Effects of food intake on pharmacokinetics of mosapride in beagle dogs.

This study was performed to determine the pharmacokinetic profile of mosapride in fasting and fed states. A single 5-mg oral dose of mosapride was administered to fasted (n = 15) and fed (n = 12) beagle dogs, and the plasma concentrations of mosapride were measured by liquid chromatography-tandem mass spectrometry. The resultant data were analyzed by noncompartmental analysis (NCA). Mosapride was absorbed in fasted and fed dogs with similar Tmax . Both Cmax and AUC were significantly higher in the fasting group than in fed dogs, being four times (10.51 µg/mL vs. 2.76 µg/mL) and 3.5 times higher (38.53 h · µg/mL vs. 10.22 h · µg/mL), respectively. These findings suggest that food intake affects the pharmacokinetics of mosapride and that the dosage regimen for this drug need to be reconsidered.

Determination of influence of food intake after a single oral dose of mosapride in beagle dogs using nonlinear mixed effect modeling.

The objective of this study was to describe the population pharmacokinetics (PK) of mosapride under fasting and fed conditions. A single 5-mg oral dose of mosapride was administered to fasted (n = 15) and fed (n = 12) beagle dogs. Plasma concentrations of mosapride were subsequently measured by liquid chromatography-tandem mass spectrometry. Data were analyzed using modeling approaches with the NONMEM 7.2 software. A one-compartment open PK model utilizing model event time (MTIME) with first-order absorption and first-order elimination was found to be more appropriate than all other PK models tested. The absorption rate constants of mosapride were significantly decreased under fed conditions, compared to fasting conditions. The observed bootstrap medians of PK parameters were generally consistent with the corresponding population mean estimates. Furthermore, with the exception of some mosapride concentrations, most of observed data fell into the range of the 5th and 95th percentiles of the simulated values. Overall, the final model was able to describe the observed mosapride concentrations reasonably well. These findings suggest that food intake affects both the rate and extent of absorption of mosapride and that the pharmacological effect of mosapride can differ significantly depending on food intake.

Stem Canker of Giant Dogwood (Cornus controversa) Caused by Fusarium lateritium in Korea.

In May 2012, a stem canker was observed on a ~20-year-old giant dogwood (Cornus controversa) in Suwon, Gyeonggi Province, South Korea, which consisted of necrotic lesions on stem bark with orange sporodochial fruiting bodies. A single fungal colony was obtained from hyphal tips that were grown out of affected tissues plated on potato dextrose agar (PDA) acidified with 0.1% lactic acid after surface sterilization with 1.0% NaOCl for 30 s and 70% ethanol for 30 s, and incubated at 25°C for 7 days in the dark. The fungal isolate was grown on PDA and carnation leaf agar (CLA) to examine its mycological characteristics. The fungal colonies grown on PDA at 25°C for 7 days had diameters of 31 to 36 mm, with the colony surface sparsely cottony or with little or no aerial mycelium, very pale brown to pink, becoming progressively lighter toward the center; the colony reverse was pinkish-white to reddish-yellow, producing very few hyaline microconidia that were ellipsoidal, mostly 1-celled, and 15.4 to 22.8 × 4.1 to 4.8 µm. It produced hyaline macroconidia that were slightly curved, frequently 3 septate, a hooked or beaked apical cell and a foot-shaped or notched basal cell, 28.0 to 35.5 × 4.0 to 5.5 µm, borne on pink sporodochia. On CLA, the colony surface was lighter toward the center with no or sparse aerial mycelium, growing to 33 to 43 mm diameter at 25°C for 7 days. Microconidia were ellipsoidal, mostly 1-celled, and 9.2 to 17.5 × 2 to 2.5 µm on CLA. Macroconidia were produced on pink sporodochia near or on carnation leaf pieces, falcate to almost straight or slightly curved, frequently 5 to 7 septate, with a hooked or beaked apical cell and a foot-shaped or notched basal cell, and 45.5 to 59 × 5.5 to 6.5 µm. Chlamydospores were rare or absent. Based on these morphological characters, the isolate was identified as Fusarium lateritium (1,2). Sequences of the internal transcribed spacer (ITS) rDNA region of the fungus (GenBank Accession No. KC453998) amplified using primers ITS1/ITS4 had 100% sequence identity to F. lateritium (JN198452). The DNA sequences of translation elongation factor-1α (EF-1α) amplified using primers EF1/EF2 (KC453997) also had 100% sequence identity to F. lateritium (AY707172 and AY707156). The culture was deposited in the Korean Collection for Type Cultures (KCTC 46029). Pathogenicity tests were conducted using 1-year-old giant dogwood seedlings grown for 3 weeks before inoculation in a 1:1:1 mixture of peat moss, perlite, and sand in 10" × 10" × 12" plastic pots. The stems of three seedlings were inoculated with the mycelial plugs from the edge of the fungal culture on PDA grown at 25°C for 7 days, which were placed on three barkless cuts per stem and sealed with Parafilm that was removed 3 weeks later. Canker symptoms on the inoculated seedlings developed after 30 days of incubation at 25 to 32°C and relative humidity of 50 to 60% in a glasshouse, from which the same fungus was isolated. Non-inoculated control seedlings showed no canker development. To our knowledge, this is the first report of stem canker on giant dogwood caused by F. lateritium in Korea and also the family Cornaceae as new host for the fungus. References: (1) D. M. Geiser et al. Mycologia 97:191, 2005. (2) J. F. Leslie and B. A. Summerall. The Fusarium Laboratory Manual. Blackwell Publishing. Ames, Iowa, 2006.

Modelling the atypical absorption of menatetrenone and the metabolism to its epoxide: effect of VKORC1 polymorphism.

WHAT IS KNOWN AND OBJECTIVE: This study aimed to model the atypical absorption of menatetrenone and its epoxide metabolite and to examine the influence of the single nucleotide polymorphism (SNP) of vitamin K2 epoxide reductase complex subunit 1 (VKORC1) on the pharmacokinetics. METHODS: After the administration of 30 mg of menatetrenone to 26 healthy subjects, the plasma concentrations of menatetrenone and its epoxide were measured using LC-MS/MS. For the haplotype analysis, the SNP of the VKORC1 gene was investigated in the 26 volunteers. The model parameters were estimated using the ADAPT II program. RESULTS AND DISCUSSION: A two-compartment model with Weibull-type absorption and saturable elimination described the pharmacokinetics of menatetrenone and its epoxide. The plasma concentrations of both tended to be lower in the H1/H7 genotype group than in the wild-type H1/H1 group. WHAT IS NEW AND CONCLUSION: We present the first detailed pharmacokinetic modelling of menatetrenone in relation to VKORC1 genotype. This study suggests that VKORC1 genotype is unlikely to be helpful in dose-selection because of the very high inter-individual variation in systemic exposure within each genotype group, and the small inter-group difference observed.

First Report of the Walnut Witches'-Broom Phytoplasma on Japanese and Black Walnut in Iowa.

A single Japanese walnut (Juglans ailantifolia Carrière, an ornamental, deciduous tree) with symptoms of witches'-brooms and branch dieback, consistent with those associated with the walnut witches'-broom (WWB) phytoplasma (1), was observed near Ames, IA. No other Japanese walnut trees were present in the planting and the numerous black walnut (Juglans nigra L.) trees were asymptomatic. Leaf samples were collected in September 2009 from witches'-brooms as well as from two asymptomatic branches from the Japanese walnut tree and from three branches each from two nearby (10 m and 100 m away, respectively) black walnut trees. The presence of phytoplasma was tested using DNA extracted (sodium dodecyl sulfate and potassium acetate methods) from the midvein of individual leaves and PCR with universal phytoplasma primers P1 and P7, which amplify from the beginning of the 16S rDNA to the beginning of the 23S rDNA gene (4). Each of the five symptomatic leaves yielded a PCR product, but the two asymptomatic leaves from the sole Japanese walnut tree did not. One of the three asymptomatic leaves from a black walnut tree (100 m away) was also positive. In a subsequent round of PCR, with the nested primers R16F2 and R16R2 (4), three additional asymptomatic leaves from the two black walnut trees were positive. The P1/P7 or R16F2/R16R2 products from each of the three trees were directly sequenced or cloned into a TA vector and sequenced using vector primers. The BLAST searches (v. 2.2.2.4) of these sequences most closely matched the sequences of the WWB phytoplasma and other members of the 16SrIII group (peach X-disease). The closest matches for the full P1/P7 sequence from the Japanese walnut (GenBank Accession No. HQ221553, 1,814 bp) were with those of phytoplasmas associated with WWB from two black walnut trees in Georgia (AF190227, 1,812 of 1,815 bp matching; and AF190226, 1,808 of 1,815 bp matching), spiraea stunt (AF190228, 1,808 of 1,814 bp), and western X (AF533231, 1,807 of 1,814 bp). The iPhyClassifier restriction fragment length polymorphism similarity coefficient was 0.99 for L33733 (Canadian peach X phytoplasma) and 0.98 for AF190226. Sequence HQ221553 differed by 10 bp from the sequence from the asymptomatic black walnut tree that was 100 m away (HQ221554, 1,815 bp), which matched closest to one of the black walnut samples from Georgia (AF190227, 1,807 of 1,816 bp). A 1,029-bp fragment from the second black walnut tree (10 m away) differed by 1 or 2 bp from the Georgia WWB accessions. To our knowledge, this is the first report of WWB symptoms in Iowa and the first identification of the WWB phytoplasma outside of Georgia (1). The disease, however, is more widely known (Illinois, Indiana, and Ohio) and may cause serious reduction in nut production (1-3). It can be lethal to Juglans spp., especially to exotic species such as Japanese walnut (2,3). The native black walnut is thought to be relatively resistant to tolerant of WWB (2,3) and may only show growth decline with no symptoms, except for broom production from cut surfaces (3). Care should be taken in moving planting stock of black walnut (4) because asymptomatic trees may harbor the phytoplasma. References: (1) J. Chen et al. Plant Dis. 76:1116, 1992. (2) C. E. Seliskar. For. Sci. 22:144, 1976. (3) W. A. Sinclair et al. Diseases of Trees and Shrubs. Cornell University Press, Ithaca, NY, 1987. (4) L. Ward. Juglans (Walnut). Post-Entry Quarantine Testing Manual. Biosecurity New Zealand, Ministry of Agriculture and Forestry, Auckland, 2008.

First Report of False Rust Caused by Synchytrium minutum on Kudzu in Korea.

Kudzu (Pueraria montana var. lobata (Willd.) Maesen & S. Almeida) is a weedy, fabaceous vine that is native to and widely distributed in Asia where it is used for various medicinal purposes such as treating convulsions and fever (2). In the United States, especially the southeastern states, kudzu has become a problematic invasive species that overgrows nearly every substrate on which it occurs. Thus, biological control strategies for controlling this vine are of great interest (4). From October to November 2004, a disease of kudzu was observed in Gwangju and Pyeongtaek in Gyeonggi Province, Korea. The disease appeared on leaves and stems as numerous, discrete, small galls, which enlarged, becoming yellowish orange and eventually erupting into orange, pulverulent sori. Galls were scattered or gregarious, amphigenous, predominately hypophyllous, and sometimes formed along veins as well as on petioles and stems. Sori that formed from galls were solitary but sometimes became confluent, 0.1 to 1 mm in diameter, globose to subglobose, and orange to dark orange; walls were hyaline and thin. Sporangia were copious in sori, typically polyhedral due to compression or globose, 16 to 32 µm in diameter, with smooth, hyaline walls and orange contents. Zoospores were not observed during several failed attempts to germinate sporangia. On the basis of morphological descriptions and keys (3), the fungus was identified as Synchytrium minutum (Pat.) Gäum. (Chytridiomycota), the only species of Synchytrium known to occur on Pueraria (1,3). Comparison with specimens from China and New Guinea (BPI 794733 and BPI 1109528) confirmed this identification. Portions of the nLSU and nSSU rDNA from one of the two Korean specimens deposited as voucher material in the U.S. National Fungus Collections (BPI 880898 and BPI 880899) were sequenced (GenBank Accession Nos. HQ324138 and HQ324139), and a subsequent BLAST search against GenBank confirmed placement in the genus Synchytrium with 95% similarity to S. decipiens. S. minutum is widespread in Asia and Oceania and also has been reported from California (1,3). To our knowledge, this is the first report of S. minutum in Korea (1) and is noteworthy to those interested in biological control of kudzu because S. minutum may have potential in this regard. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , September, 2010. (2) H. S. Jung. M.S. thesis. Seoul National University, Seoul, Korea, 1997. (3) J. S. Karling. Synchytrium. Academic Press Inc., New York, NY, 1964. (4) M. A. Weaver et al. Biol. Control 50:150, 2009.

First Report of Juneberry Rust Caused by Gymnosporangium nelsonii on Juneberry in Michigan.

Amelanchier alnifolia (Nutt.) Nutt. ex M. Roem., commonly known as juneberry or Saskatoon serviceberry, was historically a widely used prairie fruit that is native to the Northern Great Plains, southern Yukon and Northwest Territories (4). While juneberry is an important fruit crop in the prairie provinces of Canada, small commercial plantings also occur throughout the northern United States (2), including Michigan. On July 18, 2009, severe rust symptoms were observed on plants in a 2-year-old field of A. alnifolia 'Northline' in Northport, MI. The plants had been sourced as seedlings from a nursery in Alberta, Canada in 2007. Signs and symptoms were present on fruits and leaves on virtually all of the plants. Symptomatic fruit were still immature, and on average, more than 70% of the fruit surface was covered with tubular, whitish aecia with conspicuous orange aeciospores. Portions of twigs also showed fusiform swellings (1 to 3 cm long) covered with aecia. Aecia were hypophyllous, fructicolous and caulicolous, roestelioid, and 2 to 4 mm high. The peridium was cylindric and tapering toward the apex, dehiscent at the apex, retaining a tubular shape for a long time and at times becoming lacerated on the sides with age. Peridial cells were linear rhomboidal, 50 to 105 µm long, hyaline to brownish, outer walls smooth, inner walls with small papillae, and side walls delicately verrucose-rugose with elongate papillae having variable lengths. Aeciospores were globoid, 20 to 35 × 25 to 38 µm (average 30.7 × 32.5 µm), orange to cinnamon brown, and densely verrucose with walls 2.5 to 3.5 µm thick. On the basis of these morphological characters, the host, and comparison with a reference specimen (BPI 122010), the pathogen was identified as Gymnosporangium nelsonii Arthur (1,3). The 5' region of the 28S rDNA was sequenced (GenBank Accession No. HM591299.1), confirming the identification as a species of Gymnosporangium, one distinct from previously sequenced specimens available in GenBank. The specimen has been deposited at the U.S. National Fungus Collections (BPI 880671 and 880709). Four other species found previously on Amelanchier spp. in the Midwest differ as follows: G. clavipes and G. clavariiforme have verrucose peridial cells and different 28S rDNA sequences; G. nidus-avis has rugose peridial cells; and G. corniculans has cornute peridia that dehisce from lateral slits while apices remain intact and verrucose peridial walls with verrucae on the side walls (1). The infection was likely caused by basidiospores originating from telia on Juniperus spp. in the area surrounding the field. However, no telia of G. nelsonii were found on junipers in the immediate vicinity. To our knowledge, this is the first report of G. nelsonii on juneberry in Michigan and the Midwest. Because of the devastating impact of this disease on fruit quality, fungicide programs have been devised for disease control and were effective in 2010. Juneberry growers in the Midwest need to be aware of this disease and monitor their crop carefully for symptoms and signs. References: (1) F. D. Kern. A Revised Taxonomic Account of Gymnosporangium. Pennsylvania State University Press, University Park, 1973. (2) K. Laughlin et al. Juneberry for Commercial and Home Use on the Northern Great Plains. North Dakota State University, Fargo 1996. (3) S. K. Lee and M. Kakishima. Mycoscience 40:121, 1999. (4) G. Mazza and C. G. Davidson. Page 516 in: New Crops. Wiley, New York, 1993.

First Report of Uromyces acuminatus on Honckenya peploides, the Endangered Seabeach Sandwort.

Honckenya peploides (L.) Ehrh. (Caryophyllaceae), commonly known as seabeach sandwort, is a species of special concern in Connecticut (4). Nearly an entire population of H. peploides in New London County, CT was found to be severely infected by the aecial stage of a rust fungus in June of 2008. Representative plants in the population were infected with aecia on more than 50% of the leaves. Aecia were amphigenous, gregarious, cupulate, pulverulent, yellowish, and erumpent with a hyaline to whitish peridium having a lacerate, somewhat recurved margin. Peridial cells were rhomboidal, 26 to 31 × 25 to 29 µm, smooth to finely verrucose. Aeciospores were globose to ellipsoid, 23.5 to 29 × 20.5 to 22 µm, hyaline to pale yellowish with a verrucose surface and hyaline walls 1.5 to 2 µm thick. Morphological characters corresponded to a reference specimen (BPI 000105) of the aecial stage of Uromyces acuminatus Arthur from Nova Scotia, as well as published descriptions (1,2). Subsequently, telia of U. acuminatus were discovered on Spartina patens (Aiton) Muhl. (Poaceae) in May of 2009 in New London County, CT. Telia were adaxial, intercostal, scattered to gregarious, linear and at times elongate, dark brown to black, pulverulent, and erumpent. Teliospores were obovate to ellipsoid with rounded to acuminate apices rarely having two points, 30 to 41 × 19 to 24 µm, with a smooth surface and brownish-yellow to brown walls 9 to 14 µm thick at apex, which is sometimes paler, and 1 to 3 µm thick laterally, pedicels with a portion persisting on the teliospore that is up to 82 µm long and brownish-yellow. The ITS2 and 5' region of the 28S rDNA (998 bp) from the rust on H. peploides (GenBank Accession No. GU109282, BPI 879300) and the rust on S. patens (GenBank Accession No. GU058008, BPI 879285B) were sequenced to confirm the identification of U. acuminatus on H. peploides with the resulting sequences identical. U. acuminatus is widespread in the eastern United States and Canada (1-3). The telial stage is found on Spartina spp., while the aecial stage is found on numerous taxa including members of the Asparagaceae (formerly Ruscaceae, Liliaceae), Caryophyllaceae, Polemoniaceae, and Primulaceae (1-3). Puccinia arenariae (Schumach.) G. Winter, previously reported from H. peploides (4), is microcyclic and stages 0, I, and II are unknown. To our knowledge, this is the first report of U. acuminatus on the genus Honckenya. This report has significance to natural resource conservation managers and scientists working in endangered plant habitats because H. peploides and H. peploides subsp. robusta are listed as plants of special concern or endangered/extirpated in Connecticut, Maryland, New Hampshire, and Rhode Island (4). References: (1) J. C. Arthur. Order Uredinales. N. Am. Flora 7(3):161, 1912. (2) G. B. Cummins. The Rust Fungi of Cereals, Grasses and Bamboos. Springer-Verlag, New York, 1971. (3) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory. Online publication. ARS, USDA, 2009. (4) USDA, NRCS. The PLANTS Database. Online publication. National Plant Data Center, Baton Rouge, LA, 2009.

First Report of Japanese Apple Rust Caused by Gymnosporangium yamadae on Malus spp. in North America.

Plants in the genus Malus Mill. are grown in temperate regions for fruit crops such as apple and ornamental landscape plants such as flowering crab apple. Gymnosporangium yamadae Miyabe ex G. Yamada, cause of Japanese apple rust, is known to attack economically important species of Malus in Asia. In August 2004 and July 2008, the aecial stage of a rust fungus was observed in Wilmington, DE and nearby in Media, PA on leaves of toringo crab apple (Malus toringo (Siebold) Siebold ex de Vriese), a cultivated plant native to Asia. On the basis of morphological and molecular characteristics, the fungus was identified as G. yamadae. This pathogen has not been previously reported in North America. The identification was confirmed by morphological comparisons with specimens of G. yamadae from Asia and descriptions (1-3) as well as using the D1/D2 domain of 28S rDNA sequence data G. clavariiforme (GenBank Accession No. AR426211), G. clavipes (GenBank Accession No. DQ354545), G. cornutum (GenBank Accession No. AF426210), G. juniperi-virginianae (GenBank Accession Nos. AF522167, AY629316, and DQ354547), G. libocedri (GenBank Accession No. AF522168), G. sabinae (GenBank Accession Nos. AF426209 and AY512845) and G. yamadae (GenBank Accession Nos. FJ559373 and FJ559375). The specimens from North America included aecia of G. yamadae that are foliicolous, hypophyllous, roestelioid, and 4 to 7 mm high. The peridia are yellow-brown to brown and cornute to tubular with a closed brown tip at the apex and lacerate sides; the peridial remains often form a reticulate pattern. The peridial cells are long-narrow rhomboid, 83 to 120 µm long with smooth outer walls and verrucose to echinulate inner and side walls. The aeciospores are globose, 18 to 28 × 19 to 29 µm, with a slightly coronate surface and brown-yellow walls 2 to 3 µm thick. The telia known on Juniperus spp. were not observed. The specimens were deposited in the U.S. National Fungus Collections (BPI 878846, BPI 878847, BPI 878848, and BPI 878849). The 28S rDNA sequence of G. yamadae from BPI 878849 was deposited in GenBank as Accession No. FJ455091. Aecia of G. juniperi-virginianae, cause of cedar apple rust, differ from G. yamadae in having recurved peridial walls at maturity. References: (1) F. D. Kern. A Revised Taxonomic Account of Gymnosporangium. Pennsylvania State University Press, University Park, PA, 1973. (2) S. K. Lee and M. Kakishima. Mycoscience 40:109, 1999. (3) S. K. Lee and M. Kakishima. Mycoscience 40:121. 1999.

First Report of Gymnosporangium sabinae, European Pear Rust, on Bradford Pear in Michigan.

Bradford pear, Pyrus calleryana Decne., is well known as an ornamental plant for its flowers, leaf color in fall, and disease resistance, making it desirable as a street tree. In August and October of 2008, the aecial stage of Gymnosporangium sabinae (Dicks.) G. Winter was collected from leaves of P. calleryana in Farmington, MI (Oakland County). The one tree had foliage that was severely affected by the rust fungus. Using published descriptions of G. sabinae as the synonym of G. fuscum, type of the genus Gymnosporangium (1-4), the Michigan specimen was identified and confirmed by comparison with previously reported European and U.S. specimens (BPI 118736 and BPI 856578). The diagnostic characteristics of G. sabinae include: spermagonia epiphyllous; aecia hypophyllous, roestelioid, 3 to 6 mm high; peridium balanoid (acorn shaped), becoming elongated at maturity, pale yellow, sides opening with lateral slits but remaining attached at light brown, pointed apex; peridial cells elongated, 51 to 68 µm long, outer walls smooth, inner walls and side walls sparsely echinulate; aeciospores globose to broadly ellipsoid, somewhat angular, surface slightly coronate, 22 to 32 × 22 to 36 µm, walls orange, 3.5 to 5.5 µm thick. This species is distinguished from other species of Gymnosporangium on Pyrus by the balanoid (acorn-shaped) peridium and the late season maturation of the aecial stage (4). Telia and teliospores of G. sabinae are produced on the alternate host, various species of Juniperus sect. Sabinae, but were not observed in Michigan. The specimen from Michigan is deposited in the U.S. National Fungus Collections (BPI 878928). G. sabinae is widely distributed in Europe, extending to Asia and North Africa, but is rarely reported in North America. It was accidentally introduced into California in the aecial stage on P. communis L. and the telial stage on Juniperus chinensis L. (2), as well as introduced into Canada (British Columbia) (3,4). The only previous report of G. sabinae on P. calleryana is in Germany (1). G. sabinae is known to attack commercial pear and ornamental juniper plants in Europe, thus it seems important to prevent the further spread of this fungus in North America. References: (1) U. Braun. Feddes Repert. Beih. 93:213, 1982. (2) G. Laundon. Mycotaxon 3:133, 1975. (3) A. H. McCain and D. Y. Rosenberg. Calif. Dep. Agric. Bull. 50:13, 1961. (4) J. A. Parmelee. Fungi Canadensis No. 43. Gymnosporangium fuscum, 1974.

Nitric oxide protects PC12 cells from serum deprivation-induced apoptosis by cGMP-dependent inhibition of caspase signaling.

Although nitric oxide (NO) induces neuronal cell death under some conditions, it also can prevent apoptosis resulting from growth factor withdrawal. We investigated the molecular mechanism by which NO protects undifferentiated and differentiated PC12 cells from trophic factor deprivation-induced apoptosis. PC12 cells underwent apoptotic death in association with increased caspase-3-like activity, DNA fragmentation, poly(ADP-ribose) polymerase (PARP) cleavage, and cytochrome c release after 24 hr of serum withdrawal. The apoptosis of PC12 cells was inhibited by the addition of NO-generating donor S-nitroso-N-acetylpenicillamine (SNAP) (5-100 microM) and the specific caspase-3-like protease inhibitor Ac-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-cho) but not the YVADase (or caspase-1-like protease) inhibitor N-acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-cho). SNAP and Ac-DEVD-cho prevented the increase in DEVDase (caspase-3-like protease) activity. The SNAP-mediated suppression of DEVDase activity was only minimally reversed by the incubation of cell lysate with dithiothreitol, indicating that NO did not S-nitrosylate caspase-3-like proteases in PC12 cells. Western blot analysis showed that NO inhibited the proteolytic activation of caspase-3. The cGMP analog 8-bromo-cGMP (8-Br-cGMP) blocked apoptotic cell death, caspase-3 activity and activation, and cytochrome c release. The soluble guanylyl cyclase inhibitor 1-H-oxodiazol-[1,2,4]-[4,3-a] quinoxaline-1-one (CODQ) significantly attenuated NO-mediated, but not 8-Br-cGMP-dependent, inhibition of apoptotic cell death, PARP cleavage, cytochrome c release, and DEVDase activity. Furthermore, the protein kinase G inhibitor KT5823 reversed both SNAP- and 8-Br-cGMP-mediated anti-apoptotic events. All these apoptotic phenomena were also suppressed by NO production through neuronal NO synthase gene transfer into PC12 cells. Furthermore, similar findings were observed in differentiated PC12 cells stimulated to undergo apoptosis by NO donors and NGF deprivation. These findings indicate that NO protects against PC12 cell death by inhibiting the activation of caspase proteases through cGMP production and activation of protein kinase G.

Influence of micrometastasis on N stage in gastric cancer and clinical application.

In order to evaluate the real extent of lymph node metastasis (LNM) in gastric cancer, an immunohistochemical (IHC) analysis was performed. We examined 11173 lymph nodes removed from 355 patients with all stages of gastric carcinoma. Tissue preparations were stained with cytokeratin 18, monoclonal antibody against cytokeratin. Micrometastases were found in 2.5% of the lymph nodes and in 31.3% of patients. The incidence of the patients with LNM increased to 9.1% in T(1m) (n = 99), 31.6% in T(1sm) (n = 95, 23.1% in sm1, 34.8% in sm2), 66.7% in T2 (n = 108, 48.8% in mp, 76.5% in ss), 88.1% in T3 (n = 42), and 90.9% in T4 (n = 11) lesions. Upstage was identified in 8.5% of patients: 6.7% in T1 (4.0% in m, 7.7% in sm1, 10.1% in sm2), 14.8% in T2 (20% in mp, 11.8% in ss), 2.4% in T3, and 0% in T4. Factors related to LNM were: tumor size and lymphatic invasion in mucosal lesions; only lymphatic invasion in submucosal lesions; size and depth of tumor, and lymphatic invasion in T2 lesions. In conclusion, the incidence of micrometastasis in regional lymph nodes was higher than we imagined in T1 lesions, more than D1 lymphadenectomy for sm1 and selected cases of mucosal cancer, and D2 lymphadenectomy for sm2 are necessary.

Peptidylglycine alpha-amidating monooxygenase: a multifunctional protein with catalytic, processing, and routing domains.

Peptide alpha-amidation is a widespread, often essential posttranslational modification shared by many bioactive peptides and accomplished by the products of a single gene encoding a multifunctional protein, peptidylglycine alpha-amidating monooxygenase (PAM). PAM has two catalytic domains that work sequentially to produce the final alpha-amidated product peptide. Tissue-specific alternative splicing can generate forms of PAM retaining or lacking a domain required for the posttranslational separation of the two catalytic activities by endoproteases found in neuroendocrine tissue. Tissue-specific alternative splicing also governs the presence of a transmembrane domain and generation of integral membrane or soluble forms of PAM. The COOH-terminal domain of the integral membrane PAM proteins contains routing information essential for the retrieval of PAM from the surface of endocrine and nonendocrine cells. Tissue-specific endoproteolytic processing can generate soluble PAM proteins from integral membrane precursors. Soluble PAM proteins are rapidly secreted from stably transfected nonneuroendocrine cells but are stored in the regulated secretory granules characteristic of neurons and endocrine cells.

Neurobiology of nitric oxide.

Nitric oxide is a ubiquitous and unique biological messenger molecule. It mediates blood vessel relaxation by endothelium, immune function of macrophages, and neurotransmission of central and peripheral nervous systems. Endothelial and neuronal nitric oxide synthases are constitutively expressed and activated by calcium entry into cells, whereas the macrophage nitric oxide synthase is inducible with new RNA and protein synthesis upon immune stimulation. Nitric oxide may play a role in the neurotransmitter release, neural development, synaptic plasticity, and regulation of gene expression. Excessive production of nitric oxide is neurotoxic and is implicated in a variety of neurological disorders.

A new member of alpha 1-adrenoceptor-coupled G alpha h (transglutaminase II) family in pig heart: purification and characterization.

We previously reported an identification of a 77-kDa GTP-binding protein that co-purified with the alpha 1-adrenoceptor following ternary complex formation. In the present paper, we report on the purification and characterization of this GTP-binding protein (termed G alpha h5) isolated from pig heart membranes. After solubilization of pig heart membranes with NaCl, G alpha h5 was purified by sequential chromatographies using DEAE-Cellulose, Q-Sepharose, and GTP-agarose columns. The protein displayed high-affinity GTP gamma S binding which is Mg(2+)-dependent and saturable. The relative order of affinity of nucleotide binding by G alpha h5 was GTP > GDP > ITP >> ATP > or = adenyl-5'-yl imidodiphosphate, which was similar to that observed for other heterotrimeric G-proteins involved in receptor signaling. Moreover, the G alpha h5 demonstrated transglutaminase (TGase) activity that was blocked either by EGTA or GTP gamma S. In support of these observations, the G alpha h5 was recognized by a specific antibody to G alpha h7 or TGase II, indicating a homology with G alpha h (TGase II) family. These results demonstrate that 77-kDa G alpha h5 from pig heart is an alpha 1-adrenoceptor-coupled G alpha h (TGase II) family which has species-specificity in molecular mass.

Up-regulation and co-expression of fibroblast growth factor receptors in human gastric cancer.

Fibroblast growth factor (FGF), a key regulatory factor of cell growth and differentiation, is involved in embryonic development, angiogenesis, and tumorigenesis. To date, four different FGF receptors (FGFRs) have been cloned and characterized. We examined the expression of four FGFRs in human gastric cancer tissues and cell lines using Northern analysis, ribonuclease protection assay, and immunohistochemistry. The mRNAs of FGFR-1 (10/14), FGFR-2 (9/14), and FGFR-4 (9/14) were up-regulated in cancer compared with normal tissues. FGFR-3 mRNAs were barely detectable in both normal and cancer tissues. These FGFR mRNAs were co-expressed in various combinations of two or three in the same tissue. Immunohistochemistry confirmed specific staining of multiple FGFRs, except FGFR-3, in the cancer specimens. To investigate the functional significance of FGFR co-expression we examined the invasive property of SNU-16 cells, which exhibited gene amplification of FGFR-2, -3, and -4 as well as over-expression of keratinocyte growth factor receptor (KGFR), a splice variant of FGFR-2, and FGFR-4 mRNA. KGF plus acidic FGF (aFGF), KGF, and aFGF treatment enhanced the invasive potential of SNU-16 cells over the control by 100%, 107%, and 47%, respectively, indicating that neither additive nor synergistic effect was induced by stimulation with aFGF plus KGF. These results suggest that co-expression of FGFRs in various combinations may cause subtle changes in the progression of gastric cancer.

Involvement of endothelial nitric oxide synthase in the impaired endothelium-dependent relaxation of cavernous smooth muscle in hypercholesterolemic rabbit.

The present study was designed to evaluate whether functional impairment and/or protein expression of constitutive nitric oxide synthase (cNOS; endothelial NOS [eNOS] and neuronal NOS[nNOS]) was involved in impairment of endothelium-dependent relaxation of cavernous smooth muscle in hypercholesterolemic rabbits. New Zealand White rabbits were randomly divided into control and experimental groups. The control group (n=20) received a regular diet, while the two experimental groups (n=20 for each) were fed a 2% cholesterol diet for 4 and 8 weeks, respectively. We conducted isometric tension studies with endothelium-dependent and endothelium-independent vasodilators with or without preincubation with L-arginine and nonadrenergic, noncholinergic (NANC)-selective electrical field stimulation on isolated strips of corpus cavernosum. Expression of cNOS (eNOS and nNOS) protein was assessed by Western blot analysis. cNOS activities in both cytosolic and particulate fractions were measured by determining the conversion of L-[U-14C] arginine to L-[U-14C] citrulline. Blood levels of cholesterol were significantly higher (P < 0.01) in the experimental groups than in the control group. The relaxation responses to endothelium-dependent agents (acetylcholine and adenosine 5'-diphosphate [ADP]) were significantly reduced (P < 0.05) in both experimental groups, regardless of their incubation with L-arginine, compared with the control group. However, no differences were found among the three groups in the relaxation response to endothelium-independent agents (papaverine and nitroprusside) and to NANC-selective electrical field stimulation. There was no difference in immunoreactive nNOS from cytosolic and particulate fractions between the cavernous tissues of the control and experimental groups. nNOS protein levels in the particulate fractions were markedly lower than in the cytosolic fractions. The particulate cNOS activity was significantly decreased (P < 0.05) in the experimental groups compared with the control group, while the cytosolic cNOS activity in the experimental groups was not different from that found in the control group. Therefore, it is concluded that functional impairment of eNOS, rather than of nNOS, may lead to impairment of cavernous smooth muscle relaxation in response to endothelium-mediated stimuli in hypercholesterolemic rabbits.

Glutamate-stimulated calcium activation of Ras/Erk pathway mediated by nitric oxide.

NMDA-type glutamate receptor-mediated increases in intracellular calcium play a critical role in synaptic plasticity involved in learning and memory. Calcium-dependent activation of Ras and extracellular signal-regulated kineses (Erks) may transmit the glutamate signal to the nucleus which is ultimately important for long-lasting neuronal responses. The mechanism by which changes in cytoplasmic calcium mediate NMDA-induced activation of Ras and Erk is not known. In cerebral cortical neurons, this calcium influx through NMDA receptors activates Ras and its downstream effector, Erk, via nitric oxide (NO) generation by calcium-dependent neuronal NO synthase. We propose that NO is a key link between NMDA-mediated increases in cytoplasmic calcium and activity-dependent long-term changes such as differentiation, survival and synaptic plasticity.

Identification of a distinct molecular mass G alpha(h) (transglutaminase II) coupled to alpha1-adrenoceptor in mouse heart.

Our previous studies on alpha1-adrenoceptor signaling suggested that G alpha(h) family is a signal mediator in different species. To elucidate the species-specificity of G alpha(h) family in molecular mass, we used the solubilized membranes from mouse heart and the ternary complex preparations containing alpha1-agonist/receptor/G-protein. Binding of [35S]GTPgammaS and the intensity of the [alpha-32P]GTP photoaffinity labeled protein resulting from activation of the alpha1-adrenoceptor were significantly attenuated by the antagonist, phentolamine. The molecular mass of the specific GTP-binding protein was approximately 72-kDa; homologous with G alpha(h) (transglutaminase II) family. Furthermore, immunological cross-reactivity of ternary complex from mouse heart and purified G alpha(h) from rat, guinea pig, and bovine using anti-G alpha(h7) antibody showed that their molecular masses were distinctly different and approximately 72-kDa G alpha(h) from mouse heart was the lowest molecular mass. Consistent with these observations, in co-immunoprecipitation and co-immunoadsorption of the alpha1-adrenoceptor in the ternary complex preparation by anti-G alpha(h7) antibody, the G alpha(h) family protein tightly coupled to alpha1-adrenoceptor. These results demonstrate the species-specificity of G alpha(h) family in molecular mass, especially the lowest molecular mass in mouse.

A new fusion gene NUP98-IQCG identified in an acute T-lymphoid/myeloid leukemia with a t(3;11)(q29q13;p15)del(3)(q29) translocation.

NUP98 has been involved in multiple recurrent chromosome rearrangements in leukemia. We identified a novel fusion between NUP98 and IQ motif containing G (IQCG) gene from a de novo acute T-lymphoid/myeloid leukemia harboring t(3;11)(q29q13;p15)del(3)(q29). IQCG has two putative coiled-coil domains and one IQ domain. The FG repeat from NUP98 and the coiled-coil domain from IQCG were retained in the fusion protein. We demonstrated that NUP98-IQCG could form homodimer, heterodimerize with NUP98 or IQCG, bind co-activators and/or co-repressors, and show transcriptional activity in vitro. Expression of NUP98-IQCG inhibited 32Dcl3 cell apoptosis induced by Ara-C, and partially blocked granulocyte differentiation induced by G-CSF. Colony-forming assay and serial replating assays indicated that NUP98-IQCG was able to stimulate proliferation, partially block differentiation of hematopoietic stem/progenitor cells but was unable to confer transformation alone. Taken together, our data indicate that newly identified NUP98-IQCG fusion protein may play an essential role in leukemogenesis, but by itself may not be sufficient to induce leukemia.