Effects of soman on
N-methyl-
d-aspartate (NMDA) evoked
3Hnorepinephrine (NE) release were examined in rat brain cortical slices. NMDA increased
3HNE release in a concentration-dependent manner. ...Soman could inhibit the increase evoked by NMDA, but carbachol, an agonist of cholinergic receptor, could potentiate the increase evoked by NMDA. Atropine (a selective muscarinic antagonist) attenuated the release of
3HNE induced by NMDA in the presence of carbachol or acetylcholine (ACh), but had no effect on the release of
3HNE induced by NMDA alone. Both
d-tubocurarine (an antagonist of nicotinic receptor) and atropine had no effect on the release of
3HNE induced by NMDA in the presence of soman. These results suggested that soman has a direct action at non-cholinergic sites, probably at NMDA receptors.
To study the potential roles of cytokines in development and resolution of granulomatous experimental autoimmune thyroiditis (EAT), the kinetics ofin vivoexpression of cytokine genes in thyroid ...infiltrates was analysed using reverse transcriptase-PCR (RT-PCR). Both Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-10) cytokines as well as TGF-βTNF-αIL-12 and IL-1β were detected in thyroids during both the initial phase and peak of granulomatous EAT. Maximal expression of cytokine genes generally occurred 11–14 days after cell transfer, prior to maximal EAT severity, which occurred 19–21 days after cell transfer. The relative ratios of Th1:Th2 cytokines and mouse thyroglobulin-(MTg)-specific IgG1 and IgG2a autoantibody levels were similar during both the initial phase and peak of EAT. Depletion of CD8+T cells did not decrease the severity of EAT but delayed resolution of lesions. Cytokine gene expression in thyroids was not decreased by anti-CD8 treatment. Together, these data indicate that both Th1 and Th2 cytokines produced by CD4+T cells are involved in induction and development of granulomatous EAT, and CD8-dependent resolution of granulomatous EAT is apparently not mediated by these cytokines.
Anti-calcyclin binding protein (CacyBP) monoclonal antibodies (MAb) were produced using an in vitro immunization method. BALB/c mouse splenocytes were immunized with purified 6 x His-CacyBP fusion ...protein and fused with myeloma cells using polyethylene glycol (PEG) 4000. By selection using enzyme-linked immunosorbent assay (ELISA), three anti-CacyBP MAbs were obtained. The MAb BD1, whose isotype was IgG1, interacted with the fusion protein. Western blot and immunofluorescence microscopy showed that the MAb BD1 against CacyBP could recognize CacyBP protein derived from human gastric cancer cell lines in both native and denatured forms. This MAb would act as a useful tool for the detection of CacyBP protein in future studies.
We previously demonstrated that: a) a cytotoxic T cell hybridoma (HTC2) was able to induce lysis of syngeneic macrophages pulsed with either porcine thyroglobulin (pTg) or the tryptic fragments (TF) ...from pTg less than 10 kDa (M(r)) and that b) these low M(r) pTg TF included pathogenic epitopes because their injection into CBA/J mice induces thyroid lymphocytic infiltration typical of experimental autoimmune thyroiditis. Therefore the biochemical analysis of the TF preparation from pTg less than 10 kDa M(r) was undertaken and the characterized peptides were tested for their ability to be recognized or not by HTC2 cells. The sequencing of the selected peptides showed a 70% sequence homology with a portion of human thyroglobulin (hTg). The lack of a published sequence of pTg led us to synthesize a 40-amino acid peptide (F40D) similar to that portion of hTg. This F40D peptide was able to generate lymphocytic infiltrations in CBA/J mice thyroid glands, as was the native pTg molecule. Although the lymphocytic infiltrations were similar in the pTg or F40D-immunized mice, auto-antibodies to pTg or to hTg were only detectable in mice immunized with pTg. In contrast, autoantibodies levels to F40D peptide were significantly increased in serum from mice in which EAT had been induced by the F40D peptide. This highly hydrophobic peptide shows a M(r) of 4,492 kDa; it is located at the end of the second-third of the thyroglobulin molecule and up to now represents a unique sequence from the hTg molecule inducing experimental autoimmune thyroiditis.
Our previous studies indicated that soman inhibits
N-methyl-
d-aspartate (NMDA)-stimulated
3Hnorepinephrine (NE) release from rat cortical slices by acting at a non-cholinergic site. In order to ...characterize the mechanisms, neomycin, a phospholipase C (PLC) inhibitor, and polymyxin B (PMB), a rather selective protein kinase C (PKC) inhibitor, were used to examine a possible involvement of PLC and PKC in the inhibitory effect of soman on NMDA-stimulated
3HNE release. The role of pertussis toxin (PTX)-sensitive G-protein was also investigated by application of PTX. Neomycin (0.03–1.0
m
m) inhibited the release in a concentration-dependent manner, which was inhibited by 1.0
m
m soman. However, no significant interaction between soman and neomycin was observed. In addition, PMB (1.0
μg/ml) significantly inhibited release by 15.8%. With the presence of 1.0
m
m soman, inhibition of release decreased from 29% (without PMB) to 5% (1.0
μg/ml PMB). Furthermore, both in the presence and absence of 1.0
m
m soman, no significant differences for
3HNE release were found between PTX (1.0
μg/ml)-treated and non-treated slices. These results suggest that the mechanism of the inhibitory effect of soman on NMDA-stimulated
3HNE release in cortical slices appear to involve the effect on PKC, but not PLC and PTX-sensitive G-protein.
The PyPuPu and PyPuPy intermolecular triple-stranded DNA (tsDNA) can be determined more easily by capillary electrophoresis (CE) than by traditional methods. The tsDNA and its component compounds can ...be well separated by using a sieving matrix of 1.0% hydroxypropylmethylcellulose (HPMC) containing 2.5 mM magnesium ions. Such factors as buffer pH, the concentration of triplex-forming oligonucleotide (TFO), temperature, and the concentration of magnesium cation in the formation and stabilization of triple-stranded helices have been studied with capillary electrophoresis. The triplex cannot be formed when the buffer pH is lower than 4.0. When the concentration of TFO is four times higher than that of dsDNA, all of the dsDNA molecules can be associated. The limit of capillary electrophoresis detection with good reproducibility is 0.5–1 nM (S/N = 3). The CE analysis of short tsDNA takes only 40 min, whereas gel electrophoresis needs at least 5 h.
A noncommercial Hadamard transform microscopic image analysis system was used to measure fluorescence emitted by single cells. A method for subtracting the native fluorescence of theZephyranthes ...candida(Lindl.) Herb. pollen cell was developed based on its correlation to pollen area. Acridine orange and ethidium bromide were used as fluorescence probes for pollen DNA quantitative analysis, and the results obtained by the two methods were compared.
We consider a novel 2-D graphical representation of DNA sequences according to chemical structures of bases, reflecting distribution of bases with different chemical structure, preserving information ...on sequential adjacency of bases, and allowing numerical characterization. The representation avoids loss of information accompanying alternative 2-D representations in which the curve standing for DNA overlaps and intersects itself. Based on this representation we present a numerical characterization approach by the leading eigenvalues of the matrices associated with the DNA sequences. The utility of the approach is illustrated on the coding sequences of the first exon of human β-globin gene.
Mouse-thyroglobulin (MTg)-sensitized spleen cells activated in vitro with MTg induce a lymphocytic form of experimental autoimmune thyroiditis (EAT) whereas activation of the same cell population ...with MTg in the presence of anti-interleukin 2 receptor antibody (M7/20) induces a granulomatous form of EAT. The thyroid infiltrate in both lymphocytic and granulomatous EAT includes both CD4+ and CD8+ T cells and CD4+ T cells are the primary effector cells for both forms of EAT. This investigation was undertaken to begin to define the roles of alpha 4 integrin, and intercellular adhesion molecule-1 (ICAM-1) in the migration of CD4+ and CD8+ T cells to the thyroid in EAT. The studies presented here demonstrate the expression of alpha 4 integrin and ICAM-1 on CD4+ and CD8+ T cells infiltrating the thyroid and the expression of vascular cell adhesion molecule (VCAM) and ICAM-1 on thyroid cells of mice with EAT. The effects of anti-alpha 4 and anti-ICAM mAb administration on EAT severity in recipient mice was also determined. Anti-alpha 4 administration reduced or abolished lymphocyte infiltration in the thyroid resulting in reduced severity of both lymphocytic and granulomatous EAT. In contrast, anti-ICAM mAb had little effect on EAT severity. These results suggest that these two adhesion molecules exhibit differential functional roles in the modulation of EAT disease severity and that alpha 4-VCAM interactions may be of particular importance in trafficking of effector cells to the thyroid.
The technique of Hadamard transform microscopic fluorescence imaging is described. With an Acridine Orange (AO) staining cell, some factors that influence the fluorescence intensity of a cellular ...AO-DNA complex were studied; thereby the technique was applied to measure the nuclear DNA content (ploidy) in a breast tumor cell, and some parameters were employed to evaluate the tumor malignancy. A comparative study with conventional microfluorometry indicates that our system has some outstanding advantages. By using this system, the results of malignancy evaluation for 38 cases of breast tumor specimens were concordant with pathological diagnosis. This work demonstrates that this technique has a high potential value in medicine and cytobiology and may be applied as a new method for diagnostic and prognostic studies of tumor cases.
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