Identification of distinct accumulation sites of 4-aminoquinoline in chloroquine sensitive and resistant Plasmodium berghei strains

We report the synthesis of an analogue of chloroquine (CQA) which can be used as a probe to visualize accumulation of 4-aminoquinoline by electron microscopy. A mouse monoclonal antibody against CQA was raised and used for immunodetection by the protein-A gold method on ultrathin cryosections, of CQA treated parasites. We demonstrate that in a P. berghei chloroquine(CQ)-sensitive strain (N strain) the chloroquine analogue used accumulates in the endocytic vacuoles where hemoglobin (Hb) degradation is occurring. In contrast, in a P. berghei CQ-resistant strain (RC strain) the probe was found scattered all over the cytoplasm of the parasite. This result suggests that endocytic vacuoles of the parasite could constitute the site of antimalarial action of CQ.     

Mesenchymal-epithelial conversions induced by 5-Azacytidine: Appearance of cytokeratin Endo-A messenger RNA

When mouse-teratocarcinoma-derived fibroblasts (1246 cell line) are subjected to treatment with the inhibitor of DNA methylation, 5-Azacytidine (5 AzaC), they transiently express at 55-kilodalton intermediate-filament protein recognized by the epithelial-specific monoclonal antibody, TROMA-1, although they retain a fibroblastic morphology. However, rare clones (e.g., the 1339 cell line) that permanently express the antigen recognized by TROMA-1 can be derived from the 5 AzaC-treated 1246 population, and these clones have an epithelial phenotype. In the present study, we used cloned DNA probes to demonstrate that, in 1246 fibroblasts, 5 AzaC induces the appearance of Endo-A mRNA. High levels of Endo-A mRNA were also detected in the epithelial derivative, cell line 1339. In both cases, the capping site of the Endo-A mRNA was found to be the same as that in epithelial cells which normally express this RNA.     

The dopaminergic innervation of the goldfish pituitary

Summary The dopaminergic innervation of the goldfish pituitary gland was studied by immunocytochemistry at the electron-microscope level using highly specific antibodies against dopamine coupled to bovine serum albumin with glutaraldehyde. A satisfactory preservation of the tissue was achieved after immersion in 5% glutaraldehyde in phosphate buffer containing sodium metabisulfite to prevent oxidation of the endogenous dopamine. The immunocyto-chemical procedure was performed on Vibratome sections using the preembedding method. Immunoreactivity was restricted to part of the neurosecretory type-B fibers (diameter of the secretory vesicles lower than 100 nm) in which it was found to occupy the whole cytoplasm. Labeled fibers were observed within the neurohypophysis in the different parts of the gland and in the adenohypophyseal tissue where immunoreactive profiles were detected in close apposition to the different cell types. These data are in agreement with previous results obtained by means of radioautography and further support a role for dopamine in the neuroendocrine regulation of pituitary functions in teleosts.     

Experimentally induced true hermaphroditism in genetically female fowl

Summary Two types of hermaphroditism were experimentally induced in genetically female fowls by grafting of embryonic testes in embryos. Of the 27 hermaphrodites observed during the 8 months after hatching, 20 possessed a right testis and a left ovary and 7 a right testis and a left ovotestis. The testes and ovotestes contained seminiferous tubules with a more or less developed germ cell complement, attaining in many cases the early spermatid stage. The interstitial tissue was poorly functional, as shown by the absence of male secondary sex characters. The ovary or ovarian part of the ovotestes possessed numerous small ovarian follicles. The female arrangement of the plumage and the absence of spurs demonstrated the secretion of oestrogens. A mechanism is proposed for explaining this partial masculinization of genetically female gonads, a phenomenon which occurs during the period of embryonic sex differentiation, and is responsible for this experimental true hermaphroditism.     

Kinetic studies of aminoglycoside acetyltransferase and phosphotransferase from Staphylococcus aureus RPAL. Relationship between the two activities

In the Staphylococcus aureus strain harbouring the plasmid RPAL, the resistance to aminoglycoside antibiotics results from two inactivating reactions catalyzed by a 6'-N-aminoglycoside acetyltransferase and a 2"-O-amino-glycoside phosphotransferase. These enzymes are copurified with a constant ratio between the two activities, the purification process consisting in affinity chromatography, native electrophoresis and gel exclusion chromatography. The kinetic mechanisms of each activity have been determined from studies of initial velocities, as well as product and dead-end inhibitions. Both activities follow a random rapid equilibrium mechanism. The substrates and cofactors of one reaction have been tested as effectors of the other reaction. No interaction between the two activities has been observed. However, the GTP cofactor of phosphotransferase protects, at weak concentrations, the acetyltransferase against thermal inactivation, which suggests that the two activities may be associated.     

Binding sites for monoclonal antibodies and for mRNPs on SV40 large T-antigen determined with a cleavage map

Immune complexes of simian virus 40 large T-antigen with monoclonal papovavirus protein antibodies PAb 416, PAb 402, or PAb 423 were bound to protein-A-Sepharose and then cleaved into discrete fragments by limited tryptic proteolysis. PAb 402 protected a specific cleavage site, located approximately within amino acid residues 450-500, from tryptic proteolysis; PAb 423 protected another site within residues 675-699. As shown by immunoblotting, 125I-labeled PAb 416 was bound to a 17-kDa N-terminal fragment of large T-antigen (amino acid residues 1-130), and PAb 423 was bound to several overlapping fragments derived from the C terminus of large T-antigen. These monoclonal antibodies were then used as accessibility probes to study the interaction of mRNPs with cytoplasmic large T-antigen. Whereas small T-antigen and nuclear large T-antigen were fully immunoreactive, cytoplasmic large T-antigen reacted poorly with PAb 402 or polyclonal antibodies unless the mRNP moiety was removed by treatment with EDTA/RNase A. In contrast, mRNP/T-antigen complexes were fully immunoreactive with PAb 416 or PAb 423 and did not require treatment with EDTA/RNase A. The results suggest that the binding site of PAb 402 is blocked due to the interaction with mRNPs whereas the N-terminal binding site of PAb 416 and the C-terminal binding site of PAb 423 remain accessible to antibodies.     

The key role of residue 5 in angiotensin II

The conformation–biological activity relationships in a series of angiotensin II analogs substituted in position 5 were studied. Results indicated that only analogs with β-branched residue in position 5 possess spectral and biological properties identical to that of parent angiotensin II.     

Purification from Synaptosomal Plasma Membranes of Calpain I, a Thiol Protease Activated by Micromolar Calcium Concentrations

Synaptosomal plasma membranes (SPMs) were prepared from whole rat brain and assayed for calcium-stimulated proteolytic activity. Addition of calcium to SPMs caused a dose-dependent increase in trichloroacetic acid-soluble protein. Two peaks of protease activity directed against a casein substrate were detectable when SPMs were incubated with low-ionic-strength buffer and the extract was fractionated on DEAE-cellulose. The enzyme in peak 1 required less than 1/10 the calcium concentration for activation as the peak 2 protease (Kact1 = 35 microM; Kact2 = 500 microM). The specific thiol-protease inhibitors leupeptin and antipain and the alkylator iodoacetate blocked enzyme activity. The low-sensitivity protease was converted to a high-sensitivity enzyme (Kact = 20 microM) by substrate affinity chromatography in the presence of calcium. This protease was purified 550-fold from SPMs. The high- and low-sensitivity membrane-associated calcium-dependent proteases are part of a family of enzymes, the calpains, previously reported in cytosolic fractions of several tissues.     

Combined application of misonidazole and piotron pions on mouse embryos

Mouse embryos on day 8 of gestation were irradiated with negative pions (12.5-100 rad) or 200 kV X-rays (12.5-150 rad). Misonidazole (MISO), a hypoxic cell radiosensitizer, was applied 30 min before exposure. On day 13 the fetuses were examined for lethality, growth retardation and malformation. No significant embryolethal effects were observed after irradiation alone in the dose range of 12.5-100 rad (X-rays or pions). However, MISO alone and in combination with radiation led to high rates of lethality. The frequency of growth retardation was significantly increased at 100 rad and in combined treatments at low radiation doses. MISO and irradiation with 50 rad and more induced complex damages consisting of multiple and severe malformations and growth retardation. The relative biological effectiveness (RBE) for teratogenic effects was 1.6. In conclusion, the combined application of MISO and radiation of different LET revealed a strong enhancing action compared to single treatments. The extent of enhancement depends on both radiation quality and dose.     

A mathematical theory of size distributions in tissue culture

A model for growth of a tissue culture consisting of cell clumps is given. A set of equations for following the size distribution of clumps is used to determine total biomass accumulation. Existence and uniqueness of a solution to the equations is proved, and estimates of the biomass growth is given in a number of situations.