Amphetamine injections into the nucleus accumbens affect neither acquisition/expression of conditioned fear nor baseline startle response

The acoustic startle response is enhanced during states of fear and attenuated during pleasant ones. Our question was whether pharmacological stimulation of the reward system disrupts the learning and retrieval of conditioned fear as measured by fear-potentiated startle. We therefore injected the dopamine agonist amphetamine into the nucleus accumbens (NAC) immediately before either acquisition or expression of conditioned fear and measured the effect of these injections on fear-potentiated startle and baseline startle response. This study clearly showed that amphetamine injections into the NAC had no effect on baseline startle amplitude and acquisition/expression of conditioned fear. In contrast, amphetamine injections into the nucleus accumbens clearly enhanced spontaneous motor activity. These results suggest that dopamine within the NAC is not involved in modulation of fear-potentiated startle and baseline startle.     

Identification and characterization of a conserved,stage-specific gene product of Plasmodium falciparum recognized by parasite growth inhibitory antibodies

We have identified a novel conserved protein of Plasmodium falciparum, designated D13, that is stage-specifically expressed in asexual blood stages of the parasite. The predicted open reading frame (ORF) D13 contains 863 amino acids with a calculated molecular mass of 99.7 kDa and displays a repeat region composed of pentapeptide motives. Northern blot analysis with lysates of synchronized blood stage parasites showed that D13 is highly expressed at the mRNA level during schizogony. The first N'-terminal 138 amino acids of D13 were expressed in Escherichia coli and the purified protein was used to generate anti-D13 monoclonal antibodies (MAbs). Using total lysates of blood stage parasites and Western blot analysis, these MAbs stained one single band of approximately 100 kDa, corresponding to the predicted molecular mass of ORF D13. Western blot analysis demonstrated further that D13 is expressed during schizogony, declines rapidly in early ring stages and is undetectable in trophozoites. D13 protein is localized in individual merozoites in a distinct area, as demonstrated by indirect immunofluorescence analysis. After subcellular fractionation, D13 was confined to the pelleted fraction of the parasite lysate and its extraction by alkaline carbonate buffer treatment indicated that D13 is not a membrane-integral protein. Inclusion of certain anti-D13 MAbs into in vitro cultures of blood stage parasites resulted in considerable reduction in parasite growth. The N'-terminal domain encompassing 158 amino acids is 94 and 95%, respectively, identical at the amino acid level between Plasmodium knowlesi, Plasmodium yoelii, and P. falciparum. In summary, we describe a novel stage-specifically expressed, highly conserved gene product of P. falciparum that is recognized by parasite growth inhibitory antibodies.     

Redundant function of the heparan sulfate 6-O-endosulfatases Sulf1 and Sulf2 during skeletal development

Modification of the sulfation pattern of heparan sulfate (HS) during organ development is thought to regulate binding and signal transduction of several growth factors. The secreted sulfatases, Sulf1 and Sulf2, desulfate HS on 6-O-positions extracellularly. We show that both sulfatases are expressed in overlapping patterns during embryonic skeletal development. Analysis of compound mutants of Sulf1 and Sulf2 derived from gene trap insertions and targeted null alleles revealed subtle but distinct skeletal malformations including reduced bone length, premature vertebrae ossification and fusions of sternebrae and tail vertebrae. Molecular analysis of endochondral ossification points to a function of Sulf1 and Sulf2 in delaying the differentiation of endochondral bones. Penetrance and severity of the phenotype increased with reduced numbers of functional alleles indicating redundant functions of both sulfatases. The mild skeletal phenotype of double mutants suggests a role for extracellular modification of 6-O-sulfation in fine-tuning rather than regulating the development of skeletal structures.     

Cytotoxic T cell responses to haptenated cells III. Isolation and specificity analysis of continuously growing clones

Various procedures were used to derive continuously growing cytotoxic T lymphocyte (CTL) clones from a primary culture containing responder cells from immunized mice and 3-(p-sulfophenyldiazo)-4-hydroxylphenyl acetic acid (SP)-or fluorescein isothiocyanate (FL)-coupled stimulator cells. It seems likely that CTL have to undergo some change, possibly genetic, to be able to grow continuously in T cell growth factor conditioned medium in the absence of any stimulator or filler cells. The most convenient and reliable procedure to generate CTL clones with different specificities was to establish from several aliquots of a primary culture cell populations continuously growing in medium conditioned with T cell growth factor(s). Clones with different specificities segregated in the different populations. SP-and FL-specific CTL clones restricted to H-2K^k, and H-2D^d and two FL-specific CTL clones with no apparent H-2 restriction are described.     

Epithelial cancer in Fanconi anemia complementation group D2 (Fancd2) knockout mice

Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA damage, bone marrow failure, congenital defects, and cancer. To further investigate the in vivo function of the FA pathway, mice with a targeted deletion in the distally acting FA gene Fancd2 were created. Similar to human FA patients and other FA mouse models, Fancd2 mutant mice exhibited cellular sensitivity to DNA interstrand cross-links and germ cell loss. In addition, chromosome mispairing was seen in male meiosis. However, Fancd2 mutant mice also displayed phenotypes not observed in other mice with disruptions of proximal FA genes. These include microphthalmia, perinatal lethality, and epithelial cancers, similar to mice with Brca2/Fancd1 hypomorphic mutations. These additional phenotypes were not caused by defects in the ATM-mediated S-phase checkpoint, which was intact in primary Fancd2 mutant fibroblasts. The phenotypic overlap between Fancd2-null and Brca2/Fancd1 hypomorphic mice is consistent with a common function for both proteins in the same pathway, regulating genomic stability.     

Cell surface membrane antigen phenotype of human gastrointestinal mast cells

BACKGROUND: Mast cells (MC) are important effector cells of allergic and inflammatory reactions in diverse organs. These cells interact with a number of other immune cells and structural cells in the tissues as well as with proinflammatory mediators and cytokines. The various interactions are considered to be mediated through distinct cell surface membrane receptors on MC. METHODS: In the present study, we have established the cell surface membrane phenotype of human gastrointestinal MC (HGMC) using a panel of monoclonal antibodies and indirect immunofluorescence staining techniques. RESULTS: HGMC were found to react with antibodies against CD29, CD33, CD44, CD45, CD47, CD54, CD55, CD58, CD63, CD117, CD147, CD151, CD172a, and CD203c. By contrast, HGMC did not express detectable amounts of CD1, CD2, CD4, CD5, CD14, CD15, CD16, CD22, CD24, CD25, CD26, CD27, CD28, CD31, CD32, CD34, CD35, CD88, or CD116. The alpha-chain of the IL-3 receptor (CD123) was detectable neither in resting HGMC nor in HGMC exposed to stem cell factor and interleukin-4. CONCLUSIONS: HGMC express a unique profile of surface antigens including the receptor for mast cell growth factor, adhesion-related molecules, and activation-linked membrane antigens.     

The selective processing of briefly presented affective pictures: an ERP analysis

Recent event-related potential (ERP) studies revealed the selective processing of affective pictures. The present study explored whether the same phenomenon can be observed when pictures are presented only briefly. Toward this end, pleasant, neutral, and unpleasant pictures from the International Affective Pictures Series were presented for 120 ms while event related potentials were measured by dense sensor arrays. As observed for longer picture presentations, brief affective pictures were selectively processed. Specifically, pleasant and unpleasant pictures were associated with an early endogenous negative shift over temporo-occipital sensors compared to neutral images. In addition, affective pictures elicited enlarged late positive potentials over centro-parietal sensor sites relative to neutral images. These data suggest that a quick glimpse of emotionally relevant stimuli appears sufficient to tune the brain for selective perceptual processing.     

Fully automated, internally controlled quantification of hepatitis B Virus DNA by real-time PCR by use of the MagNA Pure LC and LightCycler instruments

We report on the development of a fully automated real-time PCR assay for the quantitative detection of hepatitis B virus (HBV) DNA in plasma with EDTA (EDTA plasma). The MagNA Pure LC instrument was used for automated DNA purification and automated preparation of PCR mixtures. Real-time PCR was performed on the LightCycler instrument. An internal amplification control was devised as a PCR competitor and was introduced into the assay at the stage of DNA purification to permit monitoring for sample adequacy. The detection limit of the assay was found to be 200 HBV DNA copies/ml, with a linear dynamic range of 8 orders of magnitude. When samples from the European Union Quality Control Concerted Action HBV Proficiency Panel 1999 were examined, the results were found to be in acceptable agreement with the HBV DNA concentrations of the panel members. In a clinical laboratory evaluation of 123 EDTA plasma samples, a significant correlation was found with the results obtained by the Roche HBV Monitor test on the Cobas Amplicor analyzer within the dynamic range of that system. In conclusion, the newly developed assay has a markedly reduced hands-on time, permits monitoring for sample adequacy, and is suitable for the quantitative detection of HBV DNA in plasma in a routine clinical laboratory.     

Single nucleotide variation analysis in 65 candidate genes for CNS disorders in a representative sample of the European population

The detailed investigation of variation in functionally important regions of the human genome is expected to promote understanding of genetically complex diseases. We resequenced 65 candidate genes for CNS disorders in an average of 85 European individuals. The minor allele frequency (MAF), an indicator of weak purifying selection, was lowest in radical amino acid alterations, whereas similar MAF was observed for synonymous variants and conservative amino acid alterations. In noncoding sequences, variants located in CpG islands tended to have a lower MAF than those outside CpG islands. The transition/transversion ratio was increased among both synonymous and conservative variants compared with noncoding variants. Conversely, the transition/transversion ratio was lowest among radical amino acid alterations. Furthermore, among nonsynonymous variants, transversions displayed lower MAF than did transitions. This suggests that transversions are associated with functionally important amino acid alterations. By comparing our data with public SNP databases, we found that variants with lower allele frequency are underrepresented in these databases. Therefore, radical variants obtain distinctively lower database coverage. However, those variants appear to be under weak purifying selection and thus could play a role in the etiology of genetically complex diseases.