Species composition and distribution of Coleosporium species on the needles of Pinus densiflora at a semi-natural vegetation succession site in central Japan

Coleosporium species cause pine needle rust. Most species have heteromacrocyclic life cycles, and 12 species use Pinus densiflora as aecial hosts. To understand the biology of rust fungi and develop better methods for controlling rust diseases, it is necessary to clarify that which Coleosporium species affect pine trees. However, Coleosporium on pine trees have rarely been identified at the species level because of their morphological similarities. We used polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) to clarify the species composition, abundance, and distribution of Coleosporium in a P. densiflora forest. We surveyed a site where several Coleosporium species might complete their life cycles. PCR-RFLP revealed four species on the pines: C. asterum, C. clematidis-apiifoliae, C. lycopodis, and C. phellodendri. Coleosporium phellodendri was distributed throughout the forest and was the most abundant. Aecia of C. phellodendri formed mainly on 2-y-old needles. The abundance and distribution of C. phellodendri appeared to be affected by the longer effective dispersal range of basidiospores and the existence of abundant inoculum sources. The age of leaves where C. phellodendri form aecia mainly was thought to be influenced by the characteristic life cycle, with aecial formation requiring 2?y after basidiospore infection.     

Maternal exercise training attenuates endotoxin-induced sepsis in mice offspring

Regular exercise during pregnancy can prevent offspring from several diseases, such as cardiovascular diseases, obesity, and type II diabetes during adulthood. However, little information is available about whether maternal exercises during pregnancy protect the offspring from infectious diseases, such as sepsis and multiple organ dysfunction syndrome (MODS). This study aimed to investigate whether maternal exercise training protects the offspring from endotoxin-induced septic shock in mice. Female C57BL/6 mice performed voluntary wheel exercises during pregnancy. All dams and offspring were fed normal chow with sedentary activity during lactation and after weaning. At 10-week-old, mice were intraperitoneally injected a lethal (30 mg/kg) or nonlethal (15 mg/kg) dose of lipopolysaccharide (LPS), following which the survival of mice that were administered a lethal dose was monitored for 60 h. Plasma, lung, and liver samples were collected 18 h after the injection to evaluate the cytokine concentration or mRNA expression from those administered a nonlethal dose. Although maternal exercise training could not prevent lethality during an LPS-induced septic shock, it significantly inhibited the LPS-induced loss of body weight in female offspring. Regular maternal exercise significantly inhibited the mRNA expression of the LPS-induced inflammatory cytokines, such as interleukin-1β (IL-1β) and interferon-γ (IFN-γ), in the plasma and liver. Thus, maternal exercise inhibited the LPS-induced inflammatory response in female offspring, suggesting that regular exercise during pregnancy could be a potential candidate of the onset of sepsis and MODS in offspring.     

Reconstruction of the Human Colon Epithelium In Vivo

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Human Pancreatic Tumor Organoids Reveal Loss of Stem Cell Niche Factor Dependence during Disease Progression

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Induction of Protective Immunity against Japanese Encephalitis in Mice by Immunization with a Plasmid Encoding Japanese Encephalitis Virus Premembrane and Envelope Genes

A DNA vaccine plasmid containing the Japanese encephalitis (JE) virus premembrane (prM) and envelope (E) genes (designated pcDNA3JEME) was evaluated for immunogenicity and protective efficacy in mice. Two immunizations of 4-week-old female ICR mice with pcDNA3JEME by intramuscular or intradermal injections at a dose of 10 or 100 μg per mouse elicited neutralizing (NEUT) antibodies at titers of 1:10 to 1:20 (90% plaque reduction), and all immunized mice survived a challenge with 10,000 50% lethal doses of the P3 strain of JE virus. A single immunization with 100 μg of pcDNA3JEME did not elicit detectable NEUT antibodies but induced protective immunity. Spleen cells obtained from BALB/c mice immunized once with 10 or 100 μg of pcDNA3JEME contained JE virus-specific memory cytotoxic T lymphocytes (CTLs). BALB/c mice maintained detectable levels of memory B cells and CTLs for at least 6 months after one immunization with pcDNA3JEME at a dose of 100 μg. The CTLs induced in BALB/c mice immunized twice with 100 μg of pcDNA3JEME were CD8 positive and recognized mainly the envelope protein. These results indicate that pcDNA3JEME has the ability to induce a protective immune response which includes JE virus-specific antibodies and CTLs.     

Transmission of RNA silencing signal through grafting confers virus resistance from transgenically silenced tobacco rootstocks to non-transgenic tomato and tobacco scions

We examined the transmission of RNA silencing signal in non-transgenic tomato and tobacco scions grafted onto the tobacco Sd1 rootstocks, which is silenced in both NtTOM1 and NtTOM3 required for tobamovirus multiplication. When the non-transgenic tomato scions were grafted onto the Sd1 rootstocks, RT-PCR analysis of the scions showed the reduced level of mRNA compared with that before grafting in both LeTH3 and LeTH1, tomato homologs of NtTOM1 and NtTOM3, respectively. siRNAs from both genes were detected in the scions after grafting but not before grafting. Further tomato scions were inoculated with Tomato mosaic virus (ToMV) and used for virus infection. They showed very low level of virus accumulation. Necrotic responding tobacco to tobamovirus was grafted onto the rootstock of Sdl. RT-PCR analysis showed low level expression of both NtTOM1 and NtTOM3 in the scions but siRNA was detected after grafting. When the leaves of scions were inoculated with ToMV or Tobacco mosaic virus, they produced very few local necrotic lesions (LNLs) while the control scions did many LNLs. These results suggest that RNA silencing was transmitted to non-transgenic tomato and tobacco scions after grafting onto the Sd1 rootstocks and that virus resistance was induced in the scions.     

Synthesis and properties of malic acid-containing functional polymers.

Poly-L-lactides containing beta-alkyl alpha-malate-units were prepared by ring-opening copolymerizations of L-lactide with 3-(s)-[(benzyloxycarbonyl)methyl]- (BMD) and 3-(s)-[(dodecyloxycarbonyl)methyl]-1,4-dioxane-2,5-diones (DMD). The solution-cast films of these copolymers were alkali-treated to form a carboxyl-functionalized surface on which cell-binding Arg-Gly-Asp tripeptide (RGD) was immobilized with dicyclohexylcarbodiimide as coupling agent. For the copolymer of L-lactide and BMD the benzyl groups were removed by catalytic hydrogenolysis to obtain a fully carboxyl-functionalized copolymer (PLGM), and RGD was immobilized on the surface of its cast film. All the RGD-immobilized films thus prepared exhibited improved cell attachment compared with the original films. The cell attachment increased with increasing amount of immobilized RGD, which depended on the composition of the alpha-malate units in the copolymer. The RGD-immobilized PLGM films were degraded rapidly during the cell culture, while the RGD-immobilized films of the beta-alkyl alpha-malate-containing polymers survived the cell culture with little degradation. The rate of hydrolysis increased with increasing content of alpha-malate units for both series, depending on the structure of the protecting groups of the beta-carboxyl. These results suggest that the RGD-immobilized polymers could be a new class of functional bioresorbable polymer having improved cell-attachment and adjustable hydrolysis rate.     

2'-Pyrene modified oligonucleotide provides a highly sensitive fluorescent probe of RNA.

Oligonucleotide 9mers containing 2'-O-(1-pyrenylmethyl)uridine [U(pyr)] at the center position were synthesized by using a protected U(pyr) phosphoramidite. The UV melting behaviors indicate that the pyrene-modified oligonucleotides can bind to both their complementary DNA and RNA in aqueous solution. When compared with the unmodified oligonucleotides, the pyrene-modified oligonucleotides showed higher affinity for DNA while exhibiting lower affinity for RNA. The pyrene-modified oligonucleotides in diluted solution exhibited fluorescence typical of pyrene monomer emission [lambdamax 378 (band I) and 391 nm (band III)]. When these oligomers bound to DNA, the fluorescence intensity ratio of band III/band I was increased. With this fluorescence change, a new broad emission (lambdamax 450 nm) due to exciplex between the pyrene and an adjacent nucleobase appeared. In contrast, addition of RNA to the pyrene oligonucleotides resulted in enhancement of the pyrene monomer emission with decrease in the fluorescence band ratio. The extent of the emission enhancement was found to be highly dependent on the nucleobase adjacent to the U(pyr) in the pyrene oligomers. The pyrene oligonucleotide containing dC at the 3'-site of the modification showed remarkable increase (approximately 250 times) in fluorescence (375 nm) upon binding to complementary RNA. The present findings would open the way to the design of a highly sensitive fluorescent probe of RNA.     

Strain improvement of Lentzea sp. 7887 for higher yield per unit volume on hydroxylation of cyclosporine derivative FR901459

A Gram-positive bacterium Lentzea sp. 7887 hydroxylates a cyclosporine derivative FR901459 into AS1837812 (9-hydroxide), which is an important intermediate of candidate drugs that target the hepatitis C virus. We screened a UV-induced mutant, named M-1, which showed about 1.2-fold higher conversion yields, 2-fold higher substrate concentrations (3.69 mM), and 2.5-fold higher yield per unit volume than the wild-type strain.     

Paradoxical gain‐of‐function mutant of the G‐protein‐coupled receptor PROKR2 promotes early puberty

The human genome encodes ~750 G‐protein‐coupled receptors (GPCRs), including prokineticin receptor 2 (PROKR2) involved in the regulation of sexual maturation. Previously reported pathogenic gain‐of‐function mutations of GPCR genes invariably encoded aberrant receptors with excessive signal transduction activity. Although in vitro assays demonstrated that an artificially created inactive mutant of PROKR2 exerted paradoxical gain‐of‐function effects when co‐transfected with wild‐type proteins, such a phenomenon has not been observed in vivo. Here, we report a heterozygous frameshift mutation of PROKR2 identified in a 3.5‐year‐old girl with central precocious puberty. The mutant mRNA escaped nonsense‐mediated decay and generated a GPCR lacking two transmembrane domains and the carboxyl‐terminal tail. The mutant protein had no in vitro signal transduction activity; however, cells co‐expressing the mutant and wild‐type PROKR2 exhibited markedly exaggerated ligand‐induced Ca²⁺ responses. The results indicate that certain inactive PROKR2 mutants can cause early puberty by enhancing the functional property of coexisting wild‐type proteins. Considering the structural similarity among GPCRs, this paradoxical gain‐of‐function mechanism may underlie various human disorders.