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Introduction: Theranostic applications are currently difficult to achieve owing to the limited evaluation of suitable chelators for therapeutic nuclides, such as 225 Ac and 227 Th. With a focus on targeted α therapy and theranostics using human IgG as a drug-delivery system (i.e., combining highly cytotoxic α-particle emitter radiation with efficient tumor targeting), we developed a recombinant humanized Nd2 (hNd2) as an anti-MUC5AC antibody since MUC5AC is highly expressed in patients with pancreatic cancer. Therefore, we aimed to evaluate the performance of 89 Zr- (for diagnosis) and 225 Ac- (for therapy) labeling of these antibodies using well-controlled radioisotope (RI)-labeling technology in pancreatic cancer mouse models., Methods: 89 Zr-labeled hNd2 (NMK89) and 225 Ac-labeled hNd2 (NMT25) were manufactured by chemical conjugation using affinity peptides. A binding assay and the evaluation of plasma stability were performed in vitro to confirm the properties of NMK89 and NMT25. In vivo, we evaluated biodistribution, positron emission tomography (PET)/computed tomography (CT) imaging, antitumor effects, and toxicity. Moreover, the exposure dose in humans was estimated based on the biodistribution evaluation in normal mice., Results: NMK89 and NMT25 showed binding specificity to MUC5AC and stability with radiochemical purity ≥90% in mice and human plasma following incubation for 168 h. NMK89 showed high accumulation in tumors and low non-specific accumulation in normal tissues. The antitumor effect of NMT25 was dose-dependent and significantly suppressed tumor growth in the NMT25 treatment groups compared with the control group (p < 0.05). NMK89 and NMT25 showed similar pharmacokinetics and biodistribution characteristics. Additionally, the human estimated exposure dose of NMK89 and NMT25 was confirmed, and the effective dose of NMK89 and NMT25 was 0.33 mSv/MBq and 177.5 mSv/MBq, respectively., Conclusion: NMK89 showed specific accumulation in the MUC5AC-expressing tumors, while NMT25 showed strong antitumor effects. These results suggest NMK89 and NMT25 as promising theranostic agents for pancreatic cancer., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

"The Vision for Patient-centered Pharmacies," published by the Ministry of Health, Labour and Welfare(MHLW)in October 2015 specifies three functions of family pharmacies, including home medical care. In 1994, home-visits by pharmacists officially began; however, before then, we had already visited patients whose medications and life situations were of concern, at their homes. Based on that experience, as we were planning to undertake home-visits after their institutionalization, we conducted a study of a system that would promote home care using pharmacists handling various duties, including prescriptions and health consulting. Considering the pharmacists' years of experience and work shifts, efficiency/productivity, and the role of a family pharmacy, we developed a home medical care support system by allocating two pharmacists to each patient's home. Thus, we concluded that if the entire pharmacy would be involved in home-visiting services along with outpatient prescription dispensing services as part of continuous follow-up of patients from hospital visits to home care, the pharmacy would eventually serve as a family pharmacy in the community.

Prion diseases are caused by deposition of abnormal prion protein aggregates (PrP Sc ) in the central nervous system. This study aimed to develop in vivo imaging probes that can detect cerebral PrP Sc deposits. We synthesized several quinacrine-based acridine (AC) derivatives with 2,9-substitution and radioiodinated them. The AC derivatives were evaluated as prion-imaging probes using recombinant mouse prion protein (rMoPrP) aggregates and brain sections of mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice. The distribution of these compounds in mice was also evaluated. The 2-methoxy derivative [ 125 I]2 exhibited the highest binding affinity for rMoPrP aggregates with an equilibrium dissociation constant (K d ) value of 43.4nM. Fluorescence imaging with 2 showed clear signals at the thioflavin T (ThT)-positive amyloid deposits in the mBSE-infected mouse brain. Although a discrepancy was observed between the in vitro binding of AC derivatives to the aggregates and in vivo distribution of these compounds in the brain and we failed to identify prospective prion-imaging probes in this study, the AC derivatives may be considered a useful scaffold for the development of in vivo imaging probes. Further chemical modification of these AC derivatives may discover clinically applicable prion imaging probes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In 2006, with the admission of a new batch of students, pharmaceutical education became a 6-year course. This was a result of the urgent need to train a new generation of pharmacists to respond to increasingly advanced and intricate medical care as well as the specific need to coordinate with multiple occupational categories. Meanwhile, with Japan becoming an aged society, medical care has undergone functional differentiation, and home care is now being promoted. As part of an 11- week practical course for 5th-year practical training, students attended visits to home care patients from an early stage, making it possible for them to be present at multiple visits. This was highly significant because it allowed students to experience various disease states of different patients and increase their practical knowledge of pharmaceuticals. This study explores the case example of proposals made by pharmacy students for improving medication-related problems in home care patients during 5th-year practical training.

Unlabelled: Recently, companion diagnostics with nuclear medicine techniques have been anticipated as more suitable means than biopsy for predicting treatment efficacy. The anticancer effect of capecitabine, an orally administered chemotherapeutic agent activated by thymidine phosphorylase (TP), is positively associated with tumor TP expression levels. This study aimed to assess whether TP imaging using a radiolabeled uracil derivative, (123)I-5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil ((123)I-IIMU), could predict the efficacy of capecitabine treatment., Methods: Sensitivity to doxifluridine, a metabolite of capecitabine and direct substrate for TP, was assessed by water-soluble tetrazolium salt assays in vitro for 3 human colon cancer cell lines with different TP expression profiles. The intracellular uptake and retention of (123)I-IIMU were evaluated. Mice inoculated with each cell line were treated with capecitabine for 2 wk, and tumor growth was compared. In vivo distribution studies and SPECT/CT imaging of (123)I-IIMU were performed in inoculated mice., Results: In vitro experiments showed a positive relation between TP expression levels and doxifluridine sensitivity. In vitro studies revealed that intracellular uptake and retention of (123)I-IIMU were dependent on TP expression levels. In vivo experiments in inoculated mice showed that (123)I-IIMU accumulation in tumor tissue was in line with TP expression levels and susceptibility to capecitabine treatment. Moreover, SPECT/CT imaging of (123)I-IIMU in tumor-inoculated mice showed that (123)I-IIMU reflects TP expression levels in tumor tissues., Conclusion: (123)I-IIMU could be used as an in vivo companion diagnostic for predicting the efficacy of capecitabine treatment., (© 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Context: Although adrenal vein sampling is the standard method to distinguish unilateral from bilateral forms of primary aldosteronism, it is an invasive and technically difficult procedure. (11)C-metomidate (MTO)-positron emission tomography was reported as a potential replacement for adrenal vein sampling. However, MTO has low selectivity for CYP11B2 over CYP11B1., Objective: This study aimed to determine the selectivity of (18)F-CDP2230, a new imaging agent, for CYP11B2 over CYP11B1 and determine whether the biodistribution profile of (18)F-CDP2230 is favorable for imaging CYP11B2., Methods: The IC50 of CDP2230 for the enzymatic activities of CYP11B2 and CYP11B1 was determined using cells with stable expression of either enzyme. In vitro autoradiography of human adrenal sections with aldosterone-producing adenomas was performed to confirm the specific binding ability of (18)F-CDP2230 to CYP11B2-expressing regions. Furthermore, positron emission tomography and magnetic resonance imaging were performed to evaluate the biodistribution of (18)F-CDP2230 in rats., Results: Although CDP2230 showed a significantly lower affinity for CYP11B2 and CYP11B1 than did MTO analogues, its selectivity for CYP11B2 over CYP11B1 was higher than that of MTO analogues. In vitro autoradiography revealed that the binding of (18)F-CDP2230 to CYP11B2-expressing regions in the adrenal gland was more specific than that of (123)I-IMTO. Moreover, the biodistribution study showed that (18)F-CDP2230 accumulated in adrenal glands with low background uptake., Conclusions: Our study showed a high selectivity of (18)F-CDP2230 for CYP11B2 over CYP11B1 with a favorable biodistribution for imaging CYP11B2. (18)F-CDP2230 is a promising imaging agent for detecting unilateral subtypes of primary aldosteronism.

Background: In recent years, virtual reality has been introduced to neurorehabilitation, in particular with the intention of improving upper-limb training options and facilitating motor function recovery., Methods/design: The proposed study incorporates a quantitative part and a qualitative part, termed a mixed-methods approach: (1) a quantitative investigation of the efficacy of virtual reality training compared to conventional therapy in upper-limb motor function are investigated, (2a) a qualitative investigation of patients' experiences and expectations of virtual reality training and (2b) a qualitative investigation of therapists' experiences using the virtual reality training system in the therapy setting. At three participating clinics, 60 patients at least 6 months after stroke onset will be randomly allocated to an experimental virtual reality group (EG) or to a control group that will receive conventional physiotherapy or occupational therapy (16 sessions, 45 minutes each, over the course of 4 weeks). Using custom data gloves, patients' finger and arm movements will be displayed in real time on a monitor, and they will move and manipulate objects in various virtual environments. A blinded assessor will test patients' motor and cognitive performance twice before, once during, and twice after the 4-week intervention. The primary outcome measure is the Box and Block Test. Secondary outcome measures are the Chedoke-McMaster Stroke Assessments (hand, arm and shoulder pain subscales), the Chedoke-McMaster Arm and Hand Activity Inventory, the Line Bisection Test, the Stroke Impact Scale, the MiniMentalState Examination and the Extended Barthel Index. Semistructured face-to-face interviews will be conducted with patients in the EG after intervention finalization with a focus on the patients' expectations and experiences regarding the virtual reality training. Therapists' perspectives on virtual reality training will be reviewed in three focus groups comprising four to six occupational therapists and physiotherapists., Discussion: The interviews will help to gain a deeper understanding of the phenomena under investigation to provide sound recommendations for the implementation of the virtual reality training system for routine use in neurorehabilitation complementing the quantitative clinical assessments., Trial Registration: Cliniclatrials.gov Identifier: NCT01774669 (15 January 2013).

The aim of the present study was to characterize the binding property of quinacrine-based acridine derivatives for Aβ plaques and to evaluate this series of compounds as Aβ imaging probes. Quinacrine clearly stained Aβ plaques in the brain sections of Aβ deposition model transgenic mice (Tg2576 mice). Similarly, the quinacrine analog, 2-methoxy-9-(4-(dimethyl-1-methyl) -N-butyl) amino-6-iodo acridine (5), labeled Aβ plaques in the brain slices of Tg2576 mice. In addition, [(125)I]5 showed modest affinity for Aβ(1-42) aggregates with a K(d) value of 48 nM. Biodistribution studies using normal mice demonstrated that [(125)I]5 displayed poor initial brain uptake. Next, (125)I-labeled acridines without aliphatic amino groups were synthesized and characterized. Similar to quinacrine and 5, these compounds could detect Aβ plaques in the brain sections of Tg2576 mice. It should be noted that the acridines showed much higher binding affinity for Aβ aggregates and greater in vivo blood brain barrier permeability than [(125)I]5. Among them, 13 (6-Iodo-2-methoxy-9-methylaminoacridine) and 25 (2,9-Dimethoxy-6-iodo acridine) exhibited high affinity for the Aβ aggregates with K(i) values of 14 and 29 nM, respectively. In the in vivo studies, [(125)I]13 and [(125)I]25 showed excellent initial brain uptake (3.0 and 4.4% dose/g, respectively, at 2 min) with fast washout from the brain (0.33 and 0.37% dose/g, respectively, at 60 min). These acridine derivatives are demonstrated to be promising SPECT imaging probes for amyloid in the living brain., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

It is known that activity in secondary motor areas during observation of human limbs performing actions is affected by the observer's viewpoint, with first-person views generally leading to stronger activation. However, previous neuroimaging studies have displayed limbs in front of the observer, providing an offset view of the limbs without a truly first-person viewpoint. It is unknown to what extent these pseudo-first-person viewpoints have affected the results published to date. In this experiment, we used a horizontal two-dimensional mirrored display that places virtual limbs at the correct egocentric position relative to the observer. We compared subjects using the mirrored and conventional displays while recording over the premotor cortex with functional near-infrared spectroscopy. Subjects watched a first-person view of virtual arms grasping incoming balls on-screen; they were instructed to either imagine the virtual arm as their own [motor imagery during observation (MIO)] or to execute the movements [motor execution (ME)]. With repeated-measures anova, the hemoglobin difference as a direct index of cortical oxygenation revealed significant main effects of the factors hemisphere (P = 0.005) and condition (P ≤ 0.001) with significant post hoc differences between MIO-mirror and MIO-conventional (P = 0.024). These results suggest that the horizontal mirrored display provides a more accurate first-person view, enhancing subjects' ability to perform motor imagery during observation. Our results may have implications for future experimental designs involving motor imagery, and may also have applications in video gaming and virtual reality therapy, such as for patients following stroke., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Trial-to-trial variability is a well-known issue in brain signals measured using functional near-infrared spectroscopy (fNIRS). We aimed to investigate whether trial-to-trial variability does provide information about individual performance. Seventeen subjects observed a virtual reality grasping task in first-person view while either imagining (motor imagery during observation, MIO) or imitating (motor execution, ME) the movements. Each condition was performed with the display in one of two positions, a conventional vertical position and a mirrored horizontal position which placed the virtual arm in the correct position relative to the viewpoint. Averaged oxy-hemoglobin concentration Δ[O(2)Hb] showed that the responses could be differentiated into two distinct groups: low responders (LR) and high responders (HR). Within groups, two main sources of trial-to-trial variability were identified: (a) the Δ[O(2)Hb] amplitude, with largest amplitudes in ME conditions (group HR) and smallest amplitudes in MIO conditions (group LR), and (b) the sign of Δ[O(2)Hb], with positive responses occurring most frequently during ME (group HR) and negative responses most frequently during MIO (group LR). Furthermore, the trial-to-trial dynamics differed between groups and could be described in group LR as inverted polynomial U-shaped curve in the mirror conditions (ME-mirror, MIO-mirror). Last, trial-to-trial variability was significantly dependent on task modality, i.e. ME (group HR) versus MIO (group LR), and/or the mirrored display positions (group LR). Our results show a relationship of trial-to-trial variability to individual MI performance, which may be of significance for neurorehabilitation applications. Although the sources of trial-to-trial variability remain unknown, we suggest that they may contribute to future neurofeedback applications., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Two novel benzofuran derivatives coupled with (99m)Tc complexes were tested as probes for imaging cerebral β-amyloid plaques using single photon emission tomography. Although both derivatives bound to Aβ(1-42) aggregates, (99m)Tc-BAT-BF showed higher affinity than (99m)Tc-MAMA-BF. In sections of brain tissue from an animal model of AD, (99m)Tc-BAT-BF clearly labeled β-amyloid plaques. In biodistribution experiments using normal mice, (99m)Tc-BAT-BF displayed high uptake soon after its injection and washed out from the brain rapidly, a highly desirable feature for an imaging agent. (99m)Tc-BAT-BF may be a potential probe for imaging β-amyloid plaques in Alzheimer's brains.

We synthesized a novel series of phenylindole (PI) derivatives and evaluated their biological activities as probes for imaging Aβ plaques in vivo. The affinity for Aβ plaques was assessed by an in vitro-binding assay using pre-formed synthetic Aβ aggregates. 2-phenyl-1H-indole (2-PI) derivatives showed high affinity for Aβ42 aggregates with K(i) values ranging from 4 to 32 nM. 2-PI derivatives clearly stained Aβ plaques in an animal model of AD. In biodistribution experiments using normal mice, 2-PI derivatives displayed sufficient uptake for imaging, ranging from 1.1% to 2.6% ID/g. Although additional modifications are necessary to improve uptake by and clearance from the brain, 2-PI derivatives may be useful as a backbone structure to develop novel Aβ imaging agents., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Fireflies communicate with each other by emitting yellow-green to yellow-orange brilliant light. The bioluminescence reaction, which uses luciferin, Mg-ATP and molecular oxygen to yield an electronically excited oxyluciferin species, is carried out by the enzyme luciferase. Visible light is emitted during relaxation of excited oxyluciferin to its ground state. The high quantum yield of the luciferin/luciferase reaction and the change in bioluminescence colour caused by subtle structural differences in luciferase have attracted much research interest. In fact, a single amino acid substitution in luciferase changes the emission colour from yellow-green to red. Although the crystal structure of luciferase from the North American firefly (Photinus pyralis) has been described, the detailed mechanism for the bioluminescence colour change is still unclear. Here we report the crystal structures of wild-type and red mutant (S286N) luciferases from the Japanese Genji-botaru (Luciola cruciata) in complex with a high-energy intermediate analogue, 5'-O-[N-(dehydroluciferyl)-sulfamoyl]adenosine (DLSA). Comparing these structures to those of the wild-type luciferase complexed with AMP plus oxyluciferin (products) reveals a significant conformational change in the wild-type enzyme but not in the red mutant. This conformational change involves movement of the hydrophobic side chain of Ile 288 towards the benzothiazole ring of DLSA. Our results indicate that the degree of molecular rigidity of the excited state of oxyluciferin, which is controlled by a transient movement of Ile 288, determines the colour of bioluminescence during the emission reaction.

The carbazole 1,9a-dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 catalyzes the dioxygenation of carbazole; the 9aC carbon bonds to a nitrogen atom and its adjacent 1C carbon as the initial reaction in the mineralization pathway. The CARDO system is composed of ferredoxin reductase (CarAd), ferredoxin (CarAc), and terminal oxygenase (CarAa). CarAc acts as a mediator in the electron transfer from CarAd to CarAa. To understand the structural basis of the protein-protein interactions during electron transport in the CARDO system, the crystal structure of CarAc was determined at 1.9 A resolution by molecular replacement using the structure of BphF, the biphenyl 2,3-dioxygenase ferredoxin from Burkholderia cepacia strain LB400 as a search model. CarAc is composed of three beta-sheets, and the structure can be divided into two domains, a cluster-binding domain and a basal domain. The Rieske [2Fe-2S] cluster is located at the tip of the cluster-binding domain, where it is exposed to solvent. While the overall folding of CarAc and BphF is strongly conserved, the properties of their surfaces are very different from each other. The structure of the cluster-binding domain of CarAc is more compact and protruding than that of BphF, and the distribution of electric charge on its molecular surface is very different. Such differences are thought to explain why these ferredoxins can act as electron mediators in respective electron transport chains composed of different-featured components., (Copyright 2005 Wiley-Liss, Inc.)

To elucidate the mechanism for the regulation of aspartate kinase (AK) via feedback inhibition, we constructed several chimeric enzymes between Bacillus subtilis AK II, a lysine-sensitive mesophilic enzyme, and Thermus flavus AK, a threonine-sensitive thermostable enzyme, each having the same alpha2beta2-type tetrameric structure. A chimeric AK, named BTT, composed of the chimeric alpha subunit that comprises of the N-terminal catalytic region from B. subtilis AK II and the C-terminal region from T. flavus, and the beta subunit from T. flavus, was inhibited only by threonine. Another chimeric enzyme, BT, which has a similar structure to that of BTT but lacks the beta subunit, having alpha2-type homo-dimeric structure, was also responsive only to threonine. However, the addition of threonine enhanced the activity of BT. These results indicate the regulatory function of C-terminal region and beta subunit in AK. BTT showed extremely high thermostability comparable to that of T. flavus, suggesting that the beta subunit also contributed to the stability of the AK.

In suspension-cultured rice ( Oryza sativaL.) cells, jasmonic acid (JA) functions as a signal transducer in elicitor N-acetylchitoheptaose-induced phytoalexin production. Differential screening of a cDNA library constructed using poly(A)(+) RNA from suspension-cultured rice cells treated with JA (10(-4) M) for 2 h yielded a cDNA for a gene that responded to exogenous JA by an increase in mRNA level. Nucleotide sequence analysis indicated that the cDNA encodes an homologue of the yeast Old Yellow Enzyme. The deduced amino acid sequence was very similar to the sequences of 12-oxophytodienoic acid reductases (OPR) 1 and 2 from Arabidopsis thaliana(AtOPR1 and AtOPR2) and OPR1 from tomato ( Lycopersicon esculentum) (LeOPR1). The cDNA-encoded protein purified from recombinant Escherichia coli cells as a hexahistidine-tagged fusion protein exhibited OPR activity similar to that of AtOPR1, AtOPR2, and LeOPR1, which catalyze reduction of (-)- cis-12-oxophytodienoic acid (OPDA) preferentially over (+)- cis-OPDA, a natural precursor of JA. Thus the rice enzyme was termed OsOPR1. The physiological roles of OsOPR1 are discussed. This is the first report of the cloning of an OPR gene from a monocot plant.

Although the presence of an enzyme that catalyzes beta-decarboxylating dehydrogenation of homoisocitrate to synthesize 2-oxoadipate has been postulated in the lysine biosynthesis pathway through alpha-aminoadipate (AAA), the enzyme has not yet been analyzed at all, because no gene encoding the enzyme has been identified until recently. A gene encoding a protein with a significant amino acid sequence identity to both isocitrate dehydrogenase and 3-isopropylmalate dehydrogenase was cloned from Thermus thermophilus HB27. The gene product produced in recombinant Escherichia coli cells demonstrated homoisocitrate dehydrogenase (HICDH) activity. A knockout mutant of the gene showed an AAA-auxotrophic phenotype, indicating that the gene product is involved in lysine biosynthesis through AAA. We therefore named this gene hicdh. HICDH, the gene product, did not catalyze the conversion of 3-isopropylmalate to 2-oxoisocaproate, a leucine biosynthetic reaction, but it did recognize isocitrate, a related compound in the tricarboxylic acid cycle, as well as homoisocitrate as a substrate. It is of interest that HICDH catalyzes the reaction with isocitrate about 20 times more efficiently than the reaction with the putative native substrate, homoisocitrate. The broad specificity and possible dual function suggest that this enzyme represents a key link in the evolution of the pathways utilizing citrate derivatives. Site-directed mutagenesis study reveals that replacement of Arg(85) with Val in HICDH causes complete loss of activity with isocitrate but significant activity with 3-isopropylmalate and retains activity with homoisocitrate. These results indicate that Arg(85) is a key residue for both substrate specificity and evolution of beta-decarboxylating dehydrogenases.

FK228 is a histone deacetylase (HDAC) inhibitor, the molecular mechanism of inhibition of which has been unknown. Here we show that reduction of an intramolecular disulfide bond of FK228 greatly enhanced its inhibitory activity and that the disulfide bond was rapidly reduced in cells by cellular reducing activity involving glutathione. Computer modeling suggests that one of the sulfhydryl groups of the reduced form of FK228 (redFK) interacts with the active-site zinc, preventing the access of the substrate. HDAC1 and HDAC2 were more strongly inhibited by redFK than HDAC4 and HDAC6. redFK was less active than FK228 in inhibiting in vivo HDAC activity, due to rapid inactivation in medium and serum. Thus, FK228 serves as a stable prodrug to inhibit class I enzymes and is activated by reduction after uptake into the cells. The glutathione-mediated activation also implicates its clinical usefulness for counteracting glutathione-mediated drug resistance in chemotherapy.

In Thermus thermophilus homocitrate synthase (HCS) catalyzes the initial reaction of lysine biosynthesis through alpha-aminoadipic acid, synthesis of homocitrate from 2-oxoglutarate and acetyl-CoA. HCS is strongly inhibited by lysine, indicating that the biosynthesis is regulated by the endproduct at the initial reaction in the pathway. HCS also catalyzes the reaction using oxaloacetate in place of 2-oxoglutarate as a substrate, similar to citrate synthase in the tricarboxylic acid cycle. Several other properties of Thermus HCS and an evolutionary relationship of the biosynthetic pathway in the bacterium to other metabolic pathways are also described.

Two cDNA clones for jasmonic acid (JA)-responsive genes, RRJ1 and RRJ2, were isolated by differential screening from suspension-cultured rice cells treated with JA for 2 h. The putative RRJ1 protein is completely identical to that of a putative rice cystathionine gamma-lyase, while the putative RRJ2 protein is highly similar in sequence to a rice pyruvate decarboxylase, PDC1.

We conducted a chromosome walk to obtain a DNA fragment downstream of lysJ and found an argE homolog in a putative operon composed of lysJ-orfC-orfD-argE homologs. A knockout mutant of the argE homolog showed significantly slow growth on a minimal medium, and the growth was markedly improved by addition of lysine. We therefore termed this gene lysK. Purified LysK protein has deacetylating activities for both N(2)-acetyllysine and N(2)-acetylornithine at almost equal efficiency. These results suggest that lysK which may share an ancestor with argE functions not only for the lysine biosynthesis, but also for arginine biosynthesis in Thermus thermophilus.

Our previous studies revealed that lysine is synthesized through alpha-aminoadipate in an extremely thermophilic bacterium, Thermus thermophilus HB27. Sequence analysis of a gene cluster involved in the lysine biosynthesis of this microorganism suggested that the conversion from alpha-aminoadipate to lysine proceeds in a way similar to that of arginine biosynthesis. In the present study, we cloned an argD homolog of T. thermophilus HB27 which was not included in the previously cloned lysine biosynthetic gene cluster and determined the nucleotide sequence. A knockout of the argD-like gene, now termed lysJ, in T. thermophilus HB27 showed that this gene is essential for lysine biosynthesis in this bacterium. The lysJ gene was cloned into a plasmid and overexpressed in Escherichia coli, and the LysJ protein was purified to homogeneity. When the catalytic activity of LysJ was analyzed in a reverse reaction in the putative pathway, LysJ was found to transfer the epsilon-amino group of N(2)-acetyllysine, a putative intermediate in lysine biosynthesis, to 2-oxoglutarate. When N(2)-acetylornithine, a substrate for arginine biosynthesis, was used as the substrate for the reaction, LysJ transferred the delta-amino group of N(2)-acetylornithine to 2-oxoglutarate 16 times more efficiently than when N(2)-acetyllysine was the amino donor. All these results suggest that lysine biosynthesis in T. thermophilus HB27 is functionally and evolutionarily related to arginine biosynthesis.

A search in the Bacillus subtilis genome sequence found that the gene designated yclM encode(s) a protein showing significant identity in amino acid sequence to aspartate kinases. When yclM was introduced into Escherichia coli cells deficient in all three aspartate kinase genes, production of a protein with molecular size 50 kDa, which was similar to the value deduced from the nucleotide sequence of the gene, was observed. Expectedly, the protein purified to homogeneity had aspartate kinase activity. The enzyme was significantly inhibited by simultaneous addition of both threonine and lysine, which is a typical feature of aspartate kinase III of B. subtilis. The enzyme was very unstable in 10 mM tris-HCl (pH 7.5) buffer, but was stabilized by addition of 500 mM ammonium sulfate. Although all the aspartate kinases so far investigated are oligomeric enzymes, this aspartate kinase was suggested to be a monomer.

The nucleotide sequence of the upstream region of the aspartate kinase genes of Thermus thermophilus HB27 revealed the presence of two open reading frames in the orientation opposite to that of the aspartate kinase genes. The upstream open reading frame termed ORF375 encodes a protein composed of 375 amino acid residues, possessing amino acid sequence motifs for methylases. Another open reading frame designated as sigA encodes a protein of 423 amino acid residues which shows significant identity in amino acid sequence to the principal sigma factor, a component of the DNA-dependent RNA polymerase holoenzyme. The close proximity of the open reading frames suggested that the two genes are transcribed in a polycistronic manner. By the use of an Escherichia coli expression system, SigA was produced in a soluble form. An in vitro transcription assay of purified SigA reconstituted with the core RNA polymerase of E. coli showed that Thermus SigA functioned as a sigma factor to initiate specific transcription.

An aspartate kinase-deficient mutant of Thermus thermophilus, AK001, was constructed. The mutant strain did not grow in a minimal medium, suggesting that T. thermophilus contains a single aspartate kinase. Growth of the mutant strain was restored by addition of both threonine and methionine, while addition of lysine had no detectable effect on growth. To further elucidate the lysine biosynthetic pathway in T. thermophilus, lysine auxotrophic mutants of T. thermophilus were obtained by chemical mutagenesis. For all lysine auxotrophic mutants, growth in a minimal medium was not restored by addition of diaminopimelic acid, whereas growth of two mutants was restored by addition of alpha-aminoadipic acid, a precursor of lysine in biosynthetic pathways of yeast and fungi. A BamHI fragment of 4.34 kb which complemented the lysine auxotrophy of a mutant was cloned. Determination of the nucleotide sequence suggested the presence of homoaconitate hydratase genes, termed hacA and hacB, which could encode large and small subunits of homoaconitate hydratase, in the cloned fragment. Disruption of the chromosomal copy of hacA yielded mutants showing lysine auxotrophy which was restored by addition of alpha-aminoadipic acid or alpha-ketoadipic acid. All of these results indicated that in T. thermophilus, lysine was not synthesized via the diaminopimelic acid pathway, believed to be common to all bacteria, but via a pathway using alpha-aminoadipic acid as a biosynthetic intermediate.

To reveal the catalytic mechanism of Thermus aspartate kinase, each of 29 amino acid residues that were highly conserved in the sequenced aspartate kinases, was replaced with alanine or leucine by PCR site-directed mutagenesis. Comparison of the kinetic parameters of these mutants with those of the wild-type aspartate kinase suggested that Thr47 was involved in binding aspartate and that Lys7 and Glu74 were involved in catalysis. Analysis of the effective concentrations of magnesium ion on the activity showed that the mutants with replacements at Ser41, Thr47, Asp154 and Asp182 required higher concentrations of magnesium ion. This suggests that these four residues play important roles in the binding of magnesium ions which are required for enzymatic activity.

In this study simple reaction time (simple RT) to a visual stimulus of a single subject was measured during spontaneous and controlled breathing, in which the duration of expiration was prolonged (Asian technique). The phases of breathing were classified as the pause between expiration and inspiration, the inspiration phase, the transition from inspiration to expiration, and the expiration phase. Analysis of data from about 6000 trials indicated that RT to the stimulus was shortest during the transition from inspiration to expiration in controlled breathing.