Molecular cloning,expression and characterization of three distinctive genes encoding methionine aminopeptidases in cyanobacterium<Emphasis Type="Italic"> Synechocystis</Emphasis> sp. strain PCC6803

Methionine aminopeptidase, known to be encoded by single genes in prokaryotes, is a cobalt-dependent enzyme that catalyzes the removal of N-terminal methionine residues from nascent polypeptides. Three ORFs encoding putative methionine aminopeptidases from the genome of cyanobacterium Synechocystis sp. strain PCC6803, designated as slr0786 (map-1), slr0918 (map-2) and sll0555 (map-3) were cloned and expressed in Escherichia coli. The purified recombinant proteins encoded by map-1 and map-3 had much higher methionine aminopeptidase activity than the recombinant protein encoded by map-2. Comparative analysis revealed that the three recombinant enzymes differed in their substrate specificity, divalent ion requirement, pH, and temperature optima. The broad activities of the iso-enzymes are discussed in light of the structural similarities with other peptidase families and their levels of specificity in the cell. Potential application of cyanobacterial MetAPs in the production of recombinant proteins used in medicine is proposed. This is the first report of a prokaryote harboring multiple methionine aminopeptidases.Abbreviations map Gene encoding methionine aminopeptidase - MetAP Methionine aminopeptidase - eMetAP-Ia Escherichia coli methionine aminopeptidase type Ia - yMetAP-Ib Yeast methionine aminopeptidase type Ib - yMetAP-IIa Yeast methionine aminopeptidase type IIa - hMetAP-IIb Human methionine aminopeptidase type IIb - pfMetAP–IIa Pyrococcus furiosis methionine aminopeptidase type Ia - bst MetAP-Ia Bacillus stearothermophilus methionine aminopeptidase type Ia - c1MetAP-Ia Cyanobacterial methionine aminopeptidase type Ia encoded by map-1 - c2MetAP-Ia Cyanobacterial methionine aminopeptidase type Ia encoded by map-2 - c3MetAP-Ib Cyanobacterial methionine aminopeptidase type Ib, ncoded by map-3     

Downregulation of p‐COUMAROYL ESTER 3‐HYDROXYLASE in rice leads to altered cell wall structures and improves biomass saccharification

p‐Coumaroyl ester 3‐hydroxylase (C3′H) is a key enzyme involved in the biosynthesis of lignin, a phenylpropanoid polymer that is the major constituent of secondary cell walls in vascular plants. Although the crucial role of C3′H in lignification and its manipulation to upgrade lignocellulose have been investigated in eudicots, limited information is available in monocotyledonous grass species, despite their potential as biomass feedstocks. Here we address the pronounced impacts of C3′H deficiency on the structure and properties of grass cell walls. C3′H‐knockdown lines generated via RNA interference (RNAi)‐mediated gene silencing, with about 0.5% of the residual expression levels, reached maturity and set seeds. In contrast, C3′H‐knockout rice mutants generated via CRISPR/Cas9‐mediated mutagenesis were severely dwarfed and sterile. Cell wall analysis of the mature C3′H‐knockdown RNAi lines revealed that their lignins were largely enriched in p‐hydroxyphenyl (H) units while being substantially reduced in the normally dominant guaiacyl (G) and syringyl (S) units. Interestingly, however, the enrichment of H units was limited to within the non‐acylated lignin units, with grass‐specific γ‐p‐coumaroylated lignin units remaining apparently unchanged. Suppression of C3′H also resulted in relative augmentation in tricin residues in lignin as well as a substantial reduction in wall cross‐linking ferulates. Collectively, our data demonstrate that C3′H expression is an important determinant not only of lignin content and composition but also of the degree of cell wall cross‐linking. We also demonstrated that C3′H‐suppressed rice displays enhanced biomass saccharification.     

Effects of population density, sex morph, and tree size on reproduction in a heterodichogamous maple, Acer mono, in a temperate forest of Japan

The effects of local population density, sex morph [protogynous (PG) or protandrous (PA)], and individual tree size on the demographic processes of seed production were investigated in a heterodichogamous maple, Acer mono Maxim. var. Marmoratum (Nichols.) Hara f. dissectum, in a temperate forest of Japan. As the distance from conspecific reproductive adults increased, the percentage of immature seed fall and empty seeds increased significantly, indicating higher pollination success along with local population density. Although the difference was not distinct, pollination success was affected by the local population density of the reciprocal sex morph rather than that of both sex morphs. The trees at higher local population density sites suffered higher seed mortality due to predation and decay, and tended to produce smaller seeds. Thus, the impacts of local population density operated both positively and negatively on reproduction. As a factor of individual traits, tree size scarcely affected any demographic processes. On the other hand, sex morph did affect pollination success. Trees of PG type had lower immature seed fall than those of PA type, suggesting that the former has higher efficiency of pollen acceptance than the latter. The results on seed demography presented here partly support previous suggestions that heterodichogamous plants exhibit reciprocal cross-pollination and gender specialization as reproductive traits.     

Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling

MyD88 is a Toll/IL-1 receptor (TIR) domain-containing adapter common to signaling pathways via Toll-like receptor (TLR) family. However, accumulating evidence demonstrates the existence of a MyD88-independent pathway, which may explain unique biological responses of individual TLRs, particularly TLR3 and TLR4. TIR domain-containing adapter protein (TIRAP)/MyD88 adapter-like, a second adapter harboring the TIR domain, is essential for MyD88-dependent TLR2 and TLR4 signaling pathways, but not for MyD88-independent pathways. Here, we identified a novel TIR domain-containing molecule, named TIR domain-containing adapter inducing IFN-beta (TRIF). As is the case in MyD88 and TIRAP, overexpression of TRIF activated the NF-kappaB-dependent promoter. A dominant-negative form of TRIF inhibited TLR2-, TLR4-, and TLR7-dependent NF-kappaB activation. Furthermore, TRIF, but neither MyD88 nor TIRAP, activated the IFN-beta promoter. Dominant-negative TRIF inhibited TLR3-dependent activation of both the NF-kappaB-dependent and IFN-beta promoters. TRIF associated with TLR3 and IFN regulatory factor 3. These findings suggest that TRIF is involved in the TLR signaling, particularly in the MyD88-independent pathway.     

SUPERWOMAN1 and DROOPING LEAF genes control floral organ identity in rice

We analyzed recessive mutants of two homeotic genes in rice, SUPERWOMAN1 (SPW1) and DROOPING LEAF (DL). The homeotic mutation spw1 transforms stamens and lodicules into carpels and palea-like organs, respectively. Two spw1 alleles, spw1-1 and spw1-2, show the same floral phenotype and did not affect vegetative development. We show that SPW1 is a rice APETALA3 homolog, OsMADS16. In contrast, two strong alleles of the dl locus, drooping leaf-superman1 (dl-sup1) and drooping leaf-superman2 (dl-sup2), cause the complete transformation of the gynoecium into stamens. In these strong mutants, many ectopic stamens are formed in the region where the gynoecium is produced in the wild-type flower and they are arranged in a non-whorled, alternate pattern. The intermediate allele dl-1 (T65), results in an increase in the number of stamens and stigmas, and carpels occasionally show staminoid characteristics. In the weakest mutant, dl-2, most of the flowers are normal. All four dl alleles cause midrib-less drooping leaves. The flower of the double mutant, spw1 dl-sup, produces incompletely differentiated organs indefinitely after palea-like organs are produced in the position where lodicules are formed in the wild-type flower. These incompletely differentiated organs are neither stamens nor carpels, but have partial floral identity. Based on genetic and molecular results, we postulate a model of stamen and carpel specification in rice, with DL as a novel gene controlling carpel identity and acting mutually and antagonistically to the class B gene, SPW1.     

中枢神经细胞的高尔基复合体ACP活性分布的电镜观察

本文采用电镜金属盐法—酸性磷酸酶(ACP)细胞化学技术,用30mmol/L pipes缓冲液配制低浓度戊二醛进行固定。对成年大鼠的大脑大锥体细胞,小脑浦肯野氏细胞,脊髓前角运动细胞的高尔基复合体的ACP活性进行了实验研究和探讨。结果发现ACP活性分布在高尔基复合体的部份转移泡、浓缩泡及GERL部位。高尔基复合体呈ACP阳性反应,并显示出多种形态。     

Protoplast culture and plant regeneration of several species in the genus Dianthus

Summary Seventeen cultivars belonging to the genus Dianthus were examined for protoplast isolation, culture and shoot regeneration under the same conditions. These included D. caryophyllus, D. chinensis, D. barbatus, D. plumarius, D. superbus and D. japonicus as well as interspecific hybrid cultivars (D. caryophyllus x D. chinensis and D. chinensis x D. barbatus). In all cultivars, viable protoplasts were isolated at high yields from leaves of axenic shoot cultures and some of these protoplasts divided and formed colonies. However, shoot regeneration frequencies were markedly different among the species. High frequency shoot regeneration was obtained from D. chinensis and interspecific hybrid cultivars, while only low frequency or no shoot regeneration was obtained from other species.Abbreviations MS Murashige and Skoog (1962) - FW fresh weight - MES 2-N-morpholinoethane sulfonic acid - FDA fluoroscein diacetate - NAA 1-naphthaleneacetic acid     

Endospores of halophilic bacteria of the family Bacillaceae isolated from non-saline Japanese soil may be transported by Kosa event (Asian dust storm)

Background

Natural microbial communities are extremely complex and dynamic systems in terms of their population structure and functions. However, little is known about the in situ functions of the microbial communities.

Results

This study describes the application of proteomic approaches (metaproteomics) to observe expressed protein profiles of natural microbial communities (metaproteomes). The technique was validated using a constructed community and subsequently used to analyze Chesapeake Bay microbial community (0.2 to 3.0 μm) metaproteomes. Chesapeake Bay metaproteomes contained proteins from pI 4–8 with apparent molecular masses between 10–80 kDa. Replicated middle Bay metaproteomes shared ~92% of all detected spots, but only shared 30% and 70% of common protein spots with upper and lower Bay metaproteomes. MALDI-TOF analysis of highly expressed proteins produced no significant matches to known proteins. Three Chesapeake Bay proteins were tentatively identified by LC-MS/MS sequencing coupled with MS-BLAST searching. The proteins identified were of marine microbial origin and correlated with abundant Chesapeake Bay microbial lineages, Bacteroides and α-proteobacteria.

Conclusion

Our results represent the first metaproteomic study of aquatic microbial assemblages and demonstrate the potential of metaproteomic approaches to link metagenomic data, taxonomic diversity, functional diversity and biological processes in natural environments.     

Follistatin-derived peptide expression in muscle decreases adipose tissue mass and prevents hepatic steatosis

Myostatin, a member of the transforming growth factor (TGF)-β superfamily, plays a potent inhibitory role in regulating skeletal muscle mass. Inhibition of myostatin by gene disruption, transgenic (Tg) expression of myostatin propeptide, or injection of propeptide or myostatin antibodies causes a widespread increase in skeletal muscle mass. Several peptides, in addition to myostatin propeptide and myostatin antibodies, can bind directly to and neutralize the activity of myostatin. These include follistatin and follistatin-related gene. Overexpression of follistatin or follistatin-related gene in mice increased the muscle mass as in myostatin knockout mice. Follistatin binds to myostatin but also binds to and inhibits other members of the TGF-β superfamily, notably activins. Therefore, follistatin regulates both myostatin and activins in vivo. We previously reported the development and characterization of several follistatin-derived peptides, including FS I-I (Nakatani M, Takehara Y, Sugino H, Matsumoto M, Hashimoto O, Hasegawa Y, Murakami T, Uezumi A, Takeda S, Noji S, Sunada Y, Tsuchida K. FASEB J 22: 477-487, 2008). FS I-I retained myostatin-inhibitory activity without affecting the bioactivity of activins. Here, we found that inhibition of myostatin increases skeletal muscle mass and decreases fat accumulation in FS I-I Tg mice. FS I-I Tg mice also showed decreased fat accumulation even on a control diet. Interestingly, the adipocytes in FS I-I Tg mice were much smaller than those of wild-type mice. Furthermore, FS I-I Tg mice were resistant to high-fat diet-induced obesity and hepatic steatosis and had lower hepatic fatty acid levels and altered fatty acid composition compared with control mice. FS I-I Tg mice have improved glucose tolerance when placed on a high-fat diet. These data indicate that inhibiting myostatin with a follistatin-derived peptide provides a novel therapeutic option to decrease adipocyte size, prevent obesity and hepatic steatosis, and improve glucose tolerance.