Trematode-associated morbidity and mortality of tadpoles in Israel.

Amphibians stand at the forefront of the global biodiversity crisis. The causes of their decline are diverse and include a rise in amphibian malformations due to various factors, especially trematode infection. However, linking amphibian mortality and morbidity with trematode infection has proven to be challenging due to the complex life cycle of the trematodes and the fact that trematodes are nonfastidious in their choice of definitive hosts. In Israel, the decline in local amphibian populations has been mostly attributed to the loss and degradation of wetlands and riparian habitats. Recently, however, there have been several reports of morbidity and mortality of tadpoles with signs of edema and malformations from various localities in Israel. We collected dead and morbid tadpoles and metamorphs of Hyla savignyi and Pelophylax bedriagae, and we showed that the morbidity and the deformations observed in the field are the result of infection by trematodes. We also isolated an echinostomatid trematode from the malformed and edematous tadpoles and from the freshwater snail Bulinus truncatus, all from the same site. We further succeeded in experimentally infecting H. savignyi tadpoles by echinostomatid cercariae that were shed from the snails, and we showed that infection had significantly increased the mortality rates of these tadpoles. The combination of high trematode prevalence and their pathogenic effects suggests that in nature, the effect of echinostome infection on amphibians may be substantial and could become an emerging disease in Israel.

Enhancement of factor VIIa haemostatic efficacy by formulation with PEGylated liposomes.

Recombinant activated factor VII (rFVIIa) is an effective treatment of the haemophilia patient with inhibitors and acquired haemophilia. However, on account of its relatively short half-life (HL), achieving therapeutic efficacy with FVIIa requires repeated injections. The development of a long-acting FVIIa product would therefore be beneficial. The formulation of factor VIII with PEGylated liposomes (PEGLip) was previously shown to extend the bleeding-free period in haemophilia patients. We report here an enhancement of haemostatic efficacy by similarly formulating FVIIa with PEGLip. Surface plasmon resonance analysis indicated that FVIIa binds non-covalently but with high affinity and specificity to PEGLip. A one-stage clotting assay demonstrated that formulation of FVIIa with PEGLip does not affect FVIIa activity and stability. A pharmacokinetic study in rats demonstrated that PEGLip formulation of FVIIa extends circulation time and results in higher FVIIa levels several hours after injection. Thromboelastography experiments indicated that PEGLip-FVIIa induces faster clot formation and higher clot stability than standard formulated FVIIa. These results suggest that formulation of FVIIa with PEGLip may lead to a safe and effective long-acting FVIIa that improves the care of haemophilic patients with inhibitors and acquired haemophilia.

Two tightly linked QTLs modify tomato sugar content via different physiological pathways.

Congenic lines that differ in a single defined chromosome segment are useful for the study of complex phenotypes, as they allow isolation of the effect of a particular quantitative trait locus (QTL) from those of the entire genome. We conducted high-resolution QTL mapping of a 9-cM introgression, originating from the wild tomato species Lycopersicon pennellii, in two extremely different genetic and physiological backgrounds. In the "indeterminate" glasshouse background we identified only a single QTL ( Brix9-2-5) that affects the total soluble solids of the fruit [mainly sugars, measured in Brix units (B)]. This QTL was previously delimited within the gene for an apoplastic invertase, Lin5, that modulates sugar partitioning to the fruit. Analysis of the effects of the same chromosome segment in "determinate", open-field tomatoes, revealed two QTLs, 0.3 cM apart: the fruit-specific Brix9-2-5 that affects B only, and the shoot-specific PW9-2-5, which accounts for an altered growth habit resulting in increases in plant weight, yield, and B. This study highlights the power of the congenic approach for dissecting developmental pathways leading to complex phenotypes.

Genome-wide dissection of Fusarium resistance in tomato reveals multiple complex loci.

Resistance to different pathogenic races of Fusarium oxysporum f. sp. lycopersici (F. o. lycopersici) was explored at two genomic levels in tomato. Six independent Fusarium resistance loci were identified by comparing the responses of a complete set of 53 lines carrying different introgressed regions of the Lycopersicon pennellii genome in a L. esculentum background. The loci confer varying degrees of resistance to different races of the pathogen. Corresponding map positions from different tomato species were aligned and in some cases revealed parallel resistance to F. o. lycopersici with qualitative changes in race specificities. One of the loci identified corresponds to the previously characterized complex resistance locus I2, which is involved in resistance to F. o. lycopersici race 2. A novel member of this locus, I2C-5, which belongs to the NBS-LRR family of resistance genes, was cloned and shown to confer partial resistance in transgenic plants. Thus, at a particular complex locus gene members can confer full or partial resistance to F. o. lycopersici race 2. The results of our whole-genome mapping analysis underline the robust independent origin of resistance to a particular disease and demonstrate the conservation of resistance features at syntenic loci, together with the rapid diversification of genes for innate resistance within loci.

An alternative pathway to beta -carotene formation in plant chromoplasts discovered by map-based cloning of beta and old-gold color mutations in tomato.

Carotenoid pigments in plants fulfill indispensable functions in photosynthesis. Carotenoids that accumulate as secondary metabolites in chromoplasts provide distinct coloration to flowers and fruits. In this work we investigated the genetic mechanisms that regulate accumulation of carotenoids as secondary metabolites during ripening of tomato fruits. We analyzed two mutations that affect fruit pigmentation in tomato (Lycopersicon esculentum): Beta (B), a single dominant gene that increases beta-carotene in the fruit, and old-gold (og), a recessive mutation that abolishes beta-carotene and increases lycopene. Using a map-based cloning approach we cloned the genes B and og. Molecular analysis revealed that B encodes a novel type of lycopene beta-cyclase, an enzyme that converts lycopene to beta-carotene. The amino acid sequence of B is similar to capsanthin-capsorubin synthase, an enzyme that produces red xanthophylls in fruits of pepper (Capsicum annum). Our results prove that beta-carotene is synthesized de novo during tomato fruit development by the B lycopene cyclase. In wild-type tomatoes B is expressed at low levels during the breaker stage of ripening, whereas in the Beta mutant its transcription is dramatically increased. Null mutations in the gene B are responsible for the phenotype in og, indicating that og is an allele of B. These results confirm that developmentally regulated transcription is the major mechanism that governs lycopene accumulation in ripening fruits. The cloned B genes can be used in various genetic manipulations toward altering pigmentation and enhancing nutritional value of plant foods.

Comparative genetics of nucleotide binding site-leucine rich repeat resistance gene homologues in the genomes of two dicotyledons: tomato and arabidopsis.

The presence of a single resistance (R) gene allele can determine plant disease resistance. The protein products of such genes may act as receptors that specifically interact with pathogen-derived factors. Most functionally defined R-genes are of the nucleotide binding site-leucine rich repeat (NBS-LRR) supergene family and are present as large multigene families. The specificity of R-gene interactions together with the robustness of plant-pathogen interactions raises the question of their gene number and diversity in the genome. Genomic sequences from tomato showing significant homology to genes conferring race-specific resistance to pathogens were identified by systematically "scanning" the genome using a variety of primer pairs based on ubiquitous NBS motifs. Over 70 sequences were isolated and 10% are putative pseudogenes. Mapping of the amplified sequences on the tomato genetic map revealed their organization as mixed clusters of R-gene homologues that showed in many cases linkage to genetically characterized tomato resistance loci. Interspecific examination within Lycopersicon showed the existence of a null allele. Consideration of the tomato and potato comparative genetic maps unveiled conserved syntenic positions of R-gene homologues. Phylogenetic clustering of R-gene homologues within tomato and other Solanaceae family members was observed but not with R-gene homologues from Arabidopsis thaliana. Our data indicate remarkably rapid evolution of R-gene homologues during diversification of plant families.

The SELF-PRUNING gene of tomato regulates vegetative to reproductive switching of sympodial meristems and is the ortholog of CEN and TFL1.

Vegetative and reproductive phases alternate regularly during sympodial growth in tomato. In wild-type 'indeterminate' plants, inflorescences are separated by three vegetative nodes. In 'determinate' plants homozygous for the recessive allele of the SELF-PRUNING (SP) gene, sympodial segments develop progressively fewer nodes until the shoot is terminated by two consecutive inflorescences. We show here that the SP gene is the tomato ortholog of CENTRORADIALIS and TERMINAL FLOWER1, genes which maintain the indeterminate state of inflorescence meristems in Antirrhinum and Arabidopsis respectively. The sp mutation results in a single amino acid change (P76L), and the mutant phenotype is mimicked by overexpressing the SP antisense RNA. Ectopic and overexpression of the SP and CEN transgenes in tomato rescues the 'indeterminate' phenotype, conditions the replacement of flowers by leaves in the inflorescence and suppresses the transition of the vegetative apex to a reproductive shoot. The SELF-PRUNING gene is expressed in shoot apices and leaves from very early stages, and later in inflorescence and floral primordia as well. This expression pattern is similar to that displayed by the tomato ortholog LEAFY and FLORICAULA. Comparison of the sympodial, day-neutral shoot system of tomato and the monopodial, photoperiod-sensitive systems of Arabidopsis and Antirrhinum suggests that flowering genes that are required for the processing of floral induction signals in Arabidopsis and Antirrhinum are required in tomato to regulate the alternation between vegetative and reproductive cycles in sympodial meristems.

Activation of human lymphocytes by a monoclonal antibody to B lymphoblastoid cells; molecular mass and distribution of binding protein.

A novel monoclonal antibody (BAT) to the B-lymphoblastoid cell line activates murine lymphocytes and exhibits a striking antitumor activity in mice. In order to evaluate the potential use of this antibody against human cancer, we have investigated its immuno-stimulatory properties on human peripheral blood lymphocytes (PBL). Our findings demonstrate that BAT mAb induces proliferation and cytotoxicity in human PBL against natural-killer-cell-sensitive and natural-killer-cell-resistant tumor cell lines. Interleukin-2 at a low concentration synergizes with BAT mAb in eliciting these effects. BAT mAb binds to human peripheral T cells as revealed by a double-labelling technique using anti-CD3 and BAT mAb. The molecular mass of the antigen recognized by BAT mAb was 48-50 kDa under reducing and non-reducing conditions. This study provides a basis for future experiments to evaluate the use of BAT mAb in the immunotherapy of cancer.

Recruitment of long-term care facilities for research.

We report the successful recruitment of a stratified random sample of nursing homes in the state of Maryland into three research studies funded by the National Institute on Aging. These studies examine the prevalence of infections and urinary tract instrumentation and the incidence of antimicrobial use in nursing home residents. Following selection of a facility, the administrator was telephoned and a meeting at the home was requested. At this meeting, the project was explained in detail using a packet of promotional information which included a project summary, a listing of project staff and their qualifications, and letters of support from influential organizations. A total of 61 eligible facilities were contacted in order to achieve a group of 53 participating homes with approximately 5000 beds. One hundred percent cooperation was achieved from all strata except small (less than or equal to 50 beds) proprietary comprehensive care facilities, and homes with both comprehensive and domiciliary beds. A direct, personal approach, backed by a carefully prepared study information and the support of medical and nursing home organizations resulted in successful recruitment of 53 (87%) of 61 homes sampled.