Postnatal Development of Cholecystokinin-Like Immunoreactivity and Its mRNA Level in Rat Brain Regions

Developmental changes of preprocholecystokinin mRNA (CCK mRNA) and cholecystokinin-like immunoreactivity (CCK-LI) were examined in rat brain regions (frontal cortex, colliculi, hippocampus, striatum, and cerebellum) using RNA dot blot assays with cholecystokinin (CCK) cDNA and radioimmunoassay, respectively. The CCK-LI levels in all regions examined were very low at birth. Excluding the cerebellum, the levels in these regions increased postnatally and reached adult values at 28 days of age. In contrast to CCK-LI, CCK mRNA levels changed dramatically during development. A considerable amount of CCK mRNA was detected in the frontal cortex and hippocampus at birth. The changes in the level of CCK mRNA in the frontal cortex and colliculi paralleled those of CCK-LI, including a rapid increase from 7 to 14 days of age. The synthesis of CCK mRNA preceded the appearance of CCK-LI. CCK mRNA levels in the hippocampus and striatum exhibited a transient increase, with a peak at 14 days of age. In the adult brain, the CCK mRNA levels were high in the frontal cortex, moderate in the hippocampus and colliculi, and low in the striatum. The cerebellum contained only a negligible amount of CCK mRNA during development. The relatively high level of CCK-LI compared with the low level of CCK mRNA in the striatum supports the idea that most of the striatal CCK-LI is supplied from extrastriatal regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of la on surface charges, dielectrophoresis, and electrofusion of barley protoplasts

When dielectrophoresis and electrofusion of barley (Hordeum vulgare var Moor) leaf protoplasts were assayed in the presence of 0.1 to 1 millimolar lanthanum ion (La³⁺) in the basal medium (0.7 molar mannitol, 1 millimolar piperazine-N, N-bis[2-ethanesulfonic acid]-Na [pH 6.7], 0.1 millimolar CaCl₂), dielectrophoresis and induction of electrofusion were strongly inhibited. The latter remained inhibited and the former recovered by about 60% after washing the La³⁺ -treated protoplasts without EDTA. These inhibitions were almost completely abolished by washing the La³⁺ -treated protoplasts with 1 millimolar EDTA. Inductively coupled plasma atomic emission spectroscopic analysis revealed that protoplasts retained a considerable amount of La³⁺ after washing without EDTA and released most of the bound La³⁺ by washing with 1 millimolar EDTA. This tightly bound La³⁺ seemed responsible for the inhibition of electrofusion and dielectrophoresis that was observed in the La³⁺ -treated protoplasts after washing. ζ-potentials of protoplasts were -39.0±3.2 millivolts, -16.7 ± 2.6 millivolts, and virtually zero in media containing 0, 0.1, and 0.3 millimolar La³⁺ (I = 7.2 millimolar), respectively, and had a positive value (+ 14.2 ± 2.2 millivolts) in the presence of 1 millimolar La³⁺. These effects of La³⁺ on ζ-potentials were easily abolished by washing without EDTA. This indicates that charged species located at the surface of plasma membrane of protoplasts cannot account for the sites at which La³⁺ exerts its inhibition of dielectrophoresis and electrofusion. In contrast, the promotion of spherical fusion and the reduction of broken fusion products observed in the presence of La³⁺ were almost completely abolished by washing without EDTA. Our results also indicate that the initial induction and development of electrofusion can be studied independently.     

Primary structures of alpha- and beta-subunits of alpha-amylase inhibitors from seeds of three cultivars of Phaseolus beans

The primary structures of three -amylase inhibitors (TAI, DAI, and MAI-2) consisting of glycoprotein subunits and from the respective seeds of three cultivars of Phaseolus beans, Toramame (Phaseolus vulgaris L.), Daifukumame (Phaseolus vulgaris L.), and Murasakihanamame (Phaseolus coccineus L.) were determined by sequencing the peptide fragments derived from their enzymatic digestions. Major sugar chains of the inhibitors were also assessed by analyzing glycopeptides in the enzymatic digests. The subunits, and , were shown to be composed of 76 and 139 amino acid residues, respectively, in each inhibitor. The overall amino acid sequences of the inhibitors were slightly different from one another. Furthermore, the sequence of TAI was the same as that deduced from a cDNA clone encording -amylase inhibitor-1 from the common bean (Phaseolus vulgaris L.). It was also revealed that there were two N-glycosylation sites in each -subunit: PA-derivatives of the major N-glycans were estimated to be M6B at Asn(12) and M9A at Asn(65). Each -subunit of TAI and MAI-2 had two N-glycosylation sites, while the -subunit of DAI had only one site. The major N-glycans pyridylaminated were estimated to be M3X at Asn(63) in each -subunit and M3FX at Asn(83) in -subunits of TAI and MAI-2.     

Classification and ordination of communities of soil arthropods in an urban park of Osaka City

To assess the effect of management of a park on soil arthropods, communities of Oribatida and Collembola were analyzed at 11 sites of different vegetation in Tsurumi Park, an urban park of Osaka City. The type of canopy layer and soil density strongly affected the community parameters, such as species diversity. Ordination revealed that soil density, contents of organic matter, and shrub layer were important for variety in the oribatid community; the shrub layer was important for the collembolan community. Species richness of both arthropod groups was highest in a mixed forest and lowest on bare land, while the abundance of Collembola was highest on a lawn site. Areas of a common vegetation type had a similar oribatid community;Trichogalumma nipponica dominated in deciduous forests,Eohypochthonius crassisetiger in mixed forests and one of coniferous forests. On the other hand, collembolan communities did not correspond with the vegetation.Sminthurinus sp. was collected from every site, and the most abundant species wasCryptopygus thermophilus that exhibited an outbreak on lawn sites. A significant correlation existed between species diversities but not between abundances of Oribatida and Collembola.     

Design, synthesis, and structure-activity relationships of a series of 2-Ar-8-methyl-5-alkylaminoquinolines as novel CRF(1) receptor antagonists

We designed and synthesized a series of 2-Ar-8-methyl-5-alkylaminolquinolines as potent corticotropin-releasing factor 1 (CRF(1)) receptor antagonists. The structure-activity relationships of substituents at each position (R(3), R(5), R(5'), and R(8)) was investigated. By derivatization, three compounds (6, 14b, and 14c) were identified as orally active CRF(1) receptor antagonists.     

Compromised host defense on Pseudomonas aeruginosa biofilms: characterization of neutrophil and biofilm interactions

Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on tissues and other surfaces. We characterized the interaction of purified human neutrophils with P. aeruginosa, growing in biofilms, with regard to morphology, oxygen consumption, phagocytosis, and degranulation. Scanning electron and confocal laser microscopy indicated that the neutrophils retained a round, unpolarized, unstimulated morphology when exposed to P. aeruginosa PAO1 biofilms. However, transmission electron microscopy demonstrated that neutrophils, although rounded on their dorsal side, were phagocytically active with moderate membrane rearrangement on their bacteria-adjacent surfaces. The settled neutrophils lacked pseudopodia, were impaired in motility, and were enveloped by a cloud of planktonic bacteria released from the biofilms. The oxygen consumption of the biofilm/neutrophil system increased 6- and 8-fold over that of the biofilm alone or unstimulated neutrophils in suspension, respectively. H(2)O(2) accumulation was transient, reaching a maximal measured value of 1 micro M. Following contact, stimulated degranulation was 20-40% (myeloperoxidase, beta-glucuronidase) and 40-80% (lactoferrin) of maximal when compared with formylmethionylleucylphenylalanine plus cytochalasin B stimulation. In summary, after neutrophils settle on P. aeruginosa biofilms, they become phagocytically engorged, partially degranulated, immobilized, and rounded. The settling also causes an increase in oxygen consumption of the system, apparently resulting from a combination of a bacterial respiration and escape response and the neutrophil respiratory burst but with little increase in the soluble concentration of H(2)O(2). Thus, host defense becomes compromised as biofilm bacteria escape while neutrophils remain immobilized with a diminished oxidative potential.     

Golgi/plastid‐type manganese superoxide dismutase involved in heat‐stress tolerance during grain filling of rice

Superoxide dismutase (SOD) is widely assumed to play a role in the detoxification of reactive oxygen species caused by environmental stresses. We found a characteristic expression of manganese SOD 1 (MSD1) in a heat‐stress‐tolerant cultivar of rice (Oryza sativa). The deduced amino acid sequence contains a signal sequence and an N‐glycosylation site. Confocal imaging analysis of rice and onion cells transiently expressing MSD1‐YFP showed MSD1‐YFP in the Golgi apparatus and plastids, indicating that MSD1 is a unique Golgi/plastid‐type SOD. To evaluate the involvement of MSD1 in heat‐stress tolerance, we generated transgenic rice plants with either constitutive high expression or suppression of MSD1. The grain quality of rice with constitutive high expression of MSD1 grown at 33/28 °C, 12/12 h, was significantly better than that of the wild type. In contrast, MSD1‐knock‐down rice was markedly susceptible to heat stress. Quantitative shotgun proteomic analysis indicated that the overexpression of MSD1 up‐regulated reactive oxygen scavenging, chaperone and quality control systems in rice grains under heat stress. We propose that the Golgi/plastid MSD1 plays an important role in adaptation to heat stress.     

Neuropeptides trigger oocyte maturation and subsequent spawning in the hydrozoan jellyfish Cytaeis uchidae

Oocyte maturation and subsequent spawning in hydrozoan jellyfish are generally triggered by light‐dark cycles. To examine if the initiation of the maturation process after light stimulus is mediated by neurotransmitters, neuropeptides isolated originally from Hydra magnipapillata were applied to sexually mature female medusae of the hydrozoan jellyfish Cytaeis uchidae. Among the Hydra neuropeptides tested, Hym‐53 (NPYPGLW‐NH₂), as well as a nonphysiological peptide, CGLWamide (CGLW‐NH₂), were most effective in inducing oocyte maturation and spawning. Hym‐355 (FPQSFLPRG‐NH₂) also triggered these events, but the stimulatory effect was weaker. Since Hym‐53‐OH (NPYPGLW) and Hym‐355‐OH (FPQSFLPRG) had no effect, amidation at the C‐terminus may be critical for the stimulatory activities of the peptides. Exposure to Hym‐53 for 2 min was sufficient to trigger of oocyte maturation, and the spawned eggs were able to be fertilized and to develop normally. Transmission electron microscopy confirmed that bundles of axon‐like structures that contain dense‐core synaptic vesicles and microtubules are present in the ovarian ectodermal epithelium overlying the oocytes. In addition, immunohistological analyses revealed that some of the neurons in the ectodermal epithelium are GLWamide‐ and PRGamide‐positive. These results suggest that a neuropeptide signal transduction pathway is involved in mediating the induction of oocyte maturation and spawning in this jellyfish. Mol. Reprod. Dev. 80: 223–232, 2013. © 2013 Wiley Periodicals, Inc.     

The loss of PGAM5 suppresses the mitochondrial degeneration caused by inactivation of PINK1 in Drosophila

PTEN-induced kinase 1 (PINK1), which is required for mitochondrial homeostasis, is a gene product responsible for early-onset Parkinson's disease (PD). Another early onset PD gene product, Parkin, has been suggested to function downstream of the PINK1 signalling pathway based on genetic studies in Drosophila. PINK1 is a serine/threonine kinase with a predicted mitochondrial target sequence and a probable transmembrane domain at the N-terminus, while Parkin is a RING-finger protein with ubiquitin-ligase (E3) activity. However, how PINK1 and Parkin regulate mitochondrial activity is largely unknown. To explore the molecular mechanism underlying the interaction between PINK1 and Parkin, we biochemically purified PINK1-binding proteins from human cultured cells and screened the genes encoding these binding proteins using Drosophila PINK1 (dPINK1) models to isolate a molecule(s) involved in the PINK1 pathology. Here we report that a PINK1-binding mitochondrial protein, PGAM5, modulates the PINK1 pathway. Loss of Drosophila PGAM5 (dPGAM5) can suppress the muscle degeneration, motor defects, and shorter lifespan that result from dPINK1 inactivation and that can be attributed to mitochondrial degeneration. However, dPGAM5 inactivation fails to modulate the phenotypes of parkin mutant flies. Conversely, ectopic expression of dPGAM5 exacerbated the dPINK1 and Drosophila parkin (dParkin) phenotypes. These results suggest that PGAM5 negatively regulates the PINK1 pathway related to maintenance of the mitochondria and, furthermore, that PGAM5 acts between PINK1 and Parkin, or functions independently of Parkin downstream of PINK1.