Biomedical subjects

Hiroshi Takagi

Publications and source records attributed to Hiroshi Takagi.

At least 19 recordsLinked to original sources

PubMed2025

Companion cells with high florigen production express other small proteins and reveal a nitrogen-sensitive FT repressor.

The precise onset of flowering is crucial to ensure successful plant reproduction. The gene FLOWERING LOCUS T (FT) encodes florigen, a mobile signal produced in leaves that initiates flowering at the shoot apical meristem. In response to seasonal changes, FT is induced in phloem companion cells located in distal leaf regions. Thus far, a detailed molecular characterization of the FT-expressing cells has been lacking. Here, we used bulk nuclei RNA-seq and single nuclei RNA (snRNA)-seq to investigate gene expression in FT-expressing cells and other phloem companion cells. Our bulk nuclei RNA-seq demonstrated that FT-expressing cells in cotyledons and true leaves showed differences especially in FT repressor genes. Within the true leaves, our snRNA-seq analysis revealed that companion cells with high FT expression form a unique cluster in which many genes involved in ATP biosynthesis are highly upregulated. The cluster also expresses other genes encoding small proteins, including the flowering and stem growth inducer FPF1-LIKE PROTEIN 1 (FLP1) and the anti-florigen BROTHER OF FT AND TFL1 (BFT). In addition, we found that the promoters of FT and the genes co-expressed with FT in the cluster were enriched for the consensus binding motifs of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1). Overexpression of the paralogous NIGT1.2 and NIGT1.4 repressed FT expression and significantly delayed flowering under nitrogen-rich conditions, consistent with NIGT1s acting as nitrogen-dependent FT repressors. Taken together, our results demonstrate that major FT-expressing cells show a distinct expression profile that suggests that these cells may produce multiple systemic signals to regulate plant growth and development.

BROTHER OF FT AND TFL1↗

PubMed2025

Breeding of yeast strains with intracellular amino acid accumulation for value-added alcoholic beverages.

The yeast Saccharomyces cerevisiae converts amino acids into volatile compounds with fruity and floral aromas during fermentation. These amino acid-derived aroma compounds play a critical role in defining the taste and flavor of alcoholic beverages such as sake, beer, and wine. The productivity of amino acid-derived aroma compounds depends on the intracellular availability of their precursor amino acids. Therefore, breeding yeast strains that accumulate amino acids provides a practical approach to developing alcoholic beverages with more unique and attractive sensory characteristics. In this minireview, we describe the isolation of yeast strains that overproduce branched-chain amino acids and phenylalanine, obtained through conventional mutagenesis of industrial brewing yeasts. We also discuss the mechanisms responsible for the increased production of these amino acids in the mutant strains, including altered feedback regulation and transcriptional control of key enzymes involved in their biosynthesis. In addition, we briefly introduce a plasmid-free genome editing system that enables precise modification of metabolic pathways without the integration of foreign DNA, allowing the construction of strains that are not classified as genetically modified organisms. This method represents a promising tool that allows flexible and fine-tuned engineering of yeast metabolic pathways, including the development of strains with tailored aroma profiles.

Saccharomyces cerevisiae↗

PubMed2006

EOS1, whose deletion confers sensitivity to oxidative stress, is involved in N-glycosylation in Saccharomyces cerevisiae.

The deletion strain of Saccharomyces cerevisiae YNL080c (designed as EOS1) was identified as a strain sensitive to high-sucrose stress in our previous report [A. Ando, F. Tanaka, Y. Murata, H. Takagi, J. Shima, Identification and classification of genes required for tolerance to high sucrose stress revealed by genome-wide screening of Saccharomyces cerevisiae, FEMS Yeast Res. 6 (2006) 249-267]. Delta eos1 showed higher sensitivity to oxidative stress than to high-sucrose stress. Immunofluorescence microscopic and cellular fractionation analyses suggested that Eos1 localizes in the endoplasmic reticulum membrane. We found that the deletion of EOS1 enhances tunicamycin tolerance and that in Delta eos1 the transcription level of KAR2, which is the ER stress-inducible gene, was much lower than that in the wild-type strain (BY4741) when exposed to tunicamycin. The inhibition of the N-glycosylation of carboxypeptidase Y and invertase activity caused by the addition of tunicamycin was depressed in Delta eos1, suggesting that EOS1 may be involved in N-glycosylation of the cellular proteins.

Cell Survival↗

PubMed2006

Enhancement of stress tolerance in Saccharomyces cerevisiae by overexpression of ubiquitin ligase Rsp5 and ubiquitin-conjugating enzymes.

Yeast Saccharomyces cerevisiae cells overexpressing essential ubiquitin ligase Rsp5 or ubiquitin-conjugating enzymes (Ubc1-Ubc13) showed tolerance to various stresses. Co-overexpression of Rsp5 and Ubc1, Ubc2, Ubc3, Ubc5, Ubc6, Ubc9, Ubc10, Ubc11, Ubc12, or Ubc13 further enhanced stress tolerance. These results suggest that overexpression of ubiquitin-related enzymes might be a useful method for breeding novel stress-resistant strains.

Endosomal Sorting Complexes Required for Transport↗

PubMed2006

Metabolic pathways and biotechnological production of L-cysteine.

L-Cysteine is an important amino acid both biologically and commercially. Although most amino acids are commercially produced by fermentation, cysteine is mainly produced by protein hydrolysis. However, synthetic or biotechnological products have been preferred in the market. Biotechnological processes for cysteine production, both enzymatic and fermentative processes, are discussed. Enzymatic process, the asymmetric hydrolysis of DL-2-amino-Delta(2)-thiazoline-4-carboxylic acid to L-cysteine, has been developed and industrialized. The L-cysteine biosynthetic pathways of Escherichia coli and Corynebacterium glutamicum, which are used in many amino acid production processes, are also described. These two bacteria have basically same L-cysteine biosynthetic pathways. L-Cysteine-degrading enzymes and L-cysteine-exporting proteins both in E. coli and C. glutamicum are also described. In conclusion, for the effective fermentative production of L-cysteine directly from glucose, the combination of enhancing biosynthetic activity, weakening the degradation pathway, and exploiting the export system seems to be effective.

Corynebacterium glutamicum↗

PubMed2006

Hypo-osmotic shock induces nuclear export and proteasome-dependent decrease of UBL5.

The osmolarity of body fluid is strictly controlled through the action of diuretic hormones, which are secreted in the hypothalamus. In the mammalian brain, ubiquitin-like 5 (UBL5) is expressed in oxytocin- and vasopressin-positive neurons in the hypothalamus, and these neurons play a role in regulating osmolarity. We examined the dynamics of UBL5 levels in response to hyper- or hypo-osmotic conditions. Hypo-osmotic conditions led to significantly reduced levels of UBL5 both in brain slices from the hypothalamus and in NIH-3T3 cells. This decrease in UBL5 was transcription-independent and proteasome-dependent. Time-course immunocytochemical studies using exogenous UBL5 revealed that the protein was exported from the nucleus under hypo-osmotic conditions and decreased in a proteasome-dependent manner. This report is the first to describe changes in the intracellular and subcellular localization of UBL5 in response to hypo-osmotic conditions. Our results imply osmoregulation of UBL5.

Active Transport, Cell Nucleus↗

PubMed2006

Global gene expression analysis of yeast cells during sake brewing.

During the brewing of Japanese sake, Saccharomyces cerevisiae cells produce a high concentration of ethanol compared with other ethanol fermentation methods. We analyzed the gene expression profiles of yeast cells during sake brewing using DNA microarray analysis. This analysis revealed some characteristics of yeast gene expression during sake brewing and provided a scaffold for a molecular level understanding of the sake brewing process.

DNA, Fungal↗

PubMed2006

Identification and classification of genes required for tolerance to freeze-thaw stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains.

Yeasts used in bread making are exposed to freeze-thaw stress during frozen-dough baking. To clarify the genes required for freeze-thaw tolerance, genome-wide screening was performed using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 58 gene deletions that conferred freeze-thaw sensitivity. These genes were then classified based on their cellular function and on the localization of their products. The results showed that the genes required for freeze-thaw tolerance were frequently involved in vacuole functions and cell wall biogenesis. The highest numbers of gene products were components of vacuolar H(+)-ATPase. Next, the cross-sensitivity of the freeze-thaw-sensitive mutants to oxidative stress and to cell wall stress was studied; both of these are environmental stresses closely related to freeze-thaw stress. The results showed that defects in the functions of vacuolar H(+)-ATPase conferred sensitivity to oxidative stress and to cell wall stress. In contrast, defects in gene products involved in cell wall assembly conferred sensitivity to cell wall stress but not to oxidative stress. Our results suggest the presence of at least two different mechanisms of freeze-thaw injury: oxidative stress generated during the freeze-thaw process, and defects in cell wall assembly.

Culture Media↗

PubMed2006

Age dependence of fusion of primary occipital sutures: a radiographic study.

PURPOSE: When linear lucency is present in the occipital bone on radiographs throughout childhood, differential diagnosis becomes important because some primary sutures are similar to fractures. The authors here chronicled the normal development of ossification centers, sutures, and synchondroses in the chondrocranium by radiographic examination. METHODS: One hundred and twenty-seven children, aged from newborns to 6 years and without any skull base deformities, were referred to for radiographs of Towne's projection. RESULTS: In the occipital bone at birth, three primary sutures could be identified. At the age of 0-3 years, occipital and innominate sutures started to fuse, this being complete by 4 years, whereas mendosal sutures persisted until 6 years of age, after which no primary sutures could be seen. CONCLUSION: The complex process of skull base development features a step-wise process sutural closure for which radiographic standards allow differential diagnosis from fractures with judgment of the timing.

Aging↗

PubMed2006

Rsp5 regulates expression of stress proteins via post-translational modification of Hsf1 and Msn4 in Saccharomyces cerevisiae.

Rsp5 is an essential E3 ubiquitin ligase in Saccharomyces cerevisiae and is known to ubiquitinate plasma membrane permeases followed by endocytosis and vacuolar degradation. We previously isolated the rsp5 mutant that is hypersensitive to various stresses, suggesting that Rsp5 is involved in degradation of stress-induced abnormal proteins. Here, we analyzed the ability to refold the proteins by stress proteins in the rsp5 mutant. The transcription of stress protein genes in the rsp5 mutant was significantly lower than that in the wild-type strain when exposed to temperature up-shift, ethanol or sorbitol. Interestingly, the amounts of transcription factors Hsf1 and Msn4 were remarkably defective in the rsp5 mutant. These results suggest that expression of stress proteins are mediated by Rsp5 and that Rsp5 primarily regulates post-translational modification of Hsf1 and Msn4.

Base Sequence↗

PubMed2006

A novel enzyme conferring streptothricin resistance alters the toxicity of streptothricin D from broad-spectrum to bacteria-specific.

Streptothricins (STs) produced by Streptomyces strains are broad-spectrum antibiotics. All STs consist of a carbamoylated D-gulosamine to which the beta-lysine homopolymer (1 to 7 residues) and the amide form of the unusual amino acid streptolidine (streptolidine lactam) are attached. Although many ST-resistance genes have been identified in bacteria, including clinically isolated pathogens and ST-producing Streptomyces strains, only one resistance mechanism has been identified to date. This mechanism involves the modification of the ST molecule by monoacetylation of the moiety of the beta-lysine(s). In this study, we successfully isolated a novel ST-resistance gene (sttH) from Streptomyces albulus, which is a known ST nonproducer. The in vitro analysis of SttH demonstrated that this enzyme catalyzes the hydrolysis of the amide bond of streptolidine lactam, thereby conferring ST resistance. Interestingly, the selective toxicity of ST-D possessing 3x beta-lysine moiety was altered from broad-spectrum to bacteria-specific by the hydrolysis of streptolidine lactam, although ST-F (1 x beta-lysine) was detoxified by SttH in both prokaryotes and eukaryotes (yeasts). STs have not been clinically developed due to their toxicities; however, in this study, we showed that hydrolyzed ST-D (ST-D-acid) exhibits potent antibacterial activity even when its toxicity against eukaryotic cells is reduced by SttH. This suggests that ST-D-acid is a potential candidate for clinical development or for use as a new lead compound for drug discovery.

Bacterial Proteins↗

PubMed2006

SAD: a presynaptic kinase associated with synaptic vesicles and the active zone cytomatrix that regulates neurotransmitter release.

A serine/threonine kinase SAD-1 in C. elegans regulates synapse development. We report here the isolation and characterization of mammalian orthologs of SAD-1, named SAD-A and SAD-B, which are specifically expressed in the brain. SAD-B is associated with synaptic vesicles and, like the active zone proteins CAST and Bassoon, is tightly associated with the presynaptic cytomatrix in nerve terminals. A short conserved region (SCR) in the COOH-terminus is required for the synaptic localization of SAD-B. Overexpression of SAD-B in cultured rat hippocampal neurons significantly increases the frequency of miniature excitatory postsynaptic current but not its amplitude. Introduction of SCR into presynaptic superior cervical ganglion neurons in culture significantly inhibits evoked synaptic transmission. Moreover, SCR decreases the size of the readily releasable pool measured by applying hypertonic sucrose. Furthermore, SAD-B phosphorylates the active zone protein RIM1 but not Munc13-1. These results suggest that mammalian SAD kinase presynaptically regulates neurotransmitter release.

Animals↗

PubMed2006

Functional genomic analysis of commercial baker's yeast during initial stages of model dough-fermentation.

Gene expression profiles of baker's yeast during initial dough-fermentation were investigated using liquid fermentation (LF) media to obtain insights at the molecular level into rapid adaptation mechanisms of baker's yeast. Results showed that onset of fermentation caused drastic changes in gene expression profiles within 15 min. Genes involved in the tricarboxylic acid (TCA) cycle were down-regulated and genes involved in glycolysis were up-regulated, indicating a metabolic shift from respiration to fermentation. Genes involved in ethanol production (PDC genes and ADH1), in glycerol synthesis (GPD1 and HOR2), and in low-affinity hexose transporters (HXT1 and HXT3) were up-regulated at the beginning of model dough-fermentation. Among genes up-regulated at 15 min, several genes classified as transcription were down-regulated within 30 min. These down-regulated genes are involved in messenger RNA splicing and ribosomal protein biogenesis and in transcriptional regulator (SRB8, MIG1). In contrast, genes involved in amino acid metabolism and in vitamin metabolism, such as arginine biosynthesis, riboflavin biosynthesis, and thiamin biosynthesis, were subsequently up-regulated after 30 min. Interestingly, the genes involved in the unfolded protein response (UPR) pathway were also subsequently up-regulated. Our study presents the first overall description of the transcriptional response of baker's yeast during dough-fermentation, and will thus help clarify genomic responses to various stresses during commercial fermentation processes.

Bread↗

PubMed2006

In vivo imaging of the dendritic arbors of layer V pyramidal cells in the cerebral cortex using a laser scanning microscope with a stick-type objective lens.

In the field of neuroscience, low-invasive in vivo imaging would be a very useful method of monitoring the morphological dynamics of intact neurons in living animals. At present, there are two widely used in vivo imaging methods; one is the two-photon microscope method, and the other is the fiber optics method. However, these methods are not suitable for the in vivo imaging of deeper subcortical structures. In our study, we have developed a novel method for the in vivo imaging of pyramidal neurons in layer V of the cerebral cortex, utilizing a MicroLSM system and a stick-type objective lens that can be directly inserted into the target tissue. By using this method, we succeeded in obtaining clear images of pyramidal neurons in layer V of the cerebral cortex under a low-invasive condition. The MicroLSM system is a useful and versatile in vivo imaging system that will be applicable not only to the brain but also to other organs.

Animals↗

PubMed2006

Identification and classification of genes required for tolerance to high-sucrose stress revealed by genome-wide screening of Saccharomyces cerevisiae.

Yeasts used in bread making are exposed to high concentrations of sucrose during sweet dough fermentation. Despite its importance, tolerance to high-sucrose stress is poorly understood at the gene level. To clarify the genes required for tolerance to high-sucrose stress, genome-wide screening was undertaken using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 273 deletions that yielded high sucrose sensitivity, approximately 20 of which were previously uncharacterized. These 273 deleted genes were classified based on their cellular function and localization of their gene products. Cross-sensitivity of the high-sucrose-sensitive mutants to high concentrations of NaCl and sorbitol was studied. Among the 273 sucrose-sensitive deletion mutants, 269 showed cross-sensitivities to sorbitol or NaCl, and four (i.e. ade5,7, ade6, ade8, and pde2) were specifically sensitive to high sucrose. The general stress response pathways via high-osmolarity glycerol and stress response element pathways and the function of the invertase in the ade mutants were similar to those in the wild-type strain. In the presence of high-sucrose stress, intracellular contents of ATP in ade mutants were at least twofold lower than that of the wild-type cells, suggesting that depletion of ATP is a factor in sensitivity to high-sucrose stress. The genes identified in this study might be important for tolerance to high-sucrose stress, and therefore should be target genes in future research into molecular modification for breeding of yeast tolerant to high-sucrose stress.

Adaptation, Physiological↗

PubMed2006

Functional analysis of L-serine O-acetyltransferase from Corynebacterium glutamicum.

We report here the function of L-serine O-acetyltransferase (SAT) from the glutamic acid-producing bacterium Corynebacterium glutamicum. Based on the genome sequence of C. glutamicum and the NH(2)-terminal amino-acid sequence, the gene encoding SAT (cysE) was cloned and expressed in C. glutamicum. Deletion analysis of the 5'-noncoding region showed a putative -10 region ((-27)TTAAGT(-22) or (-26)TAAGTC(-21)) and a possible ribosome-binding site ((-12)AGA(-10)) just upstream from the start codon. We found that the SAT activity was sensitive to feedback inhibition by L-cysteine, and that SAT synthesis was repressed by L-methionine. Further, cysE-disrupted cells showed L-cysteine auxotrophy, indicating that C. glutamicum synthesizes L-cysteine from L-serine via O-acetyl-L-serine through the pathway involving SAT and O-acetyl-L-serine sulfhydrylase in the same manner as Escherichia coli.

Cloning, Molecular↗

PubMed2006

Effect of drug transporter genes on cysteine export and overproduction in Escherichia coli.

L-cysteine is an important amino acid in terms of its industrial applications. We previously found a marked production of L-cysteine from glucose in recombinant Escherichia coli cells expressing an altered cysE gene encoding feedback inhibition-insensitive serine acetyltransferase. Also, a lower level of cysteine desulfhydrase (CD) activity, which is involved in L-cysteine degradation, increased L-cysteine productivity in E. coli. The use of an L-cysteine efflux system could be promising for breeding L-cysteine overproducers. In addition to YdeD and YfiK, which have been reported previously as L-cysteine exporter proteins in E. coli, we analyzed the effects of 33 putative drug transporter genes in E. coli on L-cysteine export and overproduction. Overexpression of the acrD, acrEF, bcr, cusA, emrAB, emrKY, ybjYZ, and yojIH genes reversed the growth inhibition of tnaA (the major CD gene)-disrupted E. coli cells by L-cysteine. We also found that overexpression of these eight genes reduces intracellular L-cysteine levels after cultivation in the presence of L-cysteine. Amino acid transport assays showed that Bcr overexpression conferring bicyclomycin and tetracycline resistance specifically promotes L-cysteine export driven by energy derived from the proton gradient. When a tnaA-disrupted E. coli strain expressing the altered cysE gene was transformed with a plasmid carrying the bcr gene, the transformant exhibited more L-cysteine production than cells carrying the vector only. A reporter gene assay suggested that the bcr gene is constitutively expressed at a substantial level. These results indicate that the multidrug transporter Bcr in the major facilitator family is involved in L-cysteine export and overproduction in genetically engineered E. coli cells.

Anti-Bacterial Agents↗

PubMed2006

Yeast cell death caused by mutation of the OST2 gene encoding the epsilon-subunit of Saccharomyces cerevisiae oligosaccharyltransferase.

An essential epsilon-subunit of oligosaccharyltransferase Ost2 is a yeast homolog of mammalian highly conserved DAD1 (defender against apoptotic death). In hamster cells, the Gly38Arg mutation in DAD1 causes apoptosis at restrictive temperatures due to a defect in N-linked glycosylation. To analyze the function of Ost2 in yeast cell death, we constructed Saccharomyces cerevisiae strains expressing Gly58Arg (corresponding to the Gly38Arg mutation in hamster DAD1), Gly86Arg, and Glu113Val mutant Ost2. At elevated temperatures, ost2 mutants arrested growth by decreasing cell viability. Phosphatidylserine exposure, a phenotypic marker of apoptosis in mammalian cells, was found in ost2 mutant cells at 37 degrees C, although DNA fragmentation was not clearly detected. A high concentration of sorbitol compensates for the temperature sensitivity of the ost2 mutant. These results suggest that apoptosis-like cell death in ost2 mutants is caused by the secondary effect of overall reduced protein N-linked glycosylation.

Amino Acid Sequence↗