Mitochondrial genome diversity in dagger and needle nematodes (Nematoda: Longidoridae).

Dagger and needle nematodes included in the family Longidoridae (viz. Longidorus, Paralongidorus, and Xiphinema) are highly polyphagous plant-parasitic nematodes in wild and cultivated plants and some of them are plant-virus vectors (nepovirus). The mitochondrial (mt) genomes of the dagger and needle nematodes, Xiphinema rivesi, Xiphinema pachtaicum, Longidorus vineacola and Paralongidorus litoralis were sequenced in this study. The four circular mt genomes have an estimated size of 12.6, 12.5, 13.5 and 12.7 kb, respectively. Up to date, the mt genome of X. pachtaicum is the smallest genome found in Nematoda. The four mt genomes contain 12 protein-coding genes (viz. cox1-3, nad1-6, nad4L, atp6 and cob) and two ribosomal RNA genes (rrnL and rrnS), but the atp8 gene was not detected. These mt genomes showed a gene arrangement very different within the Longidoridae species sequenced, with the exception of very closely related species (X. americanum and X. rivesi). The sizes of non-coding regions in the Longidoridae nematodes were very small and were present in a few places in the mt genome. Phylogenetic analysis of all coding genes showed a closer relationship between Longidorus and Paralongidorus and different phylogenetic possibilities for the three Xiphinema species.

A prospective study of concomitant GLP-1 analogue and insulin use in type 2 diabetes in clinical practice.

BACKGROUND: A small number of studies have shown a significant reduction in HbA1c, weight and total daily insulin dose when a glucagon-like-peptide-1 (GLP-1) analogue was added in type 2 diabetes patients already on insulin treatment. Therefore, in a clinical setting, we investigated the effect of adding GLP-1 analogues in patients with type 2 diabetes already using insulin with respect to glycaemic control, body weight and insulin dose. METHODS: In this prospective hospital-based study, we included 125 patients suffering from type 2 diabetes, treated with insulin and with a body mass index ≥ 35 kg/m2, who had started on GLP-1 analogues (liraglutide/exenatide). HbA1c, body weight, daily insulin dose, and side effects were registered at baseline, and after three, six and 12 months. RESULTS: HbA1c and weight decreased significantly at all the timepoints (p ≤ 0.001 compared with baseline; HbA1c: -5.5 mmol/mol (-0.5%) and weight: -14.3 kg after 12 months), with the largest decrease in the first three months. No significant correlation was found between weight loss and HbA1c reduction, and between duration of diabetes and both weight loss and HbA1c reduction. After six and 12 months, the total daily insulin dose decreased significantly (p < 0.001, -75.4 IU after 12 months). Moreover, 34% of the patients were able to stop using insulin therapy after 12 months. CONCLUSION: By adding a GLP-1 analogue in obese patients with type 2 diabetes already on insulin therapy, a significant reduction of HbA1c levels and body weight, and a significant reduction in insulin dose or complete discontinuation of insulin can be achieved.

Influence of temperature on the life-cycle of Globodera spp.

The potato cyst nematodes (PCN) G. rostochiensis (Woll.) and G. pallida (Stone) are the most economically important pests of potato (Solanum tuberosum L.) in the UK and are widespread in ware potato growing regions in Europe. The new EU directive 2007/33/EC which came into effect July 1, 2010 aims to control their spread and limit further increases in populations. We are investigating the role of temperature in the life cycle of PCN to assess how this effects population multiplication in relation to managing PCN. Hatching and nematode development are stages in the life cycle that are affected by temperature and thus are important life stages that can be examined to determine the impact of temperature on the length of time required for one generation to be completed and the potential for final populations to increase on different potato genetic backgrounds. In some conditions a partial or complete second generation has also been observed within the growing season. Females have been observed on the surface of tubers and "pecking" skin damage can occur which may be a result of a second generation. We are investigating the influence of temperature on the potential for a second generation or the induction of diapause.

Real-time PCR detection and quantification of vector trichodorid nematodes and Tobacco rattle virus.

This report describes a novel diagnostic method for virus-vector trichodorid nematodes and associated Tobacco rattle virus (TRV) based on a real-time fluorogenic 5' nuclease PCR assay (TaqMan). Two independent primer/probe sets were designed targeting the 18S gene of the ribosomal cistron for the trichodorid species, Paratrichodorus pachydermus and Trichodorus similis. Assays using purified plasmid DNA containing clones of the 18S region and genomic DNA extracted from individuals from both nematode species displayed high specificity as no cros s-reaction was observed between the species or with two non-target trichodorid species Paratrichodorus anemones and Trichodorus primitivus. Relative quantification of target DNA present in unknown samples was performed by comparison of the fluorescence signals of the samples to those obtained from plasmid standard dilutions. Three primer/probe sets were also used to target TRV; one set for RNA1 and the two other sets for RNA2 of specific isolates (TRV-PpK20 and TRV-TpO1). Detection of both trichodorid species and TRV RNA1 and RNA2 from a single sample was achieved and field samples were used to demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular information in relation to risk assessment in the field.

Molecular variation in the potato cyst nematode, Globodera pallida, in relation to virulence.

The potato cyst nematode Globodera pallida poses a challenge for potato growers. The potato cyst nematodes (PCN) Globodera rostochiensis and G. pallida cause damage valued at over pounds 50m per annum in the U.K. and problems in controlling PCN are growing due to the increase in populations and spread of G. pallida, the lack of many commercially attractive cultivars with resistance to this species and the pressure to reduce nematicide use. Over 60% of potato fields in the U.K. are infected with G. pallida (Minnis et al. 2000). The Scottish Agricultural Science Agency (SASA) figures show that the incidence of both species of PCN on Scottish seed potato land, though low, has been increasing. The proportion of potato land in ware production in Scotland is also increasing and now represents 50% of the potato growing area. This situation potentially increases the risk of the spread of PCN unless it is very carefully monitored and managed.

Cytokines affect resistance of human renal tumour cells to complement-mediated injury.

Overexpression of membrane-bound complement regulatory proteins (mCRPs) on tumour cells may hamper the effect of immunotherapy with complement-activating monoclonal antibody (MoAb). Therefore, it is important to investigate whether cytokines can downregulate the expression of mCRP on tumour cells. In this study, the effect of 10 cytokines on the expression of the mCRP CD46, CD55 and CD59 and the renal tumour-associated antigen G250/MN/CAIX on four human renal tumour cell lines and proximal tubular epithelial cells was determined by flow cytometry. In addition, it was measured whether changes in the expression of the classical pathway regulatory proteins CD55 and CD59 had an effect on C3 deposition and lysis. Interleukin-1beta (IL-1beta) consistently downregulated the expression of CD46 and CD59; IL-4 consistently downregulated the expression of CD46 and transforming growth factor-beta1, consistently downregulated the expression of both CD46 and CD55. However, treatment with IL-1beta and IL-4 also decreased the expression of G250/MN/CAIX. Changes in the expression of CD55 and CD59 were associated with changes in the amount of C3 deposited and the extent of complement-mediated lysis, respectively. This suggests that clinical immunotherapy, consisting of treatment with cytokines and MoAb, may induce either up- or downregulation of CD55 or CD59 and thus affect the effectiveness of immunotherapy with MoAb.

Ribosomal Intergenic Spacer: A Polymerase Chain Reaction Diagnostic for Meloidogyne chitwoodi, M. fallax, and M. hapla.

ABSTRACT Polymerase chain reaction amplification of the intergenic spacer region between the 5S and 18S genes from Meloidogyne chitwoodi, M. fallax, and M. hapla enabled these three important temperate species to be differentiated. Length polymorphism was found between M. chitwoodi and M. fallax as a result of differing numbers of short repeats located between the 5S and 18S genes. The presence of the 5S gene within the rDNA cistrons was confirmed in the Meloidogyne spp. included in this study. The region between the 28S and 5S genes for M. chitwoodi and M. fallax was short and lacked variability in repeated sequences compared with the main tropical Meloidogyne spp. and M. hapla. Differences in the number of these repeats resulted in intraspecific length polymorphism for M.hapla.

Apomictic, polyphagous root-knot nematodes: exceptionally successful and damaging biotrophic root pathogens.

Most apomictic root-knot nematodes (RKN; Meloidogyne spp.) have host ranges that encompass the majority of flowering plants, and M. incognita is possibly the world's most damaging crop pathogen. The ancestors, age, and origins of the polyphagous RKN are obscure, but there is increasing evidence that M. incognita, M. javanica, and M. arenaria are closely related, heterogeneous species with a recent, hybrid (reticulate) origin. If so, they must owe much of their current worldwide distributions to spread by agriculture. Host resistance appears to be generally durable in the field, but laboratory studies suggest that apomixis does not prevent evolution in response to selection by a parasitic bacterium (Pasteuria penetrans) and host resistance. Maintaining general fitness may be the evolutionary priority for most populations of polyphagous RKN, and a wide host range, important in the field but not in the laboratory, may be conserved by apomixis. Several factors may help confer a wide host range, including suppression of host resistance, perhaps as a consequence of the strength of the induced susceptible response. Resistance genes effective against RKN appear not to have resulted from coevolution. Rates of juvenile invasion and/or development are low in many wild and some crop plants, with the result that they are both poor hosts and sustain less damage. Overall, it is suggested that greater coordination, particularly of fundamental research, is required.

A surface-associated retinol- and fatty acid-binding protein (Gp-FAR-1) from the potato cyst nematode Globodera pallida: lipid binding activities, structural analysis and expression pattern.

Parasitic nematodes produce at least two structurally novel classes of small helix-rich retinol- and fatty-acid-binding proteins that have no counterparts in their plant or animal hosts and thus represent potential targets for new nematicides. Here we describe a protein (Gp-FAR-1) from the plant-parasitic nematode Globodera pallida, which is a member of the nematode-specific fatty-acid- and retinol-binding (FAR) family of proteins but localizes to the surface of this species, placing it in a strategic position for interaction with the host. Recombinant Gp-FAR-1 was found to bind retinol, cis-parinaric acid and the fluorophore-tagged lipids 11-(dansylamino)undecanoic acid and dansyl-D,L-alpha-amino-octanoic acid. The fluorescence emission characteristics of the dansylated analogues indicated that the entire ligand enters the binding cavity. Fluorescence competition experiments showed that Gp-FAR-1 binds fatty acids in the range C(11) to C(24), with optimal binding at C(15). Intrinsic fluorescence analysis of a mutant protein into which a tryptophan residue had been inserted supported computer-based predictions of the position of this residue at the protein's interior and possibly also at the binding site. Of direct relevance to plant defence systems was the observation that Gp-FAR-1 binds two lipids (linolenic and linoleic acids) that are precursors of plant defence compounds and the jasmonic acid signalling pathway. Moreover, Gp-FAR-1 was found to inhibit the lipoxygenase-mediated modification of these substrates in vitro. Thus not only does Gp-FAR-1 function as a broad-spectrum retinol- and fatty-acid-binding protein, the results are consistent with the idea that Gp-FAR-1 is involved in the evasion of primary host plant defence systems.

A possible role of CD46 for the protection in vivo of human renal tumor cells from complement-mediated damage.

It is still unclear which membrane-bound regulatory proteins (mCRP) are important in vivo to protect tumor cells from complement-mediated damage. To address this question, the expression levels of CD46, CD55, and CD59 were measured semi-quantitatively in situ on renal cell carcinomas and compared with the expression level and cellular distribution of these mCRP in proximal tubuli within each patient (n = 31). It was also determined whether the expression of mCRP on tumor cells is associated with deposition of C3d and C5b-9. CD46 expression was decreased on tumor cells; in contrast, CD55 was expressed on tumor cells (12 out of 31 samples), while it was not detected on proximal tubular epithelial cells (PTEC). Also, expression of CD59 on tumor cells was increased as compared with its expression on PTEC. Furthermore, the localization on the cell surface of mCRP as observed on PTEC was altered on tumor cells. Because expression of mCRP may limit a complement-mediated anti-tumor response, we determined whether complement deposition was associated with the expression level of CD46, CD55, and CD59. The presence of C3d on tumor cells was associated with a low expression level of CD46 (p < 0.02). The expression level of CD46 was also associated with a low tumor stage (p < 0.04). The results suggest that in vivo CD46 plays a role in the protection of human renal tumor cells from complement-mediated injury.

A multipartite mitochondrial genome in the potato cyst nematode Globodera pallida.

The mitochondrial genome (mtDNA) of the plant parasitic nematode Globodera pallida exists as a population of small, circular DNAs that, taken individually, are of insufficient length to encode the typical metazoan mitochondrial gene complement. As far as we are aware, this unusual structural organization is unique among higher metazoans, although interesting comparisons can be made with the multipartite mitochondrial genome organizations of plants and fungi. The variation in frequency between populations displayed by some components of the mtDNA is likely to have major implications for the way in which mtDNA can be used in population and evolutionary genetic studies of G. pallida.

A bispecific monoclonal antibody directed against both the membrane-bound complement regulator CD55 and the renal tumor-associated antigen G250 enhances C3 deposition and tumor cell lysis by complement.

Tumor cells may inhibit the induction of a complement-mediated inflammatory response through overexpression of membrane-bound regulators of complement activation. Therefore, it is of interest to determine the most efficient approach to block these membrane-bound complement regulators on tumor cells. In the present study, we first generated a bispecific mAb directed against both CD55, using the functional blocking mAb MBC1, and the highly expressed HLA class I molecule as a model for a tumor-associated Ag, using the mAb W6/32. Tumor cells opsonized with bispecific mAb W6/32*MBC1, then exposed to complement and subsequently stained for C3 deposition, were assessed by flow cytometric analysis. We found that opsonization with W6/32*MBC1 resulted in a 92% enhancement of C3 deposition on renal tumor cells as compared with opsonization with W6/32 alone and a 17% enhancement of the C3 deposition as compared with incubation with a mixture of both parental mAb. Based on these results, we developed a bispecific mAb recognizing both CD55 and the relatively low expressed renal tumor-associated Ag G250. Increasing concentrations of the bispecific mAb G250*MBC1 resulted in a 25 to 400% increase in C3 deposition on renal tumor cells as compared with C3 deposition in the presence of the parental mAb G250 alone. G250*MBC1 enhanced C3 deposition by 21% in comparison with a mixture of both parentals. Furthermore, opsonization of tumor cells with G250*MBC1 rendered these cells more sensitive to complement-mediated lysis. In conclusion, the bispecific mAb G250*MBC1 induces deposition of C3 and tumor cell lysis more efficiently than G250 alone.

Intraspecific Variation in Ribosomal DNA in Populations of the Potato Cyst Nematode Globodera pallida.

The relationships among a number of populations of Globodera pallida from Britian, the Netherlands, Germany, Switzerland, and South America were examined using PCR amplification of the ribosomal cistron between the 18S and 28S genes that include the two intergenic spacer regions (ITS1 and ITS2) and the 5.8S gene. Amplifications produced a similar-sized product of 1150 bp from all populations. Digestion of the amplified fragment with a number of restriction enzymes showed differences among the populations. The restriction enzyme RsaI distinguished the most populations. The RFLP patterns revealed by this enzyme were complex and could have arisen from heterogeneity between individuals within populations and from differences between the repeats of an individual. Sequence analysis from six of the populations, together with RFLP analysis of PCR products, shows that there is intraspecific variation in the rDNA of G. pallida.

Comparison of British populations of potato cyst nematodes with populations from continental Europe and South America using RAPDs.

Genetic variation between populations of Globodera pallida, primarily from Britain but including populations from continental Europe and South America and two Globodera rostochiensis populations, was examined using random amplified polymorphic DNA (RAPD). Fourteen primers were used and 250 amplification products observed. A comparison was made of the similarities between the species and, within G. pallida, between populations from Britain, The Netherlands, Germany, and Switzerland, of the pathotypes Pa2 and Pa3. In addition, one Pa1 population and two others from South America were included. On the basis of the RAPD analysis, all the Pa2-Pa3 populations, except one from Scotland (Luffness), constituted a single group with no clear distinction based on pathotype designation. The Luffness population is known to be distinct in its virulence. The data indicated that the main Pa2-Pa3 group could be subdivided based on geographic origin, but this is not well supported by bootstrap analysis. The Pa1 population and the two populations from South America all formed distinct groups.

Comparison of Sequences from the Ribosomal DNA Intergenic Region of Meloidogyne mayaguensis and Other Major Tropical Root-Knot Nematodes.

The unusual arrangement of the 5S ribosomal gene within the intergenic sequence (IGS) of the ribosomal cistron, previously reported for Meloidogyne arenaria, was also found in the ribosomal DNA of two other economically important species of tropical root-knot nematodes, M, incognita and M. javanica. This arrangement also was found in M. hapla, which is important in temperate regions, and M. mayaguensis, a virulent species of concern in West Africa. Amplification of the region between the 5S and 18S genes by PCR yielded products of three different sizes such that M. mayaguensis could be readily differentiated from the other species in this study. This product can be amplified from single juveniles, females, or egg masses. The sequences obtained in this region for one line of each of M. incognita, M. arenaria, and M. javanica were very similar, reflecting the close relationships of these lineages. The M. mayaguensis sequence for this region had a number of small deletions and insertions of various sizes, including possible sequence duplications.

Expression of CD46, CD55, and CD59 on renal tumor cell lines and their role in preventing complement-mediated tumor cell lysis.

Nucleated cells are protected from complement-mediated injury by the expression of membrane-bound regulators of complement activation (mRCA) CD46, CD55, and CD59. Increased expression of these mRCA may be a mechanism by which tumor cells protect themselves from complement-mediated injury and prevent an inflammatory response. In the present study, we have investigated whether human renal tumor cell lines and cultured proximal tubular epithelial cells express CD46, CD55, and CD59 and whether these mRCA influence complement-mediated lysis of these cells. The expression of CD46, CD55, and CD59 was measured by flow cytometry. To determine the effect of mRCA on lysis, tumor cells were opsonized with complement activating anti-HLA class l mAb. Lysis was measured in the presence or absence of anti-CD46, anti-CD55 or anti-CD59 mAb and serum as a source of complement, using a 51Cr release assay. Flow cytometric analysis revealed that renal tumor cell lines and proximal tubular epithelial cells all express CD46, CD55, and CD59. Lysis of renal tumor cell lines in the presence of rabbit serum depended on the number of HLA class I molecules expressed by the tumor cells. Using human serum, complement-mediated lysis was decreased by at least one-third as compared with rabbit serum. The susceptibility of renal tumor cells for complement-mediated lysis could be increased up to the level observed with rabbit serum by inhibiting the function of CD59. Inhibition of the function of CD46 or CD55 with mAb directed against these mRCA had no substantial effect on lysis. We conclude from this work that renal tumor cells and proximal tubular epithelial cells express CD46, CD55, and CD59. Of these mRCA, CD59 is most efficient in preventing complement-mediated lysis of these cells. Expression of mRCA on tumor cells may influence the effectiveness of immunotherapy with tumor-associated mAb.

Inhibition of the influenza virus RNA-dependent RNA polymerase by antisera directed against the carboxy-terminal region of the PB2 subunit.

The influenza virus RNA polymerase consists of a heterotrimeric complex of the PB1, PB2 and PA proteins, with the PB2 subunit responsible for recognizing 5' cap structures on the host cell RNAs used as primers for virus mRNA synthesis. To investigate further the role PB2 plays in mRNA synthesis, a set of polyclonal antisera raised against defined regions of the protein were tested for their ability to inhibit the virion transcriptase. All five sera were of sufficient titre to immunoprecipitate PB2 and four were capable of recognizing polymerase complexes containing PB1 and PA. However, only the serum raised against the carboxy terminus of PB2 (F5) substantially inhibited polymerase activity. This serum drastically reduced synthesis primed by globin mRNA, but only partially inhibited transcription primed by the dinucleotide ApG, or ApG and cap analogue. The preferential inhibition of globin-primed synthesis did not result from interference with cap recognition, as serum F5 did not reduce labelling of PB2 in a photoaffinity cap-binding assay. However, IgG and Fab fragments from F5 were found to inhibit virion endonuclease activity. This suggests that the C terminus of PB2 plays a crucial role in transcription initiation and implicates PB2 in endonuclease activity.

Sequences of 10 variants of the satellite-like RNA-3 of groundnut rosette virus.

Ten cDNA clones, representing the satellite-like RNA-3 associated with five different isolates of groundnut rosette virus, were prepared and sequenced. The sequences are at least 87% identical, but differences associated both with symptom differences and with geographical origin were found. The sequences contain up to five potential open reading frames (ORFs), of which three are in the strand that is most abundant in infected plants and two are in the complementary strand. Only one of these ORFs is present in all 10 sequences. Neither the sequence of RNA-3 nor that of its potential translation products had significant similarities to sequences in the databases.

The nucleotide sequence of RNA-2 of raspberry ringspot nepovirus.

The nucleotide sequence of raspberry ringspot nepovirus (RRV) RNA-2 consists of 3928 nucleotides and a poly(A) tract at the 3' end. RNA-2 contains one open reading frame which encodes a polypeptide of M(r) 123508 (123K). Edman degradation located the N terminus of the coat protein 514 residues from the C-terminal end of the 123K protein, which suggests that the coat protein is released from the polyprotein by cleavage of a C-A bond. The RRV coat protein has some sequence similarities with the coat proteins of other nepoviruses, but is no more like any one nepovirus than another. In contrast, the portion of the 123K protein to the N-terminal side of the coat protein is similar in sequence to the corresponding parts of the polyproteins of tomato black ring and grapevine chrome mosaic nepoviruses, though not to those of other nepoviruses.