Histological studies on the therapeutic effect of sustained-release microspheres of a potent LHRH agonist (leuprorelin acetate) in an experimental endometriosis model in rats

The therapeutic effect of sustained-release microspheres of a potent LHRH agonist (leuprorelin acetate) on experimental endometriosis in female rats was examined histologically. Endometriosis was produced in rats by autotransplantation of endometrial tissue obtained from the left uterine horn into the renal subcapsular space. In the nontreated rats, the transplants were well established and had formed large cysts containing fluid. The walls of the cysts were composed of epithelium and stroma resembling that of normal endometrium. In the rats which received the microspheres of leuprorelin acetate, growth of the transplant was markedly suppressed as evidenced by the reduced size of the cystic cavity and the flattened and pyknotic epithelium. Also, the uterine and ovarian weight decreased significantly. In the ovariectomized rats, growth of the transplant was also markedly suppressed, and the uterine weight decreased. The present results clearly indicate that a single injection of the sustained-release microspheres of leuprorelin acetate markedly suppresses growth of the transplant and produces uterine and ovarian atrophy in the rats.     

Effects of selenium status and supplementary seleno-chemical sources on mouse T-cell mitogenesis

Although selenium is thought to be essential for various immune responses, the excess supplementation may have an adverse effect on certain immunological functions. The present study was designed to determine the effective chemical forms of selenium and their optimal levels on T-cell mitogenesis with splenic cells from mice given a selenium-deficient diet for 8 weeks to avoid effects of cellular selenium sources. Although selenium in tissues, except for spleen and thymus, was almost depleted by feeding selenium-deficient diet, the lymphoid organs still contained low levels of selenium. Both activities of cellular glutathione peroxidase (cGPx) and thioredoxin reductase (TR) in liver and splenic cells showed a tendency to decrease by selenium deficiency. However, splenic cells were tolerant against decrease of the selenoenzyme activities, and TR was also more tolerant than cGPx. T-cell proliferation of the selenium-insufficient splenic cells induced by concanavalin A was increased by addition of Na₂SeO₃, Na₂SeO₄, Na₂Se, seleno-dl-cystine, seleno-l-methionine and selenocystamine. Their promoting action was observed at levels lower than 0.1 μmol/L and was completely suppressed at the highest concentration (1 μmol/L), except for selenocystamine. Na₂SeO₃ was one of the efficient selenocompounds for the mitogenesis, which was concomitant with the significant induction of cGPx and TR. However, recovery of cGPx activity in the selenium-insufficient cells by supplementary Na₂SeO₃ was only partial, while TR activity was readily recovered from selenium deficiency. These results therefore indicate that only low levels of selenium is essential for T-cell mitogenesis even in selenium-insufficient splenic cells, and TR, which is readily recovered by Na₂SeO₃, may be the critical enzyme.     

Mitogenomic phylogeny of the Percichthyidae and Centrarchiformes (Percomorphaceae): comparison with recent nuclear gene-based studies and simultaneous analysis

Delineation of the fish family Percichthyidae (Percomorphaceae) has a long and convoluted history, with recent morphological-based studies restricting species members to South American and Australian freshwater and catadromous temperate perches. Four recent nuclear gene-based phylogenetic studies, however, found that the Percichthyidae was not monophyletic and was nested within a newly discovered inter-familial clade of Percomorphaceae, the Centrarchiformes, which comprises the Centrarchidae and 12 other families. Here, we reexamined the systematics of the Percichthyidae and Centrarchiformes based on new mitogenomic information. Our mitogenomic results are globally congruent with the recent nuclear gene-based studies although the overall amount of phylogenetic signal of the mitogenome is lower. They do not support the monophyly of the Percichthyidae, because the catadromous genus Percalates is not exclusively related to the freshwater percichthyids. The Percichthyidae (minus Percalates) and Percalates belong to a larger clade, equivalent to the Centrarchiformes, but their respective sister groups are unresolved. Because all recent analyses recover a monophyletic Centrarchiformes but with substantially different intra-relationships, we performed a simultaneous analysis for a character set combining the mitogenome and 19 nuclear genes previously published, for 22 centrarchiform taxa. This analysis furthermore indicates that the Centrarchiformes are divided into three lineages and the superfamily Cirrhitoidea is monophyletic as well as the temperate and freshwater centrarchiform perch-like fishes. It also clarifies some of the relationships within the freshwater Percichthyidae.     

Ionophores and weak bases inhibit phagolysosomal proteolysis in Paramecium

In a recent report we showed that ionophores and weak bases inhibit digestive vacuoles (DV) acidification primarily and lysosome-DV fusion secondarily but have no effect on lysosome-DV fusion when acidification is normal. In this study we attempted 1) to show that fluorescein isothiocyanate (FITC)-albumin taken up by phagocytosis could be used for a sensitive proteolytic assay, 2) to use this assay to determine the effect of ionophores and weak bases on proteolysis and 3) to learn how an inhibition of acidification and/or lysosome-DV fusion would affect proteolysis. When cells were pulsed with FITC-albumin and latex beads for 3 min and chased, the amount of albumin degraded increased linearly from 9 to 27 min, reaching a plateau by 30 min, and was inhibited by leupeptin and pepstatin A by 47 to 89%. These results showed that the degradation of FITC-albumin occurred in the phagolysosomes. When added before acidification had commenced, carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP), monensin and NH4Cl partially inhibited lysosome-DV fusion (25-50%) and strongly inhibited proteolysis by 64 to 79%. Added between acidification and lysosome-DV fusion, fusion was unaffected while proteolysis was reduced by 40 to 50%. Added after lysosome-DV fusion was completed, proteolysis was still reduced by the same amount. Chloroquine at 0.25 mM had no effect on proteolysis except when added before acidification, it inhibited fusion by 22% and proteolysis by 16%. These data, together with those published recently, showed that 1) ionophores and weak bases inhibited acidification first, lysosome-DV fusion second and proteolysis third, but they also inhibited proteolysis directly and independent of the prior steps and 2) the proteolysis inhibitory effects were additive.     

Arabinosylnucleoside 5'-triphosphate inhibits DNA primase of calf thymus

It has been shown that DNA primase activity is tightly associated with 10S DNA polymerase alpha from calf thymus and that the ribonucleotide-dependent DNA synthesis is more sensitive to araCTP than DNA-primed DNA synthesis (Yoshida, S., et al. (1983) Biochim. Biophys. Acta 741, 348-357). Here we measured DNA primase activity using poly(dT) template or M13 bacteriophage single-stranded DNA template and primer RNA synthesis was coupled to the reaction by Escherichia coli DNA polymerase I Klenow fragment. By this method, the primer RNA synthesis can be measured independently of the associating DNA polymerase alpha. Using poly(dT) template, it was found that arabinosyladenine 5'-triphosphate (araATP) strongly inhibited DNA primase in competition with rATP. The apparent Ki for araATP was 21 microM and the ratio of Ki/Km (for rATP) was as low as 0.015. With poly(dI, dT) or M13 DNA, it was shown that araCTP also inhibited DNA primase in the similar manner. Product analysis using [alpha-32P]rATP showed that araATP inhibited the elongation of primer RNA. However, it is not likely that arabinosylnucleotides act as chain-terminators, since incubation of primer RNA with araATP did not abolish its priming activity. From these results, it is suggested that arabinosylnucleotide inhibits the initiation as well as elongation of Okazaki fragments in mammalian cells.     

Environmental DNA analysis shows high potential as a tool for estimating intraspecific genetic diversity in a wild fish population

Environmental DNA (eDNA) analysis has recently been used as a new tool for estimating intraspecific diversity. However, whether known haplotypes contained in a sample can be detected correctly using eDNA‐based methods has been examined only by an aquarium experiment. Here, we tested whether the haplotypes of Ayu fish (Plecoglossus altivelis altivelis) detected in a capture survey could also be detected from an eDNA sample derived from the field that contained various haplotypes with low concentrations and foreign substances. A water sample and Ayu specimens collected from a river on the same day were analysed by eDNA analysis and Sanger sequencing, respectively. The 10 L water sample was divided into 20 filters for each of which 15 PCR replications were performed. After high‐throughput sequencing, denoising was performed using two of the most widely used denoising packages, unoise3 and dada2 . Of the 42 haplotypes obtained from the Sanger sequencing of 96 specimens, 38 (unoise3 ) and 41 (dada2 ) haplotypes were detected by eDNA analysis. When dada2 was used, except for one haplotype, haplotypes owned by at least two specimens were detected from all the filter replications. Accordingly, although it is important to note that eDNA‐based method has some limitations and some risk of false positive and false negative, this study showed that the eDNA analysis for evaluating intraspecific genetic diversity provides comparable results for large‐scale capture‐based conventional methods. Our results suggest that eDNA‐based methods could become a more efficient survey method for investigating intraspecific genetic diversity in the field.     

Survival rate and environmental response of current-year seedlings of the temperate liana Wisteria floribunda across a heterogeneous environment

Journal of Plant Research - Lianas have a huge influence on forest structure and function. However, it is unclear how the surrounding environment affects the establishment of liana seedlings in...     

Proteomic Study to Understand Promotive Effects of Plant-Derived Smoke on Soybean (Glycine max L.) Root Growth Under Flooding Stress

Plant-derived smoke plays a key role in plant growth. Proteomic technique was used for underlying mechanisms of plant-derived smoke on the growth of soybean (Glycine max L.) under flooding stress. The length and weight of soybean root increased with 2000 parts per million plant-derived smoke under flooding stress within 4 days. Altered proteins by plant-derived smoke treatment under flooding stress were mainly related to protein metabolism, stress, and redox. Furthermore, proteins related to mitochondrial electron transport chain decreased by flooding stress; however, they increased by addition of plant-derived smoke under flooding stress. Based on the results of proteomic analysis, confirmation experiments were performed. ATPase abundance and ATP content increased with the treatment of plant-derived smoke under flooding stress. Furthermore, the ascorbate/glutathione cycle was activated with the treatment of plant-derived smoke under flooding stress. These results suggest that plant-derived smoke improves the root growth of soybean with energy production and reactive oxygen scavenging even if it is under flooding stress, which might positively regulate soybean tolerance towards flooding stress.

     

Plant stem cell research is uncovering the secrets of longevity and persistent growth

Plant stem cells have several extraordinary features: they are generated de novo during development and regeneration, maintain their pluripotency, and produce another stem cell niche in an orderly manner. This enables plants to survive for an extended period and to continuously make new organs, representing a clear difference in their developmental program from animals. To uncover regulatory principles governing plant stem cell characteristics, our research project ‘Principles of pluripotent stem cells underlying plant vitality’ was launched in 2017, supported by a Grant-in-Aid for Scientific Research on Innovative Areas from the Japanese government. Through a collaboration involving 28 research groups, we aim to identify key factors that trigger epigenetic reprogramming and global changes in gene networks, and thereby contribute to stem cell generation. Pluripotent stem cells in the shoot apical meristem are controlled by cytokinin and auxin, which also play a crucial role in terminating stem cell activity in the floral meristem; therefore, we are focusing on biosynthesis, metabolism, transport, perception, and signaling of these hormones. Besides, we are uncovering the mechanisms of asymmetric cell division and of stem cell death and replenishment under DNA stress, which will illuminate plant-specific features in preserving stemness. Our technology support groups expand single-cell omics to describe stem cell behavior in a spatiotemporal context, and provide correlative light and electron microscopic technology to enable live imaging of cell and subcellular dynamics at high spatiotemporal resolution. In this perspective, we discuss future directions of our ongoing projects and related research fields.