Effects of DSP-8658, a novel selective peroxisome proliferator-activated receptors a/γ modulator, on adipogenesis and glucose metabolism in diabetic obese mice.

AIMS/INTRODUCTION: Peroxisome proliferator-activated receptors (PPARs) play a key regulating role in homeostasis. In this study, we investigated the effects of DSP-8658, a novel selective PPARa/γ modulator, on adipogenesis and glucose metabolism in diabetic obese mice and compared these effects to those of pioglitazone, a PPARγ full agonist. MATERIALS AND METHODS: DSP-8658 functional activity was assessed by PPARγ-target genes expression in adipose 3T3-L1 cells and its anti-diabetic efficacy evaluated in db/db mice. The effects of DSP-8658 on adipogenesis were investigated diet induced obese (DIO) KK-A(y) mice. RESULTS: DSP-8658 reduced the expression of PPARγ-target gene 11 beta hydroxysteroid dehydrogenase type 1 with an EC50 value 2.1-fold that of pioglitazone and 28.4-fold that of rosiglitazone. On the other hand, DSP-8658 increased the expression of fatty acid binding protein 4 and glycerol kinase genes with EC50 values 33-fold and >15-fold those of pioglitazone and 163-fold and >38-fold those of rosiglitazone, respectively. In db/db mice, DSP-8658, like pioglitazone, decreased blood glucose, HbA1c, and plasma triglyceride levels and increased plasma insulin concentration and pancreatic insulin contents. In DIO KK-A(y) mice, DSP-8658, unlike pioglitazone, decreased subcutaneous adipose tissue weight and mean adipocyte size. However, both DSP-8658 and pioglitazone improved blood glucose and HbA1c levels with similar efficacy. Although DSP-8658 did not change the expression levels of fatty acid transport protein 1 and glycerol kinase genes in subcutaneous adipose tissue of KK-A(y) mice, pioglitazone increased these gene expression levels. CONCLUSION: Unlike PPARγ full agonists, DSP-8658 ameliorates blood glucose without increasing adipogenesis in diabetic obesity mice.

Adult right living-donor liver transplantation with special reference to reconstruction of the middle hepatic vein.

Two hundred fifty-three consecutive living-donor liver transplant recipients with a right liver graft (RLG) were divided into three groups: an extended right liver graft (ERLG) group (n = 47) in which the middle hepatic vein (MHV) trunk was included in the graft, a modified right liver graft (MRLG) group (n = 114) in which the MHV tributaries were reconstructed with cryopreserved homologous veins and a simple RLG group (n = 92) in which the MHV tributaries were sacrificed. The volume of the anterior sector was significantly impaired in the RLG group compared to the other two groups, whereas the volume of the posterior sector was significantly improved in the RLG group, indicating that the impaired anterior sector regeneration by MHV deprivation was compensated by the posterior sector regeneration. The regeneration rate of the anterior sector was highest in the ERLG group (92%), moderate in the MRLG group (71%) and lowest in the RLG group (52%). The whole graft regeneration rate of the ERLG group was significantly higher than that of the other two groups. Poor regeneration, however, was not correlated with delayed functional recovery or long-term outcome. Short-term, the patency of reconstructed MHV tributaries was over 90%, but occlusion occurred frequently over the long-term, especially in V5.

Occurrence of multinucleated hepatocytes in cynomolgus monkeys (Macaca fascicularis) from different geographical regions.

Multinucleated hepatocytes (MNHs) have been occasionally reported in macaques, as well as chimpanzees and gorillas, as an incidental finding. However, information is sparse on variations in incidence in the cynomolgus macaque (Macaca fascicularis). A survey was conducted to assess the occurrence of MNHs in the liver of stock (nonstudy) animals from SNBL SRC (Alice, TX) and SNBL USA (Everett, WA) submitted for diagnostic purposes. A total of 215 cynomolgus monkeys originally from Cambodia (61), China (5), Indonesia (125), and Mauritius (24) were used for this investigation. From each animal, usually 2 liver samples were processed for histopathology with 2 sections in each slide. An MNH was defined as a hepatocyte with 3 or more nuclei. A threshold of 3 MNHs was selected for the Multinucleated Hepatocyte Grading System: 0 = not remarkable (≤3 MNHs counted from 2-4 liver sections), minimal = 4 to 15 MNHs, mild = 16 to 30 MNHs, moderate = 31 to 59 MNHs, and severe ≥60 MNHs. The incidence of MNHs was 60 of 86 (70%) in males and 72 of 129 (56%) in females for a total overall incidence of 132 of 215 animals (61%). Affected hepatocytes were frequently observed close to the capsule and generally had 3 to 8 nuclei per hepatocyte but as many as 15 occurred in a single cell. Awareness of the incidence of MNHs in cynomolgus monkeys is important for potential use as background data in preclinical safety and toxicity evaluation studies.

Occurrence of the Chilean devil ray Mobula tarapacana (Elasmobranchii: Batoidea: Myliobatiformes) in the Sea of Okhotsk: first record from cold temperate waters.

The northernmost record for Chilean devil ray Mobula tarapacana, a circumglobal species that occurs in tropical, subtropical and limited warm temperate waters, is described. An adult female was caught incidentally in the Sea of Okhotsk on 17 September 2011. This specimen is the first confirmed occurrence of devil rays Mobula spp. in cold temperate waters.

Expression of androgen receptor in mammary glands in ovariectomized cynomolgus monkeys.

This study investigated structural alterations and the immunohistochemical expression of androgen receptor (AR), estrogen receptor (ER), and progesterone receptor (PgR) in the mammary glands from surgically postmenopausal cynomolgus monkeys (Macaca fascicularis). Fourteen animals were divided into 2 groups. Seven animals underwent an ovariectomy (OVX), and the other 7 animals underwent a sham operation (sham). The in-life phase of study was 78 weeks. Atrophy in the mammary glands of OVX monkeys was similar to early postmenopausal atrophy of the human breast. The proportion of AR-positive cells in the OVX group was significantly higher than in the sham group, but the proportion of ER and PgR-positive cells was significantly lower. These results suggest that use of a primate model for hormone receptor expression has potential applications in basic human endocrinology, particularly in research in hormone receptor expression in mammary glands (both normal and neoplastic).

Biology and biotype determination of greenbug, Schizaphis graminum (Hemiptera: Aphididae), on seashore paspalum turfgrass (Paspalum vaginatum).

Greenbug, Schizaphis graminum (Rondani) (Hemiptera: Aphididae), was first discovered damaging seashore paspalum (Paspalum vaginatum Swartz) turfgrass in November 2003 at Belle Glade, FL. Inquiries to several golf courses with seashore paspalum turf across southern Florida indicated infestation was wide spread by April 2004. Damage symptoms progress from water soaked lesions surrounding feeding sites within 24 h to chlorosis and necrosis of leaf tips within 96 h. Problems caused by greenbug feeding were initially misdiagnosed as fertilizer, disease, other insects, or water management problems because aphids were not previously found on warm season turfgrasses. Greenbug development and fecundity studies were conducted on six seashore paspalum varieties: 'Aloha,' 'SeaDwarf,' 'SeaGreen,' 'SeaIsle,' 'SeaWay,' and 'SeaWolf.' Greenbug did not survive on 'SeaWolf.' Development rates (mean +/- SEM) ranged from 7.6 +/- 0.2 to 8.2 +/- 0.2 d on the remaining varieties. Greenbug longevity and fecundity on 'Aloha' were significantly less than on the other varieties. The estimated intrinsic rate of natural increase (r(m)) for greenbug ranged from 0.24 to 0.26 across tested varieties. Values for net reproductive rate (R(o)) ranged from 12.3 on 'Aloha' to 40.4 on 'SeaWay.' In feeding trials on indicator plants, the Florida isolate of greenbug exhibited a unique biotypic profile most commonly found on noncultivated grass hosts. It was virulent on the wheat variety GRS1201 that is resistant to the principal agricultural biotypes attacking small grains and to all currently available resistant sorghum varieties.

First Report of Brown Ring Patch Caused by Waitea circinata var. circinata on Poa trivialis in Florida.

Brown ring patch is a newly described disease of cool-season turfgrass first reported in Japan on creeping bentgrass (Agrostis palustris) (2) and later reported in California on annual bluegrass (Poa annua) (1). The disease is characterized by either patches or rings of discolored to blighted turfgrass that can range from a few centimeters to a meter in diameter. Affected turfgrass plants turn chlorotic and can be blighted from the crown to the leaf tips. Blight symptoms have been associated with fluffy white-to-cream aerial mycelium after extended incubation of the sample. Symptoms including patches of blighted turfgrass approximately 10 cm in diameter were observed on roughstalk bluegrass (Poa trivialis) that had been overseeded onto a dormant 'Tifdwarf' bermudagrass (Cynodon dactylon) putting green in Palatka, FL. A sample was submitted by the superintendent in June 2005 because symptoms were confused with dollar spot and a fungicide resistance issue was suspected. The sample produced abundant aerial mycelium after incubation. The pathogen was isolated on potato dextrose agar amended with rifampicin (100 ppm) and streptomycin (100 ppm) from Poa plants surface disinfested with 70% ethanol for 30 s. Colony and sclerotia morphology were consistent with Waitea circinata var. circinata as previously described (1,2). The teleomorph W. circinata var. circinata was not observed on plant material or culture plates. Amplified fragments of rDNA including internal transcribed spacers from the isolate were sequenced bidirectionally from four bacterial clones. The consensus sequences (GenBank Accession Nos. FJ029103, FJ029104, FJ029105, and FJ029106) matched with 99% homology (99% sequence overlap) isolate TRGC1.1 of W. circinata var. circinata described by Wong, NCBI Accession No. DQ900586 (1). Pots of 'Cypress' roughstalk bluegrass that were 1 week postemergence were inoculated with the pathogen using 10 infested wheat grains. Plants were incubated at 25°C in a sealed plastic bag with a moist paper towel in the bottom. Hyphae grew from the grains and colonized the grass. Individual plants began to turn chlorotic within 3 days and greater than 90% of the turf in pots was dead after 1 week. The fungus was reisolated from affected plants. Control pots were inoculated with uninfested wheat grains and showed no disease symptoms after 1 week. Inoculations were repeated twice more with the same results. To our knowledge, this is the first report of brown ring patch on P. trivialis in Florida. References: (1) K. A. de la Cerda et al. Plant Dis. 91:791, 2007. (2) T. Toda et al. Plant Dis. 89:536, 2005.

CYP2C76-mediated species difference in drug metabolism: a comparison of pitavastatin metabolism between monkeys and humans.

The monkey is often used to predict metabolism of drugs in humans since it generally shows a metabolic pattern similar to humans. However, metabolic profiles different from humans are occasionally seen in monkeys for some drugs including pitavastatin. Recently, we have successfully identified a monkey-specific cytochrome P450 (CYP) 2C76, which possibly accounts for a species difference between monkeys and humans because of its sequence and functional uniqueness. The present study on the role of CYP2C76 and other monkey CYP2Cs in pitavastatin metabolism, as an example, has revealed that CYP2C76 is important for the metabolism of the lactone form, indicating a major role of CYP2C76 for the difference in the metabolism of pitavastatin and possibly other drugs between monkeys and humans. The current investigation on the involvement of CYP2C76 in the metabolism of other drugs is expected to reveal further the further importance of this monkey-specific drug-metabolizing enzyme.

Categories of resistance in St. Augustinegrass lines to southern chinch bug (Hemiptera: Blissidae).

The southern chinch bug, Blissus insularis Barber (Hemiptera: Blissidae), is the most important insect pest of St. Augustinegrass, Stenotaphrum secundatum (Walt.) Kuntze, in Florida and other Gulf Coast states. Resistance to southern chinch bug was identified previously in St. Augustinegrass lines 'FX-10' and NUF-76. Choice and no-choice tests and ovipositional and developmental studies were conducted to determine the categories of resistance in FX-10 and NUF-76 to southern chinch bug. When adult chinch bugs had a choice among attached stolons of three susceptible lines ('Floratam', 'Bitter Blue', and 'Palmetto') and the two resistant lines, chinch bugs were found significantly more often over a 5-d period on the susceptible lines. This result indicates the presence of antixenosis in the resistant lines FX-10 and NUF-76. In a no-choice study, chinch bugs produced less than half as many excretory spots on FX-10 as on the susceptible lines. Significantly fewer excretory spots produced by chinch bugs confined on NUF-76 accumulated by days 3 and 5 after release; however, on the first 2 d, the accumulative number of excretory spots was not significantly less than that found on susceptible lines. The no-choice study confirmed a high level of antixenosis in FX-10, a moderate level of antixenosis in NUF-76, and possible antibiosis in NUF-76. Ovipositional and developmental studies were conducted using only Floratam (a widely planted cultivar that was formerly resistant to B. insularis) and the two resistant lines. Adults released on Floratam produced 11 and 5 times more eggs and 18 and 9 times more offspring than adults on FX-10 and NUF-76, respectively. Plant anatomical and biochemical studies are required to investigate the exact cause of antixenosis in FX-10 and NUF-76 and possible antibiosis in NUF-76.

A national surveillance of Shiga toxin-producing Escherichia coli in food-producing animals in Japan.

To assess the public health risk, the prevalence and anti-microbial resistance of Shiga toxin-producing Escherichia coli (STEC) among food-producing animals were studied throughout Japan. Faecal samples were collected from healthy animals of 272 cattle, 179 pigs, and 158 broilers on 596 farms in all 47 Japanese prefectures. STEC were isolated from 62 (23%) cattle and 32 (14%) pig samples but from no chicken samples. Of the bovine isolates, 19 belonged to serotypes frequently implicated in human disease (O157:H7/non-motile (NM)/H not typeable, O26:NM/H11/H21/H not typeable, O113:H21, and O145:NM). The eae genes were observed in 37% of bovine isolates; among them one O145:NM and all four O157 isolates possessed eae-gamma1, and one O145:NM, one O103:H11, and all five O26 isolates possessed eae-beta1 gene. Among the swine isolates, stx2e were dominant, and serotypes frequently implicated in human diseases or eae-positive isolates were not observed. Bovine isolates showed less anti-microbial resistance, but six isolates of 26:NM/H11 and O145:NM were multi-resistant and may need careful monitoring. Swine isolates showed various resistance patterns; chloramphenicol resistance patterns were more common than in bovine isolates. This first national study of STEC in the Japanese veterinary field should aid our understanding of Japan's STEC status.

Influence of Silicon and Chlorothalonil on the Suppression of Gray Leaf Spot and Increase Plant Growth in St. Augustinegrass.

Silicon, applied as calcium silicate slag (20% Si), was evaluated for its potential to suppress gray leaf spot (Magnaporthe grisea) and increase plant growth in newly planted St. Augustinegrass in the summers of 2000 and 2001. Calcium silicate was applied (1,000 kg Si/ha) to three sites that contained Si-deficient Histosols prior to sprigging St. Augustinegrass, cv. Floratam, in southern Florida. This treatment was compared with foliar sprays of chlorothalonil, calcium silicate plus chlorothalonil, and an untreated control. Immediately after planting, applications of chlorothalonil (720 g/liter) were made every 10 days for a total of seven sprays at a rate of 7.6 kg a.i./ha with a commercial sprayer. Based on area under the disease progress curve (AUDPC) values for the treatments calcium silicate alone, chlorothalonil, and calcium silicate plus chlorothalonil, gray leaf spot was reduced by 7, 65, and 68% at site one, 28, 34, and 59% at site two, and 41, 55, and 68% at site three, respectively when compared with the untreated control. The application of calcium silicate alone significantly reduced the final AUDPC of gray leaf spot when compared with the control at site two only. However, when disease severities were analyzed by week, the calcium silicate treatment significantly reduced the percentage of disease at weeks 2, 6, and 8 at site one and weeks 3 to 8 at site three when compared with the control. The final percent bare ground coverage for St. Augustinegrass was increased significantly using calcium silicate by 17 and 34% over the control at sites one and two, respectively (P ≤ 0.05). Silicon was the only element to significantly increase in the leaf tissue for treatments amended with calcium silicate. Levels of Si in leaves for treatments amended with calcium silicate were from 1.2 to 1.3%, while those not receiving calcium silicate had only 0.6 to 0.7%. Amendments with calcium silicate slag for St. Augustinegrass sod production on Si-deficient soils may be an option to reduce gray leaf spot development in newly sprigged fields and promote earlier ground coverage of grass when the environment is favorable for disease.

Oxindole derivatives as orally active potent growth hormone secretagogues.

A series of substituted oxindole derivatives was synthesized and evaluated for growth hormone (GH) releasing activity using cultured rat pituitary cells. (+)-6-Carbamoyl-3-(2-chlorophenyl)-(2-diethylaminoethyl)-4-trifluoromethyloxindole (SM-130686, 37S) was found to have potent activity (EC(50) = 3.0 nM), while the other enantiomer 37R had reduced activity. The absolute configuration of 37S was confirmed by X-ray crystallographic analysis. Compound 37S showed a good pharmacokinetic profile in rats with 28% oral bioavailability at 10 mg/kg and excellent in vivo activity as evidenced by a significant weight gain after 4 days of oral administration at 10 mg/kg twice a day. Compound 37S displaced the binding of (35)S-MK-677 to human GHS-R with an IC(50) value of 1.2 +/- 0.2 nM.

Pharmacological profile of a new orally active growth hormone secretagogue, SM-130686.

SM-130686, an oxindole derivative, is a novel orally active GH secretagogue (GHS) which is structurally distinct from previously reported GHSs such as MK-677, NN703 and hexarelin. SM-130686 stimulates GH release from cultured rat pituitary cells in a dose-dependent manner. Half-maximum stimulation was observed at a concentration of 6.3+/-3.4 nM. SM-130686-induced GH release was inhibited by a GHS antagonist, but not by a GH-releasing hormone antagonist. SM-130686 dose-dependently inhibited the binding of radiolabeled ligand, (35)S-MK-677, to human GHS receptor 1a (IC(50)=1.2 nM). This indicates that SM-130686 stimulates GH release through the GHS receptor. The effect of a single oral administration of SM-130686 on GH release in pentobarbital-anesthetized rats was studied. After treatment with 10 mg/kg SM-130686, plasma GH concentrations measured by radioimmunoassay significantly increased, reaching a peak at 20-45 min, and remained above baseline during the experimental period (60 min). The anabolic effect of repetitive SM-130686 administration was studied in rats. Rats received 10 mg/kg SM-130686 orally twice a day and were weighed every day for 9 days. At day 9 there was a significant increase in both the body weight and the fat free mass (19.5+/-2.1 and 18.1+/-7.5 g respectively). Serum IGF-I concentration was also significantly elevated 6 h after the last dose of SM-130686. An endogenous GHS ligand for the GHS receptor has recently been identified from stomach extract and designated as ghrelin. The GH-releasing activity in vitro relative to ghrelin (100%) was about 52% for SM-130686. It is likely that SM-130686 is a partial agonist for the GHS receptor. In summary, we describe here an orally active GHS, SM-130686, which acts through the GHS receptor. Repetitive administration of SM-130686 to rats, similar to repetitive administration of GH, significantly increased the fat free mass by an amount almost equal to the gain in body weight.

Synthesis of tricyclic indole-2-carboxylic [correction of caboxylic] acids as potent NMDA-glycine antagonists.

The practical synthesis of a series of tricyclic indole-2-carboxylic acids, 7-chloro-3-arylaminocarbonylmethyl-1,3,4,5-tetrahydrobenz[cd]indole-2-carboxylic acids, as a new class of potent NMDA-glycine antagonists is described. The synthetic route to the key intermediate 12a comprises a regioselective iodination of 4-chloro-2-nitrotoluene, modified Reissert indole synthesis, Jeffery's Heck-type reaction with allyl alcohol, Wittig-Horner-Emmons reaction, and iodination at the indole C-3 position. The key step in the route is an intramolecular cyclization of 12a to give the tricyclic indole structure. Two methods of cyclization, (1) an intramolecular radical cyclization of 12a and (2) a sequence of intramolecular Heck reaction of 12a followed by a 1,4-reduction, were performed. The resulting tricyclic indole diester 13a was selectively hydrolyzed to afford the desired tricyclic indole monocarboxylic acid 16 on a multihundred gram scale without any chromatographic purifications. Optical resolution of 16 to (-)-isomer 17 and (+)-isomer 18 was carried out, and the resulting isomers were derivatized, respectively. Evaluation of the optically active derivatives for affinity to the NMDA-glycine binding site using the radio ligand binding assay with [(3)H]-5,7-dichlorokynurenic acid revealed that the derivatives of (-)-isomer 17 were more potent than the others and that especially substituted anilide (-)-isomer 24 (K(i) = 0.8 nM) showed high affinity.

Isolation, characterization and cDNA cloning of a one-lobed transferrin from the ascidian Halocynthia roretzi.

Transferrin was isolated from plasma of the ascidian Halocynthia roretzi by ion-exchange chromatography. The molecular weight of the plasma transferrin was determined to be 52K by SDS-polyacrylamide gel electrophoresis and gel filtration. Ascidian plasma transferrin was found to bind one mole of iron ion per mole of protein. The reductive S-pyridylethylated transferrin was subjected to Edman degradation analysis for determination of the N-terminal amino acid sequence, and it was also subjected to proteolytic fragmentation to yield peptide fragments, whose amino acid sequences were determined by Edman degradation analysis. Using the above amino acid sequences, a cDNA clone (1880 base pairs) encoding a protein of 372 amino acids containing a signal peptide of 21 amino acids was isolated from an H. roretzi hepatopancreas cDNA library. The reduced amino acid sequence contains the same sequences of the peptide fragments. A comparison of the amino acid sequence of ascidian transferrin with those of other members of the transferrin family revealed that the ascidian transferrin is composed of only the N-terminal lobe of two-lobed vertebrate transferrins. Thus, a one-lobed transferrin is present in the ascidian H. roretzi.

Experimental thromboembolic stroke in cynomolgus monkey.

To develop an experimental model of thromboembolic stroke without intracranial surgery, an autologous blood clot was delivered to the middle cerebral artery (MCA) via the internal carotid artery in cynomolgus monkeys. Male cynomolgus monkeys, in which a chronic catheter had been earlier implanted in the left internal carotid artery, were used. The clot was flushed into the internal carotid artery under sevofluorane anesthesia. A neurologic deficit score was assigned after MCA embolization. After 24 h, cerebral infarct size and location were determined by the TTC staining method. Cerebral blood flow (CBF) was measured prior to and after MCA embolization, using positron emission tomography (PET). After embolization, long-lasting and profound extensor hypotonia of the contralateral upper and lower limbs, and mild to severe incoordination were observed. Contralateral hemiplegia was observed over the following 24 h. In gross morphologic observation of the brain, the lesions involved mostly the caudate nucleus, putamen, globus pallidus and insular cortex. CBF was maximally reduced in the left MCA territory, but not in the right MCA territory. This model is relevant to thromboembolic stroke in human in neurologic dysfunction and histopathologic brain damage.

Effects of ropinirole on motor behavior in MPTP-treated common marmosets.

The effects of ropinirole (4-[2-(dipropylamino)ethyl]-2-indolinone monohydrochloride), a nonergoline dopamine receptor agonist with a high affinity for native dopamine D(2)-like receptors, on Parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 2.5 mg/animal in common marmosets were examined and compared to the effects of bromocriptine. Ropinirole (0.1-3 mg/kg, PO) increased motor activity dose dependently and reversed akinesia or uncoordinated movement in MPTP-treated marmosets. The activities for ropinirole were very similar to those of bromocriptine. Ropinirole had, however, several properties that differed from those of bromocriptine. Ropinirole caused a more rapid onset of anti-Parkinsonian activity compared to bromocriptine, and had a potency more than five times greater than that of bromocriptine in the improvement of motor deficits. The combination of ropinirole and L-DOPA increased the effectiveness of ropinirole or L-DOPA alone, and produced a more marked additive effect on motor activity than did bromocriptine and L-DOPA. Chronic administration of ropinirole for 21 days produced a statistically significant increase in motor activity compared to the initial administration, and akinesia scores, measured through rating the quality of movements, were also improved without obvious dyskinesia. This study suggests that ropinirole is a dopamine D(2)-like receptor agonistic drug of potential use in the treatment of Parkinson's disease.

East-West development: understanding the usability and acceptance of foreign data in Japan.

The introduction in Japan of New GCP regulations, as well as the internationalization of clinical trials under the ICH process (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use), has meant that the system is now faced with a number of serious challenges. This is shown by the dramatic decrease in registration of new drugs. Identifying the actors in the new context is essential. We shall first define precisely what is the Regulatory Authority in Japan, and which are the Regulatory Requirements to be fulfilled. We shall then describe the new process for evaluation of foreign clinical data. One of the major issues will be to determine in which cases a complete clinical data package will be sufficient to support regulatory submission, and in which cases additional clinical studies or bridging studies, and what kind of bridging studies will be required.

Amyloid protofilament formation of hen egg lysozyme in highly concentrated ethanol solution.

Mutant human lysozymes (Ile56Thr & Asp67His) have been reported to form amyloid deposits in the viscera. From the standpoint of understanding the mechanism of amyloid formation, we searched for conditions of amyloid formation in vitro using hen egg lysozyme, which has been extensively studied from a physicochemical standpoint. It was found that the circular dichroism spectra in the far-ultraviolet region of the hen egg lysozyme changed to those characteristic of a beta-structure from the native alpha-helix rich spectrum in 90% ethanol solution. When the concentration of protein was increased to 10 mg/mL, the protein solution formed a gel in the presence of 90% ethanol, and precipitated on further addition of 10 mM NaCl. The precipitates were examined by electron microscopy, their ability to bind Congo red, and X-ray diffraction to determine whether amyloid fibrils were formed in the precipitates. Electron micrographs displayed unbranched protofilament with a diameter of approximately 70 A. The peak point of the difference spectrum for the Congo red binding assay was 541 nm, which is characteristic of amyloid fibrils. The X-ray diffraction pattern showed a sharp and intense diffraction ring at 4.7 A, a reflection that arises from the interstrand spacing in beta-sheets. These results indicate that the precipitates of hen egg lysozyme are amyloid protofilament, and that the amyloid protofilament formation of hen egg lysozyme closely follows upon the destruction of the helical and tertiary structures.