Genetic diversity and structure of the endangered species Megaleranthis saniculifolia in Korea as revealed by allozyme and ISSR markers

The genetic diversity and structure of 12 populations of Megaleranthis saniculifolia, a rare endemic Korean plant, were analyzed using 14 allozyme loci coding 10 enzymes and 78 ISSR loci using seven primers. The genetic diversity of M. saniculifolia at the species level was similar to that observed in out-crossing and long-lived perennials, while at the population level, it was significantly low. The high F _IS value of many populations as well as homozygote excess occurred relatively evenly in many populations in relation to the Hardy-Weinberg expectation, suggesting that inbreeding was occurring within the M. saniculifolia populations. The degree of genetic differentiation based on the two markers was high, and there was no correlation between geographic and genetic distance. Bayesian cluster analysis did not reveal any remarkable geographic trends. Positive correlations were observed between genetic diversity (H _e and h) and population size. Therefore, low genetic diversity within the population and high population differentiation of M. saniculifolia were closely related to the influence of genetic drift, particularly in highly isolated populations. In addition, the fixation of the main alleles at several loci in the opposite direction provided good evidence for genetic drift. The genetic diversity of M. saniculifolia could be compromised if the distribution area or the size of the population were further reduced. In particular, the isolated populations that are fragmented within an area could be at high risk of extinction due to accelerated inbreeding or genetic drift. Considering this, a close monitoring of the population size and of the changes in the genetic structure must be performed. Some practical measures for genetic conservation are also proposed.     

A nuclear ligand MRG15 involved in the proapoptotic activity of medicinal fungal galectin AAL (Agrocybe aegerita lectin)

Background

We have previously reported a novel fungal galectin Agrocybe aegerita lectin (AAL) with apoptosis-induced activity and nuclear migration activity. The importance of nuclear localization for AAL's apoptosis-induced activity has been established by mutant study. However, the mechanism remains unclear.

Methods

We further investigated the mechanism using a previously reported carbohydrate recognition domain (CRD) mutant protein H59Q, which retained its nuclear localization activity but lost most of its apoptotic activity. The cell membrane-binding ability of recombinant AAL (rAAL) and H59Q was analyzed by FACS, and their cellular partners were identified by affinity chromatography and mass spectroscopy. Furthermore, the interaction of AAL and ligand was proved by mammalian two-hybrid and pull down assays. A knockdown assay was used to confirm the role of the ligand.

Results

The apoptotic activity of AAL could be blocked by lactose. Mutant H59Q retained comparable cell membrane-binding ability to rAAL. Four cellular binding partners of AAL in HeLa cells were identified: glucose-regulated protein 78 (GRP78); mortality factor 4-like protein 1 (MRG15); elongation factor 2 (EEF2); and heat shock protein 70 (Hsp70). CRD region of AAL was required for the interaction between AAL/mutant AAL and MRG15. MRG15 knockdown increased the cells' resistance to AAL treatment.

Conclusion

MRG15 was a nuclear ligand for AAL in HeLa cells. These data implied the existence of a novel nuclear pathway for the antitumor activity of fungal galectin AAL.

General significance

These findings provide a novel explanation of AAL bioactivity and contribute to the understanding of mushroom lectins' antitumor activity.     

The Chemical Constituents of Endophytic Fungus Trichoderma sp. MFF‐1

A fungal strain named MFF‐1 was isolated from the flower of Pyrethrum cinerariifolium. Based on the sequence at the internal transcribed spacer (ITS) region, this strain was identified as a Trichoderma sp. Two new compounds, including a mitorubrin derivative and its potential biogenetic precursor, together with a known compound, were isolated from the cultures of the endophytic fungus. Their structures were established by spectroscopic methods and determined to be (3S*,6R*,7R*)‐3,4,5,6,7,8‐hexahydro‐7‐hydroxy‐7‐methyl‐8‐oxo‐3‐[(E)‐prop‐1‐enyl]‐1H‐isochromen‐6‐yl 2,4‐dihydroxy‐6‐methylbenzoate ( 1 ), named deacetylisowortmin, (E)‐2‐(hydroxymethyl)‐3‐(2‐hydroxypent‐3‐enyl)phenol ( 2 ), and wortmannin ( 3 ). All compounds were assayed for antimicrobial activity. Compound 3 showed activity against Candida albicans and Bacillus cereus.     

Design,expression and characterization of recombinant hybrid peptide Attacin-Thanatin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Antimicrobial peptides will be attractive and potential candidates as peptide drugs because of their efficient action against microbes and low toxicity to mammal cells. To improve their antibacterial activity, some modifications needs to be made. In this research, the hybrid peptide gene Attacin-Thanatin with 642 bp in length with preferred codons of E. coli was generated using the technology of Gene splicing by overlap extension. The gene was inserted in-frame into E. coli expression plasmid pET-32a (+) and induced to express in E. coli Rosetta. The recombinant protein was partial purified and its biological activity was determined. Analysis of the E. coli Rosetta induced with IPTG revealed that the molecular weight of fusion protein was approximately 41.8 kDa, which perfectly matched the mass calculated from the amino acid sequence. Biological activity detection showed that this peptide effectively inhibited the growth of the test bacteria including E. coli DH5α, E. coli BL21 (DE3), Salmonella choleraesuis and Staphylococcus aureus. Among these bacteria, the Gram-negative E. coli was the most sensitive. Furthermore, there was minor hemolysis activity for porcine red blood cells. So, the results indicated that the hybrid peptide Attacin-Thanatin could be served as a promising candidate for the chemical antibiotics.     

Effect of root-applied spermidine on growth and respiratory metabolism in roots of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>) seedlings under hypoxia

The effects of exogenous spermidine (Spd) application to hypoxic nutrient solution on the contents of endogenous polyamines (PAs) and respiratory metabolism in the roots of cucumber (Cucumis sativus L.) seedlings were investigated. Cucumber seedlings were grown hydroponically in control and hypoxic nutrient solutions with and without addition of Spd at a concentration of 0.05 mM. The activities of key enzymes involved in the tricarboxylic acid cycle (TCAC), such as succinate dehydrogenase (SDH) and isocitrate dehydrogenase (IDH), were significantly inhibited under root-zone hypoxia with dissolved oxygen (DO) at 1 mg/l. In contrast, the activities of enzymes involved in the process of fermentation, such as pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), and alanine aminotransferase (AlaAT), were significantly increased. Thus, aerobic respiration was inhibited and fermentation was enhanced in the roots of cucumber seedlings as a result of decreasing ATP content to inhibit the dry weight of seedlings under hypoxic stress. Moreover, the contents of free, soluble conjugated, and insoluble bound putrescine (Put), Spd, and spermine (Spm) in the roots of cucumber seedlings were significantly increased under hypoxia stress. Interestingly, application of Spd to hypoxic roots markedly suppressed the accumulation of free Put and, in contrast, promoted an increase in free Spd and Spm, as well as soluble conjugated and insoluble bound Put, Spd, and Spm contents. From these data, we deduced that exogenous Spd promotes the conversion of free Put into free Spd and Spm, and soluble conjugated and insoluble bound PAs under hypoxia stress. Furthermore, the activities of LDH, PDC, and ADH were suppressed and, in contrast, the activities of SDH and IDH were enhanced by application of exogenous Spd to hypoxic roots. As a result, aerobic respiration was enhanced but fermentation metabolism was inhibited in the roots of cucumber seedlings, leading to an increase in ATP content to alleviate the inhibited dry weight of seedlings due to hypoxia stress. These results suggest that application of Spd to hypoxic nutrient solution promoted conversion of free Put into free Spd and Spm as well as soluble conjugated and insoluble bound PAs, further enhanced IDH and SDH activities, and inhibited ethanol fermentation and lactate fermentation, resulting in increased ATP content and eventually enhanced tolerance of cucumber plants to root-zone hypoxia.     

PPAR‐β facilitating maturation of hepatic‐like tissue derived from mouse embryonic stem cells accompanied by mitochondriogenesis and membrane potential retention

Relatively little is known about mitochondria metabolism in differentiating embryonic stem (ES) cells. Present research focused on several elements of cellular energy metabolism in hepatic‐like tissue derived from mouse ES cells. We demonstrated that mitochondrial location patterns and mitochondrial membrane potential (ΔΨ_m) existed in subsequent differentiation of the tissue. Mitochondriogenesis appeared at the early stage and kept a normal ΔΨ_m in differentiated mature hepatocytes. Peroxisome proliferator‐activated receptor‐α (PPAR‐α) expression was transitorily increased at the beginning, and kept a relatively low level later, which accompanied by expression of PPAR‐γ coactivator (PGC)‐1α, a master regulator of mitochondrial biogenesis. PPAR‐β expression showed robust up‐regulation in the late differentiation course. Enhanced co‐expressions of PPAR‐β and albumin with catalysis of UDP‐glucuronosyltransferases (UGTs) were observed at mature stage. While PPAR‐γ expression changed little before and after differentiation. Mitochondriogenesis could be accelerated by PPAR‐α specific agonist WY14643 and abolished by its antagonist GW6471 at the early stage. Neither of them affected mitochondrial ΔΨ_m and albumin generation in the differentiated hepatocytes. Furthermore, maturation of hepatic‐like tissue and mitochondriogenesis in hepatocyte could be efficiently stimulated by PPAR‐β specific agonist L165041 and abolished by PPAR‐β specific antagonist GSK0660, but not affected by PPAR‐γ specific agonist GW1929. In conclusion, the derived hepatic tissue morphologically possessed cellular energy metabolism features. PPAR‐α seemed only necessary for early mitochondriogenesis, while less important for ΔΨ_m retention in the mature tissue derived. The stimulation of PPAR‐β but not ‐γ enhanced hepatogenesis, hepatocytes maturation, and mitochondriogenesis. PPAR‐β took an important role in cellular energy metabolism of hepatogenesis. J. Cell. Biochem. 109: 498–508, 2010. © 2009 Wiley‐Liss, Inc.     

HDAC6 controls autophagosome maturation essential for ubiquitin‐selective quality‐control autophagy

Autophagy is primarily considered a non‐selective degradation process induced by starvation. Nutrient‐independent basal autophagy, in contrast, imposes intracellular QC by selective disposal of aberrant protein aggregates and damaged organelles, a process critical for suppressing neurodegenerative diseases. The molecular mechanism that distinguishes these two fundamental autophagic responses, however, remains mysterious. Here, we identify the ubiquitin‐binding deacetylase, histone deacetylase‐6 (HDAC6), as a central component of basal autophagy that targets protein aggregates and damaged mitochondria. Surprisingly, HDAC6 is not required for autophagy activation; rather, it controls the fusion of autophagosomes to lysosomes. HDAC6 promotes autophagy by recruiting a cortactin‐dependent, actin‐remodelling machinery, which in turn assembles an F‐actin network that stimulates autophagosome–lysosome fusion and substrate degradation. Indeed, HDAC6 deficiency leads to autophagosome maturation failure, protein aggregate build‐up, and neurodegeneration. Remarkably, HDAC6 and F‐actin assembly are completely dispensable for starvation‐induced autophagy, uncovering the fundamental difference of these autophagic modes. Our study identifies HDAC6 and the actin cytoskeleton as critical components that define QC autophagy and uncovers a novel regulation of autophagy at the level of autophagosome–lysosome fusion.     

山羊品种间体细胞核移植研究

萨能奶山羊是著名的奶用山羊品种,波尔山羊则是世界著名的肉用山羊品种.为了研究波尔山羊体细胞在奶山羊卵母细胞中的去分化,我们将成年波尔山羊的颗粒细胞或耳皮肤成纤维细胞作为供核细胞(试验组),移入奶山羊中Ⅱ期的去核卵母细胞透明带下,经电融合和离子霉素与6-二甲基氨基嘌呤(6-DMAP)激活,直接移入同期发情奶山羊输卵管或经体内培养,将发育的重构胚移人同期发情羊子宫内.妊娠早期作B超诊断,确立妊娠的观察至足月.同时将奶山羊的35日龄胎儿成纤维细胞作供核细胞(对照组),按试验组同样方法处理,将重构胚直接移入同期发情的奶山羊输卵管内.结果试验组,波尔羊颗粒粒细胞与耳皮肤成纤维细胞的融合率分别为78.2%(115/147)、57.4%(116/202),重构胚卵裂率为85.8%(115/134),桑椹胚、囊胚的发育率38.8%(52/134),早期妊娠三头,分别于妊娠40、60、60日龄终止妊娠.对照组,融合率为89.5%(136/152),早期妊娠率为42.9%(6/14),四头受体足月分娩,产四头公羊羔,其中三头存活,一头分娩时死于肺不扩张,并体重过大,显示胎儿过大综合症.经基因型鉴定证实,这四头克隆羔羊均源于同一胎儿成纤维细胞系.以上结果表明,波尔羊体细胞核在奶山羊卵母细胞中能够去分化,并维持一定程度的发育.     

鲫鱼血清和皮肤粘液IgM的分离纯化及部分性质的鉴定

采用盐析法结合葡聚糖凝胶柱 ,分离纯化鲫鱼血清IgM ;然后制备兔抗鲫鱼血清IgM多克隆抗体 ,将其偶联到Sepharose 4B上制成亲和柱 ,用于分离纯化皮肤粘液IgM。结果表明 :33%～ 4 5 %硫酸铵溶液沉淀处理可以去除鲫鱼血清中除IgM外的很多杂蛋白 ,再经葡聚糖凝胶柱纯化 ,IgM纯度可达 80 %以上 ,其重链和轻链的分子量分别为 79和 2 5kDa ;以兔抗鲫鱼血清IgM多克隆抗体亲和柱分离皮肤粘液IgM ,分离效果良好 ,IgM重链的分子量为 88kDa ;Westernblot显示兔抗鲫鱼血清IgM多克隆抗体识别的是血清和皮肤粘液IgM的重链部分。用ELISA测定鲫鱼血清中IgM含量在一年中的变化 ,结果表明IgM在春夏季的含量高于秋冬季     

广西蚱科四新种记述(直翅目：蚱总科)

本文记述采自广西状族自治区大青山及大瑶山地区蚱科昆虫４新种,即龙州蚱Ｔｅｔｒｉｘｌｏｎｇｚｈｏｕｅｎｓｉｓ ｓｐ．ｎｏｖ．、广西拟台蚱Ｆｏｒｍｏｓａｔｅｔｔｉｘｏｉｄｅｓ ｇｕａｎｇｘｉｅｎｓｉｓ ｓｐ．ｎｏｖ．、黑胫真长背蚱Ｅｕｐａｒａｔｅｔｔｉｘ ｎｉｇｒｉｔｉｂｉｓ ｓｐ．ｎｏｖ．及长翅拟真长背蚱Ｅｕｐａｒａｔｅｔｔｉｘｏｉｄｅｓ ｌｏｎｇｉｐｅｎｎｉｓ ｓｐ．ｎｏｖ．。