The characterization of the Phlebotomus papatasi transcriptome.

As important vectors of human disease, phlebotomine sand flies are of global significance to human health, transmitting several emerging and re-emerging infectious diseases. The most devastating of the sand fly transmitted infections are the leishmaniases, causing significant mortality and morbidity in both the Old and New World. Here we present the first global transcriptome analysis of the Old World vector of cutaneous leishmaniasis, Phlebotomus papatasi (Scopoli) and compare this transcriptome to that of the New World vector of visceral leishmaniasis, Lutzomyia longipalpis. A normalized cDNA library was constructed using pooled mRNA from Phlebotomus papatasi larvae, pupae, adult males and females fed sugar, blood, or blood infected with Leishmania major. A total of 47 615 generated sequences was cleaned and assembled into 17 120 unique transcripts. Of the assembled sequences, 50% (8837 sequences) were classified using Gene Ontology (GO) terms. This collection of transcripts is comprehensive, as demonstrated by the high number of different GO categories. An in-depth analysis revealed 245 sequences with putative homology to proteins involved in blood and sugar digestion, immune response and peritrophic matrix formation. Twelve of the novel genes, including one trypsin, two peptidoglycan recognition proteins (PGRP) and nine chymotrypsins, have a higher expression level during larval stages. Two novel chymotrypsins and one novel PGRP are abundantly expressed upon blood feeding. This study will greatly improve the available genomic resources for P. papatasi and will provide essential information for annotation of the full genome.

Intergeneric transfer of genes involved in the Rhizobium-legume symbiosis.

Genes that seem to be involved in the initial steps of infection of a legume by Rhizobium have been transferred, by transformation, to mutant strains of Azotobacter vinelandii that are unable to fix nitrogen. These genes code for a surface antigen that binds specifically to a protein from the host plant.

Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils.

Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin-2, MPIF-2, or CKbeta-6, is a human CC chemokine with low amino acid sequence identity to other chemokines. Eotaxin-2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross-desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP-4, but not RANTES, MIP-1alpha, or MCP-3, can completely cross-desensitize the calcium response to eotaxin-2 on these cells, indicating that eotaxin-2 shares the same receptor used by eotaxin and MCP-4. Eotaxin-2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin-2 also displaced 125I-eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO-CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP-4. 125I-Eotaxin-2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin-2 displace the ligand with equal affinity. Eotaxin and eotaxin-2 promote a Ca2+ transient in RBL-2H3 cells stably transfected with CCR3 (RBL-2H3-CCR3) and both ligands cross-desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin-2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor.

Human osteoclasts, not osteoblasts, deposit osteopontin onto resorption surfaces: an in vitro and ex vivo study of remodeling bone.

Osteopontin is a phosphorylated glycoprotein believed to be secreted by osteoblasts and deposited into the bone matrix to facilitate osteoclasts adhesion or to initiate osteoid mineralization. Previously we have presented contradictory evidence that osteoclasts express osteopontin mRNA in human remodeling bone. The aim of this study was to ascertain whether osteoclasts synthesize and deposit osteopontin in resorption lucunae. We characterized expression of osteopontin mRNA and protein expression in both intramembranous and endochondral ossification, as well as remodeling bone, in the human osteophyte. Osteopontin mRNA was expressed in osteoclast with tartrate-resistant acid phosphatase (TRAP) positivity within resorption lacunae. The osteoclasts and immediate resorption surfaces also expressed osteopontin. However, osteopontin mRNA and protein were weak (transient) or undetectable in osteoblasts at adjacent bone formation sites; no osteopontin expression was observed in the osteoid, although occasional reactivity was observed in osteocytes and the mineral-osteoid interface. In contrast, osteopontin was highly expressed in the osteoblasts and matrix of woven bone during intramembranous and endochondral ossification. The matrix expression correlated with mineralization; however, in some instances osteopontin deposition was observed prior to mineralization. Similarly, osteopontin expression was evident in cartilage matrix, solely at foci of mineralization. Chondroclasts expressed osteopontin mRNA and protein: the surfaces of resorbed calcified cartilage also expressed osteopontin. Abnormal, unmineralized matrices apparently lacked deposited osteopontin, but were nevertheless resorbed by osteoclasts; the osteoclasts and resorbed surfaces expressed no osteopontin protein. That osteoclasts are responsible for the deposition of osteopontin was confirmed in vitro, whereby resorption pits in whale dentine and bovine bone slices, produced by isolated human osteoclasts, contained deposited osteopontin. Osteopontin may facilitate the adhesion (or detachment) of the osteoclast to the bone surface. Alternatively, the possibility that osteopontin may act as a postresorptive signal to recruit osteoblasts, or to polarize and direct the mineralization of the formed osteoid, is discussed.

High-level production of biologically active human cytosolic phospholipase A2 in baculovirus-infected insect cells.

Infection of Spodoptera frugiperda insect cells with a recombinant baculovirus expressing human cytosolic phospholipase A2 (cPLA2) resulted in the production of biologically active protein. The level of recombinant human cPLA2 production in infected insect cells was at least 50-fold higher than that observed in human monoblast U937 cells.

Photodynamic therapy in the treatment of squamous cell carcinoma of the head and neck.

Photodynamic therapy is an experimental modality for tumor treatment based on the combined action of the tumor-localizing agent, ie, hematoporphyrin derivative, and red light. From 1985 through 1989, 26 patients were treated using hematoporphyrin-derived drugs and 630-nm light delivered by a tunable dye laser. All patients had biopsy-proved squamous cell carcinoma of the head and neck, and they had either failed the traditional treatment modalities or refused conventional therapies. Histological complete responses were achieved in 20 (77%) of 26 patients and partial responses in 5 (19%) of 26 patients for periods up to 48 months. Only minimal toxic reaction was noted in the group. As a guide to treatment planning for a patient group with large tumors, we used an optical dosimetry model based on tissue optics. The rate of complete responses to this treatment was 8 (73%) of 11. Our data indicate that photodynamic therapy is capable of inducing significant clinical and histological responses in the majority of those treated, and in some patients a prolonged response is produced. In certain select head and neck malignancies, photodynamic therapy has an important role as a treatment modality.

Hospital employment under revised Medicare payment schedules.

A preliminary study of the employment effects of medicare payments based on Diagnosis Related Groups suggests that cost-cutting responses by hospitals will result in smaller, more highly skilled staff and a higher proportion of clerical to health care workers.

Widespread divergence between incipient Anopheles gambiae species revealed by whole genome sequences.

The Afrotropical mosquito Anopheles gambiae sensu stricto, a major vector of malaria, is currently undergoing speciation into the M and S molecular forms. These forms have diverged in larval ecology and reproductive behavior through unknown genetic mechanisms, despite considerable levels of hybridization. Previous genome-wide scans using gene-based microarrays uncovered divergence between M and S that was largely confined to gene-poor pericentromeric regions, prompting a speciation-with-ongoing-gene-flow model that implicated only about 3% of the genome near centromeres in the speciation process. Here, based on the complete M and S genome sequences, we report widespread and heterogeneous genomic divergence inconsistent with appreciable levels of interform gene flow, suggesting a more advanced speciation process and greater challenges to identify genes critical to initiating that process.

Restriction mapping of deletions in the nif region of the Klebsiella pneumoniae chromosome.

Chromosomal DNA restriction fragments carrying the nitrogen fixation (nif) and his genes of Klebsiella pneumoniae were identified in hybridization experiments using a plasmid derived from pRD1 as a radioactive probe. Restriction mapping of 26 genetically characterized chromosomal nif deletions provided a map showing the physical location of nif genes along the chromosome.

Asymmetric transcription of R plasmid NR1 in Proteus mirabilis.

The composite R plasmid NR1, its resistance transfer factor which specifies resistance to tetracycline (RTF-Tc component), and its r-determinants component were each denatured and centrifuged to equilibrium in CsCl density gradients containing polyuridylic acid-polyguanidylic acid. The complementary deoxyribonucleic acid strands of NR1 and the complementary strands of the RTF-Tc component could be separated by this technique because of a threefold difference in polyuridylic acid-polyguanidylic acid binding to the strands of the RTF-Tc component. The two strands of the r-determinants component bound equal amounts of polyuridylic acid-polyguanidylic acid. Hybridization of single strands of plasmid deoxyribonucleic acid with in vivo-labeled ribonucleic acid from Proteus mirabilis containing NR1 indicated that transcription within the RTF-Tc component is from the NR1 strand which preferentially binds polyuridylic acid-polyguanidylic acid, whereas transcription within the r-determinants component is predominantly from the complementary strand.

Rhizobium japonicum USDA 191 has two nodD genes that differ in primary structure and function.

Several Rhizobium genes (designated nod genes) are involved in early steps in nodule formation. Here we present the results of DNA sequence and functional analysis of two nodD genes from the symbiotic plasmid of USDA 191, a fast-growing strain that forms nitrogen-fixing nodules on soybeans. Both genes encoded full-length nodD-related polypeptides, which were 69% homologous to each other. One of these genes, nodD1, complemented a Rhizobium trifolii nodD::Tn5 mutant for clover nodulation; the other gene, nodD2, did not. The nodD1 coding region was preceded by a conserved DNA sequence previously noted in other rhizobia, but no such sequence was found in front of nodD2. Plants inoculated with a nodD1 insertion mutant appeared to be nitrogen starved and had a greatly reduced nodule number. Plants inoculated with a nodD2 mutant had a partially nitrogen-starved appearance and normal nodule number, were slightly delayed in nodule formation, and formed nodules that contained reduced levels of nodulin-35 and had fewer bacteroids per infected plant cell. Thus, both of these genes are involved in symbiosis. USDA 191 carrying extra copies of nodD2 on a plasmid vector had an altered colony morphology that suggested inhibition of exopolysaccharide synthesis. The predicted gene products of nodD1 and nodD2 both showed homology to LysR, an E. coli regulatory protein. We conclude that nodD1 probably has the same function as nodD in temperate rhizobia, namely, activation of nodABC transcription in the presence of plant signals. nodD2 may be involved in regulation of exopolysaccharide synthetic genes.

Induction of Bradyrhizobium japonicum common nod genes by isoflavones isolated from Glycine max.

The early events in legume nodulation by Rhizobium spp. involve a conserved gene cluster known as the common nod region. A broad-host-range plasmid (pEA2-21) containing a Bradyrhizobium japonicum nodDABC-lacZ translational fusion was constructed and used to monitor nod gene expression in response to soybean root extract. Two inducing compounds were isolated and identified. Analysis using ultraviolet absorption spectra, proton nuclear magnetic resonance, and mass spectrometry showed that the two inducers were 4',7-dihydroxyisoflavone (daidzein) and 4',5,7-trihydroxyisoflavone (genistein). Induction was also seen with some, but not all, of the flavonoid compounds that induce nod genes in fast-growing Rhizobium strains that nodulate clover, alfalfa, or peas. When pEA2-21 was introduced into Rhizobium trifolii, it was inducible by flavones but not by daidzein and genistein. In Rhizobium fredii, pEA2-21 was induced by isoflavones and flavones. Thus, the specificity of induction appears to be influenced by the host-strain genome.

Expression of symbiotic genes of Rhizobium japonicum USDA 191 in other rhizobia.

A 200-megadalton plasmid was mobilized from Rhizobium japonicum USDA 191 to other Rhizobium strains either that cannot nodulate soybeans or that form Fix- nodules on certain cultivars. The symbiotic properties of the transconjugants indicate that both soybean specificity for nodulation and cultivar specificity for nitrogen fixation are plasmid encoded.

CCR7 ligands, SLC/6Ckine/Exodus2/TCA4 and CKbeta-11/MIP-3beta/ELC, are chemoattractants for CD56(+)CD16(-) NK cells and late stage lymphoid progenitors.

Two human CC chemokines, SLC/6Ckine/Exodus2/TCA4 and CKbeta-11/MIP-3beta/ELC, are previously reported as efficacious chemoattractants for T- and B-cells and dendritic cells. SLC and CKbeta-11 share only 32% amino acid identity, but are ligands for the same chemokine receptor, CCR7. In this study, we examined chemotactic activity of SLC and CKbeta-11 for NK cells and lymphoid progenitors in bone marrow and thymus. It was found that these two CCR7 ligands are chemoattractants for neonatal cord blood and adult peripheral blood NK cells and cell lines. SLC and CKbeta-11 preferentially attract the CD56(+)CD16(-) NK cell subset over CD56(+)CD16(+) NK cells. SLC and CKbeta-11 also demonstrate selective chemotactic activity on late stage CD34(-)CD19(+)IgM- B-cell progenitors and CD4(+) and CD8(+) single-positive thymocytes, but not early stage progenitors. It was noted that SLC is an efficient desensitizer of CKbeta-11-dependent NK cell chemotaxis, while CKbeta-11 is a weak desensitizer of SLC-dependent chemotaxis. Taken together, these results suggest that SLC and CKbeta-11 have the potential to control trafficking of NK cell subsets and late stage lymphoid progenitors in bone marrow and thymus.

Strain-Specific Inhibition of nod Gene Induction in Bradyrhizobium japonicum by Flavonoid Compounds.

A broad-host-range plasmid, pEA2-21, containing a Bradyrhizobium japonicum nodABC'-'lacZ translational fusion was used to identify strain-specific inhibitors of the genes required for soybean nodulation, the common nod genes. The responses of type strains of B. japonicum serogroups USDA 110, USDA 123, USDA 127, USDA 129, USDA 122, and USDA 138 to nod gene inhibitors were compared. Few compounds inhibited nod gene expression in B. japonicum USDA 110. In contrast, nod gene expression in strains belonging to several other serogroups was inhibited by most of the flavonoids tested. However, the application of two of these strain-specific compounds, chrysin and naringenin, had little effect on the pattern of competition between indigenous and inoculum strains of B. japonicum in greenhouse and field trials. Preliminary studies with radiolabeled chrysin and naringenin suggest that the different responses to nod gene inhibitors may be partly due to the degree to which plant flavonoids can be metabolized by each strain.

Expression and purification of stable 33-kDa soluble human CD23 using the Drosophila S2 expression system.

CD23, a 45-kDa type II membrane glycoprotein present on B cells, monocytes, and other human immune cells, is a low-affinity receptor for IgE. The extracellular region of the membrane-bound human CD23 is processed into at least four soluble (s) CD23 forms, with apparent molecular masses of 37, 33, 29, and 25 kDa. High levels of sCD23 are found in patients with allergy, certain autoimmune diseases, or chronic lymphocytic leukemia. Therefore, inhibition of the processing of membrane-bound CD23 to control the cytokine-like effects of sCD23 offers a novel therapeutic opportunity. While the 37-, 29-, and 25-kDa forms of sCD23 have been expressed previously as recombinant proteins, the 33-kDa form has not been purified and characterized. To further investigate the multiple roles of sCD23 fragments and to devise assays to identify potent small-molecule inhibitors of CD23 processing, we have produced the 33-kDa form of sCD23 using Chinese hamster ovary (CHO) and Drosophila S2 cells. The CHO-expressed 33-kDa protein was found to undergo proteolytic degradation during cell growth and during storage of purified protein, resulting in accumulation of a 25-kDa form. The Drosophila system expressed the 33-kDa sCD23 in a stable form that was purified and demonstrated to be more active than the CHO-derived 25-kDa form in a monocyte TNFalpha release assay.

A sustained, non-insulin related, hypoglycaemic effect of electroacupuncture in diabetic Psammomys obesus.

AIM/HYPOTHESIS: Electroacupuncture has been shown to induce a short-term hypoglycaemic effect in streptozotocin diabetic rats. We designed an experiment to investigate the effect of electroacupuncture in Psammomys obesus, a model of insulin resistance and non-insulin-dependent diabetes mellitus. METHODS: We divided 29 diabetic Psammomys randomly into three groups: abdominal electroacupuncture (real, n = 11), back electroacupuncture (placebo, n = 9) and control (anaesthesia, n = 9). Electroacupuncture was carried out on days 1, 3 and 5 of the experiment. During the first week of the experiment, blood glucose was tested three times on treatment days and once on the following days. Over the next 2 weeks, blood glucose was tested every other day. Animals were weighed at the same time of blood sampling. After 3 weeks, at the end of the experiment, blood was drawn for measurement of insulin, fructosamine, cholesterol and triglycerides. RESULTS: At day 5 (end of intervention), blood glucose (as per cent of primary concentrations, means +/- SE) was 57 +/- 10, 93 +/- 13 and 89 +/- 11 for the real, placebo and control groups respectively (p = 0.02). At day 8, blood glucose 68 +/- 14, 86 +/- 16 and 97 +/- 9 for the real, placebo and control groups respectively (p = 0.04). At day 22, blood glucose was 79 +/- 11, 85 +/- 15 and 131 +/- 2 for the real, placebo and control groups (p = 0.04). Comparison of the decline in blood glucose, throughout the 3 weeks, between the real and placebo groups by ANOVA was highly significant (p < 0.0001), the difference between the placebo and control groups at the same time was not significant (p > 0.05). Animal weight gain, serum insulin, fructosamine, cholesterol and triglycerides were not significantly different between real and placebo groups. CONCLUSION/INTERPRETATION: Electroacupuncture at special abdominal acupoints induces a sustained hypoglycaemic effect in diabetic Psammomys compared with electroacupuncture at non-specific points, without weight loss. No hypoinsulinaemic effect was shown in the real and placebo groups.

Screening for high-affinity ligands to the Src SH2 domain using capillary isoelectric focusing-electrospray ionization ion trap mass spectrometry.

A solution-based microscale approach for determination of high-affinity noncovalent complexes from mixtures of compounds is presented, based on capillary isoelectric focusing coupled on-line with electrospray ionization ion trap mass spectrometry. The studies are performed using the src homology 2 domain and tyrosine-phosphorylated peptide ligands as a model system. Tight complexes are formed in solution, preconcentrated up to 2 orders of magnitude and separated on the basis of their isoelectric points. The complexes are then dissociated in the mass spectrometer and the freed ligands identified. Picomole or less amounts of protein reagent are consumed per experiment. Structural information for the ligands involved in tight complex formation may be obtained using the MSn capabilities of the ion trap. The methodology can potentially be used to screen rapidly combinatorial mixtures of compounds for high-affinity ligands.

Binding interactions of human interleukin 5 with its receptor alpha subunit. Large scale production, structural, and functional studies of Drosophila-expressed recombinant proteins.

Human interleukin 5 (hIL5) and soluble forms of its receptor alpha subunit were expressed in Drosophila cells and purified to homogeneity, allowing a detailed structural and functional analysis. B cell proliferation confirmed that the hIL5 was biologically active. Deglycosylated hIL5 remained active, while similarly deglycosylated receptor alpha subunit lost activity. The crystal structure of the deglycosylated hIL5 was determined to 2.6-A resolution and found to be similar to that of the protein produced in Escherichia coli. Human IL5 was shown by analytical ultracentrifugation to form a 1:1 complex with the soluble domain of the hIL5 receptor alpha subunit (shIL5R alpha). Additionally, the relative abundance of ligand and receptor in the hIL5.shIL5R alpha complex was determined to be 1:1 by both titration calorimetry and SDS-polyacrylamide gel electrophoresis analysis of dissolved cocrystals of the complex. Titration microcalorimetry yielded equilibrium dissociation constants of 3.1 and 2.0 nM, respectively, for the binding of hIL5 to shIL5R alpha and to a chimeric form of the receptor containing shIL5R alpha fused to the immunoglobulin Fc domain (shIL5R alpha-Fc). Analysis of the binding thermodynamics of IL5 and its soluble receptor indicates that conformational changes are coupled to the binding reaction. Kinetic analysis using surface plasmon resonance yielded data consistent with the Kd values from calorimetry and also with the possibility of conformational isomerization in the interaction of hIL5 with the receptor alpha subunit. Using a radioligand binding assay, the affinity of hIL5 with full-length hIL5R alpha in Drosophila membranes was found to be 6 nM, in accord with the affinities measured for the soluble receptor forms. Hence, most of the binding energy of the alpha receptor is supplied by the soluble domain. Taken with other aspects of hIL5 structure and biological activity, the data obtained allow a prediction for how 1:1 stoichiometry and conformational change can lead to the formation of hIL5.receptor alpha beta complex and signal transduction.