OBSERVATIONS ON THE LIFE HISTORY OF PAPENFUSSIELLA CALLITRICHA (PHAEOPHYCEAE,CHORDARIALES) IN CULTURE1

Papenfussiella callitricha (Rosenv.) Kylin from eastern Canada was studied in culture. Zoids from unilocular sporangia develop into microscopic, filamentous, dioecious gametophytes which produce isogametes in filament cells and few-chambered plurilocular gametangia. Unfused gametes germinate to reproduce the gametophytes. Fusion takes place between a settled (“female”) and a motile (“male”) gamete. The zygote gives rise to a filamentous plethysmothallus that reproduces asexually by zoids formed in thallus cells and in few-chambered plurilocular zoidangia. Erect macrothalli are produced on the plethysmothallus, beginning with the formation of upright filaments. Later on, these filaments become the terminal assimilators of the macrothalli. Further assimilatory filaments, rhizoids, and unilocular sporangia are produced in a branching region at the base of the terminal assimilator. Zoids from unilocular sporangia formed in culture germinate to reestablish the gametophyte phase. Chromosome counts yielded n = 19 ± 3 for the gametophytes, and 32 ± 6 for the sporophyte, both plethysmothallus and macrothallus.     

ADVENTIVE EMBRYOGENESIS IN CITRUS I. THE OCCURRENCE OF ADVENTIVE EMBRYOS WITHOUT POLLINATION OR FERTILIZATION

Anatomical studies of unfertilized undeveloped seeds from open- and control-pollinated fruits of ten facultative apomictic Citrus cultivars were carried out with the aid of light and epifluorescence microscopes. With or without pollination, adventive embryos autonomously developed at all positions in the nucellus in all cultivars. The adventive embryos initiated at the chalazal end of the nucellus were more vigorous than those initiated at the micropylar end. Because of the lack of endosperm and poor seed development, however, all adventive embryos within the unfertilized seeds terminated their development at the globular or early cotyledonary stages and were unable to germinate under natural conditions. The capability of unfertilized seeds to develop varied from species to species. Growth of the adventive embryos was dependent on nucellus size, but the growth rate of adventive embryos relative to nucellus size was different in different species. Neither pollination, fertilization nor subsequent zygote and endosperm development further stimulated adventive embryo initiation. Conversely, pollination and subsequent fertilization of other seeds in the same fruit slightly, but significantly, suppressed adventive embryo growth in the unfertilized seeds. These facts concerning adventive embryogenesis in unfertilized seeds indicate that neither pollination nor fertilization is essential for in vivo adventive embryogenesis and that normal endosperm is necessary for perfect development of adventive embryos initiated only in the micropylar half of the nucellus.     

The Rothmund-Thomson gene product RECQL4 localizes to the nucleolus in response to oxidative stress

Mutations in the RECQL4 helicase gene have been linked to Rothmund-Thomson syndrome (RTS), which is characterized by poikiloderma, growth deficiency, and a predisposition to cancer. Examination of RECQL4 subcellular localization in live cells demonstrated a nucleoplasmic pattern and, to a lesser degree, staining in nucleoli. Analysis of RECQL4-GFP deletion mutants revealed two nuclear localization regions in the N-terminal region of RECQL4 and a nucleolar localization signal at amino acids 376-386. RECQL4 localization did not change after treatment with the DNA-damaging agents bleomycin, etoposide, UV irradiation and gamma irradiation, in contrast to the Bloom and Werner syndrome helicases that relocate to distinct nuclear foci after damage. However, in a significant number of cells exposed to hydrogen peroxide or streptonigrin, RECQL4 accumulated in nucleoli. Using a T7 phage display screen, we determined that RECQL4 interacts with poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme that promotes genomic integrity through its involvement in DNA repair and signaling pathways. The RECQL4 nucleolar localization was inhibited by pretreatment with a PARP-1 inhibitor. The C-terminal portion of RECQL4 was found to be an in vitro substrate for PARP-1. These results demonstrate changes in the intracellular localization of RECQL4 in response to oxidative stress and identify an interaction between RECQL4 and PARP-1.     

Regulated spatial expression of fusion gene constructs with the 5′ upstream region of Halocynthia roretzi muscle actin gene in Ciona savignyi embryos

pHrMA4a-Z is a recombinant plasmid in which about 1.4 kb of the 5 flanking region of a gene for muscle actin HrMA4a from the ascidian Halocynthia roretzi is fused with the coding sequence of a bacterial gene for -galactosidase (lac-Z). In this study, we examined the expression of the fusion gene construct when it was introduced into eggs of another ascidian, namely Ciona savignyi. When a moderate amount of linearized pHrMA4a-Z was introduced into fertilized Ciona eggs, the expression of the reporter gene was evident in muscle cells of the larvae, suggesting that both species share a common machinery for the expression of muscle actin genes. The 5 upstream region of HrMA4a contains several consensus sequences, including a TATA box at -30, a CArG box at -116 and four E-boxes within a region of 200 bp. A deletion construct, in which only the 216-bp 5 flanking region of HrMA4a was fused with lac-Z, was expressed primarily in larval muscle cells. However, another deletion construct consisting of only the 61-bp upstream region of HrMA4a fused with lac-Z was not expressed at all. When pHrMA4a-Z or pHrMA4a-Z (–216) was injected into each of the muscle-precursor blastomeres of the 8-cell embryo, expression of the reporter gene was observed in larval muscle cells in a lineage-specific fashion. However, expression of the reporter gene was not observed when the plasmid was injected into non-muscle lineage. Therefore, the expression of the reporter gene may depend on some difference in cytoplasmic constituents between blastomeres of muscle and non-muscle lineage in the 8-cell embyo.     

Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells

The eukaryotic sliding DNA clamp, proliferating cell nuclear antigen (PCNA), is essential for DNA replication and repair synthesis. In order to load the ring-shaped, homotrimeric PCNA onto the DNA double helix, the ATPase activity of the replication factor C (RFC) clamp loader complex is required. Although the recruitment of PCNA by RFC to DNA replication sites has well been documented, our understanding of its recruitment during DNA repair synthesis is limited. In this study, we analyzed the accumulation of endogenous and fluorescent-tagged proteins for DNA repair synthesis at the sites of DNA damage produced locally by UVA-laser micro-irradiation in HeLa cells. Accumulation kinetics and in vitro pull-down assays of the large subunit of RFC (RFC140) revealed that there are two distinct modes of recruitment of RFC to DNA damage, a simultaneous accumulation of RFC140 and PCNA caused by interaction between PCNA and the extreme N-terminus of RFC140 and a much faster accumulation of RFC140 than PCNA at the damaged site. Furthermore, RFC140 knock-down experiments showed that PCNA can accumulate at DNA damage independently of RFC. These results suggest that immediate accumulation of RFC and PCNA at DNA damage is only partly interdependent.     

An Evidence for the Repair of Double-Strand Scissions in DNA of Micrococcus radiodurans after Alpha Bombardment

The conversion of prochaetoglobosins as plausible precursors into mycotoxin chaetoglobosin A (1) in a cell-free system of Chaetomium subaffine was unsuccessful. However, reductase activity of the 20-keto-analogues (1), and prochaetoglobosins II (5) and III (6) were found in a microsomal fraction of this fungi. Two new metabolites of chaetoglobosins, named chaetoglobosin F_ex (2) and 20-dihydro-chaetoglobosin A (3), were also isolated from the same micro-organisms. Their structures were elucidated by spectroscopic data and chemical transformation.     

Metabolism of Thiophene-2-carboxylate by a Photosynthetic Bacterium

A photosynthetic bacterium, which can grow photosynthetically on benzoate, was isolated from sewage mud. Various kinds of aromatic compounds including heterocyclic aromatic compounds were photometabolized by the washed cells grown photosynthetically on benzoate with no lag period. Among these, thiophene-2-carboxylate was metabolized most rapidly to its (+)-tetrahydro derivative. The same strain could also grow on succinate under photosynthetic conditions. However, thiophene-2-carboxylate was only photometabolized after a long lag period by the washed cells grown photosynthetically on succinate, and the metabolite was not its (+)-tetrahydro derivative but (+)-3-hydroxytetrahydrothiophene-2-carboxylate. In the presence of chloramphenicol, an inhibitor of protein synthesis, the photometabolism of thiophene-2-carboxylate by the washed cells grown photosynthetically on benzoate was not affected at all, but the photometabolism of the same substrate by the washed cells grown photosynthetically on succinate was completely inhibited. These results indicate that a reduction system of broad substrate specificity for aromatic rings is already present in the benzoate-grown cells but absent in the succinate-grown cells. It seems that such a reduction system for aromatic rings is induced by an aromatic substrate.     

Development of a novel tocopheryl ester for suppression of lipid accumulation without cytotoxicity by optimization of dicarboxylic ester moiety

Tocopheryl succinate (Tsuc) is a succinic acid ester of the well-known antioxidant α-tocopherol (T). Tsuc exhibits various biological activities, including tumor growth suppression via activation of cell signaling and prevention of lipid accumulation in mouse adipocyte 3T3-L1 cells. The latter findings suggest that Tsuc may be a drug candidate for the treatment of obesity. However, Tsuc was found to induce apoptosis of normal cells (in addition to cancer cells), demonstrating the need to reduce the cytotoxicity of Tsuc without losing the suppression effect on lipid accumulation. Based on our previous findings, we focused on the ester structure of Tsuc for controlling cytotoxicity. Herein, we examined the cytotoxicity and lipid accumulation suppression effect of various T ester derivatives. We found that the terminal carboxylic group is necessary for suppression of lipid accumulation. We synthesized tocopheryl glutarate (Tglu) and tocopheryl adipate (Tadi) by elongation of carbon atoms 1 and 2 of the dicarboxylic moiety, respectively. Tglu and Tadi did not show any cytotoxicity, and both esters suppressed lipid accumulation, although their suppression activities were weaker than that of Tsuc. Tadi showed a more potent lipid accumulation inhibitory effect than Tglu. Although Tadi inhibited lipogenesis and promoted lipolysis, lipolysis was induced at lower concentrations than inhibition of lipogenesis, suggesting that Tadi mainly affects lipolysis. Taken together, we succeeded in the reduction of cytotoxicity, without loss of the suppression effect on lipid accumulation, by elongation of the dicarboxylic moiety of Tsuc. Tadi may be a promising candidate as an anti-obesity drug.     

Interferon-Tau Attenuates Uptake of Nanoparticles and Secretion of Interleukin-1β in Macrophages

Background

Type I interferons (IFNs), including IFN-alpha (IFNA) and IFN-beta (IFNB), have anti-inflammatory properties and are used to treat patients with autoimmune and inflammatory disorders. However, little is known of the role of IFN-tau (IFNT), a type I IFN produced by ruminant animals for inflammation. Because IFNB has recently been shown to inhibit nucleotide-binding oligomerization domain-like receptor, pyrin domain-containing 3 (NLRP3) inflammasome activation and subsequent secretion of the potent inflammatory cytokine interleukin (IL)-1β, we examined the effects of ruminant IFNT on NLRP3 inflammasome-mediated IL-1β secretion in human THP-1 macrophages.

Methods and Results

IFNT dose-dependently inhibited IL-1β secretion induced by nano-silica, a well-known activators of NLRP3 inflammasomes, in human macrophages primed with lipopolysaccharide (LPS, TLR4 agonist) and Pam3CSK4 (TLR1/2 agonist). IFNT also suppressed phagocytosis of nano-silica and reactive oxygen species (ROS) generation. Western blot analysis showed that IFNT inhibited both pro-IL-1β and mature IL-1β. In addition, real-time RT-PCR analysis showed that IFNT suppressed IL-1β mRNA expression induced by LPS and Pam3CSK4. Although nano-silica particles did not induce IL-10 secretion, IFNT induced IL-10 secretion in a dose-dependent manner. Furthermore, IFNT-suppressed IL-1β secretion was restored by anti-IL-10 neutralizing antibody.

Conclusions

Ruminant IFNT inhibits NLRP3 inflammasome-driven IL-1β secretion in human macrophages via multiple pathways, including the uptake of nano-silica particles, generation of ROS, and IL-10-mediated inhibition of pro-IL-1β induction. It may be a therapeutic alternative to IFNA and IFNB.