RESUMO
The extracellular matrix (ECM) constitutes a viscoelastic environment for cells. A growing body of evidence suggests that the behavior of cells cultured in naturally-derived or synthetic ECM mimics is influenced by the viscoelastic properties of these substrates. Adaptable crosslinking strategies provide a means to capture the viscoelasticity found in native soft tissues. In this work, we present a covalent adaptable hydrogel based on thioester exchange as a biomaterial for the in vitro culture of human mesenchymal stem cells. Through control of pH, gel stoichiometry, and crosslinker structure, viscoelastic properties in these crosslinked networks can be modulated across several orders of magnitude. We also propose a strategy to alter these properties in existing networks by the photo-uncaging of the catalyst 4-mercaptophenylacetic acid. Mesenchymal stem cells encapsulated in thioester hydrogels are able to elongate in 3D and display increased proliferation relative to those in static networks.
Assuntos
Elasticidade , Ésteres/química , Hidrogéis/química , Luz , Polimerização , Compostos de Sulfidrila/química , Reagentes de Ligações Cruzadas/química , Humanos , Células-Tronco Mesenquimais/citologia , Fenilacetatos/química , Estresse Mecânico , ViscosidadeRESUMO
Proteases are involved in almost every important cellular activity, from embryonic morphogenesis to apoptosis. To study protease activity in situ, hydrogels provide a synthetic mimic of the extracellular matrix (ECM) and have utility as a platform to study activity, such as those related to cell migration, in three-dimensions. While 3-dimensional visualization of protease activity could prove quite useful to elucidate the proteolytic interaction at the interface between cells and their surrounding environment, there has been no versatile tool to visualize local proteolytic activity in real time. Here, micron-sized gels were synthesized by inverse suspension polymerization using thiolene photo-click chemistry. The size distribution was selected to avoid cellular uptake and to lower cytotoxicity, while simultaneously allowing the integration of peptide-based FRET sensors of local cell activity. Proteolytic activity of collagenase was detected within an hour via changes in fluorescence of embedded microgels; incubation of microgel sensors with A375 melanoma cells showed upregulated MMP activity in the presence of soluble fibronectins in media. The microgel sensors were readily incorporated into both gelatin and poly(ethylene glycol) (PEG) hydrogels and used to successfully detect spatiotemporal proteolytic activity of A375 melanoma cells. Finally, a tumor model was constructed from a hydrogel microwell array that was used to aggregate A375 melanoma cells, and local variations in proteolytic activity were monitored as a function of distance from the cell aggregate center.
RESUMO
BACKGROUND: As part of a project aimed at developing oviposition attractants for the control and surveillance of Phlebotomus papatasi (a vector of Old-World cutaneous leishmaniasis), we tested the hypothesis that gravid sand flies are attracted to chemical cues emanating from the growth medium of conspecific larvae - predominantly larvae-conditioned host feces that represents a suitable oviposition site. We report the results of a systematic assessment of media from various developmental stages of the sand fly using oviposition and olfactometer behavioral assays. METHODS: We conducted multiple-choice oviposition assays in 500 mL Nalgene jars. Six treatments were placed on separate filter paper discs at the bottom of the jar: 2(nd)/3(rd) larval instar medium, 4(th) larval instar/pupae medium, frass from expired colonies, larval food (aged rabbit chow and rabbit feces mix), rabbit feces, and a solvent (water) control. Fifty gravid females were introduced into each jar. Cumulative number of eggs laid on each filter paper per jar was counted at different time intervals from digital images. Attraction of gravid sand flies to these six treatments was assayed with a 3-chamber linear olfactometer. Twenty gravid females were transferred to the middle chamber of the olfactometer and their distribution in treatment and control chambers was recorded after 3 h. RESULTS: Almost no eggs were oviposited during the first 72 h following a blood-meal. Cumulative egg deposition increased drastically in the next 24 h (hours 73-96), with a slight non-significant increasing trend thereafter. Comparing mean cumulative egg deposition among the six treatments, we found that significantly more eggs were oviposited on 2(nd)/3(rd) larval rearing medium followed by 4(th) instar/pupae rearing medium. Oviposition preference did not vary over time. The olfactometer results were consistent with the oviposition assays, with 2(nd)/3(rd) larval rearing medium being the most attractive, followed by 4(th) instar/pupae rearing medium. CONCLUSION: The key finding of this study is that gravid, laboratory reared, Ph. papatasi sand flies are significantly more attracted to rearing medium of the most biologically active larval stages (2(nd)/3(rd) instar and 4(th) instar/pupae). This finding indicates that sand fly-digested host food and feces is attractive to gravid females and suggests that the larvae and larval gut microbiome may be involved in conditioning the oviposition substrate and possibly the production of oviposition attractants and stimulants.
Assuntos
Oviposição , Feromônios/metabolismo , Phlebotomus/efeitos dos fármacos , Phlebotomus/fisiologia , Animais , Biometria , Insetos Vetores/efeitos dos fármacos , Insetos Vetores/fisiologia , OlfatoRESUMO
OBJECTIVES: To document the histories, clinical findings, and management of seven puppies with laryngeal collapse occurring secondarily to brachycephalic airway syndrome. METHODS: Seven brachycephalic puppies aged between 4.5 and six months underwent surgery for management of brachycephalic airway syndrome following presentation for exercise intolerance and increased respiratory noise and effort. RESULTS: Stenotic nares of varying severity and an elongated soft palate were common to all dogs. All dogs had tracheal hypoplasia and this was severe in four dogs. Laryngeal collapse was present in all dogs. Two dogs had stage I, four dogs stage II, and one dog stage III laryngeal collapse. The dog with stage III laryngeal collapse and one dog with stage II laryngeal collapse died. There was no apparent association between the changes evident on thoracic radiographs or the degree of tracheal hypoplasia and postoperative outcome. CLINICAL SIGNIFICANCE: The development of severe secondary laryngeal changes in dogs aged six months or less supports the suggestion that immature brachycephalic dogs should undergo assessment and, if indicated, surgery as soon as any clinical signs of BAS are apparent.
Assuntos
Obstrução das Vias Respiratórias/veterinária , Doenças do Cão/diagnóstico , Doenças da Laringe/veterinária , Complicações Pós-Operatórias/veterinária , Obstrução das Vias Respiratórias/genética , Obstrução das Vias Respiratórias/cirurgia , Animais , Cruzamento , Constrição Patológica/genética , Constrição Patológica/cirurgia , Constrição Patológica/veterinária , Doenças do Cão/genética , Doenças do Cão/cirurgia , Cães , Doenças da Laringe/diagnóstico , Doenças da Laringe/genética , Doenças da Laringe/cirurgia , Cavidade Nasal/anormalidades , Cavidade Nasal/cirurgia , Palato Mole/anormalidades , Palato Mole/cirurgia , Complicações Pós-Operatórias/diagnóstico , Estudos RetrospectivosRESUMO
Rupture of follicular (epidermoid) cysts is believed to be the consequence of bacterial infection. We report a 24-year-old man with idiopathic CD4 lymphopenia and chronic Mycobacterium avium intracellulare infection who developed multiple, recurring painful abscesses over the distal extremities that increased in number and severity when systemic steroid and interferon-gamma treatment was instituted for interstitial lung disease. Cultures were consistently negative for microorganisms, but pathological examination revealed ruptured epidermoid cyst walls with human papillomavirus (HPV) viropathic changes (keratinocytes with perinuclear halos and abundant basophilic keratohyaline granules). Cutaneous examination showed numerous, widespread flat-topped papules and achromic macules over the extremities, head and neck. Nested polymerase chain reaction analysis for HPV DNA revealed that the abscess-related cyst walls harboured epidermodysplasia verruciformis (EV)-associated HPV types 20, 24, alb-7 (AY013872) and 80. His cutaneous lesions harboured HPV types 3, 8 and 80. Similar to past reports, our patient developed an EV-like eruption in the setting of immunodeficiency. In this instance, EV-associated HPV infection of the follicular infundibular epithelium or pre-existing cysts in the setting of immunodeficiency may have led to cystic growth, rupture and subsequent painful inflammation.
Assuntos
Linfócitos T CD4-Positivos , Epidermodisplasia Verruciforme/etiologia , Linfopenia/complicações , Abscesso/etiologia , Adulto , Cistos/etiologia , DNA Viral/análise , Evolução Fatal , Humanos , Linfopenia/imunologia , Masculino , Infecção por Mycobacterium avium-intracellulare/complicações , Papillomaviridae/genética , Infecções por Papillomavirus/complicações , Recidiva , Dermatopatias Virais/complicaçõesRESUMO
BACKGROUND: Postoperative residual curarization (PORC) after surgery is common and its detection has a high error rate. Artificial neural networks are being used increasingly to examine complex data. We hypothesized that a neural network would enhance prediction of PORC. METHODS: In 40 previously reported patients, neuromuscular function, neuromuscular block/antagonist usage and time intervals were recorded throughout anaesthesia until tracheal extubation by an observer uninvolved in patient care. PORC was defined as significant 'fade' (train of four <0.7) at extubation. Neuromuscular function was classified as PORC (value=1) or no PORC (value=0). A back-propagation neural network was trained to assign similar values (0, 1) for prediction of PORC, by examining the impact of (i) the degree of spontaneous recovery at reversal, and (ii) the time since pharmacological reversal, using the jackknife method. Successful prediction was defined as attainment of a predicted value within 0.2 of the target value. RESULTS: Twenty-six patients (65%) had PORC at tracheal extubation. Clinical detection of PORC had a sensitivity of 0 and specificity of 1, with an indeterminate positive predictive value and a negative predictive value of 0.35. Using the artificial neural network, one patient with residual block and one with adequate neuromuscular function were incorrectly classified during the test phase, with no indeterminate predictions, giving an artificial neural network sensitivity of 0.96 (chi(2)=44, P<0.001) and specificity of 0.92 (P=1), with a positive predictive value of 0.96 and a negative predictive value of 0.93 (chi(2)=12, P<0.001). CONCLUSIONS: Neural network-based prediction, using readily available clinical measurements, is significantly better than human judgement in predicting recovery of neuromuscular function.
Assuntos
Redes Neurais de Computação , Bloqueio Neuromuscular/efeitos adversos , Doenças da Junção Neuromuscular/diagnóstico , Adulto , Feminino , Humanos , Intubação Intratraqueal/efeitos adversos , Masculino , Junção Neuromuscular/efeitos dos fármacos , Doenças da Junção Neuromuscular/induzido quimicamente , Valor Preditivo dos Testes , Estudos RetrospectivosRESUMO
BACKGROUND: Residual paralysis following the use of neuromuscular blocking drugs remains a clinical problem. As part of departmental quality assurance, we examined the degree of postoperative residual curarization (PORC) following atracurium. METHODS: Forty patients undergoing general anaesthesia involving atracurium were studied. Quantitative neuromuscular monitoring (mechanomyography, Myograph 2000, Biometer, Denmark) was performed by assessing the response to supramaximal train-of-four (TOF) stimulation of the ulnar nerve. Anaesthesia was provided by non-participating clinicians who were blinded to the study data. A TOF ratio =0.7 at extubation was classified as PORC. RESULTS: At antagonism of neuromuscular block, 70% (28/40) of patients had a TOF ratio =0.7, and 65% (26/40) of patients had a TOF ratio =0.7 at extubation. Peripheral nerve stimulator use was associated with a longer interval from antagonism of block to extubation (P=0.01), but was not associated with differences in atracurium dosage or a reduction in PORC at extubation. Patients with TOF ratio =0.7 at extubation had surgery of shorter duration [59 (SEM 6) vs 103 (9) min, P<0.001], greater doses of atracurium relative to the duration of surgery [6 (1) vs 11 (1) micro g kg(-1) min(-1), P<0.005], and shorter intervals from administration of last dose of atracurium to antagonism of neuromuscular block [29 (2) vs 53 (9) min, P<0.005] and from antagonism to extubation [6 (1) vs 15 (4) min, P<0.01]. Duration of surgical procedure was the sole multivariate predictor of PORC [odds ratio 0.94 (95% confidence intervals 0.91-0.98), P<0.01]. CONCLUSIONS: PORC remains a clinical problem despite use of intermediate-duration neuromuscular blocking drugs and peripheral nerve stimulators. Patients undergoing procedures of short duration may be at risk of inappropriately early tracheal extubation, possibly due to work pressures. The association between suboptimal antagonism of neuromuscular blockade and short procedures needs reinforcement during postgraduate training and departmental quality assurance.
Assuntos
Atracúrio/efeitos adversos , Fármacos Neuromusculares não Despolarizantes/efeitos adversos , Paralisia/induzido quimicamente , Complicações Pós-Operatórias/induzido quimicamente , Adulto , Intervalos de Confiança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Bloqueio Nervoso , Razão de Chances , Paralisia/fisiopatologia , Nervos Periféricos/fisiopatologia , Estimulação Física , Complicações Pós-Operatórias/fisiopatologia , Fatores de TempoRESUMO
OBJECTIVES: To determine the incidence of posttreatment hypothyroidism in patients treated with surgery with or without radiotherapy for advanced-stage nonthyroid head and neck cancer and to make recommendations for its detection. DESIGN: A prospective study to assess the incidence and time frame of occurrence of hypothyroidism in patients by primary tumor site and treatment modality. Thyroid function tests were performed preoperatively, at the first postoperative visit, and then approximately every 6 months. Patients were followed up for up to 3 years. SETTING: Arthur G. James Cancer Hospital and Research Institute, Columbus, Ohio. PATIENTS: A total of 251 patients with nonthyroid head and neck cancer were originally enrolled; 198 patients with evaluable data were studied to determine the incidence of posttreatment hypothyroidism. Approximately 80% of the patients had advanced stage (III or IV) or recurrent cancer. RESULTS: The overall incidence of posttreatment hypothyroidism was 15% in 198 patients followed up for a mean of approximately 12 months. Hypothyroidism developed in 12% of patients treated with nonlaryngeal surgery and radiotherapy. The group undergoing total laryngectomy (with thyroid lobectomy) and radiotherapy had a 61% incidence of hypothyroidism. The average time to detection of hypothyroidism was 8.2 months. CONCLUSIONS: Approximately 15% of patients treated for advanced head and neck cancer with surgery and radiotherapy will develop hypothyroidism. Those treated with total laryngectomy and radiotherapy are at greatest risk.
Assuntos
Hipotireoidismo/etiologia , Neoplasias Otorrinolaringológicas/cirurgia , Complicações Pós-Operatórias/etiologia , Adulto , Idoso , Terapia Combinada , Feminino , Seguimentos , Humanos , Laringectomia , Masculino , Pessoa de Meia-Idade , Esvaziamento Cervical , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/radioterapia , Recidiva Local de Neoplasia/cirurgia , Estadiamento de Neoplasias , Neoplasias Otorrinolaringológicas/patologia , Neoplasias Otorrinolaringológicas/radioterapia , Estudos Prospectivos , Radioterapia Adjuvante , Fatores de Risco , Testes de Função Tireóidea , TireoidectomiaRESUMO
The tumor suppressor gene p16, when altered, has been shown to play a role in oncogenesis in many different tumor types including head and neck cancer. The goal of this study was to analyse alterations to p16 in squamous cell carcinoma (SCC) of the head and neck and to correlate these with clinical outcome. RNA was isolated from 26 SCC head and neck tumors and from 24 matched controls. A reverse transcription polymerase chain reaction was utilized to generate p16 cDNA, which was sequenced and analysed for alterations. In the 26 patient group 58% of the tumors had a p16 alteration, which were characterized by: 8 deletions, 1 insertion/deletion, 4 point mutations and 2 with no p16 expression. In 24 matched normal tissue samples there were no p16 alterations. Those patients with p16 alterations appear to have survival rates comparable to those without p16 alterations, although patients with p16 alterations appear to have more recurrences.
Assuntos
Carcinoma de Células Escamosas/genética , Genes p16/genética , Neoplasias de Cabeça e Pescoço/genética , Mutação , Carcinoma de Células Escamosas/mortalidade , Estudos de Casos e Controles , DNA de Neoplasias/genética , Neoplasias de Cabeça e Pescoço/mortalidade , Humanos , Recidiva Local de Neoplasia/genética , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
RNA was isolated from 22 squamous cell carcinomas (SCCs) obtained from diverse sites within the head and neck and from matched normal tissue where available. Tissue samples were then screened for expression of RNA from tumor suppressor gene p16 by utilizing semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis. p16-Specific PCR amplification products generated from tumor samples were subject to further analysis by direct DNA sequencing to determine if any tumor sample harbored a p16 mutation. The results show the presence of mutations in 10 of 22 (45%) of the tumor samples. Mutations comprise two identical point mutations, two small deletions (1 bp and 2 bp), one single-nucleotide insertion, four larger deletions, and an insertion/deletion. No mutations in p16 have been identified by analysis of PCR products generated from normal matched tissue, suggesting that p16 alterations are generated by somatic mutation and are not germline in origin. All 22 samples were analyzed additionally by immunohistochemistry for nuclear expression of the retinoblastoma (RB) tumor suppressor gene product. Results show lack of RB nuclear expression in only one sample, suggesting that mutation of RB is an infrequent event in the development of SCC of the head and neck (SCCHN).
Assuntos
Carcinoma de Células Escamosas/genética , Genes p16/genética , Neoplasias de Cabeça e Pescoço/genética , Mutação Puntual , Técnicas de Cultura , Inibidor p16 de Quinase Dependente de Ciclina/genética , Humanos , Retinoblastoma/genética , Retinoblastoma/patologiaRESUMO
The safety and in vitro effectiveness of applying silver to polyethylene terephthalate fabric mechanical heart valve (MHV) sewing cuffs for the prevention of prosthetic valve endocarditis (PVE) were evaluated. PVE is an infrequent but grave complication of cardiac surgery associated with mortality rates potentially exceeding 50%. A poor response to antibiotic therapy is partly responsible for the high mortality rates. Silver is a well known antimicrobial agent with broad effectiveness. Preliminary in vitro microbial challenge studies of the coated fabric using the New York State 63 bacteriostatic test and Dow Corning Shake Flask test showed a > or = 97% reduction for most organisms tested. Sheep mitral valve replacement studies suggest comparable tissue ingrowth of uncoated and coated fabric with a more organized, thinner pannus formed on silver coated fabric. Low levels of silver were present in the serum at all time periods. These results indicate MHVs with silver coated cuffs may provide additional protection against PVE.
Assuntos
Anti-Infecciosos , Próteses Valvulares Cardíacas , Polietilenotereftalatos , Prata , Têxteis , Animais , Anti-Infecciosos/sangue , Aderência Bacteriana , Endocardite Bacteriana/prevenção & controle , Estudos de Avaliação como Assunto , Próteses Valvulares Cardíacas/efeitos adversos , Humanos , Técnicas In Vitro , Microscopia Eletrônica de Varredura , Valva Mitral , Ovinos , Prata/sangueRESUMO
OBJECTIVE: To evaluate the safety and efficacy of p55 tumor necrosis factor receptor fusion protein, a recombinant chimeric protein of human p55 (type I) tumor necrosis factor receptor (CD120a) extracellular domain and IgG1 sequences (referred to as p55-IgG), in the treatment of patients with severe sepsis or septic shock. DESIGN: Randomized, prospective, multicenter, double-blind, placebo-controlled clinical trial. SETTING: Forty-four community and university-affiliated hospitals in the United States and Europe. PATIENTS: There were 498 patients enrolled in this clinical trial. INTERVENTION: Patients prospectively stratified within each site into refractory shock or severe sepsis groups were randomized to receive a single infusion of p55-IgG, 0.083 mg/kg, 0.042 mg/kg, or 0.008 mg/kg, or placebo. Patients received standard aggressive medical/surgical care during the 28-day postinfusion period. OUTCOME MEASURE: Twenty-eight-day all-cause mortality. RESULTS: The distribution of variables describing demographics, organ system dysfunction or failure, infecting microorganisms, predicted mortality, plasma interleukin 6 levels, and plasma tumor necrosis factor alpha (TNF-alpha) levels were similar among patients in the p55-IgG and placebo treatment arms. A planned interim analysis was performed after 201 patients were enrolled. Because a statistically nonsignificant trend toward increased mortality was present in patients who had received 0.008 mg/kg, this treatment arm was discontinued, and the study continued with 3 arms. Among all infused patients, there was a statistically nonsignificant trend toward reduced 28-day all-cause mortality in those who received p55-IgG compared with placebo-treated patients (5% reduction, 0.042 mg/kg vs placebo; 15% reduction, 0.083 mg/kg vs placebo; P=.30). However, in patients with severe sepsis and early septic shock (n=247), therapy with p55-IgG, 0.083 mg/kg, was associated with a 36% reduction in 28-day all-cause mortality compared with placebo (P=.07): 20 (23%) of 87 patients died among those treated with p55-IgG, 0.083 mg/kg; 30 (37%) of 82 among those treated with p55-IgG, 0.042 mg/kg; and 28 (36%) of 78 in the placebo group. A prospectively planned logistic regression analysis to assess treatment effect on 28-day all-cause mortality by means of predicted mortality and serum interleukin 6 levels as continuous covariates demonstrated a significant improvement in outcome for the patients with severe sepsis treated with p55-IgG, 0.083 mg/kg, compared with placebo (P=.01). Serious adverse events, including death and the development of new organ system dysfunction, were reported in 65% of patients infused with placebo, with no increased frequency (56%) present in the 2 p55-IgG treatment arms. There were no reports of immediate hypersensitivity reactions caused by p55-IgG. CONCLUSIONS: In this dose-finding study, there was no decrease in mortality between placebo and p55-IgG in all infused patients. In the prospectively defined population of patients with severe sepsis who received p55-IgG, 0.083 mg/kg, there was a trend toward reduced mortality at day 28 that became significant when predicted mortality and plasma interleukin 6 levels were included in a logistic regression analysis.
Assuntos
Cadeias Pesadas de Imunoglobulinas/uso terapêutico , Receptores do Fator de Necrose Tumoral , Sepse/tratamento farmacológico , APACHE , Adulto , Idoso , Método Duplo-Cego , Feminino , Humanos , Cadeias gama de Imunoglobulina , Interleucina-6/sangue , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Insuficiência de Múltiplos Órgãos , Estudos Prospectivos , Proteínas Recombinantes de Fusão/uso terapêutico , Sepse/fisiopatologia , Choque Séptico/tratamento farmacológico , Choque Séptico/fisiopatologia , Análise de Sobrevida , Fator de Necrose Tumoral alfa/metabolismoRESUMO
We have isolated the gene for a protein designated CCA1. This protein can bind to a region of the promoter of an Arabidopsis light-harvesting chlorophyll a/b protein gene, Lhcb1*3, which is necessary for its regulation by phytochrome. The CCA1 protein interacted with two imperfect repeats in the Lhcb1*3 promoter, AAA/cAATCT, a sequence that is conserved in Lhcb genes. A region near the N terminus of CCA1, which has some homology to the repeated sequence found in the DNA binding domain of Myb proteins, is required for binding to the Lhcb1*3 promoter. Lines of transgenic Arabidopsis plants expressing antisense RNA for CCA1 showed reduced phytochrome induction of the endogenous Lhcb1*3 gene, whereas expression of another phytochrome-regulated gene, rbcS-1A, which encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, was not affected. Thus, the CCA1 protein acts as a specific activator of Lhcb1*3 transcription in response to brief red illumination. The expression of CCA1 RNA was itself transiently increased when etiolated seedlings were transferred to light. We conclude that the CCA1 protein is a key element in the functioning of the phytochrome signal transduction pathway leading to increased transcription of this Lhcb gene in Arabidopsis.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/genética , Proteínas de Transporte/genética , Regulação da Expressão Gênica de Plantas , Complexo de Proteínas do Centro de Reação Fotossintética , Complexo de Proteína do Fotossistema II , Fitocromo/fisiologia , Proteínas de Plantas , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Sequência Conservada , DNA Complementar , Complexos de Proteínas Captadores de Luz , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myb , Homologia de Sequência de AminoácidosAssuntos
Aspergilose/etiologia , Dermatomicoses/etiologia , Adulto , Aspergilose/complicações , Aspergilose/diagnóstico , Biópsia por Agulha/efeitos adversos , Dermatomicoses/complicações , Humanos , Pneumopatias Fúngicas/complicações , Pneumopatias Fúngicas/diagnóstico , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicaçõesRESUMO
We analyzed a mutant of Arabidopsis with a severely reduced level of cab140 RNA. This mutant, named lct for low level of cab140 transcript, was obtained during a selection for phytochrome signal transduction mutants. The selection was based on reduced expression of the tumor morphology shoots gene (tms2), an introduced counter-selectable marker under the control of the cab140 promoter. Expression of the introduced cab140::tms2 gene was also greatly reduced in lct, but surprisingly, expression of other phytochrome-regulated genes was not comparably affected. Furthermore, the lct phenotype could not be separated genetically from the T-DNA insert; thus, we suggest that this phenotype was caused by cosuppression of the introduced construct and the endogenous cab140 gene, and that the mutation causing the cosuppression was located on the T-DNA insert. In vitro nuclear transcription experiments demonstrated that the suppression was occurring at the level of transcription. We also found that the suppressed cab140 genes were not significantly more methylated than the nonsuppressed cab140 genes.
Assuntos
Arabidopsis/genética , Complexos de Proteínas Captadores de Luz , Mutação , Complexo de Proteínas do Centro de Reação Fotossintética/genética , RNA Mensageiro/genética , Supressão Genética , Amidoidrolases/genética , Mapeamento Cromossômico , DNA Bacteriano/genética , Genes de Plantas , Metilação , Fenótipo , Regiões Promotoras Genéticas , Transcrição Gênica , Transformação GenéticaRESUMO
We have identified and partially purified a DNA binding protein from Arabidopsis that interacts specifically with the phytochrome-responsive promoter of the Arabidopsis cab140 gene. Promoter deletion analyses in transgenic tobacco showed that, if a region that includes the sequence interacting with this protein was deleted, both expression and phytochrome responsiveness were lost. The protein protected a cytosine- and adenine-rich region from DNase I digestion, and therefore it has been called Ca-1. CA-1 was shown to be a phosphoprotein, and dephosphorylation changed the migration of the protein-DNA complex in DNA mobility shift assays. The data suggested that the protein has an apparent molecular weight of 70,000. The CA-1-protected region of the cab140 promoter included an ACGT motif that has been found in the target sequences of a number of bZIP transcription factors, but the binding behavior of CA-1 differed from those factors. CA-1 binding activity was present in plants grown in either white light or darkness, and no differences in the binding activity were detected in the dark-grown plants after short red or white light treatments. However, the CA-1 binding activity was not detectable in extracts of seedlings bearing the det1 mutation grown in the dark and given the same illumination treatments as wild type. In contrast to wild type, the mutant seedlings express cab RNA at a high level when grown in complete darkness, and we found no further increase in cab140 mRNA in response to brief red illumination. The lack of CA-1 activity in the det1 mutant suggests that it may function as a transcriptional repressor regulating the expression of the cab140 gene in Arabidopsis.
Assuntos
Arabidopsis/genética , Proteínas de Plantas/genética , Arabidopsis/metabolismo , Sequência de Bases , Sítios de Ligação , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Genes de Plantas , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
We have investigated the effects of human immunodeficiency virus type-1 (HIV-1) infection on constitutive and lipopolysaccharide (LPS)-induced expression of interleukin-6 (IL-6) in cultured blood monocyte-derived macrophages. Highly productive and cytopathic infection of macrophages was established with the macrophage-tropic HIV-1 BaL strain. On Days 14-28 post infection, infected and mock-infected cells were activated with LPS or control medium for 6-24 hours before harvesting culture supernatants and cellular RNA. IL-6 bioactivity in culture supernatants was measured with the IL-6-dependent B9 cell line. IL-6 mRNA levels were quantitated by Northern blot analysis with scanning densitometry. In the absence of LPS activation, IL-6 activity was near or below the limit of detection in supernatants from both infected and uninfected cultures. Similarly, without LPS stimulation, IL-6 mRNA was not detectable in either infected or uninfected macrophages. After activation with LPS, marked increases in IL-6 mRNA levels and supernatant bioactivity were evident in both infected and uninfected cultures, but the response to LPS was consistently greater in infected macrophages. LPS-induced IL-6 mRNA levels and supernatant bioactivity were 7.4- and 4.4-fold higher, respectively, in infected compared with uninfected macrophages (n = 5, p less than .05). These studies demonstrate that highly productive HIV-1 infection does not increase constitutive IL-6 expression in macrophages, but does prime macrophages for an augmented IL-6 response to LPS. These findings may help define the mechanisms responsible for increased IL-6 production in patients with HIV-1 infection.
Assuntos
HIV-1/fisiologia , Interleucina-6/biossíntese , Macrófagos/microbiologia , Células Cultivadas , Células Gigantes , Humanos , Cinética , Lipopolissacarídeos , Macrófagos/imunologia , Testes de Neutralização , RNA Mensageiro/metabolismo , Replicação ViralRESUMO
Cells from the human monocytic cell-line THP1 were incubated prior to activation with IFN-gamma or LPS with varying amounts of p24, the main product of the HIV gag gene and the major component of the virus core. The IFN-gamma-dependent increase of mRNA for HLA-DR and for the heavy chain of cytochrome b was markedly decreased by p24 but not by gp120. This effect was abrogated by anti-p24 antibodies. On the other hand, preincubation of THP1 cells with p24 did not affect the accumulation of the LPS-dependent mRNA for TNF alpha and IL1-beta. These results indicate that p24 at concentrations similar to those found in the serum of HIV-infected individuals specifically affects IFN-gamma-induced activation markers.
Assuntos
Grupo dos Citocromos b/genética , Produtos do Gene gag/farmacologia , HIV-1/imunologia , Antígenos HLA-DR/genética , Interferon gama/antagonistas & inibidores , Monócitos/fisiologia , Proteínas do Core Viral/farmacologia , Northern Blotting , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Proteína do Núcleo p24 do HIV , Humanos , Interleucina-1/genética , Interleucina-1/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos , Monócitos/microbiologia , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Persistence of Helicobacter pylori after duodenal ulcer healing is associated with high rates of ulcer relapse. We compared colloidal bismuth subcitrate alone with CBS combined with one of four antibiotic regimens in the treatment of duodenal ulcers. Endoscopy and antral biopsies were performed before treatment and four weeks afterwards. Biopsy specimens were examined for histological evidence of gastritis and by Gram stain and culture for H pylori infection. Altogether 141 patients were allocated to one of five treatment groups. Giving CBS and metronidazole (400 mg tid for 7 days) with and without amoxycillin (500 mg tid) achieved higher clearance rates of H pylori than treatment with CBS alone (p less than 0.01). These two combinations also achieved higher rates of antral gastritis healing than CBS alone (p less than 0.01 and p less than 0.05 respectively). Susceptibility to metronidazole was tested in 29 isolates before and in seven isolates after treatment with metronidazole by disc diffusion test and minimum inhibitory concentration assay. Twenty seven (93%) of the isolates were sensitive before treatment while six of seven (86%) were resistant afterwards. Four of the six resistant strains had acquired resistance during treatment and one of these had acquired metronidazole resistance despite concomitant treatment with amoxycillin, to which it remained sensitive. CBS with adjuvant metronidazole at a dose of 400 mg tid for seven days significantly improves the eradication of H pylori compared with CBS alone. Acquired metronidazole resistance, however, seems to be an important cause of failure to eradicate H pylori.