Cooperation between the chloroplast psbA 5′‐untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region

Chloroplast mRNA translation is regulated by the 5′‐untranslated region (5′‐UTR). Chloroplast 5′‐UTRs also support translation of the coding regions of heterologous genes. Using an in vitro translation system from tobacco chloroplasts, we detected no translation from a human immunodeficiency virus tat coding region fused directly to the tobacco chloroplast psbA 5′‐UTR. This lack of apparent translation could have been due to rapid degradation of mRNA templates or synthesized protein products. Replacing the psbA 5′‐UTR with the E. coli phage T7 gene 10 5′‐UTR, a highly active 5′‐UTR, and substituting synonymous codons led to some translation of the tat coding region. The Tat protein thus synthesized was stable during translation reactions. No significant degradation of the added tat mRNAs was observed after translation reactions. These results excluded the above two possibilities and confirmed that the tat coding region prevented its own translation. The tat coding region was then fused to the psbA 5′‐UTR with a cognate 5′‐coding segment. Significant translation was detected from the tat coding region when fused after 10 or more codons. That is, translation could be initiated from the tat coding region once translation had started, indicating that the tat coding region inhibits translational initiation but not elongation. Hence, cooperation/compatibility between the 5′‐UTR and its coding region is important for translational initiation.     

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.     

Determination of the structural ensemble of the molten globule state of a protein by computer simulations

We have used computer simulations to investigate the structural nature of the molten globule (MG) state of canine milk lysozyme. To sample the conformational space efficiently, we performed replica-exchange umbrella sampling simulations with the radius of gyration as a reaction coordinate. We applied the Weighted Histogram Analysis Method to the trajectory of the simulations to obtain the potential of mean force, from which we identified representative structures corresponding to local minima in the free energy surface. The representative structures obtained in this way are in accord with the characteristics of the MG state reported previously by experimental studies. We conjecture that the MG state comprises a series of partially structured states undergoing relatively fast conformational interchange.     

The Role of Sonic Hedgehog Signaling in Osteoclastogenesis and Jaw Bone Destruction

Sonic hedgehog (SHH) and its signaling have been identified in several human cancers, and increased levels of its expression appear to correlate with disease progression and metastasis. However, the role of SHH in bone destruction associated with oral squamous cell carcinomas is still unclear. In this study we analyzed SHH expression and the role played by SHH signaling in gingival carcinoma-induced jawbone destruction. From an analysis of surgically resected lower gingival squamous cell carcinoma mandible samples, we found that SHH was highly expressed in tumor cells that had invaded the bone matrix. On the other hand, the hedgehog receptor Patched and the signaling molecule Gli-2 were highly expressed in the osteoclasts and the progenitor cells. SHH stimulated osteoclast formation and pit formation in the presence of the receptor activator for nuclear factor-κB ligand (RANKL) in CD11b⁺ mouse bone marrow cells. SHH upregulated phosphorylation of ERK1/2 and p38 MAPK, NFATc1, tartrate-resistant acid phosphatase (TRAP), and Cathepsin K expression in RAW264.7 cells. Our results suggest that tumor-derived SHH stimulated the osteoclast formation and bone resorption in the tumor jawbone microenvironment.     

The association between the phytoplankton, Rhopalosolen species (Chlorophyta; Chlorophyceae), and Anopheles gambiae sensu lato (Diptera: Culicidae) larval abundance in western Kenya

Algae are important food resources of the larvae of the African malaria vectors, Anopheles gambiae Giles and Anopheles arabiensis Patton (Anopheles gambiae sensu lato), and other zooplankton, but empirical evidence remains meager about the agal flora in ephemeral water bodies. The animals present in natural aquatic habitats in western Kenya were sampled from July to November 2002 to study abiotic and biotic environmental factors determining A. gambiae sl larval abundance. The five highest concentrations of third and fourth instars and pupae (hereafter referred to as old-stage larvae) were sampled in conjunction with the unicellular epizoic green algae, Rhopalosolen species (Chlorophyta; Chlorophyceae). Canonical correspondence analysis revealed that the presence of Rhopalosolen species was the most important determinant of the animal assemblage. The density of old-stage A. gambiae sl larvae was positively correlated with the presence of Rhopalosolen species, but the density of first and second instars of A. gambiae sl was not. The water bodies with Rhopalosolen sp. yielded larger mosquitoes in spite of the higher density of larvae. We demonstrated that the productivity of water bodies in terms of the larvae of malaria vectors can differ in magnitude depending on the agal flora. We discuss phytoplankton as a regulator of mosquito larval populations.     

43Ca NMR studies of Ca2+-Tetrahymena calmodulin complexes

⁴³Ca NMR spectra of Ca²⁺-Tetrahymena calmodulin(Tet. CaM.) complexes have been observed under various conditions. Off-rate of Ca²⁺ from Tet. CaM. is estimated to be approx. 2.7 × 10³ s^?1 under a certain assumption. Relaxation rates of ⁴³Ca NMR of Ca²⁺-Tet. CaM. are remarkably increased(by one order in magnitude) by adding trifluoperazine(TFP), a potent calmodulin antagonist. Relaxation parameters estimated suggest that Ca²⁺ mobility is reduced by the TFP binding. A stoichiometry of TFP is two moles per Tet. CaM. molecule. The relaxation rates of ⁴³Ca NMR signals are increased by adding excessive Mg²⁺ to the Ca²⁺-Tet. CaM. solutions. The addition of Mg²⁺ to the Ca²⁺-Tet. CaM. complex decreases apparent pKa value of the complex as well.