Proceedings of the National Academy of Sciences

Klein, George

doi: N/Apmid: N/A

As stated in Bredberg's letter (1), most tumors are, indeed, monoclonal. This does not mean, however, that the neoplastic change occurs in a single step. It is now well recognized that tumors evolve by multiple genetic and epigenetic changes that affect regulatory pathways of cell division, apoptosis, and differentiation (2 …

Ohmi, Kazuhiro; Kudo, Lili C.; Ryazantsev, Sergey; Zhao, Hui-Zhi; Karsten, Stanislav L.; Neufeld, Elizabeth F.

doi: N/Apmid: 19416848

Sanfilippo syndrome type B (mucopolysaccharidosis III B, MPS III B) is an autosomal recessive, neurodegenerative disease of children, characterized by profound mental retardation and dementia. The primary cause is mutation in the NAGLU gene, resulting in deficiency of α-N-acetylglucosaminidase and lysosomal accumulation of heparan sulfate. In the mouse model of MPS III B, neurons and microglia display the characteristic vacuolation of lysosomal storage of undegraded substrate, but neurons in the medial entorhinal cortex (MEC) display accumulation of several additional substances. We used whole genome microarray analysis to examine differential gene expression in MEC neurons isolated by laser capture microdissection from Naglu−/− and Naglu+/− mice. Neurons from the lateral entorhinal cortex (LEC) were used as tissue controls. The highest increase in gene expression (6- to 7-fold between mutant and control) in MEC and LEC neurons was that of Lyzs, which encodes lysozyme, but accumulation of lysozyme protein was seen in MEC neurons only. Because of a report that lysozyme induced the formation of hyperphosphorylated tau (P-tau) in cultured neurons, we searched for P-tau by immunohistochemistry. P-tau was found in MEC of Naglu−/− mice, in the same neurons as lysozyme. In older mutant mice, it was also seen in the dentate gyrus, an area important for memory. Electron microscopy of dentate gyrus neurons showed cytoplasmic inclusions of paired helical filaments, P-tau aggregates characteristic of tauopathies—a group of age-related dementias that include Alzheimer disease. Our findings indicate that the Sanfilippo syndrome type B should also be considered a tauopathy.

Schönfeld, D.; Matschiner, G.; Chatwell, L.; Trentmann, S.; Gille, H.; Hülsmeyer, M.; Brown, N.; Kaye, P. M.; Schlehuber, S.; Hohlbaum, A. M.; Skerra, A.

doi: N/Apmid: 19416843

Biomolecular reagents that enable the specific molecular recognition of proteins play a crucial role in basic research as well as medicine. Up to now, antibodies (immunoglobulins) have been widely used for this purpose. Their predominant feature is the vast repertoire of antigen-binding sites that arise from a set of 6 hypervariable loops. However, antibodies suffer from practical disadvantages because of their complicated architecture, large size, and multiple functions. The lipocalins, on the other hand, have evolved as a protein family that primarily serves for the binding of small molecules. Here, we show that an engineered lipocalin, derived from human Lcn2, can specifically bind the T cell coreceptor CTLA-4 as a prescribed protein target with subnanomolar affinity. Crystallographic analysis reveals that its reshaped cup-like binding site, which is formed by 4 variable loops, provides perfect structural complementarity with this “antigen.” Furthermore, comparison with the crystal structure of the uncomplexed engineered lipocalin indicates a pronounced induced-fit mechanism, a phenomenon so far considered typical for antibodies. By recognizing the same epitope on CTLA-4 that interacts with the counterreceptors B7.1/B7.2 on antigen-presenting cells the engineered Lcn2 exhibits strong, cross-species antagonistic activity, as evidenced by biological effects comparable with a CTLA-4-specific antibody. With its proven stimulatory activity on T cells in vivo, the CTLA-4 blocking lipocalin offers potential for immunotherapy of cancer and infectious disease. Beyond that, lipocalins with engineered antigen-binding sites, so-called Anticalins, provide a class of small (≈180 residues), structurally simple, and robust binding proteins with applications in the life sciences in general.

Vieites, David R.; Wollenberg, Katharina C.; Andreone, Franco; Köhler, Jörn; Glaw, Frank; Vences, Miguel

doi: N/Apmid: 19416818

Amphibians are in decline worldwide. However, their patterns of diversity, especially in the tropics, are not well understood, mainly because of incomplete information on taxonomy and distribution. We assess morphological, bioacoustic, and genetic variation of Madagascar's amphibians, one of the first near-complete taxon samplings from a biodiversity hotspot. Based on DNA sequences of 2,850 specimens sampled from over 170 localities, our analyses reveal an extreme proportion of amphibian diversity, projecting an almost 2-fold increase in species numbers from the currently described 244 species to a minimum of 373 and up to 465. This diversity is widespread geographically and across most major phylogenetic lineages except in a few previously well-studied genera, and is not restricted to morphologically cryptic clades. We classify the genealogical lineages in confirmed and unconfirmed candidate species or deeply divergent conspecific lineages based on concordance of genetic divergences with other characters. This integrative approach may be widely applicable to improve estimates of organismal diversity. Our results suggest that in Madagascar the spatial pattern of amphibian richness and endemism must be revisited, and current habitat destruction may be affecting more species than previously thought, in amphibians as well as in other animal groups. This case study suggests that worldwide tropical amphibian diversity is probably underestimated at an unprecedented level and stresses the need for integrated taxonomic surveys as a basis for prioritizing conservation efforts within biodiversity hotspots.

Alian, Akram; Griner, Sarah L.; Chiang, Vicki; Tsiang, Manuel; Jones, Gregg; Birkus, Gabriel; Geleziunas, Romas; Leavitt, Andrew D.; Stroud, Robert M.

doi: N/Apmid: 19416821

HIV-1 integration into the host cell genome is a multistep process catalyzed by the virally-encoded integrase (IN) protein. In view of the difficulty of obtaining a stable DNA-bound IN at high concentration as required for structure determination, we selected IN–DNA complexes that form disulfide linkages between 5′-thiolated DNA and several single mutations to cysteine around the catalytic site of IN. Mild reducing conditions allowed for selection of the most thermodynamically-stable disulfide-linked species. The most stable complexes induce tetramer formation of IN, as happens during the physiological integration reaction, and are able to catalyze the strand transfer step of retroviral integration. One of these complexes also binds strand-transfer inhibitors of HIV antiviral drugs, making it uniquely valuable among the mutants of this set for understanding portions of the integration reaction. This novel complex may help define substrate interactions and delineate the mechanism of action of known integration inhibitors.

Kim, Yeong C.; Wu, Qingfa; Chen, Jun; Xuan, Zhenyu; Jung, Yong-Chul; Zhang, Michael Q.; Rowley, Janet D.; Wang, San Ming

doi: N/Apmid: 19416867

Studying gene expression at different hematopoietic stages provides insights for understanding the genetic basis of hematopoiesis. We analyzed gene expression in human CD34+ hematopoietic cells that represent the stem-progenitor population (CD34+ cells). We collected >459,000 transcript signatures from CD34+ cells, including the de novo-generated 3′ ESTs and the existing sequences of full-length cDNAs, ESTs, and serial analysis of gene expression (SAGE) tags, and performed an extensive annotation on this large set of CD34+ transcript sequences. We determined the genes expressed in CD34+ cells, verified the known genes and identified the new genes of different functional categories involved in hematopoiesis, dissected the alternative gene expression including alternative transcription initiation, splicing, and adenylation, identified the antisense and noncoding transcripts, determined the CD34+ cell-specific gene expression signature, and developed the CD34+ cell-transcription map in the human genome. Our study provides a current view on gene expression in human CD34+ cells and reveals that early hematopoiesis is an orchestrated process with the involvement of over half of the human genes distributed in various functions. The data generated from our study provide a comprehensive and uniform resource for studying hematopoiesis and stem cell biology.

Yu, Jong W.; Jeffrey, Philip D.; Shi, Yigong

doi: N/Apmid: 19416807

Cellular FLICE-inhibitory protein (c-FLIPL) is a key regulator of the extrinsic cell death pathway. Although widely regarded as an inhibitor of initiator caspase activation and cell death, c-FLIPL is also capable of enhancing procaspase-8 activation through heterodimerization of their respective protease domains. However, the underlying mechanism of this activation process remains enigmatic. Here, we demonstrate that cleavage of the intersubunit linker of c-FLIPL by procaspase-8 potentiates the activation process by enhancing heterodimerization between the two proteins and vastly improving the proteolytic activity of unprocessed caspase-(C)8. The crystal structures of the protease-like domain of c-FLIPL alone and in complex with zymogen C8 identify the unique determinants that favor heterodimerization over procaspase-8 homodimerization, and induce the latent active site of zymogen C8 into a productive conformation. Together, these findings provide molecular insights into a key aspect of c-FLIPL function that modulates procaspase-8 activation to elicit diverse responses in different cellular contexts.

Weaver, Timothy D.; Hublin, Jean-Jacques

doi: N/Apmid: 19380728

Childbirth is complicated in humans relative to other primates. Unlike the situation in great apes, human neonates are about the same size as the birth canal, making passage difficult. The birth mechanism (the series of rotations that the neonate must undergo to successfully negotiate its mother's birth canal) distinguishes humans not only from great apes, but also from lesser apes and monkeys. Tracing the evolution of human childbirth is difficult, because the pelvic skeleton, which forms the margins of the birth canal, tends to survive poorly in the fossil record. Only 3 female individuals preserve fairly complete birth canals, and they all date to earlier phases of human evolution. Here we present a virtual reconstruction of a female Neandertal pelvis from Tabun, Israel. The size of Tabun's reconstructed birth canal indicates that childbirth was about as difficult in Neandertals as in present-day humans, but the canal's shape indicates that Neandertals had a more primitive birth mechanism. A significant shift in childbirth apparently occurred quite late in human evolution, during the last few hundred thousand years. Such a late shift underscores the uniqueness of human childbirth and the divergent evolutionary trajectories of Neandertals and the lineage leading to present-day humans.

Felices, Martin; Yin, Catherine C.; Kosaka, Yoko; Kang, Joonsoo; Berg, Leslie J.

doi: N/Apmid: 19416854

In conventional αβ T cells, the Tec family tyrosine kinase Itk is required for signaling downstream of the T cell receptor (TCR). Itk also regulates αβ T cell development, lineage commitment, and effector function. A well established feature of Itk−/− mice is their inability to generate T helper type 2 (Th2) responses that produce IL-4, IL-5, and IL-13; yet these mice have spontaneously elevated levels of serum IgE and increased numbers of germinal center B cells. Here we show that the source of this phenotype is γδ T cells, as normal IgE levels are observed in Itk−/−Tcrd−/− mice. When stimulated through the γδ TCR, Itk−/− γδ T cells produce high levels of Th2 cytokines, but diminished IFNγ. In addition, activated Itk−/− γδ T cells up-regulate costimulatory molecules important for B cell help, suggesting that they may directly promote B cell activation and Ig class switching. Furthermore, we find that γδ T cells numbers are increased in Itk−/− mice, most notably the Vγ1.1+Vδ6.3+ subset that represents the dominant population of γδ NKT cells. Itk−/− γδ NKT cells also have increased expression of PLZF, a transcription factor required for αβ NKT cells, indicating a common molecular program between αβ and γδ NKT cell lineages. Together, these data indicate that Itk signaling regulates γδ T cell lineage development and effector function and is required to control IgE production in vivo.

Lin, Yu; Mao, Wendy L.; Mao, Ho-kwang

doi: N/Apmid: 19416809

We studied ammonia borane (AB), NH3BH3, in the presence of excess hydrogen (H2) pressure and discovered a solid phase, AB(H2)x, where x ≈1.3–2. The new AB–H2 compound can store an estimated 8–12 wt % molecular H2 in addition to the chemically bonded H2 in AB. This phase formed slowly at 6.2 GPa, but the reaction rate could be enhanced by crushing the AB sample to increase its contact area with H2. The compound has 2 Raman H2 vibron peaks from the absorbed H2 in this phase: one (ν1) at frequency 70 cm−1 below the free H2 vibron, and the other (ν2) at higher frequency overlapping with the free H2 vibron at 6 GPa. The peaks shift linearly over the pressure interval of 6–16 GPa with average pressure coefficients of dν1/dP = 4 cm−1/GPa and dν2/dP = 6 cm−1/GPa. The formation of the compound is accompanied by changes in the N–H and B–H stretching Raman peaks resulting from the AB interactions with H2 which indicate the structural complexity and low symmetry of this phase. Storage of significant amounts of additional molecular H2 in AB increases the already high hydrogen content of AB, and may provide guidance for developing improved hydrogen storage materials.