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1.  Clinical endocrinological evaluation of the gonadal axis (testosterone, LH and FSH) in prostate cancer patients switched from a GnRH antagonist to a LHRH agonist 
To investigate the levels of testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prostate-specific antigen (PSA) in prostate cancer patients before and after the switch from degarelix to leuprolide treatments.
We enrolled 40 treatment-naïve prostate cancer patients who were treated initially with degarelix and were later switched to leuprolide. The subjects were divided into three groups depending on when they were switched to leuprolide: the 3-month group (3m; switched after 84 days, n=10), the 2-month group (2m; 56 days, n=10), and the 1-month group (1m; 28 days, n=20). Patient symptoms and hormone levels were measured after switching therapy. The castration level was defined as a serum testosterone level ≤50 ng/dl.
Thirty-nine subjects (97.5%) achieved castration levels of testosterone (11±5.8 ng/dl) 2 weeks after degarelix was first administered, and the characteristics of these patients were investigated. Testosterone levels increased and exceeded the castration level in one subject each of the 3m (142 ng/dl), 2m (72 ng/dl), and 1m groups (63 ng/dl). All subjects achieved the castration level by day 5. In contrast to testosterone levels, the LH and FSH surge on day 2 was significantly higher in the 1m group than in the other groups. The clinical symptoms were not exacerbated before or after switching in any patients.
A testosterone surge was observed in 8.3 % of the study patients; however, it was very short-lived and mild. LH and FSH levels were significantly higher 1 month after administration compared with 2 or 3 months after degarelix administration.
PMCID: PMC4490683  PMID: 26146562
Prostate cancer; GnRH-antagonist; LHRH-agonist; Testosterone
2.  Immunostimulatory Lipid Nanoparticles from Herbal Medicine 
Chemical biology & drug design  2014;83(4):493-497.
Reproducibility is an important issue in biological characterization of drug candidates and natural products. It is not uncommon to encounter cases in which supposedly the same sample exhibits very different biological activities. During our characterization of macrophage-stimulatory lipids from herbal medicine, it was found that the potency of these lipids could vary substantially from experiment to experiment. Further analysis of this reproducibility issue led to the discovery of solvent-dependent nanoparticle formation by these lipids. While larger nanoparticles (approximately 100 nm) of these lipids showed modest macrophage-stimulatory activity, smaller nanoparticles (<10 nm) of the same lipids exhibited substantially higher potency. Thus, the study revealed an unexpected link between nanoparticle formation and macrophage-stimulatory activity of plant lipids. Although nanoparticles have been extensively studied in the context of vehicles for drug delivery, our finding indicates that drugs themselves can form nanoassemblies, and their biological properties may be altered by the way they assemble.
PMCID: PMC4078915  PMID: 24495243
atomic force microscopy; dynamic light scattering; Juzen-taiho-to; macrophages; macrophages; nanoparticles; β-sitosteryl β-D-glucoside
3.  A phase 1 multiple-dose study of orteronel in Japanese patients with castration-resistant prostate cancer 
Orteronel (TAK-700) is a non-steroidal, selective, reversible inhibitor of 17,20-lyase. We evaluated the safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor effect of orteronel with or without prednisolone in Japanese patients with castration-resistant prostate cancer (CRPC).
We conducted a phase 1 study in men with progressive and chemotherapy-naïve CRPC. Patients received orteronel orally at doses of 200–400 mg twice daily (BID) with or without oral prednisolone (5 mg BID). Dose-limiting toxicity (DLT) was assessed during Cycle 1 (28 days). Patients could continue study treatment until any of criteria for treatment discontinuation were met. Gonadotropin-releasing hormone therapy was continued in patients without prior orchidectomy.
Fifteen patients were enrolled and administered at least one dose of orteronel. No DLTs were reported during Cycle 1 in this study. Adverse events (AEs) were reported in all 15 patients. Most common AEs (>30 %) were hyperlipasemia (47 %), hyperamylasemia (40 %), and constipation (33 %). Acute pancreatitis (Grades 2 and 3) and pancreatitis (Grade 1) were complicated in three patients during the study. Dose-dependent increase in plasma orteronel concentrations was indicated over the 200–400 mg BID dose range. Prednisolone coadministered did not alter PK of orteronel. Serum testosterone was rapidly suppressed below the lower limit of quantification across all doses. Of 15 subjects, 13 achieved at least a 50 % reduction from baseline in prostate-specific antigen.
Orteronel at doses up to 400 mg BID was tolerable in Japanese CRPC patients. The present results support further evaluation of orteronel with or without prednisolone.
PMCID: PMC4305367  PMID: 25537627
Orteronel; Castration-resistant prostate cancer; 17,20-Lyase inhibitor; Phase 1
4.  Negative differential resistance in ZnO coated peptide nanotube 
We investigate the room temperature electronic transport properties of a zinc oxide (ZnO) coated peptide nanotube contacted with Au electrodes. Current–voltage (I–V ) characteristics show asymmetric negative differential resistance (NDR) behavior along with current rectification. The NDR phenomenon is observed in both negative and positive voltage sweep scans, and found to be dependent on the scan rate and humidity. Our results suggest that the NDR is due to protonic conduction arising from water molecule redox reaction on the surface of ZnO coated peptide nanotubes rather than the conventional resonant tunneling mechanism.
PMCID: PMC4240313  PMID: 25419052
5.  Rational strategy for shaped nanomaterial synthesis in reverse micelle reactors 
Nature communications  2014;5:3870.
The shape-controlled synthesis of nanoparticles was established in single-phase solutions by controlling growth directions of crystalline facets on seed nanocrystals kinetically; however, it was difficult to rationally predict and design nanoparticle shapes. Here we introduce a methodology to fabricate nanoparticles in smaller sizes by evolving shapes thermodynamically. This strategy enables a more rational approach to fabricate shaped nanoparticles by etching specific positions of atoms on facets of seed nanocrystals in reverse micelle reactors where the surface energy gradient induces desorption of atoms on specific locations on the seed surfaces. From seeds of 12 nm palladium nanocubes, the shape is evolved to concave nanocubes and finally hollow nanocages in the size ~10 nm by etching the center of {200} facets. The high surface area-to-volume ratio and the exposure of a large number of palladium atoms on ledge and kink sites of hollow nanocages are advantageous to enhance catalytic activity and recyclability.
PMCID: PMC4112590  PMID: 24828960
6.  Suppression of Microbial Metabolic Pathways Inhibits the Generation of the Human Body Odor Component Diacetyl by Staphylococcus spp 
PLoS ONE  2014;9(11):e111833.
Diacetyl (2,3-butanedione) is a key contributor to unpleasant odors emanating from the axillae, feet, and head regions. To investigate the mechanism of diacetyl generation on human skin, resident skin bacteria were tested for the ability to produce diacetyl via metabolism of the main organic acids contained in human sweat. l-Lactate metabolism by Staphylococcus aureus and Staphylococcus epidermidis produced the highest amounts of diacetyl, as measured by high-performance liquid chromatography. Glycyrrhiza glabra root extract (GGR) and α-tocopheryl-l-ascorbate-2-O-phosphate diester potassium salt (EPC-K1), a phosphate diester of α-tocopherol and ascorbic acid, effectively inhibited diacetyl formation without bactericidal effects. Moreover, a metabolic flux analysis revealed that GGR and EPC-K1 suppressed diacetyl formation by inhibiting extracellular bacterial conversion of l-lactate to pyruvate or by altering intracellular metabolic flow into the citrate cycle, respectively, highlighting fundamentally distinct mechanisms by GGR and EPC-K1 to suppress diacetyl formation. These results provide new insight into diacetyl metabolism by human skin bacteria and identify a regulatory mechanism of diacetyl formation that can facilitate the development of effective deodorant agents.
PMCID: PMC4229079  PMID: 25390046
7.  Impedimetric Detection of Mutant p53 Biomarker-Driven Metastatic Breast Cancers under Hyposmotic Pressure 
PLoS ONE  2014;9(6):e99351.
In cancer cells, the oncogenic mutant p53 (mtp53) protein is present at high levels and gain-of-function (GOF) activities with more expression of mtp53 proteins contribute to tumor growth and metastasis. Robust analytical approaches that probe the degree of metastasis of cancer cells in connection with the mtp53 activity will be extremely useful not only for establishing a better cancer prognosis but also understanding the fundamental mechanism of mtp53 oncogenic action. Here we assessed the influence of mtp53 in breast cancers to the mechanical property of breast cancer cells. Recently, ovarian and kidney cancer cell lines have been shown to have higher cellular elasticity as compared to normal cells assessed by monitoring the degree of deformation under hyposmotic pressure. To make fast detection in large scale, the impedance measurement was applied to monitor the swelling ratio of cells with time. The results showed that knockdown of mtp53 leads to decrease in cell swelling. In addition, by means of two types of impedimetric detection systems we consistently detected enhancement of impedance signal in mtp53-expressing breast cancer cells. Based on this observation we hypothesize that highly expressed mtp53 in metastatic mutant breast cancers can promote tumor progression by making cells more deformable and easier to spread out through extracellular matrix. The identification via the electric measurement can be accomplished within 10 minutes. All results in this report suggest that electric probing for the extent of the mtp53 expression of breast cancer cells may serve as a meaningful fingerprint for the cancer diagnostics, and this outcome will also have an important clinical implication for the development of mtp53-based targeting for tumor detection and treatment.
PMCID: PMC4060997  PMID: 24937470
8.  New Autonomous Motors of Metal-Organic Framework (MOF) Powered by Reorganization of Self-Assembled Peptides at interfaces 
Nature materials  2012;11(12):1081-1085.
There have developed a variety of microsystems that harness energy and convert it to mechanical motion. Here we developed new autonomous biochemical motors by integrating metal-organic framework (MOF) and self-assembling peptides. MOF is applied as an energy-storing cell that assembles peptides inside nanoscale pores of the coordination framework. The robust assembling nature of peptides enables reconfiguring their assemblies at the water-MOF interface, which is converted to fuel energy. Re-organization of hydrophobic peptides could create the large surface tension gradient around the MOF and it efficiently powers the translation motion of MOF. As a comparison, the velocity of normalized by volume for the DPA-MOF particle is faster and the kinetic energy per the unit mass of fuel is more than twice as large as the one for previous gel motor systems. This demonstration opens the new application of MOF and reconfigurable molecular self-assembly and it may evolve into the smart autonomous motor that mimic bacteria to swim and harvest target chemicals by integrating recognition units.
PMCID: PMC3505225  PMID: 23104155
9.  Assemblies of Functional Peptides and Their Applications in Building Blocks for Biosensors 
Advanced functional materials  2011;21(6):1018-1026.
We highlight our recent applications of functional peptide nanotubes, self-assembled from short peptides with recognition elements, as building blocks to develop sensors. Peptide nanotubes with high aspect ratios are excellent building blocks for directed assembly into device configurations, and their combining structures with the nanometric diameters and the micrometric lengths enables to bridge the nano-world and the micro-world.
PMCID: PMC3584348  PMID: 23459763
peptide nanotube; biosensor; self-assembly; pathogens; heavy metals; bionanotechnology; electrochemistry
10.  Biomimetic Fabrication of Genetically-Engineered Collagen Peptide-Assembled Freestanding Films Reinforced by Quantum Dot Joints 
Soft matter  2012;8(26):6871-6875.
Genetically-engineered collagen peptides were assembled into freestanding films when QDs are co-assembled as joints between collagen domains. These peptide based films show excellent mechanical properties with Young’s modulus of ~20 GPa, much larger than most of multi-composite polymer films and previously reported freestanding nanoparticle-assembled sheets, and it is even close to the bone tissue in nature. These films show little permanent deformation under small indentation while the mechanical hysteresis becomes remarkable when the load approaches near and beyond the rupture point, which is also characteristic to the bone tissue.
PMCID: PMC3439209  PMID: 22982983
11.  Simple method for preventing inguinal hernias after radical retropubic prostatectomy 
Prostate International  2013;1(2):76-80.
Inguinal hernias often occur after radical retropubic prostatectomy (RRP). We present a novel and simple technique for preventing inguinal hernias after RRP, which any surgeon can complete within a few minutes.
A total of 230 Japanese prostate cancer patients underwent RRP between January 2007 and September 2011. From July 2009, 115 patients underwent inguinal hernia prevention procedures at the same time as RRP. In this procedure, we released approximately 5 cm of the bilateral vas deferens and spermatic vessels from the peritoneum. In cases in which the processus vaginalis had spread into the abdomen, we ligated it close to the peritoneal cavity and then transected it. The remaining 115 patients who underwent RRP but did not undergo the hernia prevention procedure were used as the control group. The incidence rate of postoperative inguinal hernia was compared between the 2 groups.
Inguinal hernias developed during the postoperative follow-up period in 18 of the 115 control patients (15.7%) (median duration, 50 months). The hernia-free survival rate of this group was 89.6% and 84.1% at 1 and 2 postoperative years, respectively. In contrast, only 1 of the 115 patients (0.87%) who underwent the hernia prevention procedure developed an inguinal hernia during the follow-up period (median duration, 27 months). The hernia-free survival rate of this group was 100% at both 1 and 2 postoperative years (P<0.0001).
We developed a simple method for preventing post-RRP inguinal hernias. The procedure is easy to perform and produces excellent outcomes.
PMCID: PMC3814116  PMID: 24223406
Inguinal hernia; Control and prevention; Prostate neoplasms; Prostatectomy
12.  Chlormadinone acetate is effective for hot flush during androgen deprivation therapy 
Prostate International  2013;1(3):113-116.
To investigate the clinical efficacy of low-dose chlormadinone acetate (CMA) in prostate cancer patients who suffer from hot flushes that is a major side effect of androgen deprivation therapy.
Our study included 32 prostate cancer patients who had severe hot flush after undergoing hormone therapy for more than 3 months. The average age of the patients was 72.5 years. In the beginning, patients received CMA at 100 mg orally per day. We defined the hot flush as disappeared, improved, or not improved. In patients with disappeared or improved symptoms, we decreased CMA dose to 50 mg per day, and after we reevaluated the effect, we decreased CMA dose to 25 mg per day. When hot flush appeared again at 25 mg per day, we returned the dose of CMA to 50 mg per day. In cases with no change for more than two months, we canceled the treatment of CMA.
Hot flush disappeared in 17 patients, improved in 10 patients, and did not improve in 5 patients (reduction in 84% of hot flush patients). The median time to hot flush reduction was 1.16 months. The effect of CMA was maintained at 25 mg per day in 19 patients and at 50 mg per day in 8 patients. No patients had prostate-specific antigen failure in the treatment of CMA.
When hot flush appears during treatment with luteinizing hormone-releasing hormone agonist for prostate cancer, it seems that CMA can improve it immediately in most patients.
PMCID: PMC3814123  PMID: 24223412
Hormonal antineoplastic agents; Chlormadinone acetate; Hot flashes; Prostate neoplasms
13.  Androgen receptor coactivator p120 subtype β is highly expressed in prostate cancer 
Prostate International  2013;1(1):10-15.
The β form of p120 is reported to be a strong coactivator of the androgen receptor. We investigated the gene expression profiles of the α and β forms of p120 in prostate cancer cell lines, benign prostatic hyperplasia (BPH), nontreated prostate cancer (NTPC), and prostate cancer after androgen deprivation therapy (PCA-ADT).
We obtained 154 prostate needle biopsy specimens (81 in BPH, 51 in NTPC, and 22 in PCA-ADT). Levels of p120α and β expression were determined by multiplex real-time polymerase chain reaction.
Prostate cancer cell lines, LNCaP, PC-3, DU-145, and LNCaP-LA, which is a derivative of LNCaP under androgen deprivation, expressed both p120α and p120β. p120α expression levels were significantly higher than those of p120β in all cell lines examined. In human prostate tissues, p120α expression was significantly higher than that of p120β in BPH and NTPC. p120α expression in BPH was significantly higher than in other groups. In contrast, p120β expression was significantly higher in NTPC and PCA-ADT than in BPH. Expression of the two forms of p120 was not correlated with age, prostate-specific antigen, or Gleason score.
The expression profiles of p120α and p120β significantly differ in cancerous and benign prostatic tissues.
PMCID: PMC3821517  PMID: 24223396
Androgen receptor; Nuclear coactivator; Prostatic neoplasms
14.  Catalytic Peptides for Inorganic Nanocrystal Synthesis Discovered by New Combinatorial Phage Display Approach 
PMCID: PMC3517003  PMID: 21928455
catalytic peptides; biomineralization; phage display; nanocrystals; ZnO; bionanotechnology
15.  Comparison of Electrical Properties of Viruses Studied by AC Capacitance Scanning Probe Microscopy 
Capacitances of five types of viruses, adenovirus type 5 (AV 5) herpes simplex virus type 1 (HSV1), simian virus 40 (SV40), vaccinia (MVA), and cowpea mosaic virus (CPMV) were compared by AC capacitance scanning probe microscopy. This technique, using a Pt-coated AFM tip as an electrode to probe capacitance of materials between the tip and a bottom electrode, has been applied to study surface structures of semiconductors and polymers with nanometer spacial resolution, however biological samples at the nanoscale have not been explored by this technique yet. Because most biological cells are poor conductors, this approach to probe electric properties of cells by capacitance is logical. This scanning probe technique (SPM) showed that all of these viruses have distinguishable and characteristic capacitances, respectively. Series of control experiments were carried out using mutant viruses in order to validate the origin of the characteristic capacitance responses for different viruses. A mutation on the capsid in HSV1 virus with green fluorescence proteins (GFP) increased capacitance from 9×10−6 F/cm2 to 1×10−5 F/cm2 at the frequency of 104 Hz. HSV2 virus decreased capacitance when its envelope and glycoproteins were chemically extracted. These control experiments indicate that dielectric properties of capsid proteins and envelope glycoproteins significantly influence overall dielectric constants of viruses. Because those capsid proteins and glycoproteins are characteristic to the virus strain, this technique could be applied to detect and identify viruses at the single viron level using their distinct capacitance spectra as fingerprints without labeling.
PMCID: PMC3474603  PMID: 18092777
AC impedance; Bionanotechnology; Capacitance; Virus; Sensor; Label-free detection
17.  Label-free cancer cell detection with impedimetric transducers 
Analytical chemistry  2009;81(24):10167-10171.
While cancer is still an implacable disease, many cancers can be cured if they are diagnosed in an early stage. Recently, it was reported that the transformation from normal cells to cancer cells can change their mechanoelastic properties to become softer and more deformable. If some cancer cells are more deformable, then a progressive increase of the volume of softer cancer cells should be induced as an abrupt change in osmolarity is applied. Based on this hypothesis, we developed a sensor that can electronically monitor the volume increase of cancer cells under hyposmotic pressure. By this methodology, K:Molv NIH 3T3 cells, 786-O human kidney carcinoma cells, and MPSC-1 ovarian cancer cells were successfully detected within 30 minutes using on the order of 10 cells. These cancer cells could be detected with the same sensitivity even in the presence of a vast excess of the respective non-cancerous cells (NIH 3T3 cells, Human Embryonic Kidney (HEK) 293 cells, ovarian surface epithelial (OSE) cells). Since the proposed impedimetric sensor could be useful for detecting cancer cells fast and reliably, it could be further implemented in the screening of large populations of tissue samples and the detection of circulating tumor cells for point-of-care applications.
PMCID: PMC2797081  PMID: 19911810
18.  Biomimetic conformation-specific assembly of proteins at artificial binding sites nano-patterned on silicon 
Journal of the American Chemical Society  2009;131(40):14180-14181.
Biomolecules such as enzymes and antibodies possess binding sites where the molecular architecture and the physicochemical properties are optimum for their interaction with a particular target, in some cases even differentiating between stereoisomers. Here, we mimic this exquisite specificity via the creation of a suitable chemical environment by fabricating artificial binding sites for the protein calmodulin (CaM). By downscaling well-known surface chemical modification methodologies to the nanometer scale via silicon nanopatterning, the Ca2+-CaM conformer was found to selectively bind the biomimetic binding sites. The methodology could be adapted to mimic other protein-receptor interactions for sensing and catalysis.
PMCID: PMC2774499  PMID: 19757782
19.  Single-Cell Pathogen Detection with a Reverse-Phase Immunoassay on Impedimetric Transducers 
Analytical chemistry  2009;81(18):7732-7736.
The risk of infectious diseases has compelled some industries to establish a zero-tolerance standard for the presence of microorganisms in a given sample. Here, we address this issue with a novel reverse-phase immunoassay on impedimetric transducers for the specific detection of extremely low numbers of pathogens (less than 10 cells). After simply spotting the sample onto the electrodes, physisorbed analytes were targeted with urease-labeled antibodies, and the urease on the pathogens hydrolyzed urea to ionic species with a concomitant decrease of the resistivity of the solution. By this methodology, the limit of detection (LOD) based on the 3σ criterion was 1 Escherichia coli cell with an assay time under 1 h. However, the precise number of cells present in highly diluted samples is uncertain, making it difficult to assess the final LOD of the sensor. We overcome this problem by using an atomic force microscope to deposit and image in situ the exact number cells on the transducer. After performing the immunoassay, a single E. coli cell was successfully detected without ambiguity in the number of cells even in the presence of a 104 excess of a competing microorganism, thus demonstrating the outstanding LOD and selectivity of the proposed reverse-phase immunoassay.
PMCID: PMC2888025  PMID: 19746997
20.  Selective Detection of Live Pathogens via Surface-Confined Electric Field Perturbation on Interdigitated Silicon Transducers 
Analytical chemistry  2009;81(10):3830-3835.
Detection of physical changes of cells is emerging as a new diagnostic approach to determine their phenotypical features. One of such changes is related to their viability; live (viable) cells are more voluminous than the dead ones, and monitoring this parameter in tissue cells becomes essential in fields such as drug discovery and hazard evaluation. In the area of pathogen detection, an analytical system capable of specifically detecting viable cells with the simple sample preparation and detection process would be highly desirable since live microorganisms can rapidly increase their numbers even at extremely low concentration and become a severe health risk. However, current sensing strategies cannot clearly determine the viability of cells, and hence they are susceptible to false-positive signals from harmless dead pathogens. Here we developed a robust electronic immunoassay that uses a pair of polycrystalline silicon interdigitated electrodes for the rapid detection of pathogens with high specificity for live cells. After bacterial cells were specifically anchored to the surface of the antibody-modified electrode, the characteristic geometry of the transducer enables the selective detection of viable cells with a limit of detection of 3 × 102 cfu/mL and an incubation time of only 1 h. The CMOS compatible fabrication process of the chip along with the label-free, reagentless electronic detection and the easy electrode regeneration to recycle for another impedance measurement make this approach an excellent candidate for oncoming economical in-field viable-cell detection systems, fully integrable with sophisticated signal processing circuits.
PMCID: PMC2888026  PMID: 19334738
21.  Biomineralization Nanolithography: Combination of Bottom-Up and Top-Down Fabrications to Grow Arrays of Monodisperse Au Nanoparticles along Peptide Lines on Substrates 
Biomineralization Nanolithography: Combination of Bottom-Up and Top-Down Fabrications to Grow Arrays of Monodisperse Au Nanoparticles along Peptide Lines on Substrates
Combination of the top-down (peptide nanolithography) and the bottom-up fabrications (biomineralization) yielded arrays of monodisperse Au nanoparticles on the peptide lines on substrates. The number of particle lines was simply determined by the width of peptide pattern.
PMCID: PMC2776757  PMID: 19226591
Biomineralization; Nanolithography; Bionanotechnology; Nanoparticle; Peptide
22.  Effect of Nitric Oxide on the Oxygen Metabolism and Growth of E. faecalis 
Gastro-intestinal mucosal cells have a potent mechanism to eliminate a variety of pathogens using enzymes that generate reactive oxygen species and/or nitric oxide (NO). However, a large number of bacteria survive in the intestine of human subjects. Enterococcus faecalis (E. faecalis) is a Gram-positive bacterium that survives not only in the intestinal lumen but also within macrophages generating NO. It has been reported that E. faecalis generated the superoxide radical (O2−). To elucidate the role of O2− and NO in the mechanism for the pathogen surviving in the intestine and macrophages, we studied the role and metabolism of O2− and NO in and around E. faecalis. Kinetic analysis revealed that E. faecalis generated 0.5 µmol O2−/min/108 cells in a glucose-dependent manner as determined using the cytochrome c reduction method. The presence of NOC12, an NO donor, strongly inhibited the growth of E. faecalis without affecting in the oxygen consumption. However, the growth rate of NOC12-pretreated E. faecalis in NO-free medium was similar to that of untreated cells. Western blotting analysis revealed that the NOC12-treated E. faecalis revealed a large amount of nitrotyrosine-posititive proteins; the amounts of the modified proteins were higher in cytosol than in membranes. These observations suggested that O2− generated by E. faecalis reacted with NO to form peroxinitrite (ONOO−) that preferentially nitrated tyrosyl residues in cytosolic proteins, thereby reversibly inhibited cellular growth. Since E. faecalis survives even within macrophages expressing NO synthase, similar metabolism of O2− and NO may occur in and around phagocytized macrophages.
PMCID: PMC2654474  PMID: 19308272
Enterococcus faecalis; Superoxide; nitric oxide; peroxynitrite; nitro-tyrosine
23.  Crystallization and preliminary X-ray crystallographic studies of Pz peptidase A from Geobacillus collagenovorans MO-1 
Pz peptidase A has been cocrystallized with a phosphine peptide inhibitor (PPI) that selectively inhibits thimet oligopeptidase and neurolysin.
Pz peptidase A is an intracellular M3 metallopeptidase found in the thermophile Geobacillus collagenovorans MO-1 that recognizes collagen-specific tripeptide units (Gly-Pro-Xaa). Pz peptidase A shares common reactions with mammalian thimet oligopeptidase (TOP) and neurolysin, but has extremely low primary sequence identity to these enzymes. In this work, Pz peptidase A was cocrystallized with a phosphine peptide inhibitor (PPI) that selectively inhibits TOP and neurolysin. The crystals belong to space group P21, with unit-cell parameters a = 56.38, b = 194.15, c = 59.93 Å, β = 106.22°. This is the first crystallographic study of an M3 family peptidase–PPI complex.
PMCID: PMC2330125  PMID: 17277461
Pz peptidase A; M3 metallopeptidases; collagen degradation; Geobacillus collangenovorans MO-1
24.  Biomimetic and Aggregation-Driven Crystallization Route for Room-Temperature Material Synthesis: Growth of β-Ga2O3 Nanoparticles Using Peptide Assemblies as Nanoreactors 
The room temperature synthesis of β-Ga2O3 nanocrystal was examined by coupling two biomimetic crystallization techniques, the enzymatic peptide nano-assembly templating and the aggregation-driven crystallization. The catalytic template of peptide assembly nucleated and mineralized primary β-Ga2O3 crystals, and then fused them to grow single-crystalline and monodisperse nanoparticles in the cavity of the peptide assembly at room temperature. In this work, the peptide assembly was exploited as a nano-reactor with an enzymatic functionality catalyzing the hydrolysis of gallium precursors. In addition, the characteristic ring-structure of peptide assembly is expected to provide an efficient dehydration pathway and the crystallization control over the surface tension, which are advantageous for the β-Ga2O3 crystal growth. This multifunctional peptide assembly could be applied for syntheses of a variety of nanomaterials that are kinetically difficult to grow at room temperature.
PMCID: PMC2597381  PMID: 17302413
Self-assembly; Bionanotechnology; Biomineralization; Peptide; Nanoreactors
25.  Overexpression, purification, crystallization and preliminary X-ray cystallographic studies of a proline-specific aminopeptidase from Aneurinibacillus sp. strain AM-1 
Preliminary X-ray crystallographic study of a proline-specific aminopepitdase from Aneurinibacillus sp, strain AM-1 was carried out.
To elucidate the structure and molecular mechanism of a characteristic proline-specific aminopeptidase produced by the thermophile Aneurinibacillus sp. strain AM-1, its gene was cloned and the recombinant protein was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.8 Å resolution from the recombinant aminopeptidase crystal. The crystals belong to the orthorhombic space group P21212, with unit-cell parameters a = 93.62, b = 68.20, c = 76.84 Å. A complete data set was also obtained from crystals of SeMet-substituted aminopeptidase. Data in the resolution range 20–2.1 Å from the MAD data set from the SeMet-substituted crystal were used for phase determination.
PMCID: PMC2225360  PMID: 17142913
proline-specific aminopeptidase; Aneurinibacillus sp. strain AM-1; thermophiles

Results 1-25 (32)