Role of Rho kinase isoforms in murine allergic airway responses.

Inhibition of Rho-associated coiled-coil forming kinases (ROCKs) reduces allergic airway responses in mice. The purpose of this study was to determine the roles of the two ROCK isoforms, ROCK1 and ROCK2, in these responses. Wildtype (WT) mice and heterozygous ROCK1 and ROCK2 knockout mice (ROCK1(+/-) and ROCK2(+/-), respectively) were sensitised and challenged with ovalbumin. ROCK expression and activation were assessed by western blotting. Airway responsiveness was measured by forced oscillation. Bronchoalveolar lavage was performed and the lungs were fixed for histological assessment. Compared with WT mice, ROCK1 and ROCK2 expression were 50% lower in lungs of ROCK1(+/-) and ROCK2(+/-) mice, respectively, without changes in the other isoform. In WT lungs, ROCK activation increased after ovalbumin challenge and was sustained for several hours. This activation was reduced in ROCK1(+/-) and ROCK2(+/-) lungs. Airway responsiveness was comparable in WT, ROCK1(+/-), and ROCK2(+/-) mice challenged with PBS. Ovalbumin challenge caused airway hyperresponsiveness in WT, but not ROCK1(+/-) or ROCK2(+/-) mice. Lavage eosinophils and goblet cell hyperplasia were significantly reduced in ovalbumin-challenged ROCK1(+/-) and ROCK2(+/-) versus WT mice. Ovalbumin-induced changes in lavage interleukin-13, interleukin-5 and lymphocytes were also reduced in ROCK1(+/-) mice. In conclusion, both ROCK1 and ROCK2 are important in regulating allergic airway responses.

Pioglitazone attenuates fatty acid-induced oxidative stress and apoptosis in pancreatic beta-cells.

AIMS: Thiazolidinediones (TZDs), ligands for peroxisome proliferator-activated receptor gamma, are antidiabetic agents that improve hyperglycemia by decreasing insulin resistance in obese diabetic animal models and patients with type 2 diabetes. We have studied whether pioglitazone, a TZD, can exert a direct effect against pancreatic beta-cell lipoapoptosis. METHODS: MIN6 cells were cultured in medium containing either 5.6 (low glucose) or 25 mM glucose (high glucose) in the presence or absence of 0.5 mM palmitate for 48 h. We examined the effect of 10 microM pioglitazone on MIN6 cells on glucose-stimulated insulin secretion, cellular ATP, uncoupling protein-2 (UCP-2) mRNA expression, intracellular triglyceride content, reactive oxygen species production, the number of apoptotic cells and nuclear factor-kappaB (NF-kappaB) activity. RESULTS: Pioglitazone recovered partly impaired glucose-stimulated insulin secretion and cellular ATP in MIN6 cell exposed to high glucose with 0.5 mM palmitate. Pioglitazone suppressed intracellular triglyceride accumulation in cells exposed to high glucose with 0.5 mM palmitate. Palmitate-induced upregulation of UCP-2 mRNA levels was suppressed by pioglitazone in a dose-dependent manner. Pioglitazone decreased palmitate-induced reactive oxygen species production in MIN6 cells by 24% and in mouse islet cells by 53%. Pioglitazone also decreased palmitate-induced NF-kappaB activity by 40% and protected beta-cells from palmitate-induced apoptosis by 22% in MIN6 cell. CONCLUSIONS: Pioglitazone attenuated fatty acid-induced oxidative stress and apoptosis in pancreatic beta-cells. TZDs might be used as a mean for maintaining beta-cell survival and preserving capacity of insulin secretion in patients with diabetes mellitus.

Effects of tamoxifen on estrogen-induced enhancement or inhibition of tumor growth in mouse Leydig cell tumor lines.

In order to avoid the complex dual effects of estrogen and antiestrogen, the attempt was made to establish the tumor lines in which estrogens show either stimulatory or inhibitory property in terms of the tumor growth. The administration of estrogen to host mice bearing one of the mouse Leydig cell tumor lines, called T 124958-R, resulted in marked enhancement of the tumor growth even at pharmacological levels of estrogen. On the other hand, estrogenization of host mice almost completely inhibited the growth of the other tumor line (T 22137) without detectable stimulatory effects. The physiocochemical properties of the cytosol estrogen receptor in both sublines were found to be similar in relation to the affinity toward ligands, steroid specificity, sedimentation profile, and the dissociation rate kinetics. Using these tumor lines, the action mechanism of tamoxifen on the tumor growth was examined. Daily administration of this compound (30 micrograms/mouse) led to enhanced tumor growth in T 124958-R, while the growth of T 22137 was inhibited by the same procedure. In the combination experiments, tamoxifen was found to be unable to antagonize estrogen-induced enhancement or inhibition of the growth in these tumors. In addition, both tumors contained similar levels of the antiestrogen binding sites. These results suggest that tamoxifen modulated the tumor growth through its estrogenic potency.

Characterization of a unique nuclear estrogen-binding component in an estrogen-responsive mouse Leydig cell tumor.

An estrogen-responsive mouse Leydig cell tumor line (Tumor 124958) has been shown to contain only a low-affinity binder for estradiol in the cytosol fraction. This differed from the putative estrogen receptor in terms of its hormone-binding specificity as well as affinity. In addition, the possibility that an estrogen receptor-like molecule exists in the nuclei even without hormonal stimuli was examined using purified nuclei. Scatchard plot analyses showed that these nuclei possessed a large amount of estrogen binder having a high affinity for estradiol and diethylstilbestrol. The content of this nuclear binding component was not diminished by using molybdate, a potent inhibitor for receptor activation, and in vitro incubation of collagenase-dispersed cells with estradiol did not cause significant increase in the number of nuclear binding sites when compared with the values obtained by direct incubation of isolated nuclei with estradiol. These results support the view that this nuclear estrogen binder is not due to artificial migration of the cytosol receptor into nuclei during homogenization. The characterization of this nuclear binding component under cell-free conditions revealed that its affinity for estradiol in Mg2+-containing buffer was temperature dependent (Kd 3 nM at 30 degrees and 12 nM at 0 degrees) without significant alteration in the number of maximum binding sites. Introduction of a chelating agent (ethylenediaminetetraacetate) into the buffer system abolished the temperature effect on the affinity, resulting in high affinity for estradiol at both low and high temperatures. These Mg2+ and temperature effects were reversible. In addition, when compared with putative nuclear estrogen receptors, this nuclear binding was observed to be relatively resistant to high salt or micrococcal nuclease treatments in relation to solubilization from nuclei. However, trypsin digestion was found to result in a marked decrease in the nuclear binding sites, indicating that this unique nuclear binding component contains a protein unit(s). These results suggest the possibility that this tumor line contains a unique unoccupied nuclear estrogen binder which might be able to transmit estrogen signals to tumor cell nuclei with regard to tumor growth.

An IF-FISH Approach for Covisualization of Gene Loci and Nuclear Architecture in Fission Yeast.

Recent genomic studies have revealed that chromosomal structures are formed by a hierarchy of organizing processes ranging from gene associations, including interactions among enhancers and promoters, to topologically associating domain formations. Gene associations identified by these studies can be characterized by microscopic analyses. Fission yeast is a model organism, in which gene associations have been broadly mapped across the genome, although many of those associations have not been further examined by cell biological approaches. To address the technically challenging process of the visualization of associating gene loci in the fission yeast nuclei, we provide, in detail, an IF-FISH procedure that allows for covisualizing both gene loci and nuclear structural markers such as the nuclear membrane and nucleolus.

Chronic estrogen treatment causes an alteration in uterine estrogen receptor dynamics of rats.

Estrogen receptor content and dynamics in the uteri obtained from chronically estrogenized rats were analyzed. 12 day treatment with a subcutaneous implantation of a diethylstilbestrol pellet resulted in maximal stimulation of uteri with regard to wet tissue weight, DNA content, as well as progesterone receptor content without significant alteration of the estrogen receptor level. Estrogen receptor dynamics in just ovariectomized or ovariectomized and diethylstilbestrol-stimulated rats elicited by a single injection of estradiol were next examined using the exchange methods. The cytosol receptor content rapidly declined, with a small and temporary accumulation of the nuclear receptor in the uterus from rats continuously exposed to diethylstilbestrol during the preceding 12 days. A relatively rapid cytosol receptor replenishment was also observed in rats pretreated with diethylstilbestrol. This was accompanied by a rapid decrease in the nuclear receptor level to 70% of the preinjection value at 5 h after estradiol administration. These data are in contrast to findings on uteri of ovariectomized and nonestrogen-treated rats, in which a single injection of estradiol resulted in a prolonged nuclear receptor retention and a delayed cytosol receptor replenishment. Adrenalectomy did not result in a significant change of receptor dynamic patterns, suggesting that adrenal steroids do not play a role in the alteration of receptor dynamics elicited by continuous stimulation with diethylstilbestrol. These observations suggest that a continuous exposure of rat uteri to the estrogen causes an altered regulation of estrogen receptor dynamics by the homologous steroid compared to those in chronically estrogen-deprived rats.

Subacute and long term effect of swimming training on blood pressure in young and old spontaneously hypertensive rats.

The effects of swimming training (three weeks' training with the duration increasing up to a maximum of 180 min per day, at a water temperature of 36 degrees C) on arterial blood pressure were studied in 4, 11, and 18 month old spontaneously hypertensive rats. In addition, in the 11 month old rats the change in blood pressure after individual exercise was determined. The significance of a training induced loss of body weight in lowering blood pressure was assessed by pair feeding of sedentary age matched spontaneously hypertensive rats. Blood pressure was reduced by approximately 50 mmHg within 8-10 days, except in the oldest rats, which tolerated the physical activity poorly and had, if any, only a moderate fall in blood pressure. It was possible to distinguish between subacute transient effects lasting for not more than one day and long term effects. Blood pressures were 20-25 mmHg lower after individual swimming routines than those before exercise when measured on the ninth day of the training programme. On cessation of training, blood pressures approached those of sedentary rats within two weeks. It seems that the loss of body weight was of minor importance in lowering blood pressure under these experimental conditions.

Effect of molybdate on activation and stabilization of steroid receptors.

The effects of molybdate on activation and stability of glucocorticoid, androgen, and estrogen receptors were studied. The activation of steroid receptors by heating or dialysis was inhibited by molydate, but molybdate had no effect on the nuclear binding of previously activated steroid-receptor complexes. The inhibitory effect of molybdate on activation was concentration dependent and was reversed when molybdate was removed by dialysis or gel filtration on Sephadex G-25. The steroid-binding capacities of the unoccupied glucocorticoid and androgen receptors were markedly reduced by removal of a small molecular weight factor(s) from the cytosol by dialysis or gel filtration on Sephadex G-25, even at 0 C. Molybdate blocked both this loss of steroid-binding ability on dialysis and also the heat-induced destabilization of the receptors. The dialysate of rat liver cytosol and molybdate had synergistic effects in increasing the stability of the glucocorticoid receptor in the cytosol after gel filtration at 25 C. These findings suggest that activation and destabilization have a common mechanism which involves a small molecular weight component(s) and that molybdate has a specific inhibitory effect on this mechanism.

A modulator which converts activated estrogen receptor to a biologically inactive aggregated form.

The in vitro nuclear binding of rat uterine estrogen-receptor complexes has been studied. Heating cytosol from mature rat uterus at 25 C for various times in the presence of 0.15 M KCl resulted in a transient increase in nuclear binding activity, followed by irreversible loss of this activity. The molecular state of these complexes heated at 25 C in the presence of 0.15 M KCl was determined using Sephadex G-200 chromatography and sucrose density centrifugation at high ionic strength (0.4 M KCl). Gel filtration resulted in steroid-binding activity in the void volume. Sucrose density gradient analysis revealed a broad peak, ranging from approximately 5-20S. When cytosol was heated at 25 C in the presence of 10 mM molybdate to block the temperature-induced activation of receptor, nuclear binding ability was easily recovered by dialysis, while heating already activated estrogen receptor in the presence of 0.15 M KCl and 10 mM molybdate caused irreversible loss of nuclear binding ability. When cytosols prepared from immature rats (19-23 days old) were heated at 25 C in the presence of 0.15 M KCl, only a minimum loss of nuclear binding ability was shown. The radioactive peak in a high salt sucrose density gradient appeared almost exclusively in the 5S region. However, the addition of receptor-free mature uterine cytosol to estrogen-receptor complexes from immature rat uterus caused a marked loss of nuclear binding utility, with a resultant receptor aggregation, whereas rat liver cytosol had no effect on this reaction. Furthermore, heating liver glucocorticoid receptor did not cause a loss of nuclear binding ability even in the presence of receptor-free adult rat uterine cytosol. These observations suggest that there is a factor(s) in rat uterus which recognizes only activated estrogen receptor and induces receptor aggregation and a rapid loss of the nuclear binding ability of receptor in a KCl concentration- and temperature-dependent manner. Preliminary characterization indicates that this factor is macromolecular in nature and resistant to RNase and trypsin treatment, but labile at 100 C.

Effects of estrogen and vanadate on the proliferation of newly established transformed mouse Leydig cell line in vitro.

A permanent cell line (B-1-A-2) derived from an estrogen-responsive mouse Leydig cell tumor (T 124958-R) was established in culture. Although this cell line showed estrogen-induced enhancement of cell proliferation in vitro, a moderately high concentration (10(-8) M) of estradiol was required to stimulate maximum growth. A whole cell binding assay revealed that the B-1-A-2 cells contained estrogen receptors with relatively low affinity for estradiol (Kd, 10(-9)-10(-8) M). This estrogen receptor was found to have a mol wt of 65,000. In addition, incubation of the cells with [32P]orthophosphate and subsequent purification revealed that the estrogen receptor is phosphorylated. This finding prompted us to study the effect of phosphatase inhibitors on cell proliferation. The inclusion of vanadate (25 microM) in the culture medium resulted in a significant increase in estrogen sensitivity, showing a maximal growth stimulatory effect of estradiol at concentrations of 10(-10)-10(-9) M. Simultaneously, the conversion of the low affinity to the high affinity receptor (Kd, 10(-10)-10(-9) M) was induced by treatment of cells with vanadate. These data suggest that estrogen directly stimulates the growth of B-1-A-2 cells in culture, and that the sensitivity to estrogen can be altered by modulating the estrogen receptor possibly via a phosphorylation-dephosphorylation mechanism.

Tnat1 and Tnat2 from Arabidopsis thaliana: novel transposable elements with tandem repeat sequences.

A computer-aided homology search of databases found that the nucleotide sequences flanking ATLN44, a non-LTR retrotransposon (LINE) from Arabidopsis thaliana, are repeated in the A. thaliana genome. These sequences are homologous to flanking sequences of 664 bp with terminal inverted repeat sequences of about 70 bp. The 664-bp sequence and most of the 14 homologues identified were flanked by direct repeat sequences of 9 bp. These findings indicate that the repeated sequence, named Tnat1, is a transposable element that duplicates a 9-bp sequence at the target site on transposition and that ATLN44 is inserted in one Tnat1 member. Interestingly, all of the Tnat1 members had tandem repeats comprised of several units of a 60-bp sequence, the number of repeats differing among Tnat1 members. Of the Tnat1 members identified, one was inserted into another sequence repeated in the A. thaliana genome: that sequence is about 770 bp long and has terminal inverted repeat sequences of about 110 bp. The sequence is flanked by direct repeats of a 9-bp sequence, indicating that it is another transposable element, named Tnat2, from A. thaliana. Moreover, Tnat2 members had a tandem repeat about 240 bp long. Tnat1 and Tnat2 with tandem repeats in their internal regions show no homology to each other or to any of the elements identified previously; therefore they appear to be novel transposable elements.

A new class of LINEs (ATLN-L) from Arabidopsis thaliana with extraordinary structural features.

The Arabidopsis thaliana genome has about 250 copies of LINEs (here called ATLNs). Of these, some, called ATLN-Ls, have an extra sequence of about 2 kb in the region downstream of two consecutive open reading frames, orf1 and orf2. Interestingly, the extra sequences in these ATLN-L members have another open reading frame, designated as orf3. Each member is flanked by direct repeats of a target site sequence, showing that ATLN-L members with the three open reading frames have retrotransposed as a unit. The ATLN-L members are also distinct from other ATLN members: orf1 terminates with TAA (or TAG) and is located in the same frame as orf2, and the ATG initiation codon of orf2 is not present in the proximal region. A sequence that may form a pseudoknot structure in ATLN-L mRNA was present in the proximal region of orf2, therefore the TAA (or TAG) termination codon of orf1 is assumed to be suppressed to produce an Orf1-Orf2 transframe protein during the translation of the ATLN-L mRNA. The region between orf2 and orf3 is several hundred bp long, suggesting that orf3 expression is independent of orfl-orf2. The amino acid sequences of the proteins Orf1 and Orf3 are highly homologous in their N-terminal half regions that have a retroviral zinc-finger motif for RNA binding. Orf3, however, has a leucine-zipper motif in addition to the zinc-finger motif. The C-terminal regions of the Orf1 and Orf3 proteins have poor homology, but seem to have nuclear localization signals, suggesting that these proteins are involved in the transfer of ATLN-L mRNA to nuclei. A phylogenetic tree shows that Orf3 proteins form a branch distinct from the branches of the Orf1 proteins encoded by ATLN-L members. This indicates that an ancestor element of ATLN-Ls has incorporated the orf1 frame carried by another ATLN member into its distal region to orf1-orf2 during evolution.

ATLN elements, LINEs from Arabidopsis thaliana: identification and characterization.

Non-LTR retrotransposons (LINEs) are ubiquitous elements in the plant kingdom. Two hundred and nineteen LINE homologues (named ATLN) were identified in the A. thaliana genome, about 90% of which have been sequenced by a computer-aided homology search. Of these, the structures of 62 were analyzed in detail. Most, including those truncated for the 5' regions, were flanked by direct repeats of a sequence of 7-21 bp long, the target site sequence duplicated upon retrotransposition of each member. Thirty ATLN members had two consecutive open reading frames, corresponding to orf1 and orf2 essential for retrotransposition. The phylogenetic tree constructed from the amino acid sequences of the endonuclease domains of the Orf2 proteins showed that the ATLN members were grouped in two families (I and II) and that the members of each family could be further divided into several subfamilies. The members of each subfamily had several unique structural features in common in the intergenic region between orf1 and orf2 as well as in the downstream regions of orf2. Interestingly, orf2 in almost all the ATLN members is located in the -1 frame relative to orf1, indicative of the existence of such translational control mechanisms as translational coupling or frameshifting to produce an amount of Orf2 protein appropriate to that of Orf1. Moreover, the most proximal sequences in the 5' untranslated regions were non-homologous, even in members with the highest homology, unlike the LINEs in animals. The non-homologous sequences in the 5' untranslated regions might be acquired at or after transcription during retrotransposition of the ATLN elements.

Sodium chloride loading does not alter endothelium-dependent vasodilation of forearm vasculature in either salt-sensitive or salt-resistant patients with essential hypertension.

The purpose of this study was to determine whether a high NaCl intake impairs endothelium-dependent and -independent vasodilation of forearm circulation in salt sensitive (SS) patients with essential hypertension. We evaluated the effects of intra-arterial acetylcholine (ACh) and isosorbide dinitrate (ISDN) on forearm hemodynamics in 29 patients with essential hypertension, while consuming a low NaCl (50 mmol/d) or high Na Cl (340 mmol/d) diet for 1 week. The forearm blood flow (FBF) was measured by strain-gauge plethysmography. Patients were classified as SS (n=12) or salt resistant (SR; n=17) based on salt-induced changes in blood pressures. The FBF responses of ACh and ISDN were similar in the SS and SR patients while on either NaCl diet, and was not altered by salt loading (ACh, SS: low NaCl 22.8+/-4.3 vs. high NaCl 21.1+/-3.6 ml/min per 100 ml, SR: low NaCl 22.5+/-4.0 vs. high NaCl 23.3+/-4.1 ml/min per 100 ml; ISDN, SS: low NaCl 13.9+/-2.1 vs. high NaCl 14.1+/-2.2 ml/min per 100 ml, SR: low NaCl 13.8+/-2.3 vs. high NaCl 14.0+/-2.2 ml/min per 100 ml). There were no significant differences in the vascular responses to ACh and ISDN in the presence of N(G)-monomethyl-L-arginine, a nitric oxide synthase inhibitor, in either group for either NaCl diet. These findings suggest that forearm resistance artery endothelial function may not be influenced by salt loading in either SS patients which finding may play a role in determining salt sensitivity in patients with essential hypertension or SR patients.

Profile of T-cell receptor V beta gene usage of cytotoxic T cells induced by intrapleural administration of a streptococcal preparation, OK432, in malignant effusions.

T-cell receptor (TCR) gene rearrangements were analyzed in tumor-infiltrating lymphocytes (TIL) using the reverse transcription-polymerase chain reaction (RT-PCR) to determine whether oligoclonal expression of TCRV beta occurs in TIL, and if so, whether it is involved in the clinical response and mechanisms of locoregional immunotherapy using a streptococcal preparation, OK432. Patients with malignant effusion of various origins were treated with intrapleural administration of OK432, and clinical responses were assessed by cytological and chest X-ray examinations. Pleural exudate cells (PEC), obtained before and after the administration of OK432 (designated as pre- and OK432-PEC, respectively), were subjected to TCR analysis. Both pre-PEC and OK432-PEC showed highly diverse expressions of TCRV beta gene usage in either type of PEC. The frequency of TCRV beta 20 gene expression in OK432-PEC was significantly higher than in pre-PEC. Moreover, the over-expression of the TCRV beta 20 gene usage was also induced in the peripheral blood lymphocytes and pre-PEC of patients by in vitro OK432 stimulation, but not in the PBL of one healthy volunteer. Single-strand conformational polymorphism (SSCP) analysis revealed the clonotypes of these TCRV beta 20 genes. Autologous tumor-specific killing activity could be detected in OK432-PEC and was significantly reduced by treatment with a TCRV beta 20-specific monoclonal antibody. These findings suggest that the rearrangement of TCRV beta 20 gene usage may be involved in the autologous tumor-specific action of malignant effusions in the treatment with OK432.

Identification and characterization of two tandem repeat sequences (TrsB and TrsC) and a retrotransposon (RIRE1) as genome-general sequences in rice.

Three kinds of DNA sequences (here called TrsB, TrsC and RIRE1) have been previously reported to be those repeated in tandem specifically in the wild rice species with FF, CC or EE genome, respectively. To characterize these genome type-specific sequences, we carried out PCR using a pair of primers, which hybridize to a restricted region in the repeating unit sequence and prime DNA synthesis in both directions. Gel electrophoresis and DNA sequencing revealed that PCR using primers for TrsB (or TrsC) amplified the fragments with an integral series of a unit length not only from total DNA of the rice strain with FF (or CC) genome, but also from those of the rice strains with non-FF (or non-CC) genome. TrsB or TrsC was, however, found to be repeated in an extraordinary number of copies in the species with FF or CC genome, respectively, in which the TrsB (or TrsC) sequence has been originally identified. PCR using primers for RIRE1 produced various sizes of fragments from total DNA of the rice strains with EE genome. The fragments, however, showed no progression at interval of the unit length characteristic for tandem repeats. Nucleotide sequencing of the amplified fragments revealed that they were not the sequences repeated in tandem, but were those interspersed as an element having partial homology with the LTR sequences of retrotransposons, Wis-2-1A in wheat and BARE-1 in barley. RIRE1 was present in the rice species with any types of genomes, but in the species with EE genome in an extraordinary number of copies.

RIRE1, a retrotransposon from wild rice Oryza australiensis.

RIRE1 is a retrotransposon present in wild rice Oryza australiensis in an extraordinary number of copies, and only a portion of the LTR sequence has been determined previously. Here, we isolated and sequenced DNA segments of various portions of RIRE1, revealing that the sequences of LTR and the internal region were 1523 and 5277 bp in length, respectively. The internal region shows homology with the pol region in copia, a Drosophila retrotransposon, indicating that RIRE1 is a copia-like retrotransposon. The internal region of RIRE1 contained an open reading frame coding for genes, gag, pro, int, rt and rh, like copia and retroelements related to it. A clone screened from a library of the O. australiensis genomic DNA contained solo LTR, which was flanked by direct repeats of a 5-bp sequence. This suggests that RIRE1 generates a duplication of the target sequence of 5 bp upon retroposition. We observed that many RIRE1 members were nested by another RIRE1 member. This indicates that these RIRE1 members have received another RIRE1 to make an extraordinary number of copies in the O. australiensis genome without giving a deleterious effect on the growth of rice cells.

Identification and structural analysis of SINE elements in the Arabidopsis thaliana genome.

An insertion sequence was found in a Mu homologue in the genome of Arabidopsis thaliana. The insertion sequence had poly(A) at the 3' end, and promoter motifs (A- and B-boxes) recognized by RNA polymerase III. The sequence was flanked by direct repeats of a 15-bp sequence of the Mu homologue, which appears to be a target-site sequence duplicated upon insertion. These findings indicate that the insertion sequence is a retroposon SINE, and it was therefore named AtSN (A. thaliana SINE). Many members of the AtSN family were identified through a computer-aided homology search of databases and classified into two subfamilies, AtSN1 and AtSN2, having consensus sequences 159 and 149 bp in length, respectively. These had no homology to SINEs in other organisms. About half of AtSN members were truncated through loss of a region at either end of the element. Most of them were truncated at the 5' end, and had a duplication of the target-site sequence. This suggests that the ones with 5' truncation retroposed by the same mechanism as those without truncation. Members of the AtSN1 or AtSN2 subfamilies had many base substitutions when compared with the consensus sequence. All of the members examined were present in three different ecotypes of A. thaliana (Columbia, Landsberg erecta, and Wassilewskija). These findings suggest that AtSN members had proliferatedbefore the A. thaliana ecotype strains diverged.

Down-regulation of IL-10 enhances the efficacy of locoregional immunotherapy using OK-432 against malignant effusion.

To determine the significance of interleukin (IL)-10 in antitumor immune response, the effect of the down-regulation of tumor-derived IL-10 on locoregional immunotherapy was investigated. C3H/HeN mice were intraperitoneally (i.p.) inoculated with IL-10-producing murine breast cancer cell line, FM3A, and treated with locoregional administration of OK-432 with or without anti-IL-10 monoclonal antibody (mAb). Anti-IL-10 mAb did not affect the in vitro growth of FM3A cells. Administration of OK-432 plus anti-IL-10 mAb remarkably delayed the retention of malignant ascites and prolonged the survival of mice compared with the administration of OK-432 alone. Spleen cells which were collected from mice treated with OK-432 plus anti-IL-10 mAb and further stimulated in vitro with inactivated FM3A cells exhibited significantly higher cytotoxicity against FM3A cells than those from mice treated with OK-432 alone or from the control mice. The expression of major histocompatibility complex (MHC) class II molecules on spleen cells was up-regulated in vitro by the addition of OK-432 and anti-IL-10 mAb. Using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), cytokine mRNA levels of peritoneal exudate cells (PEC) and spleen cells were assessed on day 7 (before treatment) and day 14 (after treatment). In PEC, increased expression of IL-2 was observed with the administration of OK-432 plus anti-IL-10 mAb. In spleen cells, the expression of IL-2, IL-12 and IFN-gamma were strongly induced, and IL-4 expression was reduced by the administration of OK-432 plus anti-IL-10 mAb. It is suggested that down-regulation of tumor-derived IL-10 induces the up-regulation of the T helper type (Th) 1 population, resulting in an enhancement of the efficacy of locoregional immunotherapy with OK-432.

T-cell receptor V beta gene usage of human cytotoxic T-cell clones obtained from gastric cancer patients.

Nine T-cell clones have been established from tumor-infiltrating lymphocytes (TIL) isolated from ascitic fluid of a gastric cancer patient. Five of them retained cytotoxicity against autologous tumor cells (AuTu), and were all CD4+. Each clone had different usage of T-cell receptor (TCR) V beta gene as assessed by Southern blot analysis. Using AuTu and two allogeneic gastric cancer cell lines as targets, we selected three clones with unique cytotoxic properties. Two of these clones (Clone 1 and 2) preferentially lysed AuTu, but showed no or marginal cytotoxicity against allogeneic gastric cancer cells, and one clone (Clone 7) showed appreciable cytotoxicity against AuTu and allogeneic gastric cancer cells. In the detailed analysis of TCRV beta gene usage, Clone 1, 2, and 7 expressed V beta 13.1/D beta 1/J beta 1.5/C beta 1, V beta 3/D beta 2/J beta 2.4/C beta 2, and V beta 9/D beta 1/J beta 1.4/C beta 1, respectively, and the primary structures of the three TCRVb genes did not share any common features, neither in the sizes of their complementarity determining region 3 (CDR3) nor in their amino acid compositions. Interestingly, PBL of the same patient expressed CDR3 identical to that of Clone 2 and 7, but not that of Clone 1. CDR3 identical to that of Clone 2 and 7 were also detected in TIL of other gastric cancer patients. These results show that some AuTu-specific CTL included in TIL are circulating in peripheral blood, and that the CDR3 identical to that of the CTL is expressed extensively in TIL among different gastric cancer patients. Screening of the expression of the CDR3 in other gastric cancer patients is recommended to develop an immuno-therapy of gastric cancer based on antigenic peptide.