3H-azidopine photoaffinity labeling of high molecular weight proteins in chloroquine resistant falciparum malaria

Using 3H-azidopine, we have succeeded in labeling proteins from chloroquine resistant (CR) human falciparum malaria parasites in the molecular weight range of 155-170 kd. Vinblastine does not compete, but azidopine blocks the labeling using 3H-azidopine. Relatively little or no labeling of the 155-170 kd protein is seen in the chloroquine sensitive strain using 3H-azidopine. Further competition can be seen with nicardipine and reserpine (71%) respectively and verapamil (61%), chloroquine (48%), quinacrine (56%), trifluoperazine (32%) and chlorpromazine (33%). We speculate that this may be the glycoprotein responsible for the resistance to chloroquine in falciparum malaria.     

Photoaffinity labeling of P-glycoprotein in multidrug resistant cells with photoactive analogs of colchicine

Two photoactive radiolabeled analogs of colchicine, N-(p-azido[3,5-[3H]benzoyl)aminohexanoyldeacetylcolchicine ([3H]NABC]) and N-(p-azido-[3-125I]salicyl)aminohexanoyldeacetylcolchicine ([125I]NASC) were synthesized and used to identify colchicine-specific acceptor(s) in membrane vesicles from multidrug resistant (MDR) variant DC-3F/VCRd-5L Chinese hamster lung cells. Both [3H]NABC and [125I]NASC specifically photolabeled a prominent 150-180 kDa polypeptide in membrane vesicles from DC-3F/VCRd-5L cells. The photolabeled polypeptide was immunoprecipitated by monoclonal antibody C219 specific for the MDR-related P-glycoprotein (P-gp) indicating the identity of this protein with P-gp. Colchicine at 1000 microM reduced [3H]NABC photolabeling of P-gp by 72%. Furthermore, 100 microM of colchicine, vincristine, vinblastine, doxorubicin and actinomycin D inhibited [125I]NASC photolabeling by 45, 88.8, 91.1, 61.5, and 51% respectively. However, methotrexate did not affect the [125I]NASC photolabeling of P-gp, indicating the multidrug specificity of the P-gp colchicine acceptor for drugs to which these cells are resistant.     

Relationship between Wnt-1 and En-2 expression domains during early development of normal and ectopic met-mesencephalon.

Grafting a met-mesencephalic portion of neural tube from a 9.5-day mouse embryo into the prosencephalon of a 2-day chick embryo results in the induction of chick En-2 (ChickEn) expression in cells in contact with the graft (Martinez et al., 1991). In this paper we investigate the possibility of Wnt-1 being one of the factors involved in En-2 induction. Since Wnt-1 and En-2 expression patterns have been described as diverging during development of the met-mesencephalic region, we first compared Wnt-1 and En-2 expression in this domain by in situ hybridization in mouse embryos after embryonic day 8.5. A ring of Wnt-1-expressing cells is detected encircling the neural tube in the met-mesencephalic region at least until day 12.5. This ring consistently overlapped with the En-2 expression domain, and corresponds to the position of this latter gene's maximal expression. We subsequently studied ChickEn ectopic induction in chick embryos grafted with various portions of met-mesencephalon. When the graft originated from the level of the Wnt-1-positive ring, ChickEn induction was observed in 71% of embryos, and in these cases correlated with Wnt-1 expression in the grafted tissue. In contrast, this percentage dropped significantly when the graft was taken from more rostral or caudal parts of the mesencephalic vesicle. Taken together, these results are compatible with a prolonged role of Wnt-1 in the specification and/or development of the met-mesencephalic region, and show that Wnt-1 could be directly or indirectly involved in the regulation of En-2 expression around the Wnt-1-positive ring during this time. We also provide data on the position of the Wnt-1-positive ring relative to anatomical boundaries in the neural tube, which suggest a more general role for the Wnt-1 protein as a positional signal involved in organizing the met-mesencephalic domain.     

Specific binding of concanavalin A to free inositol and liposomes containing phosphatidylinositol

We previously reported that concanavalin A could bind specifically to liposomes containing phospholipids and lacking glycoconjugates (Biochem. Biophys. Res. Comm. 74, 208, 1977). In the present study we show that the binding of concanavalin A to the liposomes was greatly increased (up to 5 fold) by the presence of phosphatidylinositol in the liposomes. Furthermore, the binding of concanavalin A to phosphatidylinositol-liposomes was specific and could be inhibited by either alpha-methyl mannoside or by myo-inositol. We also found that concanavalin A-induced lymphocyte mitogenesis could be inhibited either by alpha-methyl mannoside or by myo-inositol. Simultaneous addition of both inhibitors to concanavalin A and liposomes showed that inhibition was non-competitive: alpha-methyl mannoside was more inhibitory to liposomes lacking phosphatidylinositol, and myo-inositol was more inhibitory to liposomes containing phosphatidylinositol. This suggests that the binding site for inositol might be different than that for mannose. Equilibrium dialysis and Scatchard plots revealed 4 binding sites each for inositol and mannose at neutral pH. The binding constants of concanavalin A were 0.13 X 10(4) and 0.25 X 10(4) liters/mole respectively for inositol and mannose. We conclude that concanavalin A binds specifically to the inositol portion of phosphatidylinositol.     

Hyalomma (Hyalommina) arabica: nymphal and larval identity and spiny mouse hosts in Saudi Arabia (Acarina: Ixodoidea: Ixodidae)

Nymphal and larval Hyalomma (Hyalommina) arabica Pegram, Hoogstraal, and Wassef, 1982, described herein, closely resemble those of Hyalomma (Hyalommina) rhipicephaloides Neumann, differing chiefly in body size (both stages), nymphal basis capituli and scutal proportional dimensions, and distinctness of larval coxal spurs. Females of these species are also structurally similar but males differ in major critical characters. Nymphs and larvae of both species parasitize the spiny mouse (Acomys spp.), but immatures of the third African- Arabian species of this subgenus, Hyalomma (Hyalommina) punt Hoogstraal, Kaiser, and Pedersen, are unknown. Hyalomma (Hyalommina) arabica occurs in valleys and hills of western Saudi Arabia and western Yemen; H. (H.) rhipicephaloides in the Red Sea and Dead Sea areas; and H. (H.) punt in northeastern Somalia and eastern Ethiopia. The ibex (Capra ibex nubiana Cuvier) is probably the original host of adult H. (H.) arabica and H. (H.) rhipicephaloides; the related domestic goat is an important host of adults of the 3 species, which also parasitize domestic sheep. Gazelles are recorded hosts of adults of H. (H.) rhipicephaloides and H. (H.) punt and the latter is also recorded from goats, sheep, camels and cattle.     

Motilin and the vagus in dogs

The aim of this work was to determine the influence of the vagus on the circulating levels of immunoreactive (IR) motilin. Five mongrel dogs were equipped with chronically implanted electrodes in the small intestine to record the myoelectrical activity. The release of IR motilin during fasting, after a meal, and during an infusion of insulin was studied before and after truncal vagotomy at the diaphragmatic level. When tested at least two weeks after the operation, the motility pattern of the small intestine and the secretion of IR motilin remained unaltered by vagal section. Cyclic increases in IR motilin associated with phase III's of the interdigestive myoelectric complexes were still observed after vagotomy (maximum levels of IR motilin: 250 +/- 37 versus 239 +/- 19 fmol X mL-1, not significant), and they were still abolished by feeding or by insulin. However, an inhibitory influence can probably be mediated by the vagus since, in normal animals, vagal stimulation by a "modified sham feeding" (tease feeding or presentation of food) at the beginning of a period of phase III activity promptly interrupted this part of the complex and decreased significantly the release of IR motilin by about 20%. The release of motilin is not chronically altered by distal vagotomy in dogs.     

Response of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumor cells to tamoxifen, in vitro

Primary cell cultures from a density-defined cell subpopulation of the DMBA-induced rat mammary tumors were exposed to tamoxifen during their log phase of growth. Growth inhibition and the ultrastructure of surviving cells were examined along with the influence of this antiestrogen on the secreted proteins as determined by pulse labeling with [35S]methionine and fluorography. Cell growth was remarkably inhibited at clinically achievable concentrations. However, ultrastructural changes in the surviving cells were minimal, the most noteworthy being the accumulation of myelin bodies. Protein secretion was affected in the defined subpopulation of several tumors by the reduced production of a high-molecular-weight protein. These tumors may represent a population of estrogen-sensitive tumors within the DMBA-induced mammary tumor model.     

Thyroid fine needle aspiration: the morphological features on ThinPrep® slide preparations. Eighty cases with histological control

This study had several purposes: to define cytomorphological features of thyroid cells that might be modified by alcohol fixation; to optimize May-Grünwald–Giemsa (MGG) staining on ThinPrep^® (TP; Cytyc Inc., Bexborough, MA, USA) slides and to compare the diagnostic accuracy of slides prepared by a liquid-based method with those obtained by conventional technique. This study included 120 cases of ultrasound-guided fine needle aspiration (FNA) of the thyroid and 55 FNAs performed on surgically resected thyroid specimens. Histological control was available in 80 cases. In the first group of 120 FNAs, a split-sample technique was used for the TP. Three screenings were performed: first, an individual screening of the conventional smears (CS) and of the TP, a second screening to compare cells observed on the TP with the histological control and a third screening to assess the previously defined diagnostic criteria. Twenty-seven TP cases (22%) were considered unsatisfactory for diagnosis compared with 10 in CS (8%). The high rate of unsatisfactory cases with TP is likely to be due to the use of the split-sample technique. The sensitivity was 94% for CS and 81% for TP. The specificity was 67% and 60% for CS and TP, respectively. Two occult papillary carcinomas were missed by both methods. As for the MGG staining, the modified technique used for TP resulted in the same quality as the standard procedure. Conversely, TP did however induce uncommon morphological features. In this study, sensitivity and specificity levels are higher for CS than for TP; the difference may be explained by the fact that the methanol fixative used for TP induces some cytological alterations, especially in oncocytic tumours and lymphocytic thyroïditis.     

Azidopine noncompetitively interacts with vinblastine and cyclosporin A binding to P-glycoprotein in multidrug resistant cells.

It is believed that P-glycoprotein (P-gp) is an energy-dependent drug efflux pump responsible for decreased drug accumulation in multidrug resistant (MDR) cells. In this study, we investigated whether azidopine, a photoactive dihydropyridine calcium channel blocker, is transported by P-gp in MDR Chinese hamster lung cells, DC-3F/VCRd-5L, and whether its binding site(s) on P-gp are distinct from those of Vinca alkaloids and cyclosporins. The efflux of azidopine from MDR cells was energy-dependent and inhibited by the cytotoxic agent vinblastine (VBL). Cyclosporin A (CsA), a modulator of MDR, also increased azidopine accumulation in MDR cells by decreasing the energy-dependent efflux of azidopine. P-gp in these cells was the only protein specifically bound to [3H]azidopine in photoaffinity experiments. The specific photoaffinity labeling of P-gp by [3H]azidopine was inhibited by CsA, SDZ 33-243, nonradioactive azidopine, and VBL with median concentrations (IC50) of 0.5, 0.62, 1.7, and 25 microM, respectively. The equilibrium binding of azidopine to plasma membranes of MDR variant DC-3F/VCRd-5L cells showed a single class of specific binding sites having a dissociation constant of 1.20 microM and a maximum binding capacity of 4.47 nmol/mg of protein. Kinetic analysis indicated that the inhibitory effect of VBL and CsA on azidopine binding to plasma membranes of MDR cells was noncompetitive, indicating that azidopine binds to P-gp at a binding site(s) different from the binding site(s) of these drugs.