从绿色荧光蛋白的发现谈如何提出新颖的科研问题

为了使研究生在获得专业知识的同时,还可以依据文献提出新的科研问题,并设计实验方案来解决提出的问题,我们以绿色荧光蛋白为案例教学,谈谈在其发现过程中是如何提出问题以及提出问题所用的思维方式。启发研究生运用求异和置换的思维方式来提出新颖的科研问题,并设计实验来回答提出的问题,从而为研究生完成学术论文奠定良好的基础。     

不同管理方式下橡胶林土壤氮动态特征

对西双版纳割胶、未割胶条件下橡胶林种植带土壤及其保护带土壤氮素动态变化特征进行了研究,并比较了不同保护带种植方式(距瓣豆绿肥覆盖与野生杂草覆盖)对土壤氮素动态的影响。结果表明,橡胶林种植带土壤全氮、碱解氮和硝态氮含量低于保护带。橡胶林土壤全氮、碱解氮、铵态氮、硝态氮均呈现明显的动态变化,种植距瓣豆绿肥覆盖与野生杂草生长覆盖的橡胶林土壤氮变化趋势一致。土壤全氮随时间逐渐下降,碱解氮含量先升后降,铵态氮和硝态氮含量变化幅度较大。橡胶林土壤全氮和碱解氮含量呈现表层(0～20cm)>中层(20～40cm)>底层(40～60cm)的趋势,且未割胶处理全氮和碱解氮含量>割胶处理,而保护带为距瓣豆绿肥覆盖的割胶橡胶林>杂草生长覆盖的橡胶林。距瓣豆绿肥覆盖的保护带土壤硝态氮含量高于杂草生长覆盖。碱解氮与铵态氮含量呈显著的负相关、与硝态氮呈显著正相关。割胶橡胶林土壤氮养分含量最低。橡胶林土壤种植豆科距瓣豆绿肥能够改善土壤氮素肥力。     

佛山市顺德中心城区城市绿地生态网络规划

以佛山市顺德区中心城区为研究样区,基于景观连通性定量评价方法确定绿地系统核心斑块,并采用最小费用路径法构建了绿地系统潜在的生态廊道,同时还对绿地生态网络各组分的景观格局特征进行了分析。结果表明:斑块连通性指数距离阈值设定为500m时能够合理反映各绿地斑块重要性程度,并以连通性指数重要值排序结果确定了20个绿地斑块作为绿地生态网络的核心斑块;通过最小费用路径法构建的研究区31条潜在生态廊道,符合绿地系统分布特征并能够较好地连通各绿地核心斑块;生态网络景观格局分析表明,生态廊道缓冲区内斑块类型较复杂,景观格局破碎化程度较高。     

N-terminal extended conjugates of the agonists and antagonists of both bradykinin receptor subtypes: structure-activity relationship, cell imaging using ligands conjugated with fluorophores and prospect for functionally active cargoes

Peptide agonists and antagonists of both bradykinin (BK) B(1) and B(2) receptors (B(1)R, B(2)R) are known to tolerate to a certain level N-terminal sequence extensions. Using this strategy, we produced and characterized the full set of fluorescent ligands by extending both agonists and antagonist peptides at both receptor subtypes with 5(6)-carboxyfluorescein (CF) and the ε-aminocaproyl (ε-ACA) optional spacer. Alternatively, kinin receptor ligands were extended with another carboxylic acid cargo (chlorambucil, biotinyl, pentafluorocinnamoyl, AlexaFluor-350 (AF350), ferrocenoyl, cetirizine) or with fluorescein isothiocyanate. N-terminal extension always reduced receptor affinity, more importantly for bulkier substituents and more so for the agonist version compared to the antagonist. This loss was generally alleviated by the presence of the spacer and modulated by the species of origin for the receptor. We report and review the pharmacological properties of these N-terminally extended peptides and the use of fluorophore-conjugated ligands in imaging of cell receptors and of angiotensin converting enzyme (ACE) in intact cells. Antagonists (B(1)R: B-10376: CF-ε-ACA-Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-BK; B(2)R: B-10380: CF-ε-ACA-D-Arg-[Hyp(3), Igl(5), D-Igl(7), Oic(8)]-BK and fluorescein-5-thiocarbamoyl (FTC)-B-9430) label the plasma membrane of cells expressing the cognate receptors. The B(2)R agonists CF-ε-ACA-BK, AF350-ε-ACA-BK and FTC-B-9972 are found in endosomes and model the endosomal degradation of BK in a complementary manner. The uneven surface fluorescence associated to the B(1)R agonist B-10378 (CF-ε-ACA-Lys-des-Arg(9)-BK) is compatible with a particular form of agonist-induced receptor translocation. CF-ε-ACA-BK binds to the carboxydipeptidase ACE with an affinity identical to that of BK. Metal- or drug-containing cargoes further show the prospect of ligands that confer special signaling to kinin receptors.     

Transferrin receptor-1 suppresses neurite outgrowth in neuroblastoma Neuro2A cells

Transferrin receptor-1 (TfR1) is a cell membrane-associated glycoprotein responsible for incorporation of the iron bound to transferrin through an endocytotic process from the circulating blood. Iron is believed to play a dual role as an active center of the electron transfer system in mitochondria and as an endogenous cytotoxin through promoted generation of reactive oxygen species in different eukaryotic cells. In this study, we evaluated expression profiles of different genes related to iron mobilization across plasma membranes in neuronal cells. Marked mRNA expression was seen for various iron-related genes such as TfR1 in cultured mouse neocortical neurons, while TfR1 mRNA levels were more than doubled during culture from 3 to 6days. In mouse embryonal carcinoma P19 cells endowed to differentiate into neuronal and astroglial lineages, a transient increase was seen in both mRNA and corresponding protein for TfR1 in association with neuronal marker expression during culture with all-trans retinoic acid (ATRA). In neuronal Neuro2A cells cultured with ATRA, moreover, neurite was elongated together with increased expression of both mRNA and protein for TfR1. Overexpression of TfR1 significantly decreased the length of neurite elongated, however, while significant promotion was invariably seen in the neurite elongation in Neuro2A cells transfected with TfR1 siRNA as well as in Neuro2A cells cultured with an iron chelator. These results suggest that TfR1 would be highly expressed by neurons rather than astroglia to play a negative role in the neurite outgrowth after the incorporation of circulating transferrin in the brain.     

Nuclear localization drives α1-adrenergic receptor oligomerization and signaling in cardiac myocytes

Conventional models of G-protein coupled receptor (GPCR) signaling describe cell surface receptors binding to external ligands, such as hormones or circulating peptides, to induce intracellular signaling and a physiologic response. However, recent studies identify new paradigms indicating that GPCRs localize to and signal at the nucleus and that GPCR oligomers can influence receptor function. Previously, we reported that endogenous α1-adrenergic receptors (α1-ARs) localize to and signal at the nuclei in adult cardiac myocytes. In this study, we examined the mechanisms behind α1-AR nuclear localization and how nuclear localization impacted receptor function. We verified that endogenous α1-ARs localized to the nuclear membrane of intact nuclei isolated from wild-type adult cardiac myocytes. Next, we identified and disrupted putative nuclear localization sequences in both the α1A- and α1B-adrenergic receptors, which led to mis-localization of α1-ARs in cultured adult cardiac myocytes. Using these mutants, we demonstrated that nuclear localization was required for α1-signaling in adult cardiac myocytes. We also found that the nuclear export inhibitor leptomycin B inhibited α1-AR signaling, indicating α1-AR signaling must arise in the nucleus in adult cardiac myocytes. Finally, we found that co-localization of the α1-subtypes at the nuclei in adult cardiac myocytes facilitated the formation of receptor oligomers that could affect receptor signaling. In summary, our data indicate that α1-AR nuclear localization can drive the formation of receptor oligomers and regulate signaling in adult cardiac myocytes.     

Prevalence of oral mucosal lesions in institutionalized elderly people in Mashhad, Northeast Iran

doi: 10.1111/j.1741‐2358.2011.00588.x
Prevalence of oral mucosal lesions in institutionalized elderly people in Mashhad, Northeast Iran Objective: The aim of this study was to determine the prevalence of oral mucosal lesions in institutionalized elderly people in Mashhad, northeast of Iran. Background: This study was conducted to assess the prevalence of oral lesions in the institutionalized elderly. Methods: In this survey, we studied all of the nursing homes in Mashhad; a total of five encompassing 237 residents. The subjects were examined and lesions recorded in a designed checklist. T‐test, chi‐square and Fisher’s exact tests were used for the analysis. Results: The mean age of the patients was 79.59 ± 8.88. Ninety‐eight per cent of cases had at least one oral mucosal lesion. The most common lesions were fissured tongue (66.5%), atrophic glossitis (48.8%), sublingual varicosity (42%) and xerostomia (38%). There was no significant difference in the prevalence of denture‐related lesions (DRLs) between men and women (p > 0.05). Xerostomia was more prevalent in 70–79‐year‐old than in 60–69‐year‐old subjects. No case of oral malignant lesion was detected. Discussion: These findings revealed a higher prevalence of oral mucosal lesions in Mashhad city compared with other studies conducted in other major cities in Iran and abroad. The results emphasise the necessity of national programmes towards oral health promotion.     

Prevalence of soft tissue oral lesion in elderly and its relations with deleterious habits

doi: 10.1111/j.1741‐2358.2011.00618.x Prevalence of soft tissue oral lesion in elderly and its relations with deleterious habits Introduction: With ageing, the oral mucosa becomes more susceptible to external stimuli. Objective: To investigate the prevalence of soft tissue oral lesion in elderly and its relations with deleterious habits. Materials and methods: It was a quantitative research conducted at the dental clinic of UNIFOR, Fortaleza‐CE, in the period from 1998 to 2006. The sample consisted of all 756 records of the elderly, and the data were processed by SPSS 15.0 (SPSS, Inc., Chicago, IL, USA). Results: The majority (63.0) were women, age 60–92, mean age 67 ± 6. The most significant proportions were married (48.4%), retired (42.3%) and incomplete primary school (31.5%). It was found a prevalence of 18.3% of oral lesions, with the risk being 1.6 times higher (p = 0.030) among youngest (up to 65 years) and 1.7 times higher among smokers (p = 0.048). Although this risk is 1.6 times higher among those who drank alcohol, it was not statistically significant (p = 0.122). Conclusion: The elderly showed a high prevalence of oral lesion, being the youngest, the smokers and the alcohol consumers those most vulnerable to the emergence of these diseases. Dental services need to implement programs of elderly care and health education, essential to clarify the risks and their associations with oral diseases, aiming to promote health.     

Fed-batch mode in shake flasks by slow-release technique

Most industrial production processes are performed in fed-batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode which results in completely different physiological conditions than relevant for production conditions. This may lead to wrong selections of strains. Silicone elastomer discs containing glucose crystals were developed to realize fed-batch fermentation in shake flasks. No other device for feeding was required. Glucose was fed in this way to Hansenula polymorpha cultures controlled by diffusion. Two strains of H. polymorpha were investigated in shake flasks: the wild-type strain (DSM 70277) and a recombinant strain pC10-FMD (P(FMD)-GFP). The oxygen transfer rate (OTR) and respiratory quotient (RQ) of the cultures were monitored online in shake flasks with a Respiration Activity Monitoring System (RAMOS). Formation of biomass and green fluorescent protein (GFP), pH-drift and the metabolite dynamics of glucose, ethanol and acetic acid were measured offline. With the slow-release technique overflow metabolism could be reduced leading to an increase of 85% in biomass yield. To date, 23.4 g/L cell dry weight of H. polymorpha could be achieved in shake flask. Biomass yields of 0.38-0.47 were obtained which are in the same magnitude of laboratory scale fermentors equipped with a substrate feed pump. GFP yield could be increased by a factor of 35 in Syn6-MES mineral medium. In fed-batch mode 88 mg/L GFP was synthesized with 35.9 g/L fed glucose. In contrast, only 2.5 mg/L with 40 g/L metabolized glucose was revealed in batch mode. In YNB mineral medium over 420-fold improvement in fed-batch mode was achieved with 421 mg/L GFP at 41.3 g/L fed glucose in comparison to less than 1 mg/L in batch mode with 40 g/L glucose.