Factors that bind to adeno-associated virus terminal repeats.

We have identified and characterized a DNA-protein complex that forms with the adeno-associated virus (AAV) terminal repeats. The complex formed only if the terminal palindrome was in the covalently closed or hairpin configuration; little if any binding was detected with the open duplex form of the terminal repeat. This fact suggested that both secondary structure and primary sequence are essential elements of recognition. DNase I protection studies indicated that virtually all of the A-A' palindrome and significant portions of the B-B' and C-C' palindromes are protected. The postulated terminal resolution site of AAV also is protected. Restriction mapping of the sequences necessary for binding indicated that almost all of the terminal palindrome must be present for binding to occur. Hairpins which are similar in size and shape to the AAV termini did not exhibit competition for binding, and the complex formed only if AAV-infected extracts were used. Thus, the binding reaction is specific for AAV sequences. The viral-coded nonstructural proteins Rep78 and Rep68 comigrated with the DNA-protein complex on neutral acrylamide gels, suggesting that one or both of these proteins are components of the complex. The characteristics of the complex suggested that it has a role in AAV DNA replication.     

Mercury-tolerant Transgenic Poplars Expressing Two Bacterial Mercury-metabolizing Genes

Mercury is one of the most toxic metals to various organisms, including humans. Genes involved in mercury metabolism have been cloned fromStaphylococcus aureus, and were modified here to be expressed in plants. Transgenic poplars containing both chimeric genes (p35S-merA andp35S-merB) were developed via two rounds of transformation usingnos-nptll andnos-hpt genes as selectable markers. Although expression levels varied among transgenic lines, tolerance to either ionic mercury or organic mercury matched well with the degree of expression revealed by northern hybridization. In culture, these trees were tolerant to 50 μM HgCl₂ and 2 μM CH₃HgCI. Variations in mercury tolerance among the transgenic lines indicates that vigorous selection is required to select the best clones for use in phytoremediation.     

Expression of CD1d molecules by human schwann cells and potential interactions with immunoregulatory invariant NK T cells

CD1d-restricted NKT cells expressing invariant TCR alpha-chains (iNKT cells) produce both proinflammatory and anti-inflammatory cytokines rapidly upon activation, and are believed to play an important role in both host defense and immunoregulation. To address the potential implications of iNKT cell responses for infectious or inflammatory diseases of the nervous system, we investigated the expression of CD1d in human peripheral nerve. We found that CD1d was expressed on the surface of Schwann cells in situ and on primary or immortalized Schwann cell lines in culture. Schwann cells activated iNKT cells in a CD1d-dependent manner in the presence of alpha-galactosylceramide. Surprisingly, the cytokine production of iNKT cells stimulated by alpha-galactosylceramide presented by CD1d+ Schwann cells showed a predominance of Th2-associated cytokines such as IL-5 and IL-13 with a marked deficiency of proinflammatory Th1 cytokines such as IFN-gamma or TNF-alpha. Our findings suggest a mechanism by which iNKT cells may restrain inflammatory responses in peripheral nerves, and raise the possibility that the expression of CD1d by Schwann cells could be relevant in the pathogenesis of infectious and inflammatory diseases of the peripheral nervous system.     

The Role of Interlayer Chemistry in Li‐Metal Growth through a Garnet‐Type Solid Electrolyte

Securing the chemical and physical stabilities of electrode/solid‐electrolyte interfaces is crucial for the use of solid electrolytes in all‐solid‐state batteries. Directly probing these interfaces during electrochemical reactions would significantly enrich the mechanistic understanding and inspire potential solutions for their regulation. Herein, the electrochemistry of the lithium/Li₇La₃Zr₂O₁₂‐electrolyte interface is elucidated by probing lithium deposition through the electrolyte in an anode‐free solid‐state battery in real time. Lithium plating is strongly affected by the geometry of the garnet‐type Li₇La₃Zr₂O₁₂ (LLZO) surface, where nonuniform/filamentary growth is triggered particularly at morphological defects. More importantly, lithium‐growth behavior significantly changes when the LLZO surface is modified with an artificial interlayer to produce regulated lithium depositions. It is shown that lithium‐growth kinetics critically depend on the nature of the interlayer species, leading to distinct lithium‐deposition morphologies. Subsequently, the dynamic role of the interlayer in battery operation is discussed as a buffer and seed layer for lithium redistribution and precipitation, respectively, in tailoring lithium deposition. These findings broaden the understanding of the electrochemical lithium‐plating process at the solid‐electrolyte/lithium interface, highlight the importance of exploring various interlayers as a new avenue for regulating the lithium‐metal anode, and also offer insight into the nature of lithium growth in anode‐free solid‐state batteries.     

Mutually constructive roles of Ail and LPS in Yersinia pestis serum survival

The outer membrane is a key virulence determinant of gram-negative bacteria. In Yersinia pestis, the deadly agent that causes plague, the protein Ail and lipopolysaccharide (LPS)⁶ enhance lethality by promoting resistance to human innate immunity and antibiotics, enabling bacteria to proliferate in the human host. Their functions are highly coordinated. Here we describe how they cooperate to promote pathogenesis. Using a multidisciplinary approach, we identify mutually constructive interactions between Ail and LPS that produce an extended conformation of Ail at the membrane surface, cause thickening and rigidification of the LPS membrane, and collectively promote Y. pestis survival in human serum, antibiotic resistance, and cell envelope integrity. The results highlight the importance of the Ail–LPS assembly as an organized whole, rather than its individual components, and provide a handle for targeting Y. pestis pathogenesis.     

Predicting permanent and transient protein–protein interfaces

Protein–protein interactions (PPIs) are involved in diverse functions in a cell. To optimize functional roles of interactions, proteins interact with a spectrum of binding affinities. Interactions are conventionally classified into permanent and transient, where the former denotes tight binding between proteins that result in strong complexes, whereas the latter compose of relatively weak interactions that can dissociate after binding to regulate functional activity at specific time point. Knowing the type of interactions has significant implications for understanding the nature and function of PPIs. In this study, we constructed amino acid substitution models that capture mutation patterns at permanent and transient type of protein interfaces, which were found to be different with statistical significance. Using the substitution models, we developed a novel computational method that predicts permanent and transient protein binding interfaces (PBIs) in protein surfaces. Without knowledge of the interacting partner, the method uses a single query protein structure and a multiple sequence alignment of the sequence family. Using a large dataset of permanent and transient proteins, we show that our method, BindML+, performs very well in protein interface classification. A very high area under the curve (AUC) value of 0.957 was observed when predicted protein binding sites were classified. Remarkably, near prefect accuracy was achieved with an AUC of 0.991 when actual binding sites were classified. The developed method will be also useful for protein design of permanent and transient PBIs. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Variation of acharan sulfate and monosaccharide composition and analysis of neutral <Emphasis Type="Italic">N</Emphasis>-glycans in African giant snail (<Emphasis Type="Italic">Achatina fulica</Emphasis>)

Acharan sulfate content from African giant snail (Achatina fulica) was compared in eggs and snails of different ages. Acharan sulfate was not found in egg. Acharan sulfate disaccharide →4)-α-d-GlcNpAc (1→4)-α-l-IdoAp2S(1→, analyzed by SAX (strong-anion exchange)–HPLC was observed soon after hatching and increases as the snails grow. Monosaccharide compositional analysis showed that mole % of glucosamine, a major monosaccharide of acharan sulfate, increased with age while mole % of galactose decreased with age. These results suggest that galactans represent a major energy source during development, while acharan sulfate appearing immediately after hatching, is essential for the snail growth. The structures of neutral N-glycans released from eggs by peptide N-glycosidase F (PNGase F), were next elucidated using ESI-MS/MS, MALDI-MS/MS, enzyme digestion, and monosaccharide composition analysis. Three types of neutral N-glycan structures were observed, truncated (Hex_2–4-HexNAc₂), high mannose (Hex_5–9-HexNAc₂), and complex (Hex₃-HexNAc_2–10) types. None showed core fucosylation.     

Small molecules that recapitulate the early steps of urodele amphibian limb regeneration and confer multipotency

In urodele amphibians, an early step in limb regeneration is skeletal muscle fiber dedifferentiation into a cellulate that proliferates to contribute new limb tissue. However, mammalian muscle cannot dedifferentiate after injury. We have developed a novel, small-molecule-based method to induce dedifferentiation in mammalian skeletal muscle. Muscle cellularization was induced by the small molecule myoseverin. Candidate small molecules were tested for the induction of proliferation in the cellulate. We observed that treatment with the small molecules BIO (glycogen synthase-3 kinase inhibitor), lysophosphatidic acid (pleiotropic activator of G-protein-coupled receptors), SB203580 (p38 MAP kinase inhibitor), or SQ22536 (adenylyl cyclase inhibitor) induced proliferation. Moreover, these proliferating cells were multipotent, as confirmed by the chemical induction of mesodermal-derived cell lineages. Microarray analysis showed that the multipotent, BIO-treated cellulate possessed a markedly different gene expression pattern than lineage-restricted C2C12 myoblasts, especially for genes related to signal transduction and differentiation. Sequential small molecule treatment of the muscle cellulate with BIO, SB203580, or SQ22536 and the aurora B kinase inhibitor, reversine, induced the formation of cells with neurogenic potential (ectodermal lineage), indicating the acquirement of pluripotency. This is the first demonstration of a small molecule method that induces mammalian muscle to undergo dedifferentiation and rededifferentiation into alternate cell lineages. This method induces dedifferentiation in a simple, stepwise approach and has therapeutic potential to enhance tissue regeneration in mammals.