D2/D3 dopamine receptor heterodimers exhibit unique functional properties.

Evidence for heterodimerization has recently been provided for dopamine D(1) and adenosine A(1) receptors as well as for dopamine D(2) and somatostatin SSTR(5) receptors. In this paper, we have studied the possibility that D(2) and D(3) receptors interact functionally by forming receptor heterodimers. Initially, we split the two receptors at the level of the third cytoplasmic loop into two fragments. The first, containing transmembrane domains (TM) I to V and the N-terminal part of the third cytoplasmic loop, was named D(2trunk) or D(3trunk), and the second, containing the C-terminal part of the third cytoplasmic loop, TMVI and TMVII, and the C-terminal tail, was named D(2tail) or D(3tail). Then we defined the pharmacological profiles of the homologous (D(2trunk)/D(2tail) and D(3trunk)/D(3tail)) as well as of the heterologous (D(2trunk)/D(3tail) and D(3trunk)/D(2tail)) cotransfected receptor fragments. The pharmacological profile of the cross-cotransfected fragments was different from that of the native D(2) or D(3) receptors. In most cases, the D(3trunk)/D(2tail) was the one with the highest affinity for most agonists and antagonists. Moreover, we observed that all of these receptor fragments reduced the expression of the wild type dopamine D(2) and D(3) receptors, suggesting that D(2) and D(3) receptors can form complexes with these fragments and that these complexes bind [(3)H]nemonapride less efficiently or are not correctly targeted to the membrane. In a second set of experiments, we tested the ability of the split and the wild type receptors to inhibit adenylyl cyclase (AC) types V and VI. All of the native and split receptors inhibited AC-V and AC-VI, with the exception of D(3), which was unable to inhibit AC-VI. We therefore studied the ability of D(2) and D(3) to interact functionally with one another to inhibit AC-VI. We found that with D(2) alone, R-(+)-7-hydroxydypropylaminotetralin hydrobromide inhibited AC-VI with an IC(50) of 2.05 +/- 0.15 nm, while in the presence of D(2) and D(3) it inhibited AC-VI with an IC(50) of 0.083 +/- 0.011 nm. Similar results were obtained with a chimeric cyclase made from AC-V and AC-VI. Coimmunoprecipitation experiments indicate that D(2) and D(3) receptors are capable of physical interaction

Predicting and controlling the reactivity of immune cell populations against cancer

Heterogeneous cell populations form an interconnected network that determine their collective output. One example of such a heterogeneous immune population is tumor-infiltrating lymphocytes (TILs), whose output can be measured in terms of its reactivity against tumors. While the degree of reactivity varies considerably between different TILs, ranging from null to a potent response, the underlying network that governs the reactivity is poorly understood. Here, we asked whether one can predict and even control this reactivity. To address this we measured the subpopulation compositions of 91 TILs surgically removed from 27 metastatic melanoma patients. Despite the large number of subpopulations compositions, we were able to computationally extract a simple set of subpopulation-based rules that accurately predict the degree of reactivity. This raised the conjecture of whether one could control reactivity of TILs by manipulating their subpopulation composition. Remarkably, by rationally enriching and depleting selected subsets of subpopulations, we were able to restore anti-tumor reactivity to nonreactive TILs. Altogether, this work describes a general framework for predicting and controlling the output of a cell mixture

Salivary characteristics of diabetic children

Salivary components may suffer variations that can be detected by chemical determinations. The aim of this work was to determine physical and biochemical characteristics of the saliva of a group of diabetic children compared to those of a control group. Relation to oral health indices was also determined. Twenty diabetic children (3-15-years-old) and 21 control children (5-12-years-old) were included in this study. Total proteins, sugars and calcium were determined by colorimetric methods, and glucose, urea, alpha-amylase and acid phosphatase by enzymatic methods. Our results demonstrated that acidic pH, diminished salivary flow rate and excess foam are usually present in saliva of diabetic children. Total sugars, glucose, urea and total proteins were greater in diabetic patients than controls, while calcium values were decreased. These differences were confirmed by the discrimination test. Diabetic children have higher DMFT-dmft-deft and DMFS-dmfs-defs values compared to those of the control children despite their lower sugar intake. Some salivary components in addition to the diminished flow rate could be involved in the characterization of the oral health state of diabetic children

Mechanism of action of drugs of abuse

Drug abuse is one of the most notorious socioeconomic problems of our time. In addition to the direct human affliction, the use of drugs of abuse (such as opiates and cannabinoids) is a major factor in urban criminality and in the spread of infectious diseases, including AIDS. Moreover, both opiates and cannabinoids have important beneficial medical properties (e.g. morphine in surgery), but the use of such drugs is currently legally restricted due to their addictive properties, thus limiting their medical use. A. Regulation of adenylyl cyclase by acute and chronic opiate exposure Little is known about the molecular mechanisms by which chronic opiate exposure leads to the development of opiate tolerance and dependence, and to the subsequent phase of opiate withdrawal (which is the major reason for the reluctance by abusers to give up drugs). Our goals are to characterize the biochemical changes accompanying chronic opiate treatment and the withdrawal phase. Along these lines, we were able to show (using CHO cells transfected with µ or κ opiate receptors) that while acute exposure to opiate agonists leads to adenylyl cyclase (AC) inhibition, chronic exposure results in AC superactivation (particularly evident upon withdrawal of the inhibitory opiate agonist). Nine types of AC isozymes, which differ in their tissue distribution and in their stimulation/inhibition patterns, are currently known. Utilizing COS-7 cells transfected with µ-opiate receptor and the individual AC isozymes, we found that the AC isozymes could be divided into three groups: (i) AC-I, V, VI and VIII are inhibited by acute and superactivated by chronic opiate exposure, with AC-V (known to be localized to brain areas involved in drug addiction) yielding the largest superactivation; (ii) AC-II, IV and VII are stimulated by acute opiate exposure and do not show superactivation; and (iii) AC-III is not significantly affected by opiate exposure. Moreover, we showed that the phenomenon of AC superactivation is of a general nature, and AC-I, V, VI and VIII are superactivated following chronic activation by agonists of other Gi/o-coupled receptors (e.g. CB1 cannabinoid, D 2 -dopaminergic, and m 2 -and m 4 -muscarinic). The second step of this research work involved investigating the role of G βγ dimers (released from G αi/o upon receptor activation) in AC regulation. Using transfected COS cells, we confirmed that AC-I is inhibited and AC-II is stimulated by G β1γ2 . However, we found that different G β subunits differ in their regulation of AC activity, with G β1 stimulating, and G β5 (an isoform abundant in brain) inhibiting AC-II. G β1 was also found to inhibit AC-V and VI (a weaker inhibition was observed with G β5 ). Moreover, G βγ scavengers increased the activity of AC-V and VI and prevented AC superactivation, suggesting that endogenous G βγ tonically inhibits the activity of these AC isozymes, and that this inhibition is reversed by chronic agonist exposure Future Plans: Employing molecular biological techniques, we plan to map the G βγ binding site(s) and to study the role of the G βγ subunits in AC superactivation. We will also study the effects of chronic opiates on other signaling pathways. B. Properties of endogenous cannabinoid ligands In collaboration with Professor Raphael Mechoulam (Hebrew University, Jerusalem), we searched for endogenous materials in brain and other tissues which interact with cannabinoid receptors. This search led to the discovery of two families of endogenous cannabinoid ligands, including that of anandamide (arachidonoylethanolamide) and of 2-arachidonoyl-glycerol. We found that these materials bind and activate the "brain cannabinoid receptor" (CB1), located on neural cells, and to a lesser extent the "peripheral cannabinoid receptor" (CB2), expressed in cells of the immune system