RESUMO
The "Metastasis type V", "Envelope" theory and other concepts have been proposed to make gastric surgeons have a full understanding of the importance of mesogastrium, and the correct identification of the boundary of the mesogastrium is the premise of the complete mesogastrium excision(CME), and also the best way to achieve truly standardized D2 lymph node dissection for gastric cancer. In this paper, the boundary of the mesogastrium during complete mesogastrium excision for gastric cancer and how to achieve this operation in practice were studied, which help gastric surgeons finally find the mesogastrium tissue distributed throughout the gastric cancer surgery to achieve the best surgical treatment effect.
Assuntos
Gastrectomia , Neoplasias Gástricas , Humanos , Excisão de Linfonodo , Mesentério/patologia , Mesentério/cirurgia , Medicina de Precisão , Neoplasias Gástricas/patologia , Neoplasias Gástricas/cirurgiaRESUMO
Gastric cancer (GC) is one of the malignant tumors with both high morbidity and mortality in China. Immunotherapy is expected to improve its prognosis. Molecular classification of GC based on multiple levels of assessment such as genes and tumor immune microenvironment (TiME), which can precisely screen the population with potential benefit from immunotherapy. It is helpful to make the treatment decision-making, and to further improve the efficacy of immunotherapy.
Assuntos
Neoplasias Gástricas , China , Humanos , Imunoterapia , Prognóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/terapia , Microambiente TumoralRESUMO
Objective: To compare the short-term clinical effect between laparoscopic distal pancreatectomy (LDP) and open distal pancreatectomy (ODP). Methods: We performed a retrospective study on 161 patients who underwent pancreatectomy between September 2017 to December 2018 in the Department of Pancreatic and Gastric Surgery, Cancer Hospital of Chinese Academy of Medical Sciences. According to the mode of operation, the patients were divided into the LDP group (n=43) and the ODP group (n=118). To compare the short-term clinical effect and safety between the LDP group and the ODP group, the preoperative clinical data, intraoperative related index, postoperative complication, postoperative recovery index, preoperative and postoperative inflammatory index were analyzed. Results: The preoperative clinical characteristics between the LDP group and the ODP group were not statistically different (P>0.05). The intraoperative blood loss in LDP group was (194.19±241.83) ml, significantly less than (315.17±295.94) ml in ODP group (P<0.05), and the postoperative exhaust time in LDP group was (3.00±0.72) days, significantly shorter than (4.05±0.97) days in OPD group (P<0.001). The time to get out of bed in LDP group was (3.14±1.01) days, significantly shorter than (3.55±1.05) days in OPD group (P<0.05). The postoperative eating time in LDP group was (3.88±1.61) days, significantly shorter than (5.11±1.56) days in ODP group (P<0.001). The time of the drainage tube removal in LDP group was (8.44±1.93) days, significantly shorter than (9.82±3.70) days in ODP group (P<0.05). The postoperative hospital stay in LDP group was (9.65±3.57) days, significantly shorter than (11.99±6.57) days in ODP group (P<0.05). The mean operation time in LDP group was (168.65±55.45) min, shorter than (171.23±65.61) min in ODP group, but without significant difference (P>0.05). The incidences of non-pancreatic fistula-related complications in LDP group and ODP group were 16.3% and 11.0%, respectively, without statistical significance (P>0.05). The incidences of pancreatic fistula in LDP group and ODP group were 16.3% and 19.5%, respectively, without statistical significance (P>0.05). The total incidences of complications in LDP group and ODP group were 32.6% and 30.5%, respectively, without statistical significance (P>0.05). The preoperative and postoperative inflammatory indexes between these two groups were not statistically different (P>0.05). Conclusions: Compared with ODP, LDP has the advantages of less intraoperative blood loss, faster postoperative recovery, shorter postoperative hospital stays, without increased postoperative complications and prolonged operation time. LDP is a safe and feasible operation method, and its short-term clinical effect is better than that of ODP.
Assuntos
Laparoscopia/métodos , Pancreatectomia/métodos , Neoplasias Pancreáticas/cirurgia , Humanos , Tempo de Internação , Duração da Cirurgia , Fístula Pancreática , Neoplasias Pancreáticas/patologia , Complicações Pós-Operatórias , Estudos Retrospectivos , Resultado do TratamentoRESUMO
The highly contagious novel coronavirus pneumonia (COVID-19) that broke out in December 2019 has brought huge threats and losses to human society, so it has been the concern of every countries government. Presently, there are no specific drugs for COVID-19; however, a variety of potentially effective antiviral drugs, vaccines, cell therapies, traditional Chinese medicine and other methods are in clinical trials. Liver injury is a common complication of patients receiving COVID-19 treatment and its possible high incidence may affect the outcome of the disease. The pathogenesis of COVID-19 combined with liver injury in existing studies is still unclear, and relevant guidelines and expert consensuses are insufficient for clinical diagnosis and treatment. Therefore, the relevant progress and issues are now reviewed here.
Assuntos
Betacoronavirus , Infecções por Coronavirus , Hepatopatias/etiologia , Pandemias , Pneumonia Viral , COVID-19 , Infecções por Coronavirus/complicações , Infecções por Coronavirus/tratamento farmacológico , Humanos , Fígado , Pneumonia Viral/complicações , SARS-CoV-2 , Tratamento Farmacológico da COVID-19RESUMO
Giant magnetic circular dichroism (MCD) that shows a different response to incident wave with left or right-handed circular polarization under external magnetic field is promising for magneto-optical sensing, revealing symmetry and degeneracy information of electronic states. However, traditional methods and materials that are used to obtain significant MCD involve highly strong external magnetic field. Based on the excitation of subradiant plasmonic mode and Fano resonance in graphene oligomers in the mid-infrared region, we numerically demonstrate that MCD is enhanced three times larger than the previously reported method, based on the resonance of electric dipole plasmonic mode. This giant MCD is attributed to the remarkably different excitation efficiency of subdradiant plasmonic mode due to the interparticle coupling under left or right-handed circular polarization incidence and external magnetic field. Our results offer an effective mechanism to enhance MCD signal at the nanoscale, which facilitates the sensing, spintronic, nanophotonics and other such fields.
RESUMO
Objective: To assess the effects of different nutritional support methods on postoperative recovery in patients with gastric cancer. Methods: 98 patients who received radical gastrectomy were divided into three groups: parenteral nutrition group (PN group) (n=36), early enteral nutrition group (EEN group) (n=33) and early oral feeding group (EON group) (n=29). Tolerance of enteral nutrition, postoperative recovery and economic indicators were compared. Results: The number of laparoscopic-assisted surgeries was 18, 17 and 25 in PN group, EEN group and EON group, respectively. There was no significant difference in sex, age and body mass index(BMI) among the three groups. Gastrointestinal function recovered slowly in 3 cases, including 2 cases in EEN group and 1 case in EON group. 1 case in EON group had abdominal hemorrhage. Median postoperative hospital stay in PN, EEN and EON group was 11.0, 11.0 and 8.0 days respectively, and significant reduction can be found in EON group(P<0.001). The complication rates were 30.5% (11 cases), 12.1% (4 cases), and 13.8% (4 cases), respectively, with no statistically significant difference(P=0.102). The median nutritional support costs for PN group, EEN group, and EON group were 4 543.3, 974.2, and 265.0 yuan, respectively. The median albumin consumption was 90.0, 40.0, and 0 g, respectively. The EON groups were significantly lower (P<0.001). The results of the laparoscopic assisted subgroup and the ones of whole group were consistent. Conclusion: Compared with parenteral nutrition and early enteral nutrition, early oral feeding can reduce the amount of albumin consumption, decrease the cost of nutrition support and shorten the average hospital stay after surgey without increasing the incidence of complications.
Assuntos
Gastrectomia/reabilitação , Apoio Nutricional/métodos , Neoplasias Gástricas/reabilitação , Neoplasias Gástricas/cirurgia , Convalescença , Gastrectomia/efeitos adversos , Humanos , Laparoscopia , Apoio Nutricional/efeitos adversos , Cuidados Pós-Operatórios , Recuperação de Função FisiológicaRESUMO
Objective: To assess the safety, feasibility and short-term outcome of totally laparoscopic distal gastrectomy(TLDG). Methods: Seventy-five patients who underwent laparoscopic distal gastrectomy in Cancer Hospital of Chinese Academy of Medical Science between August 2015 and April 2018 were enrolled in this study. A total of 46 laparoscopy-assisted distal gastrectomy (LADG) cases and 29 TLDG cases were included. The Short-term outcomes and safeties of the two groups were compared. Results: The operation time of TLDG group was significantly longer than that of LADG group (207±41 vs. 156±34 min, P<0.001), while the length of wound was shorter in the TLDG group (3.6±0.6 vs. 5.8±0.8 cm, P<0.001). The time to first flatus in TLDG group was (3.3±0.6) days, significantly shorter than (3.7±0.8) days in LADG group (P=0.034). There were no significant differences between the two groups in the estimated blood loss, intraoperative blood transfusion, extraction of gastric tube, drainage tube removal, interval of the first time to eat semi-liquid food, postoperative hospital stays, surgical complications, number of retrieved lymph nodes, proximal and distal resection margin lengths (all P>0.05). The white blood cell count at postoperative day 1 in the TLDG group was (10.96±1.96) ×10(9)/L, significantly lower than (12.49±3.46)×10(9)/L of the LADG group (P=0.017). While the CRP level at postoperative day 1 in the TLDG group were lower than that of LADG group, no statistical difference was observed (P=0.072). Conclusions: Our study shows that TLDG is safe and feasible. TLDG has better cosmesis, less blood loss, and faster recovery compared to LADG.
Assuntos
Gastrectomia/métodos , Laparoscopia/métodos , Neoplasias Gástricas/cirurgia , Perda Sanguínea Cirúrgica , Estudos de Viabilidade , Gastrectomia/efeitos adversos , Humanos , Laparoscopia/efeitos adversos , Tempo de Internação , Duração da Cirurgia , Complicações Pós-Operatórias , Segurança , Resultado do TratamentoRESUMO
Objective: To identify the feasibility and efficacy of indocyanine green (ICG) used in laparoscopic gastrectomy for advanced gastric cancer patients. Methods: From December 2018 to August 2019, the clinical data of 82 patients preoperatively diagnosed as advanced gastric cancer undergoing laparoscopic radical gastrectomy were retrospectively analyzed. These patients were divided into ICG group(n=38) and a historical control group (non-ICG group, n=44). The number of retrieved lymph nodes, operation time, blood loss, hospital stay, fever time, evacuation time and complications were compared between these two groups. Results: The operation time [(172.8±45.8) min vs (162.6±45.7) min], blood loss [(80.1±91.9) ml vs (78.6±89.8) ml], hospital stay [(7.0±2.0) d vs (7.5±2.4) d], fever time [(2.3±1.2) d vs (2.9±1.9) d], evacuation time [(3.4±0.8) d vs (3.4±1.1) d] and incidence of complications (5.3% vs 9.1%) were not significantly different between the ICG and historical control groups (P>0.05). The number of retrieved lymph nodes in ICG group was significantly increased compared with that of the historical control group (46.5 vs 33.0, P=0.005). Conclusions: The ICG method applied in lymph node dissection of laparoscopic radical gastrectomy is safe. Moreover, ICG might elevate the efficiency of regional lymph node dissection.
Assuntos
Gastrectomia , Verde de Indocianina/administração & dosagem , Laparoscopia/métodos , Neoplasias Gástricas/cirurgia , Estudos de Viabilidade , Humanos , Excisão de Linfonodo , Estudos Retrospectivos , Neoplasias Gástricas/patologia , Resultado do TratamentoRESUMO
OBJECTIVE: To evaluate the safety and efficacy of allogeneic natural killer (NK) cells in the treatment of primary hepatocellular carcinoma (HCC), and to elucidate the mechanism of NK cells therapy. METHODS: Twenty-one patients with primary HCC treated with allogeneic NK cells at the Fifth Medical Center of the PLA General Hospital were followed up for 1 year. Peripheral blood mononuclear cells (PBMCs) were isolated from patient-related donors and cultured in vitro for 15 days and infused to the patients in two consecutive days. Clinical data and laboratory data were collected and analyzed, including survival, clinical features, imaging changes, hematology, immunology, and biochemical indicators to evaluate the safety and efficacy of allogeneic NK cell therapy. The changes of peripheral blood lymphocyte subsets after treatment were also analyzed to explore the possible anti-tumor mechanisms. RESULTS: (1) Of the 21 patients with primary HCC, 11 patients were treated once, 5 patients were treated twice, and 5 patients were treated 3 times. After allogeneic NK cells infusion, 10 patients had fever, 1 patient had slight hepatalgia and 1 patient had slight headache, no other adverse events occurred including acute and chronic graft-versus-host disease (GVHD). They resolved spontaneously within 8 hours without other treatment. (2) The total disease control rate was 76.2% during one-year follow-up. Among them, the patients with Barcelona clinic liver cancer (BCLC) stage A had a disease control rate of 100%, stable disease (SD) in 10 cases; BCLC stage B patients had a disease control rate of 60%, partial response (PR) in 1 case, and SD 2 in cases; BCLC stage C patients had a disease control rate of 50%, complete response (CR) in 1 case, and 2 cases of PR. (3) The frequencies of NK cells and CD8+ T cells in peripheral blood were significantly lower than that before at 24 hours after treatment, and the frequencies of CD4+ T cells and CD4/CD8 were significantly higher than the baseline. CONCLUSION: Allogeneic NK cells have good safety and efficacy in the treatment of primary HCC. The anti-tumor effect of the allogeneic NK cells may play an important role in the activation of the patient's natural immune system and delay disease progression, suggesting that allogeneic NK cells combined with sorafenib may be a very effective treatment for advanced HCC, and further large-sample multicenter randomized controlled clinical trials are needed to validate this result.
Assuntos
Carcinoma Hepatocelular , Doença Enxerto-Hospedeiro , Neoplasias Hepáticas , Humanos , Células Matadoras Naturais , Leucócitos MononuclearesRESUMO
Objective: To investigate the safety and efficacy of the Weitan Waifu patch on the postsurgical gastroparesis syndrome (PGS) of gastrointestinal cancer. Methods: The multi-center, double-blind, randomized controlled trial was conducted with superiority design. Patients with PGS of gastrointestinal cancer diagnosed in 4 AAA hospitals and the abdominal symptom manifested as cold syndrome by Chinese local syndrome differentiation were recruited. These patients were randomly divided into two groups according to 1â¶1 proportion. Placebo or Weitan Waifu patch was applied in control group or intervention group, respectively, based on the basic treatments, including nutrition support, gastrointestinal decompression, promoting gastric dynamics medicine.Two acupuncture points (Zhongwan and Shenque) were stuck with placebo in control group or patch in treatment group. The intervention course was 14 days or reached the effective standard. Results: From July 15, 2013 to Jun 3, 2015, 128 participants were recruited and 120 eligible cases were included in the full analysis set (FAS), and 60 cases in each group. 88 cases were included in the per-protocol set (PPS), including 45 cases in the treatment group and 43 cases in the control group. In the FAS, the clinical effective rate in the treatment group was 68.3%, significantly superior than 41.7% of the control group (P=0.003). The medium time of effective therapy in the treatment group was 8 days, significantly shorter than 10 days in the control group (P=0.017). In the FAS, 3 adverse events occurred in the treatment group, including mild to moderate decrustation, pruritus and nausea. The incidence rate of adverse events was 5.0% (3/60) and these symptoms were spontaneously remitted after drug withdrawal. No severe adverse events were observed in the control group. There was no significant difference between these two groups (P=0.244). Conclusion: Weitan Waifu patch is a safely and effectively therapeutic method for patients with PGS (cold syndrome) of gastroenterological cancer. Trial registration: International Standard Randomized Controlled Trial Number Register, ISRCTN18291857.
Assuntos
Medicamentos de Ervas Chinesas/uso terapêutico , Neoplasias Gastrointestinais/cirurgia , Gastroparesia/tratamento farmacológico , Complicações Pós-Operatórias/tratamento farmacológico , Adesivo Transdérmico , Pontos de Acupuntura , Método Duplo-Cego , Humanos , Síndrome , Adesivo Transdérmico/efeitos adversos , Resultado do TratamentoRESUMO
AIMS: To screen and identify safe micro-organisms used during Douchi fermentation, and verify the feasibility of producing high-quality Douchi using these identified micro-organisms. METHODS AND RESULTS: PCR-denaturing gradient gel electrophoresis (DGGE) and automatic amino-acid analyser were used to investigate the microbial diversity and free amino acids (FAAs) content of 10 commercial Douchi samples. The correlations between microbial communities and FAAs were analysed by statistical analysis. Ten strains with significant positive correlation were identified. Then an experiment on Douchi fermentation by identified strains was carried out, and the nutritional composition in Douchi was analysed. Results showed that FAAs and relative content of isoflavone aglycones in verification Douchi samples were generally higher than those in commercial Douchi samples. CONCLUSIONS: Our study indicated that fungi, yeasts, Bacillus and lactic acid bacteria were the key players in Douchi fermentation, and with identified probiotic micro-organisms participating in fermentation, a higher quality Douchi product was produced. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report to analyse and confirm the key micro-organisms during Douchi fermentation by statistical analysis. This work proves fermentation micro-organisms to be the key influencing factor of Douchi quality, and demonstrates the feasibility of fermenting Douchi using identified starter micro-organisms.
Assuntos
Fungos/isolamento & purificação , Fungos/metabolismo , Glycine max/microbiologia , Lactobacillaceae/metabolismo , Aminoácidos/metabolismo , Eletroforese em Gel de Gradiente Desnaturante , Fermentação , Fungos/classificação , Fungos/genética , Isoflavonas/metabolismo , Lactobacillaceae/classificação , Lactobacillaceae/genética , Lactobacillaceae/isolamento & purificação , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
The rate of heat output is one of the suitable measurements of metabolic activity of the organism or its parts, down to the cellular or even the sub-cellular levels. In this paper, microcalorimetry was first applied to study the metabolic activity of microbial in both alginate-polylysine-alginate and alginate-chitosan-alginate microencapsulated cultures as well as in free non-encapsulated culture. The organisms used for the measurements were Escherichia coli and Saccharomyces cerevisiae. As a result of this work, it was found that, despite E. coli cell in free non-encapsulated culture has the highest metabolic rate due to the highest value of heat output, the proliferation of the cells terminates quickly with a lowest biomass formed. And we found also an obviously longer stationary phase in microencapsulated culture. As far as S. cerevisiae was concerned, it was found that there was also the highest value of heat output in free non-encapsulated culture, but the cell density was lower than that in microencapsulated culture. On account of the microcalorimetric and metabolic measurements, it can be concluded that more substrate can be used to convert to biomass in microencapsulated culture which means a higher biomass yield existed.
Assuntos
Calorimetria/métodos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Alginatos , Técnicas Bacteriológicas/métodos , Biomassa , Cápsulas , Quitosana , Polilisina/análogos & derivadosRESUMO
The LH/CG receptor is a member of the family of G protein-coupled receptors and consists of a large N-terminal extracellular domain (which is responsible for binding hormone) attached to a region that spans the plasma membrane seven times, ending with an intracellularly located C-terminus. Binding of LH or human CG (hCG) to the LH/CG receptor causes a stimulation of adenylyl cyclase, presumably via activation of Gs. The binding of hormone also leads to its subsequent internalization by receptor-mediated endocytosis. In order to investigate the role of the cytoplasmic tail of this receptor in these events, we prepared a series of mutants in which progressively larger portions of the cytoplasmic tail were deleted. Deletion of 58 amino acids from the C-terminus, in which only 11 cytoplasmic residues remain, resulted in a receptor that was not expressed on the plasma membrane. Receptors rat LHR (rLHR)-t653 and rLHR-t631, in which 21 or 43 amino acids were removed, respectively, were properly expressed. These results suggest that a region(s) between residues 616 and 631 of the rLH/CG receptor are required for proper insertion and/or targeting of the receptor into the plasma membrane. Cells expressing rLHR-t653 or rLHR-t631 bound hCG with the same high affinity as cells expressing the full-length receptor, and basal levels of cAMP were the same among the cells. However, cells expressing the truncated receptors responded to hCG with approximately 2-fold greater levels of maximal cAMP accumulation than cells expressing the full-length receptor. Deletion of up to 43 amino acids from the C-terminus of the rLH/CG receptor had no deleterious effect on hCG internalization. In fact, mutants lacking 21 and 43 amino acids exhibited progressively faster rates of hCG internalization as compared to the full-length receptor. Once internalized, hCG was also degraded at a faster rate in cells expressing the truncated LH/CG receptors. Since hCG-stimulated cAMP stimulation and hCG internalization are retained by rLHR-t631, it can be concluded that the residues, not necessarily the same, required for these functions reside within the 26 amino acids of the cytoplasmic tail closest to the seventh transmembrane helix and/or residues within the intracellular loops. Our data show, however, that both hCG-stimulated cAMP production and hCG internalization are enhanced by the removal of the distal portion of the cytoplasmic tail.
Assuntos
Gonadotropina Coriônica/metabolismo , Receptores do LH/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , AMP Cíclico/biossíntese , Citoplasma/metabolismo , Regulação para Baixo/fisiologia , Endocitose/fisiologia , Humanos , Dados de Sequência Molecular , Mutação , Ratos , Relação Estrutura-AtividadeAssuntos
Baculoviridae/genética , Receptores Androgênicos/genética , Animais , Western Blotting , Células Cultivadas , DNA , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Mariposas , Ratos , Receptores Androgênicos/análise , Proteínas Recombinantes/análise , Proteínas Recombinantes/genéticaRESUMO
The lutropin-choriogonadotropin (LH/CG) receptor is a cell surface receptor comprised of two domains of roughly equivalent size. The amino-terminal half of the receptor is relatively hydrophilic and is located extracellularly, whereas the carboxyl-terminal half of the receptor shares amino acid homology with other receptors that couple to G proteins and is similarly thought to span the plasma membrane seven times, ending with a relatively short carboxyl-terminal tail. In order to test the role of the extracellular domain in binding hormone, we constructed a mutated rat luteal LH/CG receptor cDNA (termed pCLHR-D2), which encodes for only the extracellular domain, and used it to transiently transfect human kidney 293 cells. Here we report that the expressed extracellular domain of the LH/CG receptor is capable of binding human CG with a high affinity, comparable with that of the full-length receptor. Thus, not only is the extracellular domain of the glycoprotein hormone receptors involved in binding hormone, but it alone is capable of conferring high affinity binding. Unexpectedly, it was also found that this truncated receptor is not secreted into the culture media but remains trapped within the cells.
Assuntos
Receptores do LH/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Gonadotropina Coriônica/metabolismo , Clonagem Molecular , DNA/genética , Expressão Gênica , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Receptores do LH/genética , Receptores do LH/ultraestrutura , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
A full-length rat androgen receptor cDNA was used to produce a recombinant baculovirus (AcrAR) by homologous recombination. Spodoptera frugiperda (Sf9) cells infected with this virus expressed a 110-kDa polypeptide that amounted up to about one-third of total cell protein. Studies with AR antibodies confirmed that this protein was indeed rAR. Only a minor portion of the recombinant AR was soluble in buffers without ionic detergents, but its complete solubilization was achieved in 6 M guanidine HCl (GdnHCl). Electron microscopy of cell pellets revealed that AR was localized to electron-dense cytoplasmic aggregates. The soluble cytosolic receptor was biologically active, in that it bound [3H]mibolerone with high affinity and specificity and interacted with an androgen-responsive element. The functions of the GdnHCl-solubilized AR were partially restored by a 20-50-fold dilution. The solubilized receptor was purified to an apparent homogeneity in a single step by gel filtration on a Sephacryl S-400 column in the presence of 6 M GdnHCl. The homogeneous AR protein could be renatured to bind [3H]mibolerone, interact specifically with a DNA element, and be recognized by receptor antibodies. Receptor-DNA interaction was stabilized by an antibody directed against the N-terminal part and abolished by an antibody against the hinge region of the receptor Zn2+ ions were essential for the purified receptor to refold into a specific DNA-binding form during the renaturation, with the optimal ZnCl2 concentration being 50-100 microM depending on the buffer conditions. Cd2+ ions were also capable of restoring the receptor's DNA-binding activity and did so at concentrations 10-fold lower than those of the Zn2+ ions.
Assuntos
Androgênios/metabolismo , DNA/metabolismo , Expressão Gênica , Receptores Androgênicos/genética , Animais , Baculoviridae/genética , Sequência de Bases , Western Blotting , Cátions Bivalentes , Linhagem Celular/ultraestrutura , Cromatografia em Gel , DNA/genética , Eletroforese em Gel de Poliacrilamida , Genes Virais , Microscopia Eletrônica , Dados de Sequência Molecular , Mariposas/citologia , Nandrolona/análogos & derivados , Nandrolona/metabolismo , Desnaturação Proteica , Ratos , Receptores Androgênicos/isolamento & purificação , Receptores Androgênicos/metabolismo , Especificidade por Substrato , Congêneres da Testosterona/metabolismo , TransfecçãoRESUMO
To facilitate detailed studies of androgen receptor, we have produced a full-length receptor protein and some of its deletion mutants in Spodoptera frugiperda (Sf9) insect cells, using the baculovirus expression system. Recombinant baculovirus DNA-infected Sf9 cells expressed these proteins in very high quantities, which represented as much as 30-40% of total insect cell protein at 72 h after infection. Only < 1% of the recombinant protein was soluble in low-salt buffers; the majority formed electron-dense cytoplasmic aggregates 30-40 nm in diameter. These aggregates could be solubilized in 6 mol/L guanidine HCl, and biologically active receptor was generated by diluting the guanidine HCl preparation 20- to 50-fold. The full-length receptor, expressed either in a soluble or aggregated form, had characteristics typical of a native receptor: it bound steroids with high affinity and specificity, interacted with DNA in a sequence-specific fashion, and was recognized by domain-specific receptor antibodies. Androgen-receptor protein purified to homogeneity in guanidine HCl required the presence of Zn2+ ions during the refolding to reconstitute its DNA-binding form; ZnCl2 was not, however, needed to restore the receptor's steroid-binding activity.