Pulmonary immune responses induced in BALB/c mice by Paracoccidioides brasiliensis conidia

Knowledge concerning the host–Paracoccidioides brasiliensis interactions is abundant. Yet, most of the experimental studies have used yeast cells to prepare the corresponding inoculum. As these cells do not represent the naturally infecting propagules, the corresponding experiments by-pass the earlier stages of such interactions. Studies done in patients, who also harbour yeast cells, suffer from the same bias. The review presented below focuses on the immune responses of BALB/c mice infected with conidia obtained from P. brasiliensis mycelial form cultures, the fungal stage most probably existing in nature. As such, the corresponding experiments would copy the onset and course of the human infection. A large number of experimental studies done by the CIB Medical and Experimental Mycology Unit in a period of almost 25 years have been revised and extracted so as to present a comprehensive record on the immune responses induced when mice are infected intranasally with the conidia. The establishment of this mouse model has permitted the analysis of the immune responses taking place during the early and late stages post-challenge. This unique model has made possible to characterize the course of the experimental disease including the inflammatory reaction, the expression of cytokines and of the various molecules associated to these responses, all of which lead to granuloma formation. The latter structure serves as a nest for the development of fibrosis. Thus, we have also obtained a glimpse on the complexity that accompanies the fibrosis, the most common sequelae of paracoccidioidomycosis. Additionally, a concerted effort has been made to appraise the whole gamut of immune factors and related molecules that directly or indirectly, contribute to shape the pathogenesis of this Latin American mycosis.     

Identification and characterization of Paracoccidioides lutzii proteins interacting with macrophages

Paracoccidioidomycosis (PCM), caused by thermodimorphic fungi of the Paracoccidioides genus, is a systemic disorder that involves the lungs and other organs. The adherence of pathogenic microorganisms to host tissues is an essential event in the onset of colonization and spread. The host–pathogen interaction is a complex interplay between the defense mechanisms of the host and the efforts of pathogenic microorganisms to colonize it. Therefore, the identification of fungi proteins interacting with host proteins is an important step understanding the survival strategies of the fungus within the host. In this paper, we used affinity chromatography based on surface proteomics (ACSP) to investigate the interactions of pathogen proteins with host surface molecules. Paracoccidioides lutzii extracts enriched of surface proteins were captured by chromatographic resin, which was immobilized with macrophage cell surface proteins, and identified by mass spectrometry. A total of 215 proteins of P. lutzii were identified interacting with macrophage proteins. In silico analysis classified those proteins according to the presence of sites for N- and O-glycosylation and secretion by classical and non-classical pathways. Serine proteinase (SP) and fructose-1,6-bisphosphate aldolase (FBA) were identified in our proteomics analysis. Immunolocalization assay and flow cytometry both showed an increase in the expression of these two proteins during host–pathogen interaction.     

基于蛋白质组芯片的结核分枝杆菌系统生物学研究进展

近些年全球结核病疫情愈发严重,耐药性结核病使其雪上加霜。一个重要原因是结核病新药的匮乏以及结核分枝杆菌相关基础研究的不足。因此迫切需要开发新的技术以促进结核病系统生物学基础研究,并在此基础上研究新机制,发现新靶标,开发新药物。结核分枝杆菌功能蛋白质组芯片的出现旨在促进结核病相关研究工作。考虑到结核分枝杆菌高毒力、复制周期长和需要在生物安全三级实验室中开展研究等特点和难点,该工具为结核病相关研究人员提供了一个强有力的武器。目前这一技术手段的应用已经使我们对结核分枝杆菌-宿主相互作用、小分子-蛋白结合以及抗生素耐药性机制等关键生物过程有了更深入的了解。为了更好地帮助同行了解这一有效的工具,本文综述了结核分枝杆菌功能蛋白组芯片的几种主要应用,期望同行专家能更好地将其应用于结核病相关的基础研究中。     

芳香烃龙胆酸降解途径蛋白质组学的研究

芳香烃是一类重要的环境污染物,微生物降解是其主要的处理方法。研究显示降解过程中产生保守型和诱导型的各一组同工酶。目前,仅有保守型的龙胆酸加双氧酶（GDOI）及其下游片段被克隆。产碱假单胞菌NCIB9867（P25X）的突变株-SNZ28 GDOI被打断,在龙胆酸诱导的情况下,该突变株仍能检测到龙胆酸加双氧酶活性。采用二维蛋白电泳分析突变株SNZ28在有和没有龙胆酸诱导条件下的蛋白质表达差异。电泳结果显示了两者存在有15个蛋白点的差异。通过MALDI-TOF和Q—TOF分析,其中的12个蛋白质点与数据库中已知多肽片段有同源性。其中,P4点与青枯菌（Ralstonia species）龙胆酸1,2加双氧酶同源。该结果在蛋白质组学上证实了GDOII的存在。     

Proteome investigation of the global regulatory role of sigma 54 in response to gentisate induction in Pseudomonas alcaligenes NCIMB 9867

Pseudomonas alcaligenes NCIMB 9867 (strain P25X) utilizes the gentisate pathway for the degradation of aromatic hydrocarbons. The gene encoding the alternative sigma (sigma) factor sigma(54), rpoN, was cloned from strain P25X and a rpoN knock-out strain, designated G54, was constructed by insertional inactivation with a kanamycin resistance gene cassette. The role of sigma(54) in the physiological response of P. alcaligenes P25X to gentisate induction was assessed by comparing the global protein expression profiles of the wild-type P25X with the rpoN mutant strain G54. Analysis of two-dimensional polyacrylamide gel electrophoresis gels showed that 39 out of 355 prominent protein spots exhibited differential expression as a result of the insertional inactivation of rpoN. Identification of the protein spots by matrix-assisted laser desorption/ionization-time of flight/time of flight revealed a wide diversity of proteins that are affected by the sigma(54) mutation, the largest group being proteins that are involved in carbon metabolism. The strictly inducible gentisate 1,2-dioxygenase, one of two isofunctional copies of the key enzyme in the gentisate pathway, and enzymes of the TCA cycle, pyruvate metabolism and gluconeogenesis were part of this group. Other proteins that are part of the sigma(54) regulon include enzymes implicated in nitrogen metabolism, transport proteins, stress-response proteins and proteins involved in cell motility. The results of this study showed that sigma(54) plays a global regulatory role in the expression of a wide variety of genes in P. alcaligenes, including the wild-type response to the presence of the aromatic inducer, gentisate.     

Characteristics of the conidia produced by the mycelial form of Paracoccidioides brasiliensis

The sporulation capacities of the mycelial form of Paracoccidioides brasiliensis were determined. Five different culture media were used and four human isolates studied. Conidia were produced in three agar media, namely water-agar, glucose-salts and yeast-extract. Corn meal and Sabouraud dextrose agars failed to induce sporulation. Various types of spores were characterized with peculiar bulging arthroconidia and single-celled, pear-shaped conidia predominating. The size of these conidia varied from 3.6 to 4.6 micron in length. It is concluded that the mycelial form of P. brasiliensis produces characteristic spores if the proper culture media are employed.     

中国被毛孢分生孢子形成过程显微结构的研究

分生孢子是中国被毛孢(H sinensis)生活史中极为重要的一个环节,其在冬虫夏草侵染和形成过程中有着十分关键的作用.本实验以中国被毛孢纯培养菌种为实验材料,采用显微镜观察了其菌丝体经营养生长产分生孢子过程中显微结构变化,结果表明:中国被毛孢在固体培养条件下,可以形成分生孢子,其形态和产生方式与微循环产孢过程中形成的分生孢子一致,同时,在产孢过程中,还存在瓶梗结构纵裂形成对生分生孢子的现象.     

乳腺增生组织蛋白质组的凝胶内差异显示电泳分析 `

目的：建立具有高分辨率和稳定性的乳腺增生组织蛋白质组的双向电泳图谱,并对其进行差异蛋白质组分析。方法：取乳腺增生病患者增生部位及正常部位乳腺组织,匀浆提取乳腺组织总蛋白,分别用Cy3或Cy5标记,每一对Cy3和Cy5标记样品都与一个Cy2标记的内标等量混合,上样于同一胶中进行电泳分离,经不同光激发下扫描得到不同样品的蛋白质组图谱。所获得的图谱经DeCyder软件进行分析。结果：在乳腺增生病增生的组织中,有12个蛋白质表达水平显著增加,另外3个蛋白质表达水平显著下降。结论：利用DIGE技术可以作胶内时比分析,也可以根据内标消除胶与胶之间的差异,提高统计的可信度;分析所得的15个差异蛋白质可能与乳腺增生疾病的发生与发展有关。     

Proteome analysis of bronchoalveolar lavage in lung diseases

The proteomic approach is complementary to genomics and enables protein composition to be investigated under various clinical conditions. Its application to the study of bronchoalveolar lavage (BAL) is extremely promising. BAL proteomic studies were initially based on two-dimensional electrophoretic separation of complex protein samples and subsequent identification of proteins by different methods. With the techniques available today it is possible to attain many different research objectives. BAL proteomics can contribute to the identification of proteins in alveolar spaces with possible insights into pathogenesis and clinical application for diagnosis, prognosis and therapy. Many proteins with different functions have already been identified in BAL. Some could be biomarkers that need to be individually confirmed by correlation with clinical parameters and validation by other methods on larger cohorts of patients. The standardization of BAL sample preparation and processing for proteomic studies is an important goal that would promote and facilitate clinical applications. Here, we review the principal literature on BAL proteomic analysis applied to the study of lung diseases.     

Scanning electron microscopy of the conidia produced by the mycelial form of Paracoccidioides brasiliensis

The conidia produced by the mycelial form of Paracoccidioides brasiliensis were examined by scanning electron microscopy for the first time. Several different conidial types were characterized. These included intercalary arthroconidia, several types of septate conidia that are formed from other conidia, pedunculate conidia, and terminal hyphal conidia. In addition, the ultrastructure of the supporting pedestal of the pedunculate conidium was found to be separated from the mother conidium by a septum in some instances, and at other times it was not.     

Proteome analysis of chick embryonic cerebrospinal fluid

During early stages of embryo development, the brain cavity is filled with embryonic cerebrospinal fluid (E-CSF), a complex fluid containing different protein fractions that contributes to the regulation of the survival, proliferation and neurogenesis of the neuroectodermal stem cells. Using 2-DE, protein sequencing and database searches, we identified and analyzed the proteome of the E-CSF from chick embryos (Gallus gallus). We identified 26 different gene products, including proteins related to the extracellular matrix, proteins associated with the regulation of osmotic pressure and metal transport, proteins related to cell survival, MAP kinase activators, proteins involved in the transport of retinol and vitamin D, antioxidant and antimicrobial proteins, intracellular proteins and some unknown proteins. Most of these gene products are involved in the regulation of developmental processes during embryogenesis in systems other than E-CSF. Interestingly, 14 of them are also present in adult human CSF proteome, and it has been reported that they are altered in the CSF of patients suffering neurodegenerative diseases and/or neurological disorders. Understanding these molecules and the mechanisms they control during embryonic neurogenesis is a key contribution to the general understanding of CNS development, and may also contribute to greater knowledge of these human diseases.     

几种植物油对莱氏野村菌分生孢子萌发的影响

【目的】研究从银纹夜蛾(鳞翅目:夜蛾科)[Argyrogramma agnata Staudinger(Lepidoptera:Noctuidae)]幼虫上分离得到的莱氏野村菌菌株Nr0815分生孢子,于不同温度下在8种植物油中萌发率和萌发速率的差异。【方法】采用紫外分光光度计测定了Nr0815分生孢子在8种植物油中的吸光度,并用载玻片萌发法测定了72 h内不同温度下分生孢子在不同植物油中的萌发率以及不同观察时间段的萌发速率。【结果】莱氏野村菌分生孢子在供试植物油中分散良好。在10–30°C内,莱氏野村菌分生孢子在各植物油中均能正常萌发,其中在大豆油中累积萌发率均最高。从孢子萌发速率来看,23°C时除大豆油和菜籽油外,其他植物油中孢子的萌发速率在72 h内均随着时间的延长而上升,在48–72 h时的萌发速率最大。10°C时的萌发速率最小,48 h后才开始萌发。从孢子萌发后芽管生长来看,23°C下孢子萌发后72 h时大豆油中的芽管长度最大,为(24.89±1.04)μm,葵花籽油中芽管最短,仅为(9.10±1.00)μm。【结论】明确了温度和植物油对莱氏野村菌孢子萌发的影响,也为该菌油剂开发利用提供理论基础。     

Proteome profile of functional mitochondria from human skeletal muscle using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS

Mitochondria can be isolated from skeletal muscle in a manner that preserves tightly coupled bioenergetic function in vitro. The purpose of this study was to characterize the composition of such preparations using a proteomics approach. Mitochondria isolated from human vastus lateralis biopsies were functional as evidenced by their response to carbohydrate and fat-derived fuels. Using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS, 823 unique proteins were detected, and 487 of these were assigned to the mitochondrion, including the newly characterized SIRT5, MitoNEET and RDH13. Proteins detected included 9 of the 13 mitochondrial DNA-encoded proteins and 86 of 104 electron transport chain (ETC) and ETC-related proteins. In addition, 59 of 78 proteins of the 55S mitoribosome, several TIM and TOM proteins and cell death proteins were present. This study presents an efficient method for future qualitative assessments of proteins from functional isolated mitochondria from small samples of healthy and diseased skeletal muscle.     

A Review of the Salivary Proteome and Peptidome and Saliva-derived Peptide Therapeutics

Saliva is a glandular secretion that is vital in the maintenance of healthy oral tissues. In this review we outline the high abundance salivary proteins, summarise the status of the salivary proteome and peptidome, the genetic origin and recognised functions of these proteins, the diseases associated with salivary disorders, and the emerging saliva-derived peptide therapeutics. Different proteomic approaches have reported the identification of over 1,300 proteins in saliva. However there are fewer than 100 high abundance proteins, identified by multiple methods including, two-dimensional polyacrylamide gel electrophoresis and HPLC combined with mass spectrometry. Analysis of the genes coding for the salivary proteins demonstrated a non-uniform chromosomal distribution with chromosome 4 having the largest proportion of genes expressed in salivary glands. Several diseases are associated with salivary disorders including Sjögren’s syndrome, Prader-Willi syndrome, dental caries and stress related disorders. Saliva as a diagnostic medium for various biochemical tests has provided a non-invasive and accessibility advantage over other more regularly tested body fluids such as blood and urine. To-date the emerging saliva-based therapeutics include artificial salivas and antimicrobial agents based on histatins and mucins.     

衫木叶片蛋白质组的双向电泳技术优化

为建立适用于杉木(Cunninghaimia lanceolata)叶片蛋白质组研究的双向电泳技术,对杉木叶片蛋白质的溶解方法、上样量、IEF及SDS-PAGE电泳等关键步骤进行了优化。结果表明,杉木叶片蛋白质主要分布在pH4-7范围;裂解液中含有硫脲(2mmol/L)才能较充分地溶解蛋白,DTT浓度为60mmol/L、上样量1.5mg时得到的图谱分辨率较好且蛋白斑点分布均匀、清晰,拖尾现象明显减少,平衡液Ⅱ中碘代乙酰胺浓度为450mg(15ml)-1时能提高图谱分辨率;采用与质谱兼容的考马斯亮兰进行染色,得到近700个蛋白点。