Oral immunization with a live recombinant attenuated Salmonella typhimurium protects mice against Toxoplasma gondii.

The natural site of infection for T. gondii is the mucosal surface of the intestine, so the protective immunity obtained after natural infection with T. gondii points to the importance of developing a vaccine that stimulates mucosal defences. In this study, an aroA- and aroD- attenuated strain of Salmonella typhimurium (BRD509) has been used to deliver the recombinant eukaryotic plasmid pSAG(1-2)/CTA2/B expressing a multi-antigenic gene encoding SAG1 and SAG2 of T. gondii linked to A2/B subunits of cholera toxin as a candidate oral T. gondii vaccine. Immunoblot analysis showed compound gene expression in HeLa cells in vitro and intragastric immunization of mice with the recombinant salmonella resulted in the induction of humoral and Th1 type cellular immune responses and afforded protection against RH strain T. gondii challenge. Anti-T. gondii IgG values increased markedly in the BRD509/pSAG(1-2)-CTA2/B immunized group; these values were significantly higher than those in the negative controls (P = 0.008). With CTA2/B genetic adjuvant, the T. gondii-specific response was predominantly Th1, indicating that the CTA(2)/B genetic adjuvant was able to overcome the strong Th2-bias of the antigen (IgG2a >> IgG1). Antigen-specific T cell proliferative responses and CTL activity were significantly enhanced when cholera toxin CTA2/B genetic adjuvant was used (P = 0.009; P = 0.006). Culture supernatants from antigen-stimulated splenocytes from mice in these groups were also examined by ELISA for Th1- and Th2-type cytokines; mean IFN-gamma levels produced after oral immunization with BRD509/pSAG(1-2)-CTA2/B were about nine-fold higher than after immunization with BRD509/pSAG(1-2) (P = 0.007). On the other hand, the levels of IL-4 were low for all groups and no increase was seen in the presence of CTA2/B genetic adjuvant. When the immunized mice were intraperitoneally challenged with 10(3) tachyzoites of the highly virulent RH strain, the survival time of the mice immunized with BRD509/pSAG(1-2)-CTA2/B was markedly longer than other groups (P = 0.003) and a 40% survival rate was achieved. This is the first report that demonstrates that an oral attenuated salmonella DNA vaccine can induce protective immunity against the acute phase of T. gondii infection.

Bioorganic chemistry of cyclic ADP-ribose (cADPR).

The objective of this brief review is to present an overview of the bioorganic chemistry of cyclic-ADP-ribose (cADPR) with special emphasis on the methodology used for the synthesis of analogues of cADPR. New structural analogues of cADPR can be prepared using either the biomimetic method or ADP-ribosyl cyclase from Aplysia californica. For the most part, both procedures give similar product profiles, but higher yields are generally obtained with the enzymatic method. These synthetic methodologies have allowed the transformation of a variety of structurally modified analogues of NAD+ into their corresponding cyclic nucleotides. Several of these novel analogues are more potent than cADPR in inducing calcium release and are also more stable towards degradative enzymes. They could serve as valuable affinity probes for the isolation of cADPR-binding proteins.

Probing the phosphoinositide binding site of the clathrin assembly protein AP-2 with photoaffinity labels.

The relative binding specificities of the subunitsof bovine assembly protein AP-2 for the phosphatidylinositol polyphosphates (PtdInsPn) and inositol polyphosphates (InsPn) were determined by photoaffinitylabeling. Three types of benzophenone-containing photoprobes were employed: (i) the water-solubleP-1- or P-2-tethered p-benzoyldihydrocinnamoyl-InsPn (BZDC-InsPn) analogs, (ii) P-1-linked phosphotriester PtdInsPn analogs that sampled the interface between the water and lipid phases, and (iii) sn-1-O-acyl-linked PtdInsPn analogs that interacted with proteins penetrating the bilayer. The InsPn and PtdInsPn probes bind with highest selectivity and affinity to the two alpha subunit isoforms, with certain probes and conditions resulting in strong labeling of the 50-kDa mu subunit. Three main conclusions were reached: (i) head group recognition predominated over acyl chain recognition, (ii) the PtdInsPn binding site of alpha-AP-2 prefers more highly phosphorylated species, and (iii) the protein-acyl chain interactions showed high capacity but low selectivity.

Probing the phosphoinositide 4,5-bisphosphate binding site of human profilin I.

BACKGROUND: Profilin is a widely and highly expressed 14 kDa protein that binds actin monomers, poly(L-proline) and polyphosphoinositol lipids. It participates in regulating actin-filament dynamics that are essential for many types of cell motility. We sought to investigate the site of interaction of profilin with phosphoinositides. RESULTS: Human profilin I was covalently modified using three tritium-labeled 4-benzoyldihydrocinnamoyl (BZDC)-containing photoaffinity analogs of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). The P-1-tethered D-myoinositol 1,4,5-trisphosphate (Ins(1,4,5)P3) modified profilin I efficiently and specifically; the covalent labeling could be displaced by co-incubation with an excess of PtdIns(4,5)P2 but not with Ins(1,4,5)P3. The acyl-modified PtdIns(4,5)P2 analog showed little protein labeling even at very low concentrations, whereas the head-group-modified PtdIns(4,5)P2 phosphotriester-labeled monomeric and oligomeric profilin. Mass spectroscopic analyses of CNBr digests of [3H]BZDC-Ins(1,4,5)P3-modified recombinant profilin suggested that modification was in the amino-terminal helical CNBr fragment. Edman degradation confirmed Ala1 of profilin I (residue 4 of the recombinant protein) was modified. Molecular models show a minimum energy conformation in which the hydrophobic region of the ligand contacts the amino-terminal helix whereas the 4,5-bisphosphate interacts with Arg135 and Arg136 of the carboxy-terminal helix. CONCLUSIONS: The PtdIns(4,5)P2-binding site of profilin I includes a bisphosphate interaction with a base-rich motif in the carboxy-terminal helix and contact between the lipid moiety of PtdIns(4,5)P2 and a hydrophobic region of the aminoterminal helix of profilin. This is the first direct evidence for a site of interaction of the lipid moiety of a phosphoinositide bisphosphate analog with profilin.

Specific interaction of Golgi coatomer protein alpha-COP with phosphatidylinositol 3,4,5-trisphosphate.

The phosphoinositide binding selectivity of Golgi coatomer COPI polypeptides was examined using photoaffinity analogs of the soluble inositol polyphosphates Ins(1,4,5)P3, Ins(1,3,4,5)P4, and InsP6, and of the polyphosphoinositides PtdIns(3,4,5)P3, PtdIns(4,5)P2, and PtdIns(3,4)P2. Highly selective Ins(1,3,4,5)P4-displaceable photocovalent modification of the alpha-COP subunit was observed with a p-benzoyldihydrocinnamide (BZDC)-containing probe, [3H]BZDC-Ins(1,3,4,5)P4. A more highly phosphorylated probe, [3H]BZDC-InsP6 probe labeled six of the seven subunits, with only beta, beta', delta, and epsilon-COP showing competitive displacement by excess InsP6. Importantly, [3H]BZDC-triester-PtdIns(3,4,5)P3, the lipid with the same phosphorylation pattern as Ins(1,3,4,5)P4, showed specific, PtdIns(3,4,5)P3-displaceable labeling of only alpha-COP. Labeling by the PtdIns(4,5)P2 and PtdIns(3,4)P2 photoaffinity probes was less intense and showed no discrimination based on PtdInsPn ligand. Thus, both the D-3 and D-5 phosphates are critical for the alpha-COP-PtdIns(3,4,5)P3 interaction, suggesting an important role for this polyphosphoinositide in vesicular trafficking.

Phosphoinositide binding specificity among phospholipase C isozymes as determined by photo-cross-linking to novel substrate and product analogs.

We tested for the presence of high-affinity phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and PI(3,4,5)P3 binding sites in four phospholipase C (PLC) isozymes (delta1, beta1, beta2, and beta3), by probing these proteins with analogs of inositol phosphates, D-Ins(1,4,5)P3, D-Ins(1,3,4,5)P4, and InsP6, and polyphosphoinositides PI(4,5)P2 and PI(3,4,5)P3, which contain a photoactivatable benzoyldihydrocinnamide moiety. Only PLC-delta1 was specifically radiolabeled. More than 90% of the label was found in tryptic and chymotryptic fragments which reacted with antisera against the pleckstrin homology (PH) domain, whereas less than 5% was recovered in fragments that encompassed the catalytic core. In separate experiments, the isolated delta1-PH domain was also specifically labeled. Equilibrium binding of D-Ins(1,4,5)P3 to PLC-delta1 indicated the presence of a single, high-affinity binding site; binding of D-Ins(1,4,5)P3 to PLC-beta1, -beta2, or -beta3 was not detected. The catalytic activity of PLC-delta1 was inhibited by the product D-Ins(1,4,5)P3, whereas no inhibition of PLC-beta1, -beta2, or -beta3 activity was observed. These results demonstrate that the PH domain is the sole high-affinity PI(4,5)P2 binding site of PLC-delta1 and that a similar site is not present in PLC-beta1, -beta2, or -beta3. The data are consistent with the idea that the PH domain of PLC-delta1, but not the beta isozymes, directs the catalytic core to membranes enriched in PI(4,5)P2 and is subject to product inhibition.

Efficient peptide ladder sequencing by MALDI-TOF mass spectrometry using allyl isothiocyanate.

A new modification of the peptide ladder sequencing technique is described in which allyl isothiocyanate (AITC) replaces trifluoroethyl isothiocyanate as the volatile amine-modification reagent. AITC is commercially available, readily purified, stable up to 80 degrees C and reacts cleanly and rapidly with all amino groups of polypeptides. Several model peptides and two side chain-modified peptides were sequentially degraded using AITC and the cleavage reagent heptafluorobutyric acid (HFBA) up to seven amino acids from the N-terminus. Matrix-assisted laser-desorption and ionization coupled with time-of-flight (MALDI-TOF) mass spectroscopy of the peptide mixture provided a clear ladder-like mass profile with consecutive molecular ions corresponding to each shortened peptide at picomole range. The results indicate the general utility of this analytical protocol by the use of AITC as the amine-coupling reagent.

Calcium-dependent switching of the specificity of phosphoinositide binding to synaptotagmin.

The synaptic vesicle membrane protein synaptotagmin (tagmin) is essential for fast, calcium-dependent, neurotransmitter release and is likely to be the calcium sensor for exocytosis, because of its many calcium-dependent properties. Polyphosphoinositides are needed for exocytosis, but it has not been known why. We now provide a possible connection between these observations with the finding that the C2B domain of tagmin I binds phosphatidylinositol-4,5-bisphosphate (PIns-4,5-P2), its isomer phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate (PIns-3,4,5-P3). Calcium ions switch the specificity of this binding from PIns-3,4,5-P3 (at calcium concentrations found in resting nerve terminals) to PIns-4,5-P2 (at concentration of calcium required for transmitter release). Inositol polyphosphates, known blockers of neurotransmitter release, inhibit the binding of both PIns-4,5-P2 and PIns-3,4,5-P3 to tagmin. Our findings imply that tagmin may operate as a bimodal calcium sensor, switching bound lipids during exocytosis. This connection to polyphosphoinositides, compounds whose levels are physiologically regulated, could be important for long-term memory and learning.

Isolation and identification of C-terminal peptides of proteins.

A method for the isolation and identification of C-terminal peptides of proteins has been developed. The procedure entails the racemization of the C-terminal amino acid by reaction of the N-trifluoroacetylated protein with acetic anhydride and pyridine. After deprotection, the protein is fragmented to yield a mixture of peptides, which in turn are digested with carboxypeptidases. All peptides are hydrolyzed to L-amino acids except the C-terminal peptide with the terminal D-amino-acid residue. It is resistant to the action of carboxypeptidases and is readily identified by peptide mapping.

[Pharmacokinetic study of pseudohainanensine with deuteriumlabeled analogue as internal standard].

Pseudohainanensine (HH08) is a synthetic compound which is active against L-1210. In order to study the pharmacokinetic characteristics of this compound in rats, 3', 4'-dideuteropseudohainanensine (DH08) was synthesized and used as internal standard in GC-MS determination for quantitative analysis of alkaloid HH08 in the blood of rats that had been given HH08 at a dosage of 10 mg/kg intravenously. Experiments demonstrated that the detection limit of HH08 was 3 micrograms/ml and the peak concentration in the blood was 13.1 +/- 0.2 micrograms/ml after a single intravenous injection (one min. after injection). The biological half life time can be divided into two phases; the fast phase (alpha) 2.61 min, and the slow phase (beta) 42.59 min. Two hours after the injection no drug could be detected in the blood.

Evaluation of N-4-(hydroxycarbophenyl) retinamide as a cancer prevention agent and as a cancer chemotherapeutic agent.

In this review, a retinoid analogue (N-4-hydroxycarbophenyl) retinamide (R11) has been evaluated for its pharmacological and clinical properties. R11 has been studied both as a cancer prevention agent, as well as, a cancer chemotherapeutic agent. R11 alone and in combination with other agents can be used for the treatment of cervical dysplasia or leukemia. This review will examine novel strategies and mechanisms of action for retinoids in the treatment and prevention of cancer.

[Pharmacokinetic study of 4-(hydroxycarbophenyl) retinamide (RII) in cancer patients].

RII, a new analog of retinoic acid, is an effective anticarcinogenic drug. The pharmacokinetic characteristics of RII were studied by HPLC in thirteen cancer patients after oral administration. The sensitivity to detect RII was greater than 3 ng and CV values of duplicate samples were 1.88-6.62. The results showed that peak concentration of RII in plasma appeared at 8 hrs after oral administration of 200 mg of the drug, and the mean peak concentration in serum was 3.612 +/- 1.099 micrograms/ml. Half life time (T1/2) was 6.62 +/- 0.81 hrs. The area under the drug-time curve (AUC) was 35.70 +/- 14.46 micrograms hr/ml. The results indicate that it is advisable to take RII twice a day.

Antibody patterns in the serological diagnosis of acute lymphadenopathic toxoplasmosis.

Sera from six patients at various stages of acute lymphadenopathic toxoplasmosis were tested using nine different types of test currently available for the serological diagnosis of the disease. A diagnosis was made on positive serology in three of the patients. The antibody patterns of two of these patients were studied over a period of six months and the third was studied over two months. A diagnosis was made on lymph node morphology in the other three cases. Two serum samples were tested from each of these patients. The results obtained suggest that a rationalisation of the types of test used should be encouraged and that an enzyme-linked immunosorbent assay (ELISA) for parasite-specific IgG and an antibody class capture ELISA for parasite-specific IgM are sufficient to confirm the diagnosis of acute lymphadenopathic toxoplasmosis.

Effect of (+/-)-, (+)- and (-)-gossypol on the lactate dehydrogenase-X activity of rat testis.

The effect of (+/-)-, (+)- and (-)-gossypol on testicular lactate dehydrogenase-X (LDH-X) was studied in vitro and in vivo. It was found that racemic gossypol and the two optical enantiomers had similar inhibitory effects on rat testicular LDH-X in vitro. However, neither racemic gossypol nor the enantiomers exhibited an inhibitory effect on testicular LDH-X in vivo. It is concluded that inhibition of testicular LDH-X is not likely to be the mechanism of the antifertility action of gossypol. The inhibition of testicular LDH-X in vitro by all three preparations of gossypol is probably non-specific.