Cross Species Genomic Analysis Identifies a Mouse Model as Undifferentiated Pleomorphic Sarcoma/Malignant Fibrous Histiocytoma

Undifferentiated pleomorphic sarcoma/Malignant Fibrous Histiocytoma (MFH) is one of the most common subtypes of human soft tissue sarcoma. Using cross species genomic analysis, we define a geneset from the LSL-Kras^G12D; Trp53^Flox/Flox mouse model of soft tissue sarcoma that is highly enriched in human MFH. With this mouse geneset as a filter, we identify expression of the RAS target FOXM1 in human MFH. Expression of Foxm1 is elevated in mouse sarcomas that metastasize to the lung and tissue microarray analysis of human MFH correlates overexpression of FOXM1 with metastasis. These results suggest that genomic alterations present in human MFH are conserved in the LSL-Kras^G12D; p53^Flox/Flox mouse model of soft tissue sarcoma and demonstrate the utility of this pre-clinical model.     

Effects of divalent cations on bovine testicular hyaluronidase catalyzed transglycosylation of chondroitin sulfates

Two tyrosine residues (Tyr⁴ and Tyr⁷⁶) of succinyl-CoA:3-oxoacid CoA transferase (SCOT) are sensitive to nitric oxide (NO) stress, as assessed by mass spectrometry and site-direct mutagenesis. However, monitoring the SCOT nitration in tissue or cells is challenging. Herein, we describe the development of an assay to detect nitrated SCOT directly using site-specific antibodies; the monoclonal antibodies were generated and screened against nitrated peptides of SCOT. After stringent filtration, two antibodies, anti-SCOT4N and anti-SCOT76N, which specifically recognise Tyr⁴ or Tyr⁷⁶ of SCOT, respectively, were successfully selected. In a cell model over-expressing iNOS in the mitochondria, nitrated SCOT was significantly increased compared with control cells. In addition, in a mouse model of diabetes, nitrated Tyr⁴ and Tyr⁷⁶ in the heart and kidney were higher compared to the control animals. Our results using monoclonal antibodies against nitrated SCOT peptides are in good agreement with the proteomic data.     

Evaluation of spice and herb as phyto-derived selective modulators of human retinaldehyde dehydrogenases using a simple in vitro method

Selective modulation of retinaldehyde dehydrogenases (RALDHs)—the main aldehyde dehydrogenase (ALDH) enzymes converting retinal into retinoic acid (RA), is very important not only in the RA signaling pathway but also for the potential regulatory effects on RALDH isozyme-specific processes and RALDH-related cancers. However, very few selective modulators for RALDHs have been identified, partly due to variable overexpression protocols of RALDHs and insensitive activity assay that needs to be addressed. In the present study, deletion of the N-terminal disordered regions is found to enable simple preparation of all RALDHs and their closest paralog ALDH2 using a single protocol. Fluorescence-based activity assay was employed for enzymatic activity investigation and screening for RALDH-specific modulators from extracts of various spices and herbs that are well-known for containing many phyto-derived anti-cancer constituents. Under the established conditions, spice and herb extracts exhibited differential regulatory effects on RALDHs/ALDH2 with several extracts showing potential selective inhibition of the activity of RALDHs. In addition, the presence of magnesium ions was shown to significantly increase the activity for the natural substrate retinal of RALDH3 but not the others, while His-tag cleavage considerably increased the activity of ALDH2 for the non-specific substrate retinal. Altogether we propose a readily reproducible workflow to find selective modulators for RALDHs and suggest potential sources of selective modulators from spices and herbs.     

Partial Purification and Characterization of An ATPase in Mung Bean Hypocotyl Plasma Membrane: Suggestion for A New Type of Higher Plant Plasma Membrane ATPase

A vanadate-sensitive and nitrate-resistant ATPase was solubilizedwith Zwittergent 3–14 from a highly purified plasma membranefraction of mung bean hypocotyls and partially purified by glyceroldensity gradient centrifugation and phenyl-Sepharose columnchromatography. Either phosphatidylcholine or phosphatidylserinein addition to Mg^{2 +} was required for the enzyme activity, whereasK⁺, phosphatidylethanolamine and lysophosphatidylcholine hadno effect on the activity. The purified enzyme preparation containedtwo major polypeptides with molecular masses of 67 and 55 kDaas analyzed by SDS-polyacrylamide gel electrophoresis. Whenthe plasma membrane fraction was incubated with [-³²P]ATP, a45-70-kDa polypeptide(s) was labeled, and the label could berapidly chased with cold ATP. When the fraction was incubatedwith [¹⁴C]N,N'-dicyclohexylcarbodiimide, an inhibitor for theATPase, a 15-20-kDa polypeptide was labeled. We propose thatthe enzyme is a new type of higher plant plasma membrane ATP-aseand is composed of 67- and 55-kDa subunits and probably alsoa 15-20-kDa subunit. ¹Present address: Takarazuka Institute, Sumitomo Chemical IndustriesLtd., Takatsukasa, Takarazuka, Hyogo 665, Japan (Received September 2, 1987; Accepted May 20, 1988)     

The Influence of a High‐Fat Dietary Environment in the Fetal Period on Postnatal Metabolic and Immune Function

Few reports show whether a high‐fat (HF) dietary environment in the fetal period affects immune function or the development of lifestyle‐related disease at maturity. We examined the influence of an HF dietary environment in the fetal period on postnatal metabolic and immune function. A total of 16 pregnant mice were given control (CON) diet and 16 were given HF diet in the gestational period, from mating to delivery. After delivery lactating mice were given either CON or HF diet, resulting in four groups. After weaning, the offspring mice were given the same diet that their mothers received during lactation. HF dietary intake in the postnatal period increased fat pad weights, serum glucose, and leptin levels. An HF diet in the fetal period resulted in fewer splenic lymphocytes, a thinner thymic cortex, and impaired antigen‐specific immune reactions. Furthermore, tumor necrosis factor (TNF)‐α production and serum triglyceride levels were elevated in the fetal HF group. In addition, the HF‐HF group showed a consistent decrease in ovalbumin (OVA)‐specific IgG and elevation of IgE, associated with advanced fatty changes in the liver. Results from this study suggest that HF environment during the fetal period induces epigenetic propensity toward obesity and immunological burden in part due to increased adipose tissue mass, significant reduction in the number of immune cells and decreased activities of immune cells.     

Cloning and characterization of naringenin 8-prenyltransferase, a flavonoid-specific prenyltransferase of Sophora flavescens

Prenylated flavonoids are natural compounds that often represent the active components in various medicinal plants and exhibit beneficial effects on human health. Prenylated flavonoids are hybrid products composed of a flavonoid core mainly attached to either 5-carbon (dimethylallyl) or 10-carbon (geranyl) prenyl groups derived from isoprenoid (terpenoid) metabolism, and the prenyl groups are crucial for their biological activity. Prenylation reactions in vivo are crucial coupling processes of two major metabolic pathways, the shikimate-acetate and isoprenoid pathways, in which these reactions are also known as a rate-limiting step. However, none of the genes responsible for the prenylation of flavonoids has been identified despite more than 30 years of research in this field. We have isolated a prenyltransferase gene from Sophora flavescens, SfN8DT-1, responsible for the prenylation of the flavonoid naringenin at the 8-position, which is specific for flavanones and dimethylallyl diphosphate as substrates. Phylogenetic analysis shows that SfN8DT-1 has the same evolutionary origin as prenyltransferases for vitamin E and plastoquinone. The gene expression of SfN8DT-1 is strictly limited to the root bark where prenylated flavonoids are solely accumulated in planta. The ectopic expression of SfN8DT-1 in Arabidopsis thaliana resulted in the formation of prenylated apigenin, quercetin, and kaempferol, as well as 8-prenylnaringenin. SfN8DT-1 represents the first flavonoid-specific prenyltransferase identified in plants and paves the way for the identification and characterization of further genes responsible for the production of this large and important class of secondary metabolites.     

Optimization of a Widefield Structured Illumination Microscope for Non-Destructive Assessment and Quantification of Nuclear Features in Tumor Margins of a Primary Mouse Model of Sarcoma

Cancer is associated with specific cellular morphological changes, such as increased nuclear size and crowding from rapidly proliferating cells. In situ tissue imaging using fluorescent stains may be useful for intraoperative detection of residual cancer in surgical tumor margins. We developed a widefield fluorescence structured illumination microscope (SIM) system with a single-shot FOV of 2.1×1.6 mm (3.4 mm²) and sub-cellular resolution (4.4 µm). The objectives of this work were to measure the relationship between illumination pattern frequency and optical sectioning strength and signal-to-noise ratio in turbid (i.e. thick) samples for selection of the optimum frequency, and to determine feasibility for detecting residual cancer on tumor resection margins, using a genetically engineered primary mouse model of sarcoma. The SIM system was tested in tissue mimicking solid phantoms with various scattering levels to determine impact of both turbidity and illumination frequency on two SIM metrics, optical section thickness and modulation depth. To demonstrate preclinical feasibility, ex vivo 50 µm frozen sections and fresh intact thick tissue samples excised from a primary mouse model of sarcoma were stained with acridine orange, which stains cell nuclei, skeletal muscle, and collagenous stroma. The cell nuclei were segmented using a high-pass filter algorithm, which allowed quantification of nuclear density. The results showed that the optimal illumination frequency was 31.7 µm⁻¹ used in conjunction with a 4×0.1 NA objective ( = 0.165). This yielded an optical section thickness of 128 µm and an 8.9×contrast enhancement over uniform illumination. We successfully demonstrated the ability to resolve cell nuclei in situ achieved via SIM, which allowed segmentation of nuclei from heterogeneous tissues in the presence of considerable background fluorescence. Specifically, we demonstrate that optical sectioning of fresh intact thick tissues performed equivalently in regards to nuclear density quantification, to physical frozen sectioning and standard microscopy.     

piggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus

Transgenic insects have been artificially produced to study functions of interesting developmental genes, using insect transposons such as piggyBac. In the case of the cricket, however, transgenic animals have not yet been successfully artificially produced. In the present study, we examined whether the piggyBac transposon functions as a tool for gene delivery in embryos of Gryllus bimaculatus. We used either a piggyBac helper plasmid or a helper RNA synthesized in vitro as a transposase source. An excision assay revealed that the helper RNA was more effective in early Gryllus eggs to transpose a marker gene of eGFP than the helper plasmid containing the piggyBac transposase gene driven by the G. bimaculatus actin3/4 promoter. Further, only when the helper RNA was used, somatic transformation of the embryo with the eGFP gene was observed. These results suggest that the piggyBac system with the helper RNA may be effective for making transgenic crickets.     

The diageotropica mutation and synthetic auxins differentially affect the expression of auxin-regulated genes in tomato.

The effect of a tomato (Lycopersicon esculentum) mutation, diageotropica (dgt), on the accumulation of mRNA corresponding to tomato homologs of three auxin-regulated genes, LeAux, LeSAUR, and Lepar, was examined. The dgt mutation inhibited the induction of LeAux and LeSAUR mRNA accumulation by naphthalene acetic acid (NAA) but had no effect on NAA-induced Lepar mRNA accumulation. The effect of two synthetic auxins, NAA and 3,7-dichloro-8-quinoline carboxylic acid (quinclorac), on the accumulation of LeAux, LeSAUR, and Lepar mRNA was also examined. Quinclorac induced the expression of each of the auxin-regulated genes, confirming its proposed mode of herbicidal action as an auxin-type herbicide. Concentrations of quinclorac at least 100-fold higher than NAA were required to induce LeAux and LeSAUR mRNA accumulation to similar levels, whereas Lepar mRNA accumulation was induced by similar concentrations of NAA and quinclorac. Collectively, these data suggest the presence of two auxin-dependent signal transduction pathways: one that regulates LeSAUR and LeAux mRNA accumulation and is interrupted by the dgt mutation and a second that regulates Lepar mRNA accumulation and is not defective in dgt tomato hypocotyls. These two auxin-regulated signal transduction pathways can be further discriminated by the action of two synthetic auxins, NAA and quinclorac.     

brachyenteron is necessary for morphogenesis of the posterior gut but not for anteroposterior axial elongation from the posterior growth zone in the intermediate-germband cricket Gryllus bimaculatus

In the long-germband insect Drosophila, all body segments and posterior terminal structures, including the posterior gut and anal pads, are specified at the blastoderm stage. In short- and intermediate-germband insects, however, posterior segments are sequentially produced from the posterior growth zone, a process resembling somitogenesis in vertebrates, and invagination of the posterior gut starts after anteroposterior (AP) axial elongation from the growth zone. The mechanisms underlying posterior segmentation and terminal patterning in these insects are poorly understood. In order to elucidate these mechanisms, we have investigated the roles of the Brachyury/brachyenteron (Bra/byn) homolog in the intermediate-germband cricket Gryllus bimaculatus. Loss-of-function analysis by RNA interference (RNAi) revealed that Gryllus byn (Gb'byn) is not required for AP axial elongation or normal segment formation, but is required for specification of the posterior gut. We also analyzed Gryllus caudal (Gb'cad) RNAi embryos using in situ hybridization with a Gb'byn probe, and found that Gb'cad is required for internalization of the posterior gut primordium, in addition to AP axial elongation. These results suggest that the functions of byn and cad in posterior terminal patterning are highly conserved in Gryllus and Drosophila despite their divergent posterior patterning. Moreover, because it is thought that the progressive growth of the AP axis from the growth zone, controlled by a genetic program involving Cdx/cad and Bra/byn, might be ancestral to bilaterians, our data suggest that the function of Bra/byn in this process might have been lost in insects.