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1.
bioRxiv ; 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38076974

RESUMO

The role extracellular matrix (ECM) in multiple events of morphogenesis has been well described, little is known about its specific role in early eye development. One of the first morphogenic events in lens development is placodal thickening, which converts the presumptive lens ectoderm from cuboidal to pseudostratified epithelium. This process occurs in the anterior pre-placodal ectoderm when the optic vesicle approaches the cephalic ectoderm. Since cells and ECM have a dynamic relationship of interdependence and modulation, we hypothesized that the ECM evolves with cell shape changes during lens placode formation. This study investigates changes in optic ECM including both protein distribution deposition, extracellular gelatinase activity and gene expression patterns during early optic development using chicken and mouse models. In particular, the expression of Timp2 , a metalloprotease inhibitor, corresponds with a decrease in gelatinase activity within the optic ECM. Furthermore, we demonstrate that optic ECM remodeling depends on BMP signaling in the placode. Together, our findings suggest that the lens placode plays an active role in remodeling the optic ECM during early eye development.

2.
Zhonghua Er Ke Za Zhi ; 61(8): 719-725, 2023 Aug 02.
Artigo em Chinês | MEDLINE | ID: mdl-37528013

RESUMO

Objective: To investigate the risk factors for airway mucus hypersecretion in childhood pneumonia infected by different pathogens. Method: A retrospective cohort included 968 children who were hospitalized for Mycoplasma pneumoniae pneumonia (MPP), respiratory syncytial virus (RSV) pneumonia, adenovirus pneumonia and underwent bronchoscopy in Respiratory Department of Children's Hospital of Chongqing Medical University from January 2019 to December 2021 was conducted. The children were divided into two groups distinguished by airway mucus secretion according to the airway mucus hypersecretion score which were scored according to the mucus secretion under the bronchoscope. The demographic characteristics, clinical characteristics, laboratory tests and disease severity of the two groups were compared. And the risk factors for the development of airway mucus hypersecretion in two groups were analyzed. Chi square test, Mann-Whithey U test and Fisher exact test were used to analyze the differences between the two groups, and multivariate Logistic regression was used to analyze the influencing factors. Result: There were 559 males and 409 females in the 968 children, with an age of 4.0 (1.4, 6.0) years. Among the 642 children with MPP, 185 cases were in the hypersecretion group and 457 cases were in the non-hypersecretion group. There were 41 cases in the hypersecretion group and 160 cases in the non-hypersecretion group of 201 children with RSV pneumonia. In the 125 children with adenovirus pneumonia, there were 39 cases in the hypersecretion group and 86 cases in the non-hypersecretion group. In these children, the age of children in the hypersecretion group was older than that in the non-hypersecretion group (6.0 (4.0, 7.0) vs. 5.0 (3.0, 7.0) years old, 1.5 (0.5, 3.6) vs. 0.8 (0.4, 1.6) years old, 2.0 (1.2, 4.5) vs. 1.3 (0.8, 2.0) years old, U=35 295.00, 2 492.00, 1 101.00, all P<0.05). Through multivariate Logistic regression analysis it found that increased risk of airway mucus hypersecretion was present in childhood MPP with increase in peripheral blood white blood cell count (OR=3.30, 95%CI 1.51-7.93, P=0.004) or increase in neutrophil ratio (OR=2.24, 95%CI 1.16-4.33, P=0.016) or decrease in lymphocyte count (OR=3.22, 95%CI 1.66-6.31, P<0.001) or decrease in serum albumin (OR=2.00, 95%CI 1.01-3.98, P=0.047). The risk of airway mucus hypersecretion was increased in children with RSV pneumonia combined with elevated peripheral blood eosinophils (OR=3.04, 95%CI 1.02-8.93, P=0.043). Meanwhile, airway mucus hypersecretion was associated with severe pneumonia (OR=2.46, 95%CI 1.03-6.15, P=0.047) in children with RSV pneumonia. Older age was associated with increased risk of airway mucus hypersecretion in children with adenovirus pneumonia (OR=1.02, 95%CI 1.00-1.04, P=0.026). In these children with occurrence of pulmonary rales, wheezes or sputum sounds (OR=3.65, 95%CI 1.22-12.64, P=0.028) had an increased risk of airway mucus hypersecretion. Neutrophils in bronchoalveolar lavage fluid (BALF) demonstrated higher ratio in hypersecretion group from children with MPP (0.65 (0.43, 0.81) vs. 0.59 (0.34, 0.76), U=24 507.00, P<0.01), while the proportion of macrophages in BALF was lower (0.10 (0.05, 0.20) vs. 0.12 (0.06, 0.24), U=33 043.00, P<0.05). Nucleated cell count and neutrophil ratio in BALF were higher in hypersecretion group of children with RSV pneumonia (1 210 (442, 2 100)×106 vs. 490 (210, 1 510)×106/L, 0.43 (0.26, 0.62) vs. 0.30 (0.13, 0.52), U=2 043.00, 2 064.00, all P<0.05). Conclusions: The increase in peripheral blood white blood cell count, neutrophil ratio and decrease in lymphocyte count, serum albumin in children with MPP is related to the development of airway mucus hypersecretion. In children with RSV pneumonia, the abnormal increase of eosinophils in peripheral blood has relationship with hypersecretion. The appearance of lung rale, wheezing, and sputum rale are associated with airway mucus hypersecretion in children with adenovirus pneumonia. In addition, local neutrophil infiltration in the respiratory tract is closely related to the occurrence of airway mucus hypersecretion caused by Mycoplasma pneumoniae and RSV infection.


Assuntos
Pneumonia por Mycoplasma , Pneumonia Viral , Infecções por Vírus Respiratório Sincicial , Criança , Masculino , Feminino , Humanos , Lactente , Pré-Escolar , Estudos Retrospectivos , Sons Respiratórios , Pulmão , Muco , Fatores de Risco
3.
Methods Mol Biol ; 2599: 227-239, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36427153

RESUMO

Gene expression reporter assays measure the relevance of cis-regulatory elements and DNA-binding proteins in modulating transcriptional activity. Commonly, they are performed in cell lines. However, regulation of transcriptional activity during development is complex and dynamic, and not many cell lines reproduce the embryonic conditions. Thus, conclusions derived from cell line data provide limited information about embryonic development. On the other hand, one of the major hurdles for embryonic assays is delivering reporter plasmids in a tissue-specific manner. In this sense, the chick embryo is a good model system to perform these assays. Electroporation of chick embryos provides temporal and spatially controlled plasmid delivery. Further, it is a well-established, easy, and an economical procedure. Here, we describe in detail how to measure in the chick neural tube (1) enhancer activity with GFP, (2) enhancer activity with luciferase, and (3) 3'UTR activity with luciferase.


Assuntos
Embrião de Mamíferos , Sequências Reguladoras de Ácido Nucleico , Embrião de Galinha , Animais , Feminino , Sequências Reguladoras de Ácido Nucleico/genética , Bioensaio , Desenvolvimento Embrionário , Proteínas de Ligação a DNA
4.
Eur Rev Med Pharmacol Sci ; 25(19): 6101-6108, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34661270

RESUMO

OBJECTIVE: The purpose of this study was to explore the relationship between the number of endothelial progenitor cells (EPCs) and coronary heart disease (CHD). PATIENTS AND METHODS: A total of 24 patients with CHD were chosen from Lanzhou City and Xianyang City, and then, 24 healthy controls who matched the CHD group in gender, age and address were chosen as control group. C-reactive protein (CRP) and c-reaction protein (hs-CRP) were detected. The levels of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), homocysteine (Hcy), hypoxia-inducible factor-1 (HIF-1α) and stromal cell-derived factor 1 (SDF-1α) were detected. RESULTS: The number of EPCs in control groups was both increased compared with CHD group (p<0.05). The number of EPCs in Xianyang control group was increased compared with Lanzhou control group (p<0.05). Compared with the control group, the levels of TC, LDL and CRP in the CHD group were higher (p<0.05). Compared with Lanzhou control group, Hcy level was decreased in Lanzhou CHD group (p<0.05). Compared with Xianyang control group, the levels of IL-8 and VEGF were increased, but the levels of HIF-1α and Hcy were decreased in the Xianyang CHD group (p<0.05). The expressions of IL-8, VEGF, Hcy and HIF-1α were increased in Lanzhou control group than the Xianyang control group (p<0.05). In Lanzhou CHD group, Spearman correlation analysis showed that the number of EPCs was negatively related to hs-CRP content (r=-0.631, p<0.05). CONCLUSIONS: The decrease of EPCs caused by high altitude may increase the expressions of various cytokines, leading to the occurrence of CHD.


Assuntos
Altitude , Doença das Coronárias/etiologia , Citocinas/metabolismo , Células Progenitoras Endoteliais/citologia , Adulto , Idoso , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Doença das Coronárias/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco
5.
Dev Dyn ; 250(2): 249-262, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32562595

RESUMO

BACKGROUND: During embryonic development, complex changes in cell behavior generate the final form of the tissues. Extension of cell protrusions have been described as an important component in this process. Cellular protrusions have been associated with generation of traction, intercellular communication or establishment of signaling gradients. Here, we describe and compare in detail from live imaging data the dynamics of protrusions in the surface ectoderm of chick and mouse embryos. In particular, we explore the differences between cells surrounding the lens placode and other regions of the head. RESULTS: Our results showed that protrusions from the eye region in mouse embryos are longer than those in chick embryos. In addition, protrusions from regions where there are no significant changes in tissue shape are longer and more stable than protrusions that surround the invaginating lens placode. We did not find a clear directionality to the protrusions in any region. Finally, we observed intercellular trafficking of membrane puncta in the protrusions of both embryos in all the regions analyzed. CONCLUSIONS: In summary, the results presented here suggest that the dynamics of these protrusions adapt to their surroundings and possibly contribute to intercellular communication in embryonic cephalic epithelia.


Assuntos
Extensões da Superfície Celular , Ectoderma/citologia , Animais , Embrião de Galinha , Camundongos , Morfogênese
6.
Int J Dev Biol ; 65(4-5-6): 235-244, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32930358

RESUMO

For over 100 years, the vertebrate eye has been an important model system to understand cell induction, cell shape change, and morphogenesis during development. In the past, most of the studies examined histological changes to detect the presence of induction mechanisms, but the advancement of molecular biology techniques has made exploring the genetic mechanisms behind lens development possible. Despite the particular emphasis given to the induction of the lens placode, there are still many aspects of the cell biology of lens morphogenesis to be explored. Here, we will revisit the classical detailed description of early lens morphological changes, correlating it with the cell biology mechanisms and with the molecules and signaling pathways identified up to now in chick and mouse embryos. A detailed description of lens development stages helps better understand the timeline of the events involved in early lens morphogenesis. We then point to some key questions that are still open.


Assuntos
Cristalino , Animais , Embrião de Galinha , Cristalino/embriologia , Camundongos , Morfogênese
7.
Zhonghua Wei Chang Wai Ke Za Zhi ; 23(1): 20-25, 2020 Jan 25.
Artigo em Chinês | MEDLINE | ID: mdl-31958926

RESUMO

Adenocarcinoma of esophagogastric junction (AEG) has a special anatomical position. In clinical practice, there are many overplays among thoracic surgeons, gastrointestinal surgeons, gastroenterologists and oncologists. In recent years, AEG has attracted more and more clinical attention with its increasing incidence. It has a tendency to be gradually separated from esophageal cancer and gastric cancer and be defined as a new special type of tumor. At present, there are still many controversies in the definition, classification, TNM staging, surgical approach, extent of resection, lymph node dissection, digestive tract reconstruction and neoadjuvant therapy of AEG. Meanwhile many problems still need to be solved, which is in a stage of gradual improvement and standardization. This article mainly reviews the important research progress in the field of AEG in 2019, summarizes the current clinical hotspots of AEG, especially the surgical treatment hotspots and the current application status of related new technologies, and aims the future development. We suggest that communication and cooperation among multiple disciplines should be strengthened. Through more clinical researches, basic experimental researches, and innovation and application of new technologies, personalized and accurate diagnosis and treatment will be carried out for patients with different conditions to ultimately achieve the common goal of maximizing the benefits of patients.


Assuntos
Adenocarcinoma/cirurgia , Neoplasias Esofágicas/cirurgia , Junção Esofagogástrica/cirurgia , Neoplasias Gástricas/cirurgia , Adenocarcinoma/classificação , Adenocarcinoma/diagnóstico , Adenocarcinoma/patologia , Neoplasias Esofágicas/classificação , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/patologia , Junção Esofagogástrica/patologia , Humanos , Excisão de Linfonodo/tendências , Estadiamento de Neoplasias , Neoplasias Gástricas/classificação , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/patologia
8.
Zhonghua Yi Xue Za Zhi ; 96(22): 1758-61, 2016 Jun 14.
Artigo em Chinês | MEDLINE | ID: mdl-27356644

RESUMO

OBJECTIVE: To evaluated the 3 sedation regimen for patients with septic shock. METHODS: The randomized controlled trial wan conducted. Forty-five patients with septic shock were assigned to 3 groups (midazolam group, propofol group, and dexmedetomidine group) randomly. The basic characteristics of patients, the duration of mechanical ventilation, the length of stay in the ICU, the death rate for 28 days and the regulatory cell (Treg) in peripheral blood were observed. The control group for Treg test was consisted of 20 healthy volunteers. RESULTS: There were no significant differences between the groups in the death rate for 28 days and the duration of mechanical ventilation. The length of stay in the ICU in dexmedetomidine group was shorter than that in midazolam group(15.21±5.55 vs.19.67±5.7 days, P<0.05). The Treg of 3 groups was higher than that of control group (11.82±4.93 vs.3.69±1.71, 11.30±3.42 vs. 3.69±1.71, 12.83±6.17 vs. 3.69±1.71) at the first day of ICU. The Treg after 3 ICU days in dexmedetomidine group and the Treg after 5 ICU days in propofol group and in midazolam group have no difference with control group. CONCLUSION: For the patients with septic shock, dexmedetomidine could decrease the length of stay in the ICU and the duration of immune suppression.


Assuntos
Dexmedetomidina/uso terapêutico , Hipnóticos e Sedativos/uso terapêutico , Midazolam/uso terapêutico , Propofol/uso terapêutico , Choque Séptico/tratamento farmacológico , Anestesia , Sedação Consciente , Dexmedetomidina/efeitos adversos , Feminino , Humanos , Hipnóticos e Sedativos/efeitos adversos , Unidades de Terapia Intensiva , Tempo de Internação , Masculino , Midazolam/efeitos adversos , Propofol/efeitos adversos , Respiração Artificial , Fatores de Tempo , Resultado do Tratamento
9.
Eur Rev Med Pharmacol Sci ; 20(4): 715-20, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26957275

RESUMO

OBJECTIVE: Rheumatic heart disease (RHD) results due to the cross reaction of the host immune system when it develops immunity against group A streptococcal infection. This autoimmune disease progress with different pathological conditions and the genes associated with it are still less understood. MATERIALS AND METHODS: To understand the role of NKX2-5 and Smad-6 in developing an RHD, we successfully developed RHD model using BALB/c mice and we evaluate the expression of NKX2-5 and Smad-6 in different conditions. RESULTS: The disease conditions are confirmed through histological sectioning of RHD heart tissue with its associated Aschoff bodies. The histological of control heart tissue in the absence of NKX2-5 looks abnormal with an enlarged nucleus and in the absence of Smad-6 the solid nature of heart tissue loosens. The mice developed a complex form of acute RHD with tissue hardening in the absence of either NKX2-5 or Smad-6 which are confirmed in NKX2-5 or Smad-6 null mice. Immunohistochemical studies reveal that the NKX2-5 and Smad-6 expression get down regulated on developing with RHD. Through experiments, we detected that both Nkx2-5 and Smad-6 are both inter-dependable and it negatively regulated each other by inhibiting them. In the absence of NKX2-5 or Smad-6, a severe form of RHD is observed together with down-regulation of either NKX2-5 or Smad-6. CONCLUSIONS: The present investigation of NKX2-5 and Smad-6 in RHD provides a new insight of data that helps to understand the disease pathogenesis.


Assuntos
Proteínas de Homeodomínio/biossíntese , Cardiopatia Reumática/metabolismo , Proteína Smad6/biossíntese , Fatores de Transcrição/biossíntese , Animais , Feminino , Regulação da Expressão Gênica , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Cardiopatia Reumática/genética , Cardiopatia Reumática/patologia , Proteína Smad6/genética , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/patologia , Fatores de Transcrição/genética
10.
J Vis Exp ; (93): e51951, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25406837

RESUMO

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.


Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Medula Espinal/fisiologia , Animais , Embrião de Galinha , Dissecação , Eletroporação/métodos , Expressão Gênica , Medula Espinal/embriologia , Medula Espinal/metabolismo , Medula Espinal/cirurgia , Transfecção/métodos
11.
J Neurogenet ; 28(3-4): 270-81, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24914768

RESUMO

The Rst-Neph family comprises an evolutionarily conserved group of single-pass transmembrane glycoproteins that belong to the immunoglobulin superfamily and participate in a wide range of cell adhesion and recognition events in both vertebrates and invertebrates. In mammals and fish, three Rst-Neph members, named Neph1-3, are present. Besides being widely expressed in the embryo, particularly in the developing nervous system, they also contribute to the formation and integrity of the urine filtration apparatus in the slit diaphragm of kidney glomerular podocytes, where they form homodimers, as well as heterodimers with Nephrin, another immunoglobulin-like cell adhesion molecule. In mice, absence of Neph1 causes severe proteinuria, podocyte effacement and perinatal death, while in humans, a mutated form of Nephrin leads to congenital nephrotic syndrome of the Finnish type. Intriguingly, neither Nephrin nor Neph3 are present in birds, which nevertheless have typical vertebrate kidneys with mammalian-like slit diaphragms. These characteristics make, in principle, avian systems very helpful for understanding the evolution and functional significance of the complex interactions displayed by Rst-Neph proteins. To this end we have started a systematic study of chicken Neph embryonic and post-embryonic expression, both at mRNA and protein level. RT-qPCR mRNA quantification of the two Neph paralogues in adult tissues showed that both are expressed in heart, brain, and retina. Neph1 is additionally present in kidney, liver, pancreas, lungs, and testicles, while Neph2 mRNA is barely detected in kidney, testicles, pancreas and absent in liver and lungs. In embryos, mRNA from both genes can already be detected at as early as stage HH14, and remain expressed until at least HH28. Finally, we used a specific antibody to examine the spatial dynamics and subcellular distribution of ggNeph2 between stages HH20-28, particularly in the mesonephros, dermomyotomes, developing heart, and retina.


Assuntos
Moléculas de Adesão Celular/metabolismo , Adesão Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Proteínas de Membrana/metabolismo , Animais , Embrião de Galinha , Galinhas , Bases de Dados de Proteínas
12.
Genet Mol Res ; 12(3): 3806-12, 2013 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-24085442

RESUMO

Prostate cancer is one of the most common malignancies in men. The multidrug resistance 1 gene (MDR1) is an important candidate gene for prostate cancer. The aim of this study was to evaluate the association between MDR1 gene polymorphisms and the risk of prostate cancer. MDR1 gene polymorphism and its association with the risk of prostate cancer were investigated in 357 Chinese men. A novel c.1465C>T polymorphism was detected with created restriction site-polymerase chain reaction and DNA sequencing. We found a significantly increased risk of prostate cancer in the homozygote comparison [TT vs CC: odds ratio (OR) = 2.300, 95% confidence interval (95%CI) = 1.261-4.196, chi-square = 7.53, P = 0.007], heterozygote comparison (TC vs CC: OR = 1.667, 95%CI = 1.049-2.648, chi-square = 4.71, P = 0.030), dominant model (TT/TC vs CC: OR = 1.835, 95%CI = 1.197-2.815, chi-square = 7.81, P = 0.005), recessive model (TT vs TC/CC: OR = 1.776, 95%CI = 1.023- 3.085, chi-square = 4.23, P = 0.041), and allele contrast (T vs C: OR = 1.625, 95%CI = 1.199-2.202, chi-square = 9.87, P = 0.002). These findings suggested that the c.1465C>T polymorphism of MDR1 may be risk factors for prostate cancer in Chinese men.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Povo Asiático/genética , Polimorfismo de Nucleotídeo Único , Neoplasias da Próstata/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP , Idoso , Idoso de 80 Anos ou mais , Alelos , Estudos de Casos e Controles , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Razão de Chances , Fatores de Risco , Análise de Sequência de DNA
13.
Braz J Med Biol Res ; 46(9): 752-7, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24068190

RESUMO

One of the challenges of the postgenomic era is characterizing the function and regulation of specific genes. For various reasons, the early chick embryo can easily be adopted as an in vivo assay of gene function and regulation. The embryos are robust, accessible, easily manipulated, and maintained in the laboratory. Genomic resources centered on vertebrate organisms increase daily. As a consequence of optimization of gene transfer protocols by electroporation, the chick embryo will probably become increasingly popular for reverse genetic analysis. The challenge of establishing chick embryonic electroporation might seem insurmountable to those who are unfamiliar with experimental embryological methods. To minimize the cost, time, and effort required to establish a chick electroporation assay method, we describe and illustrate in great detail the procedures involved in building a low-cost electroporation setup and the basic steps of electroporation.


Assuntos
Eletroporação/economia , Eletroporação/instrumentação , Eletroporação/métodos , Regulação da Expressão Gênica/genética , Técnicas de Transferência de Genes/instrumentação , Animais , Embrião de Galinha , Eletrodos , Desenho de Equipamento , Proteínas de Fluorescência Verde
14.
Braz. j. med. biol. res ; 46(9): 752-757, 19/set. 2013. graf
Artigo em Inglês | LILACS | ID: lil-686571

RESUMO

One of the challenges of the postgenomic era is characterizing the function and regulation of specific genes. For various reasons, the early chick embryo can easily be adopted as an in vivo assay of gene function and regulation. The embryos are robust, accessible, easily manipulated, and maintained in the laboratory. Genomic resources centered on vertebrate organisms increase daily. As a consequence of optimization of gene transfer protocols by electroporation, the chick embryo will probably become increasingly popular for reverse genetic analysis. The challenge of establishing chick embryonic electroporation might seem insurmountable to those who are unfamiliar with experimental embryological methods. To minimize the cost, time, and effort required to establish a chick electroporation assay method, we describe and illustrate in great detail the procedures involved in building a low-cost electroporation setup and the basic steps of electroporation.


Assuntos
Animais , Embrião de Galinha , Eletroporação/economia , Eletroporação/instrumentação , Eletroporação/métodos , Regulação da Expressão Gênica/genética , Técnicas de Transferência de Genes/instrumentação , Eletrodos , Desenho de Equipamento , Proteínas de Fluorescência Verde
15.
Appl Biochem Biotechnol ; 160(5): 1301-8, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19455432

RESUMO

Two aromatic substrates, paeonol (1) and emodin (2), were biotransformed by using transgenic crown galls of Panax quinquefolium. Four biotransformed products (3-6) were isolated and identified by physicochemical and spectral methods. A beta-glucoside (3, 73.2% of biotransformation yield) and a 1-(2,4-dimethoxyphenyl)- ethanone (4, 8.03%) were isolated from the suspension cultures after 7-day incubation of substrate 1. Upon administration of substrate 2, another beta-glucoside [emodin-6-O-beta-D: -glucopyranoside (5), 19.2%] and a hydroxylated derivative, citreorosein (6, 54.6%), were also obtained. The results demonstrate that transgenic crown galls of P. quinquefolium have the capacities to catalyze glycosylation, hydroxylation, and methylation reactions in the plant cells on those aromatic compounds.


Assuntos
Acetofenonas/metabolismo , Emodina/metabolismo , Panax/genética , Tumores de Planta/genética , Acetofenonas/química , Biotransformação , Emodina/química , Glicosilação , Hidroxilação , Metilação , Plantas Geneticamente Modificadas , Fatores de Tempo
16.
Braz. j. med. biol. res ; 40(11): 1455-1464, Nov. 2007. ilus, tab
Artigo em Inglês | LILACS | ID: lil-464306

RESUMO

The retina is a highly differentiated tissue with a complex layered structure that has been extensively characterized. However, most of the previous studies focused on the histology of the central retina while little is known about the cellular composition, organization and function of the marginal retina. Recent research has identified a subpopulation of multipotential progenitor cells in the marginal regions of the retina, closest to the ciliary body ("ciliary marginal zone"). These cells are capable of differentiation in response to an appropriate stimulus. Thus, it is possible that the structure and composition of the marginal retina are distinct from those of the central retina to accommodate the potential addition of newly formed neurons. To characterize the cellular profile of the chick marginal retina, we labeled it immunohistochemically for markers whose staining pattern is well established in the central retina: calbindin, calretinin, protein kinase C, and choline acetyltransferase. Calbindin was present at very low levels in the marginal retina putative photoreceptor layer. Calretinin-positive horizontal cells were also sparse close to the ciliary marginal zone. The bipolar cells in the marginal outer plexiform layer were positive for anti-protein kinase C antibodies, but the density of labeling was also decreased in relation to the central retina. In contrast, the marginal starburst cholinergic amacrine cell pattern was very similar to the central retina. From these data we conclude that the structure of the marginal retina is significantly different from that of the central retina. In particular, the expression of late retina markers in the marginal retina decreased in comparison to the central retina.


Assuntos
Animais , Corpo Ciliar/citologia , Proteínas do Olho/análise , Retina/química , Células Ganglionares da Retina/citologia , Animais Recém-Nascidos , Biomarcadores/análise , Proliferação de Células , Galinhas , Colina O-Acetiltransferase/análise , Imuno-Histoquímica , Proteína Quinase C/análise , Retina/citologia , Retina/enzimologia , /análise
17.
Braz J Med Biol Res ; 40(11): 1455-64, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17934642

RESUMO

The retina is a highly differentiated tissue with a complex layered structure that has been extensively characterized. However, most of the previous studies focused on the histology of the central retina while little is known about the cellular composition, organization and function of the marginal retina. Recent research has identified a subpopulation of multipotential progenitor cells in the marginal regions of the retina, closest to the ciliary body ("ciliary marginal zone"). These cells are capable of differentiation in response to an appropriate stimulus. Thus, it is possible that the structure and composition of the marginal retina are distinct from those of the central retina to accommodate the potential addition of newly formed neurons. To characterize the cellular profile of the chick marginal retina, we labeled it immunohistochemically for markers whose staining pattern is well established in the central retina: calbindin, calretinin, protein kinase C, and choline acetyltransferase. Calbindin was present at very low levels in the marginal retina putative photoreceptor layer. Calretinin-positive horizontal cells were also sparse close to the ciliary marginal zone. The bipolar cells in the marginal outer plexiform layer were positive for anti-protein kinase C antibodies, but the density of labeling was also decreased in relation to the central retina. In contrast, the marginal starburst cholinergic amacrine cell pattern was very similar to the central retina. From these data we conclude that the structure of the marginal retina is significantly different from that of the central retina. In particular, the expression of late retina markers in the marginal retina decreased in comparison to the central retina.


Assuntos
Corpo Ciliar/citologia , Proteínas do Olho/análise , Retina/química , Células Ganglionares da Retina/citologia , Animais , Animais Recém-Nascidos , Biomarcadores/análise , Calbindina 2 , Calbindinas , Proliferação de Células , Galinhas , Colina O-Acetiltransferase/análise , Imuno-Histoquímica , Proteína Quinase C/análise , Retina/citologia , Retina/enzimologia , Proteína G de Ligação ao Cálcio S100/análise
18.
Braz. j. med. biol. res ; 40(10): 1333-1338, Oct. 2007. ilus
Artigo em Inglês | LILACS | ID: lil-461359

RESUMO

The dorsoventral axis of the eye is determined prior to optic cup invagination. A variety of signaling pathways have been implicated in the maintenance of the optic dorsoventral axis, including, but not limited to, bone morphogenetic protein 4, Sonic Hedgehog and retinoic acid. Here, we investigated the possible contribution of Wnt ligands to the establishment or maintenance of the optic axis by analyzing their expression pattern during early chick optic development. We performed in situ hybridization of Wnt-1, Wnt-3a, Wnt-4, and Wnt-5a during the optic vesicle, early optic cup and established optic cup stages and focused our analysis on the optic region. Our data showed that Wnt-5a, but none of the others, is expressed in the dorsal region of the eye starting from the Hamburger and Hamilton stage 14 (HH14). These results are supported by cryosections of the labeled optic region, which further reveal that Wnt-5a is expressed only in the dorsal retinal pigmented epithelium. Thus, we propose that Wnt-5a is a marker for dorsal retinal pigmented epithelium in chick embryos from HH14 to HH19.


Assuntos
Animais , Embrião de Galinha , Feminino , Padronização Corporal , Olho/embriologia , Proteínas Wnt/metabolismo , Olho/metabolismo , Hibridização In Situ , Ligantes , Transdução de Sinais
19.
Braz J Med Biol Res ; 40(10): 1333-8, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17713656

RESUMO

The dorsoventral axis of the eye is determined prior to optic cup invagination. A variety of signaling pathways have been implicated in the maintenance of the optic dorsoventral axis, including, but not limited to, bone morphogenetic protein 4, Sonic Hedgehog and retinoic acid. Here, we investigated the possible contribution of Wnt ligands to the establishment or maintenance of the optic axis by analyzing their expression pattern during early chick optic development. We performed in situ hybridization of Wnt-1, Wnt-3a, Wnt-4, and Wnt-5a during the optic vesicle, early optic cup and established optic cup stages and focused our analysis on the optic region. Our data showed that Wnt-5a, but none of the others, is expressed in the dorsal region of the eye starting from the Hamburger and Hamilton stage 14 (HH14). These results are supported by cryosections of the labeled optic region, which further reveal that Wnt-5a is expressed only in the dorsal retinal pigmented epithelium. Thus, we propose that Wnt-5a is a marker for dorsal retinal pigmented epithelium in chick embryos from HH14 to HH19.


Assuntos
Padronização Corporal , Olho/embriologia , Proteínas Wnt/metabolismo , Animais , Embrião de Galinha , Olho/metabolismo , Feminino , Hibridização In Situ , Ligantes , Transdução de Sinais
20.
Chemosphere ; 67(11): 2148-55, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17275882

RESUMO

We investigated concentrations of Hg, Cd, Pb, Zn, Cu, As, Ni, and Cr in samples of soil, cereal, and vegetables from Yangzhong district, China. Compared to subsoils, the sampled topsoils are enriched in Hg, Cd, Cu, Pb, Zn, and As. High levels of Cd and Hg are observed in most agricultural soils. Concentrations of Cr and Ni show little spatial variation, and high Cu, Pb, and Zn contents correspond well to areas of urban development. High As contents are primarily recorded at the two ends of the sampled alluvion. The contents of Cd, Hg, and total organic carbon (TOC) increase gradually to maximum values in the upper parts of soil profiles, while Cr and Ni occur in low concentrations within sampled profiles. As, Pb, Cu, and Zn show patterns of slight enrichment within the surface layer. Compared to data obtained in 1990, Cd and Hg show increased concentrations in 2005; this is attributed to the long-term use of agrochemicals. Cr and Ni contents remained steady over this interval because they are derived from the weathering of parent material and subsequent pedogenesis. The measured As, Cu, Pb, and Zn contents show slight increases over time due to atmospheric deposition of material sourced from urban anthropogenic activity. Low concentrations of heavy metals are recorded in vegetables and cereals because the subalkaline environment of the soil limits their mobility. Although the heavy metal concentrations measured in this study do not pose a serious health risk, they do affect the quality of agricultural products.


Assuntos
Agricultura , Metais Pesados/análise , Poluentes do Solo/análise , Fenômenos Químicos , Físico-Química , China , Grão Comestível/química , Monitoramento Ambiental , Análise de Alimentos , Concentração de Íons de Hidrogênio , Estações do Ano , Verduras/química
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