Dopamine D2/3 Receptor Availabilities in Striatal and Extrastriatal Regions of the Adult Human Brain: Comparison of Four Methods of Analysis.

Values of binding potentials (BPND) of dopamine D2/3 receptors differ in different regions of the brain, but we do not know with certainty how much of this difference is due either to different receptor numbers, or to different affinities of tracers to the receptors, or to both. We tested the claim that both striatal and extrastriatal dopamine D2/3 receptor availabilities vary with age in vivo in humans by determining the values of BPND of the specific radioligand [11C]raclopride. We determined values of BPND in striatal and extrastriatal volumes-of-interest (VOI) with the same specific receptor radioligand. We estimated values of BPND in individual voxels of brains of healthy volunteers in vivo, and we obtained regional averages of VOI by dynamic positron emission tomography (PET). We calculated average values of BPND in caudate nucleus and putamen of striatum, and in frontal, occipital, parietal, and temporal cortices of the forebrain, by means of four methods, including the ERLiBiRD (Estimation of Reversible Ligand Binding and Receptor Density) method, the tissue reference methods of Logan and Logan-Ichise, respectively, and the SRTM (Simplified Reference Tissue Method). Voxelwise generation of parametric maps of values of BPND used the multi-linear regression version of SRTM. Age-dependent changes of the binding potential presented with an inverted U-shape with peak binding potentials reached between the ages of 20 and 30. The estimates of BPND declined significantly with age after the peak in both striatal and extrastriatal regions, as determined by all four methods, with the greatest decline observed in posterior (occipital and parietal) cortices (14% per decade) and the lowest decline in caudate nucleus (3% per decade). The sites of the greatest declines are of particular interest because of the clinical implications.

Cloning, Purification, and Characterization of Tripeptidyl Peptidase from Streptomyces herbaricolor TY-21.

Tripeptidyl peptidase (TPP) is an exopeptidase that sequentially hydrolyzes tripeptides from the N-terminus of oligopeptides or polypeptides. We performed screening for isolating novel TPP-producing microorganisms from soil samples. TPP activity was observed in the culture supernatant of Streptomyces herbaricolor TY-21 by using Ala-Ala-Phe-p-nitroanilide (pNA) as the substrate. TPP from the culture supernatant was purified to approximately 790-fold. It was shown to cleave oxidized insulin B-chain, thereby with releasing tripeptide units, but not the N-terminal-protected peptide, Cbz-Ala-Ala-Phe-pNA. The TPP gene, designated tpp, was isolated from a partial genomic DNA library of S. herbaricolor TY-21. The TPP gene consisted of 1488 bp, and encoded a 133-amino acid pre-pro-peptide and a 362-amino acid mature enzyme containing conserved amino acid residues (Asp-36, His-77, and Ser-282) similar to the catalytic residues in subtilisin. TY-21 TPP belonged to the peptidase S8A family in the MEROPS database. The mature TY-21 TPP showed approximately 49% identity with tripeptidyl peptidase subtilisin-like (TPP S) from Streptomyces lividans strain 66.

GfsA is a ß1,5-galactofuranosyltransferase involved in the biosynthesis of the galactofuran side chain of fungal-type galactomannan in Aspergillus fumigatus.

Previously, we reported that GfsA is a novel galactofuranosyltransferase involved in the biosynthesis of O-glycan, the proper maintenance of fungal morphology, the formation of conidia and anti-fungal resistance in Aspergillus nidulans and A. fumigatus (Komachi Y et al., 2013. GfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O-glycan in Aspergillus nidulans and Aspergillus fumigatus. Mol. Microbiol. 90:1054-1073). In the present paper, to gain an in depth-understanding of the enzymatic functions of GfsA in A. fumigatus (AfGfsA), we established an in vitro assay to measure galactofuranosyltransferase activity using purified AfGfsA, UDP-α-d-galactofuranose as a sugar donor, and p-nitrophenyl-ß-d-galactofuranoside as an acceptor substrate. LC/MS, 1H-NMR and methylation analyses of the enzymatic products of AfGfsA revealed that this protein has the ability to transfer galactofuranose to the C-5 position of the ß-galactofuranose residue via a ß-linkage. AfGfsA requires a divalent cation of manganese for maximal activity and consumes UDP-α-d-galactofuranose as a sugar donor. Its optimal pH range is 6.5-7.5 and its optimal temperature range is 20-30°C. 1H-NMR, 13C-NMR and methylation analyses of fungal-type galactomannan extracted from the ∆AfgfsA strain revealed that AfGfsA is responsible for the biosynthesis of ß1,5-galactofuranose in the galactofuran side chain of fungal-type galactomannan. Based on these results, we conclude that AfGfsA acts as a UDP-α-d-galactofuranose: ß-d-galactofuranoside ß1,5-galactofuranosyltransferase in the biosynthetic pathway of galactomannans.

Indicators of the need for insulin treatment and the effect of treatment for gestational diabetes on pregnancy outcomes in Japan.

This study assessed indicators of the need for insulin therapy and the effect of treatment on pregnancy outcomes in Japanese patients with gestational diabetes mellitus (GDM). All patients diagnosed with GDM were hospitalized for three days. Plasma glucose profiles in patients under strict dietary management and the characteristics of GDM patients with high daily glucose levels were investigated. Patients who failed to achieve glycemic targets were treated with insulin. Indicators of the need for insulin treatment were investigated. Pregnancy outcomes in patients prescribed dietary management and patients prescribed insulin treatment were compared. The study included 112 patients with GDM. GDM patients with high daily glucose levels in the hospital exhibited significantly higher 1-h and 2-h plasma glucose levels in oral glucose tolerance tests (OGTTs) at diagnosis. In our hospital, 102 GDM patients with singleton pregnancies were followed until delivery; 32 (31.3%) were treated with insulin. Univariate analysis identified significant associations of insulin requirement with family history of diabetes and with 1-h and 2-h OGTT values at diagnosis. Multivariate analysis showed that the 1-h OGTT plasma glucose level at diagnosis was an independent predictor of the need for insulin. In perinatal outcomes, insulin treatment was associated with low birth weight.

Isolation, sequencing, and heterologous expression of the Paecilomyces variotii gene encoding S-hydroxymethylglutathione dehydrogenase (fldA).

The filamentous fungus Paecilomyces variotii NBRC 109023 (teleomorph: Byssochlamys spectabilis NBRC 109023) degrades formaldehyde at concentrations as high as 2.4 % (w/v). In many prokaryotes and in all known eukaryotes, formaldehyde degradation is catalyzed by S-hydroxymethylglutathione (S-HMGSH) dehydrogenase. We report here the isolation and characterization of the gene encoding S-HMGSH dehydrogenase activity in P. variotii. The 1.6-kb fldA gene contained 5 introns and 6 exons, and the corresponding cDNA was 1143 bp, encoding a 40-kDa protein composed of 380 amino acids. FldA was predicted to have 74.3, 73.7, 68.5, and 67.4 % amino acid identity to the S-HMGSH dehydrogenases of Hansenula polymorpha, Candida boidinii, Saccharomyces cerevisiae, and Kluyveromyces lactis, respectively. The predicted protein also showed high amino acid similarity (84∼86 %) to the products of putative fldA genes from other filamentous fungi, including Aspergillus sp. and Penicillium sp. Notably, the P. variotii fldA gene was able to functionally complement a Saccharomyces cerevisiae strain (BY4741 ∆sfa1) lacking the gene for S-HMGSH dehydrogenase. The heterologous expression construct rendered BY4741 ∆sfa1 tolerant to exogenous formaldehyde. Although BY4741 (parental wild-type strain) was unable to degrade even low concentrations of formaldehyde, BY4741 ∆sfa1 harboring Paecilomyces fldA was able to degrade 4 mM formaldehyde within 30 h. The findings from this study confirm the essential role of S-HMGSH dehydrogenase in detoxifying formaldehyde.

Cloning and characterization of a unique cytotoxic protein parasporin-5 produced by Bacillus thuringiensis A1100 strain.

Parasporin is the cytocidal protein present in the parasporal inclusion of the non-insecticidal Bacillus thuringiensis strains, which has no hemolytic activity but has cytocidal activities, preferentially killing cancer cells. In this study, we characterized a cytocidal protein that belongs to this category, which was designated parasporin-5 (PS5). PS5 was purified from B. thuringiensis serovar tohokuensis strain A1100 based on its cytocidal activity against human leukemic T cells (MOLT-4). The 50% effective concentration (EC50) of PS5 to MOLT-4 cells was approximately 0.075 µg/mL. PS5 was expressed as a 33.8-kDa inactive precursor protein and exhibited cytocidal activity only when degraded by protease at the C-terminal into smaller molecules of 29.8 kDa. Although PS5 showed no significant homology with other known parasporins, a Position Specific Iterative-Basic Local Alignment Search Tool (PSI-BLAST) search revealed that the protein showed slight homology to, not only some B. thuringiensis Cry toxins, but also to aerolysin-type ß-pore-forming toxins (ß-PFTs). The recombinant PS5 protein could be obtained as an active protein only when it was expressed in a precursor followed by processing with proteinase K. The cytotoxic activities of the protein against various mammalian cell lines were evaluated. PS5 showed strong cytocidal activity to seven of 18 mammalian cell lines tested, and low to no cytotoxicity to the others.

Draft Genome Sequence of the Formaldehyde-Resistant Fungus Byssochlamys spectabilis No. 5 (Anamorph Paecilomyces variotii No. 5) (NBRC109023).

Byssochlamys spectabilis no. 5 (anamorph Paecilomyces variotii no. 5) (NBRC109023) was isolated from a soil sample in 2001 in Kumamoto Prefecture, Japan. This fungus is highly resistant to formaldehyde. Here, we report a draft genome sequence of P. variotii no. 5; this draft was produced with the intent of investigating the mechanism of formaldehyde resistance. This is the first report of the genome sequence of any Paecilomyces species.

GfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O-glycan in Aspergillus nidulans and Aspergillus fumigatus.

The cells walls of filamentous fungi in the genus Aspergillus have galactofuranose (Galf)-containing polysaccharides and glycoconjugates, including O-glycans, N-glycans, fungal-type galactomannan and glycosylinositolphosphoceramide, which are important for cell wall integrity. Here, we attempted to identify galactofuranosyltransferases that couple Galf monomers onto other wall components in Aspergillus nidulans. Using reverse-genetic and biochemical approaches, we identified that the AN8677 gene encoded a galactofuranosyltransferase, which we called GfsA, involved in Galf antigen biosynthesis. Disruption of gfsA reduced binding of ß-Galf-specific antibody EB-A2 to O-glycosylated WscA protein and galactomannoproteins. The results of an in-vitro Galf antigen synthase assay revealed that GfsA has ß1,5- or ß1,6-galactofuranosyltransferase activity for O-glycans in glycoproteins, uses UDP-d-Galf as a sugar donor, and requires a divalent manganese cation for activity. GfsA was found to be localized at the Golgi apparatus based on cellular fractionation experiments. ΔgfsA cells exhibited an abnormal morphology characterized by poor hyphal extension, hyphal curvature and limited formation of conidia. Several gfsA orthologues were identified in members of the Pezizomycotina subphylum of Ascomycota, including the human pathogen Aspergillus fumigatus. To our knowledge, this is the first characterization of a fungal ß-galactofuranosyltransferase, which was shown to be involved in Galf antigen biosynthesis of O-glycans in the Golgi.

Study of the distribution by age group of serum cross-linked C-terminal telopeptide of type I collagen and procollagen type I N-propeptide in healthy Japanese women to establish reference values.

Osteoporosis prevention is an important public health goal. Bone turnover markers are clinically measured to assess bone strength. C-terminal telopeptide of type I collagen (CTX) is released when collagens degrade and serves as an indicator of bone resorption. Simple CTX immunoassays are now available. However, serum CTX (sCTX) reference ranges for Japanese women are lacking. Procollagen type I N-propeptide (intact P1NP) reflects osteoblast activity, serving as a marker of bone formation. Because sCTX and intact P1NP are clinically applied as bone turnover markers, we determined reference ranges for both sCTX and intact P1NP in healthy Japanese women. We collected 228 blood samples from healthy Japanese women aged 19-83 years, grouped by age and menopausal status. We measured sCTX and intact P1NP and examined their correlation. sCTX values differed significantly between the two consecutive decade groups encompassing 19-39 years of age, intact P1NP values between 20 and 30 s, between post-menopausal 50 and 60 s, and between pre-and post-menopausal women in their 50 s. The mean sCTX of 91 healthy pre-menopausal women was 0.255 (0.100-0.653) ng/mL, the intact P1NP in 90 women 33.2 (17.1-64.7) µg/L. Corresponding values for post-menopausal women were 0.345 (0.115-1.030) ng/mL and 41.6 (21.9-79.1) µg/L. sCTX correlated with intact P1NP. Bone resorption markers are measured to assess anti-resorption agents, bone formation markers to assess the effects of bone-forming agents. The sCTX and intact P1NP reference values determined herein, in healthy Japanese women, are expected to be useful for osteoporosis treatment, assessment of fracture risk, and other clinical applications.

Purification and properties of S-hydroxymethylglutathione dehydrogenase of Paecilomyces variotii no. 5, a formaldehyde-degrading fungus.

S-hydroxymethylglutathione dehydrogenase from Paecilomyces variotii No. 5 strain (NBRC 109023), isolated as a formaldehyde-degrading fungus, was purified by a procedure that included ammonium sulfate precipitation, DEAE-Sepharose and hydroxyapatite chromatography and isoelectrofocusing. Approximately 122-fold purification was achieved with a yield of 10.5%. The enzyme preparation was homogeneous as judged by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the purified enzyme was estimated to be 49 kDa by SDS-PAGE and gel filtration, suggesting that it is a monomer. Enzyme activity was optimal at pH 8.0 and was stable in the range of pH 7.0-10. The optimum temperature for activity was 40°C and the enzyme was stable up to 40°C. The isoelectric point was pH 5.8. Substrate specificity was very high for formaldehyde. Besides formaldehyde, the only aldehyde or alcohol tested that served as a substrate was pyruvaldehyde. Enzyme activity was enhanced by several divalent cations such as Mn2+ (179%), Ba2+ (132%), and Ca2+ (112%) but was completely inhibited by Ni2+, Fe3+, Hg2+, p-chloromercuribenzoate (PCMB) and cuprizone. Inactivation of the enzyme by sulfhydryl reagents (Hg2+ and PCMB) indicated that the sulfhydryl group of the enzyme is essential for catalytic activity.

Characterization of endoplasmic reticulum-localized UDP-D-galactose: hydroxyproline O-galactosyltransferase using synthetic peptide substrates in Arabidopsis.

We characterized peptidyl hydroxyproline (Hyp) O-galactosyltransferase (HGT), which is the initial enzyme in the arabinogalactan biosynthetic pathway. An in vitro assay of HGT activity was established using chemically synthesized fluorescent peptides as acceptor substrates and extracts from Arabidopsis (Arabidopsis thaliana) T87 cells as a source of crude enzyme. The galactose residue transferred to the peptide could be detected by high-performance liquid chromatography and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analyses. HGT required a divalent cation of manganese for maximal activity and consumed UDP-D-galactose as a sugar donor. HGT exhibited an optimal pH range of pH 7.0 to 8.0 and an optimal temperature of 35 degrees C. The favorable substrates for the activity seemed to be peptides containing two alternating imino acid residues including at least one acceptor Hyp residue, although a peptide with single Hyp residue without any other imino acids also functioned as a substrate. The results of sucrose density gradient centrifugation revealed that the cellular localization of HGT activity is identical to those of endoplasmic reticulum markers such as Sec61 and Bip, indicating that HGT is predominantly localized to the endoplasmic reticulum. To our knowledge, this is the first characterization of HGT, and the data provide evidence that arabinogalactan biosynthesis occurs in the protein transport pathway.

Salvage chemotherapy with a combination of irinotecan hydrochloride and mitomycin C in elderly Japanese patients with gynecological malignancies: a pilot study.

The efficacy and toxicity of salvage chemotherapy with a combination of irinotecan hydrochloride (CPT-11) and mitomycin C (MMC) in elderly Japanese patients with gynecological malignancies are reported. Six patients, aged 75 years or older, two with stage IV uterine cervical cancer and four with platinum- and taxane-resistant ovarian cancer, were treated. CPT-11 (120 mg/m(2)) was administered intravenously for 4 h with 500 ml of normal saline solution, followed by 3.5 mg/m(2) of MMC with 100 ml of normal saline, administered for 60 min, on days 1 and 15 and every 5 weeks thereafter. Six patients received a total of 24 courses of treatment, which provided an opportunity to explore, in a preliminary manner, the efficacy and toxicity of this approach. All patients demonstrated reasonable tolerance to this management strategy. There were no deaths attributable to toxicity. In a carefully selected patient population, CPT-11 and MMC chemotherapy could be administered to elderly Japanese patients with gynecological malignancies. Phase I and II studies are needed to confirm the feasibility of this treatment.

Magnetic resonance imaging of synovial proliferation in temporomandibular disorders with pain.

OBJECTIVE: The objective of this study was to assess the clinical significance of synovial proliferation in patients with painful temporomandibular disorders based on magnetic resonance imaging findings. METHODS: The current study was conducted in 100 joints of 100 patients with unilateral painful temporomandibular disorders. One hundred joints on the contralateral side of patients with unilateral disease were used as nonpain group. Areas in the articular space that showed a low signal intensity on T1-weighted imaging, a high signal intensity on T2-weighted imaging, and high signal intensity on gadolinium-enhanced fat-suppressed T1-weighted imaging were judged to be regions of synovial proliferation. RESULTS: Synovial proliferation alone was observed in 8.0% of the pain group, but in none of the nonpain group. Synovial proliferation + effusion was observed in 33.0% of the pain group and in 7.0% of the nonpain group. Effusion alone was observed in 7.0% of the pain group and in 3.0% of the nonpain group. The mean visual analog scale value of pain was in the order of synovial proliferation alone > synovial proliferation + effusion > effusion alone. The incidence rates of anterior displacement of the disk were 100% for synovial proliferation alone, 93.9% for synovial proliferation + effusion, 57.1% for effusion alone, and 57.7% for "without synovial proliferation/effusion." CONCLUSIONS: Strong correlations were observed between synovial proliferation, pain, and disk displacement. It is considered that evaluating effusion alone provides only limited information on the disease state in painful temporomandibular disorders. Thus, it is essential to include enhanced T1-weighted imaging as a means to judge the disease state as well as to assess disease progression.

Compatible solutes protect against chaotrope (ethanol)-induced, nonosmotic water stress.

Water stress is one of the major stresses experienced by cellular systems and can take a number of distinct forms. In response to turgor-related osmotic stress, cells produce compatible solutes that are macromolecule protectants and also counteract the outflow of water from stressed cells. In this report we show that the germination of conidia of Aspergillus nidulans, a sensitive indicator of water stress, in the presence of ethanol is correlated with the intracellular concentration of the compatible solutes glycerol and erythritol, which protect against both osmotic and nonturgor forms of water stress.

[Multi-center study for the evaluation of clinical usefulness of attenuation and scatter correction on 201Tl myocardial SPECT].

The aim of this study was to evaluate the clinical usefulness of attenuation and scatter correction (AC, SC) on a 201Tl myocardial single-photon emission computed tomography (201Tl SPECT) as a multi-center trial. With a dual-detecter and a triple-detector SPECT systems with a 99mTc transmission source, simultaneous transmission/emission tomography (TCT/ECT) was performed on 38 patients with angiographically coronary heart disease (CHD) and 26 patients without evidence of CHD. Stress and delayed attenuation and scatter corrected images (SAC) and uncorrected images (NC) were reconstructed. On NC images of normal cases, influence of attenuation was greater in male than female. In comparison of 201Tl distribution between male and female, significant decrease in 201Tl activity was observed in the inferoposterior wall in male and that was observed in the anterobasal wall of the left myocardium in female. Such a difference in 201Tl distribution between male and female disappeared on SAC images. On the diagnostic performance for the identification of CHD, SAC images demonstrated improved specificity and accuracy values in the right coronary arterial territory (RCA) with visual analysis statistically. Sensitivity value in the RCA was also improved, but it was not statistically significant. Sensitivity value in the left circumflex arterial territory (LCX) increased without decrease in specificity value on SAC images. In the left anterior descending arterial territory (LAD), sensitivity value increased on SAC images. Although specificity value decreased on SAC images in LAD territory, it was not statistically significant. The difference in 201Tl distribution between male and female is improved in normal cases by attenuation and scatter correction on 201Tl myocardial SPECT. Diagnostic performance of CHD is also improved by attenuation and scatter correction, especially in territories of which specificity in assessing the absence of disease have been suboptimal. In conclusion, attenuation and scatter correction on 201Tl myocardial SPECT is considered to be clinically useful.

Acetic Acid Production by an Electrodialysis Fermentation Method with a Computerized Control System.

In acetic acid fermentation by Acetobacter aceti, the acetic acid produced inhibits the production of acetic acid by this microorganism. To alleviate this inhibitory effect, we developed an electrodialysis fermentation method such that acetic acid is continuously removed from the broth. The fermentation unit has a computerized system for the control of the pH and the concentration of ethanol in the fermentation broth. The electrodialysis fermentation system resulted in improved cell growth and higher productivity over an extended period; the productivity exceeded that from non-pH-controlled fermentation. During electrodialysis fermentation in our system, 97.6 g of acetic acid was produced from 86.0 g of ethanol; the amount of acetic acid was about 2.4 times greater than that produced by non-pH-controlled fermentation (40.1 g of acetic acid produced from 33.8 g of ethanol). Maximum productivity of electrodialysis fermentation in our system was 2.13 g/h, a rate which was 1.35 times higher than that of non-pH-controlled fermentation (1.58 g/h).