Trypanosoma brucei undergoes genetic exchange in its insect vector by an unknown mechanism. To visualize the production of hybrids in the fly, a tetracycline (Tet)-inducible expression system was adapted. One parental trypanosome clone was transfected with the gene encoding Green Fluorescent Protein (GFP) under control of the Tet repressor in trans; transfection with these constructs also introduced genes for resistance to hygromycin and phleomycin, respectively. An experimental cross with a second parental clone carrying a gene for geneticin resistance produced fluorescent hybrids with both hygromycin and geneticin resistance. These results are consistent with the meiotic segregation and reassortment of the GFP and repressor genes. Fluorescent hybrids were visible in the salivary glands of the fly, but not the midgut, confirming that genetic exchange occurs among the trypanosome life cycle stages present in (or possibly en route to) the salivary glands. In conclusion, the experimental design has successfully produced fluorescent hybrids which can be observed directly in the salivary glands of the fly, and it has been shown that the recombinant genotypes were most probably the result of meiosis.

Manduca sexta is a nicotine-insensitive insect, the larval form of which feeds on tobacco. It has been postulated that its nicotine insensitivity may reflect the presence of a modified nicotinic acetylcholine receptor whose alpha subunits lack the amino acid residues necessary for binding nicotine: we have performed ligand binding assays and molecular cloning to examine this hypothesis. [125I]alpha-bungarotoxin bound specifically to both larval and adult membranes, with Kd values of 7.6 and 6.5 nM and Bmax values of 119 and 815 fmol/mg protein, respectively. The pharmacological profile of [1251]alpha-bungarotoxin binding was similar in both tissues. In particular, nicotine (Ki values: 1.6 microM and 2 microM for larvae and adults, respectively) competed with an affinity similar to that found for nicotine-sensitive insects. No alpha-bungarotoxin-insensitive binding sites labelled by [3H]epibatidine could be detected. Using the alpha-like subunit from the locust Schistocerca gregaria to probe two cDNA libraries, and by inverse PCR on circularized genomic DNA from Manduca sexta, we have obtained overlapping cDNA clones that contain the complete coding sequence of a putative nicotinic subunit from Manduca sexta (MARA1). No other alpha-subunit cDNAs were isolated using this probe, although it hybridized to multiple bands on Southern blots. The sequence of MARA1 is consistent with an alpha-like subunit capable of binding alpha-bungarotoxin, and it retains all those amino acids implicated in nicotine binding to vertebrate nicotinic receptors. Taken together, these findings provide no support for the hypothesis that the nicotine insensitivity of Manduca sexta is the result of a nicotinic receptor with diminished nicotine binding.

Fetal and neonatal alloimmune thromboctyopenia due to maternal human platelet antigen (HPA)-1a antibodies affects primigravidas. Immunization must occur early in pregnancy before fetal platelets enter maternal blood via fetomaternal hemorrhage. The HPA-1a antigen is located on platelet glycoprotein (GP)IIIa (CD61, beta3 integrin), which is also present on the placental syncytiotrophoblast (ST) and in direct contact with maternal blood. Since ST debris is shed into maternal blood during pregnancy, this material might be immunogenic in vivo. For experimental purposes, we prepared and characterized ST microparticles (STMPs) in vitro from term placentas. Phenotype analysis by flow cytometry and Western blotting showed that STMP expressed more placental alkaline phosphatase (PLAP) than GPIIIa. Quantitative real-time PCR demonstrated expression of human placental lactogen (HPL), human chorionic gonadotrophin (HCG), and GPIIIa by STMP, in the order HPL > HCG > GPIIIa. PLAP, HPL, and HCG are trophoblast-specific proteins. These STMPs may be a useful model for studying the natural ST debris in plasma of pregnant women.

SummaryFetal and neonatal alloimmune thrombocytopenia (FNAIT) occurs most frequently when human platelet antigen (HPA)-1a-positive fetal platelets are destroyed by maternal HPA-1a immunoglobulin (Ig)G antibodies. Pregnancies at risk are treated by administration of high-dose intravenous Ig (IVIG) to women, but this is expensive and often not well tolerated. Peptide immunotherapy may be effective for ameliorating some allergic and autoimmune diseases. The HPA-1a/1b polymorphism is Leu/Pro33 on β3 integrin (CD61), and the anti-HPA-1a response is restricted to HPA-1b1b and HLA-DRB3*0101-positive pregnant women with an HPA-1a-positive fetus. We investigated whether or not HPA-1a antigen-specific peptides that formed the T cell epitope could reduce IgG anti-HPA-1a responses, using a mouse model we had developed previously. Peripheral blood mononuclear cells (PBMC) in blood donations from HPA-1a-immunized women were injected intraperitoneally (i.p.) into severe combined immunodeficient (SCID) mice with peptides and HPA-1a-positive platelets. Human anti-HPA-1a in murine plasma was quantitated at intervals up to 15 weeks. HPA-1a-specific T cells in PBMC were identified by proliferation assays. Using PBMC of three donors who had little T cell reactivity to HPA-1a peptides in vitro, stimulation of anti-HPA-1a responses by these peptides occurred in vivo. However, with a second donation from one of these women which, uniquely, had high HPA1a-specific T cell proliferation in vitro, marked suppression of the anti-HPA-1a response by HPA-1a peptides occurred in vivo. HPA-1a peptide immunotherapy in this model depended upon reactivation of HPA-1a T cell responses in the donor. For FNAIT, we suggest that administration of antigen-specific peptides to pregnant women might cause either enhancement or reduction of pathogenic antibodies.

The observation that different rPrP production protocols influence QuIC performance indicates that caution should be exercised when comparing inter-laboratory QuIC results.

This volume of the "Methods in Molecular Biology" series contains a collection of methodologies that will enable the researcher to elucidate the G-protein sub-units involved with particular receptors and evaluate their effects. It is divided into three parts covering: characterization of receptors, mechanisms of receptor coupling, and regulation and function of G-protein receptor/sub-units.Techniques covered include: binding assays; mutagenesis studies; antibody production and subsequent use in immunolocalization of receptors; heterologous expression of G-protein receptors; in situ hybridization; antisense work to determine sub-unit function; Northern blots; RNAse protection assays; phosphorylation studies; GPR kinase assays using rhodopsin; and receptor internalization studies.Particularly noteworthy is the chapter on radioligand binding assays, which is easy to comprehend and has a good discussion of experimental design and data analysis. Similarly, there is an extensive chapter on the use of 35-S GTPγS for the autoradiographic localization of G-proteins/receptors with extensive notes on applications, limitations and expected results.There are good overviews of mutagenesis studies including both stable and transient expression techniques. However, with limited discussion on both the choices of expression vectors and the methods available for performing transient expression.The standard techniques for generating receptor selective antibodies, using peptides and fusion proteins, are clearly presented. However, modern adjuvants and software packages for analyzing potential antigens are omitted. Similarly, the immunolocalization chapter provides a very good overview with comprehensive notes on suitable controls and interpretation of results. A novel technique using paramagnetic beads to deliver antisense RNA or DNA into cells is also well detailed.This book would suit those already familiar with basic molecular biological and biochemical techniques.

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