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We describe the Blue Native (BN)-PAGE retardation assay for the detection of interactions of biomolecules with protein complexes. Potential interactors of proteins are included in the BN gel matrix, resulting in retardation of proteins that interact with the added molecule. After validation using the T-cell antigen receptor, we applied the assay for a general identification of dextran interactors in combination with mass spectroscopy. The proteomic screen revealed triosephosphate isomerase oligomer as a dextran-binding, high M(R) complex.

Recently, we have described a bispecific PSMA x CD3 diabody with one binding site for the T-cell antigen receptor (TCR-CD3) and another for the Prostate Specific Membrane Antigen (PSMA). It effectively eliminates human prostate cancer cells by redirecting T-lymphocytes in vitro and in vivo. Here, we show that activation of the T-cells and killing of the tumor cells, only occurred when the T-cells were coincubated with PSMA-positive tumor cells and the PSMA x CD3 diabody. Both CD4+ and CD8+ human T-lymphocytes were activated. Surprisingly, they were equally potent in their cytotoxic activity, proliferation, and up-regulation of activation markers. Both, CD4+, and CD8+ T-cells mainly used the perforin-granzyme- based pathway and to a somewhat lesser extent the FasL pathway to lyse tumor cells. When Jurkat T-cells were stimulated with the diabody alone, the TCR-CD3 was not triggered. In contrast, when the diabody was clustered with a secondary antibody the TCR-CD3 was stimulated as detected by Ca(2+)-influx and Erk, IkappaB, and linker of activated T-cell phosphorylation. Clustering of the diabody could also be achieved by the dimeric PSMA antigen expressed on tumor cells. Thus, although the diabody binds to all T-cells, only those in contact with PSMA-expressing cancer cells are activated. In conclusion, the PSMA x CD3 diabody is suitable for a controlled polyclonal T-cell therapy of prostate cancer.

Summary: Costimulatory receptors and ligands trigger the signaling pathways that are responsible for modulating the strength, course, and duration of an immune response. High-resolution structures have provided invaluable mechanistic insights by defining the chemical and physical features underlying costimulatory receptor:ligand specificity, affinity, oligomeric state, and valency. Furthermore, these structures revealed general architectural features that are important for the integration of these interactions and their associated signaling pathways into overall cellular physiology. Recent technological advances in structural biology promise unprecedented opportunities for furthering our understanding of the structural features and mechanisms that govern costimulation. In this review, we highlight unique insights that have been revealed by structures of costimulatory molecules from the immunoglobulin and tumor necrosis factor superfamilies and describe a vision for future structural and mechanistic analysis of costimulation. This vision includes simple strategies for the selection of candidate molecules for structure determination and highlights the critical role of structure in the design of mutant costimulatory molecules for the generation of in vivo structure-function correlations in a mammalian model system. This integrated 'atoms-to-animals' paradigm provides a comprehensive approach for defining atomic and molecular mechanisms.

The killer cell lectin-like receptor G1 (KLRG1) is an inhibitory receptor expressed by memory T cells and NK cells in man and mice. It is frequently used as a cell differentiation marker and members of the cadherin family are ligands for KLRG1. The present study provides new insights into the interaction of mouse KLRG1 with E-cadherin. Firstly, we demonstrate that co-engagement of KLRG1 and CD3/TCR in a spatially linked manner was required for inhibition arguing against the notion that KLRG1-ligation per se transmits inhibitory signals. Secondly, experiments with T cells carrying Y(7)F-mutant KLRG1 molecules with a replacement of the tyrosine residue to phenylalanine in the single ITIM indicated that the inhibitory activity of KLRG1 is counteracted to some degree by increased interaction of KLRG1(+) T cells with E-cadherin expressing target cells. Thirdly, we demonstrate that deletion of the first or the second external domain of E-cadherin abolished reactivity in KLRG1-reporter cell assays. Finally, we made the intriguing observation that KLRG1 formed multimeric protein complexes in T cells in addition to the previously described mono- and dimeric molecules.

Background: North American and European genome-wide association scans have identified ATG16L1 and IL23R as novel inflammatory bowel disease (IBD) susceptibility genes and subsequent reports confirmed these findings in large independent populations. The aims of this study were to investigate the association and examine genotype-phenotype relationships in a Hungarian IBD cohort., Methods: 415 unrelated IBD patients (CD: 266, age: 35.2+/-12.1 years, duration: 8.7+/-7.5 years and UC: 149, age: 44.4+/-15.4 years, duration: 10.7+/-8.9 years) and 149 healthy subjects were investigated. IL23R Arg381Gln (R381Q, rs11209026) and ATG16L1 Thr300Ala (T300A, rs2241880) polymorphisms were tested using LightCycler allele discrimination method. Detailed clinical phenotypes were determined by reviewing the medical charts., Results: The association between IL23R rs11209026, ATG16L1 rs2241880 and CD was confirmed (OR(IL23R381Q): 0.38, 95% CI: 0.16-0.87; OR(ATG16L1300AA): 1.86, 95% CI: 1.04-3.40). No difference was found between patients with UC and either controls or CD. In CD, IL23R 381Gln heterozygosity was associated with inflammatory disease (70% vs. 34%, p=0.037), while disease restricted to the colon was more prevalent in patients with the ATG16L1 300Ala/Ala homozygosity (33.3% vs. 21.1%, p=0.036). In addition, carriage of the variant alleles did not predict response to steroids, infliximab or need for surgery., Conclusions: We confirmed that ATG16L1 and IL23R are susceptibility loci for CD in Hungarian CD patients. Further studies are needed to confirm the reported phenotype-genotype associations found in this study.

Background: Since the first report of the significant gene-environment interaction (G x E) in depression published by Caspi et al., the literature is considerably contradictory in this field. To clarify this question, we analyzed the interaction between the serotonin transporter gene (SLC6A4) and threatening life events (TLE) on Zung Self-Rating Depression Score (ZSDS)., Methods: Five markers tagging the whole SLC6A4 gene (5-HTTLPR and 4 single nucleotide polymorphisms: rs2020942, rs140700, rs3798908, rs1042173) were genotyped in 567 nonclinical individuals. Generalized linear models were used to analyze single marker associations, and likelihood ratio tests and score tests were used for haplotype analysis., Results: Haplotype analysis revealed a significant global effect of haplotypes on ZSDS score in high TLE subgroup (p = .008). Besides the 5-HTTLPR, rs140700 tagging the middle region of the gene had significant effects. Subjects carrying the A allele of rs140700 scored lower on ZSDS independently of 5-HTTLPR carrier status. Explained variances for depressive phenotype were 1%, 4%, and 6% when 5-HTTLPR, 5-HTTLPR x TLE and 5-HTTLPR x rs140700 x TLE were included in the model, respectively., Conclusions: Our results demonstrate heterogeneity of individuals carrying S alleles of 5-HTTLPR in association with high TLE providing possible explanation for the inconsistency of previous studies. In addition to the promoter, the middle region of the SLC6A4 gene carries the G x G x E interaction for mood, and this new model provided a higher explained variance. We report the first evidence for the significant effects of haplotypes of the SLC6A4 gene and threatening life events on depressive phenotype.

The recreational drug ecstasy [3,4-methylenedioxymethamphetamine (MDMA)], has been found to selectively damage brain serotonin neurons in experimental animals, and probably in human MDMA users, but detailed morphometric analyses and parallel functional measures during damage and recovery are missing. Since there is evidence that serotonin regulates sleep, we have compared serotonergic markers parallel with detailed analysis of sleep patterns at three time-points within 180 d after a single dose of 15 mg/kg MDMA in male Dark Agouti rats. At 7 d and 21 d after MDMA treatment, significant(30-40%), widespread reductions in serotonin transporter (5-HTT) density were detected in the cerebral cortex, hippocampus, most parts of the hypothalamus, and some of the brainstem nuclei. With the exception of the hippocampus, general recovery was observed in the brain 180 d after treatment. Transient increases followed by decreases were detected in 5-HTT mRNA expression of dorsal and median raphe nuclei at 7 d and 21 d after the treatment. Significant reductions in rapid eye movement (REM) sleep latency, increases in delta power spectra in non-rapid eye movement sleep and increased fragmentation of sleep were also detected, but all these alterations disappeared by the 180th day. The present data provide evidence for long-term, albeit, except for the hippocampus, transient changes in the terminal and cellular regions of the serotonergic system after this drug. Reduced REM latency and increased sleep fragmentation are the most characteristic alterations of sleep consistently described in depression using EEG sleep polygraphy.

Monoclonal antibodies (MAbs) to a cell surface histone on Histoplasma capsulatum modify murine infection and decrease the growth of H. capsulatum within macrophages. Without the MAbs, H. capsulatum survives within macrophages by modifying the intraphagosomal environment. In the present study, we aimed to analyze the affects of a MAb on macrophage phagosomes. Using transmission electron and fluorescence microscopy, we showed that phagosome activation and maturation are significantly greater when H. capsulatum yeast are opsonized with MAb. The MAb reduced the ability of the organism to regulate the phagosomal pH. Additionally, increased antigen processing and reduced negative costimulation occur in macrophages that phagocytose yeast cells opsonized with MAb, resulting in more-efficient T-cell activation. The MAb alters the intracellular fate of H. capsulatum by affecting the ability of the fungus to regulate the milieu of the phagosome.

MRP2 (ABCC2) is an efflux transporter expressed on the apical membrane of polarized cells. This protein has a major role in the biliary elimination of toxic compounds from the liver. As MRP2 transports many endogenous compounds, including LTC4 as well as xenobiotics and toxic phase II metabolites, blockade of this transporter may cause the accumulation of these compounds in the hepatocyte, resulting in hepatotoxicity. The vesicular transport assay is a great tool to study drug-drug and drug-endogenous compound interactions of ABC transporters. In this assay, inside-out membrane vesicles are used, so the test compound can readily access the transporter. As MRP2 transports many ionic compounds that are difficult to investigate in a whole-cell system because of permeability reasons, the vesicular transport assay is a good choice for screening MRP2-mediated interactions. LTC4 is not an optimal substrate for high-throughput screening for MRP2 interactors, even though it is an important MRP2 substrate. Therefore, the transport of a drug surrogate, 5(6)-carboxy-2,'7'-dichlorofluorescein (CDCF), by MRP2 was characterized using the vesicular transport assay. The data indicate that CDCF proves to be an ideal substrate for MRP2 vesicular transport assay with its optimal detection and transport properties.

Many antigen receptors of the immune system belong to the family of multichain immune recognition receptors (MIRRs). Binding of ligand (antigen) to MIRR results in receptor phosphorylation, triggering downstream signaling pathways and cellular activation. How ligand binding induces this phosphorylation is not yet understood. In this Chapter, we discuss two models exploring the possibility that kinases and phosphatases are intermingled on the cell surface. Thus, in resting state, MIRR phosphorylation is counteracted by dephosphorylation. Upon ligand binding, phosphatases are removed from the vicinity of the MIRR and kinases, such that phosphorylated MIRRs can accumulate (segregation models). In the first model, clustering of MIRRs by multivalent ligand leads to their concentration in lipid rafts where kinases, but not phosphatases, are localized. The second model takes into account that the MIRR-ligandpair needs dose apposition of the two cell membranes, in cases where ligand is presented by an antigen-presenting cell. The intermembrane distance is too small to accommodate transmembrane phosphatases, which possess large ectodomains. Thus, phosphatases become spatially separated from the MIRRs and kinases (kinetic-segregation model).

The rocker mice are hereditary ataxic mutants that carry a point mutation in the gene encoding the CaV2.1 (P/Q-type) Ca2+ channel alpha1 subunit, and show the mildest symptoms among the reported CaV2.1 mutant mice. We studied the basic characteristics of the rocker mutant Ca2+ channel and their impacts on excitatory synaptic transmission in cerebellar Purkinje cells (PCs). In acutely dissociated PC somas, the rocker mutant channel showed a moderate reduction in Ca2+ channel current density, whereas its kinetics and voltage dependency of gating remained nearly normal. Despite the small changes in channel function, synaptic transmission in the parallel fiber (PF)-PC synapses was severely impaired. The climbing fiber inputs onto PCs showed a moderate impairment but could elicit normal complex spikes. Presynaptic function of the PF-PC synapses, however, was unexpectedly almost normal in terms of paired-pulse facilitation, sensitivity to extracellular Ca2+ concentration and glutamate concentration in synaptic clefts. Electron microscopic analyses including freeze-fracture replica labeling revealed that both the number and density of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors substantially decreased without gross structural changes of the PF-PC synapses. We also observed an abnormal arborization of PC dendrites in young adult rocker mice (approximately 1 month old). These lines of evidence suggest that even a moderate dysfunction of CaV2.1 Ca2+ channel can cause substantial changes in postsynaptic molecular composition of the PF-PC synapses and dendritic structure of PCs.

Oligomeric state makes important contributions to the signaling mechanisms of costimulatory molecules. In this study we address the biological relevance of the disulfide-linked dimeric structure of CD28. Fluorescence Resonance Energy Transfer (FRET) demonstrates that removal of the interdomain disulfide bond (C123) does not interfere with the formation of CD28 oligomers on the cell surface. Although the C123S mutant shows 40% lower binding affinity to the ligand B7-1, it is able to costimulate anti-CD3-induced IL-2 production but at a lower level (150%) compared to the wild-type (270%). Interestingly, binding to B7-2 was not affected. Thus, the covalently linked dimeric structure of CD28 represents an important mechanistic determinant for the optimal costimulatory activity in the immunological synapse.

Two forms of long-term depression (LTD), triggered by activation of NMDA receptors (NMDARs) and metabotropic glutamate receptors (mGluRs), respectively, can be induced at CA1 synapses in the hippocampus. Compared with NMDAR-LTD, relatively little is known about mGluR-LTD. Here, we show that protein tyrosine phosphatase (PTP) inhibitors, orthovanadate and phenylarsine oxide, selectively block mGluR-LTD induced by application of the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG-LTD), because NMDAR-LTD is unaffected by these inhibitors. Furthermore, DHPG-LTD measured using whole-cell recording is similarly blocked by either bath-applied or patch-loaded PTP inhibitors. These inhibitors also block the changes in paired-pulse facilitation and coefficient of variation that are associated with the expression of DHPG-LTD. DHPG treatment of hippocampal slices was associated with a decrease in the level of tyrosine phosphorylation of GluR2 AMPA receptor (AMPAR) subunits, an effect blocked by orthovanadate. Finally, in dissociated hippocampal neurons, orthovanadate blocked the ability of DHPG to reduce the number of AMPA receptor clusters on the surface of dendrites. Again, the effects of PTP blockade were selective, because NMDA-induced decreases in surface AMPAR clusters was unaffected by orthovanadate. Together, these data suggest that activation of postsynaptic PTP results in tyrosine dephosphorylation of AMPARs and their removal from the synapse.

Mechanisms of long-term potentiation and depression (LTP and LTD) change considerably during development, but the importance of these changes and the factors that control them is not clear. We found that visual experience triggered a switch in mechanisms of LTD in rat perirhinal cortex, an area critical for visual recognition memory. Thus, changes in synaptic plasticity mechanisms were correlated with the changing physiological demands on the CNS.

Serious efforts have been made to develop anxiolytics with improved clinical utility and reduced side effects. 5-Hydroxytryptamine (5-HT)(2C) receptor antagonists are potential anxiolytics; however, their effects on vigilance are not well characterized. To compare the effects of benzodiazepines and subtype-selective 5-HT(2C) receptor antagonists on anxiety, vigilance, and electroencephalogram (EEG) power density, social interaction test and polygraphic recordings were performed in male Sprague-Dawley rats after chlordiazepoxide (CDP; 4.0 mg/kg i.p.) and SB-242084 (6-chloro-5-methyl-1-[2-(2-methylpyrid-3-yloxy)-pyrid-5-yl carbamoyl] indoline) (0.1, 0.3, and 1.0 mg/kg i.p.) treatment. CDP and SB-242084 (0.3 and 1.0 mg/kg) had similar anxiolytic effects. Spectral analysis of EEG in wakefulness (W) and paradoxical sleep (PS) showed an opposite effect on activity (5-9 Hz); it decreased after CDP, whereas it increased after SB-242084 (even at 0.1 mg/kg). In addition, CDP significantly decreased slow-wave activity (0.5-4 Hz) in deep slow-wave sleep (SWS-2) and increased power at frequencies above 12 Hz mainly in W and PS. A markedly increased intermediate stage of sleep was also found after CDP treatment. At the highest dose, SB-242084 increased W and decreased SWS-2. In summary, low but potent anxiolytic doses of the subtype-selective 5-HT(2C) receptor antagonist SB-242084 did not affect vigilance states but caused an increased activity in W, raising the possibility of a cognitive-enhancing effect of the drug. In contrast, acute CDP administration, based on spectral analysis of the EEG, produced a more superficial sleep along with a decreased activity.

The divergently transcribed DIT1 and DIT2 genes of Saccharomyces cerevisiae, which belong to the mid-late class of sporulation-specific genes, are subject to Ssn6-Tup1-mediated repression in mitotic cells. The Ssn6-Tup1 complex, which is required for repression of diverse sets of coordinately regulated genes, is known to be recruited to target genes by promoter-specific DNA-binding proteins. In this study, we show that a 42-bp negative regulatory element (NRE) present in the DIT1-DIT2 intergenic region consists of two distinct subsites and that a multimer of each subsite supports efficient Ssn6-Tup1-dependent repression of a CYC1-lacZ reporter gene. By genetic screening procedures, we identified DFG16, YGR122w, VPS36, and the DNA-binding proteins Rim101 and Nrg1 as potential mediators of NRE-directed repression. We show that Nrg1 and Rim101 bind simultaneously to adjacent target sites within the NRE in vitro and act as corepressors in vivo. We have found that the ability of Rim101 to be proteolytically processed to its active form and mediate NRE-directed repression not only depends on the previously characterized RIM signaling pathway but also requires Dfg16, Ygr122w, and components of the ESCRT trafficking pathway. Interestingly, Rim101 was processed in bro1 and doa4 strains but was unable to mediate efficient repression.

The number of ionotropic receptors in synapses is an essential factor for determining the efficacy of fast transmission. We estimated the number of functional AMPA receptors at single postsynaptic sites by a combination of two-photon uncaging of glutamate and the nonstationary fluctuation analysis in immature rat Purkinje cells (PCs), which receive a single type of excitatory input from climbing fibers. Areas of postsynaptic membrane specialization at the recorded synapses were measured by reconstruction of serial ultrathin sections. The number of functional AMPA receptors was proportional to the synaptic area with a density of approximately 1280 receptors/microm2. Moreover, highly sensitive freeze-fracture replica labeling revealed a homogeneous density of immunogold particles for AMPA receptors in synaptic sites (910 +/- 36 particles/microm2) and much lower density in extrasynaptic sites (19 +/- 2 particles/microm2) in the immature PCs. Our results indicate that in this developing synapse, the efficacy of transmission is determined by the synaptic area.

Activation of group I and group II metabotropic glutamate receptors (mGluRs) is thought to be required for long-term depression (LTD) of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor-mediated synaptic transmission in the perirhinal cortex. However, little is known about how activation of mGluRs leads to this form of synaptic plasticity. AMPA receptor phosphorylation has been implicated in several forms of modulation of synaptic transmission. In the CA1 area of the hippocampus, N-methyl-d-aspartate (NMDA) receptor-dependent LTD is associated with the reduced phosphorylation of the GluR1 AMPA receptor subunit at serine 845 (GluR1-S845). Immunoblot analysis of perirhinal cortical neurons using GluR1 and GluR1-S845 phosphorylation state specific antibodies showed that stimulation of adenylyl cyclase (AC) with forskolin (FSK) dramatically increased PKA-mediated phosphorylation of GluR1-S845. However, selective or simultaneous application of mGluR5 agonist (S)-3,5-dihydroxyphenylglycine (CHPG) and mGluR2/3 agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) did not produce detectable changes in GluR1-S845 phosphorylation. These results indicate that in the perirhinal cortex mGluR activation does not alter the phosphorylation state of GluR1-S845. Therefore, it is likely that the process involved in the modification of AMPA receptors in mGluR activation dependent LTD in the perirhinal cortex is mechanistically distinct from NMDA receptor-mediated LTD described in hippocampal neurons.

It is widely believed that long-term depression (LTD) and its counterpart, long-term potentiation (LTP), involve mechanisms that are crucial for learning and memory. However, LTD is difficult to induce in adult cortex for reasons that are not known. Here we show that LTD can be readily induced in adult cortex by the activation of NMDA receptors (NMDARs), after inhibition of glutamate uptake. Interestingly there is no need to activate synaptic NMDARs to induce this LTD, suggesting that LTD is triggered primarily by extrasynaptic NMDA receptors. We also find that de novo LTD requires the activation of NR2B-containing NMDAR, whereas LTP requires activation of NR2A-containing NMDARs. Surprisingly another form of LTD, depotentiation, requires activation of NR2A-containing NMDARs. Therefore, NMDARs with different synaptic locations and subunit compositions are involved in various forms of synaptic plasticity in adult cortex.

Glial precursor cells (GPCs) are present in the adult human central nervous system (CNS) and they can be isolated and maintained in culture for in vitro studies. This study analysed expression of mGluR3 and mGluR5 metabotropic glutamate receptor (mGluR) mRNAs in GPCs. A2B5 surface antigen positive GPCs were isolated using immunomagnetic selection from dissociated temporal lobe subcortical white matter cells. The separated GPCs were maintained in cultures and characterised by immunoreactivity for the differentiation markers A2B5 and human platelet-derived growth factor-alpha receptor (PDGFalphaR). Reverse transcription followed by multiplex PCR analysis showed that the GPCs expressed both mGluR3 and mGluR5a mRNAs. Double immunostaining for glial progenitor markers and mGluR5 proteins demonstrated that all A2B5 and PDGFalphaR-positive cells were also positive for mGluR5. The results indicate that GPCs present in the adult human CNS express mGluR3 and mGluR5a. These neurotransmitter receptors may be involved in the proliferation and differentiation of glial cells.

Rationale: Despite the well documented neurochemical actions of 3,4-methylenedioxymethamphetamine (MDMA), acute effects in rats previously exposed to the drug have not been extensively explored., Objective: To examine motor activity and vigilance effects of MDMA in drug-naive rats and in rats exposed to the drug 3 weeks earlier., Methods: MDMA (15 mg/kg, i.p.) was administered to Dark Agouti rats. Motor activity, wakefulness, light slow wave sleep (SWS-1), deep slow wave sleep (SWS-2) and paradoxical sleep (PS), sleep and PS latencies were measured. Acrophases and amplitudes of the 24 h cycles were calculated by cosinor analysis. In parallel groups, local cerebral glucose utilization (lCMRglu) and (3H)-paroxetine binding were measured in motor areas of the brain., Results: In drug-naive rats MDMA caused marked increases in motor activity and wakefulness for at least 5-6 h. Circadian patterns of motor activity and sleep/vigilance parameters were altered up to 5 days after treatment. Despite most parameters tending to return to normal, there were still significant effects of MDMA on motor activity, wakefulness, and SWS-2 28 days later. Acute MDMA administration caused significant increases in lCMRglu, but after 3 weeks lCMRglu was decreased in the same brain areas. No significant change in [3H]paroxetine binding was observed in motor areas, although significant reductions were seen elsewhere (neocortex -81%). In rats exposed to MDMA 3 weeks earlier, most acute effects induced by MDMA administration were similar to those in drug-naive rats, but shorter duration of the acute effects were found in motor activity and vigilance., Conclusions: Our findings provide evidence that MDMA use can lead to long-term changes in regulation of circadian rhythms, motor activity and sleep generation.

There is a growing body of evidence supporting the theory that cutaneous dysplastic naevus syndrome patients are at increased risk of developing not only skin but also uveal melanoma. The relationship between dysplastic naevus syndrome and ocular naevi needs to be clarified. In this study we investigated the ocular pigmented findings in patients with dysplastic naevus syndrome and compared the results with a control group (subjects without atypical moles) in order to investigate the frequency of ocular naevi among dysplastic naevi-bearing patients. A total of 152 dysplastic naevus syndrome patients were enrolled in our investigation. The control group consisted of 142 sex-, age- and skin type-matched healthy volunteers without cutaneous dysplastic naevi or skin melanoma. Conjunctival and uveal pigmented findings and iris colour were recorded during a detailed ophthalmic examination. A greater number of conjunctival naevi (3.2% versus 0%), iris naevi (5.2% versus 1.4%), iris freckles (17% versus 5.6%) and choroidal naevi (5.2% versus 0.7%) were detected in the dysplastic naevus syndrome group compared with the controls. The difference reached statistical significance in the case of conjunctival naevi, choroidal naevi and iris freckles. Our results confirm the hypothesis that dysplastic naevus syndrome patients might have overstimulation of their melanocytic system not only in the skin but also in the uvea, leading to increased benign (as well as rarely malignant) melanocytic proliferation. Dysplastic naevus syndrome patients should be screened by ophthalmologists because of the increased frequency of different ocular pigmented alterations.

Long-term depression (LTD) induction relies upon receptor cross-talk between group I and group II metabotropic glutamate receptors (mGluRs) in perirhinal cortex. The molecular mechanism of this mGluR interplay is not clear. Here, we show that the mGluR subtypes postulated to be involved in this mechanism are developmentally regulated and mGluR2 has a preferential role over mGluR3 in the synergistic interaction with mGluR5. We have identified a >70% reduction in basal cAMP levels following mGluR2 stimulation, which could lead to increased mGluR5 function via reduced PKA mediated phosphorylation and decreased desensitisation of mGluR5. To further investigate the roles of mGluRs in downstream intracellular signalling, we have examined the effects of mGluRs on the phosphorylation state of cAMP response element-binding protein (CREB). Both group I and group II agonists increased the phosphorylation of CREB, which indicates a cAMP- and PKA-independent signalling mechanism. These results suggest a convergence of signalling mechanisms from surface mGluRs to CREB-mediated transcription.

An analytical method was developed for determining macrolide antibiotics in treated wastewater effluents and in ambient water based on solid-phase extraction and LC/MS analysis as well as on LC/MS/MS for structural confirmation. In wastewater treatment plants (WWTPs) macrolides are only partly eliminated and can therefore reach the aquatic environment. In treated effluents from three WWTPs in Switzerland, clarithromycin, roxithromycin, and erythromycin-H2O, the main degradation product of erythromycin, were found. The most abundant, clarithromycin, reflects the consumption pattern of macrolide antibiotics. Summer concentrations of clarithromycin varied between 57 and 330 ng/L in treated WWTP effluents. In the WWTP Kloten-Opfikon seasonal differences revealed a load two times higher in winter than in summer. The higher abundance of erythromycin-H2O in the effluent of WWTP Kloten-Opfikon can be explained by distinct consumption patterns due to the main international airport of Switzerland in the catchment area. In the Glatt River clarithromycin reached concentrations of up to 75 ng/L. Mass flux determinations in treated effluents and in river water in the Glatt Valley watershed showed that elimination of clarithromycin along the river stretch of 36 km is insignificant (<20%). Investigations in the Glatt River before and after the diversion of the largest WWTP revealed an observable decrease in clarithromycin loads.

Kainate receptors (KARs) modulate synaptic transmission at both pre-synaptic and post-synaptic sites. The overlap in the distribution of KA-2 and GluR6/7 subunits in several brain regions suggests the co-assembly of these subunits in native KARs. The molecular mechanisms that control the assembly and surface expression of KARs are unknown. Unlike GluR5-7, the KA-2 subunit is unable to form functional homomeric KAR channels. We expressed the KA-2 subunit alone or in combination with other KAR subunits in HEK-293 cells. The cell surface expression of the KAR subunit homo- and heteromers were analysed using biotinylation and agonist-stimulated cobalt uptake. While GluR6 or GluR7 homomers were expressed on the cell surface, KA-2 alone was retained within the endoplasmic reticulum. We found that the cell surface expression of KA-2 was dramatically increased by co-expression with either of the low-affinity KAR subunits GluR5-7. However, co-expression with other related ionotropic glutamate receptor subunits (GluR1 and NR1) does not facilitate the cell surface expression of KA-2. The analysis of subcellular fractions of neocortex revealed that synaptic KARs have a relatively high KA-2 content compared to microsomal ones. Thus, KA-2 is likely to contain an endoplasmic reticulum retention signal that is shielded on assembly with other KAR subunits.

We investigated the expression of metabotropic glutamate receptor (mGluR) isoforms in CG-4 rodent oligodendroglial progenitor cells (OPC) and rat brain oligodendrocytes. Our RT-PCR analysis detected mRNAs for mGluR3 and mGluR5 isoforms in OPCs. Although neurons express both mGluR5a and mGluR5b splice variants, only mGluR5a was identified in OPCs. Antibodies to mGluR2/3 and mGluR5 detected the corresponding receptor proteins in immunoblots of OPC membrane fractions. Furthermore, immunocytochemical analysis identified mGluR5 in oligodendrocyte marker O4-positive OPCs. The expression of mGluR5 was also demonstrated in oligodendrocyte marker (O4 and O1) positive cells in white matter of postnatal 4- and 7-day-old rat brain sections using immunofluorescent double labelling and confocal microscopy. The mGluR5 receptor function was assessed in CG-4 OPCs with fura-2 microfluorometry. Application of the mGluR1/5 specific agonist (S)-3,5-dihydroxyphenylglycine (DHPG) induced calcium oscillations, which were inhibited by the selective mGluR5 antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP). The DHPG induced calcium oscillations required Ca2+ release from intracellular stores. In OPCs the group II mGluR agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) decreased forskolin-stimulated cAMP synthesis, indicating the presence of functional mGluR3. The newly identified mGluR3 and mGluR5a may be involved in the differentiation of oligodendrocytes, myelination and the development of white matter damage.

During development neuronal circuitries are refined by activity. Here we studied the role of spontaneous electrical activity in the regulation of synapse formation in the intact newborn (Postnatal Day 3; P3) rat hippocampus in vitro. The blockade of the spontaneous network activity with TTX led to an increase in the number of functional excitatory synapses in the CA3 area of the developing hippocampus. In parallel, there was a substantial increase in the expression levels of the presynaptic markers synaptophysin, synaptotagmin, and synapsin I and of GluR1 AMPA receptor subunits. These changes were associated with an increase in the frequency and amplitude of AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs). Our correlated immunocytochemical, electronmicroscopical, and electrophysiological experiments indicate that in the developing hippocampus spontaneous network activity controls the number of functional synapses.

Glutamatergic synapses are the primary source of excitatory transmission in the central nervous system (CNS), and their formation is critical in the establishment of neuronal connections. The refinement of these connections occurs during development and also it is postulated during learning and memory. Recent progress in understanding the molecular components of synaptic junctions, together with advances in imaging techniques, has started to offer new insights into the development of excitatory synapses. Studies performed on low-density primary neuronal cultures have enabled dissection of the temporal sequence of events, which have lead to the differentiation of pre- and postsynaptic components. A central feature of the development of excitatory synapses is the accumulation of glutamatergic receptors (GluRs) at the postsynaptic site. These receptors need to be localized and fixed opposite nerve terminals that release glutamate. But for this to occur, neurons require intracellular anchoring molecules, as well as mechanisms that ensure the efficient turnover and transport of receptor proteins. This review focuses on some of the developmental changes observed in the subcellular distribution and molecular organization of AMPA and NMDA type ionotropic GluRs (iGluRs), which mediate the majority of fast excitatory neurotransmission in the CNS.

Aims/hypothesis: The neurotransmitters glutamate and gamma-aminobutyric acid (GABA) could participate in the regulation of the endocrine functions of islets of Langerhans. We investigated the role of the metabotropic glutamate (mGluRs) and GABA(B) (GABA(B)Rs) receptors in this process., Methods: We studied the expression of mGluRs and GABA(B)Rs in rat and human islets of Langerhans and in pancreatic alpha-cell and beta-cell lines using RT-PCR and immunoblot analysis. Effects of mGluR and GABA(B) R agonists on insulin secretion were determined by radioimmunoassays and enzyme-linked immunoadsorbent assays (ELISAs)., Results: We detected mGluR3 and mGluR5 (but not mGluR1, 6 and 7) mRNAs in all of the samples examined. Trace amount of mGluR2 was found in MIN6 beta cells; mGluR4 was identified in rat islets; and mGluR8 expression was detected in rat islets, RINm5F and MIN6 cells. GABA(B)R1 a/b and 2 mRNAs were identified in islets of Langerhans and MIN6 cells. The expression of mGluR3, mGluR5, GABA(B)R1 a/b and GABA(B)R2 proteins was confirmed using specific antibodies. Group I (mGluR1/5) and group II (mGluR2/3) specific mGluR agonists increased the release of insulin in the presence of 3 to 10 mmol/l or 3 to 25 mmol/l glucose, respectively, whereas a group III (mGluR4/6-8) specific agonist inhibited insulin release at high (10-25 mmol/l) glucose concentrations. Baclofen, a GABA(B)R agonist, also inhibited the release of insulin but only in the presence of 25 mmol/l glucose., Conclusion/interpretation: These data suggest that mGluRs and GABA(B)Rs play a role in the regulation of the endocrine pancreas with mechanisms probably involving direct activation or inhibition of voltage dependent Ca(2+)-channels, cAMP generation and G-protein-mediated modulation of K(ATP) channels.

Glutamate receptors mediate the majority of excitatory responses in the central nervous system. The establishment and refinement of glutamatergic synaptic connections depend on the concerted actions of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), N-methyl-D-aspartate (NMDA), and kainate (KA) type ionotropic glutamate receptors (iGluRs) and G-protein coupled metabotropic receptors. While a lot remains to be clarified, the most is known about the mechanisms by which the iGluR subtypes are targeted and how this is influenced by synaptic activity on both short and long time scales. Changes in their subunit compositions are also input specific and developmentally regulated. The identification of key molecular components of the postsynaptic density (PSD) and novel proteins that influence receptor targeting and clustering have started to reveal the underlying molecular mechanisms of the trafficking and targeting of iGluRs. Here we discuss the evidence that these basic mechanisms are used during developmental synaptic plasticity.

Histidine decarboxylase (HDC) is the single enzyme responsible for histamine synthesis. HDC-deficient mice (HDC(-/-)) have no histamine in their tissues when kept on a histamine-free diet. Therefore, the HDC(-/-) mice provide a suitable model to investigate the involvement of histamine in the regulation of histamine receptor expression. Gene expression of H1 and H2 histamine receptors was studied in several organs of HDC(-/-) mice and compared to standard (HDC(+/+)) mice. In many tissues, prolonged absence of histamine induced down-regulation of the H2 receptor subtype. The expression of the H1 receptor was less sensitive to histamine deficiency. Exogenous histamine present in the diet abolished the differences observed in H2 receptor expression. These results suggest that the expression of mouse H2 receptor is under the control of histamine in a tissue-specific manner.

Long-term potentiation (LTP) of synaptic transmission is under intense investigation. It is believed that the mechanisms involved in its induction and expression are critically involved in synaptic processes that are important for learning and memory and other physiological functions. A reliable means of inducing LTP in dissociated cultured neurones would facilitate investigations into the molecular basis of LTP but has been hard to achieve. Here we report a mechanism for inducing LTP in postnatal dissociated hippocampal neurones using transient depolarisation. This form of LTP is prevented by NMDA receptor antagonists and by chelating Ca2+ in the postsynaptic neurone. It is manifest primarily as an increase in the frequency of mEPSCs.

Histidine decarboxylase (HDC) is expressed by the cells of melanoma, in which the histamine content tends to be relatively high. This study shows that elevated expression of HDC was found by western blot analysis of primary and metastatic melanoma tissue using a polyclonal HDC specific antibody. The specificity of anti-HDC antibody was confirmed by inhibition of HDC translation (i.e., immunopositivity) in melanoma cells by HDC-specific antisense oligonucleotide. Moreover, the decrease in proliferation caused by HDC antisense oligonucleotides indicates considerable functional relevance of histamine synthesis in melanoma growth and suggests a possible in situ application of specific antisense oligonucleotides for HDC in melanoma therapy.

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA), acts at ionotropic (GABA(A) and GABA(C)) and metabotropic (GABA(B)) receptors. Functional GABA(B) receptors are heterodimers of GABA(B(1)) and GABA(B(2)) subunits. Here we show a robust, direct, and specific interaction between the coiled-coil domain present in the C-terminus of the GABA(B(1)) subunit and the transcription factor ATF4 (also known as CREB2). ATF4 and GABA(B(2)) binding to the GABA(B(1)) subunit were mutually exclusive. In rat hippocampal neurons native GABA(B(1)) showed surprisingly little similarity to GABA(B(2)) in its subcellular distribution. GABA(B(1)) and ATF4, however, were highly colocalized throughout the cell and displayed a punctate distribution within the dendrites. Activation of GABA(B) receptors in hippocampal neurons caused a dramatic translocation of ATF4 out of the nucleus into the cytoplasm. These data suggest a novel neuronal signaling pathway that could regulate the functional expression of GABA(B) receptors and/or modulate gene transcription., (Copyright 2001 Academic Press.)

AMPA and NMDA receptors mediate most excitatory synaptic transmission in the CNS. We have developed antibodies that recognize all AMPA or all NMDA receptor variants on the surface of living neurons. AMPA receptor variants were identified with a polyclonal antibody recognizing the conserved extracellular loop region of all four AMPA receptor subunits (GluR1-4, both flip and flop), whereas NMDA receptors were immunolabeled with a polyclonal antibody that binds to an extracellular N-terminal epitope of the NR1 subunit, common to all splice variants. In non-fixed brain sections these antibodies gave labeling patterns similar to autoradiographic distributions with particularly high levels in the hippocampus. Using these antibodies, in conjunction with GluR2-specific and synaptophysin antibodies, we have directly localized and quantified surface-expressed native AMPA and NMDA receptors on cultured living hippocampal neurons during development. Using a quantitative cell ELISA, a dramatic increase was observed in the surface expression of AMPA receptors, but not NMDA receptors, between 3 and 10 d in culture. Immunocytochemical analysis of hippocampal neurons between 3 and 20 d in vitro shows no change in the proportion of synapses expressing NMDA receptors (approximately 60%) but a dramatic increase (approximately 50%) in the proportion of them that also express AMPA receptors. Furthermore, over this period the proportion of AMPA receptor-positive synapses expressing the GluR2 subunit increased from approximately 67 to approximately 96%. These changes will dramatically alter the functional properties of hippocampal synapses.

Introduction: It is a well known fact that there is no invasive uterine carcinoma which was not a "carcinoma in situ" in the initial stage. On the basis of this knowledge it is evident that active systematic detection of malignant uterine neoplasms is of great importance. Incidence of cervical carcinoma had been at the first place for years, but in the last 10 years the number of diagnosed cancer of corpus uteri has significantly increased. Uterine hemorrhage is a problem at any age, but at menopause and senium it is of special importance, because at this age every hemorrhage must be considered carcinomatous, unless something else is established (1). Out of all methods, histopathological analysis of the biopsy material is the most reliable, but only complete, not fractional, uterine abrasion has a full diagnostic value. Explorative curettage was performed in all cases of polypectomies and cervix biopsies (2,3,4)., Material and Methods: A retrospective study included all histopathological findings of curettages, polypectomies and biopsies performed due to hemorrhage in the period from 1965 to 1998. During this period 48,606 women were hospitalized at the Gynecology Department of the General Hospital in Senta. 9,475 (19.49%) underwent explorative curettage, partly for therapeutic and partly for diagnostic reasons. The biopsy material was sent for histopathological analysis, disregarding the quantity and macroscopic appearance., Results: The greatest number of explorative curettages were performed in women from 41-50 years of age (41.35%). The number of interventions decreased in the sixth and seventh decade, with sudden increase over 70 years of age. The greatest number of functional bleeding occurred in women from 21 to 50 years of age (33.59%), whereas endometrial hyperplasia was diagnosed in 22.6% of patients 41-50 years of age. Biopsies and polypectomies with explorative curettages were most frequent in patients 61-70 years of age. Uterine carcinoma was diagnosed in 470 patients: out of this number cervical carcinoma was diagnosed in 234 patients, and corpus uteri carcinoma in 236 women. Cervical carcinoma occurred most frequently in the group of patients 41-50 years of age, whereas corpus uteri carcinoma was most frequent in the group 51-70 years of age. It is important to point out the correlation between cervical carcinoma and corpus uteri carcinoma being 1:1.04., Conclusion: 1. In order to detect corpus uteri carcinoma, especially in women over 40 years of age, it is necessary to pay special attention to all kinds of uterine bleedings. 2. Explorative curettage of the whole uterine cavity and histopathological analysis of biopsy material are considered to be the most reliable diagnostic methods for detection of carcinoma. 3. The correlation between cervical carcinoma and corpus uteri carcinoma is 1:1.04. It points to a difference in regard to corpus uteri carcinoma established in previous studies.

Here, we show that disruption of N-ethylmaleimide-sensitive fusion protein- (NSF-) GluR2 interaction by infusion into cultured hippocampal neurons of a blocking peptide (pep2m) caused a rapid decrease in the frequency but no change in the amplitude of AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs). N-methyl-D-aspartate (NMDA) receptor-mediated mEPSCs were not changed. Viral expression of pep2m reduced the surface expression of alpha-amino-3-hydroxy-5-methyl-isoxazolepropionate (AMPA) receptors, whereas NMDA receptor surface expression in the same living cells was unchanged. In permeabilized neurons, the total amount of GluR2 immunoreactivity was unchanged, and a punctate distribution of GluR2 was observed throughout the dendritic tree. These data suggest that the NSF-GluR2 interaction is required for the surface expression of GluR2-containing AMPA receptors and that disruption of the interaction leads to the functional elimination of AMPA receptors at synapses.

Introduction: Authors have investigated the incidence and distribution of hormonally active ovarian tumors in a 30-year surgical material. Out of 552 ovarian tumors, there were 28 hormonally active tumors (5.07%): 18 granulosa cell tumors, 4 thecomas, 2 arrhenoblastomas, 2 malignant v. Kalden folliculomas. There were laso 2 benign tumors: ectopic adrenal ovarian tumor, and ovarian tube hydatidiform mole. They secrete estrogen and testosterone and their effects are evident: prior to puberty (Praecox pubertas), during the reproductive period, but most often in menopause. In order to make the diagnosis, when uterine hemorrhage occurs, it is necessary to perform explorative curettage, but the tumors must be surgically removed. It is necessary to perform that kind of surgery, which corresponds to the age of the woman. Recurrences may appear even 35 years later., Material and Methods: Hysterectomy with bilateral adnexectomy was performed in 18 (64.28%) postmenopausal women. In 4 (14.29%) women of reproductive age unilateral adnexectomy was performed, while explorative laparotomy was performed also in 4 (14.29%) women. 5-year survival in the first stage of the disease was 75% (20 women). During the first three years 5 (17.86%) women decreased in the IV stage of the disease, as well as one woman (3.57%) in the II b stage, which makes a total of 6 women (21.43%) with fatal outcome. Ovarian tumors also include hormonally active tumors which secrete female and male sex hormones, and whose effects are evident on hormonal receptors. They are divided into benign or malignant, whereas they are all potentially malignant. We differentiate two groups: 1. Feminizing mesenchymomas, which secrete female sex hormones and 2. Virilizing tumors, which secrete male sex hormones. These tumors may occur prior puberty, during the reproductive period and in the postmenopausal period--senium. They can cause minor, long-term or hemorrhages similar to menstruation, as well as hypertrophy of the myometrium. Glandular cystic hyperplasia occurs often, whereas proliferation is rare. Histopathological findings after surgery (for example of myoma) are often surprising, because they reveal hormonally active undetected ovarian tumors or glandular cystic endometrial hyperplasia. All hormonally active tumors are potentially malignant. The aim of this retrospective study was to compare our prior attitudes and interventions with newer attitudes and to make changes on behalf of our patients., Results: Our investigation included 552 adnexal tumors in a 30-year material. Each woman with uterine hemorrhage had undergone explorative curettage and the material was sent for histopathological analysis. During the investigated period 28 hormonally active tumors were found: 2 benign and 26 malignant or potentially malignant tumors (Table 2). There were 18 cases (40.91%) with granulosa cell tumors, 6 cases (13.64%) with cystadenocarcinoma ovarii serosum; 3 cases (6.82%) with borderline tumors; 4 cases (9.09%) with thecoma of the ovarii. Pseudomucious adenocarcinoma was found in 2 cases (4.55%) as well as endometrial adenocarcinoma, malignant mesenchymoma, arrhenoblastoma, malignant folliculoma v. Kalden; whereas malignant teratoma was established in 1 case (2.27%), as well as anaplastic carcinoma, metastatic carcinoma--Krunberg. It is obvious that hormonally active tumors make almost half of the cases (46.43%) and occur mostly at the age of 50-59 years of age (3.4%). Out of 28 patients, 18 underwent hysterectomy and bilateral adnexectomy (64.28%); 4 (14.29%) underwent unilateral adnexectomy that is explorative laparotomy for taking bioptic samples for histological examination (14.29%). Out of 28 patients, 4 were women of reproductive age. All of them underwent adnexectomy and are alive. Hemorrhages usually occur a few years after menopausal period. In one case it occurred 35 years after menopause (arrhenoblastoma), in another 25 years after menopause due to endometrial carcinoma asso

It has been suggested that some glutamatergic synapses lack functional AMPA receptors. We used quantitative immunogold localization to determine the number and variability of synaptic AMPA receptors in the rat hippocampus. Three classes of synapses show distinct patterns of AMPA receptor content. Mossy fiber synapses on CA3 pyramidal spines and synapses on GABAergic interneurons are all immunopositive, have less variability, and contain 4 times as many AMPA receptors as synapses made by Schaffer collaterals on CA1 pyramidal spines and by commissural/ associational (C/A) terminals on CA3 pyramidal spines. Up to 17% of synapses in the latter two connections are immunonegative. After calibrating the immunosignal (1 gold = 2.3 functional receptors) at mossy synapses of a 17-day-old rat, we estimate that the AMPA receptor content of C/A synapses on CA3 pyramidal spines ranges from <3 to 140. A similar range is found in adult Schaffer collateral and C/A synapses.

Here, we show that N-ethylmaleimide-sensitive fusion protein (NSF) interacts directly and selectively with the intracellular C-terminal domain of the GluR2 subunit of AMPA receptors. The interaction requires all three domains of NSF but occurs between residues Lys-844 and Gln-853 of rat GluR2, with Asn-851 playing a critical role. Loading of decapeptides corresponding to the NSF-binding domain of GluR2 into rat hippocampal CA1 pyramidal neurons results in a marked, progressive decrement of AMPA receptor-mediated synaptic transmission. This reduction in synaptic transmission was also observed when an anti-NSF monoclonal antibody (mAb) was loaded into CA1 neurons. These results demonstrate a previously unsuspected direct interaction in the postsynaptic neuron between two major proteins involved in synaptic transmission and suggest a rapid NSF-dependent modulation of AMPA receptor function.

The postsynaptic apparatus is associated with a number of glycoproteins with apparent molecular masses of 180, 116, and 110 kDa, which are highly concentrated in and may be uniquely associated with this structure. These glycoproteins, purified by concanavalin A lectin-affinity chromatography, showed immunoreactivity in the present study with subunit-specific antibodies to glutamate receptors as follows: GP 180, NMDA receptor subunits NR2A/NR2B; GP 116, NMDA receptor NR1 (1a); and GP 110, pan-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (pan-AMPA) receptors. Sensitivities to the glycosidases peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase H on both western blots and silver-stained gels suggested that the glutamate receptors were at least major constituents of the glycoprotein bands. Similar detailed glycosylation was observed for all three glycoproteins, with neutral oligosaccharides being dominant. Oligomannosidic glycans (with from five to nine mannoses) accounted for approximately 50% of the neutral sugars, with Man 5 (at almost 20% of the neutral sugars) always the major glycan. Other abundant neutral oligosaccharides were of the complex type. Similar sensitivities to peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase H were observed for cell line-expressed NMDA receptor subunits, suggesting that irrespective of the glycosylation processing available, the least highly processed oligosaccharides will be expressed. This may be indicative of glycosylation sites in these receptors that are inaccessible to the later processing enzymes and favours the oligomannosidic class of glycans in functional roles.

We have used immunocytochemistry to examine the distribution of raf protein kinases in sensory neurons of the adult rat. In lumbar and trigeminal sensory ganglia, cells of all size ranges appeared to be raf immunoreactive and this was confirmed by double labeling using subpopulation specific markers. Almost all cells labeled with Griffonia simplicifolia IB4 (a small cell marker) or immunostained by using a large cell marker (RT97) showed raf immunoreactivity. These two markers label cells known to differ in their expression of neurotrophin receptors (trk). Thus raf kinases are not confined to cells expressing only certain trk subtypes. In the dorsal horn of the spinal cord, raf immunoreactivity was present in scattered neurons. However, sensory axons identified by IB4 histochemistry were devoid of raf immunostaining. Lectin-labeled nerve fibers in the cornea, lower lip and glans penis were also not immunoreactive. Ligation of the sciatic nerve did not produce any accumulation of raf immunoreactivity, confirming that raf kinases are not axonally transported to the peripheral processes of sensory neurons. Surgical dissection of the sciatic nerve caused the normal homogeneous cytoplasmic raf immunoreactivity to be replaced in some cells by a staining concentrated predominantly under the plasma membrane. One possibility is that this represents activation of raf in these cells.

It is estimated that world-wide approximately 13 million adults and at least 1 million children already have the HIV virus. Projections call for another twenty or thirty million new infections in the 1990s. Stresses that generally accompany chronic illnesses have been well identified. Also well known are the unique series of concerns that adult AIDS patients and their caretakers experience. Of children who are born to HIV infected mothers, the prevalent mode of transmission of the HIV infection, 30 per cent have been found to be infected and of these approximately 12-15 per cent will develop AIDS. This paper focuses on children at risk in urban environments, where other social problems such as poverty and drug use complicate the picture. It reviews psycho-social issues associated with AIDS, such as, knowledge and "disclosure" of infection status, problems of healthy siblings, "kinship care", problems of professionals engaged with this population, etc. Ethical guidelines as well as recommendations for policy and services are also presented which can protect and assist affected children and their families, and the professionals working with them. For children, there is a unique inter-connectedness, medical, developmental and psychasocial needs at all ages, that has to be taken into account as the illness modifies developmental progress and creates an altered social milieu.