Identification of a new adhesin-like protein from Lactobacillus mucosae ME-340 with specific affinity to the human blood group A and B antigens.

AIMS: To identify and characterize a new adhesin-like protein of probiotics that show specific adhesion to human blood group A and B antigens. METHODS AND RESULTS: Using the BIACORE assay, the adhesion of cell surface components obtained from four lactobacilli strains that adhered to blood group A and B antigens was tested. Their components showed a significant adhesion to A and B antigens when compared to the bovine serum albumin (BSA) control. The 1 mol l(-1) GHCl fraction extracted from Lactobacillus mucosae ME-340 contained a 29-kDa band (Lam29) using SDS-PAGE. The N-terminal amino acid sequence and homology analysis showed that Lam29 was 90% similar to the substrate-binding protein of the ATP-binding cassette (ABC) transporter from Lactobacillus fermentum IFO 3956. The complete nucleotide sequence (858 bp) of Lam29 was determined and encoded a protein of 285 amino acid residues. Phylogenetic analysis and multiple sequence alignments indicated this protein may be related to the cysteine-binding transporter. CONCLUSIONS: The adhesion of ME-340 strain to blood group A and B antigens was mediated by Lam29 that is a putative component of ABC transporter as an adhesin-like protein. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus mucosae ME-340 expressing Lam29 may be useful for competitive exclusion of pathogens via blood group antigen receptors in the human gastrointestinal mucosa and in the development of new probiotic foods.

DDBJ with new system and face.

DDBJ (http://www.ddbj.nig.ac.jp) collected and released 1 880 115 entries or 1 134 086 245 bases in the period from July 2006 to June 2007. The released data contains the high-throughput cDNAs of cricket and high-quality draft genome of medaka among others. Our computer system has been upgraded since March 2007. Another new aspect is an efficient data retrieval tool that has recently been equipped and served at DDBJ. It is called All-round Retrieval for Sequence and Annotation, which enables the user to search for keywords also in the Feature/Qualifier of the International Nucleotide Sequence Database Collaboration (http://www.insdc.org/). We will also replace our home page with a more efficient one by the end of 2007.

DDBJ in collaboration with mass-sequencing teams on annotation.

In the past year, we at DDBJ (DNA Data Bank of Japan; http://www.ddbj.nig.ac.jp) collected and released 1,066,084 entries or 718,072,425 bases including the whole chromosome 22 of chimpanzee, the whole-genome shotgun sequences of silkworm and various others. On the other hand, we hosted workshops for human full-length cDNA annotation and participated in jamborees of mouse full-length cDNA annotation. The annotated data are made public at DDBJ. We are also in collaboration with a RIKEN team to accept and release the CAGE (Cap Analysis Gene Expression) data under a new category, MGA (Mass Sequences for Genome Annotation). The data will be useful for studying gene expression control in many aspects.

DNA Data Bank of Japan (DDBJ) in XML.

The DNA Data Bank of Japan (DDBJ, http://www.ddbj.nig.ac.jp) has collected and released more entries and bases than last year. This is mainly due to large-scale submissions from Japanese sequencing teams on mouse, rice, chimpanzee, nematoda and other organisms. The contributions of DDBJ over the past year are 17.3% (entries) and 10.3% (bases) of the combined outputs of the International Nucleotide Sequence Databases (INSD). Our complete genome sequence database, Genome Information Broker (GIB), has been improved by incorporating XML. It is now possible to perform a more sophisticated database search against the new GIB than the ordinary BLAST or FASTA search.

DDBJ in the stream of various biological data.

In the past year we at DDBJ (http://www.ddbj.nig. ac.jp) have made a steady increase in the number of data submissions with a 50.6% increment in the number of bases or 46.5% increment in the number of entries. Among them the genome data of man, ascidian and rice hold the top three. Our activity has extended to providing a tool that enables sequence retrieval using regular expressions, and to launching our SOAP server and web services to facilitate the acquisition of proper data and tools from a huge number of biological data resources on websites worldwide. We have also opened our public gene expression database, CIBEX.

DNA Data Bank of Japan (DDBJ) for genome scale research in life science.

The DNA Data Bank of Japan (DDBJ, http://www.ddbj.nig.ac.jp) has made an effort to collect as much data as possible mainly from Japanese researchers. The increase rates of the data we collected, annotated and released to the public in the past year are 43% for the number of entries and 52% for the number of bases. The increase rates are accelerated even after the human genome was sequenced, because sequencing technology has been remarkably advanced and simplified, and research in life science has been shifted from the gene scale to the genome scale. In addition, we have developed the Genome Information Broker (GIB, http://gib.genes.nig.ac.jp) that now includes more than 50 complete microbial genome and Arabidopsis genome data. We have also developed a database of the human genome, the Human Genomics Studio (HGS, http://studio.nig.ac.jp). HGS provides one with a set of sequences being as continuous as possible in any one of the 24 chromosomes. Both GIB and HGS have been updated incorporating newly available data and retrieval tools.

Domain dislocation: a change of core structure in periplasmic binding proteins in their evolutionary history.

Periplasmic binding proteins (PBPs) serve as receptors for various water-soluble ligands in ATP-binding cassette (ABC) transport systems, and form one of the largest protein families in eubacterial and archaebacterial genomes. They are considered to be derived from a common ancestor, judging from their similarities of three-dimensional structure, their mechanism of ligand binding and the operon structure of their genes. Nevertheless, there are two types of topological arrangements of the central beta-sheets in their core structures. It follows that there must have been differentiation in the core structure, which we call "domain dislocation", in the course of evolution of the PBP family. To find a clue as to when the domain dislocation occurred, we constructed phylogenetic trees for PBPs based on their amino acid sequences and three-dimensional structures, respectively. The trees show that the proteins of each type clearly cluster together, strongly indicating that the change in the core structure occurred only once in the evolution of PBPs. We also constructed a phylogenetic tree for the ABC proteins that are encoded by the same operon of their partner PBP, and obtained the same result. Based on the phylogenetic relationship and comparison of the topological arrangements of PBPs, we obtained a reasonable genealogical chart of structural changes in the PBP family. The present analysis shows that the unidirectional change of protein evolution is clearly deduced at the level of protein three-dimensional structure rather than the level of amino acid sequence.

Quantitative in vivo analysis of benzodiazepine binding sites in the human brain using positron emission tomography.

Central-type benzodiazepine binding sites were characterized in a single normal human subject, using positron emission tomography (PET) and the radiolabelled benzodiazepine antagonist, carbon-11 labelled flumazenil ([11C] Ro 15-1788). The subject was scanned using tracer alone and tracer plus 4 different concentrations of unlabelled Ro 15-1788, including one concentration of unlabelled Ro 15-1788, chosen to produce maximum displacement of [11C] Ro 15-1788 from specific binding sites. Concentrations of free, unmetabolized [11C] Ro 15-1788 in plasma were estimated using a simple extraction and ultrafiltration method. Radioactivity in the regional exchangeable pool in brain was estimated under non-saturation conditions from the ratio of radioactivity in brain to plasma, under saturation conditions and the kinetics of free ligand in plasma. The specific binding was, then, estimated by the difference between the total radioactivity in brain and exchangeable pool radioactivity. Scatchard analyses were performed to yield Bmax and Kd values under pseudo-equilibrium conditions, which was observed as an increase of specific binding/free with reduction in specific binding. In cerebral cortex and cerebellum, the Bmax values were about 62-73 nmol/l and the Kd values were 3.6-6 nM in the estimation of free ligand in plasma and 12-15 nM in the estimation of exchangeable pool in brain, as free in brain.

Visualization of specific binding sites of benzodiazepine in human brain.

Using 11C-labeled Ro15-1788 and positron emission tomography, studies of benzodiazepine binding sites in the human brain were performed on four normal volunteers. Rapid and high accumulation of 11C activity was observed in the brain after i.v. injection of [11C]Ro15-1788, the maximum of which was within 12 min. Initial distribution of 11C activity in the brain was similar to the distribution of the normal cerebral blood flow. Ten minutes after injection, however, a high uptake of 11C activity was observed in the cerebral cortex and moderate uptake was seen in the cerebellar cortex, the basal ganglia, and the thalamus. The accumulation of 11C activity was low in the brain stem. This distribution of 11C activity was approximately parallel to the known distribution of benzodiazepine receptors. Saturation experiments were performed on four volunteers with oral administration of 0.3-1.8 mg/kg of cold Ro15-1788 prior to injection. Initial distribution of 11C activity following injection peaked within 2 min and then the accumulation of 11C activity decreased rapidly and remarkably throughout the brain. The results indicated that [11C] Ro15-1788 associates and dissociates to specific and nonspecific binding sites rapidly and has a high ratio of specific receptor binding to nonspecific binding in vivo. Carbon-11 Ro15-1788 is a suitable radioligand for the study of benzodiazepine receptors in vivo in humans.

Kinetics of [11C]N,N-dimethylphenylethylamine in mice and humans: potential for measurement of brain MAO-B activity.

Carbon-11-labeled N,N-dimethylphenylethylamine ([11C]DMPEA) was synthesized by the reaction of N-methylphenylethylamine with [11C]methyl iodide. This newly synthesized radiotracer was developed for the purpose of in vivo measurement of monoamine oxidase-B activity in the brain using a metabolic trapping method. Initially, biodistribution was investigated in mice. The rapid and high uptake of 11C radioactivity in the brain was observed following intravenous injection of [11C]DMPEA, the peak of which was reached at 1 min, followed by a decrease at 1-5 min and slowly thereafter. The kinetics of [11C]DMPEA in the human brain were determined using positron emission tomography (PET) and showed that 11C radioactivity increased gradually over 60 min following initial rapid uptake of 11C radioactivity, with basal ganglia and thalamus showing high accumulation.

An acute effect of triazolam on muscarinic cholinergic receptor binding in the human brain measured by positron emission tomography.

An acute effect of triazolam, a potent benzodiazepine agonist, on cholinergic receptor binding in the human brain was measured by PET (positron emission tomography) using [11C]N-methyl-4-piperidylbenzilate ([11C]NMPB), a potent muscarinic cholinergic receptor antagonist. Two PET scans were performed in each subject: (1) control scan; (2) after oral administration of 0.5 mg triazolam or placebo. The previously discussed amnestic effect of triazolam was measured by immediate and delayed recall of meaningful and meaningless syllables. A compartment model employing the radioactivity in the cerebellum as an input function was used for the quantification of receptor binding. The binding parameter, k3, was decreased after triazolam administration in all measured regions, whereas no change was observed after placebo treatment. The reduction compared to the control study varied from 8.6 +/- 3.7% in the temporal cortex to 16.3 +/- 6.3% in the thalamus. Triazolam administration impaired both immediate and delayed recall of syllables, whereas placebo administration had no effects. Benzodiazepine agonists are reported to decrease the cortical acetylcholine release. The decrease of acetylcholine release in the synaptic cleft might be the explanation for the decreased binding of [11C]NMPB.

D1 dopamine receptor binding in mood disorders measured by positron emission tomography.

D1 dopamine receptor binding in mood disorders was studied by positron emission tomography (PET) using 11C-SCH23390. Ten patients with bipolar mood disorders and 21 normal controls were studied in the drug-free state. The patients were in euthymic (N = 6), depressed (N = 3) and manic (N = 1) states. Regional radioactivity in the brain was followed for 40 min by PET. A two-compartment model was used to obtain the binding potential (k3/k4) for the striatum and frontal cortex. The binding potentials for the frontal cortex for the patients were significantly lower than those for normal controls, whereas those for striatum were not significantly different. These findings suggest that D1 dopamine receptors in the frontal cortex may be in a different state in patients with bipolar mood disorders.

Age-related changes in human D1 dopamine receptors measured by positron emission tomography.

The effects of age on the binding parameters of 11C-SCH23390, the highly selective ligand for central D1 dopamine receptors, at specific binding sites in the brain were studied. Seventeen healthy male volunteers (20-72 years old) participated. Regional radioactivity in the brain was followed for 40 min by positron emission tomography (PET). A high accumulation of radioactivity was observed in the striatum and there was a conspicuous accumulation in the neocortex. A two-compartment model was used to obtain quantitative estimates of rate constants of association (K3) and dissociation (k4). The binding potential (k3/k4) of the dopamine D1 receptors in the striatum and frontal cortex decreased by 35% and 39%, respectively, with age. The value of k3 decreased by 58% in the striatum and 83% in the frontal cortex, whereas the value of k4 decreased by 35% in the striatum and 72% in the frontal cortex with age.

Detection of benzodiazepine receptor occupancy in the human brain by positron emission tomography.

Benzodiazepine receptor occupancy in the brain following oral administration of clonazepam (CZP) with a dose of 30 micrograms/kg in six healthy young men and a further dose of 50 micrograms/kg in one of the subjects was estimated by carbon-11 labeled Ro15-1788 and positron emission tomography (PET). The effects of CZP on the latency of auditory event-related potentials (P300) were also studied. Overall brain 11C uptake was depressed and the % inhibition of 11C uptake in the gray matter of the brain at 30 min after [11C]Ro15-1788 injection was 15.3-23.5% (mean, n = 6) following 30 micrograms/kg CZP when compared with that in the control experiment without any previous treatment. The 11C uptake in the cerebral cortex in the subject who received both doses decreased in a dose-related manner after 30 micrograms/kg and 50 micrograms/kg CZP. The P300 latency was prolonged significantly by 30 micrograms/kg CZP [31.6 +/- 16.3 ms (mean +/- SD, n = 6), P less than 0.05]. The P300 latency in the same subject was prolonged in a dose-related manner by 30 micrograms/kg and 50 micrograms/kg CZP. The technique using [11C]Ro15-1788 and PET permits comparison of the pharmacological effects with the percentage of receptor sites which benzodiazepines occupy in the human brain. P300 also seems to be useful to investigate the pharmacological effects of benzodiazepines.

Lesion of the nucleus basalis magnocellularis does not affect cerebral cortical blood flow in rats.

The effects of a unilateral ibotenic acid lesion of the nucleus basalis magnocellularis (NBM) on blood flow of the cerebral cortex and striatum were studied at 2, 4, 8 and 16 weeks after the lesion in conscious rats. In the cerebral cortex, no side-to-side difference in blood flow was observed, though cholinergic enzyme activity was markedly reduced on the side of the lesion. The results suggest that NBM lesion produces disturbance of cholinergic neurons in the cerebral cortex without significant alteration of blood flow.

Age-related changes in human muscarinic acetylcholine receptors measured by positron emission tomography.

The effects of age on the binding parameters of [11C]N- methyl-4-piperidylbenzilate ([11C]NMPB), a specific muscarinic cholinergic receptor ligand, were studied. Eighteen healthy male volunteers (18-75 years old) participated. Regional radioactivity in the brain was followed for 60 min by positron emission tomography (PET). Uptake of [11C]NMPB continuously increased in all brain areas with the exception of the cerebellum. For the quantification of receptor binding, a compartment model, in which radioactivity in the cerebellum was used as an input function, was used. The binding parameter, K3, of muscarinic acetylcholine receptors in eight brain regions (pons, hippocampus, frontal cortex, striatum, temporal cortex, thalamus, occipital cortex, parietal cortex) showed an age-related decrease of about 45% over the age range.

No age-related changes in human benzodiazepine receptor binding measured by PET with [11C]Ro 15-4513.

The effects of age on the binding of [11C]Ro 15-4513, a partial inverse agonist of the central benzodiazepine receptor, were studied. Sixteen healthy male volunteers (21-78 years old) participated. Regional radioactivity in the brain was followed for 45 min by positron emission tomography after a bolus injection of [11C]Ro 15-4513. Similar tracer kinetics were observed in both young and old subjects. For the quantification of receptor binding in vivo, a compartment model, in which radioactivity in the pons was used as an input function, was applied. There were no significant changes in the binding potentials with age (P > 0.1) in ten brain regions. These observations delineate an interesting difference between central benzodiazepine (BZ) receptors and other neurotransmitter receptors in the human brain measured by PET that have been shown to have a reduction with age.

In vivo binding of [11C]Ro15-4513 in human brain measured with PET.

Ro15-4513, an azide derivative of benzodiazepine antagonist flumazenil (Ro15-1788), and Ro15-1788 were labelled with carbon 11. Sequential PET scans following injection of [11C]Ro15-4513 or [11C]Ro15-1788 into normal male healthy volunteers were measured, and kinetic analysis using pons as a reference region was performed. [11C]Ro15-4513 was highly accumulated in frontal cortex, temporal cortex, hippocampus and relatively lower accumulation in occipital cortex, whereas almost homogeneous distribution of Ro15-1788 throughout cortex area was seen. The kinetic analysis revealed that such differences of regional distribution in brain between two labelled ligands were mainly due to the regional difference of the dissociation rate constants in vivo (k4). [11C]Ro15-4513 may be a useful tool for the in vivo study of benzodiazepine receptors in human brain.

Serotonin 5-HT2 receptors in schizophrenic patients studied by positron emission tomography.

Using positron emission tomography (PET) and [11C]N-methylspiperone (NMSP), we examined 5-HT2 receptors in the cortex of schizophrenic patients in whom we previously observed decreased prefrontal D1 receptor binding. The subjects were 10 neuroleptic-naive schizophrenic patients, 7 schizophrenic patients who were drug-free but had previously been treated with neuroleptics, and 12 normal controls. A non-significant trend towards decreased prefrontal [11C]NMSP binding was observed in the neuroleptic-treated patients, suggesting a possible effect of previous neuroleptic treatment on the alteration in cortical 5-HT2 function. However, the neuroleptic-naive patients showed no noticeable difference in cortical [11C]NMSP binding compared to controls. Our results do not rule out the role of 5-HT2 function as a crucial site of therapeutic activity of schizophrenia, but they do suggest that cortical 5-HT2 receptors might not be primarily involved in the pathophysiology of schizophrenia.

Dopamine D2 and serotonin S2 receptors in susceptibility to methamphetamine psychosis detected by positron emission tomography.

Positron emission tomography (PET) was used to assess the role of dopamine D2 receptors in the striatum and serotonin S2 receptors in the frontal cortex in the susceptibility to methamphetamine-induced psychosis. Subjects were six men who had previously experienced methamphetamine psychosis (methamphetamine subjects) and 10 age- and sex-matched control subjects. The radiotracer used was 11C-N-methylspiperone. Although binding availability, assessed by dynamic analysis, in the two regions did not differ between the two groups, the ratio of binding availability in the striatum to that in the frontal cortex significantly decreased in the methamphetamine subjects as compared with the control subjects. These findings suggest that an imbalance in the activity of these two receptors may be related to the susceptibility to methamphetamine psychosis.