiferana serpin-1 gene were cloned from an epidermis cDNA library. Analysis of the deduced amino acid sequences indicated that the cloned cDNAs encode four different proteins displaying identical N- but distinct C-termini, the latter region containing the inhibitory loop. The entire CfSerpin-1 gene is transcribed while the variants are generated. Antibodies generated against the purified recombinant serpins cross-reacted with the other three. Each of the four Cfserpin-1 cDNA variants was transcribed throughout larval development, from the 4th to the 6th instar, but transcript levels during the intermolt phases were generally higher than during the molting phase. The epidermis and fat body had higher levels of Cfserpin-1 transcripts than the midgut. Cfserpin-1 proteins, detected with the Cfserpin-1a antibody, were found in the epidermis, midgut, fat body, plasma and molting fluid of 6th instar larvae and pre-pupae. Prepupal and pupal insects had higher levels of the proteins than the 6th instar feeding larvae, despite a drop in transcript levels. Cfserpin-1a could bind with the serine proteinase elastase and form a complex in vitro. We hypothesize that the cloned serpins could be involved in the regulation of cuticle degradation during the insect molting cycle.

The bugs that came in from the cold: molecular adaptations to low temperatures in insects.

The widespread distribution of insects over many ecological niches is a testimony to their evolutionary success. The colonization of environments at high latitudes or altitudes required the evolution of biochemical strategies that reduced the impact of cold or freezing stress. This review focuses on our current interests in some of the genes and proteins involved in low temperature survival in insects. Although the most widespread form of protection is the synthesis of low molecular weight polyol cryoprotectants, proteins with intrinsic protective properties, such as the thermal hysteresis or antifreeze proteins are also important. These have been cloned and characterized in certain moths and beetles. Molecular techniques allowing the isolation of genes differentially regulated by low temperatures have revealed that heat shock proteins, cold stress proteins, membrane protectants, as well as ice nucleators and other less well characterized proteins likely also play a role in cold hardiness.

Seed, soil and vegetative transmission contribute to the spread of pecluviruses in Western Africa and the Indian sub-continent.

Peanut clump and sugarcane red leaf mottle diseases are caused by viruses of the genus Pecluvirus. Indian peanut clump virus occurs in the Indian sub-continent and Peanut clump virus in West Africa. A feature of these viruses is that they are both seed and soil transmitted. Both modes of transmission contribute to long-term persistence and field spread. Data on seed transmission in pearl millet, virus movement within the plant and virus diversity based on RNA-1 partial sequences are presented. This study emphasizes that pecluviruses are also viruses of cereals infecting sorghum and pearl millet, and highlights a correlation between the countries cultivating these two crops and the virus distribution. Ways of controlling pecluviruses and their vector, Polymyxa graminis, taking into account the virus dissemination routes, are proposed.

Cloning, expression, and localization of a molt-related beta-N-acetylglucosaminidase in the spruce budworm, Choristoneura fumiferana.

A beta-N-acetylglucosaminidase cDNA (CfGlcNAcase) was cloned from the spruce budworm, Choristoneura fumiferana. Western blotting analysis of developmental CfGlcNAcase expression revealed high levels of expression of the gene on the last day of the 5th instar larvae and the first day in the 6th instar larvae, followed by a decrease to background levels during the intermolt of the 6th instar. CfGlcNAcase was detected again from the last day of the 6th instar to day 2 of pupal stage. CfGlcNAcase expression was induced by tebufenozide at 24 h post treatment and remained at high levels until 72 h. Immunohistochemical localization analysis of CfGlcNAcase indicated that CfGlcNAcase was present in the molting fluid, epidermis, trachea, and hemolymph in prepupae during the transformation from larva to pupa. CfGlcNAcase cDNA was expressed into a recombinant protein in bacterial and baculovirus systems and the protein expressed in the baculovirus system had a higher chitinolytic activity than in the bacterial system and appeared to be secreted.

Transcription of antifreeze protein genes in Choristoneura fumiferana.

Antifreeze proteins (AFPs) are encoded by approximately 17 genes in the spruce budworm, Choristoneura fumiferana. Northern analysis using 6 different cDNA probes showed isoform-specific patterns that varied during development. Transcripts for the majority of isoforms were most abundant in the second instar overwintering stage, but some were also detected in first instar and even in egg stages. In situ hybridization using riboprobes corresponding to two 9 kDa protein isoforms showed differential AFP expression even in second instars; CfAFP10 RNA was detected in all tissues, but CfAFP337 RNA distribution was more limited. Two genomic regions encoding three AFP genes have been isolated. Presumptive regulatory regions conferred transcriptional activity when placed upstream of a luciferase reporter sequence and transfected into a C. fumiferana cell line. The CfAFP2.26 core promoter is an 87 bp sequence containing a TATA box, whereas the CfAFP2.7 core promoter is a 76 bp sequence with both a TATA box and CAAT box, which directed higher reporter activities when tested in vitro. Reporter activity was not enhanced with five different hormones, although lower activities were observed with all intron-containing constructs. AFP message half-life, as assessed using reporter assays, was not appreciably influenced by isoform-specific-3'UTRs. These studies successfully demonstrate the temporal and spatial diversity of AFP expression encoded by this small gene family, and underscore the complexity of their regulation.

Analysis of antifreeze proteins within spruce budworm sister species.

Spruce budworm (Choristoneura) species survive sub-zero winter temperatures by producing antifreeze proteins (AFPs) encoded by a multigene family of short and long isoforms. We report in this study the first analysis of antifreeze proteins from related Choristoneura sister species. The additional thirty amino acid insert found in the longer AFP isoforms maintains the proteins beta-helix and original fifteen amino acid (Thr-X-Thr) repeat motif. Analysis of the beta-helix region shows more divergent residues surround the conserved Thr residues. Maintaining the beta-helix structure and conserved Thr residues appear to be paramount for AFP function and surviving sub-zero winter temperatures. Two other species within the same lepidopteran clade, Ditrysia, do not appear to contain any AFP-like sequences.

Foaming and interfacial properties of hydrolyzed beta-lactoglobulin.

beta-lactoglobulin (beta-lg) was hydrolyzed with three different proteases and subsequently evaluated for its foaming potential. Foam yield stress (tau0) was the primary variable of interest. Two heat treatments designed to inactivate the enzymes, 75 degrees C/30 min and 90 degrees C/15 min, were also investigated for their effects on foam tau0. Adsorption rates and dilatational rheological tests at a model air/water interface aided data interpretation. All unheated hydrolysates improved foam tau0 as compared to unhydrolyzed beta-lg, with those of pepsin and Alcalase 2.4L(R) being superior to trypsin. Heat inactivation negatively impacted foam tau0, although heating at 75 degrees C/30 min better preserved this parameter than heating at 90 degrees C/15 min. All hydrolysates adsorbed more rapidly at the air/water interface than unhydrolyzed beta-lg, as evidenced by their capacity to lower the interfacial tension. A previously observed relationship between interfacial dilatational elasticity (E') and tau0 was generally confirmed for these hydrolysates. Additionally, the three hydrolysates imparting the highest tau0 not only had high values of E' (approximately twice that of unhydrolyzed beta-lg), they also had very low phase angles (essentially zero). This highly elastic interfacial state is presumed to improve foam tau0 indirectly by improving foam stability and directly by imparting resistance to interfacial deformation.

Structure-function relationships in spruce budworm antifreeze protein revealed by isoform diversity.

The spruce budworm, Choristoneura fumiferana, produces antifreeze protein (AFP) to assist in the protection of the overwintering larval stage. AFPs are thought to lower the freezing point of the hemolymph, noncolligatively, by interaction with the surface of ice crystals. Previously, we had identified a cDNA encoding a 9-kDa AFP with 10-30 times the thermal hysteresis activity, on a molar basis, than that shown by fish AFPs. To identify important residues for ice interaction and to investigate the basis for the hyperactivity of the insect AFPs, six new spruce budworm AFP cDNA isoforms were isolated and sequenced. They differ in amino-acid identity as much as 36% from the originally characterized AFP and can be divided into three classes according to the length of their 3' untranslated regions (UTRs). The new isoforms have at least five putative 'Thr-X-Thr' ice-binding motifs and three of the new isoforms encode larger, 12-kDa proteins. These appear to be a result of a 30 amino-acid insertion bearing two additional ice-binding motifs spaced 15 residues apart. Molecular modeling, based on the NMR structure of a short isoform, suggests that the insertion folds into two additional beta-helix loops with their Thr-X-Thr motifs in perfect alignment with the others. The first Thr of the motifs are often substituted by Val, Ile or Arg and a recombinantly expressed isoform with both Val and Arg substitutions, showed wild-type thermal hysteresis activity. The analysis of these AFP isoforms suggests therefore that specific substitutions at the first Thr in the ice binding motif can be tolerated, and have no discernible effect on activity, but the second Thr appears to be conserved. The second Thr is thus likely important for the dynamics of initial ice contact and interaction by these hyperactive antifreezes.

A polydnavirus from the spruce budworm parasitoid, Tranosema rostrale (Ichneumonidae).

The calyx epithelium of the campoplegine wasp, Tranosema rostrale, contains typical ichneumonid polydnaviruses (PVs) that display an apparently uncommon association with the egg chorion. The latter structure features fine hair-like projections, longest around the egg's apices. In the lumen of the ovary, T. rostrale virus becomes lodged between these projections and forms a particulate coat around the egg. In the host, Choristoneura fumiferana, projections and associated virions are observed in close contact with basement membranes of fat body and muscle tissues, to which the eggs rapidly become attached following introduction into the host hemocoel. We discuss the implications of this unusual virus-chorion association in terms of immune protection, delivery of virus to specific host tissues, and the evolution of PVs.

The antifreeze potential of the spruce budworm thermal hysteresis protein.

Antifreeze proteins (AFP) inhibit ice growth by surface adsorption that results in a depression of the freezing point below the melting point. The maximum level of this thermal hysteresis shown by the four structurally unrelated fish AFP is approximately 1.5 degrees C. In contrast, hemolymph and crude extracts from insects can have 5 degrees to 10 degrees C of thermal hysteresis. Based on the isolation, cloning, and expression of a thermal hysteresis protein (THP) from spruce budworm (Choristoneura fumiferana), the vastly greater activity is attributable to a 9 kDa protein. This novel, threonine- and cysteine-rich THP has striking effects on ice crystal morphology, both before and during freezing. It is also 10 to 30 times more active than any known fish AFP, offering the prospect of superior antifreeze properties in cryoprotective applications.

Toxicologic profile of iobitridol, a new nonionic low-osmolality contrast medium.

PURPOSE: The toxicologic profile of iobitridol, a new nonionic low-osomolality contrast medium, was evaluated in compliance with the current regulatory requirements in Europe, the USA and Canada. MATERIAL AND METHODS: The toxicity of iobitridol was tested following acute or repeated i.v. administration in several different species (mouse, rat, dog); single oral administration in the mouse and intracisternal injection in the rat. Furthermore, teratogenicity and mutagenicity were evaluated in the rat and rabbit. Local perivenous toxicity was assessed in the rabbit. RESULTS: The acute toxicity of iobitridol in the mouse is equivalent to that of iohexol, a reference product tested under the same conditions. Chronic administration (daily injections i.v. injection over 4 weeks) in the rat and dog did not demonstrate any particular toxicity for iobitridol. It should be noted that, unlike iohexol, iobitridol did not provoke any vacuolization of the renal tubular cells in the rat following repeated injections. Furthermore, this contrast agent did not show any teratogenic or mutagenic potential. The typical local inflammatory signs observed following perivenous injection in the rabbit were low in intensity and reversible. CONCLUSION: The toxicologic profile of iobitridol appears to be favorable and does not show any particular risk for clinical use under the usual indications of water soluble iodinated contrast agents.

Pharmacologic profile of iobitridol, a nonionic iodinated contrast medium.

The present article combines and summarizes the preclinic studies carried out in vitro and in vivo to determine the pharmacologic and biochemical profile of iobitridol, a new nonionic iodinated low-osmolality contrast medium (CM). The effects of this product on the main hemodynamic, bronchopulmonary, neurologic, renal, blood chemistry and electrophysiologic parameters and RBC morphology were studied in detail in comparison with CM in the same chemical category or with reference substances of the same osmolality. The in vivo studies were performed under conditions resembling clinical use. Iobitridol showed an excellent pharmacologic and biochemical profile, which was identical or superior to that of other products in its category.

[Electrocorticographic evaluation of the neurologic tolerability of iobitridol (Xenetix), a new non-ionic contrast medium in rabbits]. / Evaluation électrocorticographique de la tolérance neurologique de l'iobitridol (Xenetix), un nouveau produit de contraste non-ionique, chez le lapin.

Electro-corticographic tracings (two longitudinal leads, bipolar assembly) were recorded from curarized rabbits (5/group) receiving selective internal carotid artery injections of either iobitridol, a new non-ionic contrast medium, or iohexol, the high osmolar diatrizoate or hypertonic mannitol (isotonic to the non-ionic agents). A further group was submitted to the surgical preparation but was not injected. The solutions were injected at a dose of 2.5 ml during 30 seconds. The animals were anaesthetized (halothane) during the surgical period. The permeability of the blood-brain barrier was assessed by means of the extravasation of Evans'blue. Tracings were visually assessed and a semi-quantitative method for blind evaluation of fast/slow rhythms was used. This method was pharmacologically validated by the use of pentobarbital and pentylenetetrazole. Diatrizoate and iohexol induced respectively 3 and 2 paroxystic tracings during or immediately after the injection period. Iobitridol and hypertonic mannitol did not cause such effect. Paroxystic tracings in the iohexol group were not associated with extravasation of Evans'blue in the cerebral parenchyma. Blood brain barrier was disrupted in all rabbits receiving the high osmolar agent diatrizoate. Tracings of the control group were characterized by a progressive increase of fast rhythms, as those of the iobitridol and mannitol groups. On the contrary, iohexol and especially diatrizoate induced an increase in the proportion of slow waves. Taken together, these data suggest that iobitridol shows an excellent tolerability potential for clinical use.

Modulation of the renal effects of contrast media by endothelium-derived nitric oxide in the rat.

RATIONALE AND OBJECTIVES: A possible involvement of endothelium derived relaxing nitric oxide (NO) in the pathogenesis of iodinated contrast media (CM)-induced nephrotoxicity was investigated in the rat. METHODS: Male rats (6 to 12 per group) were uninephrectomized. Six days later, the aorta was clamped above the renal artery and a low-osmolar contrast medium (CM), ioxaglate, was injected (1 mL/min; 3 minutes) via an aortic puncture in the single remaining kidney. Contrast medium was injected with or without the NO-synthase inhibitor L-NAME (100 mg/kg intravenously [i.v.] 5 minutes before CM). One group received L-Arginine, the physiological precursor of NO (100 mg/kg i.v.), 5 minutes before L-NAME. Phenylephrine (300 micrograms/kg; 30 min) was used as a vasoconstrictive NO-independent control. The effects of iohexol, another low-osmolar CM, on creatinine clearance (CrCl) were also studied with and without pretreatment with L-NAME. A control group was subjected to a 3-minute renal ischemia only. Creatinine clearance and urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion were determined before, and 24 and 48 hours after CM administration. Blinded histologic analysis was carried out after completion of the study. RESULTS: When administered alone, neither L-NAME nor L-arginine modified CrCl. Ioxaglate mildly but significantly decreased CrCl at 24 hours (-26.5% of preinjection value). This was similar to the effect observed in the control group subjected to ischemia only. When associated with L-NAME, ioxaglate markedly decreased CrCl (-58 + 11% at 24 hours, P < .05 vs. ioxaglate alone). A similar interaction was noted in the case of iohexol. L-NAME also markedly increased ioxaglate-induced urinary NAG excretion. Phenylephrine had a similar impact on renal function. L-arginine pretreatment reduced the increase in serum creatinine induced by L-NAME+ioxaglate (68 + 17 mumol/L vs. 175 + 59 mumol/L for L-NAME+ioxaglate; P < .05) and urinary NAG excretion. Ioxaglate alone induced only tubular epithelial vacuolization. When associated with L-NAME, this CM induced tubular and vascular lesions, as well as necrosis in the outer medulla. Such histologic effects were clearly inhibited by L-arginine. CONCLUSION: These data indicate that L-NAME, a specific inhibitor of NO-synthase, and phenylephrine, accentuate the nephrotoxicity of CM in the rat. This is consistent with results from the literature showing that CM-toxicity is enhanced by renal ischemia.

Pharmacokinetic study of gadolinium-DOTA in control and streptozocin diabetic rats.

The pharmacokinetics of gadolinium tetraazacyclododecanetetraacetic acid (Gd-DOTA), a contrast agent used in magnetic resonance imaging, have been evaluated in control and streptozocin-diabetic rats of different ages. In control rats, an age-related decrease in the Gd-DOTA elimination rate was noted, supported by a significantly lower apparent total body clearance and a significantly higher mean residence time. In diabetic rats, a similar but less important age-related change in the mean residence time and the apparent total body clearance was observed. Regardless of age-related differences in the pharmacokinetic parameters, a diabetic state induced several alterations in the Gd-DOTA pharmacokinetic parameters. The apparent total body clearance was significantly higher and the mean residence time significantly lower in diabetic rats indicating a higher elimination rate of Gd-DOTA. An important age-related increase in the volume of distribution at steady-state was noted in diabetic rats.

[Pharmacotoxicological profile of a non-ionic LOCM at high hydrophilicity: ioversol]. / Profil pharmacotoxicologique d'un LOCM non ionique à haute hydrophilie: ioversol.

The authors summarize the results of the main preclinical studies conducted to evaluate the tolerance of ioversol (Optiray), a new non ionic Low Osmolality Contrast Medium (LOCM). The various studies conducted by different routes of administration, in a number of animal species and at different dosages led to the conclusion that the product was equivalent or superior to the other non ionic agents used as references. This high inertia towards the biological systems particularly well evidenced in the neurologic tolerance studies has to be relied to the non ionic LOCM character of the agent but also to its highest hydrophilicity in its class and to the good distribution of this hydrophilicity around the molecule. These physiochemical characteristics which differentiate ioversol from the other products of its class make it a promising product for clinical use in humans.