Proteomic profiling of giant skeletal muscle proteins

Introduction: Distinct subtypes of contractile fibres are highly diverse in their proteomic profile and greatly adaptable to physiological or pathological challenges. A striking biochemical feature of heterogeneous skeletal muscle tissues is the presence of a considerable number of extremely large protein species, which often present a bioanalytical challenge for the systematic separation and identification of muscle proteomes during large-scale screening surveys.

Areas covered: This review outlines the proteomic characterization of skeletal muscles with a special focus on giant proteins of the sarcomere, the cytoskeleton and the sarcoplasmic reticulum. This includes an overview of the involvement of large muscle proteins, such as titin, nebulin, obscurin, plectin, dystrophin and the ryanodine receptor calcium release channel, during normal muscle functioning, swift adaptations to changed physiological demands and changes in relation to pathobiochemical insults.

Expert commentary: The proteomic screening and characterization of total muscle extracts and various subcellular fractions has confirmed the critical role of large skeletal muscle proteins in the regulation of ion homeostasis, the maintenance of contraction-relaxation cycles and fibre elasticity, and the stabilisation of supramolecular complexes of the muscle periphery and cytoskeletal networks of contractile fibres. These findings will be helpful for the future functional systems analysis of giant muscle proteins.     

大熊猫幼兽腹泻粪便分离出的轮状病毒鉴定

11 只5 ～ 11 月龄的断奶大熊猫幼兽先后患一种急性传染性、顽固性腹泻病,1 只大熊猫幼兽在发病4 d 后死亡,相同饲养条件下由母兽哺育的同龄大熊猫幼兽未见发病。取发病大熊猫幼兽粪便、血液以及呕吐物进行细菌学和寄生虫学检查,未检出病原;用轮状病毒粪便抗原ELISA 快速诊断,受检的8 只大熊猫幼兽腹泻便都多次检出阳性结果。采集发病大熊猫的病料进行病毒病原分离,从腹泻大熊猫幼兽的粪便中分离出病毒颗粒,通过电镜形态、理化特性和中和试验鉴定,确认分离病毒为轮状病毒。      

Non-invasive giant panda paternity exclusion

Giant panda hair samples obtained by noninvasive methods served as a source of DNA for amplification of seven giant panda microsatellite loci utilizing the polymerase chain reaction. Thirteen giant pandas held in Chinese zoos were tested for identification of paternity. Some males listed as sires have been excluded as the biological father of captive-born giant pandas. Because of the death of some potential sires, paternity is still not assigned for some giant pandas, although there is a high likelihood that paternity assignment could be made if postmortem samples are available for genetic analysis. The DNA microsatellite variation assayed by the test we have developed provides a rapid, highly informative, and noninvasive method for paternity identification in giant pandas. © 1994 Wiley-Liss, Inc.     

大熊猫和小熊猫粪便DNA提取的简易方法

采集了大熊猫和小熊猫的新鲜粪便样品 ,使用 1 0 0 %乙醇保存。通过重复离心富集研究动物的肠道脱落细胞 ,并使用乙醇和双蒸水洗涤以除去抑制物。用 1 %的SDS快速裂解细胞 ,离心除去残渣后 ,向裂解液中加入蛋白酶进行消化。消化结束后使用等体积的酚 /氯仿抽提 ,乙醇沉淀DNA。用双蒸水溶解粪便DNA后 ,使用PCR产物纯化试剂盒对粪便DNA进行纯化。电泳检测结果显示 ,从乙醇保存的大、小熊猫粪便样品中抽提到高质量的粪便DNA。对线粒体控制区、细胞色素b基因、 1 2SrRNA基因的PCR扩增反应以及测序结果也证实了样品保存方法和DNA抽提方法可靠而高效。此方法使用实验室内常用的分子生物学试剂 ,不仅克服了分子粪便学研究中常见的抑制物粪便DNA微量降解严重等障碍 ,与商业化的粪便抽提试剂盒 (QIAampDNAStoolMiniKit,Qiagen)相比还是一种经济的试验方法 (抽提反应成本为试剂盒的 1 / 5 )。文中还对粪便DNA内细菌基因组等背景DNA可能对分子粪便学试验结果的影响进行了探讨。在基于PCR技术的遗传学研究中 ,对于植食性动物而言 ,粪便内的背景DNA对目标动物DNA片断的扩增和序列测定未见影响 ;但对于肉食性动物 ,则必须考虑被捕食者基因组对试验可能产生的影响 ,应谨慎对待     

Composition of captive giant panda milk

Eleven milk samples were collected from three female giant pandas (Ailuropoda melanoleuca) during 23 days after delivery. Concentrations of crude protein (CP), lactose (Lac), and three vitamins (VA, VD, VE) in these samples were tested. Concentration of CP, Lac, VA, VD, and VE of these samples in wet basis averaged 6.77±0.78%, 3.23±0.54%, 0.073±0.043 mg/L, 0.24±0.08 mg/L, and 6.76±1.01 mg/L, respectively. Results demonstrated that nutrients concentrations of milk samples had no significant difference among different pandas (P>0.05). The average content of milk protein between 3–6 days and 7–23 days had significant difference (P<0.01). Results indicate that panda milk is similar to that of other carnivores. It has higher protein but lower lactose than milk in domestic ungulates and humans. Zoo Biol 0:1–6, 2005. © 2005 Wiley‐Liss, Inc.     

Functional annotation from the genome sequence of the giant panda

The giant panda is one of the most critically endangered species due to the fragmentation and loss of its habitat. Studying the functions of proteins in this animal, especially specific trait-related proteins, is therefore necessary to protect the species. In this work, the functions of these proteins were investigated using the genome sequence of the giant panda. Data on 21,001 proteins and their functions were stored in the Giant Panda Protein Database, in which the proteins were divided into two groups: 20,179 proteins whose functions can be predicted by GeneScan formed the known-function group, whereas 822 proteins whose functions cannot be predicted by GeneScan comprised the unknownfunction group. For the known-function group, we further classified the proteins by molecular function, biological process, cellular component, and tissue specificity. For the unknown-function group, we developed a strategy in which the proteins were filtered by cross- Blast to identify panda-specific proteins under the assumption that proteins related to the panda-specific traits in the unknown-function group exist. After this filtering procedure, we identified 32 proteins (2 of which are membrane proteins) specific to the giant panda genome as compared against the dog and horse genomes. Based on their amino acid sequences, these 32 proteins were further analyzed by functional classification using SVM-Prot, motif prediction using MyHits, and interacting protein prediction using the Database of Interacting Proteins. Nineteen proteins were predicted to be zinc-binding proteins, thus affecting the activities of nucleic acids. The 32 panda-specific proteins will be further investigated by structural and functional analysis.     

大熊猫的生境选择特征

基于王朗国家级自然保护区1997—2009年的连续监测数据,利用分布频率法和Bai-ley法,从地形因子、森林群落结构和主食竹3个方面研究了大熊猫的生境选择特征.结果表明:王朗国家级自然保护区的大熊猫对生境具有明显的选择性.在地形上,多选择海拔在2500～3000 m的山体脊部、上部和中部的均匀坡和凸坡,坡向西南,坡度在6°～30°,与水源距离>300 m的环境;森林群落结构上,多选择起源为次生林、针阔混交林,微生境为竹林的生境,乔木平均高度在20～29 m,灌木盖度在0～24%;主食竹多选择平均高度在2～5 m,竹丛盖度>50%,混生,生长状况良好的缺苞箭竹.     

Increased adipogenicity of cells from regenerating skeletal muscle

Adipose tissue development is observed in some muscle pathologies, however, mechanisms that induce accumulation of this tissue as well as its cellular origin are unknown. The adipogenicity of cells from bupivacaine hydrochloride (BPVC)-treated and untreated muscle was compared in vitro. Culturing cells from both BPVC-treated and untreated muscles in adipogenic differentiation medium (ADM) for 10 days resulted in the appearance of mature adipocytes, but their number was 3.5-fold higher in cells from BPVC-treated muscle. Temporal expressions of PPARgamma and the presence of lipid droplets during adipogenic differentiation were examined. On day 2 of culture in ADM, only cells from BPVC-treated muscle were positive both for PPARgamma and lipid droplets. Pref-1 was expressed in cells from untreated muscle, whereas its expression was absent in cells from BPVC-treated muscle. In ADM, the presence of insulin, which negates an inhibitory effect of Pref-1 on adipogenic differentiation, was required for PPARgamma2 expression in cells from untreated muscle, but not for cells from BPVC-treated muscle. These results indicate that BPVC-induced degenerative/regenerative changes in muscle lead to increased adipogenicity of cells, and suggest that this increased adipogenicity not only involves an increase in the number of cells having adipogenic potential, but also contributes to the progression of these cells toward adipogenic differentiation.     

Efficient generation of iPS cells from skeletal muscle stem cells

Reprogramming of somatic cells into inducible pluripotent stem cells generally occurs at low efficiency, although what limits reprogramming of particular cell types is poorly understood. Recent data suggest that the differentiation status of the cell targeted for reprogramming may influence its susceptibility to reprogramming as well as the differentiation potential of the induced pluripotent stem (iPS) cells that are derived from it. To assess directly the influence of lineage commitment on iPS cell derivation and differentiation, we evaluated reprogramming in adult stem cell and mature cell populations residing in skeletal muscle. Our data using clonal assays and a second-generation inducible reprogramming system indicate that stem cells found in mouse muscle, including resident satellite cells and mesenchymal progenitors, reprogram with significantly greater efficiency than their more differentiated daughters (myoblasts and fibroblasts). However, in contrast to previous reports, we find no evidence of biased differentiation potential among iPS cells derived from myogenically committed cells. These data support the notion that adult stem cells reprogram more efficiently than terminally differentiated cells, and argue against the suggestion that "epigenetic memory" significantly influences the differentiation potential of iPS cells derived from distinct somatic cell lineages in skeletal muscle.     

Culture of mature trophoblastic giant cells from bovine placentomes

The mostly binucleate trophoblast giant cells (TGC) found in bovine placentomes, in addition to synthesizing and releasing hormones play an important role in fetal development and maternal adaptation to pregnancy. Placentomes from early gestation were collected, and for isolation of mature TGC, three cellular disaggregation methods, mechanical (MECH), enzymatic by trypsin (TRYP) or collagenase (COLL) were compared to each other. Further on, the cell survival in culture medium (DMEM) supplemented with either 10% fetal calf serum (FCS) or 10% serum replacement (SR) on culture plates free of any substrate was evaluated over a period of 90 days by trypan blue exclusion. The cells were further characterized by HOECHST 33342 nuclear staining, and immunocytochemical staining with monoclonal antibodies against vimentin and cytokeratin. A mean total rate of TGC survival of 82.56% was recorded. Statistical analysis showed significantly higher survival rates after enzymatic disaggregation with COLL (86.23%) than following MECH (80.38%) or TRYP (80.91%) treatment. Supplementation of DMEM with FCS resulted in significantly higher cellular survival rates (87.13%) when compared to the addition of SR (77.73%). Analysis of the influence of both, disaggregation method and medium supplementation on TGC survival revealed statistically significant differences between the following groups: MECH-SR (71.09%) was significantly lower than all other groups; TRYP-SR (78.03%) was significantly different from all other groups; TRYP-FCS (83.43%) and COLL-SR (84.08%) were significantly lower than MECH-FCS (89.98%) which together with COLL-FCS (88.25%) showed the highest cellular survival rate. In summary, our results show that TGC isolated from early gestation placentomes may be viable for more than 90 days of culture. However, whether these TGC produce placental lactogen throughout this period has yet to be determined.     

Sox andZfx genes in giant panda

By using PCR cloning techniques, the DNA sequences of the HMG box regions of sixSox genes (pSox) and the zinc finger domains of twoZfz genes (pZfx) in the giant panda were identified. The giant pandaSox genes fell into two subfamilies,SOX-S1 andSOX-S2. ThepSox andpZfx genes of the giant panda were highly homologous to the corresponding genes in mammals and revealed close substitution rates to those in the primates.     

大熊猫粪便乳酸菌的分离鉴定

大熊猫作为国家保护动物,其健康问题备受瞩目。为了维护大熊猫的肠道健康,本研究从大熊猫肠道内分离出适宜于大熊猫肠道环境的乳酸菌菌株,有望将其制成熊猫肠道微生物制剂,从而改善大熊猫肠道菌群环境。从雅安市宝兴县蜂桶寨自然保护区选取圈养与野生大熊猫的粪便,通过体外培养分离出9个菌株。分离菌株经过革兰氏染色镜检、过氧化氢产气、菌落形态观察等方法与技术初步鉴定为乳酸菌。对这9株乳酸菌进行耐酸试验、耐胆盐试验、抑菌能力试验和产酸能力等测试,筛选出了3个适应性较强,有望制成调节大熊猫肠道内环境平衡作用的微生态菌剂的菌株。16S rRNA基因序列分析表明:分离菌株J1、J2和J4分别为融合魏斯氏菌（Weissella confusa）,海氏肠球菌（Enterococcus heynei）和非解乳糖链球菌（Streptococcus alactolyticus）,有望被应用于大熊猫肠道微生态制剂的研究。     

Characterization of the giant myofiber in bovine skeletal muscle

Semitendinosus (ST) muscle samples were excised pre- and postrigor from 32 experimental animals (Bos taurus). Four intact and 4 castrate males were exsanguinated when they reached age endpoints of 8, 12, 16, and 20 months. The samples were sectioned, histochemically fiber-typed for SDH and ATPase activity, and examined for giant myofibers. Of the 32 muscles analyzed, only one (8-month intact male) postrigor ST contained a giant myofiber that was hematoxylin and eosin positive, SDH negative, ATPase positive, and 2 X the size of the surrounding fibers. Subsequent studies demonstrated these giant type II white myofibers are an anomaly of muscle contraction and not a distinct fiber type as has been previously reported. This is a first report of giant white or type II myofibers in postrigor bovine skeletal muscle.     

大熊猫的生境选择特征

基于王朗国家级自然保护区1997—2009年的连续监测数据,利用分布频率法和Bailey法,从地形因子、森林群落结构和主食竹3个方面研究了大熊猫的生境选择特征.结果表明：王朗国家级自然保护区的大熊猫对生境具有明显的选择性.在地形上,多选择海拔在2500～3000 m的山体脊部、上部和中部的均匀坡和凸坡,坡向西南,坡度在6°～30 °,与水源距离＞300 m的环境;森林群落结构上,多选择起源为次生林、针阔混交林,微生境为竹林的生境,乔木平均高度在20～29 m,灌木盖度在0～24%;主食竹多选择平均高度在2～5 m,竹丛盖度＞50%,混生,生长状况良好的缺苞箭竹.     

Gut microbiota in reintroduction of giant panda

Reintroduction is a key approach in the conservation of endangered species. In recent decades, many reintroduction projects have been conducted for conservation purposes, but the rate of success has been low. Given the important role of gut microbiota in health and diseases, we questioned whether gut microbiota would play a crucial role in giant panda's wild‐training process. The wild procedure is when captive‐born babies live with their mothers in a wilderness enclosure and learn wilderness survival skills from their mothers. During the wild‐training process, the baby pandas undergo wilderness survival tests and regular physical examinations. Based on their performance through these tests, the top subjects (age 2–3 years old) are released into the wild while the others are translocated to captivity. After release, we tracked one released panda (Zhangxiang) and collected its fecal samples for 5 months (January 16, 2013 to March 29 2014). Here, we analyzed the Illumina HiSeq sequencing data (V4 region of 16S rRNA gene) from captive pandas (n = 24), wild‐training baby pandas (n = 8) of which 6 were released and 2 were unreleased, wild‐training mother pandas (n = 8), one released panda (Zhangxiang), and wild giant pandas (n = 18). Our results showed that the gut microbiota of wild‐training pandas is significantly different from that of wild pandas but similar to that of captive ones. The gut microbiota of the released panda Zhangxiang gradually changed to become similar to those of wild pandas after release. In addition, we identified several bacteria that were enriched in the released baby pandas before release, compared with the unreleased baby pandas. These bacteria include several known gut‐health related beneficial taxa such as Roseburia, Coprococcus, Sutterella, Dorea, and Ruminococcus. Therefore, our results suggest that certain members of the gut microbiota may be important in panda reintroduction.     

亚成体大熊猫肠道真菌多样性

【目的】分析5只亚成体大熊猫肠道真菌的多样性。【方法】采用基于ITS基因的RFLP(Restriction Fragment Length Polymorphism)方法,对肠道真菌总DNA进行ITS-RFLP分析,构建ITS克隆文库,然后用HhaI、HaeIII进行酶切指纹图谱分析,测序并绘制系统进化树。【结果】研究表明,5只亚成体大熊猫肠道真菌主要由Ascomycota(平均占46.24%)、Basidiomycota(平均占15.79%)2个门和一些未分类(平均占29.14%)、未培养(平均占8.83%)的真菌。其中,Ascomycota主要以Saccharomycetes(平均占63.74%)和Dothideomycetes(平均占35.91%)2个纲为主;Basidiomycota主要以Tremellomycetes(平均占65.80%)和Microbotryomycetes(平均占33.15%)2个纲为主。而这4个纲分别则主要以Candida、Debaryomyces;Pleosporales、Myriangium;Cystofilobasidium、Trichosporon;Leucosporidium、Leucosporidiella八个菌属为主,各样品中所占比例不等。【结论】亚成体大熊猫肠道内存在一定比例的真菌菌群,且ITS-RFLP技术能够很好地对其多样性进行分析。真菌的发现扩大了我们对大熊猫肠道微生物结构的了解,同时也有助于我们进一步研究真菌能否帮助大熊猫消化高纤维素食物。