DISC1 causes associative memory and neurodevelopmental defects in fruit flies.

Originally found in a Scottish family with diverse mental disorders, the DISC1 protein has been characterized as an intracellular scaffold protein that associates with diverse binding partners in neural development. To explore its functions in a genetically tractable system, we expressed the human DISC1 in fruit flies (Drosophila melanogaster). As in mammalian neurons, DISC1 is localized to diverse subcellular domains of developing fly neurons including the nuclei, axons and dendrites. Overexpression of DISC1 impairs associative memory. Experiments with deletion/mutation constructs have revealed the importance of amino-terminal domain (46-290) for memory suppression whereas carboxyl domain (598-854) and the amino-terminal residues (1-45) including the nuclear localization signal (NLS1) are dispensable. DISC1 overexpression also causes suppression of axonal and dendritic branching of mushroom body neurons, which mediate a variety of cognitive functions in the fly brain. Analyses with deletion/mutation constructs reveal that protein domains 598-854 and 349-402 are both required for the suppression of axonal branching, while amino-terminal domains including NLS1 are dispensable. In contrast, NLS1 was required for the suppression of dendritic branching, suggesting a mechanism involving gene expression. Moreover, domain 403-596 is also required for the suppression of dendritic branching. We also show that overexpression of DISC1 suppresses glutamatergic synaptogenesis in developing neuromuscular junctions. Deletion/mutation experiments have revealed the importance of protein domains 403-596 and 349-402 for synaptic suppression, while amino-terminal domains including NLS1 are dispensable. Finally, we show that DISC1 functionally interacts with the fly homolog of Dysbindin (DTNBP1) via direct protein-protein interaction in developing synapses.

Emerging roles for the FCRL family members in lymphocyte biology and disease.

Members of the extended Fc receptor-like (FCRL) family in humans and mice are preferentially expressed by B cells and possess tyrosine-based immunoregulatory function. Although the majority of these proteins repress B cell receptor-mediated activation, there is an emerging evidence for their bifunctionality and capacity to counter-regulate adaptive and innate signaling pathways. In light of these findings, the recent discovery of ligands for several of these molecules has begun to reveal exciting potential for them in normal lymphocyte biology and is launching a new phase of FCRL investigation. Importantly, these fundamental developments are also setting the stage for defining their altered roles in the pathogenesis of a growing number of immune-mediated diseases. Here we review recent advances in the FCRL field and highlight the significance of these intriguing receptors in normal and perturbed immunobiology.

Nuclear DISC1 regulates CRE-mediated gene transcription and sleep homeostasis in the fruit fly.

Disrupted-in-schizophrenia-1 (DISC1) is one of major susceptibility factors for a wide range of mental illnesses, including schizophrenia, bipolar disorder, major depression and autism spectrum conditions. DISC1 is located in several subcellular domains, such as the centrosome and the nucleus, and interacts with various proteins, including NudE-like (NUDEL/NDEL1) and activating transcription factor 4 (ATF4)/CREB2. Nevertheless, a role for DISC1 in vivo remains to be elucidated. Therefore, we have generated a Drosophila model for examining normal functions of DISC1 in living organisms. DISC1 transgenic flies with preferential accumulation of exogenous human DISC1 in the nucleus display disturbance in sleep homeostasis, which has been reportedly associated with CREB signaling/CRE-mediated gene transcription. Thus, in mammalian cells, we characterized nuclear DISC1, and identified a subset of nuclear DISC1 that colocalizes with the promyelocytic leukemia (PML) bodies, a nuclear compartment for gene transcription. Furthermore, we identified three functional cis-elements that regulate the nuclear localization of DISC1. We also report that DISC1 interacts with ATF4/CREB2 and a corepressor N-CoR, modulating CRE-mediated gene transcription.

Intraarticular administration of platelet-rich plasma with biodegradable gelatin hydrogel microspheres prevents osteoarthritis progression in the rabbit knee.

OBJECTIVE: To investigate the therapeutic potential of administration of gelatin hydrogel microspheres containing platelet-rich plasma (PRP), by examining its effects on progression of osteoarthritis (OA) in a rabbit model. METHODS: PRP and platelet-poor plasma (PPP) were prepared from rabbit blood. Adult rabbit chondrocytes were cultured in the alginate beads with the presence of 3% PRP or 3% PPP. Glycosaminoglycan (GAG) synthesis was quantified using dimethylmethylene blue assay. To confirm the anabolic effect of PRP in vivo, cartilage matrix gene expression was examined after intraarticular administration of PRP contained in gelatin hydrogel microspheres. The PRP contained in gelatin hydrogel microspheres was administered into the rabbit knee joint twice with an interval of 3 weeks, beginning 4 weeks after anterior cruciate ligament transection (ACLT). Ten weeks after ACLT, gross morphological and histological examinations were performed. RESULTS: PRP significantly stimulated chondrocyte GAG synthesis in vitro. In the knee joint, expression of proteoglycan core protein mRNA in the articular cartilage increased after administration of PRP contained in microspheres. Intraarticular injections of PRP in gelatin hydrogel microspheres significantly suppressed progression of OA in the ACLT rabbit model morphologically and histologically. CONCLUSION: The present findings indicate that sustained release of growth factors contained in PRP has preventive effects against OA progression. These preventive effects appear to be due to stimulation of cartilage matrix metabolism, caused by the growth factors contained in PRP.

Isolation of the chicken NF-kappa B p65 subunit-encoding cDNA and characterization of its products.

NF-kappa B is a heterodimeric transcription factor consisting of subunits of 50 kDa (p50) and 65 kDa (p65). cDNA clones encoding the chicken NF-kappa B p65 subunit were isolated. Sequence analysis showed that chicken p65 is approximately 55% identical to the mouse and human p65 proteins, and contains the Rel homology domain (RHD) in its N-terminal 286 amino acids (aa) and the putative transactivation domain in its C-terminal region. The RHD is particularly highly conserved between the chicken and mammalian p65 proteins. Northern blot hybridization analysis detected the expression of a 2.6-kb transcript of p65 in various organs, with the highest level in spleen. A fusion protein containing the RHD of chicken p65 was found to bind to a consensus kappa B-site in an electrophoretic mobility shift assay (EMSA). This binding was specifically inhibited by the presence of fusion proteins containing the C-terminal ankyrin repeats domain (ARD) of chicken p105, the precursor protein for the p50 subunit. Immunoprecipitation analysis showed that p65 formed a complex(es) with multiple cellular proteins, including p50, p105 and c-Rel in chicken spleen cells.

Adipogenesis inhibitory factor. A novel inhibitory regulator of adipose conversion in bone marrow.

Recombinant adipogenesis inhibitory factor (AGIF) was purified to homogeneity from the conditioned medium of COS-1 cells transfected with human AGIF cDNA. The amino-terminal sequence analysis of the mature AGIF revealed that AGIF was produced as a precursor consisting of 199 amino acids and processed into a mature form of 178 amino acids by a cleavage between Ala(-1) and Pro(+1). The purified AGIF inhibited the process of adipogenesis in mouse 3T3-L1 preadipocytes, indicating that AGIF directly acts on the cells. AGIF acted as an adipogenic antagonist not only on the extramedullary cell line 3T3-L1 but also on the mouse bone marrow stroma-derived cell line H-1/A, suggesting that this cytokine may regulate adipogenesis in bone marrow.

Molecular cloning of cDNA encoding adipogenesis inhibitory factor and identity with interleukin-11.

A cDNA encoding a novel adipogenesis inhibitory factor (AGIF) that inhibits the process of adipogenesis in mouse 3T3-L1 preadipocytes was cloned from a cDNA library of the human bone marrow-derived stromal cell line KM-102. The cloned cDNA contains an open reading frame coding for an AGIF precursor of 199 amino acids. Analysis of the sequence of this cDNA revealed identity of this factor with a recently reported novel cytokine, designated interleukin-11 (IL-11). AGIF/IL-11 may play an important role in stromal cell-associated hematopoeisis through its regulatory action on adipocyte differentiation in the bone marrow microenvironment.

Heterogeneity of T cell clones specific for a single indirect alloantigenic epitope (I-Ab/H-2Kd54-68) that mediate transplant rejection.

BACKGROUND: One of the complexities of solid organ allograft rejection is the inherent diversity of the specific T cell antigenic epitopes that participate in this response, including the role of direct alloantigen recognition and indirect recognition of donor-derived peptides in recipient antigen-presenting cells. To probe the role of distinct T cell receptor (TCR) avidity differences and the role of cytokine expression patterns of different effector T cells that may participate in allograft rejections, we have identified a dominant allopeptide derived from the H-2Kd molecule, recognized by H-2b CD4 T cells in the context of syngeneic I-Ab. METHODS: To identify a stimulatory peptide derived from the H-2Kd molecule, a panel of synthetic overlapping peptides was screened for immunogenicity and a panel of T cell clones established. These clones were characterized for TCR Vbeta usage by mAb staining and/or reverse transcribed-polymerase chain reaction analysis, peptide dose sensitivity as a marker of TCR avidity, cytokine expression phenotype in vitro, and their ability to mediate rejection of a vascularized cardiac allograft after adoptive transfer to immunodeficient mice. RESULTS: The H-2Kd54-68 peptide was identified as a dominant stimulatory peptide by the ability of T cells from C57BL/6 (H-2b) mice primed by a combination of allogeneic spleen cell injection and mixed peptide immunization to mount an in vitro proliferative response and interferon-gamma production by peptide stimulation. Furthermore, direct immunization with synthetic H-2Kd54-68 peptide of normal C57BL/6 mice resulted in accelerated rejection of both skin and cardiac allografts from B10.D2 (H-2d) mice, but not 3rd party B10.BR (H-2k) grafts. A panel of 15 distinct CD4+ clones specific for H-2Kd54-68 peptide were established and shown to utilize a variety of TCR Vbeta and different apparent TCR avidities to H-2Kd54-68 peptide when stimulated in vitro. To characterize these clones further, two clones were chosen based on the difference of avidity to H-2Kd54-68 peptide. The cytokine expression pattern was determined and indirect alloantigen specificity confirmed by analysis of responses to purified peptide and B10.D2 spleen cells using normal H.2b and I-Abeta chain knockout mice as APC donors. Both of these T cell clones were able to mediate rejection of B10.D2, but not B10.BR hearts, in immunodeficient mice, but the morphological pattern of T cell infiltration was distinct. CONCLUSIONS: These results demonstrate the potential importance of fine dissection of the alloantigeneic response to solid organ transplants and provide unique insights into the role of TCR avidity and cytokine expression patterns in different morphological patterns of transplant rejection.

Immunosuppression by inhibition of cellular adhesion mediated by leukocyte function-associated antigen-1/intercellular adhesion molecule-1 in murine cardiac transplantation.

BACKGROUND: Donor alloantigen-specific tolerance to vascularized allografts can be induced by several treatments, but the immunological mechanism(s) of these effects remain unclear. One hypothesis is that allograft unresponsiveness is correlated with a shift in the pattern of expression of the T helper 1 versus T helper 2 T-cell cytokines. We report here an extensive analysis of murine cardiac allografts, during normal first set rejection and in mice treated with anti-adhesion molecule monoclonal antibodies (mAbs), a regimen that results in prolonged unresponsiveness. METHODS: A combination of immunohistochemical staining with a panel of mAbs, and in situ hybridization with a panel of digoxigenin-labeled riboprobes, was performed on frozen-tissue sections of cardiac allografts. RESULTS: In several strain combinations, injection of anti-leukocyte function-associated antigen-1 and anti-intercellular adhesion molecule-1, from day 0 to day 6 after transplantation, results in significant long-term survival. Examination of tolerated cardiac allografts by in situ hybridization and immunohistochemical staining shows an altered cytokine expression pattern, although the frequency of CD3 and CD4 cells is not dramatically reduced. These allografts show a decreased frequency of interferon-gamma and interleukin (IL)-2-expressing cells and a slightly increased frequency of cells expressing IL-4 and IL-10, compared with unmodified acute rejection. A direct role of these changes in T-cell cytokine expression is demonstrated by reversal of tolerance induction and rejection of the allograft by in vivo injection of either anti-IL-10 or anti-IL-4 mAb. CONCLUSIONS: Although there are significant differences in the frequency of different cellular subsets and patterns of cytokine gene expression, these differences are quantitatively subtle, suggesting a delicately balanced immune response that can develop a pattern of specific unresponsiveness, with relatively minor alterations in the specific T-cell response.

Partial purification and characterization of lathosterol 5-desaturase from rat liver microsomes.

The terminal oxidase of the NADH-dependent lathosterol 5-desaturation system was solubilized from rat liver microsomes with 2% Triton X-100, and partially purified approximately 18-fold with 19% yield after DEAE-cellulose and 6-aminohexyl-Sepharose column chromatography. The final enzyme preparation was free from other electron transfer components and phospholipids in microsomes, and the desaturation reaction was reconstituted with the following components: NADH, molecular oxygen, phospholipids and three proteins, i.e., NADH-cytochrome b5 reductase, cytochrome b5 and the terminal oxidase. Omission of one of these components led to an almost complete loss of the desaturase activity. Under the reconstitution conditions, the desaturase activity was significantly inhibited by potassium cyanide but was not affected by -SH reagents such as N-ethylmaleimide and dithiothreitol.

Foreign serum-induced bile duct lesion (BDL) in athymic BALB/c nude mice.

To investigate a role of cellular immunity in foreign serum-induced bile duct lesion (BDL) in mice, athymic BALB/c nude (nu/nu) mice were intraperitoneally injected with swine serum (SS) twice a week up to 8 weeks and were compared with euthymic BALB/c heterozygote (nu/+) and wild-type (+/+) mice treated with SS in the same way for 4 weeks. All immunized nu/+ and +/+ mice developed marked BDL, and their sera showed high anti-SS IgE and IgG1 antibody titers, whereas no immunized nu/nu mice developed lesions, and their sera showed no elevation of antibody titers. Next, nu/nu mice were reconstituted with splenocytes derived from nu/+ mice, and then were intraperitoneally injected with SS twice a week for 3 weeks. Most of the reconstituted nu/nu mice developed BDL, and their sera showed the elevation of anti-SS IgE and IgG antibody titers. These results suggest that cellular immunity may play a pivotal role in the pathogenesis of swine serum-induced BDL.

Modification of swine serum-induced bile duct lesion in BALB/c mice by cyclophosphamide.

Effects of cyclophosphamide (CY) on the antibody titer level and incidence and severity of swine serum (SS)-induced bile duct lesion (BDL) were examined. BDL induced by 0.2 ml of SS per head twice a week for 2 weeks was characterized by hyperplasia of biliary epithelial cells, proliferation of mucous glands, and periductal infiltration of eosinophils with mild fibrosis. CY showed no significant influence on the above-mentioned parameters at the dose levels of 140 and 210 mg/kg. On the other hand, CY lowered the antibody titer level and decreased the severity of BDL at the dose level of 280 mg/kg, and it suppressed the antibody response and BDL at the dose level of 280 x 2 mg/kg. Thus the antibody titer level and the severity of BDL were closely related each other.

GABA may function tonically via GABA(A) receptors to inhibit hypotension and bradycardia by L-DOPA microinjected into depressor sites of the nucleus tractus solitarii in anesthetized rats.

We have proposed that DOPA is a transmitter of the primary baroreceptor afferents terminating in the rat nucleus tractus solitarii (NTS). GABA is a putative inhibitory neuromodulator for baroreflex inputs in the NTS. GABA may inhibit DOPAergic transmission. Drugs were microinjected into depressor sites of the NTS in anesthetized rats. DOPA (10-60 ng) elicited dose-dependent depressor responses. GABA (3-300 ng) elicited dose-dependent pressor responses. Nipecotic acid (100 ng) elicited pressor responses. Bicuculline (10 ng) elicited depressor responses. Responses to DOPA (30 ng) were inhibited by pretreatment with GABA and nipecotic acid, but potentiated by bicuculline, when vascular responses to pretreated drugs returned to basal levels. DOPA ME, a competitive DOPA antagonist, did not displace specific [3H]GABA binding. Prior DOPA ME (1 microg) inhibited by one-half pressor responses to 300 ng GABA. GABA seems to inhibit tonically via GABA(A) receptors depressor responses to DOPA and to elicit pressor responses partially by inhibition of tonic function of endogenous DOPA to activate depressor sites in the NTS. These findings further support the above proposal.

The evidence for tonic GABAergic regulation of basal L-DOPA release via activation of inhibitory GABA(A) receptors in the nucleus tractus solitarii of anesthetized rats.

We have proposed that DOPA is a neurotransmitter of the primary baroreceptor afferents terminating in the rat nucleus tractus solitarii (NTS). GABA is a putative inhibitory neuromodulator for baroreflex inputs in the NTS. Thus, GABA may inhibit DOPAergic transmission in the NTS. We tried to clarify whether basal DOPA release is inhibited by muscimol, a GABA(A) agonist, and facilitated by bicuculline, a GABA(A) antagonist, during microdialysis of the NTS in anesthetized rats. DOPA release was consistently detectable. Muscimol 10-100 microM perfused via probes gradually inhibited concentration-dependently DOPA release. Peak 30% inhibition occurred 2 h after perfusion. Muscimol (30 microM)-induced inhibition was antagonized by non-effective 10 microM bicuculline. Bicuculline (30 microM) elicited peak 30% facilitation of DOPA release 2 h after perfusion. Endogenous GABA seems to regulate tonically basal DOPA release via activation of inhibitory GABA(A) receptors in the rat NTS. These findings further support the above proposal.

Chromosome spreading techniques for primary gastrointestinal tumors.

The requirement for well spread out chromosomes for the cytogenetic analysis of primary gastrointestinal tumors led us to develop new techniques. These techniques involved two main procedures: (1) preliminary incubation with culture medium in the presence of collagenase, Dispase, and colcemid, for 3 h, and (2) treatment with an extremely hypotonic solution (0.044M KCl) for 30 min. The techniques were applied to 11 gastrointestinal malignancies (including 1 early gastric cancer and 1 metastatic liver lesion of colon cancer) and significant increases (P < 0.01) in the number of metaphases of analyzable karyotypes were obtained, compared with a previous method in which the standard hypotonic molarity of KCL (0.075 M) was employed. The mean value for metaphase numbers of the analyzable karyotypes was 37.0 +/- 3.7% in the 5 gastric cancers and 44.7 +/- 4.8% in the 5 colon cancers and 1 metastatic lesion. These values were three times and more than twice, respectively, the values obtained by the previous method. A fluorescence in situ hybridization (FISH) study was carried out on one cologenic tumor, the alpha-satellite centromere-specific probe 17 being used. Deletion of the long arm of chromosome 17 was demonstrated. The method proposed here could yield a sufficient number of metaphases without the use of tissue culture that might cause alteration of karyotype. It can be employed with small biopsy specimens and in studies utilizing the FISH technique.

Process of carboxylation of glutamic acid residues in the gla domain of human des-gamma-carboxyprothrombin.

In the absence of vitamin K (VK) or in the presence of VK antagonists, hepatic VK-dependent carboxylase activity is inhibited and des-gamma-carboxyprothrombin (DCP) is released into the blood. We analyzed the number of glutamic acid (Glu) residues and their positions in the Gla domain (GD) of DCP to investigate the gamma-carboxylation mechanism of VK-dependent carboxylase. Several DCPs were found in each subject studied. The 10 Gla residues of human prothrombin were carboxylated in order from the N-terminal (residues 26, 25, 16, 29, 20, 19, 14, 32, 7 and 6). The process of Glu carboxylation seemed to proceed three-dimensionally from inside to outside the molecule.

Detection of circulating carcinoembryonic antigen (CEA) by counter-immunoelectrophoresis.

A sensitive method is described for detecting circulating carcinoembryonic antigen (CEA) by combined use of counter-immunoelectrophoresis and intensifiers. Perchloric acid extraction of serum was followed by counter-immunoelectrophoresis against monospecific antiserum to CEA. A precipitin line formed was intensified further by electrophoretic binding of anti-gammaG gamma-globulin and Amido Black staining. Less than 10 ng CEA/ml was detectable. Using this method, a total of 84 sera from patients with or without malignancy was examined. The results obtained were in general agreement with those reported by radioimmunoassay. The method appears to be useful as a diagnostic aid, since it requires neither labeled preparations of purified CEA nor special equipment for assay.

Sequence heterogeneity of the ten rRNA operons in Clostridium perfringens.

We have cloned and sequenced rRNA operons of Clostridium perfringens strain 13 and analyzed the sequence structure in view of the phylogenesis. The organism had ten copies of rRNA operons all of that comprised of 16S, 23S and 5S rDNAs except for one operon. The operons clustered around the origin of replication, ranging within one-third of the whole genome sequence as it is arranged in a circle. Seven operons were transcribed in clockwise direction, and the remaining three were transcribed in counter clockwise direction assuming that the gyrA was transcribed in clockwise direction. Two of the counter clockwise operons contained tRNA(Ile) genes between the 16S and 23S rDNAs, and the other had a tRNA(Ile) genes between the 16S and 23S rDNAs and a tRNA(Asn) gene in the place of the 5S rDNA. Microheterogeneity was found within the rRNA structural genes and spacer regions. The length of each 16S, 23S and 5S rDNA were almost identical among the ten operons, however, the intergenic spacer region of 16S-23S and 23S-5S were variable in the length depending on loci of the rRNA operons on the chromosome. Nucleotide sequences of the helix 19, helix 19a, helix 20 and helix 21 of 23S rDNA were divergent and the diversity appeared to be correlated with the loci of the rRNA operons on the chromosome.