RESUMO
Enterotoxigenic Escherichia coli (ETEC) is the causative agent of a wide range of diseases, which are the important cause of illness and mortality in piglets. ETEC strains expressing F4 fimbriae are frequently associated with post-weaning diarrhea (PWD) and lead to great economic losses in swine production industry worldwide. The aim of this study was to establish a rapid and effective isothermal amplification method for detection of F4 fimbriae. Loop-mediated isothermal amplification (LAMP), Polymerase spiral reaction (PSR) and cross-priming ampli- fication (CPA) were used to develop and optimize the detection method first time. Subsequently, the specificity and sensitivity of these methods were evaluated, and the clinical samples were detected with these methods. All the F4-positive samples could produce ladder-like amplifica- tions products and lead the chromogenic substrate SYBR Green I produce green fluorescence, while in blank control and negative samples lack of this pattern or remained orange. The sensi- tivity of LAMP and CPA were 10 times higher than PSR method. Meanwhile, these three methods were validated with clinical samples, 7 were found positive, while 125 samples were negative, the testing results were consisted with the real-time PCR method. These findings suggested that the isothermal amplification based on the F4 fimbriae is a rapid, effective and sensitive method under resource constrains.
Assuntos
Antígenos de Bactérias/metabolismo , Escherichia coli Enterotoxigênica/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Antígenos de Bactérias/genética , DNA Bacteriano/genética , Escherichia coli Enterotoxigênica/genética , Proteínas de Escherichia coli/genética , Proteínas de Fímbrias/genética , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: It is well verified that lncRNA are emerging as imperative regulators in various tumors. LncRNA CALML3-AS1 (CALML3-AS1), a freshly discovered lncRNA, has been confirmed as a tumor promoter in bladder cancer. This present study aimed to explore the biological functions and molecular mechanisms of CALML3-AS1 in cervical cancer (CC). PATIENTS AND METHODS: We analyzed RNA sequencing data from The Cancer Genome Atlas (TCGA) datasets to determine dysregulated lncRNAs in CC. Real Time-Polymerase Chain Reaction (RT-PCR) was applied for the assays of CALML3-AS1 amplification in CC samples and cell lines. Kaplan-Meier analysis and multivariate assays were carried out for determination of the prognostic values. The functions of CALML3-AS1 on cell proliferation, invasion, migration, and apoptosis were determined by a series of cells experiments by knocking down CALML3-AS1. MRNA and protein expressions of signaling pathways were examined using Western blot. RESULTS: We found that CALML3-AS1 was upregulated in CC tissues and this upregulation was associated with FIGO stage, histological grade, and reduced overall survival. Multivariate assays indicated that high CALML3-AS1 expression was an independent prognostic parameter indicating poorer clinical outcome for CC patients. Functional assays suggested that knockdown of CALML3-AS1 suppressed the proliferation, migration, and invasion of CC cells, and induced apoptosis. Mechanistic investigations revealed that inhibiting the expression of CALML3-AS1 decreased the levels of ß-catenin, cyclin D1, and c-myc via Western blot. CONCLUSIONS: Our study revealed that CALML3-AS1 could be an oncogenic lncRNA promoting the growth and metastasis of CC by modulating Wnt/ß-catenin pathway, suggesting that CALML3-AS1 may be an important contributor to CC progression.
Assuntos
Proliferação de Células , RNA Longo não Codificante/metabolismo , Regulação para Cima , Neoplasias do Colo do Útero/patologia , Via de Sinalização Wnt/genética , Adulto , Idoso , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/mortalidade , beta Catenina/metabolismoRESUMO
OBJECTIVE: To identify factors affecting xenograft growth of epithelial ovarian cancer (EOC) cells in nude mice and to detect characteristic mutations occurring in the xenografts following serial passage. MATERIALS AND METHODS: A total of 64 human EOCs were subcutaneously inoculated in Balb/c nude mice in order to obtain a series of xenografts. Whole-exome sequencing was analyzed with Agilent SureSelect targeted enrichment capture system and Illumina Solexa Hiseq 2000 sequencing platform. Mutations were confirmed by comparison against the reference genome build 37.3. RESULTS: The tumor take rate was 50% (32/64). TP53 mutation was detected in nine often Type II tumors. BRAF and CTNNB1 were not mutated in any of the samples, and PTEN mutation occurred in only one sample. The present data indicate that advanced stage serous EOCs and early stage non-serous EOCs were easy to grow in nude mice, and xenografts maintained the characteristic mutations. CONCLUSIONS: Advanced stage serous EOCs and early stage non-serous EOCs were easy to grow in nude mice, and xenografts maintained the characteristic mutations. Xenografts in nude mice are useful in vivo models for the study of human EOCs.
Assuntos
Adenocarcinoma/genética , Xenoenxertos/metabolismo , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Adenocarcinoma/patologia , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/patologia , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/patologia , Animais , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/patologia , Carcinoma Epitelial do Ovário , Modelos Animais de Doenças , Feminino , Xenoenxertos/patologia , Humanos , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Neoplasias Epiteliais e Glandulares/patologia , Proteínas Nucleares/genética , Neoplasias Ovarianas/patologia , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , beta Catenina/genética , Proteínas ras/genéticaRESUMO
AIM: Colorectal cancer (CRC) is the second commonest cause of cancer death in Taiwan. Although numerous genes have been associated with tumorigenesis in colorectal cancer, only a few have been validated and used as biomarkers for predicting clinical outcome. The aim of this study was to analyse the association of APC gene mutation and miR-21 expression with clinical outcome in CRC patients. METHOD: In total, 195 colorectal cancer patients were enrolled in a single medical centre between 2003 and 2007. APC gene mutation and expression of APC and miR-21 were analysed by direct DNA sequencing and real-time reverse transcription polymerase chain reaction. The primary outcome included 5-year overall survival and univariate (Kaplan-Meier) and multivariate (Cox regression) analyses of prognostic factors. RESULTS: The results showed that 66 (33.8%) of 195 tumour tissues contained an APC mutation. The predominant APC gene variations were deletion mutations (50.0%). APC gene expression was low in CRC and negatively correlated with miR-21 expression and gene mutation. In advanced-stage cancer, patients with APC mutation/high miR-21 had poorer overall survival rates than those with APC mutation/low miR-21, APC wild-type/high miR-21 and APC wild-type/low miR-21. CONCLUSION: In Taiwan, downregulation of the APC gene in CRC correlated with gene mutation and miR-21 upregulation. APC mutation and miR-21 expression could be used to predict the clinical outcome of CRC, especially in patients with advanced disease.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Genes APC , MicroRNAs/genética , Idoso , Neoplasias Colorretais/química , Regulação para Baixo , Feminino , Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , MicroRNAs/análise , Mutação , Estadiamento de Neoplasias , Modelos de Riscos Proporcionais , RNA Mensageiro/análise , Taxa de Sobrevida , Via de Sinalização WntRESUMO
Elevated nerve growth factor (NGF) is believed to play a role in many types of pain. An NGF-blocking antibody (muMab 911) has been shown to reduce pain and hyperalgesia in pain models, suggesting a novel therapeutic approach for pain management. Since NGF also plays important roles in peripheral nervous system development and sensory nerve outgrowth, we asked whether anti-NGF antibodies would adversely impact peripheral nerve regeneration. Adult rats underwent a unilateral sciatic nerve crush to transect axons and were subcutaneously dosed weekly for 8weeks with muMab 911 or vehicle beginning 1day prior to injury. Plasma levels of muMab 911 were assessed from blood samples and foot print analysis was used to assess functional recovery. At 8-weeks post-nerve injury, sciatic nerves were prepared for light and electron microscopy. In a separate group, Fluro-Gold was injected subcutaneously at the ankle prior to perfusion, and counts and sizes of retrogradely labeled and unlabeled dorsal root ganglion neurons were obtained. There was no difference in the time course of gait recovery in antibody-treated and vehicle-treated animals. The number of myelinated and nonmyelinated axons was the same in the muMab 911-treated crushed nerves and intact nerves, consistent with observed complete recovery. Treatment with muMab 911 did however result in a small decrease in average cell body size on both the intact and injured sides. These results indicate that muMab 911 did not impair functional recovery or nerve regeneration after nerve injury in adult rats.
Assuntos
Anticorpos Monoclonais/farmacologia , Fator de Crescimento Neural/antagonistas & inibidores , Regeneração Nervosa/fisiologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Nervo Isquiático/fisiologia , Envelhecimento , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Compressão Nervosa , Regeneração Nervosa/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/lesõesRESUMO
Nonlinear, three-photon double excitation of He in intense extreme ultraviolet free-electron laser fields (â¼24.1 eV, â¼5 TW/cm2) is presented. Resonances to the doubly excited states converging to the He+ N=3 level are revealed by the shot-by-shot photoelectron spectroscopy and identified by theoretical calculations based on the time-dependent Schrödinger equation for the two-electron atom under a laser field. It is shown that the three-photon double excitation is enhanced by intermediate Rydberg states below the first ionization threshold, giving a greater contribution to the photoionization yields than the two-photon process by more than 1 order of magnitude.
RESUMO
Inflammatory events may contribute to the pathogenesis of Parkinson's disease (PD) and interleukin 1 (IL-1) may exert both neurotoxic and neuroprotective effects. We conducted a case-control study in a cohort of 493 PD cases and 388 ethnically matched controls to investigate the association of IL-1alpha C-889T and IL-1beta C-511T polymorphisms with the risk of PD. No significant difference in the genotype distribution of the analyzed polymorphisms was found between PD and controls. However, after stratification by age, individuals over 70 years of age carrying IL-1alpha-889 C/T genotype demonstrated a significant decrease in risk of developing PD (OR = 0.44; 95% CI = 0.22-0.88, p = 0.021) and the decrease is strengthened by IL-1beta-511 T-carrying genotype (OR = 0.28; 95% CI = 0.11-0.71, p = 0.008). Our data suggest that IL-1alpha, acting synergistically with IL-1beta, plays role in PD susceptibility among Taiwanese people older than 70 years of age.
Assuntos
Interleucina-1alfa/genética , Doença de Parkinson/epidemiologia , Doença de Parkinson/genética , Polimorfismo Genético/genética , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Predisposição Genética para Doença/epidemiologia , Humanos , Interleucina-1beta/genética , Masculino , Pessoa de Meia-Idade , Taiwan/epidemiologiaRESUMO
Aluminum coordination in the framework of USY and ZSM-5 zeolites containing charge-compensating cations (NH4+, H+, or Cu+) was investigated by Al K-edge EXAFS and XANES. This work was performed using a newly developed in-situ cell designed especially for acquiring soft X-ray absorption data. Both tetrahedrally and octahedrally coordinated Al were observed for hydrated H-USY and H-ZSM-5, in good agreement with 27Al NMR analyses. Upon dehydration, water desorbed from the zeolite, and octahedrally coordinated Al was converted progressively to tetrahedrally coordinated Al. These observations confirmed the hypothesis that the interaction of water with Brønsted acid protons can lead to octahedral coordination of Al without loss of Al from the zeolite lattice. When H+ is replaced with NH4+ or Cu+, charge compensating species that absorb less water, less octahedrally coordinated Al was observed. Analysis of Al K-edge EXAFS data indicates that the Al-O bond distance for tetrahedrally coordinated Al in dehydrated USY and ZSM-5 is 1.67 angstroms. Simulation of k3chi(k) for Cu+ exchanged ZSM-5 leads to an estimated distance between Cu+ and framework Al atoms of 2.79 angstroms.
Assuntos
Alumínio/química , Cobre/química , Análise Espectral/métodos , Zeolitas/química , Prótons , Raios XRESUMO
Selection line rats congenitally high or low for autotomy in the neuroma model of neuropathic pain (HA and LA rats) were found to be correspondingly high and low in a second type of neuropathic pain, the Chung model, which employs an alternative phenotypic endpoint, tactile allodynia. It has been proposed that both phenotypes reflect ectopic hyperexcitability in axotomized primary sensory neurons. To test this hypothesis we made in vitro recordings from sensory neurons in the L4 and 5 dorsal root ganglia. Baseline excitability was similar in HA and LA rats, and axotomy caused an increase in both lines. However, in the one neuronal subclass previously linked to neuropathic pain in these models the increase was significantly greater in HA than LA rats, and only at the time when pain scores in the two lines were diverging. Heritable differences in electrical response to axotomy in a specific afferent cell type appear to be a fundamental determinant of neuropathic pain.
Assuntos
Potenciais de Ação/fisiologia , Gânglios Espinais/fisiopatologia , Hiperalgesia/fisiopatologia , Neuralgia/fisiopatologia , Neurônios Aferentes/fisiologia , Doenças do Sistema Nervoso Periférico/fisiopatologia , Animais , Relógios Biológicos/genética , Modelos Animais de Doenças , Feminino , Gânglios Espinais/patologia , Predisposição Genética para Doença , Hiperalgesia/genética , Hiperalgesia/patologia , Masculino , Compressão Nervosa , Neuralgia/genética , Neuralgia/patologia , Neuroma/genética , Neuroma/patologia , Neuroma/fisiopatologia , Neurônios Aferentes/patologia , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/patologia , Fenótipo , Ratos , Automutilação/genética , Nervos Espinhais/patologia , Nervos Espinhais/fisiopatologiaRESUMO
New features are revealed in the low-energy photoionization spectrum of Ar by critically combining high photon resolution and differential photoelectron spectroscopic techniques. Two LS-forbidden doubly excited resonances are seen in the 3p(-1)(3/2, 1/2) partial cross sections which exhibit mirroring profiles, resulting in complete cancellation in the total photoionization cross section, as was predicted by Liu and Starace [Phys. Rev. A 59, R1731 (1999)]. These results demonstrate that a new class of weakly spin-orbit induced, mirroring resonances should be observable in partial, but not in total, collisional cross sections involving atoms, molecules, and solids in general.
RESUMO
Primary sensory neurons with myelinated axons were examined in vitro in excised whole lumbar dorsal root ganglia (DRGs) taken from adult rats up to 9 days after tight ligation and transection of the L(5) spinal nerve (Chung model of neuropathic pain). Properties of subthreshold membrane potential oscillations, and of repetitive spike discharge, were examined. About 5% of the DRG neurons sampled in control DRGs exhibited high-frequency, subthreshold sinusoidal oscillations in their membrane potential at rest (V(r)), and an additional 4.4% developed such oscillations on depolarization. Virtually all had noninflected action potentials (A(0) neurons). Amplitude and frequency of subthreshold oscillations were voltage sensitive. A(0) neurons with oscillations at V(r) appear to constitute a population distinct from A(0) neurons that oscillate only on depolarization. Axotomy triggered a significant increase in the proportion of neurons exhibiting subthreshold oscillations both at V(r) and on depolarization. This change occurred within a narrow time window 16-24 h postoperative. Axotomy also shifted the membrane potential at which oscillation amplitude was maximal to more negative (hyperpolarized) values, and lowered oscillation frequency at any given membrane potential. Most neurons that had oscillations at V(r), or that developed them on depolarization, began to fire repetitively when further depolarized. Spikes were triggered by the depolarizing phase of oscillatory sinusoids. Neurons that did not develop subthreshold oscillations never discharged repetitively and rarely fired more than a single spike or a short burst, on step depolarization. The most prominent spike waveform parameters distinguishing neurons capable of generating subthreshold oscillations, and hence repetitive firing, was their brief postspike afterhyperpolarization (AHP) and their low single-spike threshold. Neurons that oscillated at V(r) tended to have a more prolonged spike, with slower rise- and fall-time kinetics, and lower spike threshold, than cells that oscillated only on depolarization. The main effects of axotomy were to increase spike duration, slow rise- and fall-time kinetics, and reduce single-spike threshold. Tactile allodynia following spinal nerve injury is thought to result from central amplification ("central sensitization") of afferent signals entering the spinal cord from residual intact afferents. The central sensitization, in turn, is thought to be triggered and maintained in the Chung model by ectopic firing originating in the axotomized afferent neurons. Axotomy by spinal nerve injury enhances subthreshold membrane potential oscillations in DRG neurons, augments ectopic discharge, and hence precipitates neuropathic pain.
Assuntos
Potenciais de Ação/fisiologia , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Neuralgia/fisiopatologia , Nervos Espinhais/lesões , Animais , Axotomia , Eletrofisiologia , Masculino , Neurônios Aferentes/fisiologia , Periodicidade , Ratos , Ratos Wistar , Nervos Espinhais/fisiologiaRESUMO
Spike activity in dorsal root ganglion (DRG) neurons depolarizes passive neighbors that share the same ganglion. We asked whether age or prior nerve injury affect this 'cross-depolarization' signal. Intracellular recordings made from excised DRGs in vitro revealed that the prevalence and duration of cross-depolarization were no greater in adult than in young rats, and that its amplitude was significantly smaller in adults. The amplitude of cross-depolarization was not affected by nerve injury. The decrease in membrane input resistance (R(in)) observed during cross-depolarization was less than that expected from equivalent depolarization alone. This affirms prior evidence that the neural process underlying cross-depolarization causes a net increase in R(in).
Assuntos
Comunicação Celular/fisiologia , Gânglios Espinais/lesões , Gânglios Espinais/fisiologia , Neurônios Aferentes/fisiologia , Fatores Etários , Animais , Feminino , Masculino , Dor , Terminações Pré-Sinápticas/fisiologia , Ratos , Ratos WistarRESUMO
This study was designed to investigate the effect of nicotine on spontaneous and amphetamine (AMP)-induced motor activity in rats with or without tolerance to nicotine. Tolerance were induced by treating the rats with nicotine (0.3 mg/kg, s.c.) 2 hr before receiving challenge doses. Motor activity including locomotion and stereotypy was monitored automatically by videocamera every 15 min for 90 min. The results indicated that: (1) Nicotine increased spontaneous locomotion at 0.15 or 0.3 mg/kg (s.c.) in naive rats and at 0.6 mg/kg in tolerant rats. Nicotine also slightly affected AMP-induced locomotion at 0.15, 0.3 or 0.6 mg/kg in both naive and tolerant rats, and (2) Nicotine increased spontaneous stereotypy at 0.3 or 0.6 mg/kg in naive rats only and showed no effect on AMP-induced stereotypy in either naive or tolerant rats. Comparing the results of spontaneous motor activity between naive and tolerant rats, it revealed behavioral desensitization in locomotion at low doses (0.15 or 0.3 mg/kg) and hyperlocomotion at higher dose (0.6 mg/kg), and revealed desensitization in stereotypy at 0.3 or 0.6 mg/kg. Moreover, nicotine had temporary (at 0-15 min interval) attenuating effect on AMP-induced locomotion in naive rats but showed a potentiating effect on AMP-induced locomotion in tolerant rats. The present results indicated that acute tolerance modified the action of nicotine in both spontaneous and AMP-induced locomotion, while stereotypy was changed only in the spontaneous one but not in the AMP-induced one. In other words, acute tolerance modified the effect of nicotine on locomotion-related dopaminergic system, and it affected the stereotypy-related dopaminergic system only in the spontaneous one but not in the AMP-induced one.
Assuntos
Anfetamina/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Atividade Motora/efeitos dos fármacos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Monofosfato de Adenosina/farmacologia , Animais , Relação Dose-Resposta a Droga , Tolerância a Medicamentos , Feminino , Ratos , Ratos Wistar , Comportamento Estereotipado/efeitos dos fármacosRESUMO
Recent studies have shown that grass genomes have very similar gene compositions and regions of conserved gene order, as exemplified by collinear genetic maps of DNA markers. We have begun the detailed study of sequence organization in large (100-500 kb) segments of the nuclear genomes of maize, sorghum and rice. Our results indicate collinearity of genes in the regions homoeologous to the maize adh1 and sh2-a1 genes. Comparable genes were found to be physically closer to each other in grasses with small genomes (rice and sorghum) than they are in maize. In several instances, we have found evidence of tandem and 'distantly tandem' duplications of segments containing maize and sorghum genes. These duplications complicate characterizations of microcollinearity and could also interfere with some map-based approaches to gene isolation.
Assuntos
Evolução Biológica , Sequência Conservada , Genes de Plantas , Poaceae/genética , Passeio de Cromossomo , Grão Comestível/genética , Oryza/genética , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência , Zea mays/genéticaRESUMO
A cluster of five alpha-zein subfamily 4 (alpha-zein SF4) genes are present in a 56 kb region of the maize W22 genome. Two types of alpha-zein SF4 genes are in the cluster. One of the genes, termed a type 1 (T1) alpha-zein SF4 gene, contains no early in-frame stop codons. Four of the genes, termed type 2 (T2) alpha-zein SF4 genes, contain one or two early in-frame stop codons. The base sequence of the T1 alpha-zein SF4 gene is similar (> 90%) to the sequences of any of the four T2 alpha-zein SF4 genes. However, their sequences differ markedly at distances greater than -875 bp upstream from the translation initiation codon of the alpha-zein coding region. This region of dissimilarity is well inside the functional 5'-flanking region for the genes since a 1.8 kb transcript is initiated in this region and the sequences of the T2 alpha-zein SF4 genes are similar in this region. Two sizes of mRNA transcripts, 1.8 kb and 0.9 kb, were detected in a gene specific manner for 4 of the 5 genes in this alpha-zein SF4 gene cluster. One of the T2 alpha-zein SF4 genes had only the 0.9 kb transcript. The RNA level for the 0.9 kb transcript of the T1 alpha-zein SF4 gene was 5- to 10-fold higher than the transcript levels of any of the T2 alpha-zein SF4 genes. In each case, the amount of the 0.9 kb transcript detected was at least 5-fold higher than the amount of the 1.8 kb transcript. A cDNA clone with a sequence identical to a T2 alpha-zein SF4 gene was isolated, providing the first direct evidence for the transcription of T2 alpha-zein genes containing early in-frame stop codon(s) in maize endosperm.
Assuntos
Genes de Plantas/genética , Família Multigênica/genética , Transcrição Gênica , Zea mays/genética , Zeína/genética , Sequência de Bases , Clonagem Molecular , Variação Genética , Dados de Sequência Molecular , Pseudogenes/genética , RNA Mensageiro/genética , Sementes/genética , Sementes/metabolismo , Análise de Sequência de DNARESUMO
Transient expression of GUS in rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens was characterized using binary vectors containing gusA genes that express minimal (pKIWI105 and pCNL1) or no (p35S-GUS-INT and pCNL56) GUS activity in bacteria. Four-day old seedlings obtained from seeds or immature embryos of rice were cut into shoot, root, and seed remnants and inoculated with various strains of A. tumefaciens. Transient GUS expression events were quantitated histochemically by determining the frequency of explants exhibiting blue spots indicative of GUS at four to six days after cocultivation with A. tumefaciens. A. tumefaciens strains that did not contain the gusA gene (At643) or a Ti-plasmid (At563 and At657) did not elicit any blue staining characteristic of GUS activity. Several parameters were important in obtaining efficient transient expression of GUS in rice mediated by A. tumefaciens. The growth regulator 2,4-D inhibited GUS expression if present during the seed germination period, but the presence of 6 mg/l 2,4-D during cocultivation of the explants with A. tumefaciens slightly enhanced GUS expression efficiency. All 21 rice cultivars tested expressed GUS after co-cultivation with A. tumefaciens. The GUS expression frequency was highest amongst the indica cultivars. The frequencies of GUS expression in japonica cultivars and in Oryza glaberrima cultivars (grown primarily in Africa) were generally one-half to one-third the level found for indica varieties. Leaf explants were more susceptible to A. tumefaciens-facilitated GUS expression than were roots or seed remnants. The vir genes of an agropine-type Ti-plasmid of A. tumefaciens were most effective in directing transient GUS expression in rice, whereas those of a nopaline-type and an octopine-type plasmid were less effective. We have also found that the frequency of transient expression of GUS was higher with pBIN19 as the precursor cloning vector than with pEND4K as the precursor cloning vector. Reasons for differences in effectiveness of these binary vectors are discussed. Using the conditions described here, A. tumefaciens-mediated frequencies of transient GUS expression in four-day old shoots of several rice cultivars were routinely in excess of 50%.
Assuntos
Agrobacterium tumefaciens/genética , Glucuronidase/genética , Oryza/genética , Ácido 2,4-Diclorofenoxiacético/farmacologia , Células Cultivadas , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Técnicas In Vitro , Plasmídeos , RNA Mensageiro/genéticaRESUMO
In an effort to improve the T-DNA-mediated transformation frequency of economically important crops, we investigated the possible enhancement effect of multiple copies of virG genes contained in Agrobacterium tumefaciens strains upon the transient transformation of celery, carrot and rice tissues. Four days after A. tumefaciens infection, we performed histochemical beta-glucuronidase (GUS) assays to determine the frequency of transient transformation of calli from celery and carrot, and explants from rice and celery. Additional copies of octopine- and agropine-type virG genes in A. tumefaciens strains containing an agropine-type Ti-plasmid enhanced the frequency of transient transformation of celery and rice. This enhancement ranged from 25% to five-fold, depending upon the source of the virG gene and the plant tissues inoculated. For both rice and celery, we observed a greater enhancement of transformation using A. tumefaciens strains containing additional copies of an octopine-type virG gene than with strains harboring additional copies of an agropine-type virG gene. Multiple copies of virG genes contained in A. tumefaciens strains harboring a nopaline-type Ti-plasmid had a smaller enhancing effect upon the transformation of celery tissues, and no enhancing effect upon the transformation of rice. In contrast, we obtained a three-fold increase in the transient transformation frequency of carrot calli using an A. tumefaciens strain harboring a nopaline-type Ti-plasmid and additional copies of an octopine-type virG gene. Our results show that multiple copies of virG in A. tumefaciens can greatly enhance the transient transformation frequency of celery, carrot and rice tissues, and that this enhancement is influenced by both the type of Ti-plasmid harbored by A. tumefaciens and by the infected plant species.
Assuntos
Agrobacterium tumefaciens/genética , Genes Bacterianos , Vetores Genéticos , Oryza/genética , Plantas Geneticamente Modificadas , Verduras/genética , Arginina/análogos & derivados , Arginina/genética , Células Cultivadas , Técnicas de Cultura , Glucuronidase/genética , Oxazinas/metabolismo , Plasmídeos , Transformação GenéticaRESUMO
Five genes of the alpha-zein subfamily four (SF4) are located in a 56 kb genomic region of the maize inbred line W22. Their nucleotide and deduced amino acid sequences have been determined. The sequences define two types of alpha-zein SF4 genes-type 1 (T1) and type 2 (T2). The single T1 alpha-zein SF4 gene codes for an alpha-zein protein with a M(r) of about 22,000. This is the first alpha-zein SF4 gene sequenced that contains no early in-frame stop codons in its coding sequence. The four T2 alpha-zein SF4 genes in this cluster contain one or two early in-frame stop codons. In addition, our T1 and T2 genes differ markedly in the base sequences of their distal 5' non-translated flanking regions. The nucleotide and the deduced amino acid sequences of these two types of alpha-zein SF4 genes are similar (greater than 90%) to one another and to all known alpha-zein SF4 genes and cDNAs. Of the known W22 alpha-zein SF4 genes, only one in six does not contain an early in-frame stop codon. If the number of alpha-zein SF4 genes is 15-20, then we estimate that only about 4 of the W22 alpha-zein SF4 genes are without in-frame early stop codons.
Assuntos
Família Multigênica/genética , Zea mays/genética , Zeína/genética , Sequência de Aminoácidos , Sequência de Bases , Códon/genética , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico/genética , Mapeamento por RestriçãoRESUMO
It is well recognized that meniscal tear in the avascular portion is difficult to heal. The purpose of this study is to evaluate the ability of periosteum and fibrin adhesive system (FAS) in promoting the meniscal repair in the avascular zone. Full-thickness defects of 1 x 3 mm were created in the avascular portion of lateral menisci in adult mongrels. The control menisci were left without any treatment. The experimental defects were either repaired with autogenous periosteum or the combination of periosteum and FAS. Histologic and ultrastructural evaluation of the reparative tissue were compared at 4, 8, and 16 weeks postoperatively. The reparative tissue in the control group remained fibrous even at week 16, whereas that in the grafted and graft/FAS groups resembled normal adjacent fibrocartilage. Moreover, the regenerated chondrocytes in the graft/FAS group appeared more mature than those in the grafted group. Our results suggest that periosteum is a potential source of primitive cells from which chondrocytes could be differentiated and developed during the reparative process of meniscal fibrocartilage. As to FAS, it not only acted as a hemostatic agent and a fixant, but also could indirectly accelerate the chondrogenesis of periosteum in the meniscal defects. It is sufficient to conclude that the combination of periosteum and FAS in promoting meniscal defects or severe tears in the avascular portion bears its potential in future clinical application.