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We argue that conjunction analysis does not solve the problems with subtraction analyses that it sets out to solve and that it has additional problems of its own. We argue that detailed analyses of the tasks set to identify cognitive functions are required for both conjunction and subtraction analyses to achieve their goals of localizing cognitive operations using functional neuroimaging techniques.
The primate cingulate cortex is structurally and functionally complex. Although no studies have investigated the regional densities of multiple neurotransmitter receptor systems, such information would be useful for assessing its functions and disease vulnerabilities. We quantified nine different receptors in five transmitter systems by in vitro autoradiographic mapping of the cingulate cortex of macaque monkeys with the aim to link cytoarchitectonic regions and functional specialization. Receptor mapping substantiated the subdivision of the cingulate cortex into anterior versus posterior regions. In anterior cingulate cortex (ACC) AMPA glutamatergic receptors and GABA(A) inhibitory receptors were present in significantly higher concentrations than the modulatory alpha-adrenergic and muscarinic receptors. These differences were absent in the posterior cingulate cortex (PCC). By contrast, NMDA receptor densities were significantly higher than AMPA receptor densities in PCC, but not in ACC. The midcingulate area 24' shared more features with ACC than PCC. This area was characterized by the highest ratios of NMDA receptors to alpha-adrenergic, muscarinic and 5-HT2 receptors among all cingulate regions. Compared to rostrocaudal divisions, the differences between dorsoventral subdivisions a-c were small in all regions of cingulate cortex, and only muscarinic and alpha-adrenergic receptor densities followed the degree of cytoarchitectonic differentiation. We conclude that multiple receptor mapping reveals a highly differentiated classification of cingulate cortex with a characteristic predominance of fast ionotropic excitatory and inhibitory receptors in ACC, but a strong and varied complement of NMDA and metabotropic receptors in PCC.
Functional imaging has demonstrated the specific involvement of the human middle-temporal complex (hMT/V5+) during processing of moving stimuli. Some studies applied transcranial magnetic stimulation (TMS) to investigate the causal relevance of hMT/V5+ for motion perception. Although the studies used similar visual stimuli and TMS parameters, the critical time point of functionally relevant hMT/V5+ activity differed by 100 ms and more. The present study aimed to elucidate further the temporal characteristics of motion processing in hMT/V5+ by investigating all critical time windows currently debated in the literature. In contrast to previous studies, we used TMS neuronavigation based on individual fMRI results of five participants to target hMT/V5+, applying single-pulse TMS at 24 different time windows (-50 till +200 ms relative to stimulus onset). We revealed that TMS significantly impaired motion perception when applied over hMT/V5+ at 40 to 30 ms before as well as 130 to 150 ms after onset of the moving stimuli. While the late effective time window conforms to results from previous experiments, we did not find evidence for an early time window around 0 ms that has been reported in other studies. Our neuronavigation approach enabled us to quantify the interindividual variance in the exact location of hMT/V5+ and the respective TMS target position on the skull of the participants. Considering that shifting the TMS coil position only by a few millimeters can already lead to a complete loss of TMS effects, our study clearly demonstrates the utility of neuronavigated TMS when investigating specific neuronal effects as in the case of motion processing.
Functional magnetic resonance imaging (fMRI) studies of the human brain have suggested that low-frequency fluctuations in resting fMRI data collected using blood oxygen level dependent (BOLD) contrast correspond to functionally relevant resting state networks (RSNs). Whether the fluctuations of resting fMRI signal in RSNs are a direct consequence of neocortical neuronal activity or are low-frequency artifacts due to other physiological processes (e.g., autonomically driven fluctuations in cerebral blood flow) is uncertain. In order to investigate further these fluctuations, we have characterized their spatial and temporal properties using probabilistic independent component analysis (PICA), a robust approach to RSN identification. Here, we provide evidence that: i. RSNs are not caused by signal artifacts due to low sampling rate (aliasing); ii. they are localized primarily to the cerebral cortex; iii. similar RSNs also can be identified in perfusion fMRI data; and iv. at least 5 distinct RSN patterns are reproducible across different subjects. The RSNs appear to reflect "default" interactions related to functional networks related to those recruited by specific types of cognitive processes. RSNs are a major source of non-modeled signal in BOLD fMRI data, so a full understanding of their dynamics will improve the interpretation of functional brain imaging studies more generally. Because RSNs reflect interactions in cognitively relevant functional networks, they offer a new approach to the characterization of state changes with pathology and the effects of drugs.
Neuroimaging studies of normal young adults have consistently found right prefrontal cortex (RPFC) activity during the performance of recency memory tasks. However, it is unclear whether the involvement of RPFC during these tasks reflects retrieval processes or executive processes such as: strategic ordering or monitoring. In the current study, we distinguish between those PFC regions that are more related to retrieval processes, versus strategic ordering processes. An event-related fMRI study was conducted in which eight young subjects were scanned while performing verbal episodic retrieval tasks (recognition and recency memory tasks), and verbal non-memory strategic organizing control tasks (reverse alphabetizing of words). The fMRI results show that young subjects engaged right dorsolateral PFC during recency and reverse alphabetizing control tasks. Left ventral PFC was engaged across all tasks; however, a subset of voxels within this region was more active during retrieval tasks. Left dorsolateral and right ventral PFC activity was more related to the performance of reverse alphabetizing tasks, respectively. We conclude that right dorsolateral PFC activity during recency memory reflects more general strategic organizational or monitoring processes, and is not EM-specific.
In this study, we explored the protective anti-tumor potency of mouse (self) Hsp70 or Hsp110-based DNA vaccination approach targeting a tumor-associated antigen, human papilloma virus (HPV) type 16 E7 protein. Linkage of E7 to the N-terminus of the mouse Hsp70 not only elicits an E7-specific cytotoxic T cell (CTL) response, but also protects mice against challenge with E7 expressing tumors. CD8+ T-cells are crucial in both priming and effector phases for the induction of tumor immunity, whereas CD4+ T-cells and NK cells do not appear to play a major role. Furthermore, the ATP-binding domain deletion mutant Hsp70(382-641), when fused to E7, was immunologically effective, suggesting that the peptide-binding region, not the ATPase domain of Hsp70, is required for the vaccine activity of the E7-Hsp70 DNA. This study demonstrates that autologous Hsp70 is highly potent in enhancing antigen-specific immune responses. Functional domain mapping and orientation of the E7 and Hsp70 in the fusion gene may have clinical implications for the design and optimization of Hsp70-based DNA vaccines.
Deletions in the plasmid pMC874 take place in resting cells incubating on McConkey's or minimal lactose agar and are time rather than generation dependent. These deletions join the km(r) promoter to a promoterless lac operon giving rise to Lac(+) papillae on McConkey's lactose agar, and can occur in the absence of sequence homologies such as direct or inverted repeats. Using this as a selective screen we isolated 31 mutants designated dli (for deletion increase), which enhanced to different extents the frequency of this unusual class of deletions. Six of these were characterized by phenotypic tests and their ability to stimulate other deletion events such as the excision of Tn10 from various chromosomal sites and the loss of cloned fragments between two EcoR1 sites in the gene for chloramphenicol resistance (cat) of plasmid pBR325. Two of them showed contrasting phenotypes and were studied further: one (dli1) stimulated Lac(+) deletions in pMC874 in resting cells but not Tn10 excision from chromosomal locations in log phase cells, and the other one (dli2) did exactly the reverse, i.e. it enhanced Tn10 excision but not Lac(+) deletion incidence. Mapping and complementation tests showed that dli1 is a null mutation in recC and was renamed recC2251. This is strong evidence that resting phase deletions in pMC874 are stimulated by the absence of a functional RecBCD enzyme. The dli2 mutation was identified by mapping and phenotypic tests as a mutation in uvrD, the gene for helicase II, and it was tentatively designated uvrD(-)dli2. These results show that (1) pMC874 is an excellent system to select mutants for genetic functions involved in the generation of resting phase deletions, and (2) there are at least two major deletion pathways in E. coli, one active in resting and the other in actively dividing cells.
The objective of this review is to examine the role of neuronal angiotensin II (Ang II) receptors in vitro. Two types of G protein-coupled Ang II receptors have been identified in cardiovascularly relevant areas of the brain: the AT1 and the AT2. We have utilized neurons in culture to study the signaling mechanisms of AT1 and AT2 receptors. Neuronal AT1 receptors are involved in norepinephrine (NE) neuromodulation. NE neuromodulation can be either evoked or enhanced. Evoked NE neuromodulation involves AT1 receptor-mediated, losartan-dependent, rapid NE release, inhibition of K+ channels and stimulation of Ca2+ channels. AT1 receptor-mediated enhanced NE neuromodulation involves the Ras-Raf-MAP kinase cascade and ultimately leads to an increase in NE transporter, tyrosine hydroxylase and dopamine beta-hydroxylase mRNA transcription. Neuronal AT2 receptors signal via a Gi protein and are coupled to activation of PP2A and PLA2 and stimulation of K+ channels. Finally, putative cross-talk pathways between AT1 and AT2 receptors will be discussed.
The objective of this review is to examine the role of neuronal angiotensin II (Ang II) receptors in vitro. Two types of G protein-coupled Ang II receptors have been identified in cardiovascularly relevant areas of the brain: the AT1 and the AT2. We have utilized neurons in culture to study the signaling mechanisms of AT1 and AT2 receptors. Neuronal AT1 receptors are involved in norepinephrine (NE) neuromodulation. NE neuromodulation can be either evoked or enhanced. Evoked NE neuromodulation involves AT1 receptor-mediated, losartan-dependent, rapid NE release, inhibition of K+ channels and stimulation of Ca2+ channels. AT1 receptor-mediated enhanced NE neuromodulation involves the Ras-Raf-MAP kinase cascade and ultimately leads to an increase in NE transporter, tyrosine hydroxylase and dopamine beta-hydroxylase mRNA transcription. Neuronal AT2 receptors signal via a Gi protein and are coupled to activation of PP2A and PLA2 and stimulation of K+ channels. Finally, putative cross-talk pathways between AT1 and AT2 receptors will be discussed.
The Interleukin Enhancer Binding Factor 3 (ILF3) gene has been mapped to chromosome 19 in humans and to chromosome 9 in mice. Several reported double-stranded RNA binding proteins including NF90, ILF3, MPP4 and DRBP76 have been suggested to be isoforms of the ILF3 gene but this has not been clearly established. We isolated several ilf3 transcripts from a melanoma cDNA library and two corresponding genomic fragments, and report alternative splicing and polyadenylation site selection in the human ILF3 gene. We show the existence of an alternative splice site responsible for the sequence divergence in the 3' part of the transcripts. Another alternative splicing event at a site between the two double-stranded RNA binding motifs leads to the additional presence in some cases of a four amino acids NVKQ peptide. We also describe the utilization of three distinct polyadenylation signals and the generation of an ilf3 transcript with a long extended 3' UTR. The expression of the different transcripts was evaluated. We used a GenBank sequence for the part of chromosome 19 corresponding to the ILF3 gene to determine the exon-intron organization of the entire gene which spans 38 kb and is divided into 21 exons.
Few magnetic resonance imaging studies of schizophrenia have investigated brain tissue volumes and their relation to clinical symptoms in patients with an early age at illness onset. The twofold purpose of the study was to investigate both gray and white matter volumes in schizophrenic men with an early age at illness onset, and to determine whether clinical features correlated with tissue volume changes, using an automated voxel-by-voxel image analysis procedure. Twenty male patients with DSM-IV diagnoses of schizophrenia, and an early age at onset (m+/-SD=19+/-2) were compared with 20 age-matched health men. Magnetic resonance (1.5-T) scans were obtained with an Inversion-Recovery prepared fast gradient echo sequence enhancing gray and white matter contrast. Statistical Parametric Mapping was used for image segmentation and comparison. Patients had significant gray matter reductions in medial frontal gyri, left insula, left parahippocampus, and left fusiform gyrus; bilateral white matter reductions in frontal lobes, and increased total cerebrospinal fluid volume were also observed. Negative symptom scores were negatively related to white matter volumes in cingulate regions, and in the right internal capsule. These findings emphasize a pattern of left-hemisphere gray matter abnormalities, and suggest that fronto-paralimbic connectivity may be altered in men with early onset schizophrenia.
The purpose of the present study was to evaluate the differential activation of the motor cortex during finger tapping in patients with schizophrenia using the newly available imaging method of functional magnetic resonance imaging (fMRI). Nine patients with DSMIII-R schizophrenia and 9 well-matched healthy volunteer subjects underwent fMRI examination on a conventional MR unit; activation of the primary motor cortex was evaluated during performance of a finger motion task. Localized activation of the motor cortex was observed in 17 of 18 subjects during fMRI. Patients and controls were, however, indistinguishable with respect to signal intensity or area thereof within the motor cortex. fMRI did not reveal motor cortical dysfunction in schizophrenia. Despite its infancy, fMRI holds considerable promise to advance understanding of the neurodynamics of psychiatric disorders, particularly schizophrenia.
The characteristics of blood oxygenation level-dependent (BOLD) fMRI and magnetoencephalographic (MEG) responses to vibrotactile stimuli in humans were studied and compared. The stimuli, presented with interstimulus intervals (ISIs) ranging from 1 to 5 s, yielded highly reproducible MEG responses, with current dipoles in the primary somatosensory (SI) cortex in all subjects. BOLD fMRI responses to similar stimuli showed substantial intrasubject variation in the activation sites around the SI cortex. BOLD responses were detected in all subjects in the secondary somatosensory (SII) cortices as well, with comparable BOLD response amplitudes to those in the SI cortex. Current dipoles, used to model the MEG signals, were stronger at longer ISIs than shorter ISIs. The BOLD response amplitudes did not show a similar dependence on ISI, but the activated brain area was larger when longer ISIs or longer stimuli were applied. Our results support the view that combined use of brain mapping methods provides complementary information and should be considered in functional brain examinations.
We have prepared glycosylated analogues of the principal neutralizing determinant of gp120 and studied their conformations by NMR and circular dichroism spectroscopies. The 24-residue peptide from the HIV-1IIIB isolate (residues 308-331) designated RP135, which contains the immunodominant tip of the V3 loop, was glycosylated with both N- and O-linked sugars. The structures of two glycopeptides, one with an N-linked beta-glucosamine (RP135NG) and the other with two O-linked alpha-galactosamine units (RP135digal), were studied by NMR and circular dichroism spectroscopies. Molecular dynamics calculations based on the NMR data obtained in water solutions were performed to explore the conformational substates sampled by the glycopeptides. The data showed that covalently linking a carbohydrate to the peptide has a major effect on the local conformation and imparts additional minor changes at more distant sites of partially defined secondary structure. In particular, the transient beta-type turn comprised of the -Gly-Pro-Gly-Arg- segment at the "tip" of the V3 loop is more highly populated in RP135digal that in the native peptide and N-linked analogue. Binding data for the glycopeptides with 0.5beta, a monoclonal antibody mapped to the RP135 sequence, revealed a significant enhancement in binding for RP135digal as compared with the native peptide, whereas binding was reduced for the N-linked glycopeptide. These data show that glycosylation of V3 loop peptides can affect their conformations as well as their interactions with antibodies. The design of more ordered and biologically relevant conformations of immunogenic regions from gp120 may aid in the design of more effective immunogens for HIV-1 vaccine development.
We present a resonant optical nanoantenna positioned at the end of a metal-coated glass fiber near-field probe. Antenna resonances, excitation conditions, and field localization are directly probed in the near field by single fluorescent molecules and compared to finite integration technique simulations. It is shown that the antenna is equivalent to its radio frequency analogue, the monopole antenna. For the right antenna length and local excitation conditions, antenna resonances occur that lead to an enhanced localized field near the antenna apex. Direct mapping of this field with single fluorescent molecules reveals a spatial localization of 25 nm, demonstrating the importance of such antennas for nanometer resolution optical microscopy.
Colchicine is frequently employed as a pharmacologic tool to enhance perikaryal neuropeptide concentrations, in order to facilitate mapping of functional neuron populations in the brain. However, it is not clear if effects of colchicine on central neurons include transcriptional activation. The following studies utilized immunocytochemical techniques to evaluate the effects of intracerebroventricular (i.c.v.) drug treatment on Fos-like immunoreactivity (Fos-li) in preoptic and hypothalamic neurons. Since colchicine is administered orally in the treatment of gout-associated arthritis, additional experiments examined whether intragastric delivery of colchicine elicits protooncogene expression by neurons in the brain. Groups of adult male rats were treated with colchicine by i.c.v. injection (150 micrograms/3.0 microliters 0.9% saline) or by gavage (4.0 mg/1.0 ml 0.9% saline); vehicle-treated controls received saline alone. All animals were sacrificed 24 hr after drug or vehicle treatment. Serial 25 microns brain sections were processed for Fos-like immunoreactivity using anti-human Fos4-17 antibodies (Ab-2, Oncogene Sciences) in conjunction with avidin-biotin immunoperoxidase cytochemistry. These studies revealed negligible immunolabeling for Fos 24 hr after vehicle treatment, 24 hr after intracerebral delivery of colchicine, Fos-li was observed within the medial preoptic area, the arcuate nucleus, the supraoptic nucleus, and parvocellular neurons in the paraventricular nucleus. Animals treated with colchicine by gavage exhibited Fos-immunopositive neurons in the same sites, but additional immunolabeling for Fos was also observed within the median preoptic nucleus, suprachiasmatic nucleus, dorsomedial nucleus, and magnocellular neurons in the paraventricular nucleus. These results suggest, first of all, that neuronal responses to colchicine exposure include the synthesis of Fos-like proteins in a number of brain sites, at least over the time frame examined here. The present findings that protooncogene expression occurs within central neurons in response to intragastric drug administration suggest that neuronal activation may in response to drug-induced neural afferent and/or endocrine stimuli of peripheral origin.
The Polycomb group (PcG) of proteins conveys epigenetic inheritance of repressed transcriptional states. In Drosophila, the Polycomb repressive complex 1 (PRC1) maintains the silent state by inhibiting the transcription machinery and chromatin remodelling at core promoters. Using immunoprecipitation of in vivo formaldehyde-fixed chromatin in phenotypically diverse cultured cell lines, we have mapped PRC1 components, the histone methyl transferase (HMT) Enhancer of zeste (E(z)) and histone H3 modifications in active and inactive PcG-controlled regions. We show that PRC1 components are present in both cases, but at different levels. In particular, active target promoters are nearly devoid of E(z) and Polycomb. Moreover, repressed regions are trimethylated at lysines 9 and 27, suggesting that these histone modifications represent a mark for inactive PcG-controlled regions. These PcG-specific repressive marks are maintained by the action of the E(z) HMT, an enzyme that has an important role not only in establishing but also in maintaining PcG repression.
We examined the interplay between the insulin/IGF-1- and beta-catenin-regulated pathways, both of which are suspected to play a role in hepatocarcinogenesis. Insulin and IGF-1 stimulated the transcription of a Lef/Tcf-dependent luciferase reporter gene by 3-4-fold in HepG2 cells. This stimulation was mediated through the activation of phosphatidylinositol 3-kinase (PI 3-K)/Akt and the inhibition of glycogen synthase kinase-3beta (GSK-3beta) since the effects of insulin and IGF-1 were inhibited by dominant-negative mutants of PI 3-K or Akt and an uninhibitable GSK-3beta. Together with inhibiting GSK-3beta, insulin and IGF-1 increased the cytoplasmic levels of beta-catenin. The PI 3-K/Akt/GSK-3beta pathway was not the sole to mediate insulin and IGF-1 stimulation of Lef/Tcf-dependent transcription. The Ras signalling pathway was also required as (i) the stimulatory effects of insulin and IGF-1 were inhibited by dominant-negative Ras or the MEK1 inhibitor PD98059 and (ii) activated Ha-Ras or constitutively active MEK1 synergized with catalytically inactive GSK-3beta to stimulate Lef/Tcf-dependent transcription. This study provides the first evidence that insulin and IGF-1 stimulate the beta-catenin pathway through two signalling cascades bifurcating downstream of PI 3-K and involving GSK-3beta inhibition and Ras activation. These findings demonstrate for the first time the ability of insulin and IGF-1 to activate the beta-catenin pathway in hepatoma cells and thereby provide new insights into the role of these factors in hepatocarcinogenesis.