Different effects of serotonin antagonists on 3H-mianserin and 3H-ketanserin recognition sites

In minces prepared from the frontal cortex of rats treated with ketanserin (10 mg/kg i.p.) or mianserin (5 mg/kg i.p.) twice daily for 21 days, the Vmax of the adenylate cyclase stimulated by NE (100 microM) is attenuated, suggesting that ketanserin and mianserin share with a number of antidepressants the ability to attenuate the adenylate cyclase stimulation by NE. Ketanserin, given with the above mentioned dose schedule for 7 consecutive days, reduced the Bmax of 5HT2 recognition sites but failed to change either the Bmax or the apparent Kd of H-mianserin binding. A significant decrease in the Bmax of 5HT2 binding sites is elicited also by a single injection of mianserin (1). This drug also down-regulates its own binding when given twice daily for 3 weeks. From this and other information (2,3), it is concluded that ketanserin and mianserin bind to distinct recognition sites. The possibility that 5HT2 and mianserin recognition sites are functionally related and that serotonergic synapses are modulated by multiple chemical signals might be considered.     

Adaptation of a gamma-glutamyl transpeptidase assay to microtiter plates

A kinetic assay for measuring gamma-glutamyl transpeptidase (GGT) activity has been adapted to microtiter plates and an automated microtiter plate reader. This method permits the simultaneous analysis of enzyme activity in a large number of samples incubated with the chromogenic GGT substrate gamma-glutamyl-p-nitroanilide. A major advantage of this assay over previously reported methods is the substantial reduction in the time needed for measuring sample enzyme activity. In addition, reduction of the total assay volume to 0.28 ml conserves both sample and reagents. This method has been calibrated at 23 degrees C using purified GGT, and used to analyze GGT activity in human sera. The assay is sensitive over a range of 3-200 U/liter.     

The formation and biotransformation of cysteine conjugates of halogenated ethylenes by rabbit renal tubules

The nephrotoxicity of chlorotrifluoroethylene (CTFE) was examined using isolated rabbit renal tubules suspensions. Exposure of the tubules to CTFE resulted in consumption of CTFE, formation of a glutathione conjugate and inhibition of active organic acid transport. Synthetic cysteine, N-acetylcysteine or glutathione conjugates of CTFE inhibited transport indicating S-conjugation as a possible toxic pathway. 1,2-dichlorovinyl glutathione (DCVG), a model synthetic glutathione conjugate, was used to examine the degradation and toxicity of these conjugates. DCVG inhibited rabbit renal tubule transport in vivo and in vitro. The DCVG was found to be degraded with the evolution of glutamine and glycine to produce the ultimate nephrotoxicant, dichlorovinyl cysteine. Dichlorovinyl cysteine is then bioactivated with the release of ammonia. This sequential degradation explains the latency of DCVG-induced renal transport inhibition relative to dichlorovinyl cysteine. It is now evident that certain halogenated ethylenes are capable of being biotransformed to glutathione conjugates in the kidney with their subsequent hydrolysis to nephrotoxic cysteine conjugates.     

Characterization of proteins secreted by sheep oviduct epithelial cells and their function in embryonic development

The role in early development of proteins secreted by oviduct epithelial cells has been investigated. Secreted proteins devoid of serum contamination have been produced by the surgical removal and immediate incubation of oviduct cells in [35S]methionine-containing medium. After electrophoretic separation, secreted polypeptides could be divided into those that were secreted uniformly throughout the oestrous cycle and a second class that showed a cyclical pattern of secretion. The first class of proteins represented a small proportion of total output whilst the predominant second class was composed mainly of polypeptides of Mr 92 and 46 x 10(3), respectively. Both of these polypeptide species, referred to as sheep oviduct proteins 92 and 46 (SOP 92, SOP 46), are detected only during the first 4 to 5 days after oestrus when the embryos are located in the oviduct. Oviduct cells collected at oestrus and maintained thereafter in culture secrete the same pattern of proteins and follow the same time course as their counterparts in vivo. The interaction between the oviduct proteins and the developing embryo was studied firstly by determining whether any of the secreted proteins bound to the zona pellucida. The results of iodination studies showed that two polypeptides of Mr 92 and 46 x 10(3), respectively, were bound to the zona pellucida of eggs removed from the oviduct but were absent from eggs that had not had contact with the oviduct epithelium. That these newly acquired proteins represent SOP 92 and 46 is suggested by their electrophoretic mobility and their ability to bind to the zona of follicular eggs when added in vitro and by the fact that they both disappear from the zonae of embryos after exit from the oviduct. The collection of unlabelled secreted proteins enabled us to produce a monoclonal antibody, which was used in the second series of experiments on oviduct-embryo interactions. The results confirmed that SOP 92 binds to the zona pellucida and moreover showed that this protein crosses the zona and becomes associated with the individual blastomeres of the developing embryo. These findings provide evidence that the mammalian oviduct probably plays a direct role in supporting embryonic development through specific polypeptides produced by its epithelium.     

Study of propidium iodide binding to DNA in intact cells by flow cytometry.

We studied the in situ binding of propidium iodide to DNA in fixed human lymphocytes, using flow cytometry. Experimental data of fluorescence emission vs dye concentration and vs cell concentration were obtained. Data were interpreted by means of two different mathematical models specific for the staining reaction, and the binding parameters were obtained by "best-fitting" of the data. A model based on two classes of binding sites with different affinity constants gave the most satisfactory fitting. The accessibility of the in situ chromatin turned out to be reduced with respect to the non in situ accessibility for ethidium bromide as reported in the literature. The present study shows the usefulness of the flow-cytometric technique for probing DNA structure in intact cells.     

Chromatographic resolution of amino acid adducts of aliphatic halides

Numerous xenobiotics are known to be bioactivated and to covalently bind to proteins, but the resulting amino acid adducts (AAAs) are unknown. In this study the AAAs of twelve ¹⁴C-labeled aliphatic halides were examined after formation in an in vitro microsomal system. After exhaustive solvent extraction of the precipitated microsomal protein, the AAAs were isolated by Pronase digestion, followed by filtration through a 500 mol. wt. exclusion membrane. The liberated AAAs were applied to a constant flow DC-4A cation exchange column, resolved by stepwise buffer elution, collected and counted for radioactivity. Column recovery for applied radioactivity was 100 ± 4%. Generally, 1–4 different AAAs (defined by eluting radioactivity) were resolved, with each organohalogen displaying a characteristic elution profile. Methyl iodide, trichloroethylene and 1,2-dichloroethylene had a single major AAA while bromotrichloromethane, 1,2-dibromoethane, 1,1,1-trichloroethane, 1,2-dichloroethane, 1,1,2-trichloroethane, 2-bromo-2-chloro-1,1,1-trifluoroethane, chloroform and carbon tetrachloride had up to 4 AAAs or more, indicating combinations of binding site(s) and reactive intermediate(s). The single AAA formed following incubation of methyl iodide with the microsomes was identified as S-methylcysteine. Thus, this method appears capable of resolving binding sites and is the initial isolation step for identifying specific adducts to proteins.     

Reversible binding of multivalent antigen in the control of B lymphocyte activation.

A model is proposed describing multiple binding of multivalent antigen to specific receptors of immunocompetent cells. The rate at which these multiple bonds can dissociate and the effective valence of the antigen appear to be of the greatest importance in determining the degree of receptor occupancy at equilibrium. In particular, as antigen concentration decreases, only antigens with high number of determinants per molecule maintain the ability to form multiple bonds even if the dissociation rate is relatively high. Assuming that multiple binding is directly linked to lymphocyte activation, this may result in an inverse relation between antigen valence and ability to induce selection of clones with higher affinities.     

The extent of whole‐genome copy number alterations predicts aggressive features in primary melanomas

Recent evidence indicates that melanoma comprises distinct types of tumors and suggests that specific morphological features may help predict its clinical behavior. Using a SNP‐array approach, we quantified chromosomal copy number alterations (CNA) across the whole genome in 41 primary melanomas and found a high degree of heterogeneity in their genomic asset. Association analysis correlating the number and relative length of CNA with clinical, morphological, and dermoscopic attributes of melanoma revealed that features of aggressiveness were strongly linked to the overall amount of genomic damage. Furthermore, we observed that melanoma progression and survival were mainly affected by a low number of large chromosome losses and a high number of small gains. We identified the alterations most frequently associated with aggressive melanoma, and by integrating our data with publicly available gene expression profiles, we identified five genes which expression was found to be necessary for melanoma cells proliferation. In conclusion, this work provides new evidence that the phenotypic heterogeneity of melanoma reflects a parallel genetic diversity and lays the basis to define novel strategies for a more precise prognostic stratification of patients.