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Regulatory Mechanisms of Cellulolytic and Hemicellulolytic Enzyme Gene Expression in Filamentous Fungi: Toward Elucidating the Regulatory Mechanisms of Biomass-Degrading Enzyme Production in Filamentous Fungi Emi KUNITAKE, [ABSTRACT FROM AUTHOR]

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We investigated the effect of long-term administration of kale (Brassica oleracea L. var. acephala) on hippocampal gene expression in senescence-accelerated mouse prone 8, SAMP8. The mice were fed the AIN-93M diet containing 0.05% (w/w) kale extract (KE) for 31 weeks. The long-term KE administration resulted in significant decreases in escape latency compared to control group in the Barnes maze test. According to the DNA microarray analysis, KE administration significantly up-regulated various genes of G protein-coupled receptors pathways in the hippocampus. In contrast, KE administration significantly down-regulated genes of complement and coagulation cascades and focal adhesion-PI3K-Akt-mTOR-signaling pathway. Up-regulated pathways included genes involved in the modulation of neurotransmission and synaptic plasticity. Down-regulated pathways included genes related to the thrombus formation. These results could provide partial information about one of the molecular mechanisms underlying the suppressive effect of KE intake against cognitive decline. [ABSTRACT FROM AUTHOR]

Temperate plants enhance their freezing tolerance before winter to survive in freezing environment. Plants use the cytosolic transient Ca2+ concentration change as a kind of signal to regulate gene expression in cold acclimation process. We developed the experimental system for observation of Ca2+ signal during cooling, and observed Ca2+ signal to understand “how plants sense cold and seasonal changes.” We found several characteristics of Ca2+ signal and cold sensing. For example, plants adjust the Ca2+ signal to the ambient temperature to response small temperature decrease, and then small temperature fluctuation induces the cold-inducive transcription factor DREB1/ CBFs expression via Ca2+ signals. [ABSTRACT FROM AUTHOR]

BACKGROUND：Non-steroidal anti-inflammatory drugs (NSAIDs) have been widely used for the management of pain and inflammation. However, little remains known about the effects of NSAIDs on synovitis of the human temporomandibular joint (TMJ). Cyclooxygenase (COX) inhibitor are one of main NSAIDs. This study investigate the effects of COX inhibitor on fibroblast-like synoviocytes (FLS) derived from the human TMJ. METHODS：Human synovial tissue was obtained from a patient with internal derangement who underwent arthroscopy of the TMJ. FLSs were prepared from the tissues using the outgrowth method. A COX inhibitor (indometacin or celecoxib) was added to the IL-1β stimulating cells in culture. Gene expression profiling was performed using microarray analysis. RESULTS：The expressions of IL-23A, IL-11, and IL-33, which were up-regulated in FLSs stimulated by IL-1xxxwere down-regulated in the cells treated by COX inhibitors. CONCLUSION：COX inhibitors such as indomethacin and celecoxib reduce the gene expression of inflammatory cytokines induced by IL-1β Our results suggest that these COX inhibitors are useful for treating synovitis in TMJ. [ABSTRACT FROM AUTHOR]

The term “RNA sequencing (RNA-Seq)” often means the sequencing of cDNA generated from RNA as well as the direct sequencing of RNA. RNA-Seq has become well known as next generation sequencers emerge, particularly because their high throughput nature of data collection makes RNA-Seq a powerful tool for genome-wide gene expression analysis. In this review, I introduce, mainly from a technical point of view, a basic scheme of RNA sequencing, its development, and its application in several interesting studies. [ABSTRACT FROM AUTHOR]

Energy-conversion systems mediated by bacterial metabolism have recently attracted much attention, and therefore, demands for tuning of bacterial metabolism are increasing. It is widely recognized that intracellular redox atmosphere is one of the important factors influencing on bacterial metabolism. Although intracellular redox atmosphere had been conventionally tuned by dissolved oxygen concentration or by appropriate selection of an electron acceptor for respiration, we are developing novel method based on electrochemical technique for on-line (dynamic) tuning. Here we review our recent studies on electrochemical tuning of intracellular redox environment toward the control of bacterial metabolic pathway and activity. [ABSTRACT FROM AUTHOR]

SCG is a 6-branched 1,3-β-D-glucan, and is a major cell wall structural component in fungi. The leukocytes from DBA/1 and DBA/2 mice are highly sensitive to SCG, producing cytokines, such as GM-CSF, IFN-γ nd TNF-α. GM-CSF plays a key biological role in this activity. We analyzed factors induced by SCG in splenocytes from DBA/2 mice by DNA microarray analysis on the condition of high sensitivity to β-glucan. Splenocytes were stimulated with SCG at 0, 24 or 30 h, and then supernatant was collected at 48 h to measure cytokines. SCG stimulated splenocytes to produce GM-CSF, IFN-γ and TNF-αin all the supernatants of 0, 24, and 30h. The amount of IFN-γ production thus stimulated at 24 h was comparable to that at 0 h. Cytokine induction was observed at 4 h after SCG-stimulation even in the splenocytes pre-cultured for 36 h. The gene expression induced by SCG was analyzed with DNA microarray in the splenocytes in this condition. SCG up-regulated the expression of genes including Edn1 and Ptgs2 as well as genes associated with cytokine and chemokine. PGE2 was detected in the medium of splenocytes stimulated with SCG. Taken together, these results indicated that splenocytes enhanced the sensitivity to SCG in earlier culture periods, and then responded to SCG to induce not only the cytokines but also various other factors. [ABSTRACT FROM AUTHOR]

Molecular biological approaches have recently begun to be applied to molecular genetics studies of dermatophytes. High-throughput gene analysis methodologies, such as EST sequencing, differential cDNA screening, and cDNA based microarray analysis have been used to obtain information on many dermatophyte genes and their expression profiles under different experimental conditions. In addition, whole genome sequencing projects are underway for several important dermatophytes, such as Trichophyton rubrum and Microsporum canis. These studies will provide large amounts of valuable information for elucidating the molecular basis of host invasion by dermatophytes and their virulence. Targeted gene disruption by homologous recombination is one of the most common approaches for determining the functions and roles of numerous genes isolated from pathogenic fungi. However, the difficulty genetic manipulation due to low transformation frequency of dermatophytes may limit the successful production of null mutants by targeted gene disruption via holologous recombination. To overcome these problems, our group has developed useful genetic manipulation systems for dermatophytes using the clinically important dermatophyte, T. mentagrophytes. [ABSTRACT FROM AUTHOR]

One of the major goals in evolutionary developmental biology is to elucidate the relationship between gene regulatory networks and the diverse morphologies. Segmentation in arthropod embryogenesis represents a well-known example of body plan diversity. Striped patterns of gene expression that lead to the future body segments appear simultaneously or sequentially, respectively. To reveal the basic differences in the network structure, we have numerically evolved hundreds of gene regulatory networks. By analyzing the topologies of the generated networks, we show that the characteristics of striped pattern are determined by Feed-Forward Loops (FFLs) and negative Feed-Back Loops (nFBLs). [ABSTRACT FROM AUTHOR]

This study investigated the effect of hyperbaric oxygen (HBO)on the gene expression of the extracellular matrix proteins of the ruptured anterior cruciate ligament (ACL). In each of 80 Sprague-Dawley rats, ACL was lacerated at the tibial insertion. Forty rats were exposed to 100% oxygen at 2.5 ATA for 2 hours for 5 days a week, beginning the day of surgery(Group H), whereas the remaining 40 control rats were housed in normal room air(Group C). The animals were sacrificed at 3. 7, 14, and 28 days postoperatively, and ACLs were harvested after macroscopic examination. RNA was extracted from the harvested ACL and the gene expression of type I and type HI procollagen, three kinds of matrix metalloproteinases(MMP) (collagenase 3, gelatinase A and B), and tissue inhibitor of matrix metalloproteinase (TIMP) l and 2 was examined semi-quantitatively by the RT-PCR method. None of the lacerated ACL healed macroscopically. The gene expression of type I and type III procollagen and TIMP 1 and 2 in Group H was higher than that in Group C, whereas there was no significant difference in the gene expression of each MMP between the groups. It is suggested that the administration of HBO enhances collagen synthesis and inhibits tissue degradation by decreasing the ratio of MMP to TIMP in ruptured ACL. Although it is unlikely that the administration of HBO heals the ruptured ACL by itself, it may be useful as an adjunct after primary repair of ACL. [ABSTRACT FROM AUTHOR]

Repetitive behavior, a form of compulsivity, is a component of several neuropsychiatric disorders, including obsessive-compulsive disorder and addiction. Dysfunction of dopaminergic modulation in the striatum is thought to be a key neural mechanism underlying compulsive behavior repetition; however, the mechanistic links between dopaminergic abnormalities and compulsivity remain unclear. This review discusses our recent work demonstrating the contribution of the NOX1 isoform of the superoxide-producing enzyme, nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase), to compulsive-like repetitive behavior in mice that received repeated stimulation of D 2 receptors. Nox1 deficiency inhibited compulsive-like repetitive behaviors, as assessed by observation of spontaneous behavior patterns and perseveration in the reversal learning test. Repeated stimulation of D 2 receptors also upregulated expression of Nox1 in the central striatum (CS), and induced excitatory synaptic potentiation in CS indirect pathway medium spiny neurons. Such synaptic potentiation required recruitment of β-arrestin and was blocked by Nox1 deficiency or acute pharmacological inhibition of NOX1. Furthermore, upregulation of NOX1 in the CS contributed to accumulation of activated Src kinase following stimulation of D 2 receptors. Local inhibition of NOX1 or neuron-specific Nox1-knockdown in the CS was sufficient to reduce repetitive behavior. Collectively, these results reveal a novel role for NOX1 in D 2 receptor-mediated excitatory synaptic potentiation in the striatum, suggesting the potential of NOX1 inhibition as a treatment for compulsivity.

Myeloproliferative neoplasms (MPN) are caused by somatic mutations in hematopoietic stem/progenitor cells and result in excessive increase in the blood cell mass in the peripheral blood and/or fibrosis in the bone marrow. JAK2, CALR, and MPL mutations are well-known driver mutations of MPN and are widely applied as diagnostic markers of MPN. Moreover, several studies using massive parallel sequencing technologies have shown that mutations in ASXL1, EZH2, SRSF2, and IDH1/2 affect the prognosis of overt primary myelofibrosis and have further clarified that the mutation order may influence the MPN phenotype. More recently, our group identified that CREB3L1 mRNA was overexpressed in a platelet- and megakaryocyte-specific manner in driver mutation positive MPN and that the quantitation of this gene expression can be used as a diagnostic marker for MPN. In this educational lecture, we discuss the clinical impacts of the mutations frequently identified in MPN patients.

Blood vessels supply oxygen and nutrients to all the cells in a living body, and provide essential transport routes for collecting waste products. For these functions, blood vessel networks should be appropriately formed in each tissue. Therefore, blood vessels are one of the earliest organs formed during the developmental process. Development of the blood vessel system promotes tissue differentiation and organ morphogenesis, allowing each organ to maintain its unique functions under changing metabolic conditions. Blood vessels have a relatively simple structure, consisting of endothelial cells covering the inner layer, and pericytes or smooth muscle cells surrounding the outside. The structure of the vascular network is extremely diverse, with blood vessels uniquely organized depending on the tissues they serve, to create tissue-specific microenvironments. How are such tissue-specific vascular environments generated? Over the years, anatomical findings have accumulated to confirm this vascular diversity. However, the molecular basis for this diversity has remained unclear. In the present article, we review the mechanisms of coordinated developmental control of the vascular and neural systems in the cerebral cortex from the viewpoint of the accurate expression control of vascular endothelial growth factor (VEGF) signaling, and describe future perspectives.

Providing plausible strategies for brain aging protection should be a critical concern for countries with large elderly populations including Japan. Age-related cognitive impairments and movement disorders, such as Alzheimer's and Parkinson's diseases, are caused by neurodegeneration that primarily initiates in the hippocampus and the midbrain substantia nigra, respectively. Neurons are postmitotic, and therefore, the accuracy of cellular metabolism should be crucial for maintaining neural functions throughout their life. Thus accuracy of protein synthesis is a critical concern in discussing mechanisms of aging. The essence of the so-called "error catastrophe theory" of aging was on the fidelity of ribosomal translation and/or aminoacylation of tRNA. There is evidence that reduced protein synthesis accuracy results in neurodegeneration. Similarly, reduced proteostasis via autophagy and proteasomes in aging is crucial for protein quality control and well documented as a risk for aging. In both neurodegeneration and protein quality controls, various proteins are involved in their regulation, but recent evidence suggests that repressor element-1 silencing transcription factor (REST) could be a master regulatory protein that is crucial for orchestrating the neural protecting events in human brain aging. REST is induced in the aged brain, and protects neurons against oxidative stress and protein toxicity. Interestingly, REST is identical with neuron-restrictive silencer factor (NRSF), the master regulator of neural development. Thus NRSF/REST play important roles in both neurogenesis and neurodegeneration. In this review, I summarize the interesting scientific crossover, and discuss the potential use of NRSF/REST as a pharmaceutical target for controlling aging, particularly in relation to brain aging.

Recently, mRNAs and microRNAs (miRNAs) have been identified in exosomes, which can be taken up by neighboring or distant cells. These exosomal-miRNAs may regulate gene expression in recipient cells. miRNAs are a type of non-coding RNA that induce post-transcriptional gene silencing of their target genes and regulate a wide range of biological processes, including apoptosis, differentiation, metabolism, and cell proliferation. According to recent reports, aberrant expression of miRNAs is associated with most pathological disease processes, including carcinogenesis. Therefore circulating onco-miRs are considered as significant therapeutic targets for cancer therapy. However, there is no report to regulate the function of miRNAs in exosomes. In this study, we developed novel drug delivery system using anti-exosome antibody-oligonucleotide conjugates (ExomiR-Tracker) for functional inhibition of circulating miRNAs. The "ExomiR-Tracker" is the world's first innovative molecule that has targeting property for exosome-recipient cells and specifically delivers nucleic acid medicines to the target cells. We found that ExomiR-Tracker can bind to the surface of exosomes and that the complexes are introduced into exosome-recipient cells then inhibit the activity of miRNA. We showed that ExomiR-Tracker can accumulate in cancerous tumors after intravenous administration. Existing technologies have difficulties for introducing anti-miR into exosomes and extremely low possibility to deliver anti-miR to exosome-recipient cells after intravenous administration. However, we successfully developed useful inhibition technology against exosomal-miRNA.

Recently, aging is becoming an important social problem in many developed countries including Japan. It is socially and universally important to unveil the impact of aging and extend healthy life expectancy. Here we show our recent finding that dedicator of cytokinesis 11 (DOCK11, also known as Zizimin2) may be involved in immunosenescence of B cells. DOCK11 was identified as a guanine nucleotide exchange factor for a small GTPase called cell division cycle 42. Expression of DOCK11 is restricted to lymphoid tissues, and becomes downregulated with age. Thus we examined the involvement of DOCK11 in immunosenescence of B-1a B cells as an example. B-1a cells are the main source of antibodies at steady state, and function as the first line of defense against infection. Although DOCK11 was expressed by B-1a cells, the expression levels declined with age. Furthermore, production of anti-pneumococcal immunoglobulin M antibodies was suppressed in aged mice, and was recovered by adoptive transfer with B-1a cells in a DOCK11-dependent manner. Thus DOCK11 may be involved in immunosenescence of B-1a cells.

Because the liver is the primary target organ for chemicals and pharmaceuticals, evaluation of these substances' liver toxicity is of critical importance. New evaluation methods without animal testing (i.e., in vitro and/or in silico) are eagerly anticipated, both for animal welfare and for decreasing cost. Also, the importance of mechanistic interpretation of the output derived from non-animal testing has been increasing. Accordingly, we investigated the potential for evaluating liver toxicity by applying the adverse outcome pathway (AOP) concept using gene set enrichment analysis (GSEA) from gene expression (GEx) data. A case study targeting hepatocellular fatty degeneration (HFD) is reported and discussed. We first identified the events detectable in an in vitro system by comparing the GEx data from the rat primary hepatocyte (in vitro) and rat liver (in vivo) treated with a chemical with the ability to induce HFD as one of the phenotypes in a 28-day repeated-dose toxicity test. Then, the scores based on GSEA were calculated after establishing the gene sets for each event leading to HFD. As a result, the mechanistic information leading to HFD was obtained from the score calculated based on the GSEA and the usefulness of the transcriptome-driven evaluation using AOP was demonstrated.

The author has described two new functions of endothelial cells for efficient delivery of drugs to tissues. First, it was indicated that tight junction (TJ)-associated protein, claudin-1, exerts potent paracellular barrier function in cultured mouse lung microvascular endothelial cells (LMECs). This barrier was instantly and reversibly opened by reduction of TJ proteins expression via histamine H 1 and H 2 receptors. Histamine was biosynthesized by l-histidine decarboxylase from uptaken l-histidine, and biotransformed by type B of monoamine oxidase, suggesting that histamine concentration is controlled in rat brain MECs (BMECs) and LMECs. Moreover, uptake of l-histidine into BMECs and LMECs markedly increased with addition of ZnSO 4 . Second, it was suggested that drug-metabolizing enzymes such as CYP and flavin-containing monooxygenase exist in vascular endothelial cells exposed to blood and to aerobic conditions. These cells have the same ability to metabolize drugs as hepatocytes, demonstrating that vascular endothelial cells are a metabolic barrier against tissue transfer of drugs. From these results, it was suggested that reversible opening of TJ and selective inhibition of drug metabolism in vascular endothelial cells may be efficient delivery strategies of drugs to tissues. Finally, I hope that this research will lead to development of new drugs and possible re-evaluation of discontinued drugs.

DNA preserves and inherits genetic information. 7,8-dihydro-8-oxoguanine (G O ) and abasic sites are some of the most common DNA lesions generated endogenously in living organisms and they induce mutations. The resultant mutations in our DNA cause diseases such as cancers. G O and abasic sites are known to induce mutations at the positions of the lesions. We revealed G O induced mutations at points distant from a lesion besides mutations at the lesion site in human cells when WRN helicase or DNA polymerase λ was knocked down. In addition, an abasic site analog, tetrahydrofuran, also induced the same type of mutations and large deletions. Thus, these endogenous DNA damages could induce more diverse mutations than previously thought. Recently, much research toward the development of gene therapy approaches has been carried out to apply gene therapy in a clinical setting. In this study, we found that the usual plasmid DNA with suitable transcription regulatory sequences achieved durably expressed transgenes in mouse liver. In addition, we successfully improved gene-correction efficiency with tailed duplex DNA fragments by introducing a second mismatch. These results give us important information to apply a transgene expression approach and tailed duplexes in a clinical setting.

Caloric restriction (CR) improves whole-body metabolism, suppresses various age-related pathophysiological changes, and extends lifespan. The beneficial actions of CR are regulated in growth hormone (GH)/insulin-like growth factor-1 (IGF-1) signal-dependent and -independent manners. To clarify the GH/IGF-1-independent mechanism, we compared gene expression profiles in white adipose tissue (WAT) between CR and GH/IGF-1 suppression, and found that CR upregulated sterol regulatory element-binding protein 1c (SREBP-1c) regulatory gene expression. To validate the impact of SREBP-1c as a beneficial mediator of CR, we compared the responses to CR between wild-type and SREBP-1c knockout (KO) mice. CR extended lifespan, upregulated gene expression involved in FA biosynthesis, activated mitochondrial biogenesis, and suppressed oxidative stress predominantly in WAT. In contrast, most of these findings were not observed in KO mice. Furthermore, SREBP-1c was implicated in CR-associated mitochondrial activation through upregulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis. Sirtuin-3 (SIRT3) regulates mitochondrial quality and is also involved in the beneficial actions of CR. We observed that CR upregulated the mature form of SIRT3 protein and mitochondrial intermediate peptidase (MIPEP), a mitochondrial signal peptidase (MtSPase), in WAT. MIPEP cleaved precursor form of SIRT3 to mature form, and activated certain mitochondrial matrix proteins, suggesting that MIPEP might contribute to maintenance of mitochondrial quality during CR via SIRT3 activation. Taken together, CR induces SREBP-1c-dependent metabolic remodeling, including enhancement of FA biosynthesis and mitochondrial activation, via PGC-1α, and improvement of mitochondria quality via Mipep in WAT, resulting in beneficial actions.

Immune cells such as T cells, macrophages and dendritic cells express various cholinergic system components, including muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs, respectively) and choline acetyltransferase (ChAT), depending on the status of the immune system. The cholinergic system which these components comprise has important effects on the regulation of immune and inflammatory responses. α7 nAChR is a neuronal-type nAChR composed of a homopentamer of the α7 subunit and is characterized by high permeability to Ca 2+ . It is also expressed in immune cells. For example, α7 nAChRs expressed in B cells suppress IgG production by suppressing B cell maturation into plasma cells. In addition, α7 nAChRs expressed in macrophages suppress production and release of tumor necrosis factor (TNF)-α in a mouse lipopolysaccharide (LPS)-induced sepsis model, thereby protecting the mice from lethal shock. In this review, we summarize the functions of α7 nAChRs expressed in CD4 + helper T (Th) cells and antigen-presenting cells (APCs), such as dendritic cells and macrophages. We focus in particular on their role in Th cell differentiation. α7 nAChRs on APCs interfere with antigen presentation, which leads to suppression of Th cell differentiation. By contrast, α7 nAChRs on naïve Th cells enhance their differentiation. These distinct roles of α7 nAChRs expressed in APCs and Th cells could be useful for development of drugs and therapeutic strategies for the treatment of immune- and inflammation-related diseases and cancers.