A Crucial Role of Bone Morphogenetic Protein Signaling in the Wound Healing Response in Acute Liver Injury Induced by Carbon Tetrachloride

Background. Acute liver injury induced by administration of carbon tetrachloride (CCl4) has used a model of wound repair in the rat liver. Previously, we reported transient expression of bone morphogenetic protein (Bmp) 2 or Bmp4 at 6–24 h after CCl4 treatment, suggesting a role of BMP signaling in the wound healing response in the injured liver. In the present study, we investigated the biological meaning of the transient Bmp expression in liver injury. Methods. Using conditional knockout mice carrying a floxed exon in the BMP receptor 1A gene, we determined the hepatic gene expressions and proliferative activity following CCl4-treated liver. Results. We observed retardation of the healing response in the knockout mice treated with CCl4, including aggravated histological feature and reduced expressions of the albumin and Tdo2 genes, and a particular decrease in the proliferative activity shown by Ki-67 immunohistochemistry. Conclusion. Our findings suggest a crucial role of BMP signaling in the amelioration of acute liver injury

Function Control of Anti-microRNA Oligonucleotides Using Interstrand Cross-Linked Duplexes

MicroRNA (miRNA)-guided argonaute (Ago) controls gene expression upon binding to the 3′ UTR of mRNA. The miRNA function can be competitively inhibited by single-stranded anti-miRNA oligonucleotides (AMOs). In this study, we constructed a novel type of AMO flanked by interstrand cross-linked 2′-O-methylated RNA duplexes (CLs) that confer a stable helical conformation. Compared with other structured AMOs, AMO flanked by CLs at the 5′ and 3′ termini exhibited much higher inhibitory activity in cells. Anti-miRNA activity, nuclease resistance, and miRNA modification pattern distinctly differed according to the CL-connected positions in AMOs. Moreover, we found that the 3′-side CL improves nuclease resistance, whereas the 5′-side CL contributes to stable binding with miRNA in Ago upon interaction with the 3′ part of miRNA. These structure-function relationship analyses of AMOs provide important insights into the function control of Ago-miRNA complexes, which will be useful for basic miRNA research as well as for determining therapeutic applications of AMO

Root-knot nematodes associated with carrot crop in Federal District region, and reaction of cultivars

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Fitopatologia, Programa de Pós-Graduação em Fitopatologia, 2017.A cenoura é uma das hortaliças mais importantes no mercado brasileiro, principalmente, para a comercialização in natura. A produção dessa hortaliça pode ser severamente afetada pela infecção de nematoides parasitas de plantas, sendo que mais de 90 espécies deste grupo de patógenos já foram associadas à referida cultura. O nematoide das galhas (Meloidogyne spp.) é o grupo que causa maiores perdas na cultura da cenoura. Os objetivos desse trabalho foram: (1) identificar as espécies de Meloidogyne presentes em áreas de cultivo de cenoura no Distrito Federal e no Município de Cristalina (GO); (2) avaliar a resposta de cultivares de cenoura quanto aos níveis naturais de infecção por espécies de Meloidogyne nas safras de 2013 e 2015, e (3) avaliar a reação de cultivares de cenoura em condições de campo („Brasília-CNPH‟, „Brasília-Agrocinco‟ e „Nantes‟) e em condições de casa de vegetação („Brasília-CNPH‟, „Brasília-Agrocinco‟ e „Kuronan‟) aos nematoides M. incognita, M. javanica e M. enterolobii (avaliada somente em condições controladas em casa de vegetação). Amostras de solo foram coletadas em campos comerciais de cenoura do Distrito Federal (DF) e de Cristalina, Goiás (GO). Ao todo foram coletadas 32 amostras compostas (22 amostras no DF e dez amostras em Cristalina, GO). Foi possível detectar nematoides das galhas em 22 amostras e identificar as espécies dos mesmos em 19 amostras. Por meio do fenótipo de esterase, foram identificadas as espécies M. javanica e M. incognita, ambas ocorreram isoladamente ou em mistura populacional. Meloidogyne javanica foi a espécie predominante nas áreas amostradas. Em relação à avaliação de cultivares comerciais quanto à produtividade, verificou-se diferença estatística significativa tanto entre as cultivares de cenoura dentro de cada safra, como entre as safras. As cultivares mais produtivas foram: „Brasília-CNPH‟, „Brasília-Horticeres‟, „BRS Planalto-CNPH‟, „BRS Planalto-Agrocinco‟, „Brasília-Agrocinco‟ e „Brasília-Tecnoseed‟. A cultivar Brasília-CNPH obteve produtividade semelhante nas duas safras. Quanto à reação ao nematoide das galhas, foi observada diferença estatística para o fator de aumento/redução da população de juvenis do nematoide (J2) no solo (FA), entre as cultivares avaliadas nas duas safras. A população „CNPH 1212554‟ foi a que apresentou maior FA na safra 2013 e a cultivar „Brasília-Tecnoseed‟ na safra 2015. Nas duas safras, as cultivares que apresentaram menor quantidade de nematoides por grama de casca de raiz foram: „Brasília-CNPH‟ e „Brasília-Agrocinco‟. Quanto à reação das cultivares ao nematoide das galhas verificou-se que para as variáveis: índice de massa de ovos (IMO), índice de galhas (IG) e fator de reprodução (FR), as cultivares „Kuronan‟, „Brasília-Agrocinco‟ e „Brasília-CNPH‟ comportaram-se como suscetiveis ao ataque de M. incognita, M. incognita + M. javanica e resistentes à espécie M. enterolobii em condições de casa de vegetação. No campo, somente a cultivar „Nantes‟ mostrou suscetibilidade frente aos nematoides M. incognita e M. incognita + M. javanica e as cultivares „Brasília- Agrocinco‟ e „Brasília-CNPH‟ comportaram-se como resistentes, de acordo com os valores de IMO e IG.Carrot is among the most important vegetable crops in Brazil, mainly, for the fresh root market. The production of this vegetable can be severely affected by the infection of plant parasitic-nematodes, with more than 90 species being associated with this crop.The root-knot nematodes (Meloidogyne spp.) is the main group of these soil-borne pathogens that causes yield and quality losses in the carrot crop. The main objectives of this study were: (1) to identify Meloidogyne species with natural occurrence in carrot growing areas in the Federal District and in Cristalina, State of Goiás (GO); (2) to evaluate reaction of carrot cultivars to Meloidogyne species under field conditions in 2013 and 2015 crop seasons; (3) to evaluate a subset of carrot cultivars under field („Brasília-CNPH‟, „Brasília-Agrocinco‟ and „Nantes‟) and under greenhouse conditions („Brasília-CNPH‟, „Brasília-Agrocinco‟ and „Kuronan‟) to M. incognita, M. javanica and M. enterolobii (evaluated only in controlled assays under greenhouse conditions). Soil samples were collected in carrot fields from the Federal District (DF) and from Cristalina (GO). A total of 32 samples were collected (22 samples in the Federal District and ten samples in Cristalina, GO). It was possible to detect root-knot nematodes in 22 samples and to identify the species in 19 samples. Analyses using the esterase phenotype, indicated the presence of the species M. javanica and M. incognita, which appeared either isolated or in a population mix. Meloidogyne javanica was the predominant species in the sampled areas. The cultivars with the highest yields were: „Brasília-CNPH‟, „Brasília-Horticeres‟, „BRS Planalto-CNPH‟, „BRS Planalto-Agrocinco‟, „Brasília-Agrocinco‟ and „Brasília-Tecnoseed‟. The cultivar „Brasília CNPH‟ displayed similar root yields in both crop seasons. It was observed statistical difference for the factor of increase/decrease of juveniles in soil (FA) between cultivars evaluated in the two crop seasons. The population „CNPH 1212554‟ was the one with the highest FA in the 2013 crop season and cv. Brasília-Tecnoseed in the 2015 crop season. The cultivars with the lowest number of nematodes per gram of root skin in the two crop seasons were: „Brasília-CNPH‟ and „Brasília-Agrocinco‟. As to the reaction of the carrot cultivars to the root-knot nematode, it was verified that for the variables: egg mass index (IMO), gall index (GI) and reproduction factor (FR): cultivars „Kuronan‟, „Brasília-Agrocinco‟ and „Brasília-CNPH‟ displayed a susceptible reaction to the attack of M. incognita, M. incognita + M. javanica and showed resistant reaction to M. enterolobii under greenhouse. Under field conditions, only cultivar „Nantes‟ showed susceptibility to M. incognita and to M. incognita + M. javanica, whereas „Brasília-Agrocinco‟ and „Brasília-CNPH‟ cultivars displayed a resistant reaction, according to IMO and GI values

In Vivo Determination of Vitamin D Function Using Transgenic Mice Carrying a Human Osteocalcin Luciferase Reporter Gene

Vitamin D is an essential factor for ossification, and its deficiency causes rickets. Osteocalcin, which is a noncollagenous protein found in bone matrix and involved in mineralization and calcium ion homeostasis, is one of the major bone morphogenetic markers and is used in the evaluation of osteoblast maturation and osteogenic activation. We established transgenic mouse line expressing luciferase under the control of a 10-kb osteocalcin enhancer/promoter sequence. Using these transgenic mice, we evaluated the active forms of vitamins D2 and D3 for their bone morphogenetic function by in vivo bioluminescence. As the result, strong activity for ossification was observed with 1α,25-hydroxyvitamin D3. Our mouse system can offer a feasible detection method for assessment of osteogenic activity in the development of functional foods and medicines by noninvasive screening

Identification of the Control Region of Pancreatic Expression of Bmp4 In Vitro and In Vivo

<div><p>Bone morphogenetic protein 4 (Bmp4) was recently shown to be related to glucose homeostasis in mouse adult pancreas through the regulation of insulin production. We previously revealed the predominant expression of <i>Bmp4</i> in adult pancreas by <i>in vivo</i> imaging of transgenic mice. However, the control regions for predominant <i>Bmp4</i> expression in the adult pancreas are unclear. In this study, we established transgenic (Tg) mice that allow real time <i>in vivo</i> bioluminescence imaging of the enhancer/promoter activity of the <i>Bmp4</i> gene. Tg mice expressing firefly luciferase with a 7 kb upstream region and 5′-non-coding sequence (three exons and two introns) of the <i>Bmp4</i> gene showed pancreatic expression of bioluminescence, while the Tg mice bearing luciferase with the 7 kb upstream region alone did not show pancreatic expression of the reporter gene. Interestingly, pancreatic expression of bioluminescence was also present in Tg mice harboring the truncated promoter without exon IA and IB, indicating the presence of a cryptic promoter in front of exon II. Furthermore, the bioluminescence signal was not detected in embryonic pancreas, but increasing signals were observed in neonatal and infantile Tg mice depending on the genotypes observed. These results suggested that a novel mechanism of transcription is involved in pancreatic expression of the <i>Bmp4</i> gene.</p></div

Establishment of 7 kb-Bmp4Luc transgenic mice.

<p>(A) Genomic PCR of transgenic mouse lines (#1, #8 and #17) produced by pro-nuclear microinjection of linearized p7kb-Bmp4Luc. Arrowheads indicate primer sets in the left panel. Template DNA was prepared from tails of each transgenic line and from wild-type mice (Non-Tg) and plasmids (pBmp4-Luc). (B) Fluorescence in situ hybridization (FISH) assays were performed using fibroblast cells isolated from the tail of each transgenic mouse line. p7kb-Bmp4Luc plasmid DNA was used as a probe (yellow signals with arrows). Chromosomal DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue).</p

Luciferase activity of Bmp4Luc reporter constructs <i>in vitro</i>.

<p>(A) Mouse <i>Bmp4</i> is located on the long arm of mouse chromosome 14, and consists of four exons (IA, IB, II, III and IV) shown by boxes with two transcription start sites (TSS1, TSS2). Dark boxes show the coding sequence. The position of each element is counted from TSS1 as +1. Triangles denote splicing spans. (B) Luciferase activity of Bmp4Luc reporter constructs. NIT1 and NIH3T3 cells were transiently cotransfected with reporter plasmids and <i>Renilla</i> luciferase vector (pRL-TK) as a transfection control. The <i>x</i> axes show relative luciferase activities: firefly luciferase (FL) from the reporter plasmid was normalized to <i>Renilla</i> (RL) luciferase activity from the control vector. All data are the mean ± SE from three independent experiments. **Represents a significant difference (p<0.01) by Student’s <i>t</i>-test.</p