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Presents a review of the roles of chloroplast RNA polymerase sigma factors in chloroplast development and stress response in higher plants. Two types of polymerases that performed chloroplast transcription; Assertion that PEP is a eubacteria-type multisubunit enzyme whose catalytic core subunits are encoded by genplast genome, whereas NEP is the nuclear encoded T7 phage-type single subunit enzyme; importance of PEP for the biogenesis and maintenance of chloroplasts and finely tuned by the nuclear encoded sigma units; Involvement of SIG2 in the transcription of several chloroplasts tRNA genes; Factors that make target genes and the physiological role of each sigma subunit clearer.

A eubacteria-type RNA polymerase (PEP) plays crucial roles for chloroplast development in higher plants. The core subunits are encoded on plastid DNA (rpo genes) while the regulatory sigma factors are encoded on the nuclear DNA (SIG genes). However, the definite gene specificity of each sigma factor is unknown. We recently identified an Arabidopsis recessive pale-green mutant abc1 in which T-DNA is inserted in SIG2 (sigB). In this mutant, almost normal etioplasts were developed under dark conditions while the small chloroplasts with poor thylakoid membranes and stacked lamellar were developed under light conditions. The sig2-1 mutant was deficient in accumulating enough photosynthetic and photosynthesis-related proteins as well as chlorophyll. However, mRNAs of their structural genes were not significantly reduced. Further analyses revealed that several plastid-encoded tRNAs including trnE-UUC that has dual function for protein and ALA biosyntheses were drastically reduced in the sig2-1 mutant. In contrast, nucleus-encoded T7 phage-type RNA polymerase (NEP)-dependent gene transcripts were steadily accumulated in the mutant. These results indicate that progress of chloroplast development requires SIG2-dependent expression of plastid genes, particularly some of the tRNA genes. [ABSTRACT FROM AUTHOR]

The quorum-sensing system in bacteria is a well-known regulatory system that controls gene expression in a cell density-dependent manner. A transcriptional regulator (LuxR homologue), signal synthase (LuxI homologue) and autoinducer (acyl homoserine lactone) are indispensable for this system in most Gram-negative bacteria. In this study, we found that SdiA, an Escherichia coli LuxR homologue, is a negative regulator of the expression of virulence factors EspD and intimin in enterohaemorrhagic E. coli (EHEC) O157:H7. The expression of EspD and intimin was inhibited at the RNA level upon SdiA overexpression. SdiA has a DNA-binding motif in its C-terminal part and can bind to the promoter regions of the esp and eae genes in vitro. Extracellular factors, which accumulate in culture supernatants of O157:H7 at the stationary phase of growth and inhibit EspD and intimin synthesis, bind to the N-terminal part of SdiA in vivo and in vitro. O157:H7 overproducing the N-terminal part of SdiA exhibited hypertranscription of EspD and intimin, suggesting that the overproduced N-terminal part had inhibited the activity of intact SdiA through titration of the extracellular factors. These results indicate that a quorum-sensing system including the SdiA protein controls colonization by O157:H7. [ABSTRACT FROM AUTHOR]

In Arabidopsis thaliana, a mature mesophyll cell contains approximately 100 chloroplasts. Although 12 arc mutants (accumulation and replication of chloroplasts) and two chloroplast division genes homologous to eubacterial ftsZ have been isolated from A. thaliana, the molecular mechanism underlying the chloroplast division is still unclear. We characterized AtMinD1, a eubacterial minD homolog, for chloroplast division in A. thaliana. AtMinD1-green fluorescent protein targeted to the chloroplasts and possibly associated with the envelope membranes in vivo. During the seed germination, the AtMinD1 transcripts were accumulated twice, just after release from cold treatment and at the beginning of rapid greening, in similar fashion to AtFtsZs. Furthermore the transcript level in a severest chloroplast division mutant, arc6, was 3–5-fold higher than that in wild-type. [ABSTRACT FROM PUBLISHER]

In plant cells, plastid DNA is transcribed by at least two types of RNA polymerase, plastid-encoded RNA polymerase (PEP) and nuclear-encoded RNA polymerase (NEP). PEP is homologous to eubacterial transcription machinery, but its regulatory subunit, sigma (σ) factor, is not encoded on the plastid DNA. We previously cloned the three nuclear-encoded sigma factor genes from Arabidopsis thaliana and designated them as sigA, sigB, and sigC. By means of RFLP mapping, sigA and sigB were mapped on chromosome I and sigC on the chromosome III. Based on comparison of the genomic structure of the three sig genes, intron sites in the 3′ half of the genes were shown to be identical between sigB and sigC but divergent in sigA, consistent with the phylogenetic relevance of the three gene products. A transient expression assay of GFP fusions in Arabidopsis protoplasts showed that the N-termini of all three sig gene products functioned as chloroplast-targeting signals. We also constructed transgenic Arabidopsis lines harboring the sigA-promoter or the sigB-promoter uidA fusion. Both the sigA- and sigB-promoters were similarly activated at cotyledons, hypocotyls, rosette leaves, cauline leaves, sepals, and siliques but not at roots, seeds, or other flower organs. In addition, the two promoters were repeatedly activated in young seedlings under continuous light, possibly in an oscillated fashion. [ABSTRACT FROM AUTHOR]

Presents a study on the CspA family in Escherichia coli, which are cold shock proteins. Temperature of E. coli which alters changes in growing cells; Names of genes which are cold shock inducible; Names of genes found in the CspA family of proteins; Results of study.

CYP2C19 is a member of the human microsomal cytochrome P450 (CYP). Significant variation in CYP2C19 levels and activity can be attributed to polymorphisms in this gene. Wildtype CYP2C19 and 13 mutants (CYP2C19.1B, CYP2C19.5A, CYP2C19.5B, CYP2C19.6, CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.11, CYP2C19.13, CYP2C19.16, CYP2C19.19, CYP2C19.23, CYP2C19.30, and CYP2C19.33) were coexpressed with NADPH‐cytochrome P450 reductase in Escherichia coli. Hydroxylase activity toward testosterone and progesterone was also examined. Ten CYP2C19 variants showed Soret peaks (450 nm) typical of P450 in the reduced CO‐difference spectra. CYP2C19.11 and CYP2C19.23 showed higher testosterone 11α, 16α‐/17‐ and progesterone 6β‐,21‐,16α‐/17α‐hydroxylase activities than CYP2C19.1B. CYP2C19.6, CYP2C19.16, CYP2C19.19, and CYP2C19.30 showed lower activity than CYP2C19.1B. CYP2C19.9, CYP2C19.10. CYP2C19.13, and CYP2C19.33 showed different hydroxylation activities than CYP2C19.1B. These results indicated that CYP2C19 variants have very different substrate specificities for testosterone and progesterone. [ABSTRACT FROM AUTHOR]

CYP2C9 is a human microsomal cytochrome P450c (CYP). Much of the variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild-type CYP2C9 and mutants were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. The hydroxylase activities toward 7-ethoxycoumarin, flavanone and steroids were examined. Six CYP2C9 variants showed Soret peaks (450 nm) typical of P450 in reduced CO-difference spectra. CYP2C9.38 had the highest 7-ethoxycoumarin de-ethylase activity. All the CYP2C9 variants showed lower flavanone 6-hydroxylation activities than CYP2C9.1 (the wild-type). CYP2C9.38 showed higher activities in testosterone 6β-hydroxylation, progesterone 6β−/16α-hydroxylation, estrone 11α-hydroxylation and estradiol 6α-hydroxylation than CYP2C9.1. CYP2C9.40 showed higher testosterone 17-oxidase activity than CYP2C9.1; CYP2C9.8 showed higher estrone 16α-hydroxylase activity and CYP2C9.12 showed higher estrone 11α-hydroxylase activity. CYP2C9.9 and CYP2C9.10 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.9 and CYP2C9.10 was not changed, but CYP2C9.8, CYP2C9.12 and CYP2C9.40 showed different substrate specificity toward steroids compared with CYP2C9.1; and especially CYP2C9.38 displayed diverse substrate specificities towards 7-ethoxycoumarin and steroids. [ABSTRACT FROM AUTHOR]

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) make up the core machinery that mediates membrane fusion. SNAREs, syntaxin, synaptosome-associated protein (SNAP), and synaptobrevin form a tight SNARE complex that brings the vesicle and plasma membranes together and is essential for membrane fusion. The cDNAs of SNAP-25, VAMP2, and Syntaxin 1A from Bombyx mori were inserted into a plasmid, transformed into Escherichia coli, and purified. We then produced antibodies against the SNAP-25, VAMP2, and Syntaxin 1A of Bombyx mori of rabbits and rats, which were used for immunohistochemistry. Immunohistochemistry results revealed that the expression of VAMP2 was restricted to neurons in the pars intercerebralis (PI), dorsolateral protocerebrum (DL), and central complex (CX) of the brain. SNAP-25 was restricted to neurons in the PI and the CX of the brain. Syntaxin 1A was restricted to neurons in the PI and DL of the brain. VAMP2 co-localized with SNAP-25 in the CX, and with Syntaxin 1A in the PI and DL. VAMP2, SNAP-25, and Syntaxin 1A are present in the CA. Bombyxin-immunohistochemical reactivities (IRs) of brain and CA overlapped with VAMP2-, SNAP-25, and Syntaxin 1A-IRs. VAMP2 and Syntaxin 1A are present in the prothoracicotropic hormone (PTTH)-secretory neurons of the brain. [ABSTRACT FROM AUTHOR]

In the tetrapyrrole biosynthetic pathway of higher plants, 5-aminolevulinic acid (ALA) is metabolized by ALA dehydratase (ALAD). Here, we isolated ALAD1 cDNA from common wheat ( Triticum aestivum L.) and its diploid progenitors, and produced transgenic tobacco plants expressing the wheat ALAD1 gene. The ALAD1 genes were highly conserved among wheat relatives, and three homoeologous loci of wheat ALAD1 ( TaALAD1) were equally transcribed in common wheat. A transient expression assay of a TaALAD1-GFP (green fluorescent protein) fusion protein suggested that TaALAD1 is localized in chloroplasts. Overexpression of TaALAD1 in transgenic tobacco resulted in a significant increase in ALAD activity in leaves. Moreover, the transgenic tobacco showed vigorous growth and increased survival rate on medium containing ALA at herbicidal concentrations. These results indicate that wheat ALAD1 has catalytic activity in metabolizing ALA in plastids, and that ectopic expression of TaALAD1 in transgenic plants increases their tolerance to ALA application at high concentrations. [ABSTRACT FROM AUTHOR]

Rab proteins are low-molecular weight (20-25 kDa) monomeric GTPases that are central to the control and regulation of vesicle trafficking. RabX6 is an insect-specific Rab protein that has no close homolog in vertebrates. However, little information about insect-specific Rab proteins is available. In this study, RabX6 was expressed in Escherichia coli and subsequently purified. Antibodies against Bombyx mori RabX6 were produced in rabbits and rats for western immunoblotting and immunohistochemistry. Western blotting of testis tissues revealed two bands, at positions corresponding to a molecular weight of approximately 26 kDa. RabX6-like immunohistochemical reactivity (RabX6-ir) was identified at the face of the testis, not in the spermatogonia, and was specifically detected at a pair of tritocerebral cells of the male brain. Furthermore, RNA interference of RabX6 was shown to decrease testicular growth. These findings suggest that RabX6 is involved in the regulation of testicular growth and male-specific neuropeptide secretion in the brain of B. mori. [ABSTRACT FROM AUTHOR]

Abstract: CYP2C9 is a human microsomal cytochrome P450c (CYP). Much variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild‐type CYP2C9 and ten mutants were co‐expressed with NADPH‐cytochrome P450 reductase in Escherichia coli. The hydroxylase activities toward steroids were examined. CYP2C9.2, CYP2C9.3, CYP2C9.4, CYP2C9.16, CYP2C9.28, CYP2C9.48 and CYP2C9.52 had higher testosterone 6β‐hydroxylation than CYP2C9.1. CYP2C9.4 showed higher progesterone 6β‐hydroxylation activity than CYP2C9.1. CYP2C9.28 and CYP2C9.48 showed higher progesterone 11α‐hydroxylation activity than CYP2C9.1. CYP2C9.48 showed higher progesterone 16α‐hydroxylation activity than CYP2C9.1. CYP2C9.2, CYP2C9.3, CYP2C9.16 and CYP2C9.30 had higher estrone 16α‐hydroxylation activity than CYP2C9.1. CYP2C9.3 had higher estrone 11α‐hydroxylation activity than CYP2C9.1. CYP2C9.39 and CYP2C9.57 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.39 and CYP2C9.57 was not changed, but CYP2C9.2, CYP2C9.3, CYP2C9.4, CYP2C9.16, CYP2C9.28, CYP2C9.30, CYP2C9.48 and CYP2C9.52 showed different hydroxylation activities toward steroids compared with CYP2C9.1. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, Rab guanosine triphosphate-ases serve as key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab3, Rab6, and Rab27 control the regulatory secretory pathway of neuropeptides and neurotransmitters. The cDNAs of Rab3, Rab6, and Rab27 from B. mori were inserted into a plasmid, transformed into Escherichia coli, and then subsequently purified. We then produced antibodies against Rab3, Rab6, and Rab27 of Bombyx mori in rabbits and rats for use in western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue revealed a single band at approximately 26 kDa. Immunohistochemistry results revealed that Rab3, Rab6, and Rab27 expression was restricted to neurons in the pars intercerebralis and dorsolateral protocerebrum of the brain. Rab3 and Rab6 co-localized with bombyxin, an insect neuropeptide. However, there was no Rab that co-localized with prothoracicotropic hormone. The corpus allatum secretes neuropeptides synthesized in the brain into the hemolymph. Results showed that Rab3 and Rab6 co-localized with bombyxin in the corpus allatum. These findings suggest that Rab3 and Rab6 are involved in neurosecretion in B. mori. This study is the first to report a possible relationship between Rab and neurosecretion in the insect corpus allatum. [ABSTRACT FROM AUTHOR]

Rice straw is a promising resource for bioethanol production. Because the glucose content of pretreatment liquid hydrolysates is highly correlated with ethanol yield, the selection of appropriate rice cultivars is essential. The glucose content in liquid hydrolysates of pretreated rice straws of 208 diverse cultivars was evaluated in natural field in 2013 and 2014 using a novel high-throughput system. The glucose content of the rice straw samples varied across cultivars and was affected by environmental factors such as temperature and solar radiation. Several high-quality cultivars exhibiting high glucose content in both years were identified. The results of this study can aid in development of novel rice cultivars suitable as both feedstocks for bioethanol production and cooking. The rice straw glucose content after pretreatment varied by cultivar. Thus selecting suitable cultivars is crucial for efficient ethanol production. [ABSTRACT FROM AUTHOR]

CYP2A6 is a major hepatic member of the cytochrome P450 family in humans. Much variation in CYP2A6 levels and activity can be attributed to genetic polymorphisms of this gene. CYP2A6*25 comprises an amino acid substitution, F118L. To clarify the effect of the leucine substitution at position 118 in CYP2A6.25, this variant, wild type CYP2A6 and three additional variants consisting of artificial mutations at the substrate binding site (position 481) suggested by earlier reports using random mutagenesis studies [CYP2A6.1, CYP2A6.25, CYP2A6.1(F118A), CYP2A6.1 (A481G) and CYP2A6.25(A481G)], were co-expressed with NADPH-cytochrome P450 reductase in E. coli. The hydroxylase activity of these variants toward 7-ethoxycoumarin, coumarin, flavone, α-naphthoflavone, flavanone and hydroxyflavanone were examined. All the mutants had lower activities for coumarin 7-hydroxylation than the wild type. All the mutants showed higher activities for flavone and α-naphthoflavone compared with CYP2A6.1. CYP2A6.1 had the highest flavanone 2'-hydroxylase activity, whereas CYP2A6.25 had the highest 6- and 4'-hydroxylase activities. CYP2A6.1(F118A), CYP2A6.1(A481G) and CYP2A6.25(A481G) had higher flavanone 3'-hydroxylase activities than CYP2A6.1 and CYP2A6.25. Furthermore, 4'-hydroxyflavanone was metabolized by CYP2A6.25. These results indicate that the CYP2A6.25 mutation confers new substrate specificity towards flavonoids. [ABSTRACT FROM AUTHOR]

We evaluated the metabolism of several herbicides and progesterone by two P450 proteins (CYP1A9 and CYP1C1) from Japanese eel ( Anguilla japonica ). Expression vectors harboring CYP1A9 and CYP1C1 sequences were introduced into Escherichia coli . E. coli membrane fractions were incubated with each substrate, and the metabolites were analyzed. CYP1A9 and CYP1C1 deethylated 7-ethoxycoumarin and phenacetin, and demethylated chlorotoluron, diuron, and linuron. CYP1C1 specifically hydroxlyated progesterone at the 6β and 16α positions. Five amino acids of CYP1A9 related to substrate binding were selected for mutation analyses [CYP1A9(F128A), CYP1A9(F229A), CYP1A9(F263A), CYP1A9(V387A), and CYP1A9(I391A)]. Two variants, CYP1A9(F229A) and CYP1A9(F128A), changed the ratio of 16α hydroxyprogesterone to 6β hydroxyprogesterone. Among all the variants, CYP1A9(F263A) showed the highest activity towards substrates used. CYP1A9(V387A) and CYP1A9(I391A) showed higher activities than that of CYP1A9 toward progesterone. The substrate specificity of CYP1A9 may be altered by replacing an amino acid related to substrate binding. [ABSTRACT FROM AUTHOR]

Rab guanosine triphosphatases in eukaryotic cells are key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab7 regulates traffic from early to late endosomes and from late endosomes to vacuoles/lysosomes. The Rab7-interacting lysosomal protein (RILP) was extracted from the silkworm, Bombyx mori ( B. mori), and expressed in Escherichia coli ( E. coli), followed by its purification. The glutathione sulfotransferase pull-down assay revealed that Rab7 of B. mori interacted with RILP of B. mori. We then produced antibodies against RILP of B. mori in rabbits for their use in Western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue for RILP revealed a single band, at approximately 50 kD. RILP-like immunohistochemical reactivity (RILP-ir) was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Furthermore, RILP-ir was colocalized with the eclosion hormone-ir and bombyxin-ir. However, RILP-ir was not colocalized with prothoracicotropic hormone-ir. These results were similar to those of Rab7 from our previous study. These findings suggest that RILP and Rab7 are involved in the neurosecretion in a restricted subtype of neurons in B. mori. Thus, our study is the first to report of a possible relationship between an insect Rab effector and neurosecretion. [ABSTRACT FROM AUTHOR]

Abstract: The cyclic nucleotides cGMP and cAMP have been reported to play key roles in the regulation of plant processes and responses. We have previously reported that several genes encoding flavonoid biosynthetic enzymes, including chalcone synthase (CHS) in soybean (Glycine max L.), were induced by cGMP but not cAMP. The soybean genome contains nine CHS gene copies (GmCHS1–9). We investigated the responsiveness of several GmCHS genes to cGMP, cAMP, NO, and white light. Quantitative RT-PCR analysis showed that the transcript levels of GmCHS7 and GmCHS8 were increased by 3.6- and 3.8-fold, respectively, with cGMP whereas the transcript levels of GmCHS2 remained constant. Although cAMP had no effect on the transcript levels of the three genes, NO had an activation effect on all three. White light activated the three genes in a transient manner, with GmCHS2, GmCHS7, and GmCHS8 transcript levels increasing 3-fold after 3 h and decreasing to basal levels after 9 h. The GmCHS8 promoter contains several important cis-elements, including the G-box and H-box forming the Unit-I-like sequence and the MYB binding sequence, a target of the GmMYB176 transcription factor regulating the expression of GmCHS8. A transient gene expression assay revealed the activation of the Unit-I-like sequence, but not of the MYB binding sequence, by cGMP. The combination of G-box and H-box was necessary for cGMP responsiveness. Taken together, these results suggest that the Unit-I-like sequence in the promoters of GmCHS7 and GmCHS8 is a cGMP responsive cis-element in these genes and that NO exerts its effect via cis-elements other than the Unit-I-like sequence. [Copyright &y& Elsevier]

ABSTRACT CYP 2A6 is a human enzyme that metabolizes many xenobiotics including coumarin, indole, nicotine and carcinogenic nitrosamines. The gene for CYP2A6 is polymorphic. There are few data available to clarify the relationship between P450 genetic variants and the metabolism of materials in food. The CYP 2A6 wild-type protein and 13 mutants (CYP2A6.1, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.7, CYP2A6.8, CYP2A6.11, CYP2A6.15, CYP2A6.16, CYP2A6.17, CYP2A6.18, CYP2A6.21, CYP2A6.23 and CYP2A6.25) were co-expressed with NADPH-cytochrome P450 reductase in E. coli. The hydroxylase activities toward 7-ethoxycoumarin, coumarin, safrole, flavanone and hydroxyflavanone were examined. Ten types of CYP2A6 variants except for CYP2A6.2, CYP2A6.5 and CYP2A6.6 showed Soret peaks (450 nm) typical of P450 in the reduced CO-difference spectra and had 7-ethoxycoumarin O-deethylase activities. CYP2A6.15 and CYP2A6.18 showed higher activities for safrole 1′-hydroxylation than CYP2A6.1. CYP2A6.25 and CYP2A6.7 had lower safrole 1′-hydroxylase activities. CYP2A6.7 had lower flavanone 6- and 2′-hydroxylase activities, whereas CYP2A6.25 had higher 6-hydroxylase activity and lower 2′-hydroxylase activity. Hydroxyflavanone was metabolized by CYP2A6.25, but was not metabolized by wild-type CYP2A6.1. These results indicate that CYP2A6.25 possessed new substrate specificity toward flavonoids. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Rab proteins are small GTPases that play essential roles in vesicle transport. In this study, we examined the expression of Rab proteins and neuropeptide hormones in the brain of the silkworm, Bombyx mori. We produced antibodies against B. mori Rab1 and Rab14 in rabbits. Immunoblotting of samples of brain tissue from B. mori revealed a single band for each antibody. Rab1 and Rab14 immunohistochemical labeling in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Rab1, Rab7 and Rab14 co-localized with bombyxin. Rab1 and Rab7 co-localized with eclosion hormone. Rab1 co-localized with prothoracicotropic hormone. These results suggest that Rab1, Rab7 and Rab14 may be involved in neuropeptide transport in the brain of B. mori. This is the first report on the specificity of Rab proteins for the secretion of different neuropeptides in insects. [ABSTRACT FROM AUTHOR]

Abstract: Through the use of a number of bioconversion experiments we demonstrated that P450 proteins (CYP1A9 and CYP1C1) from Japanese eel (Anguilla japonica) metabolized a number of herbicides and the drug phenacetin. We performed bioconversion experiments in which substrates were added directly to incubation medium. The resulting metabolites were extracted and analyzed by high-performance liquid chromatography. Proteins CYP1A9 and CYP1C1 metabolized 50nmol of the drug phenacetin to yield 12.1 and 1.1nmol of product (acetaminophen), respectively. Further incubation of CYP1A9 with 50nmol of the herbicides chlorotoluron, diuron, linuron, simazine, or atrazine yielded 16.5, 18.5, 7.3, 1.6, or 0.8nmol of product, respectively. CYP1C1 also metabolized linuron, diuron, and simazine yield 5.4, 4.6, or 0.7nmol of product, respectively. Next, polyclonal antibody was isolated by immunizing with two conjugated-peptides (amino acid residues 272–290 and 294–310) of CYP1A9. This antibody did not recognize human CYP1A2 or CYP1C1. Western blotting using the antibody revealed one band in the livers of Japanese eel and tilapia (Oreochromis niloticus). Theses results suggest that CYP1A9 and CYP1C1 metabolize herbicides, and that CYP1A9 is an useful biomarker of contamination when detected with this antibody. [Copyright &y& Elsevier]

Small GTPases of the Rab family are key regulators of membrane trafficking. We produced antibodies against the Rab7 protein of Bombyx mori (BRab7) in rabbits, and against the Rab11 protein of B. mori (BRab11) in mice. The antibodies recognized BRab7 and BRab11 proteins, but did not recognize other Rab proteins. Immunoblotting of samples from brain tissue of B. mori revealed a single band for each antibody. Rab11 was expressed in most tissues, whereas Rab7 was expressed in the brain, ovary, and testis. Immunohistochemical reactivity of Rab7 and Rab11 in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Double-labeling experiments demonstrated that immunohistochemical reactivity of Rab7 co-localized with that of Rab11 and partially with that of Rab8. Immunohistochemical reactivity of Rab11 and Rab8 co-localized with that of PERIOD, one of the proteins associated with circadian rhythm. These findings suggest that Rab7, Rab8, and Rab11 are involved in protein transport in the neurons of the brain of B. mori and might play a role in the control of circadian rhythm. [ABSTRACT FROM AUTHOR]

Cucumisin is a subtilisin-like serine protease (subtilase) that is found in the juice of melon fruits (Cucumis melo L.). It is synthesized as a preproprotein consisting of a signal peptide, NH2-terminal propeptide, and 67-kDa protease domain. We investigated the role of this propeptide (88 residues) in the cucumisin precursor. Complementary DNAs encoding the propeptides of cucumisin, two other plant subtilases (Arabidopsis ARA12 and rice RSP1), and bacterial subtilisin E were expressed in Escherichia coli independently of their mature enzymes. The cucumisin propeptide strongly inhibited cucumisin in a competitive manner with a Ki value of 6.2 ± 0.55 nM. Interestingly, cucumisin was also strongly inhibited by ARA12 and RSP1 propeptides but not by the subtilisin E propeptide. In contrast, the propeptides of cucumisin, ARA12, and RSP1 did not inhibit subtilisin. Deletion analysis clearly showed that two hydrophobic regions, Asn32-Met38 and Gly97-Leu103, in the cucumisin propeptide were important for its inhibitory activity. Site-directed mutagenesis also confirmed the role of a Val36-centerd hydrophobic cluster within the Asn32-Met38 region in cucumisin inhibition. Circular dichroism spectroscopy revealed that the cucumisin propeptide had a secondary structure without a cognate protease domain and that the thermal unfolding of the propeptide at 90 °C was only partial and reversible. A tripeptide, Ile35-Val36-Tyr37, in the Asn32-Met38 region was thought to contribute toward the formation of a proper secondary structure necessary for cucumisin inhibition. This is the first report on the function and structural information of the propeptide of a plant serine protease. [ABSTRACT FROM AUTHOR]

Small GTPases of the Rab family act as essential regulators of vesicle transport pathways. Five Rab cDNA clones (BRab1, 7, 8, 11 and 14) from Bombyx mori were expressed in Escherichia coli as a thioredxin or glutathione sulfotransferase fusion protein. After purification, the fusion protein was tested for phosphorylation using protein kinase C (PKC). Results indicate that all of them were phosphorylated in vitro. The phosphorylation site of BRab1 was determined by mass-spectrometric analysis, which identified that Ser-17 of BRab1 was phosphorylated by PKC. Deletion and site-directed mutagenesis indicated that Ser-111of BRab8, in addition to Ser-17, was newly phosphorylated. Further immunohistochemical analysis using antibodies against Rab8 indicated that there are some Rab8 immunoreactive cells close to the neuropeptide secreting cells. This result suggests that in insects Rab proteins are regulated by phosphorylation and at least some of them are involved in neuropeptide secretion. [ABSTRACT FROM AUTHOR]

The article presents a study that examines the localization and functionality of Arabidopsis thaliana MinD (AtMinD1) fused with a dual hemagglutinin epitope (dHA) or a yellow fluorescent protein (YFP). It used the wildtype Arabidopsis thaliana to provide a basis of MinD function and localization in chloroplast division. Results say that AtMinD1-dHA successfully complements the arc11/atminD1 mutant phenotype and is located at the envelope membrane and the mid-chloroplast constriction site.

Abstract: We indicated that two P450s (1A9 and 1C1) from Japanese eel (Anguilla japonica) metabolized 7-ethoxycoumarin, 7-ethoxyresorufin, and flavanone. At first, we constructed expression vectors for two types of P450 (1A9 and 1C1). The reduced CO-difference spectra of Escherichia coli cells transformed with these plasmids showed Soret peaks (450 nm) that were typical of P450s. We performed bioconversion experiments in which substrates were added directly to incubation medium. The resulting metabolite(s) were extracted and analyzed by high-performance liquid chromatography and spectrofluorometer. Incubation of 50 nmol 7-ethoxyresorufin with P450 1C1 yielded 0.773 nmol of deethylated product, whereas 50 nmol 7-ethoxycoumarin resulted in 4.76 nmol. P450 1A9 metabolized 50 nmol of 7-ethoxyresorufin and 7-ethoxycoumarin to yield 6.54 and 20.9 nmol of deethylated product, respectively. Incubation of 50 nmol flavanone with P450 1C1 yielded 1.46 nmol and 0.69 nmol of products, whereas 50 nmol flavanone with P450 1A9 resulted in 1.10 nmol. In this system, 4′-hydroxy flavanones were formed by P450 1A9 and P450 1C1. P450 1A9 also metabolized 50 nmol of 17 beta-estradiol to yield 4.25 nmol of product. In this system, 2-hydroxy estradiol was formed by P450 1A9 using 17 beta-estradiol as a substrate. This study is the first to identify the substrates that P450 1C1 and 1A9 metabolize. [Copyright &y& Elsevier]

Small GTPases of the Rab family are key regulators of membrane trafficking. Monoclonal antibodies are useful tools for identifying proteins that interact with other proteins and for examining their tissue distribution. We selected a monoclonal antibody against Rab8 of Bombyx mori L. It specifically recognized amino acid residues 30-109, which are conserved among Rab8 proteins, and did not recognize any other Rab proteins. Western blotting using the antibody revealed one band in the brains of B. mori and rat. Far-Western blotting analysis detected three proteins interacting with Rab8. These results indicate that this antibody is useful for clarifying the physiological function of Rab8 of B. mori and other species. This is a report of a study on a monoclonal antibody against insect Rab protein. [ABSTRACT FROM AUTHOR]

We developed a system for bioconverting diverse compounds using P450s produced in Escherichia coli. Vectors for the expressing various P450 cDNAs quickly and easily in E. coli were developed by using several restriction enzyme sites. Three types of P450 (2C2, 2C29, and 2D22) were produced using these plasmids. Substrates were directly added to the incubation medium and metabolized. To obtain pure product from the medium, we first tried production of P450 in synthetic medium. The amount of another P450 2C43 produced in the synthetic medium was similar to the amount produced in Luria broth (LB) medium. Next, estradiol, a steroid, was added as a substrate, incubated, and the metabolite was extracted and analyzed by high-performance liquid chromatography. The metabolite extracted from synthetic medium was purer than that obtained from LB medium. Three P450s (2C29, 2C2, and 2A4) metabolized testosterone at different positions. P450 2C29 metabolized 7-ethoxycoumarin, androstendione, and dehydroepiandrosterone in this medium. P450s produced in the synthetic medium may be useful for producing various modified compounds for high-throughput screening. [ABSTRACT FROM AUTHOR]

The article presents a study that examines cloning and characterization of the recombinant inhibitor expressed in Escherichia coli. The possible role in the regulation of seed germination in the prevention of precocious germination had been discussed. Nucleotide sequence of the cDNA and its gene that encode a bifunctional rice amylase/subtillisin inhibitor (RASI) were analyzed. Based on the results, it has been suggested that RASI might function in the defense of the seed against microorganisms.

Transcription in higher plant plastids is performed by two types of RNA polymerases called NEP and PEP, and expression of photosynthesis genes in chloroplasts is largely dependent on PEP, a eubacteria-type multi-subunit enzyme. The transcription specificity of PEP is modulated by six nuclear-encoded sigma factors (SIG1 to SIG6) in Arabidopsis thaliana. Here, we show that one of the six sigma factors, SIG5, is induced under various stress conditions, such as high light, low temperature, high salt and high osmotic conditions. Interestingly, transcription from the psbD blue light-responsive promoter (psbD-BLRP) was activated by not only light but also various stresses, and the transcription and the transcriptional activation of psbD-BLRP were abolished in a sig5-2 mutant. This suggests that the PEP holoenzyme containing SIG5 transcribes the psbD-BLRP in response to multiple stresses. Since the seed germination under saline conditions and recovery from damage to the PSII induced by high light were delayed in the sig5-2 mutant, we postulate that SIG5 protects plants from stresses by enhancing repair of the PSII reaction center. [ABSTRACT FROM PUBLISHER]

Presents an analysis of plastid gene expression in an Arabidopsis mutant deficient in a plastid transcription factor sigma, SIG2. Multiple functions of the genes in the plastid genome of higher plants; Mechanism trough which plastid transcription is achieved; Significance of the expression of these plastid-encoded genes for plastid functions.

Background: 5′-Aminolevulinic acid (ALA) is widely used in the pharmaceutical industry, healthcare, and food production, and is a substrate for the biosynthesis of heme, which is required for respiration and photosynthesis. Enhancement of ALA biosynthesis has never been developed in Saccharomyces cerevisiae, which is a well-known model microorganism used for bioproduction of many value-added compounds. Results: We demonstrated that metabolic engineering significantly improved ALA production in S. cerevisiae. First, we found that overexpression of HEM1, which encodes ALA synthetase, increased ALA production. Furthermore, addition of an optimal amount of glycine, a substrate for ALA biosynthesis, or levulinic acid, an inhibitor of ALA dehydrogenase, effectively increased ALA production. Next, we developed an assay for multiple metabolites including ALA and found that aconitase, encoded by ACO1 and ACO2, is the rate-limiting enzyme of ALA biosynthesis when sufficient glycine is supplied. Overexpression of ACO2 further enhanced ALA production in S. cerevisiae overexpressing HEM1. Conclusions: In this study, ALA production in S. cerevisiae was enhanced by metabolic engineering. This study also shows a strategy to identify the rate-limiting step of a target synthetic pathway by assay for multiple metabolites alongside the target product. This strategy can be applied to improve production of other valuable products in the well-studied and well-industrialized microorganism S. cerevisiae. [ABSTRACT FROM AUTHOR]