A new Bihari inequality and initial value problems of first order fractional differential equations.

We prove existence of solutions, and particularly positive solutions, of initial value problems (IVPs) for nonlinear fractional differential equations involving the Caputo differential operator of order α∈(0,1). One novelty in this paper is that it is not assumed that f is continuous but that it satisfies an Lp-Carathéodory condition for some p>1α (detailed definitions are given in the paper). We prove existence on an interval [0, T] in cases where T can be arbitrarily large, called global solutions. The necessary a priori bounds are found using a new version of the Bihari inequality that we prove here. We show that global solutions exist when f(t, u) grows at most linearly in u, and also in some cases when the growth is faster than linear. We give examples of the new results for some fractional differential equations with nonlinearities related to some that occur in combustion theory. We also discuss in detail the often used alternative definition of Caputo fractional derivative and we show that it has severe disadvantages which restricts its use. In particular we prove that there is a necessary condition in order that solutions of the IVP can exist with this definition, which has often been overlooked in the literature.

Non-local second-order boundary value problems with derivative-dependent nonlinearity.

We prove the existence of multiple positive solutions of nonlinear second-order nonlocal boundary value problems with nonlinear term having derivative dependence. We allow the nonlinearity to grow quadratically with respect to derivatives. We obtain a priori bounds for norms of derivatives by using a recently obtained Gronwall-type inequality. Three examples illustrate some of the results. This article is part of the theme issue 'Topological degree and fixed point theories in differential and difference equations'.

Streamwater acid-base chemistry and critical loads of atmospheric sulfur deposition in Shenandoah National Park, Virginia.

A modeling study was conducted to evaluate the acid-base chemistry of streams within Shenandoah National Park, Virginia and to project future responses to sulfur (S) and nitrogen (N) atmospheric emissions controls. Many of the major stream systems in the park have acid neutralizing capacity (ANC) less than 20 microeq/L, levels at which chronic and/or episodic adverse impacts on native brook trout are possible. Model hindcasts suggested that none of these streams had ANC less than 50 microeq/L in 1900. Model projections, based on atmospheric emissions controls representative of laws already enacted as of 2003, suggested that the ANC of those streams simulated to have experienced the largest historical decreases in ANC will increase in the future. The levels of S deposition that were simulated to cause streamwater ANC to increase or decrease to three specified critical levels (0, 20, and 50 microeq/L) ranged from less than zero (ANC level not attainable) to several hundred kg/ha/year, depending on the selected site and its inherent acid-sensitivity, selected ANC endpoint criterion, and evaluation year for which the critical load was calculated. Several of the modeled streams situated on siliciclastic geology exhibited critical loads <0 kg/ha/year to achieve ANC >50 microeq/L in the year 2040, probably due at least in part to base cation losses from watershed soil. The median modeled siliciclastic stream had a calculated critical load to achieve ANC >50 microeq/L in 2100 that was about 3 kg/ha/year, or 77% lower than deposition in 1990, representing the time of model calibration.

Feasibility of human trials to assess developmental immunotoxicity, and some comparison with data on New World monkeys.

Procedures to reveal 'immunotoxic' potentials of chemicals in animal experiments (mostly in rodents) have been recommended, but the selection of test systems is rather arbitrary. The predictive power of extrapolations to the possible situation in humans is unknown because human studies to confirm or to reject clues from animal data are largely lacking. End points selected in animal studies and those expected to be relevant in humans are not identical. Results of animal experiments are based on doses, generally ignoring the important species differences in pharmacokinetics. This unfavorable situation is especially pronounced when attempting to evaluate 'environmental chemicals'. Because much more information is available on many medicinal drugs, exposures can be defined and pharmacokinetic data are available or obtainable. The situation is even more complicated when attempting to assess possible adverse effects on the developing immune system: in addition to the problems mentioned, numerous different developmental periods with varying susceptibilities must be considered, and species differences in the immune response are superimposed with large differences in pre-, early post-, and later postnatal development. Simultaneously, the kinetic variables are continuously changing with time (with additional variability among species). Different results, even between rats and mice, are bound to occur. Extrapolation to the situation possibly relevant for human exposure will be almost impossible, especially from rodent data. The majority of such effects induced peri- or early postnatally may be expected to be reversible. It must also be assessed whether qualitatively different adverse effects are likely to be induced during 'development', which cannot be revealed (accepting quantitative differences) by more easily performed tests on the adult organism. Considering the intrinsic difficulties, the most promising approach would be to directly obtain data from human trials. This is feasible for medicinal drugs. Alternatively, data on nonhuman primates, the species phylogenetically closest to man, may provide useful information. The status quo for such a strategy and the possible pitfalls are discussed in this overview.

Genetic control of innate immune responses against cytomegalovirus: MCMV meets its match.

Cytomegalovirus (CMV) is a widespread pathogen that is responsible for severe disease in immunocompromised individuals and probably, associated with vascular disease in the general population. There is increasing evidence that cells of the innate immune system play a key role in controlling this important pathogen. This is particularly evident in the experimental murine CMV (MCMV) model of infection which has revealed an important role for natural killer (NK) cells in controlling early viral replication after infection with MCMV. In this model, different strains of inbred mice exhibit striking differences in their level of susceptibility to MCMV infection. Genetic studies, performed almost 10 years ago, revealed that this pattern of susceptibility/resistance can be attributed to a single genetic locus termed Cmv1 and recently several groups that have been working on the mapping and identification of Cmv1 have met with success. Interestingly, Cmv1 is allelic to a member of the Ly49 gene family, which encode activating or inhibitory transmembrane receptors present on the surface of NK cells. All Ly49 receptors characterized to date interact with MHC class I molecules on potential target cells, resulting in the accumulation of signals to the NK to either 'kill' or 'ignore' the cell based upon the repertoire of MHC class I molecules expressed. The identification of Cmv1 as Ly49H, a stimulatory member of the Ly49 family, adds an interesting twist to the Ly49 story. Although the ligand of Ly49H is not yet known, there is already compelling evidence that the ligand is upregulated on virally infected cells, resulting in specific activation of Ly49H-expressing NK cells. This review provides an historical perspective of the MCMV infection model from its inception to the discovery of the gene responsible for the phenotype and provides a basis for further experiments aimed at understanding the role of NK cells, in general, and Ly49H, in particular, in mediating resistance to cytomegalovirus.

Vaccination with plasmid DNA encoding TSA/LmSTI1 leishmanial fusion proteins confers protection against Leishmania major infection in susceptible BALB/c mice.

We have recently shown that a cocktail containing two leishmanial recombinant antigens (LmSTI1 and TSA) and interleukin-12 (IL-12) as an adjuvant induces solid protection in both a murine and a nonhuman primate model of cutaneous leishmaniasis. However, because IL-12 is difficult to prepare, is expensive, and does not have the stability required for a vaccine product, we have investigated the possibility of using DNA as an alternative means of inducing protective immunity. Here, we present evidence that the antigens TSA and LmSTI1 delivered in a plasmid DNA format either as single genes or in a tandem digene construct induce equally solid protection against Leishmania major infection in susceptible BALB/c mice. Immunization of mice with either TSA DNA or LmSTI1 DNA induced specific CD4(+)-T-cell responses of the Th1 phenotype without a requirement for specific adjuvant. CD8 responses, as measured by cytotoxic-T-lymphocyte activity, were generated after immunization with TSA DNA but not LmSTI1 DNA. Interestingly, vaccination of mice with TSA DNA consistently induced protection to a much greater extent than LmSTI1 DNA, thus supporting the notion that CD8 responses might be an important accessory arm of the immune response for acquired resistance against leishmaniasis. Moreover, the protection induced by DNA immunization was specific for infection with Leishmania, i.e., the immunization had no effect on the course of infection of the mice challenged with an unrelated intracellular pathogen such as Mycobacterium tuberculosis. Conversely, immunization of BALB/c mice with a plasmid DNA that is protective against challenge with M. tuberculosis had no effect on the course of infection of these mice with L. major. Together, these results indicate that the protection observed with the leishmanial DNA is mediated by acquired specific immune response rather than by the activation of nonspecific innate immune mechanisms. In addition, a plasmid DNA containing a fusion construct of the two genes was also tested. Similarly to the plasmids encoding individual proteins, the fusion construct induced both specific immune responses to the individual antigens and protection against challenge with L. major. These results confirm previous observations about the possibility of DNA immunization against leishmaniasis and lend support to the idea of using a single polygenic plasmid DNA construct to achieve polyspecific immune responses to several distinct parasite antigens.

Cytokine expression by murine DX5+ cells in response to IL-12, IL-18, or the combination of IL-12 and IL-18.

In this study we analyzed the response of DX5+ NK and NK T cells to in vitro stimulation with IL-12 or IL-18. Production of IFN-gamma in response to either IL-12 or IL-18 was dependent upon costimulation with either IL-2 or IL-15. DX5+ splenocytes showed a rapid (6 h) and sustained (6-72 h) accumulation of IFN-gamma transcripts followed by a delayed (12-24 h) up-regulation of IL-10 or IL-13 expression in response to IL-2 + IL-12 or IL-2 + IL-18, respectively. Incubation of DX5+ splenocytes with the combination of IL-2 + IL-12 + IL-18 resulted in up-regulation of IFN-gamma and IL-13 transcripts but down-regulation of IL-10 expression. Furthermore, two distinct populations of cells producing differing amounts of IFN-gamma were observed by intracellular staining after stimulation with IL-2 + IL-12 + IL-18. Last, we demonstrate that DX5+ cells respond to IL-18 independently of IL-12, as cells from both wild-type and IL-12Rbeta2KO mice produce IFN-gamma and IL-13 in response to IL-2 + IL-18.

Protection against cutaneous leishmaniasis induced by recombinant antigens in murine and nonhuman primate models of the human disease.

Leishmaniasis affects approximately 2 million people each year throughout the world. This high incidence is due in part to the lack of an efficacious vaccine. We present evidence that the recombinant leishmanial antigens LmSTI1 and TSA, which we identified and characterized previously, induce excellent protection in both murine and nonhuman primate (rhesus monkey) models of human cutaneous leishmaniasis. The remarkable protection induced by LmSTI1 and TSA in an animal model that is evolutionarily close to humans qualifies this antigen combination as a promising candidate subunit vaccine against human leishmaniasis.

Identification and characterization of T cell-stimulating antigens from Leishmania by CD4 T cell expression cloning.

Persistent immunity against Leishmania: infections in humans is mediated predominantly by CD4(+) T cells of the Th1 phenotype. Herein we report the expression cloning of eight Leishmania: Ags using parasite-specific T cell lines derived from an immune donor. The Ags identified by this technique include the flagellar proteins alpha- and beta-tubulin, histone H2b, ribosomal protein S4, malate dehydrogenase, and elongation factor 2, as well as two novel parasite proteins. None of these proteins have been previously reported as T cell-stimulating Ags from Leishmania: beta-tubulin-specific T cell clones generated against Leishmania: major amastigotes responded to Leishmania:-infected macrophages and dendritic cells. IFN-gamma enzyme-linked immunospot analysis demonstrated the presence of T cells specific for several of these Ags in PBMC from self-healing cutaneous leishmaniasis patients infected with either Leishmania: tropica or L. major. The responses elicited by Leishmania: histone H2b were particularly striking in terms of frequency of histone-specific T cells in PBMC (1 T cell of 6000 PBMC) as well as the percentage of responding donors (86%, 6 of 7). Ags identified by T cells from immune donors might constitute potential vaccine candidates for leishmaniasis.

Computation and visualization of regional-scale forest disturbance and associated dissolved nitrogen export from Shenandoah National Park, Virginia.

Long-term watershed research conducted in Shenandoah National Park (SNP) in Virginia and elsewhere in the eastern U.S. indicates that annual export of dissolved nitrogen (N) from gaged forested watersheds to surface waters increases dramatically in response to vegetation disturbances. Dissolved N leakage is a common, well-documented response of small forested watersheds to logging in the larger region, while recent defoliation outbreaks of the gypsy moth ( Lymantria dispar) larva in the deciduous forests of SNP have been shown to generate similar biogeochemical responses. A recent modeling analysis further suggests that a parsimonious, empirical, unit N export response function (UNERF) model can explain large percentages of the temporal variation in annual N export from a group of small gaged forested watersheds in the years following disturbance. The empirical UNERF modeling approach is completely analogous to the unit hydrograph technique for describing storm runoff, with the model representing annual N export as a linear deterministic process both in space and in time. The purposes of this analysis are to (1) test the applicability of the UNERF model using quarterly streamwater nitrate data from a group of ungaged watersheds in SNP; (2) demonstrate a park-wide application of a regional UNERF model that references the geographic distributions of bedrock geology and the timing and extent of gypsy moth defoliation over the entire SNP area; and (3) visualize the temporal and spatial patterns in vegetation disturbance and annual dissolved N export through the use of computer animation software. During water year 1992, the year of peak defoliation, our modeling study suggests that park-wide export had transiently increased by 1700% from a baseline rate of about 0.1 kg/ha/year. SNP forests appear to be characteristic of other N-limited second-growth forests in the eastern U.S. that leak little N under undisturbed conditions, despite receiving relatively large inputs of N from atmospheric deposition sources. Vegetation disturbances can apparently cause major changes in N input-output balances with potentially important ramifications for low-order forest streams and downstream receiving waters.

Differentiation of murine NK cells into distinct subsets based on variable expression of the IL-12R beta 2 subunit.

The cytokine IL-12 manifests its biological activity via interaction with a heterodimeric receptor (IL-12R) present on activated T and NK cells. The cDNAs for two IL-12R subunits have been cloned from human and mouse and designated IL-12Rbeta1 and IL-12Rbeta2. The expression of IL-12Rbeta2 on T cells is influenced by cytokines, particularly IL-4, IL-12, and IFN-gamma; however, little is known regarding regulation of IL-12R expression on NK cells. In this study we show that murine NK cells differentiate into IL-12Rbeta2(low) and IL-12Rbeta2(high) subsets after in vitro stimulation with IL-2 in the absence of exogenous polarizing cytokines. Subset development occurs gradually as NK cells expand in vitro and is generally complete by 8-12 days of culture. Once established, IL-12Rbeta2(low) and IL-12Rbeta2(high) subsets are highly stable in vitro and can be maintained for at least 20 days after FACS sorting. Formation of these NK subsets appears to be strain independent. Flow cytometric analyses demonstrate that both subsets express a number of NK-associated markers, including NK1.1, DX-5, Ly-49A, and Ly-49C, but that the Ly-49G2 class I inhibitory receptor is expressed predominantly on the IL-12Rbeta2(high) population. Both IL-12Rbeta2(low) and IL-12Rbeta2(high) NK cells respond to exogenous IL-12 by rapid production of high levels of IFN-gamma and increased lytic activity against NK-sensitive YAC-1 target cells. Analyses of cytokine gene expression by RNase protection assay indicated that similar to the recently described human NK1 subset, both IL-12Rbeta2(high) and IL-12Rbeta2(low) murine NK subsets expressed high levels of IFN-gamma, whereas neither subset expressed mRNA for the NK2-associated cytokines IL-5 and IL-13.

Assessing lymphocyte functions in neonates for revealing abnormal prenatal development of the immune system.

Because it is difficult to assess prenatally induced functional deficits of the human immune system, we developed an ex vivo method for differentiation and maturation of peripheral lymphocytes of newborn, preferentially using umbilical cord blood. Many lymphocyte subsets of newborn infants are "immature" with respect to defined surface receptors. An example of such an immaturity is the almost complete lack of "memory"-type helper T cells (also designated as helper-inducer cells), characterized by expressing the surface receptors: CD4(+)CD45R0(+)CD45RA(-)CD29(high). On the other hand, umbilical cord blood contains many "naive"-type helper T cells (often designated as suppressor-inducer cells), with the receptors: CD4(+)CD45R0(-)CD45RA(+)CD29(low). In this report, we demonstrate that the immature helper lymphocyte population of umbilical cord blood is capable of differentiating to mature cells following stimulation with pokeweed mitogen (PWM) and other stimulants ex vivo. The obtained receptor pattern is virtually indistinguishable from the one observed on the mature cells of adults. Such an extensive differentiation can only be achieved with cells of newborns. As intermediates during differentiation in culture, CD45R0(+)CD45RA(+) cells may be observed which are rather rare in vivo. Additionally, the appearance of several activation (CD25, CD69, HLA-DR) and adhesion (CD11a, CD11b, CD11c, CD18, CD49b, CD49d, CD54) receptors on CD4 cells were analyzed. With this model system evidence for the sequence of events during differentiation and maturation may be obtained. This ex vivo-model is capable of studying the capacity of lymphocytes for differentiation and activation processes barely accessible in vivo. It may also be expected to represent an interesting tool for measuring the capacity for maturation and differentiation in the blood of children of different ages under normal and pathological conditions ex vivo. In addition, substance-induced effects may be studied in vitro with this approach on immature cells from newborn, or infants during culturing. Teratogenesis Carcinog. Mutagen. 20:171-193, 2000.

Clustered randomised trial of an intervention to improve the management of asthma: Greenwich asthma study.

OBJECTIVES: To evaluate the effectiveness of an asthma resource centre in improving treatment and quality of life for asthmatic patients. DESIGN: Community based randomised controlled trial. SETTING: 41 general practices in Greenwich with a practice nurse. SUBJECTS: All registered patients aged 15-50 years. INTERVENTION: Nurse specialists in asthma who educated and supported practice nurses, who in turn educated patients in the management of asthma according to the British Thoracic Society's guidelines. MAIN OUTCOME MEASURES: Quality of life of asthmatic patients, attendance at accident and emergency departments, admissions to local hospitals, and steroid prescribing by general practitioners. RESULTS: Of 24 400 patients randomly selected and surveyed in 1993, 12 238 replied; 1621 were asthmatic of whom 1291 were sent a repeat questionnaire in 1996 and 780 replied. Of 24 400 patients newly surveyed in 1996, 10 783 (1616 asthmatic) replied. No evidence was found for an improvement in asthma related quality of life among newly surveyed patients in intervention practices compared with control practices. Neither was there evidence of an improvement in other measures of the quality of asthma care. Weak evidence was found for an improvement in quality of life in intervention practices among asthmatics registered with study practices in 1993 and followed up in 1996. Neither attendances at accident and emergency departments nor admissions for asthma showed any tendency to diverge in intervention and control practices over the study period. Steroid prescribing rates rose steadily during the study period. The average annual increase in steroid prescribing was 3% per year higher in intervention than control practices (95% confidence interval -1% to 6%, P=0.10). CONCLUSIONS: This model of service delivery is not effective in improving the outcome of asthma in the community. Further development is required if cost effective management of asthma is to be introduced.

Molecular cloning, expression, and immunogenicity of MTB12, a novel low-molecular-weight antigen secreted by Mycobacterium tuberculosis.

Proteins secreted into the culture medium by Mycobacterium tuberculosis are thought to play an important role in the development of protective immune responses. In this report, we describe the molecular cloning of a novel, low-molecular-weight antigen (MTB12) secreted by M. tuberculosis. Sequence analysis of the MTB12 gene indicates that the protein is initially synthesized as a 16.6-kDa precursor protein containing a 48-amino-acid hydrophobic leader sequence. The mature, fully processed form of MTB12 protein found in culture filtrates has a molecular mass of 12. 5 kDa. MTB12 protein constitutes a major component of the M. tuberculosis culture supernatant and appears to be at least as abundant as several other well-characterized culture filtrate proteins, including members of the 85B complex. MTB12 is encoded by a single-copy gene which is present in both virulent and avirulent strains of the M. tuberculosis complex, the BCG strain of M. bovis, and M. leprae. Recombinant MTB12 containing an N-terminal six-histidine tag was expressed in Escherichia coli and purified by affinity chromatography. Recombinant MTB12 protein elicited in vitro proliferative responses from the peripheral blood mononuclear cells of a number of purified protein derivative-positive (PPD+) human donors but not from PPD- donors.

Human and murine immune responses to a novel Leishmania major recombinant protein encoded by members of a multicopy gene family.

Vaccination of BALB/c mice with Leishmania major promastigote culture filtrate proteins plus Corynebacterium parvum confers resistance to infection with L. major. To define immunogenic components of this protein mixture, we used sera from vaccinated mice to screen an L. major amastigote cDNA expression library. One of the immunoreactive clones thus obtained encoded a novel protein of L. major with a molecular mass of 22.1 kDa. The predicted amino acid sequence of this clone exhibited significant homology to eukaryotic thiol-specific-antioxidant (TSA) proteins. Therefore, we have designated this protein L. major TSA protein. Southern blot hybridization analyses indicate that there are multiple copies of the TSA gene in all species of Leishmania analyzed. Northern blot analyses demonstrated that the TSA gene is constitutively expressed in L. major promastigotes and amastigotes. Recombinant TSA protein containing an amino-terminal six-histidine tag was expressed in Escherichia coli with the pET17b system and was purified to homogeneity by affinity chromatography. Immunization of BALB/c mice with recombinant TSA protein resulted in the development of strong cellular immune responses and conferred protective immune responses against infection with L. major when the protein was combined with interleukin 12. In addition, recombinant TSA protein elicited in vitro proliferative responses from peripheral blood mononuclear cells of human leishmaniasis patients and significant TSA protein-specific antibody titers were detected in sera of both cutaneous-leishmaniasis and visceral-leishmaniasis patients. Together, these data suggest that the TSA protein may be useful as a component of a subunit vaccine against leishmaniasis.

Molecular characterization of the heat-inducible LmSTI1 protein of Leishmania major.

We have recently isolated a cDNA encoding the Leishmania major homologue of the yeast stress-inducible protein STI1. Southern blot analyses indicate that this protein is encoded by a single copy gene in L. major and that this gene is highly conserved throughout the Leishmania genus. The STI1 gene is constitutively expressed in both L. major promastigotes and amastigotes however, STI1 transcript levels can be upregulated in promastigotes by a shift in culture temperature from 26 to 37 degrees C. Upregulation of transcript was detectable within 5' of heat shock and continued to increase for a further 8 h before returning to constitutive levels. In addition, biosynthetic incorporation of [35S]methionine followed by immunoprecipitation revealed an increase in the level of nascent STI1 protein synthesized when promastigote cultures were shifted from 26 to 37 degrees C. The L. major STI1 protein and the heat shock proteins Hsp83 and Hsp70 form a salt-sensitive complex in L. major promastigotes as evidenced by co-immunoprecipitation using an antiserum specific for L. major STI1. Furthermore, this complex can be reconstituted in vitro by adding recombinant STI1 containing an amino-terminal histidine tag to promastigote lysate and subsequent purification using metal chelate affinity chromatography.

Molecular cloning of a novel protein antigen of Leishmania major that elicits a potent immune response in experimental murine leishmaniasis.

BALB/c mice are highly susceptible to infection with the protozoan parasite Leishmania major. This susceptibility has been attributed, in part, to the expansion of parasite-specific CD4+ Th2 cells that antagonize Th1 responses and promote humoral immunity. In the present study, we have utilized sera from L. major-infected BALB/c mice to screen an L. major amastigote cDNA expression library. One of the clones detected encodes a novel Ag designated as L. major stress-inducible 1 (LmSTI1). LmSTI1 contains six copies of the tetratricopeptide consensus motif and is highly related to a family of stress-inducible proteins that is conserved from yeast to humans. Sera from L. major-infected BALB/c mice have LmSTI1-specific Ab titers in excess of 1:200,000, comprised predominantly of IgG1, IgG2A, and IgG2B isotypes. Recombinant LmSTI1 protein elicited strong proliferative responses from draining lymph node cells of L. major-infected BALB/c mice at both early (10 days) and late (28 days) stages of infection and elicited production of high levels of IFN-gamma and low levels of IL-4. In contrast, soluble leishmanial lysate elicited high levels of IL-4 and low IFN-gamma production. Thus, we have identified an Ag of Leishmania capable of eliciting a mixed cellular response that is skewed toward a Th1 phenotype in susceptible BALB/c mice with advanced infections. In addition, analyses of sera from human patients with cutaneous, visceral, and post-kala azar visceral leishmaniasis indicated that a majority of individuals from all three clinical groups mounted strong humoral responses against LmSTI1.

The gene encoding streptothricin acetyltransferase (sat) as a selectable marker for Leishmania expression vectors.

The pLEX series of vectors was developed for the stable expression of exogenous genes in the protozoan parasite Leishmania. These pUC-based constructs contain one of three independent selectable markers and a multiple cloning site inserted between the upstream and downstream untranslated regions of the previously cloned Leishmania major HEXBP gene. Selection was based on resistance to the aminoglycosides, hygromycin B and neomycin, and to nourseothricin, a novel independent selectable marker for transfection of Leishmania. The vectors were introduced into Leishmania promastigotes by electroporation and were maintained as extrachromosomal circular concatemers containing between four and eight repeat units of the pLEX monomer. To demonstrate the efficient expression of cloned exogenous genes using the pLEX system, promastigotes were transfected with a pLEX construct that contained a second drug-resistant selectable marker gene cloned into the expression site, and clones were obtained that grew on media containing two antibiotics. These vectors, together with the novel selectable marker, will further facilitate the molecular analysis of gene expression in Leishmania.

Leishmania major HEXBP deletion mutants generated by double targeted gene replacement.

The Leishmania major single-stranded DNA binding protein HEXBP contains nine 'CCHC' zinc finger motifs and binds to oligodeoxynucleotides derived from the antisense strand of the GP63 gene 5' flanking region in gel mobility shift assays and UV-crosslinking assays. In the present study a HEXBP-deficient clone of L. major was generated by double targeted gene replacement. The two HEXBP alleles were sequentially replaced with genes encoding resistance to the aminoglycoside antibiotics hygromycin B and G418 and drug-resistant clones were selected by plating on semi-solid drug-containing media. Successful deletion of both copies of the HEXBP gene implies that HEXBP is a not essential for growth of Leishmania promastigotes. Characterization HEXBP-deficient promastigotes revealed that HEXBP deficiency had no effect on the abundance of GP63 mRNA and protein in in vitro cultivated promastigotes and that HEXBP-deficient promastigotes were capable of lesion formation in BALB/c mice.

Molecular cloning and expression of a Leishmania major gene encoding a single-stranded DNA-binding protein containing nine "CCHC" zinc finger motifs.

The genes encoding GP63, the major surface glycoprotein of the protozoan parasite Leishmania, are highly conserved across diverse species of Leishmania both within the protein coding region and in the immediate 5'-untranslated region. Located between the 3' trans-spliced leader acceptor site and the translational initiation codon of the GP63 gene is an area of conserved hexanucleotide direct repeats (CTCGCC) which vary in number according to species. To determine whether these repeats represent a site of protein DNA interaction, a Leishmania major lambda gt11 expression library was screened with a radiolabeled synthetic oligodeoxynucleotide probe containing multiple CTCGCC repeats to detect clones expressing functional DNA-binding proteins. Using this approach a gene was isolated which encodes a sequence-specific DNA-binding protein referred to as HEXBP (hexamer-binding protein). Sequence analysis of the HEXBP gene showed that HEXBP contains nine cysteine-rich motifs which are identical to a consensus sequence known as the "CCHC type" zinc finger. This motif is present in a number of nucleic acid-binding proteins including the nucleocapsid protein of retroviruses. In accordance with the activity exhibited by other proteins containing the "CCHC" motif, HEXBP binds to single-stranded nucleic acids as demonstrated by gel mobility shift assays and Southwestern blot assays.