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The human genome consists of more than 20000 genes and is essential for all biological phenomena. To understand these biological phenomena, including diseases, and to be able to modify them, approaches that enable optical control of the genome may be useful. Recently, we developed an optogenetic tool, named photoactivatable Cas9 (PA-Cas9). We divided Cas9 nuclease from the CRISPR-Cas9 system into two fragments and connected photo-inducible dimerization proteins, named Magnet system, to the fragments, leading to the development of PA-Cas9 of which nuclease activity is switchable with light. PA-Cas9 allows direct editing of DNA sequences by light stimulation. Additionally, we developed a light-inducible, RNA-guided programmable system for endogenous gene activation based on the CRISPR-Cas9 system. We demonstrated that this optogenetic tool allows rapid and reversible targeted gene activation by light. Using this tool, we exemplified optical control of neuronal differentiation of human induced pluripotent stem cells (iPSCs). The CRISPR-Cas9-based, photoactivatable transcription system offers a simple and versatile approach to precise gene activation. In addition to the CRISPR-Cas9-based optogenetic tools, we developed a photoactivatable Cre-loxP system. This tool allows optical control of DNA recombination reaction in an internal organ even by external, noninvasive illumination using LED light source. To date, genome engineering technology and optogenetics technology have emerged separately as different applications. Our studies described above merge these emerging research fields together.

Interleukin-1β is widely known as an inflammatory cytokine, and is an important and indispensable factor to understand inflammation. Therefore many researchers have investigated function and property of interleukin-1β. However some questions still remain to be fully elucidated. To approach these questions, bioimaging technology has been exploited recently. As interleukin-1β is regulated by unique mechanism via transcriptional activation and protein processing, several imaging tools have been developed for analyzing interleukin-1β by applying the regulatory mechanism. In this review I introduce representative interleukin-1β-related imaging technologies including basic principle of them and findings obtained with them. Additionally I referred briefly to other imaging technologies for monitoring inflammation in the last section.

Mast cells are hematopoietic-lineage cells that participate in immunoglobulin E (IgE)-associated immune responses, including allergic reactions and parasite resistance. Recent studies have shown that zinc (Zn) ion can behave as an intracellular signaling molecule and that Zn is involved in mast cell activation. We demonstrated that mast cells stimulated through the high-affinity IgE receptor (FcεRI) rapidly release intracellular Zn from the endoplasmic reticulum (ER), and we named this phenomenon the "Zn wave". Furthermore, we found that the L-type calcium channel (LTCC) is the gatekeeper for the Zn wave. LTCC antagonists inhibited the Zn wave, and an agonist was sufficient to induce it. Notably, LTCC was mainly localized to the ER rather than to the plasma membrane in mast cells, and the Zn wave was impaired by LTCC knockdown. We also found that the LTCC-mediated Zn wave positively controlled inflammatory cytokine gene induction by enhancing the DNA-binding activity of nuclear factor-kappa B (NF-κB). These findings indicated that the LTCC has a novel function as a gatekeeper for the Zn wave, which is involved in regulating NF-κB signaling. In this review, we describe our current understanding of Zn signaling, especially with regard to the Zn wave and the role of Zn signaling in mast cells.

Artificial zinc finger proteins (ZFPs) consist of Cys2-His2-type modules composed of approximately 30 amino acids that adopt a ββα structure and coordinate a zinc ion. ZFPs recognizing specific DNA target sequences can substitute for the binding domains of various DNA-modifying enzymes to create designer nucleases, recombinases, and methylases with programmable sequence specificity. Enzymatic genome editing and modification can be applied to many fields of basic research and medicine. The recent development of new platforms using transcription activator-like effector (TALE) proteins or the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) system has expanded the range of possibilities for genome-editing technologies. These technologies empower investigators with the ability to efficiently knockout or regulate the functions of genes of interest. In this review, we discuss historical advancements in artificial ZFP applications and important issues that may influence the future of genome editing and engineering technologies. The development of artificial ZFPs has greatly increased the feasibility of manipulating endogenous gene functions through transcriptional control and gene modification. Advances in the ZFP, TALE, and CRISPR/Cas platforms have paved the way for the next generation of genome engineering approaches. Perspectives for the future of genome engineering are also discussed, including applications of targeting specific genomic alleles and studies in synthetic biology.

Gene therapy is an attractive strategy, for not only targeting nuclear genome, but the mitochondrial genome as well. Human mitochondrial DNA (mtDNA) encodes 13 subunits of the electron transport chain, 22 tRNAs, and 2 rRNAs and their mutations cause a wide range of mitochondrial diseases. Each cell contains hundreds to thousands of mtDNAs, and in the case of a diseased cell, the mitochondrion possesses both mutant mtDNA and wild-type mtDNA. It is generally accepted that the disease phenotype appears when the proportion of the pathogenic mutant mtDNA exceeds a certain threshold. Therefore, the suppression of mutant mtDNA or supplementing wild-type mtDNA will control the onset of mitochondrial disease. To achieve the transfection of an exogenous therapeutic gene to the mitochondrial matrix where mtDNA is transcribed and translated, it is necessary to transfer cargos through mitochondrial outer and inner membranes. Several methods have been examined for mitochondrial transfection, but a universal, wide-ranging transfection technique has yet not been established. We recently developed a mitochondrial targeting delivery system, namely the MITO-Porter. The MITO-Porter is liposomal nanocarrier with a mitochondrial fusogenic lipid composition. We reported that the MITO-Porter could deliver chemical compounds and proteins to the mitochondrial matrix via membrane fusion. In this review, we report (1) on the pharmacological enhancement of lecithinized superoxide dismutase (PC-SOD) using MITO-Porter, (2) the transcription activation of exogenous DNA by mitochondrial transcription factor A (TFAM), and (3) perspectives on a mitochondrial targeting device.

The gene encoding lipin 1 was identified with a positional cloning approach that localized the causative mutation in fatty liver dystrophic (fld) mice, a mouse model of lipodystrophy. The fld mouse lacks normal adipose tissue in the body, and displays metabolic dysregulation such as obesity, insulin resistance, and hypertriglyceridemia. Lipin 1 is abundantly expressed in key metabolic tissues, including adipose tissue, skeletal muscle, and liver. In the cytosol, lipin 1 acts as an Mg²⁺-dependent phosphatidate phosphatase type-1 (PAP1), catalyzing a key step in the synthesis of glycerolipids. In the nucleus, lipin 1 acts as a transcriptional coactivator through its direct interaction with peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α (PGC-1α) and PPARα. Through two distinct functions in the nucleus and cytosol, lipin 1 modulates lipid metabolism and glucose homeostasis. Here we will discuss recent developments in our understanding of the role of lipin 1 in lipid metabolism.

Heme-containing proteins, the heme proteins, are known to have physiologic functions in humans, mammalians, fish, plants, and bacteria. For example, hemoglobin and myoglobin, which belong to the globin family, have been studied in terms of their structures and functions with spectroscopic and mutagenic methods. Recently, a new class of heme proteins has been discovered, referred to as gas sensors. These are heme-based sensor proteins that play important roles in transcriptional activation, histidinekinase activities, phosphodiesterase activities, etc. CooA is a CO-sensing transcriptional activator derived from the photosynthetic bacterium Rhodospirillum rubrum. FixL is a rhizobial oxygen sensor protein, and we have targeted Bj FixL derived from Bradyrhizobium japonicum. Dos from Escherichia coli is an oxygen sensor protein, which senses oxygen in the heme-containing domain and induces phosphodiesterase activity in other domains. In previous work, we studied the axial ligands and C-helix of CooA to clarify the activation mechanism. Moreover, FixL and Dos were investigated using time-resolved spectroscopic methods. Whereas FixL has a pentacoordinate heme in the ferrous deoxy form, there are a proximal histidine (His 77) and a distal methionine (Met 95) as axial ligands to coordinate to the heme iron in EcDos. However almost all gas sensors show mono-exponential rebinding (6-7 ps), while EcDosH and full-length Dos show biexponential rebinding (7 ps and 35 ps) on the internal ligand. The results were also supported by molecular dynamic simulation. Here we discuss recent work on gas sensors with implications provided by our research.

Artificial transcription factors targeting any desired genes are very attractive from the standpoint of regulating biological functions for life science studies and clinical applications. In order to generate such transcription factors, specific DNA binding domains are required to address a single site for each gene promoter. C(2)H(2) type zinc finger motif is one of the best frameworks to create new artificial DNA binding proteins for the following features: the zinc finger motif can recognize three bases DNA, be tandemly repeated by covalent linkage, and work as a monomer. Taking advantage of these features, manifold zinc finger proteins targeting various DNA sequences have been created so far. For application to a target in sequences as complex as the human genome, the significantly strict specificity in DNA binding must be required. Conjugating multiple fingers (multi-zinc fingers) enables to recognize longer sequences which are sufficient for addressing a single site in the human genome, whereas it has become known that as the number of finger motifs increases, the equilibrium time with the target sequence is significantly longer by in vitro experiments. Our recent study showed that the multi-zinc finger type artificial transcription factor could activate the reporter gene promptly. There is much interest in creating gene regulators, and the artificial transcription factors based on multi-zinc finger motifs could be a superior scaffold.

The mechanisms of aging are still largely a mystery. However, recent studies using yeast, C. elegans, and Drosophila show that longevity can be altered by genetic manipulations. Moreover, drugs increase lifespan of these organisms have been identified. An anticonvulsant ethosuximide, antidepressants such as mianserin, antioxidants, an inhibitor of histone deacetylase, and resveratrol, a Sir2 activator, significantly extend lifespan of some organisms. Although these drugs have not been reported to extend lifespan of mammals, they provide molecular cues to elucidate aging mechanisms.

Ischemic stress can induce the expression of selective gene in the brain. In the acute phase of ischemia, immediate early gene is induced, and this is followed by the induction of heat shock proteins, apoptosis-related genes and inflammatory cytokines. Some of the genes expressed in the neuron induce neuronal death while other genes are related to neuronal survival. In the later phase of ischemia, genes related to neurogenesis and tissue remodeling are expressed in the brain. These genes may play an important role in the recovery of neurological function. Many of these genes are expressed mainly in the glial cells in this phase. Ischemic tolerance is powerful protective mechanism phenomenon against ischemic injury. Ischemic tolerance is induced 24-48 hours following sublethal ischemia. Since gene expression is altered during this period, gene expression may be involved in ischemic tolerance. It was reported that induction of favorable gene, such as those for heat shock proteins, might be a molecular mechanism playing some part in ischemic tolerance. On the other hand, DNA microarray analysis reveals that gene suppression, rather than expression, may play an important role in the molecular mechanism underlying ischemic tolerance. Gene expression profiles in ischemic brain and in ischemic tolerance have been investigated and a part of the molecular mechanism involved in those phenomena has been revealed recently. Although further study is necessary, gene expression may be an important therapeutic target in ischemic stroke.

Adipocytes and osteoblasts are derived from mesenchymal stem cells, and some adipocyte differentiation regulators suppress osteoblast differentiation. PPAR-gammaplays a pivotal role for adipocyte differentiation and glucose tolerance. PPAR-gammais a member of nuclear receptor super family and regulates mRNA expression level of target genes by binding to fatty acid derivatives and thiazolidinediones. Recently, it was found that PPAR-gammainhibits osteoblast differentiation and regulates bone metabolism. In this report, we show recent studies about the extracellular signals regulating the transactivation function of PPAR-gamma.

Transcriptional activation of metallothionein (MT) genes by heavy metals is a valuable system for understanding the functions of MT as well as the cellular response against heavy metals. Although it is now known that heavy metal signals culminating in MT induction converge upon a transcription factor MTF-1, the mechanism underlying the MTF-1 response to heavy metals has not been elucidated. To address this issue, we investigated various aspects of the in vivo response of MTF-1 against heavy metals. Chromatin immunoprecipitation assay showed that heavy metal-dependent DNA binding of MTF-1 is the critical step in vivo. MTF-1 is primarily localized in the nucleus so that heavy metal-dependent nuclear translocation demonstrated by other groups does not seem to be universal and hence may not be critical for activation of MTF-1. In the six Zn finger motifs, the hallmark of MTF-1, the third and the fourth fingers are essential for the nuclear localization of MTF-1. Furthermore, all fingers except the last are important for transcriptional activation function of MTF-1, suggesting their key role for MTF-1 function. Also, a cysteine cluster structure located in the C-terminal region of MTF-1 is critical for transactivating function of MTF-1. These results suggest a central role of the Zn-finger domain and intramolecular cooperation through a structural change of MTF-1 for its response to heavy metal challenge.

The nuclear factor-kappa B (NF-kappaB) family of transcription factors is known to play an important role in the regulation of the immune system. NF-kappaB is activated by bacterial and viral antigens, which lead to the production of proinflammatory cytokines and chemokines. The rapid detection of activated NF-kappaB by systemic inflammatory response syndrome (SIRS) is considered to be crucial for the treatment of patients with septicemia. The aim of the present study was to evaluate the sensitivity of two methods, electrophoretic mobility shift assay (EMSA) and transcription factor enzyme-linked immunoassay (TF-ELISA). TF-ELISA detected 25ng of recombinant human NF-kappaB p50 (rhp50) and 5 microg of TNFalpha-stimulated HeLa nuclear protein, while EMSA detected approximately 100 ng of rhp50 and 10 microg of HeLa nuclear protein. We found that TF-ELISA was more sensitive than EMSA in detecting NF-kappaB; however, it was judged that the 3-6 hour measuring time required in TF-ELISA was excessively long for patients with SIRS. Therefore, the development of new analytical methods with improved sensitivity and measurement time was necessary for the detection and quantification of activated NF-kappaB protein in the hospital laboratory. Consequently, we have developed a new NF-kappaB analyzer based on surface plasmon resonance (SPR), which is recognized as one of the most sensitive direct optical detection methods. This method can detect nanomolar concentrations of NF-kappaB within 15 minutes. In addition, we have developed new experimental apparatus for the detection of NF-kappaB based on fluorescence correlation spectroscopy (FCS), which is able to analyze binding between DNA and protein in the liquid phase. At present, we are carrying out clinical trials using this new transcription factor analysis apparatus for SIRS patients.

Due to the advent of multi-drug therapy (MDT) recommended by the WHO, for the treatment of leprosy, presently, leprosy is regarded as a "curable disease". The number of new cases in Japan is relatively very low, due to which the disease is likely to be neglected, but on scientific grounds, there is a necessity to perform in depth studies. Leprosy caused by M. leprae is still unclear on various aspects including transmission, immunology, nerve damage etc. Here we introduce the recent advances in the field of basic leprosy research.

The molecular and biochemical mechanisms contributing to the development and progression of prostate carcinogenesis are still unknown. Recent technologies such as, real-time PCR, microarray, and laser capture microdissection, have accelerated the study of the molecular events involved in prostate cancer. The expression and function of numerous genes have been shown to be altered in prostate cancer. Such gene regulations are involved in cell cycle, signal transduction pathways, or gene expression. Analysis of the genes during prostate cancer progression has provided a better understanding of the basis of the disease. This review summarises current knowledge of the molecular analysis for prostate cancer.

G-protein coupled receptor(GPCR) agonists are well-known inducers of cardiac hypertrophy. We found that the shedding of HB-EGF via metalloproteinase activation and subsequent transactivation of the epidermal growth factor receptor occurred when cardiomyocytes were stimulated by GPCR agonists, leading to cardiac hypertrophy. A new inhibitor of HB-EGF shedding, KB-R7785, blocked this signaling. We cloned a disintegrin and metalloprotease 12(ADAM12) as a specific enzyme to shed HB-EGF in the heart and found that dominant negative expression of ADAM12 abrogated this signaling. KB-R7785 bound directly to ADAM12, suggesting that inhibition of ADAM12 blocked the shedding of HB-EGF. In mice with cardiac hypertrophy, KB-R7785 inhibited the shedding of HB-EGF and attenuated hypertrophic changes. These data suggest that shedding of HB-EGF by ADAM12 plays an important role in cardiac hypertrophy, and that inhibition of HB-EGF shedding could be a potent therapeutic strategy for cardiac hypertrophy.

The functional study on NF-kB bindings sites in transcription-regulatory region of rat inducible type nitric oxide synthase (iNOS) has been carried out by reporter assay to learn the signaling mechanisms of actions of interleukin-1 (IL-1). By progressive excision of 5'-ends of the insert and introduction of mutagenesis of each NF-kB binding site, it has been shown that 3 kB sites are essential for IL-1-induced signaling pathway in a mutually dependent manner.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by various combinations of cerebellar ataxia, choreoathetosis, myoclonus, epilepsy, dementia and psychiatric symptoms. With the discovery of the gene for DRPLA in 1994, molecular mechanisms of neurodegeneration has been intensively investigated. Expression of truncated DRPLA proteins in cultured cell has been shown to result in aggregate body formation and apoptosis. We have developed transgenic mice for DRPLA (Q129 mice). The Q129 mice showed a severe neurological phenotype characterized by ataxia, myoclonus and seizures. Although numerous neuronal intranuclear inclusions (NIIs) were observed in the brain, obvious neuronal loss was not observed. NIIs, however, appeared much later than the phenotypic presentations, and intranuclear accumulation (diffuse nuclear staining) of mutant proteins was the earliest phenomenon. These results suggest that "neuronal dysfunction", but not "neuronal cell death", is responsible for the phenotypes. Hypothesizing that nuclear proteins interacting with expanded polyglutamine stretches are involved in the pathogenesis, we found that that expanded polyglutamine stretches preferentially bind to TAF130, a cofactor involved in CREB-dependent transcriptional activation, and strongly suppressed CREB-dependent transcriptional activation. Taken together, these findings suggest that the interference of CREB-dependent transcription by expanded polyglutamine stretches is involved in the neuronal dysfunction in polyglutamine diseases.

Androgen plays an important role in the growth of prostate cancer, but the molecular mechanism that underlies development of resistance to antiandrogen therapy remains unknown. Cyclin E has now been shown to increase the transactivation activity of the human androgen receptor (AR) in the presence of its ligand dihydrotestosterone. The enhancement of AR activity by cyclin E was resistant to inhibition by the antiandrogen 5-hydroxyflutamide. Cyclin E was shown to bind directly to the AB domain of the AR, and to enhance its AF-1 transactivation function. These results suggest that cyclin E functions as a coactivator of the AR, and that aberrant expression of cyclin E in tumors may contribute persistent activation of AR function, even during androgen ablation therapy.

In developed societies, chronic diseases such as diabetes, obesity, atherosclerosis and cancer are responsible for most deaths. These ailments have complex causes involving genetic, environmental and nutritional factors. There is evidence that a group of closely related nuclear receptors, called peroxisome proliferator-activated receptors(PPARs), may be involved in these diseases. This, together with the fact that PPAR activity can be modulated by drugs such as thiazolidinediones and fibrates, has instigated a huge research effort in to PPARs. Here we present the latest developments in the PPAR field, with particular emphasis on the physiological function of PPARs during various nutritional states, and the possible role of PPARs in several chronic disease.

Gene therapy for hypertension is very important for the next generation of antihypertensive drugs. Important question regarding vector-related limitations and suboptimal in vivo delivery systems will require expeditious attention for gene therapy to become a more widely applicable option in cardiovascular diseases including hypertension. However, the elucidation of molecular mechanisms of transcriptional activation of several important genes in the cardiovascular physiology enables us to develop clinical application of gene therapy for hypertension at the level of transcription factors. Recent studies showed that in vivo or ex vivo transfection of double-stranded oligodeoxynucleotides to block the binding of nuclear factors to specific cis-elements in the promoter regions of several genes resulted in inhibition of gene trans-activation and suppressed pathological changes in the cardiovascular system.

The clinical entity of dentatorubral-pallidoluysian atrophy (DRPLA) was discovered and established in Japan. The characteristic clinical genetic features of DRPLA including prominent anticipation prompted us to search the genes carrying CAG repeats as the candidate genes for DRPLA. Based on this approach, the DRPLA gene was discovered by two independent Japanese groups in 1994. Given that DRPLA is caused by expansion of CAG repeats, the molecular mechanisms of the anticipation and the broad clinical presentations of DRPLA depending on the age at onset are clearly understood as a function of the size and the instability of the CAG repeats. Recent studies have suggested that mutant proteins with expanded polyglutamine stretches have "gain-of-toxic" functions to neuronal cells. The molecular mechanisms of the "toxic" functions, however, have not been identified. Based on the recent findings that nuclear transport of the mutant proteins carrying expanded polyglutamine stretches is important in the pathogenesis, we screened nuclear proteins which bind to expanded polyglutamine stretches. We found that expanded polyglutamine stretches bind to TAFII130, a TATA-binding protein-associated factor, and interfere with CREB-dependent transcriptional activation. Since CREB-dependent transcriptional activation plays essential roles in neuronal survival and plasticity, such interference is expected to lead to neuronal dysfunction.

The genetic reporter system has been introduced as a tool for sensitive and convenient monitoring of gene expression. In the present study, the promoter activity of growth hormone (GH)-coding gene was sequentially monitored in GH3 cells (rat pituitary adenoma cell-line) by means of bioluminescence which was evoked by a secreted reporter, Vargula hilgendorfii (VH) luciferase. Three kinds of expression plasmids were constructed, which consisted of a rat GH promoter fragment and VH luciferase cDNA, and transfected into GH3 cells. The sensitivity of the reporter was revealed to be extremely high from the dose-response curve of VH luciferase. With the use of the stable transformant, the reporter activity as well as that of GH was sequentially measured in plate culture. The intracellular dynamics of reporter signals was analyzed through the response to inhibitors of transcription, protein synthesis and vesicle transport, which suggested that the overall time from transcription to secretion was essentially identical to those of the VH luciferase and GH although the both proteins took different time course at different intracellular stages. When the GH promoter was activated by triiodothyronine (T3), the reporter activity was enhanced, and well correlating with the amount of GH mRNA, which indicated a reliability of the reporter system. Furthermore, in the present reporter system, a synergistic effect of T3 and dexamethasone was observed not only on the GH transcription but also on the stability of mRNA. It is concluded that the bioluminescence reporter system using the VH luciferase is an excellent tool for sequential monitoring of the GH transcriptional activity.

Lysozyme is an important component of innate immunity against common pathogens at mucosal surfaces. We previously cloned and characterized the bovine lysozyme 5A (lys5A) promoter with the purpose of determining cis- and trans-acting elements controlling airway epithelial cell-specific expression. We found that such expression is controlled by protein binding to an ETS consensus sequence located approximately at -46 to -40 bp from the transcription start site. The identity of the ETS-related protein responsible for gene transactivation was unknown. In this study, we screened six ETS-related proteins by transient transfection into epithelial cells and fibroblasts. Results showed that among these factors, the myeloid Elf-I-like factor (MEF) was the most potent. Gel shift analysis of epithelial cell nuclear extracts using a lys5A probe including the ETS-binding site (-50/-31) yielded a single band with retarded mobility. This band was super-shifted by an antibody directed against MEF. Supporting the possibility that MEF is responsible for functional transactivation of lysozyme in epithelial cells, we found that antisense MEF mRNA decreased lys5A promoter activity and that MEF overexpression in stably transfected cells increased lysozyme mRNA and protein expression. We conclude that MEF is required for epithelial cell transactivation of lysozyme.

Angiotensin II (AII) receptor type 1 (AT1), a G-protein-coupled receptor, is involved in the development of cardiovascular diseases such as hypertensin, cardiac hypertrophy, and atherosclerosis. Recent reports indicate that tyrosine phosphorylation of multiple intracellular molecules is responsible for most of these AII actions mediated by AT1, similar to receptor tyrosine kinase signaling pathways. AII activates MAPK by tyrosine phosphorylating the EGF receptor by the mechanism called transactivation with subsequent Ras activation in vascular smooth muscle and cardiac fibroblast cells. In contrast, AT1 leads to MAPK activation through PKC in cardiac myocytes. In addition to these signals, JAK/STAT pathways, which mediate cytokine actions, are also important for several AII functions through AT1.

Nuclear hormone receptors are transcription factors which regulate gene transcription by lipophilic hormones. The functional domains such as transactivation, DNA binding, ligand binding and dimerization are mapped in the distinct region of the receptors. To analyze these functions, ligand binding assay, gel shift assay, transfection study, GST pull-down method, immunoprecipitation method, two hybrid system and yeast functional assay are employed. These assays have been shown to clarify the molecular basis of the disorders which are due to mutation in the receptors.

The 1,25-dihydroxyvitamin D receptor (VDR) belongs to the nuclear hormone receptor family. Mutational analysis revealed that the carboxyl-terminal region between leucine-417 and glutamic acid-420 of the human VDR is essential for the ligand-dependent transactivation. Mutant VDR at this AF-2 region exhibits only weak suppressive effect on the transactivation via the wild type receptor compared to the estrogen and vitamin A receptors, which confer the strong dominant negative effect. Using the AF-2 mutant VDR protein, we demonstrated a 65kD nuclear protein, which binds to the AF-2 region of the human VDR in a ligand dependent manner.

Nuclear receptors for steroid/thyroid hormones, vitamin A,D and fat-soluble ligands form a gene superfamily of ligand-inducible transcription factors, which plays an important roles in a wide spectrum of biological events by regulating expression of a set of target genes. Two domains (A/B and E) of nuclear receptors are demonstrated to activate transcription, designated AF-1 (A/B) and AF-2 (E), respectively. Homo- and hetero-dimers of nuclear receptors bind target enhancer elements in the target gene promoters. The target enhancer elements are referred as hormone response elements (HRE), and composed of two hexamer core motifs. The orientations and spaces between two core motifs play critical roles in discreminating the recognition of HREs among the members of nuclear receptor. DNA-bound nuclear receptors controls transcription in a ligand-binding dependent way in co-operation with a multiprotein complex containing RNA polymerase II and a series of auxiliary factors, TFIIA,B,D, E,F and H. During process of ligand-induced transactivation by nuclear receptors, nuclear co-factors interacting with the AF-1 and AF-2 seem to be involved. Several transcriptional co-activators and co-repressors have been recently identified, and their function is currently under investigation.

This review summarizes current issues on neuronal structural plasticity and its molecular marker genes in the aged brain. Neuronal growth-associated proteins (nGAPs) are introduced as potential markers of neuronal structural plasticity in the brain. The expression of genes encoding nGAPs such as GAP-43, SCG10 and stathmin is increased following striatal and hippocampal neuronal deafferentation lesions in the adult rat brain. In aged brains, the magnitude of neuronal plasticity is reduced and nGAP gene induction is limited at least in part, while the kinetics of the response remains unchanged in comparison to young brains. These results suggest that nGAPs serve as molecular markers of neuronal structural plasticity in the aging brain and that neuronal plasticity decreases, at least in certain neuronal circuits, during aging. The expression of genes encoding SCG10, stathmin and GAP-43 is also altered in age-related neurodegenerative conditions such as Alzheimer's disease in humans. Thus, studies of nGAPs are important for the elucidation of the mechanisms of the reduction of neuronal structural plasticity in aged brains.

Pit-1/GHF-1 is a pituitary specific POU-domain DNA binding factor, which transactivates the growth hormone (GH), prolactin (PRL), thyrotropin beta subunit (TSH beta) genes, and PIT1 gene itself. It is indispensable for the differentiation and the proliferation of the cells producing GH, and PRL. Loss of the PIT1 gene function (PIT1 abnormality) results in combined deficiency of GH, PRL, and TSH. PIT1 abnormality serves as a model of transcription factor abnormalities.

Activation of helper T-cells mediated by the T-cell receptor induces a series of biochemical events. Among them, both the activation of PKC/Ras- and CaM/CN-mediated pathways play a central role in the signal transduction of cytokine gene induction. Cytokines produced by non-transformed Th1 and Th2 clones were classified into three groups, based on their signal requirement patterns for their expression. Closer examination using various stimulation conditions suggested that the balance between the activities of the two signaling pathways contributes to cytokine expression. Th1 and Th2 effector functions and their development are attributable to their coordinated and differential expression of cytokines. Clarification of the mechanisms of Th1/Th2 differentiation should lead to rational strategies for manipulating pathological immune responses.

In this review, I discuss recent progress in our understanding of the role of Epstein-Barr virus on pathological conditions in Sjögren's syndrome (SS). In order to clarify the association of EBV in SS pathogenesis and further analyze the precise transcriptional mechanism of EBV reactivation, we assayed transcription of ZEBRA which is the fast transcribed EBV-encoded immediate early gene product and was an indispensable role in EBV reactivation. The ZEBRA expression was observed in the ductal epithelial cells and infiltrating B cells, and SS peripheral blood lymphocytes (PBL). On the other hand, to identify the inducer of EBV reactivation, we have used in vitro EBV reactivation model cell line. Screening of cDNA by mRNA differential display subtraction method, one clone, named "AK-1", was highly expressed in SS PBL. Nucleotide sequence analysis revealed that AK-1 was complete match with p300/CBP-associated factor (P/CAF). Furthermore, as another approach, we have partially cloned cDNA with a novel sequence containing a region homologous to the basic leucin zipper domain of CREB/ATF family by degenerate PCR. Our results together with recent reports, suggest the possibility that CREB family protein-p300-P/CAF ternary complex on ZEBRA promoter might be involved in ZEBRA transcriptional activation.