The binding of [3H]R-PIA to A1 adenosine receptors produces a conversion of the high- to the low-affinity state

Kinetic evidence for negative cooperativity on the binding of [3H]R-PIA to A1 adenosine receptors was obtained from dissociation experiments at different ligand concentrations and from the equilibrium isotherm. The dissociation curves indicate that there is an apparent ligand-induced transformation of high- to low-affinity states of the receptor. At concentrations of 18.2 nM R-PIA or higher there was only found the low-affinity state of the receptor. In view of these results equilibrium binding data were analyzed by the usual two-state model (assuming that there is an interconversion between them) and by the negative cooperativity model employing the Hill equation.     

Silver Enhancement of Nickel-Diaminobenzidine as Applied to Single and Double Immunoperoxidase Staining

A reliable and practical method is proposed for increasing sensitivity and detection efficiency of immunocytochemical techniques, based on silver enhancement of the nickel-diaminobenzidine product of the peroxidase reaction. The procedure produces a strong signal at the site of the end product of the peroxidase reaction which is visible as black grains at the light microscopic level. The method has been used to detect peroxidase labeled probes in immunocytochemical tissue preparations and blotting assays and is ideal for the purposes of double staining and photographic documentation.     

To lek or not to lek: mating strategies of male fallow deer

We studied the mating system of fallow deer (Dama dama) for6 years in central Italy. Males in this population could defendterritories that were either single, clumped in leks, or satelliteto leks. The most highly successful males in our study werein leks. When we considered all males, there were no significantdifferences in average copulatory success according to territorytype because many lek males did not achieve any copulations,which were seen in only a few lek territories. The variancein copulatory success, however, was much greater for leks thanelsewhere. Single territories were occupied for shorter timesduring the rut than lek territories. Fighting among males wasmore frequent in the lek, even when we excluded highly successfullek males from the analysis. Chases of nonterritorial malesand harem size were correlated with the number of copulationsachieved by individual males, but did not vary according toterritory type. Copulatory success of some individuals increasedwith age, but there were no age differences among males holdingdifferent types of territories. Satellite males switched tolek territoriality in the course of one rut, but switches fromsingle territory to lek territory were rare. We suggest thatmales in single territories are inferior competitors that selecta low-risk, lowbenefit strategy, whereas those in lek territorieswhere no copulations were seen may be attempting to establishthemselves on the lek to increase their copulatory success infuture years.     

Further characterization of adenosine transport in renal brush-border membranes

Adenosine transport has been further characterized in rat renal brush-border membranes (BBM). The uptake shows two components, one sodium-independent and one sodium-dependent. Both components reflect, at least partly, translocation via a carrier mechanism, since the presence of adenosine inside the vesicles stimulates adenosine uptake in the presence as well as in the absence of sodium outside the vesicles. The sodium-dependent component is saturable (Km adenosine = 2.9 microM, Vmax = 142 pmol/min per mg protein) and is abolished at low temperatures. The sodium-independent uptake has apparently two components: one saturable (Km = 4-10 microM, Vmax = 174 pmol/min per mg protein) and one non-saturable (Vmax = 3.4 pmol/min per mg protein, Km greater than 2000 microM). Inosine, guanosine, 2-chloroadenosine and 2'-deoxyadenosine inhibit the sodium-dependent and -independent transport, as shown by trans-stimulation experiments, probably because of translocation via the respective transporter. Uridine and dipyridamole inhibited only the sodium-dependent uptake. Other analogs of adenosine showed no inhibition. The kinetic parameters of the inhibitors of the sodium-dependent component were further investigated. Inosine was the most potent inhibitor with a Ki (1.9 microM) less than the Km of adenosine. This suggests a physiological role for the BBM ecto-adenosine deaminase (enzyme which extracellularly converts adenosine to inosine), balancing the amount of nucleoside taken up as adenosine or inosine by the renal proximal tubule cell.     

Experimental pulmonary paracoccidioidomycosis in mice: Morphology and correlation of lesions with humoral and cellular immune response

The present paper describes a murine model for pulmonary paracoccidioidomycosis injecting 6×10⁵ yeast forms ofParacoccidioides brasiliensis (Pb) by the direct intratracheal route. The sequential histopathology of lung and dissemination lesions together with humoral (immunodiffusion test) and cellular immune response (footpad test and macrophage inhibition factor assay — MIF assay) were investigated since the 1^st to the 360^th day after infection. All infected animal showed pulmonary Pbmycosis up to Day 30; onwards the lesions subsided being found only in one mouse at Day 360. Dissemination lesions were observed in paratracheal and cervical lymph nodes in 9 out of 68 infected animals. Histologically early lesions were rich in polymorphonuclear cells and evolved to a macrophage desquamative pneumonitis at Day 15 and to typical epithelioid granulomata from Day 30 up to Day 360. Specific precipitating antibodies were first detected 15 days after infection, peaked from Day 30 to 60 and were not observed at Day 360. Significant cell-mediated immunity to Pb was noted at Day 15 with the peak reaction at Day 60 and 90.The intratracheal route represents a highly effective way of infecting mouse with Pb. This experimental pulmonary Pbmycosis is a granulomatous inflammation which courses with specific humoral and cellular immune response. It may be a good tool for further investigation in the pathogenesis and natural history of the disease.     

Lack of expression on cultured human T-lymphocytes of a T-cell antigen shared by the molt-4 cell line and normal human thymocytes

Summary Four hematopoietic cell lines (CCRF-CEM, HSB-2, MOLT-4, and RPMI-8402), derived from acute lymphoblastic leukemia and expressing T-cell surface markers (T-HCL), were studied with two specific anti-T-cell sera. The sera were raised in rabbits against human thymocytes (anti-HTY) and against T-cells cultured in the presence of conditioned medium derived from lymphocytes stimulated with PHA (anti-CTC). Both sera were absorbed to obtain a T-cell specific pattern of reaction and were further absorbed with normal peripheral blood lymphocytes or with each of the four T-HCL. The anti-HTY sera absorbed with CEM, 8402, and HSB-2 still reacted with MOLT-4. A similar pattern of reactivity was found only with the anti-CTC absorbed with 8402, whereas, after absorptions with the other cell lines, this antiserum was unreactive against MOLT-4. After absorption with normal peripheral blood lymphocytes, anti-HTY still reacted with thymocytes and MOLT-4 but was negative on CTC. In contrast, anti-CTC absorbed with peripheral blood lymphocytes (PBL) was negative on thymocytes and MOLT-4 but still reacted against CTC. Our data confirm the existence of a T-cell antigen (probably an early T-cell differentiation antigen) shared between thymus and MOLT-4. This antigen is not expressed on CTC, although these cells express an antigenic pattern more complex than PBL. Antisera to CTC represents a source of anti-T-cell sera free of contamination with antibodies to early thymus-related antigens but containing other T-cell-related specificities. Supported in part by Naval Medical Research and Development Command, Research Task No. ZF51.524.013.1025, and National Cancer Institute Contract No. Y01-CB-00319. The opinions and assertions contained herein are the private ones of the writers and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in the current edition of the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animal Resources, National Research Council.     

Chemical composition of the plasma membrane from epimastigote forms of Trypanosoma cruzi

The chemical composition of two plasma membrane fractions from epimastigote forms of Trypanosoma cruzi is reported. Fraction M, a preparation obtained by conventional methods of cell fractionation is composed of 31% proteins, 34% lipids, 16% carbohydrates and 3% of the lipopeptidophosphoglycan. Phospholipids and sterols account for 7.5 and 9%, respectively, of the total mass. Phosphatidylethanolamine is the major phospholipid in fraction M, representing 45% of the total membrane phospholipids. The other fraction, fraction V (vesicles), was obtained by treatment of the cell with a vesiculating agent. This fraction contains 42% lipids, 20% carbohydrates, 13% proteins and 21% of the lipopeptidophosphoglycan. Phospholipids and sterols make up 17 and 8%, respectively, of the total mass of this fraction. Phosphatidylcholine and phosphatidylethanolamine are the main phospholipids found in fraction V. Phosphonolipids and sialic acid have not been detected in either membrane fraction. Sodium dodecyl sulphate polyacrylamide gel electrophoretic analysis show that the glycoproteins ABC and the lipopeptidophosphoglycan are 50- and 10-times more concentrated, respectively, in fractions V and M than in the whole cell homogenate. The high molar sterol/phospholipid ratio found in fraction M suggests that this fraction is less fluid than fraction V, perhaps reflecting a migration of certain membrane components in the presence of the vesiculating agent. Hence, fraction M is, probably, more representative of the epimastigote plasma membrane as a whole than fraction V.     

The epoxide hydrolase catalyzed hydrolysis of trans-3-bromo-1,2-epoxycyclohexane. A direct proof for a general base catalyzed mechanism of the enzymatic hydration

The hydrolysis of (±)-$\text{trans}$-3-bromo-1,2-epoxycyclohexane in the presence of rabbit liver microsomes was investigated, and found to yield, beside $\text{c}$-3-bromocyclohexane-$\text{r}$-1,$\text{t}$-2-diol, 2,3-epoxycyclohexanol. It was demonstrated that the latter compound was the only product of the enzymatic reaction, whereas the diol resulted from a non enzymatic hydration in the reaction medium. These data provide the first direct proof for a general base catalysis in the enzymatic epoxide hydration, previously hypothesized on the basis of several lines of indirect evidence, and disprove alternative mechanisms involving protonation of the oxirane oxygen.