Dual embryonic origin of the mammalian otic vesicle forming the inner ear

The inner ear and cochleovestibular ganglion (CVG) derive from a specialized region of head ectoderm termed the otic placode. During embryogenesis, the otic placode invaginates into the head to form the otic vesicle (OV), the primordium of the inner ear and CVG. Non-autonomous cell signaling from the hindbrain to the OV is required for inner ear morphogenesis and neurogenesis. In this study, we show that neuroepithelial cells (NECs), including neural crest cells (NCCs), can contribute directly to the OV from the neural tube. Using Wnt1-Cre, Pax3(Cre/+) and Hoxb1(Cre/+) mice to label and fate map cranial NEC lineages, we have demonstrated that cells from the neural tube incorporate into the otic epithelium after otic placode induction has occurred. Pax3(Cre/+) labeled a more extensive population of NEC derivatives in the OV than did Wnt1-Cre. NEC derivatives constitute a significant population of the OV and, moreover, are regionalized specifically to proneurosensory domains. Descendents of Pax3(Cre/+) and Wnt1-Cre labeled cells are localized within sensory epithelia of the saccule, utricle and cochlea throughout development and into adulthood, where they differentiate into hair cells and supporting cells. Some NEC derivatives give rise to neuroblasts in the OV and CVG, in addition to their known contribution to glial cells. This study defines a dual cellular origin of the inner ear from sensory placode ectoderm and NECs, and changes the current paradigm of inner ear neurosensory development.     

An incoming nucleotide imposes an anti to syn conformational change on the templating purine in the human DNA polymerase-iota active site

Substrate-induced conformational change of the protein is the linchpin of enzymatic reactions. Replicative DNA polymerases, for example, convert from an open to a closed conformation in response to dNTP binding. Human DNA polymerase-iota (hPoliota), a member of the Y family of DNA polymerases, differs strikingly from other polymerases in its much higher proficiency and fidelity for nucleotide incorporation opposite template purines than opposite template pyrimidines. We present here a crystallographic analysis of hPoliota binary complexes, which together with the ternary complexes show that, contrary to replicative DNA polymerases, the DNA, and not the polymerase, undergoes the primary substrate-induced conformational change. The incoming dNTP "pushes" templates A and G from the anti to the syn conformation dictated by a rigid hPoliota active site. Together, the structures posit a mechanism for template selection wherein dNTP binding induces a conformational switch in template purines for productive Hoogsteen base pairing.     

Glucocorticoid receptor-like Zn(Cys)4 motifs in BslI restriction endonuclease

BslI restriction endonuclease cleaves the symmetric sequence CCN(7)GG (where N=A, C, G or T). The enzyme is composed of two subunits, alpha and beta, that form a heterotetramer (alpha(2)beta(2)) in solution. The alpha subunit is believed to be responsible for DNA recognition, while the beta subunit is thought to mediate cleavage. Here, for the first time, we provide experimental evidence that BslI binds Zn(II). Specifically, using X-ray absorption spectroscopic analysis we show that the alpha subunit of BslI contains two Zn(Cys)(4)-type zinc motifs similar to those in the DNA-binding domain of the glucocorticoid receptor. This conclusion is supported by genetic analysis of the zinc-binding motifs, whereby amino acid substitutions in the zinc finger motifs are demonstrated to abolish or impair cleavage activity. An additional putative zinc-binding motif was identified in the beta subunit, consistent with the X-ray absorption data. These data were corroborated by proton induced X-ray emission measurements showing that full BslI contains at least three fully occupied Zn sites per alpha/beta heterodimer. On the basis of these data, we propose a role for the BslI Zn motifs in protein-DNA as well as protein-protein interactions.     

Widespread occurrence of the homologues of the early nodulin (ENOD) genes in Oryza species and related grasses

Eighty accessions representing 23 species from the genus Oryza were examined for the presence of homologues of early nodulin (ENOD) genes. Southern analyses indicated a widespread distribution of homologues of ENOD genes across all the genomes of rice as well as other monocots. The degree of cross-hybridization of the legume ENOD genes with sequences in the genomes of various species, as revealed by hybridization differentials measured in terms of signal intensities, however, suggests that the homologues of ENOD genes are conserved to varied extents in different Oryza species. The presence of homologues of ENOD genes in a wide variety of plant species denotes that the biological functions of early nodulins may be diverse, and not restricted to nodule organogenesis alone. The fact that ENOD gene homologues exist widely both in dicots and monocots provides evidence that these homologues have arisen from a common ancestral plant.     

Energetic and structural considerations for the mechanism of protein sliding along DNA in the nonspecific BamHI-DNA complex

The molecular mechanism by which DNA-binding proteins find their specific binding sites is still unclear. To gain insights into structural and energetic elements of this mechanism, we used the crystal structure of the nonspecific BamHI-DNA complex as a template to study the dominant electrostatic interaction in the nonspecific association of protein with DNA, and the possible sliding pathways that could be sustained by such an interaction. Based on calculations using the nonlinear Poisson-Boltzmann method and Brownian dynamics, a model is proposed for the initial nonspecific binding of BamHI to B-form DNA that differs from that seen in the crystal structure of the nonspecific complex. The model is electrostatically favorable, and the salt dependence as well as other thermodynamic parameters calculated for this model are in good agreement with experimental results. Several residues in BamHI are identified for their important contribution to the energy in the nonspecific binding model, and specific mutagenesis experiments are proposed to test the model on this basis. We show that a favorable sliding pathway of the protein along DNA is helical.     

Cloning and sequencing of complete tau-crystallin cDNA from embryonic lens of Crocodylus palustris

τ-Crystallin is a taxon-specific structural protein found in eye lenses. We present here the cloning and sequencing of complete τ-crystallin cDNA from the embryonic lens ofCrocodylus palustris and establish it to be identical to the α-enolase gene from non-lenticular tissues. Quantitatively, the τ-crystallin was found to be the least abundant crystallin of the crocodilian embryonic lenses. Crocodile τ-crystallin cDNA was isolated by RT-PCR using primers designed from the only other reported sequence from duck and completed by 5′- and 3′-rapid amplification of cDNA ends (RACE) using crocodile gene specific primers designed in the study. The complete τ-crystallin cDNA of crocodile comprises 1305 bp long ORF and 92 and 409 bp long untranslated 5′- and 3′-ends respectively. Further, it was found to be identical to its putative counterpart enzyme α-enolase, from brain, heart and gonad, suggesting both to be the product of the same gene. The study thus provides the first report on cDNA sequence of τ-crystallin from a reptilian species and also re-confirms it to be an example of the phenomenon of gene sharing as was demonstrated earlier in the case of peking duck. Moreover, the gene lineage reconstruction analysis helps our understanding of the evolution of crocodilians and avian species.     

Transcription corepressor CtBP is an NAD(+)-regulated dehydrogenase

The Lcd1p/Mec1p complex is crucial for normal S phase progression and for signaling DNA damage. We show that Lcd1p/Ddc2p and Mec1p in cell extracts bind to DNA ends. Although Lcd1p binds DNA independently of Mec1p, recruitment of Mec1p to DNA requires Lcd1p. DNA binding by Lcd1p is also independent of Rad9p, Rad17p, and Rad24p. Recombinant Lcd1p binds DNA, and this is impaired by Lcd1p mutations that abrogate its in vivo functions. Furthermore, Mec1p is recruited to cdc13-induced DNA damage and HO endonuclease-induced double-strand breaks in vivo. This requires Lcd1p, and recruitment of Lcd1p/Mec1p to cdc13-induced damage is abolished by Lcd1p mutations that abrogate its in vivo functions. Recruitment of Lcd1p to these lesions is independent of Mec1p and Rad9p/Rad24p. Thus, recruitment of Mec1p to DNA lesions by Lcd1p is crucial for the DNA damage response.     

Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering—A Comparison between Wild-Type Protein and a Hinge Mutant

Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS). The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations.     

Chemodiversity and α-Glucosidase Activity of Eucalyptus Species from Northwestern Himalaya,India

The aim of current work was to determine essential oils (EOs) composition from three Eucalyptus species, including E. citriodora, E. camaldulensis and E. globulus and assess their α-glucosidase inhibitory activity. The EOs were collected using the hydrodistillation technique and characterized by GC/MS, GC-FID and NMR. The isolated EOs from leaves parts of Eucalyptus species varied from 0.56 to 1.0 % on fresh weight basis. The content of the EOs was distinct according to the species. The most abundant metabolites were identified as citronellal (0–83.0 %), 1,8-cineole (0.2–44.8 %), spathulenol (0.4–16.1 %) α-pinene (0.4–15.9 %), p-cymene (3.7–11.9 %), citronellol (0–8.6 %), β-eudesmol (5.3–8.6 %) and β-pinene (0–7.1 %). The EOs obtained from targeted samples exhibited strong α-glucosidase inhibitory activity. These results are encouraging and underline that the EOs of Eucalyptus species may be a promising alternative source of natural antidiabetic.     

Induction of Krüppel‐like factor 2 reduces K/BxN serum‐induced arthritis

Krüppel‐like factor 2 (KLF2) critically regulates activation and function of monocyte, which plays important pathogenic role in progressive joint destruction in rheumatoid arthritis (RA). It is yet to be established the molecular basis of KLF2‐mediated regulation of monocytes in RA pathogenesis. Herein, we show that a class of compound, HDAC inhibitors (HDACi) induced KLF2 expression in monocytes both in vitro and in vivo. KLF2 level was also elevated in tissues, such as bone marrow, spleen and thymus in mice after infusion of HDACi. Importantly, HDACi significantly reduced osteoclastic differentiation of monocytes with the up‐regulation of KLF2 and concomitant down‐regulation of matrixmetalloproteinases both in the expression level as well as in the protein level. In addition, HDACi reduced K/BxN serum‐induced arthritic inflammation and joint destruction in mice in a dose‐dependent manner. Finally, co‐immunoprecipitation and overexpression studies confirmed that KLF2 directly interacts with HDAC4 molecule in cells. These findings provide mechanistic evidence of KLF2‐mediated regulation of K/BxN serum‐induced arthritic inflammation.