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Voltage source inverters (VSI) have been widely used in uninterruptible power supplies, unified power flow controllers or unified power quality conditioners, and distributed generation systems (DGS). VSIs are inherently efficient, compact, and economical devices used to control power flow and provide quality supply. VSIs can be classified as voltage-controlled VSIs (VCVSIs) and current-controlled VSIs (CCVSIs), depending on their control mechanism. In this paper, a detailed comparison of VCVSIs and CCVSIs for DGS applications is presented. This paper examines the advantages and limitations of each control technique in a single-phase DGS, without incorporating additional hardware and/or extra complex control techniques. Discussions on the concepts, hypotheses, and computer simulations of different VSIs in the presence of different loads and conditions are presented. The experimental results confirm the validity of the analysis and simulations outlined. The paper provides design recommendations for the use of VCVSIs and CCVSIs in various applications. Index Terms--DC-AC power conversion, energy conversion, power conditioner, power electronics.

Biochemical, genetic and gene-expression analyze Rdp3 in two different complexes, the smaller complex Rpd3C(S) shares Sin3 and Ume1 with Rpd3C(L) but contains the unique subunits Rco1 and Eaf3. Chromination immunoprecipitation and biochemical experiments indicated that the chromodomain of Eaf3 recruits Rpd3C(S) to nucleosomes methylated by Set2 on histone H3 lysine 36 leading to deacetylation of transcribed regions.

Reflexive polygons have attracted great interest both in mathematics and in physics. This paper discusses a new aspect of the existing study in the context of quiver gauge theories. These theories are 4d supersymmetric worldvolume theories of D3 branes with toric Calabi-Yau moduli spaces that are conveniently described with brane tilings. We find all 30 theories corresponding to the 16 reflexive polygons, some of the theories being toric (Seiberg) dual to each other. The mesonic generators of the moduli spaces are identified through the Hilbert series. It is shown that the lattice of generators is the dual reflexive polygon of the toric diagram. Thus, the duality forms pairs of quiver gauge theories with the lattice of generators being the toric diagram of the dual and vice versa. [ABSTRACT FROM AUTHOR]

We review three methods of counting abelian orbifolds of the form ℂ3/Γ which are toric Calabi-Yau (CY). The methods include the use of 3-tuples to define the action of Γ on ℂ3, the counting of triangular toric diagrams and the construction of hexagonal brane tilings. A formula for the partition function that counts these orbifolds is given. Extensions to higher dimensional orbifolds are briefly discussed. [ABSTRACT FROM AUTHOR]

Some subunits of the SWI/SNF complex function as tumor suppressors. However, underlying mechanisms are still incompletely defined. Here, we show that Srg3, a mouse homolog of BAF155 that function as a core subunit of this complex, suppresses tumorigenesis in vivo. DNA damage signals promoted Srg3 degradation by inducing p53. Deficiency of Srg3 promoted G1 cell-cycle arrest, but antagonized apoptotic response to DNA damage by robustly inducing p53 and p21 proteins. Srg3 heterozygous mice were prone to sarcoma formation, which was further enhanced by haploinsufficiency of p53. These tumors highly expressed p53 and p21 but lacked Srg3 expression. Our results establish a novel function of Srg3 in tumor suppression and provide insights into genetic pathways dictating tumor suppression by the SWI/SNF complex. [ABSTRACT FROM AUTHOR]

Voltage source inverters (VSI) have been widely used in uninterruptible power supplies, unified power flow controllers or unified power quality conditioners, and distributed generation systems (DGS). VSIs are inherently efficient, compact, and economical devices used to control power flow and provide quality supply. VSIs can be classified as voltage-controlled VSIs (VCVSIs) and current-controlled VSIs (CCVSIs), depending on their control mechanism. In this paper, a detailed comparison of VCVSIs and CCVSIs for DGS applications is presented. This paper examines the advantages and limitations of each control technique in a single-phase DGS, without incorporating additional hardware and/or extra complex control techniques. Discussions on the concepts, hypotheses, and computer simulations of different VSIs in the presence of different loads and conditions are presented. The experimental results confirm the validity of the analysis and simulations outlined. The paper provides design recommendations for the use of VCVSIs and CCVSIs in various applications. [ABSTRACT FROM AUTHOR]

We have investigated the cell signal distributions of ferroelectric random access memories (FeRAMs) using newly developed design scheme, hybrid bit line architecture and their reliability against imprint degradation. Since FeRAMs have relatively large signal distributions due to nonuniform ferroelectric storage capacitors, cell signal interference between neighboring bit lines severely degrades the sensing signal margin. In order to remove the cross-talk noise, hybrid bit line architecture is developed, which is a combination of the conventional folded and open bit line schemes. The lifetime against imprint degradation of FeRAM devices is also increased using hybrid bit line architecture. [ABSTRACT FROM AUTHOR]

The SNF2/SWI2 ATPase/helicase family comprises proteins from a variety of species, which serve a number of functions, such as transcriptional regulation, maintenance of chromosome stability during mitosis, and various types of DNA repair. Several proteins with unknown functions are also included in this family. The number of genes that belong to this family is rapidly expanding, which makes it easier to analyze the common biological functions of the family members. This study was designed to clone the SNF2/SWI2 helicase-related genes from the fission yeast Schizosaccharomyces pombe in the hope that this would help to elucidate the common functions of the proteins in this family. The hrp1+ ( helicase- related gene from S. p ombe) gene was initially cloned by PCR amplification using degenerate primers based on conserved SNF2 motifs within the ERCC6 gene, which encodes a protein involved in DNA excision repair. The hrp1+ ORF codes for an 1373-amino acid polypeptide with a molecular mass of 159 kDa. Like other SNF2/SWI2 family proteins, the deduced amino acid sequence of Hrp1 contains DNA-dependent ATPase/7 helicase domains, as well as a chromodomain and a DNA-binding domain. This configuration is similar to that of mCHD1 (mouse chromo-ATPase/ helicase- DNA-binding protein 1), suggesting that Hrp1 is a S. pombe homolog of mCHD1, which is thought to function in altering the chromatin structure to facilitate gene expression. Northern blot analysis showed that the hrp1+ gene produces a 4.6-kb transcript, which reaches its maximal level just before the cells enter the exponential growth phase, and then decreases gradually. DNA-damaging agents, such as MMS, MNNG and UV, decrease the rate of transcription of hrp1+. Deletion of the hrp1+ gene resulted in accelerated cell growth. On the other hand, overexpression of Hrp1 caused a reduction in growth rate. These results indicate that hrp1+ may act as a negative regulator of cellular growth. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the prevalence of lower urinary tract symptoms/benign prostatic hyperplasia in a Korean population., Materials and Methods: The Korean Prostate & Voiding Health Association provided free prostate-related community health care and conducted surveys in all regions of Korea from 2001 to 2022 with the cooperation of local government public health centers. A total of 72,068 males older than 50 were surveyed and analyzed. History taking, International Prostate Symptom Score (IPSS), transrectal ultrasonography, prostate-specific antigen (PSA) testing, uroflowmetry, and urine volume testing were performed., Results: The mean prostate volumes in males in their 50s, 60s, 70s, and 80s or above were 24.7 g, 27.7 g, 31 g, and 33.7 g, respectively. The proportion of males with high PSA greater than 3 ng/mL was 3.8% among males in their 50s, 7.7% among males in their 60s, 13.1% among males in their 70s, and 17.9% among males 80 years of age or older. The mean IPSS total scores in males in their 50s, 60s, 70s, and 80s or above were 10.7, 12.7, 14.5, and 16, respectively. Severe symptoms were reported by 27.3% of males, whereas 51.7% reported moderate symptoms. The mean Qmax in males in their 50s, 60s, 70s, and 80s or above were 20 mL/s, 17.4 mL/s, 15.4 mL/s, and 13.8 mL/s, respectively., Conclusions: In this population-based study, mean prostate volume, IPSS, PSA, and Qmax were 30.6±15.1 g, 14.8±8.2, 1.9±4.7 ng/mL, and 15.6±6.5 mL/s, respectively. Aging was significantly associated with increased prostate volume, PSA levels, and IPSS scores, and with decreased Qmax and urine volume., Competing Interests: The authors have nothing to disclose., (© The Korean Urological Association.)

Stress is adverse experience that require constant adaptation to reduce the emotional and physiological burden, or "allostatic load", of an individual. Despite their everyday occurrence, a subpopulation of individuals is more susceptible to stressors, while others remain resilient with unknown molecular signatures. In this study, we investigated the contribution of the DNA modifications, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), underlying the individual differences in stress susceptibility and resilience. Genome-wide 5mC and 5hmC profiles from 3- and 6-month adult male mice that underwent various durations of social defeat were generated. In 3-month animals, 5mC and 5hmC work in parallel and do not distinguish between stress-susceptible and resilient phenotypes, while in 6-month animals, 5mC and 5hmC show distinct enrichment patterns. Acute stress responses may epigenetically "prime" the animals to either increase or decrease their predisposition to depression susceptibility. In support of this, re-exposure studies reveal that the enduring effects of social defeat affect differential biological processes between susceptible and resilient animals. Finally, the stress-induced 5mC and 5hmC fluctuations across the acute-chronic-longitudinal time course demonstrate that the negative outcomes of chronic stress do not discriminate between susceptible and resilient animals. However, resilience is more associated with neuroprotective processes while susceptibility is linked to neurodegenerative processes. Furthermore, 5mC appears to be responsible for acute stress response, whereas 5hmC may function as a persistent and stable modification in response to stress. Our study broadens the scope of previous research offering a comprehensive analysis of the role of DNA modifications in stress-induced depression., Competing Interests: Conflicts of interest statement The author(s) declare no competing interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Wogonin, a flavonoid isolated from Scutellaria baicalensis, has attracted increasing scientific attention in recent years because of its potent anti-tumor activity. Its role during viral infection has largely been unexplored. Wogonin treatment effectively suppressed both influenza A and B virus replication in Madin-Darby Canine Kidney (MDCK) cells and human lung epithelial (A549) cells. In contrast, wogonin treatment following influenza A virus infection led to up-regulation of interferon (IFN)-induced antiviral signaling. Additionally, influenza A virus infection in A549 cells induced 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and activation in a time-dependent manner and wogonin treatment led to the suppression of AMPK phosphorylation. Furthermore, the treatment with AMPK-specific inhibitor (compound C; CC) attenuated influenza A virus replication. These data suggest that wogonin possesses a potent anti-influenza activity mediated by regulation of AMPK activation, suggesting that wogonin has the potential to be developed as an anti-influenza drug.

Self-reactive thymocytes are eliminated by negative selection, whereas competent thymocytes survive by positive selection. The strength of the T-cell receptor (TCR) signal determines the fate of thymocytes undergoing either positive or negative selection. The TCR signal strength is relatively higher in negative selection than in positive selection and induces pro-apoptotic molecules such as Nur77 and Nor-1, which are members of the orphan nuclear receptor family, that then cause TCR-mediated apoptosis. However, at the molecular level, it remains unclear how positive or negative selection is distinguished based on the TCR signal. We found that the expression of Twist2 is differentially regulated in positively and negatively selected thymocytes. In particular, TCR signal strength that elicits positive selection induces Twist2 expression via the Ca 2+ -Cacineurin-NFATc3 pathway, whereas strength of the TCR signal that results in negative selection abolishes NFATc3-dependent Twist2 induction via specific activation of the JNK pathway. Using Twist2-deficient and Twist2 transgenic mice, we also found that Twist2 determines thymocyte sensitivity to TCR-mediated apoptosis by regulating the expression of Nur77 and Nor-1. Twist2 partially retains histone deacetylase 7 (HDAC7) in the nucleus and recruits it to the Nur77 promoter region to repress Nur77 in positively selected thymocytes. Thus our results suggest a molecular mechanism of how thymocytes interpret the strength of the TCR signal and how TCR sensitivity is controlled during thymic selection.

Background: we report our technical success and complication rates in treating posterior circulation aneurysms at sites other than the basilar apex, superior cerebellar artery origin, or the posterior inferior cerebellar artery origin via endovascular embolization or sacrifice., Materials and Methods: we retrospectively reviewed case records for patients undergoing coil embolization of atypical posterior circulation aneurysms from January 2003 to December 2007., Results: thirty-two aneurysms in 32 patients were treated. Twenty-one patients (65%) presented with a subarachnoid hemorrhage. Twenty-two aneurysms were treated with coiling alone, 9 with stent-assisted coiling, and 1 with a combination of Onyx plus stent-assisted coiling. Twelve aneurysms were treated with vessel sacrifice. Immediately post procedure, 27/32 aneurysms (84%) were considered successfully treated, resulting in either vessel sacrifice, complete obliteration, or minimal neck remnant. Sixteen of 19 patients (84%) were considered successfully treated at a mean angiographic follow up of 8 months. The procedural morbidity and mortality was 15% and 6% respectively., Conclusion: endovascular embolization remains a viable and durable method of treatment for atypical posterior circulation aneurysms.

A new ursane-type triterpenoid, weigelic acid (1), and seven known compounds, ursolic acid (2), ilekudinol A (3), corosolic acid (4), ilekudinol B (5), esculentic acid (6), pomolic acid (7), and asiatic acid (8) were isolated from the leaf and stem of Weigela subsessilis. The structure of the new triterpenoid was established as 1beta,2alpha,3alpha,23-tetrahydroxyurs-12-en-28-oic acid on the basis of spectroscopic analyses. In addition, the isolated compounds were evaluated for their anti-complement activity against the classical pathway of the complement system. Of these, compounds 1-2 and 4-8 exhibited anti-complement activity with IC50 values of 152, 90, 130, 51, 56, 4, and 163 microM, respectively, whereas 3 was inactive. This shows that a carboxylic group of ursane-type triterpenoids seems to play an important role in inhibiting the hemolytic activity of human serum against erythrocytes.

Twelve compounds were isolated from the MeOH extract of the leaf and stem of the Korean endemic plant Weigela subsessilis L. H. Bailey. Their chemical structures were elucidated on the basis of physicochemical and spectroscopic data and by comparison with those of published literatures. These compounds were identified as three sterols, beta-sitosterol acetate (2), betasitosterol (3), daucosterol (11), eight triterpenoids, squalene (1), ursolic acid (4), ilekudinol A (5), corosolic acid (6), ilekudinol B (7), esculentic acid (8), pomolic acid (9), asiatic acid (10), and one iridoid glycoside, alboside I (12). This is the first report pertaining to the isolation of these compounds from Weigela subsessilis L. H. Bailey. In addition, three compounds 7, 9, and 12 were found to display a strong inhibitory effect on the production of IL-8 in the HT29 cells stimulated by TNF-alpha.

Hrp3, a paralog of Hrp1, is a novel member of the CHD1 (chromo-helicase/ATPase-DNA binding 1) protein family of Schizosaccharomyces pombe. Although it has been considered that CHD1 proteins are required for chromatin modifications in transcriptional regulations, little is known about their roles in vivo. In this study, we examined the effects of Hrp3 on heterochromatin silencing using several S. pombe reporter strains. The phenotypic analysis revealed that hrp3(+) is not an essential gene for cell viability. However, Hrp3 is required for transcriptional repression at silence loci of mat3. A chromatin immunoprecipitation assay showed that Hrp3 directly associates with mat3 chromatin. Thus, our results strongly suggest that Hrp3 is involved in heterochromatin silencing and plays a direct role as a chromatin remodeling factor at mat3 in vivo.

Srg3 (SWI3-related gene product) is a mouse homolog of yeast SWI3, Drosophila melanogaster MOIRA (also named MOR/BAP155), and human BAF155 and is known as a core subunit of SWI/SNF complex. This complex is involved in the chromatin remodeling required for the regulation of transcriptional processes associated with development, cellular differentiation, and proliferation. We generated mice with a null mutation in the Srg3 locus to examine its function in vivo. Homozygous mutants develop in the early implantation stage but undergo rapid degeneration thereafter. An in vitro outgrowth study revealed that mutant blastocysts hatch, adhere, and form a layer of trophoblast giant cells, but the inner cell mass degenerates after prolonged culture. Interestingly, about 20% of heterozygous mutant embryos display defects in brain development with abnormal organization of the brain, a condition known as exencephaly. Histological examination suggests that exencephaly is caused by the failure in neural fold elevation, resulting in severe brain malformation. Our findings demonstrate that Srg3 is essential for early embryogenesis and plays an important role in the brain development of mice.

We previously have reported that SRG3 is required for glucocorticoid (GC)-induced apoptosis in the S49.1 thymoma cell line. Activation of Notch1 was shown to induce GC resistance in thymocytes. However, the specific downstream target of Notch1 that confers GC resistance on thymocytes is currently unknown. We found that the expression level of SRG3 was critical in determining GC sensitivity in developing thymocytes. The expression of SRG3 also was down-regulated by the activated form of Notch1 (NotchIC). The promoter activity of the SRG3 gene also was down-regulated by NotchIC. Expression of transgenic SRG3 resulted in the restoration of GC sensitivity in thymocytes expressing transgenic Notch1. These results suggest that SRG3 is the downstream target of Notch1 in regulating GC sensitivity of thymocytes.

Immature double-positive thymocytes are sensitive to glucocorticoid (GC)-induced apoptosis, whereas mature single-positive T cells are relatively resistant. Thymocytes seem to acquire resistance to GCs during differentiation into mature single-positive thymocytes. However, detailed knowledge concerning what determines the sensitivity of thymocytes to GCs and how GC sensitivity is regulated in thymocytes during development is lacking. We have previously reported that the murine SRG3 gene (for SWI3-related gene) is required for GC-induced apoptosis in a thymoma cell line. Herein, we provide results suggesting that the expression level of SRG3 protein determines the GC sensitivity of T cells in mice. SRG3 associates with the GC receptor in the thymus, but rarely in the periphery. Transgenic overexpression of the SRG3 protein in peripheral T cells induces the formation of the complex and renders the cells sensitive to GC-induced apoptosis. Our results also show that blocking the formation of the SRG3-GC receptor complex with a dominant negative mutant form of SRG3 decreases GC sensitivity in thymoma cells. In addition, mice overexpressing the SRG3 protein appear to be much more susceptible to stress-induced deletion of peripheral T cells than normal mice, which may result in an immunosuppressive state in an animal.

We generated new fusion genes carrying positive- and negative-selection markers, and a reporter gene in a single reading frame. The new genes were constructed by sequentially linking the coding sequences of drug-resistance genes (hygro, or puro), a green fluorescence protein (GFP) gene (gfp), and the thymidine kinase gene (tk). The new synthetic genes (hygro/gfp/tk and puro/ gfp/tk) were inserted into retroviral vectors to test their usefulness as selective markers and reporters. The genes were functional in a positive selection in the presence of hygromycin (hygro/gfp/tk) or puromycin (puro/gfp/ tk). In addition, cells expressing the new fusion genes were clearly identifiable by their green fluorescence emitted from GFP. At the same time, these cells were sensitive to a gancyclovir treatment, allowing efficient removal of the transduced cells. The presently described synthetic genes will be valuable tools in both gene therapy and basic gene transfer studies, where positive selection of the transduced cells, monitoring gene expression, and negative selection of the transduced cells are simultaneously required.

The Schizosaccharomyces pombe DNA repair gene rhp51(+) encodes a RecA-like protein with the DNA-dependent ATPase activity required for homologous recombination. The level of the rhp51(+) transcript is increased by a variety of DNA-damaging agents. Its promoter has two cis-acting DNA damage-responsive elements (DREs) responsible for DNA damage inducibility. Here we report identification of Rdp1, which regulates rhp51(+) expression through the DRE of rhp51(+). The protein contains a zinc finger and a polyalanine tract similar to ones previously implicated in DNA binding and transactivation or repression, respectively. In vitro footprinting and competitive binding assays indicate that the core consensus sequences (NGG/TTG/A) of DRE are crucial for the binding of Rdp1. Mutations of both DRE1 and DRE2 affected the damage-induced expression of rhp51(+), indicating that both DREs are required for transcriptional activation. In addition, mutations in the DREs significantly reduced survival rates after exposure to DNA-damaging agents, demonstrating that the damage response of rhp51(+) enhances the cellular repair capacity. Surprisingly, haploid cells containing a complete rdp1 deletion could not be recovered, indicating that rdp1(+) is essential for cell viability and implying the existence of other target genes. Furthermore, the DNA damage-dependent expression of rhp51(+) was significantly reduced in checkpoint mutants, raising the possibility that Rdp1 may mediate damage checkpoint-dependent transcription of rhp51(+).

We have previously described a SWI/SNF-related protein complex (PYR complex) that is restricted to definitive (adult-type) hematopoietic cells and that specifically binds DNA sequences containing long stretches of pyrimidines. Deletion of an intergenic DNA-binding site for this complex from a human beta-globin locus construct results in delayed human gamma- to beta-globin switching in transgenic mice, suggesting that the PYR complex acts to facilitate the switch. We now show that PYR complex DNA-binding activity also copurifies with subunits of a second type of chromatin-remodeling complex, nucleosome-remodeling deacetylase (NuRD), that has been shown to have both nucleosome-remodeling and histone deacetylase activities. Gel supershift assays using antibodies to the ATPase-helicase subunit of the NuRD complex, Mi-2 (CHD4), confirm that Mi-2 is a component of the PYR complex. In addition, we show that the hematopoietic cell-restricted zinc finger protein Ikaros copurifies with PYR complex DNA-binding activity and that antibodies to Ikaros also supershift the complex. We also show that NuRD and SWI/SNF components coimmunopurify with each other as well as with Ikaros. Competition gel shift experiments using partially purified PYR complex and recombinant Ikaros protein indicate that Ikaros functions as a DNA-binding subunit of the PYR complex. Our results suggest that Ikaros targets two types of chromatin-remodeling factors-activators (SWI/SNF) and repressors (NuRD)-in a single complex (PYR complex) to the beta-globin locus in adult erythroid cells. At the time of the switch from fetal to adult globin production, the PYR complex is assembled and may function to repress gamma-globin gene expression and facilitate gamma- to beta-globin switching.

Exposure of Schizosaccharomyces pombe cells to UV light results in increased uvi15(+) gene expression at both the mRNA and protein levels, leading to elevated cell survival. This UV-induced expression of the uvi15(+) gene was reduced in Deltasty1 and Deltawis1 cells lacking the stress-activated protein kinase pathway, but not in DNA damage checkpoint mutants. To further understand the cellular mechanisms responsible for this UV-induced expression, the transcription rate and mRNA half-life were investigated. Transcription run-on assays revealed that the rate of uvi15(+) transcription was increased 1.8-fold regardless of Sty1 when cells were UV irradiated. The half-life of uvi15(+) mRNA was also increased 1.5-fold after UV irradiation, but it was decreased in the Deltasty1 background for both basal and UV-induced mRNAs, indicating that the stress-activated MAPK cascade can mediate UV-induced gene expression by increasing mRNA half-life. Deletion analyses identified a 54 nt element downstream of the distal poly(A) site, which was involved in the increased half-life of uvi15(+) mRNA. These results suggest that both Sty1 and the 3'-regulatory element regulate UV-induced expression of the uvi15(+) gene at the post-transcriptional level.

DNA topoisomerase IIalpha (topo IIalpha) is an essential nuclear enzyme required for chromosome segregation during mitosis. Consistent with its critical role in cell division is the fact that the expression of the gene for topo IIalpha is strongly regulated by the proliferation state of cells. Using a transient expression system, we determined the contribution of putative cis-acting elements in its promoter region to its basal level and cell proliferation-dependent transcription. Experiments with 5' and/or 3' serial deletion and site-directed mutation revealed that (1) maximal promoter activity resides in the fragment extending to position -663 bp from the ATG initiation codon, (2) minimal promoter activity is harboured at -195 bp, (3) the defined minimal promoter contains only two putative elements, inverted CCAAT box 4 (ICB4) (-166 to -162 bp) and the most proximal GC-rich box in the promoter (GC2) (-149 to -143 bp), and (4) ICB4 is most important in the basal-level transcription of the gene for rat topo IIalpha. The luciferase activities of the mutated reporter plasmids in G(0)-arrested and exponentially growing cells showed that proliferation-specific regulation is controlled mainly by GC2. Electrophoretic mobility-shift assays indicated that Sp1 binds specifically to the GC2 site. The extent of DNA-protein complex formation increases after the stimulation of cells to proliferate. These results indicate that the increased binding activity of Sp1 to GC2 is important in the up-regulation of the gene for topo IIalpha in growing cells.

Based on the surmise that a variety of genes might play important roles in embryonic development and tissue differentiation, and that some of them are likely to be expressed in undifferentiated ES cells, we attempted to identify new genes from the ES cell cDNA library. The modified method of expressed sequence tags (ESTs) and the examination of the expression patterns in adult tissues and in vitro differentiated ES cells were utilized in this study. We have isolated and identified several novel cDNA clones with interesting developmental expression pattern. Among the 83 clones randomly chosen, 23 clones (27.7%) have no homology to any sequences in public databases. The rest contain limited or complete sequence homology to the previously reported mammalian genes or ESTs, yet some clones have not been previously identified in the mouse. To examine the expression profile of clones during development and differentiation, sets of slot blots were hybridized with developmental stage specific or tissue specific probes. Out of 40 novel clones tested (21 totally unknown clones and 19 unidentified clones in mouse), most of them were up- or down-regulated as differentiation proceeded, and some clones showed differentiation-stage specific expression profiles. Surprisingly, a majority of genes were also expressed in adult tissues, and some clones even revealed tissue specific expression. These results demonstrate that not only was the strategy we employed in this study quite efficient for screening novel genes, but that the information gained by such studies would also be a useful guide for further analysis of these genes. It also suggests the feasibility of this approach to explore the genomewide network of gene expression during complicated biological processes, such as embryonic development and tissue differentiation.

Using the modified screening approach in combination with expressed sequence tags, we have identified several novel cDNAs from mouse embryonic stem (ES) cells, whose expression is tissue-restricted and/or developmentally regulated. One of the cDNAs, ES18, is preferentially expressed in lymph node and thymus, and contains noteworthy features of transcriptional regulator. The expression of ES18 transcript was selectively regulated during the apoptosis of T-cell thymoma S49.1 induced by several stimuli. Interestingly, the ES18 transcript was differently regulated in the mutually antagonistic process, between dexamethasone- and A23187-induced cell death of T-cells. Moreover, the message level of ES18 was selectively enhanced by staurosporine, a broad protein kinase inhibitor, but not by other protein kinase inhibitors such as GF109203X and H89. In addition, ES18 transcript was induced by C2-ceramide, which is a mediator of both dexamethasone- and staurosporine-induced apoptotic signaling. We further showed that transient overexpression of ES18 in mouse T-cell lymphoma increased the apoptotic cell death. These data suggest that ES18 may be selectively involved in specific apoptotic processes in mouse T-cells.

We show that the tumor-specific primary cytotoxic T lymphocytes (CTL) induced in vitro with the MCA205 fibrosarcoma cells transduced with the B7.1 (CD80) gene are highly effective in adoptive-transfer therapy of the parental tumors. The MCA205 fibrosarcoma cell line was transduced with the retroviral vectors encoding the B7.1 gene and tested for their efficiency as stimulators in short-term (5 days) mixed lymphocyte/tumor cell cultures with highly purified syngenic, unprimed T cells as responders. The induction of the CTL required the presence of a low dose of interleukin-2 (25 U/ml). The injection of the CTL prevented colony formation by the intravenously injected tumor cells in a lung colonization assay in which the CTL were injected after inoculation of tumor cells. We also showed that the adoptive transfer of the same T cells was effective in delaying the growth of the subcutaneously injected tumor cells. These results imply that the short-term mixed lymphocyte/tumor cell culture with the tumor cells transduced with the gene for the B7.1 costimulatory molecule is potentially a good source of CTL for adoptive-transfer therapy of tumors.

Two mRNA species with different sizes (3.8 kb and 2.8 kb) for the fyn proto-oncogene have been noticed during Northern hybridization analysis. However, the difference between the two mRNA species has not been resolved yet. By screening a phage expression library using the monoclonal antibody (mAb) B16-5 which recognizes Src homology 3 (SH3) domains of phospholipase C-gamma and Nck, we have cloned a cDNA encoding the larger species of fyn mRNA. The size of the clone was 3.5 kb and DNA sequencing analysis of the clone showed that it was fyn expressed mainly in T-cells, fyn (T), with an untranslated region 1 kb longer than the previously reported one. The 3'-end fragment of the clone hybridized only to the larger species (3.8 kb) of fyn mRNA but not to the smaller one (2.8 kb) on Northern blot analysis. Furthermore, an additional polyadenylation signal sequence was found at the end of this clone. These results indicate that the two mRNA species for fyn are produced by alternative polyadenylation.

When a T cell hybridoma, 70.7, was treated with a Ca2+ ionophore (A23187), apoptotic cell death was induced. Interestingly, we observed that the expression of Tcf-1, a T cell-specific transcription factor, mRNA was reduced by approximately 5-fold in the A23187-treated apoptotic cells compared to an ethanol-treated control. The hybridoma cells, however, did not display such a reduced expression of Tcf-1 mRNA upon treatment with buthionine sulfoxide, which is known to induce a necrosis-like cell death. When another T cell hybridoma, KMIs-8.3.5, was treated with A23187 and phorbol myristate acetate, which leads to activation-induced apoptosis, Tcf-1 expression was again greatly reduced. However, a mutant line (KCIT1-8.5) derived from KMIs-8.3.5, which produces IL-2 upon activation and is resistant to apoptosis, did not show such reduction in Tcf-1 expression. We also showed that the reduced expression level of CD3epsilon mRNA and surface TCR-CD3 complex in apoptotic T cells is caused by the reduced expression of Tcf-1. When 70.7 cells were transfected with a plasmid DNA pSVtcf-1, in which Tcf-1 gene expression is driven by the SV40 promoter, such reduction of the Tcf-1 mRNA and the surface expression of the TCR-CD3 complex were not observed upon apoptosis induction. Our results suggest that the reduced expression of Tcf-1 is specific for the apoptotic, but not for the activating, process of T cells and is also responsible for the reduced surface expression of the TCR-CD3 in apoptotic T cells.

Developing thymocytes that give rise to CD8+ (cytotoxic) and CD4+ (helper) alpha beta-TCR T lymphocytes go through progressive stages of expression of coreceptors CD8 and CD4 from being negative for both (the double-negative stage), to coexpressing both (the double-positive (DP) stage), to a mutually exclusive sublineage-specific expression of one or the other (the single-positive (SP) stage). To delineate the mechanisms underlying regulation of CD8 during these developmental transitions, we have examined expression of a series of mouse CD8 alpha gene constructs in developing T cells of conventional and CD8 alpha "knock-out" transgenic mice. Our results indicate that cis-active transcriptional control sequences essential for stage- and sublineage-specific expression lie within a 5' 40-kb segment of the CD8 locus, approximately 12 kb upstream of the CD8 alpha gene. Studies to characterize and sublocalize these cis sequences showed that a 17-kb 5' subfragment is able to direct expression of the CD8 alpha gene up to the CD3intermediate DP stage but not in more mature DP or SP cells. These results indicate that stage-specific expression of CD8 alpha in developing T cells is mediated by the differential activity of multiple functionally distinct cis-active transcriptional control mechanisms. It will be important to determine the relationship of "switching" between these cis mechanisms and selection.

We isolated a new mouse gene that is highly expressed in thymocytes, testis, and brain. This gene, SRG3, showed a significant sequence homology to SWI3, a yeast transcriptional activator, and its human homolog BAF155. SRG3 encodes 1,100 amino acids and has 33-47% identity with SWI3 protein over three regions. The SRG3 protein contains an acidic NH2 terminus, a myb-like DNA binding domain, a leucine-zipper motif, and a proline- and glutamine-rich region at its COOH terminus. Rabbit antiserum raised against a COOH-terminal polypeptide of the SRG3 recognized a protein with an apparent molecular mass of 155 kD. The serum also detected a 170-kD protein that seems to be a mouse homologue of human BAF170. Immunoprecipitation of cell extract with the antiserum against the mouse SRG3 also brought down a 195-kD protein that could be recognized by an antiserum raised against human SWI2 protein. The results suggest that the SRG3 protein associates with a mouse SWI2. The SRG3 protein is expressed about three times higher in thymocytes than in peripheral lymphocytes. The expression of anti-sense RNA to SRG3 mRNA in a thymoma cell line, S49.1, reduced the expression level of the SRG3 protein, and decreased the apoptotic cell death induced by glucocorticoids. These results suggest that the SRG3 protein is involved in the glucocorticoid-induced apoptosis in the thymoma cell line. This implicates that the SRG3 may play an important regulatory role during T cell development in thymus.