Faculty Bibliography

n-Dodecyl-β-D-maltoside solubilized glucuronide transporter (GusB), the product of gusB gene from Escherichia coli, was treated with Bio-Beads as an agent for removing the detergent from a micellar solution under suitable combination with dimyristoylphosphatidylcholine. Optimizing conditions led to a two-dimensional crystalline array formation of GusB. The crystalline arrays appear to have a hexagonal lattice with layer group P6, the unit cell dimensions of a = b = 13.8 nm and γ = 120°. Each stain-protruding periodic unit showed approximately 11.8 ± 0.3 nm in a diameter in the inverse Fourier-filtered image to have formed with pentameric GusB (5 × 49.7 kDa).

When ether vapor was allowed to diffuse into a CH(2)Cl(2) solution of an enantiomer of a hexa-peri-hexabenzocoronene (HBC) derivative carrying a chiral (BINAP)Pt(II)-appended coordination metallacycle (HBC(Py)([(R)-Pt]) or HBC(Py)([(S)-Pt])), screw-sense-selective assembly took place to give optically active nanotubes (NT(Py)([(R)-Pt]) or NT(Py)([(S)-Pt])) with helical chirality, which were enriched in either left-handed (M)-NT(Py)([(R)-Pt]) or right-handed (P)-NT(Py)([(S)-Pt]), depending on the absolute configuration of the (BINAP)Pt(II) pendant. When MeOH was used instead of ether for the vapor-diffusion-induced assembly, nanocoils formed along with the nanotubes. As determined by scanning electron microscopy, the diastereomeric excess of the nanocoils was 60% (80:20 diastereomeric ratio). Removal of the (BINAP)Pt(II) pendants from NT(Py)([(R)-Pt]) or NT(Py)([(S)-Pt]) with ethylenediamine yielded metal-free nanotubes (NT(Py)) that remained optically active even upon heating without any change in the circular dichroism spectral profile. No helical inversion took place when NT(Py) derived from HBC(Py)([(R)-Pt]) or HBC(Py)([(S)-Pt]) was allowed to complex with (BINAP)Pt(II) with an absolute configuration opposite to the original one.

BACKGROUND:The recent morphological studies on chaperonins have revealed that nearly equivalent amount of symmetric GroEL-(GroES)2 (football-shaped) and asymmetric GroEL-GroES (bullet-shaped) complexes coexist during the chaperonin reaction cycle, which prompted us to reexamine the equatorial split observed for chaperonin from Thermus thermophilus by implementing semi-empirical molecular orbital (MO) calculations, since it is now believed that the symmetric formation is a precursor to the equatorial split. METHODS:Semi-empirical MO calculations were employed to investigate the intersubunit interactions within the bullet-shaped T. thermophilus chaperonin capturing the substrate of folding intermediates. Interaction energies between each cis-GroEL subunit and closely related remaining subunits in cis-GroEL ring, or in trans-GroEL ring across the equatorial plane, and further, interaction energies between each GroES subunit and adjacent subunits in the same GroES ring and in cis-GroEL ring were simulated. RESULTS:Anisotropic intensities and energy distribution of the subunits were revealed by the calculations, which are consistent with two conformations of the subunits forming cis-GroEL ring as revealed by X-ray crystal structure, and with an anisotropic critical binding site on cis-GroEL ring for chaperonin functioning. CONCLUSIONS:This is the first application of semi-empirical MO calculations to the macromolecular complex of the native bullet-shaped chaperonin (GroEL-GroES-ADP homolog) from T. thermophilus. GENERAL SIGNIFICANCE/CONCLUSIONS:The results also appear to support the occurrence of the equatorial split for T. thermophilus chaperonin observed via electron microscopy, but has not yet been fully observed for Escherichia coli GroEL-GroES system.

The development of nanocarriers that selectively release guest molecules on sensing a particular biological signal is being actively pursued in nanomedicine for diagnostic and therapeutic purposes. Here we report a protein-based nanocarrier that opens in the presence of intracellular adenosine-5'-triphosphate (ATP). The nanocarrier consists of multiple barrel-shaped chaperonin units assembled through coordination with Mg(2+) into a tubular structure that protects guest molecules against biological degradation. When its surface is functionalized with a boronic acid derivative, the nanocarrier is able to enter cells. The hydrolysis of intracellular ATP into adenosine-5'-diphosphate (ADP) induces conformational changes of the chaperonin units, which in turns generate a mechanical force that leads to the disassembly of the tube and release of the guests. This scission occurs with a sigmoidal dependence on ATP concentration, which means that the nanocarrier can differentiate biological environments in terms of the concentration of ATP for selective guest release. Furthermore, biodistribution tests reveal preferential accumulation of the nanocarriers in a tumour tissue.

Spermatogenesis is a complex process that generates haploid germ cells or spores and implements meiosis, a succession of two special cell divisions that are required for homologous chromosome segregation. During prophase to the first meiotic division, homologous recombination (HR) repairs Spo11-dependent DNA double-strand breaks (DSBs) in the presence of telomere movements to allow for chromosome pairing and segregation at the meiosis I division. In contrast to HR, non-homologous end joining (NHEJ), the major DSB repair mechanism during the G1 cell cycle phase, is downregulated during early meiotic prophase. At somatic mammalian telomeres, the NHEJ factor Ku70/80 inhibits HR, as does the Rap1 component of the shelterin complex. Here, we investigated the role of Ku70 and Rap1 in meiotic telomere redistribution and genome protection in spermatogenesis by studying single and double knockout mice. Ku70(-/-) mice display reduced testis size and compromised spermatogenesis, whereas meiotic telomere dynamics and chromosomal bouquet formation occurred normally in Ku70(-/-) and Ku70(-/-)Rap1(Delta/Delta) knockout spermatocytes. Elevated mid-preleptotene frequencies were associated with significantly increased DNA damage in Ku-deficient B spermatogonia, and in differentiated Sertoli cells. Significantly elevated levels of gammaH2AX foci in Ku70(-/-) diplotene spermatocytes suggest compromised progression of DNA repair at a subset of DSBs. This might explain the elevated meiotic metaphase apoptosis that is present in Ku70-deficient stage XII testis tubules, indicating spindle assembly checkpoint activation. In summary, our data indicate that Ku70 is important for repairing DSBs in somatic cells and in late spermatocytes of the testis, thereby assuring the fidelity of spermatogenesis.

The Sonic Hedgehog (Shh) pathway is responsible for critical patterning events early in development and for regulating the delicate balance between proliferation and differentiation in the developing and adult vertebrate brain. Currently, our knowledge of the potential role of Shh in regulating neural stem cells (NSC) is largely derived from analyses of the mammalian forebrain, but for dorsal midbrain development it is mostly unknown. For a detailed understanding of the role of Shh pathway for midbrain development in vivo, we took advantage of mouse embryos with cell autonomously activated Hedgehog (Hh) signaling in a conditional Patched 1 (Ptc1) mutant mouse model. This animal model shows an extensive embryonic tectal hypertrophy as a result of Hh pathway activation. In order to reveal the cellular and molecular origin of this in vivo phenotype, we established a novel culture system to evaluate neurospheres (nsps) viability, proliferation and differentiation. By recreating the three-dimensional (3-D) microenvironment we highlight the pivotal role of endogenous Shh in maintaining the stem cell potential of tectal radial glial cells (RGC) and progenitors by modulating their Ptc1 expression. We demonstrate that during late embryogenesis Shh enhances proliferation of NSC, whereas blockage of endogenous Shh signaling using cyclopamine, a potent Hh pathway inhibitor, produces the opposite effect. We propose that canonical Shh signaling plays a central role in the control of NSC behavior in the developing dorsal midbrain by acting as a niche factor by partially mediating the response of NSC to epidermal growth factor (EGF) and fibroblast growth factor (FGF) signaling. We conclude that endogenous Shh signaling is a critical mechanism regulating the proliferation of stem cell lineages in the embryonic dorsal tissue.

Sonic Hedgehog (Shh/GLI) and EGFR signaling pathways modulate Neural Stem Cell (NSC) proliferation. How these signals cooperate is therefore critical for understanding normal brain development and function. Here we report a novel acute effect of Shh signaling on EGFR function. We show that during late neocortex development, Shh mediates the activation of the ERK1/2 signaling pathway in Radial Glial cells (RGC) through EGFR transactivation. This process is dependent on metalloprotease activity and accounts for almost 50% of the EGFR-dependent mitogenic response of late NSCs. Furthermore, in HeLa cancer cells, a well-known model for studying the EGFR receptor function, Shh also induces cell proliferation involving EGFR activation, as reflected by EGFR internalization and ERK1/2 phosphorylation. These findings may have important implications for understanding the mechanisms that regulate NSC proliferation during neurogenesis and may lead to novel approaches to the treatment of tumors.

Parathyroid Hormone related Protein (PTHrP) is a critical regulator of mammary gland morphogenesis in the mouse embryo. Loss of PTHrP, or its receptor, PTHR1, results in arrested mammary buds at day 15 of embryonic development (E15). In contrast, overexpression of PTHrP converts the ventral epidermis into hairless nipple skin. PTHrP signaling appears to be critical for mammary mesenchyme specification, which in turn maintains mammary epithelial identity, directs bud outgrowth, disrupts the male mammary rudiment and specifies the formation of the nipple. In the embryonic mammary bud, PTHrP exerts its effects on morphogenesis, in part, through epithelial-stromal crosstalk mediated by Wnt and BMP signaling. Recently, PTHLH has been identified as a strong candidate for a novel breast cancer susceptibility locus, although PTHrP's role in breast cancer has not been clearly defined. The effects of PTHrP on the growth of the embryonic mammary rudiment and its invasion into the dermis may, in turn, have connections to the role of PTHrP in breast cancer.