Internalization of surface anionic sites and phagosome-lysosome fusion during interaction of Toxoplasma gondii with macrophages

The participation of cell surface anionic sites on the interaction between tachyzoites of Toxoplasma gondii and macrophages and the process of phagosome-lysosome fusion were analyzed using cationized ferritin as a marker of cell surface anionic sites and albumin-colloidal gold as a marker for secondary lysosomes. Incubation of either the macrophages or the parasites with cationized ferritin before the interaction increased the ingestion of parasites by macrophages. Anionic sites of the macrophage's surface, labeled with cationized ferritin before the interaction, were internalized together with untreated parasites. However, after interaction with glutaraldehyde-fixed or specific antibody-coated parasites, the cationized ferritin particles were observed in endocytic vacuoles which did not contain parasites. Macrophages previously labeled with albumin-gold at 37 degrees C, were incubated in the presence of cationized ferritin at 4 degrees C and then incubated with untreated or specific antibody-coated parasites. After interaction with opsonized parasites, the colloidal gold particles were observed in the parasitophorous vacuoles while the cationized ferritin particles were observed in cytoplasmic vesicles. However, when the interaction was carried out with untreated parasites, the parasitophorous vacuoles exhibited ferritin particles while the colloidal gold particles were observed in cytoplasmic vesicles. These observations, in association with studies previously reported, suggest that the state of the parasite surface determines the mechanism of parasite entry into the macrophage, the composition of the membrane lining the parasitophorous vacuole and the ability of lysosomes to fuse with the vacuoles.     

Rat PHI, PHI-GLY and PHV (1-42) stimulate adenylate cyclase in six rat tissue and cell membranes

PHI and the two C-terminally extended forms PHI-GLY and PHV(1-42) coexist in rat tissues. We compared the relative potency and efficacy of these three PHI forms and of VIP to stimulate adenylate cyclase activity and, when feasible, to occupy VIP receptors in six rat tissue and cell membranes. With the exception of lung membranes, all three PHI forms were markedly less potent than VIP but all were systematically as efficacious. PHI-GLY and PHV(1-42) were never more potent than PHI itself and their relative potencies revealed four spectra, depending on the membrane preparation tested: 1) PHI = PHI-GLY = PHV(1-42) in hepatic, pulmonary and pancreatic membranes; 2) PHI greater than PHV(1-42) = PHI-GLY in membranes from circulating lymphocytes; 3) PHI = PHV(1-42) greater than PHI-GLY in membranes from the thymocyte cell line 51E; and 4) PHI greater than PHI-GLY = PHV(1-42) in anterior pituitary membranes. These results indicate that the two naturally observed C-terminal extensions of rat PHI variously affected peptide potency on 6 rat membrane preparations.     

Centrally administered neuropeptide Y enhances the hypothermia induced by peripheral administration of adrenoceptor antagonists

The distribution of neuropeptide Y in the brain includes extensive coexistence within adrenaline- and noradrenaline-containing neurons and many of its actions are often associated with adrenergic systems. Since neuropeptide Y immunoreactivity is particularly intense in the preoptic area, one of the principal sites for thermoregulation, we have tested the effects of neuropeptide Y on core temperature in normothermic rats, and rats rendered hypothermic by systemic treatment with adrenergic antagonists. In the normothermic rat, intracerebroventricular administration of 1 microgram of neuropeptide Y did not have a significant effect on core temperature. Intraperitoneal treatment with the alpha 1-adrenoceptor antagonist, prazosin, or the beta-adrenoceptor antagonist, propranolol, caused an immediate and significant hypothermia; the intracerebroventricular administration of 1 microgram of neuropeptide Y, 10 minutes after these drugs, strongly potentiated their hypothermic effect. Although intraperitoneal treatment with the alpha 2-adrenoceptor antagonist, idazoxan, had no hypothermic effect per se, the intracerebroventricular administration of NPY 10 minutes after this antagonist led to a significant decrease in core temperature.     

Somatostatin binding to dissociated cells from rat cerebral cortex

A method has been developed for the study of somatostatin (SS) binding to dissociated cells from rat cerebral cortex. Binding of [¹²⁵I][Tyr¹¹]SS to cells obtained by mechanical dissociation of rat cerebral cortex was dependent on time and temperature, saturable, reversible and highly specific. Under conditions of equilibrium, i.e., 60 min at 25°C, native SS inhibited tracer binding in a dose-dependent manner. The Scatchard analysis of binding data was linear and yielded a dissociation constant of 0.60±0.08 nM with a maximal binding capacity of 160±16 fmol/mg protein. The binding of [¹²⁵I][Tyr¹¹]SS was specific as shown in experiments on tracer displacement by the native peptide, SS analogues, and unrelated peptides.     

A new principle of cell sorting by using selective electroporation in a modified flow cytometer

When a strong electric field pulse of a few microseconds is applied to biological cells, small pores are formed in the cell membranes; this process is called electroporation. At high field strengths and/or long pulse durations the membranes will be damaged permanently. This eventually leads to cell kill. We have developed a modified flow cytometer in which one can electroporate individual cells selected by optical analysis. The first experiments with this flow cytometer were designed to use it as a damaging sorter; we used electric pulses of 10 microseconds and resulting field strengths of 2.0 and 3.2 x 10(6) V/m to kill K562 cells and lymphocytes respectively. The hydrodynamically focused cells are first optically analyzed in the usual way in a square flow channel. At the end of this channel the cells are forced to flow through a small Coulter orifice, into a wider region. If optical analysis indicates that a cell is unwanted, the cell is killed by applying a strong electric field across the Coulter orifice. The wanted living cells can be subsequently separated from the dead cells and cell fragments by a method suitable for the particular application (e.g., centrifugation, cell growth, density gradient, etc.). The results of these first experiments demonstrate that by using very simple equipment, sorting by selective killing with electric fields is possible at rates of 1,000 cells/s with a purity of the sorted fraction of 99.9%.     

Ultrastructure of E1 + 2 + 9 + 12 inversion breakpoints in Drosophila subobscura

The ultrastructure of the Drosophila subobscura chromosome regions around the breakpoints of the complex E1 + 2 + 9 + 12 gene arrangement was analyzed. This overlapping inversion is formed by the association of the E1, E2, E9, and E12 simple inversions. Ultrastructure of sections involving 58D/59A, 61C/D, 62D/63A, 64B/C, 67A/B, and 68B/C breakpoints on Est chromosomes were compared with the ultrastructure of sections involving chromosomes were compared with the ultrastructure of sections involving 58D/68B, 62D/64C, 59A/63A, 64B/68C, 67B/61C, and 67A/61B breakpoints on E1 + 2 + 9 + 12 chromosomes. No detectable changes of structural organization on banding patterns induced by the E1 + 2 + 9 + 12 inversion were found. Ultrastructural analysis of the two E12 breakpoints has, however, facilitated the analysis of the left boundary of E12 inversion. Accordingly, we propose 61B/C as a new breakpoint instead of 61C/D.     

Altered regulation of glycogen metabolism by vasopressin and phenylephrine in hepatocytes from insulin-resistant obese (fa/fa) rats. Role of protein kinase C.

The hormonal control of glycogen synthase and phosphorylase interconversion was investigated in hepatocytes isolated from lean and genetically obese (fa/fa) rats. In cells from obese animals, the inactivation of synthase by 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), phospholipase C, vasopressin and the alpha 1-adrenergic agonist phenylephrine was markedly impaired, and the property of PMA to counteract phosphorylase activation by phenylephrine was attenuated. The maximal response of phosphorylase activation to phenylephrine and vasopressin was increased in obese-rat hepatocytes, but the sensitivity to these hormones was similar to that in lean-rat hepatocytes. These observations indicate that the defect in protein kinase C that we reported previously in heart of insulin-resistant fa/fa rats [van de Werve, Zaninetti, Lang, Vallotton & Jeanrenaud (1987) Diabetes 36, 310-319] is probably also expressed in liver.     

Genetic elements involved in Tn21 site-specific integration, a novel mechanism for the dissemination of antibiotic resistance genes.

Transposon Tn21 codes for a site-specific integration system, which is probably a novel recombination mechanism, responsible for the acquisition of resistance genes in this widespread family of transposons. Using insertion and deletion mutagenesis we have identified the genetic loci of the various recombination hot-spots (RHS) and of the gene product (the integrase) that catalyses the reaction. The site of recombination has been localized in two of the RHSs to the DNA sequence GTTAG, which is present at the 3' termini of a loosely conserved palindromic sequence of approximately 59 bp. This 59 bp sequence, which flanks the inserted genes in a number of naturally occurring transposons, is the only element required in cis for the recombination reaction.     

Anti-oxidant defences and peroxidation in liver and brain of aged rats.

Old rats (28 months), when compared with young adults (9 months), did not show differences in activities of superoxide dismutase (SOD) or selenium-dependent and -independent glutathione peroxidases (GPx), or in levels of GSH, GSSG, GSSG/GSH and endogenous peroxidation in liver and brain. Rates of stimulated peroxidation in vitro were decreased in the livers of old rats. Old animals showed decreased levels of hepatic catalase and glutathione reductase. Nevertheless, when enzyme activities were referred to cytochrome oxidase activity these decreases disappeared, and GPx and SOD (brain) were even increased in old rats.