The rice <Emphasis Type="Italic">bright green leaf</Emphasis> (<Emphasis Type="Italic">bgl</Emphasis>) locus encodes OsRopGEF10, which activates the development of small cuticular papillae on leaf surfaces

Development of specialized epidermal cells and structures plays a key role in plant tolerance to biotic and abiotic stresses. In the paddy field, the bright green leaf (bgl) mutants of rice (Oryza sativa) exhibit a luminous green color that is clearly distinguishable from the normal green of wild-type plants. Transmission and scanning electron microscopy revealed that small cuticular papillae (or small papillae; SP), nipple-like structures, are absent on the adaxial and abaxial leaf surfaces of bgl mutants, leading to more direct reflection and less diffusion of green light. Map-based cloning revealed that the bgl locus encodes OsRopGEF10, one of eleven OsRopGEFs in rice. RopGEFs (guanine nucleotide exchange factors for Rop) activate Rop/Rac GTPases, acting as molecular switches in eukaryotic signal transduction by replacing the bound GDP (inactive form) with GTP (active form) in response to external or internal cues. In agreement with the timing of SP initiation on the leaf epidermis, OsRopGEF10 is most strongly expressed in newly developing leaves before emergence from the leaf sheath. In yeast two-hybrid assays, OsRopGEF10 interacts with OsRac1, one of seven OsRac proteins; consistent with this, both proteins are localized in the plasma membrane. These results suggest that OsRopGEF10 activates OsRac1 to turn on the molecular signaling pathway for SP development. Together, our findings provide new insights into the molecular genetic mechanism of SP formation during early leaf morphogenesis.     

Synthesis of double-layered rotavirus-like particles using internal ribosome entry site vector system in stably-transformed <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

We established a bicistronic expression system using an encephalomyocarditis virus (EMCV)-derived internal ribosomal entry site (IRES) element to generate stably transformed Drosophila melanogaster Schneider 2 (S2) cells expressing human rotavirus Wa capsid proteins, VP2 and VP6, for the synthesis of VP2/6 double-layered virus-like particle (DVLP). The EMCV-derived IRES permitted bicistronic translation of recombinant VP6. Recombinant VP2 and VP6 were detected in extracellular fractions of stably transformed S2 cells. A wheel-like DVLP (diam ~ 50–55 nm) with short spikes was produced from the extracellular fraction of stably transformed S2 cells. A bicistronic expression system using an EMCV-derived IRES element can thus be used to express two proteins of interest in stably transformed S2 cells. The bi-or tri-cistronic expression of recombinant VP2/6/7 using stably transformed S2 cells can also be used to produce rotavirus VLPs.     

Partial nitrification using an electrolytic aerating bioreactor with ammonia-oxidizing bacteria-dominant activated sludge

An electrolytic aerating bioreactor was used to partially nitrify ammonia from wastewater. Activated sludge was cultured for 8 months to increase the population of ammonia-oxidizing bacteria (AOB) and then used in the bioreactor. The maximum ammonia removal rate was 0.64 mM NH₃/l h in a 50 ml reactor using 5.4 g mixed liquor suspended solids per litre of AOB-dominant activated sludge.     

Generation of antibodies recognizing an aberrant glycoform of human tissue inhibitor of metalloproteinase-1 (TIMP-1) using decoy immunization and phage display

Aberrant glycosylation of human tissue inhibitor of metalloproteinase-1 (TIMP-1) by N-acetylglucosaminyltransferase-V (GnT-V) was previously reported to be related to cancer progression. Here, we report on the antibodies recognizing the structural features initiated by an addition of N-linked β(1,6)-N-acetylglucosamine (GlcNAc) branch by GnT-V on TIMP-1. Two glycoforms of TIMP-1, TIMP1-L produced in Lec4 cells without GnT-V activity and TIMP1-B in GnT-V overexpressing transfectant cells, were purified from culture supernatant and used for generation of antibodies. TIMP1-L was injected in the left hind footpad of mice as decoy and TIMP1-B in the right hind footpad as immunogen. Phage-displayed scFv library was constructed from the B cells retrieved from the right popliteal lymph nodes and subjected to panning and screening. Phage ELISA of individual clones revealed the scFv clones with preferential binding activity to TIMP1-B, and they were converted into an scFv-Fc format for further characterization of binding specificity. Glycan binding assay of an antibody, 1-9F, revealed its differential specificity toward an extension of glycan structure initiated with β(1,6)-GlcNAc linkage and terminally decorated with a sialic acid. This study demonstrates feasibility of a new strategy combining decoy immunization with phage display for the efficient generation of antibodies tracking down structural features of different glycoforms.     

Effects of Treadmill Exercise Combined with MK 801 Treatment on Neuroblast Differentiation in the Dentate Gyrus in Rats

N-methyl-d-aspartate receptor (NR) is involved in activity-dependent synaptic plasticity, such as associative long-term potentiation, and in related central functions, such as learning and memory. In this study, we observed effects of treadmill exercise on NR1 and doublecortin (DCX, a marker for neuroblast differentiation) in the subgranular zone of the dentate gyrus (DG). At 6 weeks of age, rats were put on a treadmill with or without running for 1 h/day for 5 consecutive days at 22 m/min for 5 weeks. Exercise increased NR1 immunoreactivity and protein level in the hippocampus. To identify the correlations between NR and neuroblasts, we intraperitoneally administered a NR antagonist, MK-801, to the exercised rats. MK-801 treatment reduced NR1 protein level in the hippocampus of the exercised rats. In addition, in the MK-801-treated group, the number of DCX cells was significantly decreased in the subgranular zone of the DG. These results suggest that NR may be one of the important factors that modulate neuroblast differentiation during exercise in rats.     

Comparison of Phosphorylated Extracellular Signal-Regulated Kinase 1/2 Immunoreactivity in the Hippocampal Ca1 Region Induced by Transient Cerebral Ischemia Between Adult and Aged Gerbils

In this study, the authors examined the difference of phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) in the hippocampal CA1 region (CA1) between adult and aged gerbils after 5 min of transient cerebral ischemia. Delayed neuronal death in the CA1 of the aged group was much slower than that in the adult group after ischemia/reperfusion (I/R). pERK1/2 immunoreaction was observed in the CA1 region of the sham-operated adult gerbil. pERK1/2 immunoreactivity and protein levels in the ischemic CA1 region of the adult group were markedly increased 4 days after I/R, and then reduced up to 10 days after I/R. In contrast, pERK1/2 immunoreaction was hardly detected in the CA1 region of sham-operated aged gerbils, and the immunoreactivity increased from 1 day after the ischemic insult, and still observed until 10 days post-ischemia. In addition, pERK1/2-immunoreaction was expressed in astrocytes in the ischemic CA1 region: The expression in the ischemia-operated aged gerbils was later than that in the ischemia-operated adult gerbils. These results indicate that different patterns of ERK1/2 immunoreactivity may be associated with different processes of delayed neuronal death in adult and aged animals.     

Determinants of redox sensitivity in RsrA, a zinc-containing anti-sigma factor for regulating thiol oxidative stress response

Various environmental oxidative stresses are sensed by redox-sensitive regulators through cysteine thiol oxidation or modification. A few zinc-containing anti-sigma (ZAS) factors in actinomycetes have been reported to respond sensitively to thiol oxidation, among which RsrA from Streptomyces coelicolor is best characterized. It forms disulfide bonds upon oxidation and releases bound SigR to activate thiol oxidative stress response genes. Even though numerous ZAS proteins exist in bacteria, features that confer redox sensitivity to a subset of these have been uncharacterized. In this study, we identified seven additional redox-sensitive ZAS factors from actinomycetes. Comparison with redox-insensitive ZAS revealed characteristic sequence patterns. Domain swapping demonstrated the significance of the region K(33)FEHH(37)FEEC(41)SPC(44)LEK(47) that encompass the conserved HX(3)CX(2)C (HCC) motif. Mutational effect of each residue on diamide responsive induction of SigR target genes in vivo demonstrated that several residues, especially those that flank two cysteines (E39, E40, L45, E46), contribute to redox sensitivity. These residues are well conserved among redox-sensitive ZAS factors, and hence are proposed as redox-determinants in sensitive ZAS. H37A, C41A, C44A and F38A mutations, in contrast, compromised SigR-binding activity significantly, apparently affecting structural integrity of RsrA. The residue pattern around HCC motif could therefore serve as an indicator to predict redox-sensitive ZAS factors from sequence information.     

Effects of Adrenalectomy and Replacement Therapy of Corticosterone on Cell Proliferation and Neuroblast Differentiation in the Rat Dentate Gyrus

Newly generated neurons in the dentate gyrus differentiate into mature granule cells. In the present study, we observed the effects of adrenalectomy (ADX) and corticosterone replacement therapy (CRT) on cell death, cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus (SZDG). For this, the animals received vehicle or CRT after ADX, and were sacrificed 5 or 42 days later. Plasma corticosterone levels were very low in the adrenalectomized groups, whereas CRT after ADX significant increased serum corticosterone levels at 42 days, not 5 days, after ADX. ADX induced some neuronal damage in the dentate gyrus at 5 days post-ADX. CRT did not significantly reduce the neuronal damage at 5 days post-ADX; however, neuronal damage was not shown at 42 post-ADX with CRT. Ki67 (a marker for cell proliferation) and doublecortin (DCX, a marker for neuronal differentiation) immunoreaction was detected in the SZDG. ADX transiently increased cell proliferation and neuroblast differentiation 5 days after ADX, not 42 days, after ADX, and the CRT 42 days after ADX prominently decreased cell proliferation and neuroblast differentiation in the dentate gyrus. These results suggest that adrenal corticosteroid hormone is not essential for cell proliferation and neuroblast differentiation in long-term period after ADX.     

Differential effects of treadmill exercise in early and chronic diabetic stages on parvalbumin immunoreactivity in the hippocampus of a rat model of type 2 diabetes

In the present study, we investigated the effects of treadmill exercise in early and chronic diabetic stages on parvalbumin (PV) immunoreactivity in the subgranular zone of the dentate gyrus of Zucker diabetic fatty (ZDF) and its lean control rats (ZLC). To investigate the effects, ZLC and ZDF rats at 6 or 23 weeks of age were put on a treadmill with or without running for 1 h/day/5 consecutive days at 16–22 m/min for 5 weeks or 12–16 m/min for 7 weeks, respectively. Physical exercise in pre-diabetic rats prevented onset of diabetes, while exercise in rats at chronic diabetic stage significantly reduced blood glucose levels. In addition, physical exercise in the pre-diabetic rats significantly increased PV immunoreactive fibers in the strata oriens and radiatum of the CA1-3 region and in the polymorphic and molecular layers of the dentate gyrus compared to that in sedentary controls. However, in rats at chronic stages, PV immunoreactivity was slightly increased in the CA1-3 region as well as in the dentate gyrus compared to that in the sedentary controls. These results suggest that physical exercise has differential effects on blood glucose levels and PV immunoreactivity according to diabetic stages. Early exercise improves diabetic phenotype and PV immunoreactive fibers in the rat hippocampus.