Faculty Bibliography

An 11.7-A-resolution cryo-EM map of the yeast 80S.eEF2 complex in the presence of the antibiotic sordarin was interpreted in molecular terms, revealing large conformational changes within eEF2 and the 80S ribosome, including a rearrangement of the functionally important ribosomal intersubunit bridges. Sordarin positions domain III of eEF2 so that it can interact with the sarcin-ricin loop of 25S rRNA and protein rpS23 (S12p). This particular conformation explains the inhibitory action of sordarin and suggests that eEF2 is stalled on the 80S ribosome in a conformation that has similarities with the GTPase activation state. A ratchet-like subunit rearrangement (RSR) occurs in the 80S.eEF2.sordarin complex that, in contrast to Escherichia coli 70S ribosomes, is also present in vacant 80S ribosomes. A model is suggested, according to which the RSR is part of a mechanism for moving the tRNAs during the translocation reaction

Expression of telomerase activity presumably is involved in maintaining self-replication and the undifferentiated state of stem cells. Adult mouse bone marrow mesenchymal stem cells (mMSCs) are multipotential cells capable of differentiating into a variety of lineage cell types, including adipocytes and chondrocytes. Here we show that the lacking telomerase of mMSC lose multipotency and the capacity to differentiate. Primary cultures of mMSCs were obtained from both telomerase knockout (mTR(-/-)) and wild-type (WT) mice. The MSCs isolated from mTR(-/-) mice failed to differentiate into adipocytes and chondrocytes, even at early passages, whereas WT MSCs were capable of differentiation. Consistent with other cell types, late passages mTR(-/-)MSCs underwent senescence and were accompanied by telomere loss and chromosomal end-to-end fusions. These results suggest that in addition to its known role in cell replication, telomerase is required for differentiation of mMSCs in vitro. This work may be significant for further potentiating adult stem cells for use in tissue engineering and gene therapy and for understanding the significance of telomerase expression in the process of cell differentiation

The hippocampus is critical for formation of spatial memories. Hippocampal pyramidal neurons in freely behaving animals exhibit spatially selective firing patterns, which taken together form an internal representation of the environment. This representation is thought to contribute to the hippocampal spatial memory system. Behavioral long-term memories differ from short-term memories in requiring the synthesis of new proteins. Does the development of the internal hippocampal representation also require the synthesis of new proteins? We found that blocking protein synthesis in the brain of mice by 95% does not affect short-term stability of newly formed hippocampal place fields but abolishes stability in the long term. By contrast, inhibiting protein synthesis does not affect the retention and recall of previously established fields in a familiar environment, indicating that protein synthesis-dependent reconsolidation is not required for recall. Our results indicate that place fields parallel both behavioral memories and the late phase of long-term potentiation in requiring the synthesis of new proteins for consolidation.

In both animals and plants, many developmentally important regulatory genes have complementary microRNAs (miRNAs), which suggests that these miRNAs constitute a class of developmental signalling molecules. Leaves of higher plants exhibit a varying degree of asymmetry along the adaxial/abaxial (upper/lower) axis. This asymmetry is specified through the polarized expression of class III homeodomain/leucine zipper (HD-ZIPIII) genes. In Arabidopsis, three such genes, PHABULOSA (PHB), PHAVOLUTA (PHV) and REVOLUTA (REV), are expressed throughout the incipient leaf, but become adaxially localized after primordium emergence. Downregulation of the HD-ZIPIII genes allows expression of the KANADI and YABBY genes, which specify abaxial fate. PHB, PHV and REV transcripts contain a complementary site for miRNA165 and miRNA166, which can direct their cleavage in vitro. Here we show that miRNA166 constitutes a highly conserved polarizing signal whose expression pattern spatially defines the expression domain of the maize hd-zipIII family member rolled leaf1 (rld1). Moreover, the progressively expanding expression pattern of miRNA166 during leaf development and its accumulation in phloem suggests that miRNA166 may form a movable signal that emanates from a signalling centre below the incipient leaf.

Vpu is an 81-residue integral membrane protein encoded in the HIV-1 genome that is of considerable interest because it plays important roles in the release of virus particles from infected cells and in the degradation of the cellular receptor. We report here the total chemical synthesis of full-length Vpu(1-81) as well as a site-specifically (15)N-labeled analogue, Vpu(2-81), using native chemical ligation methodologies and also report a structural and functional comparison of these constructs with recombinant protein obtained via bacterial expression. The structures of the synthetic and expressed polypeptides were similar in lipid micelles using solution NMR spectroscopy. Solid-state NMR spectra of the polypeptides in aligned hydrated lipid bilayers indicated that their overall topologies were also very comparable. Further, the channel activity of the synthetic protein was found to be analogous to that previously characterized for the recombinant protein. We have thus demonstrated that using solid phase peptide synthesis and chemical ligation it is feasible to obtain large quantities of a purified and homogeneous membrane protein in a structurally and functionally relevant form for future structural and characterization studies.

Epithelial differentiation involves the generation of luminal surfaces and of a noncentrosomal microtubule (MT) network aligned along the polarity axis. Columnar epithelia (e.g., kidney, intestine, and Madin-Darby canine kidney [MDCK] cells) generate apical lumina and orient MT vertically, whereas liver epithelial cells (hepatocytes and WIFB9 cells) generate lumina at cell-cell contact sites (bile canaliculi) and orient MTs horizontally. We report that knockdown or inhibition of the mammalian orthologue of Caenorhabditis elegans Par-1 (EMK1 and MARK2) during polarization of cultured MDCK and WIFB9 cells prevented development of their characteristic lumen and nonradial MT networks. Conversely, EMK1 overexpression induced the appearance of intercellular lumina and horizontal MT arrays in MDCK cells, making EMK1 the first known candidate to regulate the developmental branching decision between hepatic and columnar epithelial cells. Our experiments suggest that EMK1 primarily promotes reorganization of the MT network, consistent with the MT-regulating role of this gene product in other systems, which in turn controls lumen formation and position.

The growth and aging of 3T3-L1 adipocytes were investigated in a synchronized tissue-culture system. We systematically characterized several major aspects of adipocyte metabolism and functions as variables of cell age. We found that terminal differentiation of 3T3-L1 cells is followed by a near-linear hypertrophic growth (increase in triglyceride content) of the cultured adipocytes throughout a 20-day study period. However, three metabolically and functionally distinct stages are recognized. The first stage overlaps with differentiation and is represented by small immature adipocytes. The second stage is characterized by fully mature adipocytes that show peaked overall metabolic activities. The third stage is marked by cell aging, with deterioration in every major aspect of the cell's functionality except for the function of net energy storage, which is preserved even in aged adipocytes. Compared with young mature adipocytes, older cells are increasingly insulin resistant, have decreased glucose uptake and fuel consumption, and show impaired glycerokinase-mediated fatty acid reesterification. Moreover, aged adipocytes show reduced gene expression for adiponectin and leptin, each of which is important in systemic regulation of energy metabolism. The characterization of these cell age-dependent changes in adipocyte functionality provides a model for understanding dynamic changes at the tissue level and suggests that adipose tissue is modifiable via adipocyte aging.

Gamma interferon (IFN-gamma) is critical in the immune response against Mycobacterium tuberculosis. In an ongoing trial of aerosol IFN-gamma in conjunction with standard drug therapy, we have observed activation of IFN signaling in bronchoalveolar lavage (BAL) cells from tuberculosis (TB) patients. We hypothesized that aerosol IFN-gamma treatment of pulmonary TB would increase expression of genes important for the control of TB. We investigated the expression of downstream genes by measuring inducible nitric oxide synthase (iNOS) and the chemokine IFN-inducible 10-kDa protein (IP-10) by real-time quantitative reverse transcription-PCR. In vitro, M. tuberculosis induced IP-10, and IFN-gamma stimulated this further, with no effect on iNOS expression. We studied 21 patients with pulmonary TB and 7 healthy subjects. Similar to the in vitro model, IP-10 mRNA was increased in BAL cells from TB patients and was augmented after treatment with aerosolized IFN-gamma. TB was also associated with elevated iNOS mRNA, but aerosolized IFN-gamma did not further enhance expression. Genomic analysis identified 1,300 of 4,058 genes expressed in BAL cells from six TB patients before and after 1 month of therapy, including aerosolized IFN-gamma. However, only 15 genes were differentially regulated by IFN-gamma. We conclude that iNOS and IP-10 mRNA expression is increased in TB but that aerosol IFN-gamma treatment increases expression of few genes in the human lung

Corneal epithelium is traditionally thought to be a self-sufficient, self-renewing tissue implying that its stem cells are located in its basal cell layer. Recent studies indicate however that corneal epithelial stem cells reside in the basal layer of peripheral cornea in the limbal zone, and that corneal and conjunctival epithelia represent distinct cell lineages. These ideas are supported by the unique limbal/corneal expression pattern of the K3 keratin marker for corneal-type differentiation; the restriction of the slow-cycling (label-retaining) cells in the limbus; the distinct keratin expression patterns of corneal and conjunctival epithelial cells even when they are provided with identical in vivo and in vitro growth environments; and the limbal cells' superior ability as compared with central corneal epithelial cells in undergoing in vitro proliferation and in reconstituting in vivo an intact corneal epithelium. The realization that corneal epithelial stem cells reside in the limbal zone provides explanations for several paradoxical properties of corneal epithelium including its 'mature-looking' basal cells, the preponderance of tumor formation in the limbal zone, and the centripetal cellular migration. The limbal stem cell concept has led to a better understanding of the strategies of corneal epithelial repair, to a new classification of various anterior surface epithelial diseases, to the use of limbal stem cells for the reconstruction of corneal epithelium damaged or lost as a consequence of trauma or disease ('limbal stem cell transplantation'), and to the rejection of the traditional notion of 'conjunctival transdifferentiation'. The fact that corneal epithelial stem cells reside outside of the cornea proper suggests that studying corneal epithelium per se without taking into account its limbal zone will yield partial pictures. Future studies need to address the signals that constitute the limbal stem cell niche, the mechanism by which amniotic membrane facilitates limbal stem cell transplantation and ex vivo expansion, and the lineage flexibility of limbal stem cells