Early neuromodulation prevents the development of brain and behavioral abnormalities in a rodent model of schizophrenia.

The notion that schizophrenia is a neurodevelopmental disorder in which neuropathologies evolve gradually over the developmental course indicates a potential therapeutic window during which pathophysiological processes may be modified to halt disease progression or reduce its severity. Here we used a neurodevelopmental maternal immune stimulation (MIS) rat model of schizophrenia to test whether early targeted modulatory intervention would affect schizophrenia's neurodevelopmental course. We applied deep brain stimulation (DBS) or sham stimulation to the medial prefrontal cortex (mPFC) of adolescent MIS rats and respective controls, and investigated its behavioral, biochemical, brain-structural and -metabolic effects in adulthood. We found that mPFC-DBS successfully prevented the emergence of deficits in sensorimotor gating, attentional selectivity and executive function in adulthood, as well as the enlargement of lateral ventricle volumes and mal-development of dopaminergic and serotonergic transmission. These data suggest that the mPFC may be a valuable target for effective preventive treatments. This may have significant translational value, suggesting that targeting the mPFC before the onset of psychosis via less invasive neuromodulation approaches may be a viable preventive strategy.

Paradoxical augmented relapse in alcohol-dependent rats during deep-brain stimulation in the nucleus accumbens.

Case reports indicate that deep-brain stimulation in the nucleus accumbens may be beneficial to alcohol-dependent patients. The lack of clinical trials and our limited knowledge of deep-brain stimulation call for translational experiments to validate these reports. To mimic the human situation, we used a chronic-continuous brain-stimulation paradigm targeting the nucleus accumbens and other brain sites in alcohol-dependent rats. To determine the network effects of deep-brain stimulation in alcohol-dependent rats, we combined electrical stimulation of the nucleus accumbens with functional magnetic resonance imaging (fMRI), and studied neurotransmitter levels in nucleus accumbens-stimulated versus sham-stimulated rats. Surprisingly, we report here that electrical stimulation of the nucleus accumbens led to augmented relapse behavior in alcohol-dependent rats. Our associated fMRI data revealed some activated areas, including the medial prefrontal cortex and caudate putamen. However, when we applied stimulation to these areas, relapse behavior was not affected, confirming that the nucleus accumbens is critical for generating this paradoxical effect. Neurochemical analysis of the major activated brain sites of the network revealed that the effect of stimulation may depend on accumbal dopamine levels. This was supported by the finding that brain-stimulation-treated rats exhibited augmented alcohol-induced dopamine release compared with sham-stimulated animals. Our data suggest that deep-brain stimulation in the nucleus accumbens enhances alcohol-liking probably via augmented dopamine release and can thereby promote relapse.

Development and characterization of composite YSZ-PEI electrophoretically deposited membrane for Li-ion battery.

In this work, the electrophoretic-deposition (EPD) method was used to fabricate pristine and composite ceramic-polymer membranes for application in planar and 3D microbattery configurations. The major focus was on the effect of polyethyleneimine additive on the morphology, composition, and electrochemical properties of the membrane. The ionic conductivity, cycleability, and charge/discharge behavior of planar LiFePO(4)/Li cells comprising composite porous YSZ-based membrane with impregnated LiPF(6) EC:DEC electrolyte were found to be similar to the cells with commercial Celgard membrane. Conformal EPD coating of the electrode materials by a thin-film ceramic separator is advantageous for high-power operation and safety of batteries.

Homologous expression of a mutated beta-tubulin gene does not confer benomyl resistance on Trichoderma virens.

AIMS: To clone the beta-tubulins and to induce resistance to benzimidazoles in the biocontrol fungus Trichoderma virens through site-directed mutagenesis. METHODS AND RESULTS: Two beta-tubulin genes have been cloned using PCR amplification followed by the screening of a T. virens cDNA library. The full-length cDNA clones, coding for 445 and 446 amino acids, have been designated as T. virens tub1 and T. virens tub2. A sequence alignment of these two tubulins with tubulins from other filamentous fungi has shown the presence of some unique amino acid sequences not found in those positions in other beta-tubulins. Constitutive expression of the tub2 gene with a histidine to tyrosine substitution at position 6 (known to impart benomyl/methyl benzimadazol-2-yl carbamate resistance in other fungi), under the Pgpd promoter of Aspergillus nidulans, did not impart resistance to benomyl. CONCLUSIONS: The homologous expression of tub2 gene with a histidine to tyrosine mutation at position +6, which is known to impart benomyl tolerance in other fungi, does not impart resistance in T. virens. SIGNIFICANCE AND IMPACT OF THE STUDY: Unlike other Trichoderma spp., T. virens, has been difficult to mutate for benomyl tolerance. The present study, through site-directed mutagenesis, shows that a mutation known to impart benomyl tolerance in T. viride and other fungi does not impart resistance in this fungus. Understanding the mechanisms of this phenomenon will have a profound impact in plant-disease management, as many plant pathogenic fungi develop resistance to this group of fungicides forcing its withdrawal after a short period of use.

A mitogen-activated protein kinase of the corn leaf pathogen Cochliobolus heterostrophus is involved in conidiation, appressorium formation, and pathogenicity: diverse roles for mitogen-activated protein kinase homologs in foliar pathogens.

Fungal pathogens perceive and respond to molecules from the plant, triggering pathogenic development. Transduction of these signals may use heterotrimeric G proteins, and it is thought that protein phosphorylation cascades are also important. We have isolated a mitogen-activated protein kinase homolog from the corn pathogen Cochliobolus heterostrophus to test its role as a component of the transduction pathways. The new gene, CHK1, has a deduced amino acid sequence 90% identical to Pmk1 of the rice blast fungus Magnaporthe grisea and 59% identical to Fus3 of Saccharomyces cerevisiae. A series of chk1 deletion mutants has poorly developed aerial hyphae, autolysis, and no conidia. No pseudothecia are formed when a cross between two Deltachk1 mutants is attempted. The ability of Deltachk1 mutants to infect corn plants is reduced severely. The growth pattern of hyphae on a glass surface is strikingly altered from that of the wild type, forming coils or loops, but no appressoria. This set of phenotypes overlaps only partially with that of pmk1 mutants, the homologous gene of the rice blast fungus. In particular, sexual and asexual sporulation both require Chk1 function in Cochliobolus heterostrophus, in contrast to Pmk1, but perhaps more similar to yeast, where Fus3 transmits the mating signal. Chk1 is required for efficient colonization of leaf tissue, which can be compared with filamentous invasive growth of yeast, modulated through another closely related mitogen-activated protein kinase, Kss1. Ubiquitous signaling elements thus are used in diverse ways in different plant pathogens, perhaps the result of coevolution of the transducers and their targets.

The effect of dental alloys on mouse lymphocyte subpopulations.

The aim of the present study was to determine the effect of nickel-containing alloys on lymphocyte subsets in an experimental setting. Plates of alloys containing nickel (Ceramalloy, Talladium, Cerillium, Rexillium) or gold (Orion) were implanted subcutaneously into mice. The levels of CD4+ and CD8+ T-lymphocyte subpopulations and of Smig+ B lymphocytes were determined at various intervals following implantation, using monoclonal antibodies and flow cytometry. No changes were detected in the proportion of the lymphocyte subsets tested. One month after implantation, the mean fluorescence intensity of CD4, CD8 or Smig, in the peripheral blood lymphocytes (PBL) of the nickel alloy-implanted animals, was significantly higher than that prior to this procedure. Only a mild increase in CD4 and CD8 was noted after implantation of the gold alloy. The observed effects are most likely attributable to the surgical trauma, and do not indicate that nickel-containing dental alloys influence T cell subsets in this murine model.

The appearance of the CD4+CD8+ phenotype on activated T cells: possible role of antigen transfer.

Stimulation of peripheral blood lymphocytes (PBL) with phytohemagglutinin (PHA) strikingly increased the proportion of CD4+CD8+ cells. Highly purified CD4+ and CD8+ lymphocyte populations cultured in the presence of PHA consistently failed to coexpress the CD8 and CD4 markers. Similarly, exposure of highly purified CD4+ cells to PHA and recombinant interleukin-2 resulted in augmented expression of CD25 but failed to induce the expression of CD8. When purified preparations of either CD4+ or CD8+ cells were activated separately for 3 days and incubated together for an additional 5 h, a considerable proportion of CD4+CD8+ cells was found in the mixture. Cycloheximide treatment did not prevent the appearance of the CD8 marker on CD4 cells. CD4+CD8+ cells isolated from PBL exposed for 3 days to PHA lost their CD8 antigenicity within 24-48 h in the absence of PHA. Increased levels of soluble CD4 and CD8 antigens were found in supernatant fluids of PHA-stimulated cells. T cells failed, however, to bind soluble markers even after prolonged incubation in the presence of supernatant fluids. Our studies show that activation of CD4+ cells per se does not elicit the CD4+CD8+ phenotype and that soluble T cell markers do not bind to T cells. Rather, it seems that direct cell-cell contact is required for the transfer of CD8 molecules from CD8+ cells to the membrane of CD4+ cells.

Interspecific luciferase beta subunit hybrids between Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi.

Bacterial luciferase (EC 1.14.14.3) is a heterodimer composed of alpha- and beta-chains encoded by luxA and luxB, respectively. Although some interspecific combinations of these subunits lead to active enzyme, others do not. The beta subunits of Vibrio fischeri and Photobacterium leiognathi form active enzyme with the alpha subunits of V.fischeri, P.leiognathi and Vibrio harveyi, while the beta subunit from V.harveyi only complements the alpha subunit of V.harveyi. Inactivity is caused by a lack of dimerization of the beta subunit of V.harveyi with the alpha subunits of V.fischeri and P.leiognathi. These observations served as the basis for a search to discover which segment of the beta polypeptide confers the ability to dimerize with the alpha subunits of V.fischeri and P.leiognathi. Intragenic beta subunit hybrids were made between V.harveyi, V.fischeri and P.leiognathi. Unique restriction sites were introduced into the respective luxB genes to divide them into four roughly equal segments. In all, 78 hybrids were constructed by in vitro techniques. The N-terminal segment of the peptide contains the signals that differentiate between the beta subunits of V.fischeri and P. leiognathi and the beta subunit of V. harveyi, and allow the former to dimerize with their alpha subunits. The second segment has no major effect on enzyme activity but does exhibit some context effects. Important interactions were found between the third and fourth segments of the polypeptide with respect to enzymatic activity.

In vitro activation leads to the binding of T-cell markers to the surface of B-lymphocytes.

The aim of the present study was to determine whether activation of human T-cells in vitro results in the expression of markers characteristic for T-cells on the surface of B-lymphocytes and to correlate antigenic changes with the release of soluble T-cell antigens. Peripheral blood lymphocytes (PBL) were exposed in vitro to phytohemagglutinin (PHA) for 3 days. Changes in the phenotype of the cells were determined by flow cytometry and the level of soluble T-cell antigens was assessed by ELISA. PHA-activated PBL released elevated quantities of soluble CD2, CD4, and CD8 compared with control cultures. Following PHA stimulation the proportion of CD4+ CD8+ and HLA-DR+ cells increased. In addition, after 3 days of activation with PHA about 80% of the CD19+ cells (B-cells) reacted with F(ab)2 fragments of CD2 monoclonal antibodies (MoAbs). A high proportion of B-cells in activated cultures also reacted with F(ab)2 fragments of anti-CD8 MoAb and anti-CD4 MoAb. The removal of either CD4+ or CD8+ T-cells from the cultures prior to stimulations with PHA drastically reduced the proportion of B-cells expressing CD4 or CD8, respectively. The attachment of T-cell markers to the surface B-lymphocytes may constitute a new mechanism for B-cell regulation by T-cells.

Formation of active bacterial luciferase between interspecific subunits in vivo.

Interspecific complementation between luxAs and luxBs from Vibrio harveyi, Vibrio fischeri, Photobacterium leiognathi and Xenorhabdus luminescens was examined in vivo. The individual genes from these species were cloned on different compatible plasmids or amplified by PCR and brought together to yield cis combinations without extraneous DNA. The beta subunits from V. harveyi and X. luminescens form active enzyme only with alpha subunits from one of these species. All other combinations yield active enzymes. The lack of activity of the V. harveyi and X. luminescens beta subunits with the alpha subunits from V. fischeri and P. leiognathi results from a lack of association. This was shown by in vivo competition in which these beta subunits were overproduced in comparison with the beta and alpha of V. fischeri. No reduction in light was found. Overall, the in vivo results parallel those found in vitro using isolated denatured subunits and renaturation by removal of the denaturant.

[Psychosocial characteristics of phenylketonuric women: birth defect prevention].

Women with phenylketonuria (PKU) are at high risk for having offspring with mental retardation, microcephaly, heart defects and low birth weight. These adverse outcomes can be prevented by a low-phenylalanine diet started before conception and continued throughout pregnancy. In view of the frequency of poor dietary compliance in women with PKU, a psychosocial model was developed that delineates developmental stages with specific behavioral goals for them to follow. In the present study 15 women with PKU over the age of 16 were followed for 3 years and compared to groups of their healthy acquaintances and of diabetic women. Structured interviews and standard questionnaires were used to study factors hypothesized as being related to the subjects' adjustment and to achievement of their PKU-related behavioral goals. After 1 year most of the PKU subjects were not planning a pregnancy, making their main behavioral goal the prevention of an unplanned pregnancy. Their knowledge of the risks of maternal PKU and family planning was unsatisfactory. PKU subjects had more conservative attitudes about sex and contraception than the controls. The psychosocial profile of PKU subjects pinpointed their special needs and indicated the kinds of specific intervention that might help them adhere to the recommended treatment and prevent birth defects in their offspring.

Fusion of LuxA and LuxB and its expression in E. coli, S. cerevisiae and D. melanogaster.

Luciferase from Vibrio harveyi is encoded by two adjacent genes, luxA and luxB. The two genes were fused by replacing a segment extending from near the end of luxA into the N-terminal end of luxB by a synthetic oligonucleotide. The construction removed the TAA stop codon at the end of luxA, the intervening region of 26 base pairs, and the initial methionine of luxB. A Smal site was included at the junction between the two genes and an AatII site was created near the end of luxA without altering its amino acid sequence. In Escherichia coli the fused luxAB gene could be expressed to produce functional luciferase that gave about 20% of the activity in cells without the fusion. An out-of-frame ATG exists close to and preceding the ATG of the luxA gene. This was removed and the entire fused gene bracketed by several restriction enzyme sites. The fused luxAB gene was successfully expressed in Saccharomyces cerevisiae and Drosophila melanogaster by transferring it to appropriate plasmid vectors.

Modulation of the expression of CD4 on HL-60 cells by exposure to 1,25-dihydroxyvitamin D3.

The CD4 molecule functions as a receptor for the binding and infectivity of the human immunodeficiency virus (HIV). It is of interest, therefore, to develop procedures for its down-regulation. In the present study, the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the expression of cell surface antigens of the HL-60 promyelocytic leukemia cell line was analyzed. Exposure of HL-60 cells to 1,25(OH)2D3 resulted in down-regulation of CD4 as assessed by their staining with the Leu-3a monoclonal antibody (MoAb). This treatment increased the staining of HL-60 cells with the monocyte-specific 63D3 MoAb. In contrast to the rapid elimination of cell surface CD4 by exposure of HL-60 to phorbol myristate acetate (PMA), the maximal reduction of CD4 by 1,25(OH)2D3 was attained within 48 h after the beginning of the exposure.

Idiotype regulation of thymus autoantibodies.

Rabbit antisera specific for idiotypic determinants (Id) of monoclonal Thy-1 autoantibodies were tested for their capacity to elicit Id-bearing thymus autoantibodies in mice. Rabbits were immunized with the 20-10-5 monoclonal IgM Thy-1 autoantibody, and idiotype-specific antibodies (anti-Id) were obtained by affinity chromatography. BALB/c and C3H mice were immunized by repeated i.p. injections of the anti-Id reagent. Control mice received repeated injections of purified normal rabbit immunoglobulin (NRIg) sheep red blood cells (SRBC) or human serum albumin (HSA). The sera of the treated mice were examined for their reactivity with anti-Id xenoantisera and for their capacity to react with thymus cells. The injection of anti-Id sera into either BALB/c or C3H mice resulted in a significant increase in the serum concentration of Id-bearing immunoglobulins, while no such change was observed in the sera of mice injected with NRIg. The administration of anti-Id, NRIg, SRBC or HSA resulted in a gradual increase in the concentration of autoantibodies reactive with thymus cells. Incubation with anti-Id sera prevented the binding to thymus cells of autoantibodies induced by anti-Id treatment. In contrast, the binding capacity of autoantibodies induced by NRIg, SRBC or HSA was unaffected by anti-Id sera. Thus, only the thymus autoantibodies induced by anti-Id treatment expressed Id-determinants. These results demonstrate the existence of a regulatory network in the formation of thymus autoantibodies.

Group work with adolescent PKU girls and their mothers.

Group therapy was the method chosen for working with PKU girls and their mothers in improving their knowledge of maternal PKU and the prevention of unplanned pregnancies. It was found effective in relieving anxiety, and the importance of including the mothers in the group was critical.

Idiotypic determinants on monoclonal autoantibodies to the Thy-1 antigen.

Distinct monoclonal autoantibodies directed against the Thy-1 antigen differ in their reactivity with various target cells. The aim of the present study was to analyse the idiotypic specificities present on various monoclonal autoreactive Thy-1 antibodies. Xenogeneic anti-idiotypic (anti-Id) reagents were prepared by immunizing rabbits either with the 20-10-5 or with the C16-31 monoclonal Thy-1 autoantibodies. Both of the anti-Id reagents reacted with the immunizing MoAb in ELISA and inhibited the interaction of the immunizing MoAb with Thy-1-positive target cells. The anti-20-10-5 reagent reacted in ELISA inhibition tests with the C16-31 MoAb, yet failed to inhibit the binding of C16-31 MoAb to thymocytes. Anti-C16-31 Id reacted with the 20-10-5 MoAb both in ELISA assays and in cell-bound fluorescence inhibition tests. These results indicate that anti-C16-31 Id detects an Id determinant shared by C16-31 and 20-10-5 MoAb, which is a binding site-related idiotype, while anti-20-10-5 Id detects an idiotypic determinant shared by these two MoAb that is not located at the combining site of C16-31 MoAb. In contrast to the marked cross-reactivity found between C16-31 and 20-10-5 MoAb, the two anti-Id reagents showed only a weak cross-reactivity with five other Thy-1 autoantibodies tested, including those autoantibodies that showed patterns of reactivity similar to those of C16-31 and 20-10-5, with various target cells. It is concluded, therefore, that individual autoreactive Thy-1 MoAb possess distinctive idiotypic determinants, although some cross-reactivity can be detected between certain MoAbs.

A new autoreactive monoclonal antibody specific for the Thy-1 antigen.

The present study describes the development of a new IgM monoclonal autoantibody reactive with the Thy-1 antigen. The C16-31 monoclonal antibody (mAb) was considered as autoreactive because it reacted with thymus cells of both the C3H and BALB/c strains, which were involved in the development of the antibody. The antibody was reactive with thymus cells in both immunofluorescent and cytotoxic tests. It also showed a weak immunofluorescent reactivity with peripheral T-lymphocytes. The identification of the specificity detected by the C16-31 mAb as the Thy-1 antigen was based on the following criteria: C16-31 mAB displayed a preferential reactivity with Thy-1.2 bearing thymus cells, rather than with Thy-1.1 bearing thymus cells. The tissue distribution of the antigen detected by the C16-31 antibody by direct tests and by direct tests and by adsorption experiments was in accordance with that characteristic for Thy-1. It was high on brain tissue and on thymus cells, and considerably lower on peripheral T-lymphocytes. Coating of thymocytes with C16-31 antibody blocked their reactivity with other monoclonal Thy-1 antibodies. Conversely, coating of thymus cells with rabbit anti-brain serum (RABR) inhibited the binding of C16-31. The C16-31 mAb differed from the Thy-1 autoantibodies described previously in its relatively strong reactivity with brain tissue and its considerably weaker reactivity with peripheral T-lymphocytes. Moreover, C16-31 mAb showed a preferential allospecificity for Thy-1.2, only in its reactivity with thymocytes. In contrast, it reacted equally well with brain tissue from either Thy-1.2 or Thy-1.1 mice.

Establishment of the Amsalem T-cell line from a patient with acute lymphoblastic leukemia. Expression of E-receptor-associated antigens in cells incapable of forming E-rosettes.

A new T-cell line (Amsalem) was established from the peripheral blood of a patient with pre-T leukemia. Amsalem cells are unique in that they possess antigenic determinants associated with the E-receptor, yet fail to form rosettes with sheep red blood cells (SRBC). Amsalem cells were found to possess morphological and cytochemical features characteristic of T-lymphocytes, and were sensitive to the cytotoxic effect of rabbit antisera specific for T-cell antigens. In immunofluorescent tests with monoclonal antibodies, Amsalem cells showed a strong reactivity with the OKT-11A and A-22 antibodies, specific for the E-receptor. The cells were reactive with OKT-4 and showed a very weak reactivity with OKT-6 and OKT-8. No reactivity was found with the OKT-3, Leu 7, Leu 11, and OKM1 antibodies. Amsalem cells failed to form rosettes with SRBC; however, mouse anti-Amsalem serum inhibited the formation of E-rosettes. It is concluded that the Amsalem cell line is a line of pre-T leukemia cells characterized by a dissociation between its inability to form E-rosettes and the presence of antigenic constituents of the E-receptor.

The role of E receptors in the attachment of thymocytes and T lymphocytes to human target cells.

Human thymocytes, activated T lymphocytes, and neuraminidase-treated T cells possess the distinct capacity of forming conjugates with various human cell lines. The present study investigated whether E receptors, which endow human T cells with their capacity to bind sheep red blood cells (SRBC), are involved in this phenomenon. Monoclonal antibodies to human T cells and various simple sugars were studied for their effect on the attachment of human T cells to target cells. A-22, a monoclonal antibody to the E receptor, inhibited the formation of E rosettes by T cells and SRBC, and reacted in immunofluorescent-staining assays with the majority of human thymocytes and peripheral T cells, and with T-cell lines capable of forming E rosettes. When human thymus cells were treated with A-22 antibody they showed a reduction of up to 70% in their capacity to attach to the GM-4762 lymphoblast cell line and the K-562 myeloid line. Antibody treatment of the target cells, rather than of the thymus cells, had no effect on the formation of conjugates between thymus cells and target cells. Treatment of thymus cells with various monoclonal antibodies to T cells which do not react with the E receptor had no inhibitory effect. The exposure of human thymus cells to various simple sugars (D-mannose, D-fucose, galactose, and lactose) markedly reduced their capacity of forming conjugates with target cells. Exposure of neuraminidase-treated peripheral blood lymphocytes and of activated T cells to A-22 antibody inhibited their attachment to human target cells. The present study suggests that E receptors play a role in the attachment of human thymus cells and activated T cells to other human cells, and raises the possibility that these T-cell receptors may be involved in the process of recognition of "self" structures by human T lymphocytes.