Age-related decline of serotonin transporters in living human brain of healthy males

There is growing interest in serotonin transporter (5-HTT) function in the human brain, since alteration in 5-HTT has been suggested in a variety of neurophychiatric disorders. Age-related decline in postsynaptic 5-HT receptors has been demonstrated in postmortem human studies and in vivo imaging studies, and has been assumed to be related to changes in mental function in the normal aging process. However, few studies have investigated the aging effect on 5-HTT in human brain in vivo, since the availability of suitable ligands has been limited. To investigate the aging effect on 5-HTT in living human brain, we performed positron emission tomography (PET) scans with a selective ligand for 5-HTT, [11C](+)McN5652. We examined 28 healthy male volunteers aged between 20 and 79 years. The uptake was quantified in the thalamus and midbrain by graphical analysis with the cerebellum as a reference tissue, and binding potential (BP) was used for the index of 5-HTT binding. There was a significant age-related decline in BP in the thalamus and midbrain. The decline in [11C](+)McN5652 binding was 9.6% per decade in the thalamus and 10.5% per decade in the midbrain.     

Egg Yolk antibody and its application

A hen transfers her serum immumnoglobulin G to the egg yolk (IgY) and gives im|munity to her offspring. Therefore, the hen egg can be an effective supplier of a large amount of antigen specific antibody that accumulates in the egg yolk. Antigen specific antibody has been widely used for immunological analysis in the field of diagnosis as well as pure scientific research. The production and separation technology of IgY is demonstrated in the present study.     

Gut symbiotic bacteria in the cabbage bugs <Emphasis Type="Italic">Eurydema rugosa</Emphasis> and <Emphasis Type="Italic">Eurydema dominulus</Emphasis> (Heteroptera: Pentatomidae)

The cabbage bugs Eurydema rugosa Motschulsky and Eurydema dominulus (Scopoli) (Heteroptera: Pentatomidae: Strachiini) possess a number of crypts in a posterior region of the midgut, which are filled with bacterial symbiont cells. Here we characterized the gut symbionts of Eurydema stinkbugs using molecular phylogenetic and histological techniques. Specific gammaproteobacteria were consistently identified from the posterior midgut of E. rugosa representing nine populations and E. dominulus representing six populations, respectively. The bacterial 16S rRNA gene sequences were identical within the species but slightly different (98.2% sequence identity) between the species. Molecular phylogenetic analysis revealed that the Eurydema symbionts formed a well-defined monophyletic group in the Gammaproteobacteria. The symbionts were phylogenetically distinct from the gut symbionts of the stinkbug families Acanthosomatidae, Plataspidae, Parastrachiidae, Scutelleridae, and other pentatomid species, suggesting multiple evolutionary origins of the gut symbiotic bacteria among diverse stinkbugs. In situ hybridization confirmed that the symbiont is located in the cavity of the midgut crypts. Aposymbiotic insects of E. rugosa, which were produced by egg surface sterilization, were viable but suffered retarded growth, reduced body weight, and abnormal body color, suggesting the biological importance of the symbiont for the host.     

Properties of an adrenal cytochrome P-450 (P-45011β) for the hydroxylations of corticosteroids

A highly purified preparation of cytochrome P-450, designated as P-450_11β, has been obtained from bovine adrenal cortex mitochondria. The P-450_11β exhibits remarkably high steroid hydroxylase activity in the reconstituted adrenal electron-donating system from NADPH via NADPH:adrenal ferredoxin oxidoreductase (EC 1.6.7.1) and adrenal ferredoxin. The turnover numbers (moles of hydroxylated product formed per minute per mole of P-450-heme) are 110 and 18 for respective 11β- and 18-hydroxylase activity when deoxycorticosterone is the substrate. The apparent K_m value is 6 μm for both reactions. The ratio, about 6:1 between the two activities, is constant under various experimental conditions including those in the presence of competitive inhibitors of hydroxylation. In addition to deoxycorticosterone, other steroids such as 11-deoxycortisol, 4-androstene-3,17-dione and testosterone are the hydroxylatable substrates. In cases in which 4-androstene-3,17-dione, a C₁₉-steroid, is the substrate, the hydroxylatable sites appear to be its respective 11β- and 19-position. The ratio between the two activities is about 4:1. In view of these results, it is concluded that one hemoprotein species, the P-450_11β, is responsible for the hydroxylase reactions of various Corticosteroids. 2-Methyl-1,2-di-3-pyridyl-1-propanone (metyrapone) inhibits the P-450_11β-catalyzed steroid hydroxylase reactions of either deoxycorticosterone at 11β- and 18-position or 4-androstene-3,17-dione at 11β- and 19-position (K_i = 0.1-0.2 μM). The P-450_scc-catalyzed cholesterol desmolase reaction is also inhibited, although weakly (K_i = 160 μM). In addition, both adrenal cytochromes appeared to differ from each other in spectral response to metyrapone.     

Simultaneous Copy Number Losses within Multiple Subtelomeric Regions in Early-Onset Type2 Diabetes Mellitus

Genetic factors play very important roles in the onset and progression of type 2 diabetes mellitus (T2DM). However, the genetic factors correlating with T2DM onset have not as yet been fully clarified. We previously found that copy number losses in the subtelomeric region on chromosome 4p16.3 were detected in early-onset Japanese T2DM patients (onset age <35 years) at a high frequency. Herein, we additionally found two novel copy number losses within the subtelomeric regions on chromosomes 16q24.2-3 and 22q13.31-33, which have significant associations with early-onset Japanese T2DM. The associations were statistically significant by Fisher''s exact tests with P values of 5.19×10⁻³ and 1.81×10⁻³ and odds ratios of 5.7 and 4.4 for 16q24.2-3 and 22q13.31-33, respectively. Furthermore, copy number variation (CNV) analysis of the whole genome using the CNV BeadChip system verified simultaneous copy number losses in all three subtelomeric regions in 11 of our 100 T2DM subjects, while none of 100 non-diabetic controls showed the copy number losses in all three regions. Our results suggest that the mechanism underlying induction of CNVs is involved in the pathogenesis of early-onset T2DM. Thus, copy number losses within multiple subtelomeric regions are strongly associated with early-onset T2DM and examination of simultaneous CNVs in these three regions may lead to the development of an accurate and selective procedure for detecting genetic susceptibility to T2DM.     

Aceruloplasminemia, an inherited disorder of iron metabolism

Ceruloplasmin, a multi-copper ferroxidase that affects the distribution of tissue iron, has antioxidant effects through the oxidation of ferrous iron to ferric iron. Aceruloplasminemia is an inherited disorder of iron metabolism due to the complete lack of ceruloplasmin ferroxidase activity caused by mutations in the ceruloplasmin gene. It is characterized by iron accumulation in the brain as well as visceral organs. Clinically, the disease consists of the triad of retinal degeneration, diabetes mellitus, and neurological disease, which include ataxia, involuntary movements, and dementia. These symptoms reflect the sites of iron deposition. The unique involvement of the central nervous system distinguishes aceruloplasminemia from other inherited and acquired iron storage disorders. Twenty-one mutations in the ceruloplasmin gene have been reported in 24 families worldwide. In Japan, the incidence was estimated to be approximately one per 2,000,000 in the case of non-consanguineous marriages. Excess iron functions as a potent catalyst of biologic oxidation. Previously we showed that an increased iron concentration is associated with increased levels of lipid peroxidation in the serum, cerebrospinal fluid, and erythrocyte membranes. The levels of malondialdehyde and 4-hydroxynonenals, indicators of lipid peroxidation, were also elevated in the basal ganglia and cerebral cortex. Positron emission tomography showed diminished brain metabolism of glucose and oxygen. Enzyme activities in the mitochondrial respiratory chain of the basal ganglia were reduced to approximate 45% and 42%, respectively, for complexes I and IV. These findings suggest that iron-mediated free radicals causes neuronal cell damage through lipid peroxidation and mitochondrial dysfunction in aceruloplasminemia brains.     

Diversity of Wolbachia endosymbionts in heteropteran bugs

An extensive survey of Wolbachia endosymbionts in Japanese terrestrial heteropteran bugs was performed by PCR detection with universal primers for wsp and ftsZ genes of Wolbachia, cloning of the PCR products, restriction fragment length polymorphism analysis of infecting Wolbachia types, and molecular phylogenetic characterization of all the detected Wolbachia strains. Of 134 heteropteran species from 19 families examined, Wolbachia infection was detected in 47 species from 13 families. From the 47 species, 59 Wolbachia strains were identified. Of the 59 strains, 16 and 43 were assigned to A group and B group in the Wolbachia phylogeny, respectively. The 47 species of Wolbachia-infected bugs were classified into 8 species with A infection, 28 species with B infection, 2 species with AA infection, 3 species with AB infection, 5 species with BB infection, and 1 species with ABB infection. Molecular phylogenetic analysis showed little congruence between Wolbachia phylogeny and host systematics, suggesting frequent horizontal transfers of Wolbachia in the evolutionary course of the Heteroptera. The phylogenetic analysis also revealed several novel lineages of Wolbachia. Based on statistical analyses of the multiple infections, we propose a hypothetical view that, in the heteropteran bugs, interactions between coinfecting Wolbachia strains are generally not intense and that Wolbachia coinfections have been established through a stochastic process probably depending on occasional horizontal transfers.     

Novel clade of alphaproteobacterial endosymbionts associated with stinkbugs and other arthropods

Here we report a novel clade of secondary endosymbionts associated with insects and other arthropods. Seed bugs of the genus Nysius (Hemiptera: Lygaeidae) harbor the primary gammaproteobacterial symbiont Schneideria nysicola within a pair of bacteriomes in the abdomen. Our survey of Nysius species for their facultative bacterial associates consistently yielded a novel type of alphaproteobacterial 16S rRNA gene sequence in addition to those of Wolbachia. Diagnostic PCR survey of 343 individuals representing 24 populations of four Nysius species revealed overall detection rates of the alphaproteobacteria at 77.6% in Nysius plebeius, 87.7% in Nysius sp. 1, 81.0% in Nysius sp. 2, and 100% in Nysius expressus. Further survey of diverse stinkbugs representing 24 families, 191 species, and 582 individuals detected the alphaproteobacteria from an additional 12 species representing six families. Molecular phylogenetic analysis showed that the alphaproteobacteria from the stinkbugs form a distinct and coherent monophyletic group in the order Rickettsiales together with several uncharacterized endosymbionts from fleas and ticks. The alphaproteobacterial symbiont clade was allied to bacterial clades such as the endosymbionts of acanthamoebae, the endosymbionts of cnidarians, and Midichloria spp., the mitochondrion-associated endosymbionts of ticks. In situ hybridization and electron microscopy identified small filamentous bacterial cells in various tissues of N. plebeius, including the bacteriome and ovary. The concentrated localization of the symbiont cells at the anterior pole of oocytes indicated its vertical transmission route through host insect generations. The designation "Candidatus Lariskella arthropodarum" is proposed for the endosymbiont clade.     

Crystal Structure of the Parasporin-2 Bacillus thuringiensis Toxin That Recognizes Cancer Cells

Parasporin-2 is a protein toxin that is isolated from parasporal inclusions of the Gram-positive bacterium Bacillus thuringiensis. Although B. thuringiensis is generally known as a valuable source of insecticidal toxins, parasporin-2 is not insecticidal, but has a strong cytocidal activity in liver and colon cancer cells. The 37-kDa inactive nascent protein is proteolytically cleaved to the 30-kDa active form that loses both the N-terminal and the C-terminal segments. Accumulated cytological and biochemical observations on parasporin-2 imply that the protein is a pore-forming toxin. To confirm the hypothesis, we have determined the crystal structure of its active form at a resolution of 2.38 Å. The protein is unusually elongated and mainly comprises long β-strands aligned with its long axis. It is similar to aerolysin-type β-pore-forming toxins, which strongly reinforce the pore-forming hypothesis. The molecule can be divided into three domains. Domain 1, comprising a small β-sheet sandwiched by short α-helices, is probably the target-binding module. Two other domains are both β-sandwiches and thought to be involved in oligomerization and pore formation. Domain 2 has a putative channel-forming β-hairpin characteristic of aerolysin-type toxins. The surface of the protein has an extensive track of exposed side chains of serine and threonine residues. The track might orient the molecule on the cell membrane when domain 1 binds to the target until oligomerization and pore formation are initiated. The β-hairpin has such a tight structure that it seems unlikely to reform as postulated in a recent model of pore formation developed for aerolysin-type toxins. A safety lock model is proposed as an inactivation mechanism by the N-terminal inhibitory segment.     

Functional and Structural Analysis of Influenza Virus Neuraminidase N3 Offers Further Insight into the Mechanisms of Oseltamivir Resistance

The influenza virus neuraminidase H274Y substitution is a highly prevalent amino acid substitution associated with resistance to the most heavily used influenza drug, oseltamivir. Previous structural studies suggest that the group specific 252 residue (Y252 in group 1 and T252 in group 2) might be a key factor underlying H274Y resistance. However, H274Y has only been reported in N1 subtypes, which indicates that there must be additional key residues that determine H274Y resistance. Furthermore, we found that members of NA serotype N3 also possess Y252, raising the key question as to whether or not H274Y resistance may also be possible for some group 2 NAs. Here, we demonstrate that the H274Y substitution results in mild oseltamivir resistance for N3. Comparative structural analysis of N3, N1, and their 274Y variants indicates that the interaction of residue 296 (H in N1 and nonaromatic for other serotypes) with conserved W295 is another important determinant of oseltamivir resistance.