Out-of-field doses for scanning proton radiotherapy of shallowly located paediatric tumours-a comparison of range shifter and 3D printed compensator.

The lowest possible energy of proton scanning beam in cyclotron proton therapy facilities is typically between 60 and 100 MeV. Treatment of superficial lesions requires a pre-absorber to deliver doses to shallower volumes. In most of the cases a range shifter (RS) is used, but as an alternative solution, a patient-specific 3D printed proton beam compensator (BC) can be applied. A BC enables further reduction of the air gap and consequently reduction of beam scattering. Such pre-absorbers are additional sources of secondary radiation. The aim of this work was the comparison of RS and BC with respect to out-of-field doses for a simulated treatment of superficial paediatric brain tumours. EURADOS WG9 performed comparative measurements of scattered radiation in the Proteus C-235 IBA facility (Cyclotron Centre Bronowice at the Institute of Nuclear Physics, CCB IFJ PAN, Kraków, Poland) using two anthropomorphic phantoms-5 and 10 yr old-for a superficial target in the brain. Both active detectors located inside the therapy room, and passive detectors placed inside the phantoms were used. Measurements were supplemented by Monte Carlo simulation of the radiation transport. For the applied 3D printed pre-absorbers, out-of-field doses from both secondary photons and neutrons were lower than for RS. Measurements with active environmental dosimeters at five positions inside the therapy room indicated that the RS/BC ratio of the out-of-field dose was also higher than one, with a maximum of 1.7. Photon dose inside phantoms leads to higher out-of-field doses for RS than BC to almost all organs with the highest RS/BC ratio 12.5 and 13.2 for breasts for 5 and 10 yr old phantoms, respectively. For organs closest to the isocentre such as the thyroid, neutron doses were lower for BC than RS due to neutrons moderation in the target volume, but for more distant organs like bladder-conversely-lower doses for RS than BC were observed. The use of 3D printed BC as the pre-absorber placed in the near vicinity of patient in the treatment of superficial tumours does not result in the increase of secondary radiation compared to the treatment with RS, placed far from the patient.

Raman spectroscopy and PCA-SVM as a non-invasive diagnostic tool to identify and classify qualitatively glycated hemoglobin levels in vivo.

In this study we identify and classify high and low levels of glycated hemoglobin (HbA1c) in healthy volunteers (HV) and diabetic patients (DP). Overall, 86 subjects were evaluated. The Raman spectrum was measured in three anatomical regions of the body: index fingertip, right ear lobe, and forehead. The measurements were performed to compare the difference between the HV and DP (22 well controlled diabetic patients (WCDP) (HbA1c <6.5%), and 49 not controlled diabetic patients (NCDP) (HbA1c ≥6.5%)). Multivariable methods such as principal components analysis (PCA) combined with support vector machine (SVM) were used to develop effective diagnostic algorithms for classification among these groups. The forehead of HV versus WCDP showed the highest sensitivity (100%) and specificity (100%). Sensitivity (100%) and specificity (60%), were highest in the forehead of WCDP, versus NCDP. In HV versus NCDP, the fingertip had the highest sensitivity (100%) and specificity (80%). The efficacy of the diagnostic algorithm by receiver operating characteristic (ROC) curve was confirmed. Overall, our study demonstrated that the combination of Raman spectroscopy and PCA-SVM are feasible non-invasive diagnostic tool in diabetes to classify qualitatively high and low levels of HbA1c in vivo.

Nicotine-induced up regulation of α4ß2 neuronal nicotinic receptors is mediated by the protein kinase C-dependent phosphorylation of α4 subunits.

Sustained exposure to nicotine is well known to increase the cell surface density of α4ß2* neuronal nicotinic receptors both in vivo and in vitro, but the cellular mechanisms mediating this effect are equivocal. Using a pharmacological approach to investigate the effects of nicotine on receptor subunit expression and phosphorylation in SH-EP1 cells expressing human α4 and ß2 nicotinic receptor subunits, we have demonstrated that incubation with nicotine for 24 h increased the expression of immature and mature forms of both α4 and ß2 subunits in a concentration-dependent manner, and that inhibition of protein kinase C (PKC), but not cAMP-dependent protein kinase (PKA) inhibited the nicotine-induced increased expression of subunits. Incubation of cells with nicotine for 24 h also increased the phosphorylation of immature forms of α4 subunits similar to that induced by activation of either PKC or PKA. When cells were preincubated with nicotine, the PKC-mediated increased phosphorylation was inhibited; the PKA-mediated phosphorylation was unaltered. The phosphopeptide maps for immature α4 subunits following nicotine exposure or PKC activation were identical, and phosphoamino acid analyses indicated phosphorylation on serine residues only. Results indicate that nicotine-induced up regulation of α4ß2 neuronal nicotinic receptors involves a PKC-dependent mechanism and likely reflects the ability of nicotine to activate PKC, leading to the phosphorylation of immature α4 subunits, promoting subunit assembly and receptor maturation. Because up regulation of these receptors has been implicated to mediate tolerance, locomotor sensitization and addiction to nicotine, results identify a potential new target for modulating the effects of nicotine on the brain.

Theoretical and experimental study of negative LiF clusters produced by fast ion impact on a polycrystalline 7LiF target.

The emission of negative cluster ions produced by the impact of approximately 60 MeV (252)Cf fission fragments on a (7)LiF polycrystalline target is analyzed. The negative ion mass spectrum is dominated by the ((7)LiF)(n)F(-) series, n = 1 to approximately 30. The desorption yield distribution of the ((7)LiF)(n)F(-) members has a maximum at n = 2 and then decreases as the sum of two exponentials whose decay parameters are k(Fast) = 0.9 and k(Slow) = 0.08. These k values are the same as those observed for the positive series and close to others obtained for condensed gas targets. Relative cluster ion stabilities, deduced from the experimental ion abundances for the (LiF)(n)F(-) series, are proposed to be correlated with theoretical structures according to their internal energy by using the deviation plot (D-plot) methodology. A pool of candidate cluster structures was generated using a genetic algorithm and further analyzed and optimized using density functional theory (DFT) with the hybrid functional B3LYP (DFT/B3LYP) and Moller-Plesset perturbation theory (MP2). For the small clusters (n = 1 to 2), the most stable structures are found to be linear, whereas the larger clusters (n = 4 to 6) present cubic or polyhedral structures. Fragmentation energies, ionization potentials, and relative stabilities are reported for the most abundant families of the (LiF)(n)F(-) and (LiF)(n)(-) series.

A theoretical and experimental study of positive and neutral LiF clusters produced by fast ion impact on a polycrystalline LiF target.

The positive and neutral clusters produced by the impact of approximately 60 MeV (252)Cf fission fragments on a LiF polycrystalline target are analyzed. The positive ion spectrum is dominated by the (LiF)(n)Li(+) series, n = 0-7, exhibiting a total yield 2 orders of magnitude higher than that of the (LiF)(n)(+) series. The yield for the dominant (LiF)(n)Li(+) series decreases roughly as exp(-kn), where k approximately 0.9 for n = 0-3 and k approximately 0.6 for the heavier clusters (n = 4-9), while the yield of the (LiF)(n)(+) series also decreases exponentially as n increases with k approximately 0.6. Theoretical calculations were performed for the (LiF)(n)Li(0), (LiF)(n)Li(+), and (LiF)(n)(0) series for n up to 9. For the smaller clusters the structures first obtained with a genetic algorithm generator were further optimized at the DFT/B3LYP/6-311+G(3df), DFT/B3LYP/LACV3P*, and MP2/LACV3P* levels of theory. An energy criterion is used for a proper taxonomic description of the optimized cluster isomers. Cluster properties such as fragmentation energy and stability are discussed for the proposed configurations. The results show that for all three series the most stable isomers present a linear structure for small cluster size (n = 1-3), while cubic cells or polyhedral structures are preferred for larger cluster sizes (n = 4-9). Fragmentation energy results suggest that a desorbed excited (LiF)(n)Li(+) ion preferentially dissociates via a cascade of (LiF)(n)(0) units, in agreement with the slope modification in the exponential decay of the (LiF)(n)Li(+) ion abundances for n > or = 3.

Competitive and uncompetitive inhibitors simultaneously acting on an autocatalytic zymogen activation reaction.

The time course of the residual enzyme activity of a general model consisting of an autocatalytic zymogen activation process inhibited by an irreversible competitive inhibitor and an irreversible uncompetitive inhibitor has been studied. Approached analytical expressions which furnish the time course of the residual enzyme activity from the onset of the reaction depending on the rate constants and initial concentration have been obtained. The goodness and limitations of the analytical equations were checked by comparing with the results obtained from the numerical integration, i.e. with the simulated progress curves. A dimensionless parameter giving the relative contributions of both the activation and the inhibitions routes is suggested, so that the value of this parameter determines whether the activation or the inhibitions routes prevail or if both processes are balanced during the time for which the analytical expressions are valid. The effects of the initial zymogen, free enzyme and inhibitors concentrations are analysed. Finally an experimental design and kinetic data analysis is proposed to evaluate simultaneously the kinetic parameters involved and to discriminate between different zymogen activation processes which can be considered particular cases of the general model.

Consenso sobre a transdisciplinaridade na medicina periodontal 2005 / Consensus on periodontal medicine transdisciplinarity 2005

Objetivo : estabelecer diretrizes para as investigações sobre a transdisciplinaridade na Medicina Periodontal. Métodos: representantes das diferentes especialidades que estudam e pesquisam diversoa aspectos da Medicina Periodontal participaram do II Seminário de Periodontia em Saúde Coletiva - Uma Visão Transdisciplinar na Medicina Periodontal (Feira de Santana-Bahia, 2005) e prepararam este consenso, no qual estabeleceram as diretrizes para as investigações sobre a transdisciplinaridade na Medicina Periodontal, particularmente na questão da possível associação entre a doença periodontal e a prematuridade e baixo peso ao nascer. Resultados: Este consenso foi produzido após a apresentação de cada questão e extensa discussão pelos participantes do evento

[Determination of colonic transit time in healthy subjects in Mexico City. Prospective study]. / Determinación del tiempo de tránsito colónico en personas sanas de la Ciudad de México. Estudio prospectivo.

UNLABELLED: Colonic transit time (CTT) is determined by multiple factors; currently, normal values for the Mexican population are not available. In order to get an estimate one must look at the values reported in the international literature, but cultural, ethnical, nutritional and economic differences may lead to different values. OBJECTIVE: To determine the normal values of colonic transit time in healthy people in Mexico City by the use of radiopaque markers. PATIENTS AND METHODS: Prospective, longitudinal and observational study, which included healthy patients ranging from 18 to 60 years old; excluding pregnant women. The whole group of patients was given before breakfast a gelatin capsule which had 20 radiopaque markers inside -the markers were each 2mm long, and were made by the researcher-. After that, they were taken a simple abdominal X-ray film every 24 hours until they totally eliminated the markers. Their eating and defecation habits were evaluated and also the total amount of liquid they consumed. Inferential statistics were used; data was validated with both parametric and non-parametric tests, considering a significance of p < 0.05. RESULTS: A hundred patients were included in the sample in which 48% were female and 52% male, they were divided in three groups: group A (31%)from 18 to 25 years, group B (37%)from 26 to 40 and group C (32%)from 41 to 60 years; there were no important differences in their water consumption, which was in average of 1.87 lts. in 24 hours; also, there were no considerable differences regarding to their meat, vegetables and fruits' consumption, which was in average of 4.4 times a week; the whole group eliminated the markers according to X-rays which was in 54% after 72 hrs, 45% after 48 hrs and 1% after 24 hrs. We can observe an increase of the CTT related to age: in group C 94% eliminated the markers after 72 hrs and there was no significant difference (statistically) with regards to the other groups. A tendency of an increase of CTT with regards to age was observed: in group A, 80% eliminated the markers after 48 hrs, in group B 49% eliminated them after 48 hrs and 51% after 72 hrs and, in group C, 94% eliminated them after 72 hrs without any statistically significant differences among the study groups. CONCLUSION: The CTT in healthy patients is in a 100% of the cases studied lower or equal to 72 hrs with a tendency to increase in relation to age.

Characterization of human alpha4beta2 neuronal nicotinic receptors stably expressed in SH-EP1 cells.

These studies characterized human alpha4beta2 neuronal nicotinic receptors stably expressed in a human epithelial cell line (SH-EP1). Receptors in transfected SH-EPI-halpha4beta2 cells were functional, as determined by increases in intracellular Ca2+ in response to a nicotine stimulus. Nicotine increased Fura-2 fluorescence in a concentration-dependent manner with an apparent EC50 of 2.4 microM, a response that was blocked by the specific antagonist mecamylamine. When cells were incubated in 50 nM nicotine for 24 hours, the Ca2+ response inactivated by 44%, an effect that recovered within 24 hours. SH-EP1-halpha4beta2 cells expressed a single class of high affinity binding sites for [3H]cytisine with a Kd of 0.63 +/- 0.08 nM and a Bmax of 6,797 +/- 732 femtomoles/mg protein. Incubation of cells with 50 nM nicotine for 24 hours increased the Bmax by 45% without changing affinity, a concentration-dependent effect with an EC50, of 58.6 nM. The nicotine-induced up regulation was reversible, and control values were achieved within 24 hours. Results indicate that SH-EPI-halpha4beta2 cells may be a good model system to study regulation of human alpha4beta2 receptors, the most abundant nicotinic receptor subtype in brain.

Variable length representation in evolutionary electronics.

This work investigates the application of variable length representation (VLR) evolutionary algorithms (EAs) in the field of Evolutionary Electronics. We propose a number of VLR methodologies that can cope with the main issues of variable length evolutionary systems. These issues include the search for efficient ways of sampling a genome space with varying dimensionalities, the task of balancing accuracy and parsimony of the solutions, and the manipulation of non-coding segments. We compare the performance of three proposed VLR approaches to sample the genome space: Increasing Length Genotypes, Oscillating Length Genotypes, and Uniformly Distributed Initial Population strategies. The advantages of reusing genetic material to replace non-coding segments are also emphasized in this work. It is shown, through examples in both analog and digital electronics, that the variable length genotype's representation is natural to this particular domain of application. A brief discussion on biological genome evolution is also provided.

Modulation of carbachol-stimulated AP-1 DNA binding activity by therapeutic agents for bipolar disorder in human neuroblastoma SH-SY5Y cells.

Lithium, carbamazepine and sodium valproate are mood stabilizers used in the treatment of bipolar disorder, and although their mechanisms of action remain unknown, signal transduction systems and the associated modulation of gene expression may constitute significant actions. We examined if acute or chronic treatments with these agents modulated the activation of the AP-1 transcription factor or the increased intracellular calcium levels in human neuroblastoma SH-SY5Y cells caused by stimulation with carbachol. AP-1 activation stimulated by carbachol was reduced by pretreatment for 1 h, 24 h or 7 days with 1 mM lithium by 15%, 37%, and 60%, respectively, and with 0.05 mM carbamazepine by 3%, 21%, and 46%, respectively, but not by pretreatment with 0.5 mM sodium valproate. AP-1 DNA binding activity stimulated by carbachol or by phorbol ester-induced activation of protein kinase C was inhibited by the protein kinase C inhibitor Ro31-8220, but phorbol ester-stimulated AP-1 activation was unaltered by 7-day pretreatments with lithium or carbamazepine. Activation of AP-1 by carbachol was dependent on calcium, as it was inhibited by treatment with the extracellular calcium chelator EGTA, the intracellular calcium chelator BAPTA-AM, and the calcium/calmodulin kinase II inhibitor KN62. Pretreatment for 7 days with lithium or carbamazepine had no significant effect on carbachol-stimulated increases in intracellular calcium levels, but reduced the stimulation of AP-1 by the calcium ionophore ionomycin by 30% to 40%. Thus, chronic treatment with the antibipolar agents lithium and carbamazepine attenuates carbachol-stimulated AP-1 DNA binding activity, and these agents preferentially inhibit signaling cascades activated by the calcium rather than the protein kinase C arm of the phosphoinositide signaling pathway.

Selective increases in phosphoinositide signaling activity and G protein levels in postmortem brain from subjects with schizophrenia or alcohol dependence.

Comparisons of the activity of the G protein-mediated phosphoinositide signal transduction system and of G protein levels were made in two regions of frontal cortex from eight schizophrenic, alcohol-dependent, and control subjects. G protein-mediated phosphoinositide hydrolysis was measured by stimulating cortical membranes incubated with [3H]phosphatidylinositol with 0.3-10 microM guanosine 5'-O-(3-thio)triphosphate (GTPgammaS). In frontal cortex areas 8/9, GTPgammaS-induced phosphoinositide hydrolysis was 50% greater in schizophrenic than control or alcohol-dependent subjects, whereas there were no differences among these groups of subjects in the response to GTPgammaS in frontal cortex area 10. Agonists for dopaminergic, cholinergic, purinergic, serotonergic, histaminergic, and glutamatergic receptors coupled to the phosphoinositide signaling system increased [3H]phosphatidylinositol hydrolysis in a GTPgammaS-dependent manner. Responses to most agonists were similar in all three subject groups in both cortical regions, with the largest difference being a 40% greater response to dopaminergic receptor stimulation in frontal cortex 8/9 from schizophrenic subjects. Measurements of the levels of phospholipase C-beta, and of alpha-subunits of Gq, Go, Gi1, Gi2, and Gs, made by immunoblot analyses revealed no differences among the groups of subjects except for increased G alpha(o) in schizophrenic subjects and increased G alpha(o) and G alpha(i1) in alcohol-dependent subjects. These results demonstrate that schizophrenia is associated with increased activity of the phosphoinositide signal transduction system and increased levels of G alpha(o), whereas the phosphoinositide system was unaltered in alcohol dependence, but G alpha(o) and G alpha(i1) were increased.

Binding of aminoalkylindoles to noncannabinoid binding sites in NG108-15 cells.

1. Aminoalkylindoles, typified by WIN 55212-2, bind to G protein-coupled cannabinoid receptors in brain. Although cannabinoids inhibit adenylyl cyclase in NG108-15 neuroblastoma x glioma hybrid cells, cannabinoid receptor binding in these cells has not been described previously. This study compares pharmacological characteristics of [3H]WIN 55212-2 binding sites in rat cerebellar membranes and in NG108-15 membranes. 2. Although the KD of specified [3H]WIN 55212-2 binding was similar in brain and NG108-15 membranes, the Bmax was 10 times lower in NG108-15 than in cerebellar membranes. In both brain and NG108-15 membranes, aminoalkylindole analogues were relatively potent in displacing [3H]WIN 55212-2 binding. However, IC50 values for more traditional cannabinoids were significantly higher in NG108-15 membranes than in brain, e.g., the Ki values for CP55,940 were 1.2 nM in brain and > 5000nM in NG108-15 membranes. Moreover, sodium and GTP-gamma-S decreased [3H]WIN 55212-2 binding in brain but not in NG108-15 membranes. 3. These data suggest that WIN 55212-2 does not label traditional cannabinoid receptors in NG108-15 cells and that these novel aminoalkylindole binding sites are not coupled to G proteins.

Comparison of [3H]phosphatidylinositol and [3H]phosphatidylinositol 4,5-bisphosphate hydrolysis in postmortem human brain membranes and characterization of stimulation by dopamine D1 receptors.

Assessing the function of the phosphoinositide signal transduction system in membranes prepared from postmortem human brain by measuring the hydrolysis of exogenous labeled phosphoinositides has been applied to studies of a variety of CNS disorders in recent years. Two issues concerning such studies were addressed in the current investigation: how do [3H]phosphatidylinositol and [3H]phosphatidylinositol 4,5-bisphosphate compare as substrates, and how do dopamine D1 receptors influence phosphoinositide signaling? Comparisons of [3H]phosphatidylinositol and [3H] phosphatidylinositol 4,5-bisphosphate hydrolysis stimulated by guanosine-5'-O-(3-thiotriphosphate)-activated G proteins and by several receptor agonists demonstrated that in most cases each substrate gave similar relative results in membranes prepared from prefrontal cortices of six individuals. However, using optimal assay conditions, [3H]phosphatidylinositol produced a greater signal-to-noise ratio compared with [3H] phosphatidylinositol 4,5-bisphosphate. Dopamine D1 receptors were demonstrated to be directly coupled to phosphoinositide hydrolysis in human brain membranes, and this response was shown to be mediated by the G(q/11) G protein subtype and by the beta-subtype of phospholipase C. Therefore, these results demonstrate that [3H]phosphatidylinositol is a suitable substrate to measure phosphoinositide hydrolysis in human brain membranes and that dopamine D1 receptors directly stimulate this signaling system.

Phosphoinositide signaling in human brain.

The phosphoinositide signal transduction system constitutes one of the primary means for intercellular communication in the central nervous system, but only recently has this system been studied in human brain. Although some investigations have studied phosphoinositide signaling in slices from biopsied human brain, due to the limited access to such material a greater number of studies have utilized membranes prepared from postmortem human brain. With membranes exposed to exogenous labeled phosphoinositides, activation of phospholipase C with calcium, with G-proteins stimulated by GTP gamma S or NaF, or with several receptor agonists, have demonstrated that all of the components of the phosphoinositide system are retained in human brain membranes and are responsive to appropriate stimuli. Investigators have begun to examine the effects of neurological (Alzheimer's disease, epilepsy, Parkinson's disease) and psychiatric (schizophrenia, major depression, bipolar affective disorder) diseases on the activity of the phosphoinositide system. Alzheimer's disease has been studied to the greatest extent and a severe deficit in phosphoinositide signaling has been identified in most studies. In addition, brain regionally selective deficits in G-protein function associated with phosphoinositide signaling have been reported in subjects with major depression or with bipolar affective disorder, and in the latter an ameliorative effect of the therapeutic drug lithium was identified. Although significant progress has been achieved in studying the phosphoinositide system in human brain, many issues remaining to be addressed are discussed in this review. With carefully controlled studies, it appears that much will be learned in the near future about the phosphoinositide signal transduction system in human brain and the effects of a variety of disorders on its function.

The phosphoinositide signal transduction system is impaired in bipolar affective disorder brain.

The function of the phosphoinositide second messenger system was assessed in occipital, temporal, and frontal cortex obtained postmortem from subjects with bipolar affective disorder and matched controls by measuring the hydrolysis of [3H]phosphatidylinositol ([3H]PI) incubated with membrane preparations and several different stimulatory agents. Phospholipase C activity, measured in the presence of 0.1 mM Ca2+ to stimulate the enzyme, was not different in bipolar and control samples. G proteins coupled to phospholipase C were concentration-dependently activated by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and by NaF. GTP gamma S-stimulated [3H]Pl hydrolysis was markedly lower (50%) at all tested concentrations (0.3-10 microM GTP gamma S) in occipital cortical membranes from bipolar compared with control subjects. Responses to GTP gamma S in temporal and frontal cortical membranes were similar in bipolars and controls, as were responses to NaF in all three regions. Brain lithium concentrations correlated directly with GTP gamma S-stimulated [3H]Pl hydrolysis in bipolar occipital, but not temporal or frontal, cortex. Carbachol, histamine, trans-1-aminocyclopentyl-1,3-dicarboxylic acid, serotonin, and ATP each activated [3H]Pl hydrolysis above that obtained with GTP gamma S alone, and these responses were similar in bipolars and controls except for deficits in the responses to carbachol and serotonin in the occipital cortex, which were equivalent to the deficit detected with GTP gamma S alone. Thus, among the three cortical regions examined there was a selective impairment in G protein-stimulated [3H]Pl hydrolysis in occipital cortical membranes from bipolar compared with control subjects. These results directly demonstrate decreased activity of the phosphoinositide signal transduction system in specific brain regions in bipolar affective disorder.

Alterations in phosphoinositide signaling and G-protein levels in depressed suicide brain.

The function of the phosphoinositide signal transduction system and the levels of heterotrimeric G-protein alpha-subunits were examined in postmortem prefrontal cortex regions (8/9) and region (10) from suicide victims with major depression and matched control subjects without psychiatric illness. The hydrolysis of [3H]phosphatidylinositol (PI) stimulated by phospholipase C, GTP-gamma-S, NaF, and neurotransmitter receptor agonists was measured in membrane preparations from both groups. Phospholipase C-beta activity was similar in depressed suicide and control subjects in the two regions of prefrontal cortex. In prefrontal cortex (10), but not in (8/9), the GTP-gamma-S concentration-dependent stimulation of [3H]PI hydrolysis was significantly lower (30%) in the depressed suicide group compared to the control group. Receptor-coupled, G-protein-mediated [3H]PI hydrolysis induced with carbachol, histamine, trans-1-aminocyclopentyl-1, 3-dicarboxylic acid (ACPD, a glutamatergic metabotropic receptor agonist), serotonin, or 2-methylthio-adenosine triphosphate (2mATP, a purinergic receptor agonist) in the presence of GTP-gamma-S stimulated equivalent responses in the two groups of subjects in each brain region. In prefrontal cortex (10) there was a 68% increase in the level of the 45 kDa subtype of G alpha s and in prefrontal cortex (8/9) there was a significant decrease (21%) in the level of G alpha i2 in the depressed suicide group compared to the control group. Levels of other heterotrimeric G-protein alpha-subunits (G alpha q/11, G alpha i1, and G alpha o) were not different in depressed suicide and control subjects in either brain region. Moreover, there were no differences in the levels of phospholipase C-beta or protein kinase C-alpha in the two groups of subjects in either brain region examined. These results demonstrate that in the prefrontal cortex of suicide victims with major depression compared to normal control subjects there is a region-specific alteration of G-protein-induced activation of the phosphoinositide signal transduction system and in the levels of G-protein alpha-subunits involved in cyclic AMP synthesis. These findings provide direct evidence in human brain that these two important signal transduction systems are altered in suicide subjects with major depression.

Agonists and antagonists differentially regulate the high affinity state of the D2L receptor in human embryonic kidney 293 cells.

Studies with radiolabeled antagonists have revealed that both agonists and antagonists induce up-regulation of D2 dopamine receptors in cells transfected to express D2L or D2S receptors. The regulation induced by agonists, but not antagonists, was synergistic with cAMP analogues, and differences in the time courses of the effects of agonists and antagonists have been observed. These findings have been extended by using a radiolabeled agonist to investigate agonist- and antagonist-induced regulation of the high affinity state of the D2L dopamine receptor in transfected HEK 293 cells. Exposure to agonists decreased the proportion of receptors in the high affinity, agonist-preferring state. Exposure to antagonists, however, led to an increase in the density of receptors with a high affinity for agonists. The effects of both agonists and antagonists on the agonist-preferring receptors occurred without a lag and were time and dose dependent. Inhibition of forskolin-stimulated cAMP accumulation by agonists was not affected by exposure of the cells to the antagonist (-)-sulpiride. Desensitization was seen after exposing cells to the agonist quinpirole for 1.5 hr, suggesting that the rapid loss of high affinity binding sites represents an uncoupling of the receptor from the G protein that mediates inhibition of adenylyl cyclase. Pretreatment of cells with the protein synthesis inhibitor cycloheximide did not block the quinpirole-induced loss of receptors with a high affinity for agonists. The effect of (-)-sulpiride on high affinity binding sites was blocked by cycloheximide, but only after incubation of cells for sufficient time to induce an increase in the total number of receptors. After incubation of cells with (-)-sulpiride for a short time, the increase in the number of receptors with a high affinity for agonists was unaffected by cycloheximide. These results suggest that the increase in agonist binding after brief exposure to an antagonist is due to interactions of the receptor with one or more G proteins that are not coupled to inhibition of adenylyl cyclase, whereas the increase in agonist binding at later time points is associated with the antagonist-induced up-regulation.

Lack of discrimination by agonists for D2 and D3 dopamine receptors.

The affinities of D3 dopamine receptors for antagonists are similar to those of D2 receptors. D3 receptors have been reported, however, to have affinities nearly 100-fold higher than those of D2 receptors for some agonists, including (+/-)-7-hydroxy-n,n-dipropyl-aminotetralin (7-OH-DPAT) and quinpirole. This has led to the use of these agonists to try to identify functional responses mediated by D3 receptors in vivo. However, D2 receptors exist in multiple states having high and low affinities for agonists. The G protein-coupled state of D2 receptors is believed to be the functional state of these receptors. When receptors were labeled with the D2 receptor antagonist [125I]-(S)-3-iodo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5,6- dimethoxysalicylamide ([125I]-NCQ-298) under conditions that promote uncoupling of receptors from G proteins, the affinities of D3 receptors were approximately 130-fold higher than those of D2 receptors for 7-OH-DPAT and quinpirole. When receptors were labeled with the D2 receptor agonist [125I]-(R)trans-7-hydroxy-2-[N-propyl-N-(3'-iodo-2'- propenyl)-amino]tetralin ([125I]-7-OH-PIPAT) under conditions that favor interactions of receptors with G proteins, the affinities of D3 receptors were less than sevenfold higher than the affinities of D2 receptors for the same drugs. Similarly, small differences in the affinities of D2 and D3 receptors for other agonists were seen when receptors were labeled with [125I]-7-OH-PIPAT. These data demonstrate that putative D3 receptor-selective agonists also interact with a high-affinity, G protein-coupled state of D2 receptors. The similarities in affinities of the agonist-preferring state of D2 and D3 receptors means that currently available agonists cannot be used to discriminate between behavioral effects mediated by D2 and D3 receptors.