A new method for assaying adhesion of cancer cells to the greater omentum and its application for evaluating anti-adhesion activities of chemically synthesized oligosaccharides

A new ex vivo method for assaying adhesion of cancer cells to the greater omentum has been developed using mouse greater omentum and [³H]labelled human gastric and mouse colorectal cancer cells. Since the adhesion rates were found to increase up to 18 h and labelled cells seemed to be stable during the period, the present method could be useful for investigating adhesion of cancer cells to the greater omentum, which must occur at the first step of the peritoneal dissemination. The adhesion of cancer cells to the greater omentum was inhibited by a series of chemically synthesized oligosaccharides and Galβ1,3[3OMeGalβ1,4GlcNAcβ1,6]αBn was found to be the best inhibitor. The anti-tumor effect of this novel tetrasaccharide in vivo was shown in preliminary experiments using Balb/c mice and colon26 cells.     

Positional cloning of the gene(WFS1) for Wolfram syndrome

Wolfram syndrome(DIDMOAD syndrome) is an autosomal recessive neurodegenerative disorder characterized by juvenile-onset, insulin-requiring diabetes mellitus and optic atrophy. Other symptoms including diabetes insipidus, neurosensory deafness, urinary tract and neurological abnormalities are often accompanied. In patients, beta-cells are selectively lost from their pancreatic islets of Langerhans. The gene was previously mapped to 4p16.1. By haplotype analysis and recombination mapping in 5 families, we localized the gene within a region less than 250 kb on chromosome 6p. In the region, we identified a novel gene(WFS1) encoding a putative transmembrane protein. Mutations were identified in all affected members of the families and these mutations were associated with disease phenotype. This finding was further confirmed by other investigators and to date, more than 50 mutations were identified in the WFS1 gene from the patients with Wolfram syndrome. The WFS1 gene encodes a protein of 100.3 kDa with 9 to 10 putative transmembrane domains. The protein appears to be important in the survival and maintenance of normal pancreatic beta-cells and neurons. Physiological function of the WFS1 protein and mechanisms by which defective WFS1 lead to the development of Wolfram syndrome need to be clarified.     

Immunoglobulin superfamily molecules in the nervous system.

Among the various types of membrane molecules involved in cell-cell interactions in the nervous system, we have focused in this review upon membrane proteins belonging to the immunoglobulin superfamily (IgSF). IgSF molecules are distinctive in that: (1) a large percentage of known neural adhesion molecules belongs to the IgSF; (2) they are homologous in structure (Ig domain), yet exhibit large variation of function in cell-cell interactions. The structure of IgSF molecules is briefly summarized in Section II, and each member of the IgSF which has been found in the nervous system is reviewed in Section III. In Section IV, we have discussed possible properties of yet-unknown nervous system IgSF molecules, on the assumption that nervous system IgSF molecules thus far discovered comprise only a small portion of those existing. Discussion is based upon an analogy with the immune system and upon knowledge of cell-cell interactions in the development of the nervous system. Our principal aims in this review are to summarize knowledge of neural IgSF molecules and to discuss the possibility that some IgSF molecules may encode in their structures instructions for recognizing, or for being recognized by, target neural cells. Further growth of knowledge of IgSF molecules may yield insights into the patterns of cell-cell interactions underlying the formation of neuronal circuits during development.     

Cardiac ankyrin repeat protein is preferentially induced in atrophic myofibers of congenital myopathy and spinal muscular atrophy

Cardiac ankyrin repeat protein (CARP), which is structurally characterized by the presence of four ankyrin repeat motifs in its central region, is believed to be localized in the nucleus and to participate in the regulation of cardiac-specific gene expression in cardiomyocytes. However, we recently found that CARP was induced in skeletal muscle by denervation, leading us to speculate that CARP may be induced under some pathological conditions. In the present study, we immunohistochemically analyzed the expression of CARP in 11 cases of spinal muscular atrophy (SMA) and 14 cases of congenital myopathy. In SMA, CARP was expressed selectively in severely atrophic myofibers, suggesting that CARP expression may reflect the status of muscle atrophy. Furthermore, in the congenital myopathies, the expression patterns of CARP were distinct among the subtypes, which included nemaline myopathy, myotubular myopathy, central core disease, and congenital fiber type disproportion. Although CARP was preferentially expressed in severely damaged myofibers in nemaline myopathy, it was not detected in central core disease. These findings suggest that immunohistochemical evaluation of CARP may be helpful in the diagnosis of SMA and the congenital myopathies.     

Creation and use of a composite polyurethane-expanded polytetrafluoroethylene graft for hemodialysis access

The Thoratec (Vectra) polyurethane vascular access graft (TPVA) is among the most recent additions to the list of materials used to construct prosthetic grafts for vascular access during hemodialysis. We give the TPVA very high marks, and recognize the utility of such a graft for use in hemodialysis. However, the strong elasticity of this graft can lead to unexpected complications after suturing. We devised a new surgical method using a TPVA-ePTFE (expanded polytetrafluoroethylene) composite graft, substituting the anastomosis section of the TPVA with a portion of ePTFE graft material, and have been able to overcome most of the TPVA's potential problems. We herein describe the technique.     

Interleukin-2 receptor gene expression in kidney transplant recipients treated with cyclosporin A.

We examined the effects of cyclosporin A (CsA) administered in vivo on the capacity of peripheral blood mononuclear cells (PBMC) from kidney transplant recipients to express IL-2 receptor (IL-2R) gene at the level of mRNA after mitogen stimulation in vitro. There were no differences in the percentage of IL-2R+ cells among the groups of normal individuals, azathioprine-prednisolone treated, and CsA-prednisolone-treated recipients, using FITC-labelled monoclonal anti-IL-2R antibody (anti-alpha chain): 40.3 +/- 10.1% and 62.8 +/- 11.1% of normal PBMC (n = 18), 37.0 +/- 9.3% and 61.7 +/- 5.8% of PBMC from azathioprine-prednisolone-treated recipients (n = 20), and 37.7 +/- 9.6% and 60.7 +/- 12.7% of PBMC from CsA-prednisolone-treated recipients (n = 20) expressed IL-2R after 24 h and 48 h of phytohaemagglutinin stimulation, respectively. However, in a study of Northern blotting using cDNA for IL-2R (anti-alpha chain specific), both the 3500 and 1400 bp families of IL-2R mRNA were remarkably decreased in PBMC from CsA-prednisolone-treated recipients compared with azathioprine-prednisolone-treated recipients and normal individuals. These studies demonstrated that CsA could inhibit IL-2R gene expression at the level of mRNA at physiological concentration.     

Nucleotide deletion of T cell receptor V gamma 2 and J gamma 2 coding sequences at V gamma 2-J gamma 2 junctions in immature B cell lines.

Immature B cell lines transformed with a temperature-sensitive mutant of Abelson murine leukaemia virus undergo preferentially V gamma 2 to J gamma 2 joinings during culture at a non-permissive temperature (39 degrees C). Here we examined nucleotide addition and deletion at the V gamma 2-J gamma 2 junctions in these V gamma 2 to J gamma 2 joinings, in comparison with the V gamma 2-J gamma 2 junctional sequences reported previously in T cells. Forty-eight V gamma 2-J gamma 2 junctions were PCR-amplified and sequenced. Only three of 48 V gamma 2-J gamma 2 junctions had nucleotide addition. The average nucleotide deletion of V gamma 2 coding sequences at the V gamma 2-J gamma 2 junctions was 6.9 nucleotides in immature B cells and 5.1 nucleotides in T cells (p less than 0.05). Also, the average nucleotide deletion of J gamma 2 coding sequences at the V gamma 2-J gamma 2 junctions was 3.1 nucleotides in immature B cells and 1.9 nucleotides in T cells (p less than 0.01). The average of total number of the deleted nucleotides at the V gamma 2-J gamma 2 junctions was 10.0 nucleotides in immature B cells and 7.0 nucleotides in T cells (p less than 0.01). No correlation was found between the extent of the nucleotide deletion of the V gamma 2 coding sequence and that of the J gamma 2 coding sequence at each V gamma 2-J gamma 2 junction in immature B and T cells. These results demonstrated that nucleotide deletion at the V gamma 2-J gamma 2 junctions was significantly wider in immature B cells than in T cells.     

Muscarinic stimulation of guinea pig adrenal chromaffin cells stimulates catecholamine secretion without significant increase in Ca2+ uptake

In isolated guinea pig adrenal chromaffin cells, not only nicotine, but also muscarine stimulated catecholamine (CA) secretion, the stimulation by muscarine being the greater. The secretions of CA by muscarine and nicotine were both dependent on the presence of Ca2+ in the medium, but only the latter was associated with a rapid increase in 45Ca2+ uptake. Experiments with the fluorescent Ca2+ indicator quin 2, showed that muscarine caused an increase in cytoplasmic free Ca2+ concentration [( Ca2+]i). Moreover, the intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) inhibited both CA secretion and increase in [Ca2+]i induced by muscarine. These results indicate that in isolated guinea pig adrenal chromaffin cells, nicotine stimulated CA secretion by increasing Ca2+ uptake by the cells, whereas muscarine stimulated CA secretion by mobilizing Ca2+ from the intracellular pool.     

Evidence of [Ca(2+)]i elevation by anti-calreticulin immunoreactive protein in neurons

Calreticulin is a multifunctional Ca(2+)-binding protein. The effect of anti-calreticulin antibody on intracellular free calcium concentration [Ca(2+)]i was studied in cultured neurons using fura-2 based microfluorometry. Anti-calreticulin increased [Ca(2+)]i in a dose dependent manner. The anti-calreticulin antibody produced a rapid transient [Ca(2+)]i peak followed by a long slowly decaying plateau. Anti-calreticulin induced extracellular Ca(2+) influx in cultured neuron cells was blocked partially by N-methyl-D-aspartate receptor (NMDAR) antagonist 2-amino-5-phosphonovaleric acid (AP5) and spider polyamine toxin JSTX-3, which is recognized as a blocker of glutamatergic nervous system. Furthermore, anti-calreticulin induced intracellular Ca(2+) desensitized NMDAR. Dual immunofluorescent staining studies revealed that NMDAR co-localized with calreticulin in the cultured neurons. Thus, the signal transduction system of NMDA might be closely concerned with the extracellular calreticulin like protein.     

Characterization of epstein-barr virus-infected mantle cell lymphoma lines

It has been reported that Epstein-Barr virus (EBV) resides in resting B cells in vivo. However, an ideal in vitro system for studying EBV latent infection in vivo has not yet been established. In this study, a mantle cell lymphoma line, SP53, was successfully infected with a recombinant EBV containing a neomycin-resistant gene. The EBV-carrying SP53 cells were obtained by selection using G418. They expressed EBER-1, EBNAs, and LMP1; this expression pattern of the EBV genes was similar to that in a lymphoblastoid cell line (LCL). However, proliferation assay showed that the EBV-carrying SP53 cells have a doubling time of 73 h, compared with 57 h of SP53 cells. Transplantation of 10(8) SP53 cells to nude mice formed tumors in 4 of 10 mice inoculated, but the EBV-carrying SP53 cells did not. Unexpectedly, EBV infection reduced the proliferation and tumorigenicity of SP53 cells. However, the EBV-carrying SP53 cells showed higher resistance to apoptosis induced by serum starvation than did the SP53 cells. The inhibition of proliferation and the resistance to apoptosis induced in SP53 cells by EBV infection indicate that this cell line might to some extent provide a model of in vivo EBV reservoir cells.