Antagonistic growth regulation by Dpp and Fat drives uniform cell proliferation

We use the Dpp morphogen gradient in the Drosophila wing disc as a model to address the fundamental question of how a gradient of a growth factor can produce uniform growth. We first show that proper expression and subcellular localization of components in the Fat tumor-suppressor pathway, which have been argued to depend on Dpp activity differences, are not reliant on the Dpp gradient. We next analyzed cell proliferation in discs with uniformly high Dpp or uniformly low Fat signaling activity and found that these pathways regulate growth in?a complementary manner. While the Dpp mediator Brinker inhibits growth in the primordium primarily in the lateral regions, Fat represses growth mostly in the medial region. Together, our results indicate that the activities of both signaling pathways are regulated in a parallel rather than sequential manner and that uniform proliferation is achieved by their complementary action on growth.     

Notch Signaling in Osteocytes Differentially Regulates Cancellous and Cortical Bone Remodeling

Notch receptors play a role in skeletal development and homeostasis, and Notch activation in undifferentiated and mature osteoblasts causes osteopenia. In contrast, Notch activation in osteocytes increases bone mass, but the mechanisms involved and exact functions of Notch are not known. In this study, Notch1 and -2 were inactivated preferentially in osteocytes by mating Notch1/2 conditional mice, where Notch alleles are flanked by loxP sequences, with transgenics expressing Cre directed by the Dmp1 (dentin matrix protein 1) promoter. Notch1/2 conditional null male and female mice exhibited an increase in trabecular bone volume due to an increase in osteoblasts and decrease in osteoclasts. In male null mice, this was followed by an increase in osteoclast number and normalization of bone volume. To activate Notch preferentially in osteocytes, Dmp1-Cre transgenics were crossed with Rosa^Notch mice, where a loxP-flanked STOP cassette is placed between the Rosa26 promoter and Notch1 intracellular domain sequences. Dmp1-Cre^+/−;Rosa^Notch mice exhibited an increase in trabecular bone volume due to decreased bone resorption and an increase in cortical bone due to increased bone formation. Biomechanical and chemical properties were not affected. Osteoprotegerin mRNA was increased, sclerostin and dickkopf1 mRNA were decreased, and Wnt signaling was enhanced in Dmp1-Cre^+/−;Rosa^Notch femurs. Botulinum toxin A-induced muscle paralysis caused pronounced osteopenia in control mice, but bone mass was preserved in mice harboring the Notch activation in osteocytes. In conclusion, Notch plays a unique role in osteocytes, up-regulates osteoprotegerin and Wnt signaling, and differentially regulates trabecular and cortical bone homeostasis.     

The Same Major Histocompatibility Complex Polymorphism Involved in Control of HIV Influences Peptide Binding in the Mouse H-2Ld System

Single-site polymorphisms in human class I major histocompatibility complex (MHC) products (HLA-B) have recently been shown to correlate with HIV disease progression or control. An identical single-site polymorphism (at residue 97) in the mouse class I product H-2L^d influences stability of the complex. To gain insight into the human polymorphisms, here we examined peptide binding, stability, and structures of the corresponding L^d polymorphisms, Trp⁹⁷ and Arg⁹⁷. Expression of L^dW97 and L^dR97 genes in a cell line that is antigen-processing competent showed that L^dR97 was expressed at higher levels than L^dW97, consistent with enhanced stability of self-peptide·L^dR97 complexes. To further examine peptide-binding capacities of these two allelic variants, we used a high affinity pep-L^d specific probe to quantitatively examine a collection of self- and foreign peptides that bind to L^d. L^dR97 bound more effectively than L^dW97 to most peptides, although L^dW97 bound more effectively to two peptides. The results support the view that many self-peptides in the L^d system (or the HLA-B system) would exhibit enhanced binding to Arg⁹⁷ alleles compared with Trp⁹⁷ alleles. Accordingly, the self-peptide·MHC-Arg⁹⁷ complexes would influence T-cell selection behavior, impacting the T-cell repertoire of these individuals, and could also impact peripheral T cell activity through effects of self-peptide·L^d interacting with TCR and/or CD8. The structures of several peptide·L^dR97 and peptide·L^dW97 complexes provided a framework of how this single polymorphism could impact peptide binding.     

Heme ligand identification and redox properties of the cytochrome c synthetase, CcmF

Cytochrome c maturation in many bacteria, archaea, and plant mitochondria involves the integral membrane protein CcmF, which is thought to function as a cytochrome c synthetase by facilitating the final covalent attachment of heme to the apocytochrome c. We previously reported that the E. coli CcmF protein contains a b-type heme that is stably and stoichiometrically associated with the protein and is not the heme attached to apocytochrome c. Here, we show that mutation of either of two conserved transmembrane histidines (His261 or His491) impairs stoichiometric b-heme binding in CcmF and results in spectral perturbations in the remaining heme. Exogeneous imidazole is able to correct cytochrome c maturation for His261 and His491 substitutions with small side chains (Ala or Gly), suggesting that a "cavity" is formed in these CcmF mutants in which imidazole binds and acts as a functional ligand to the b-heme. The results of resonance Raman spectroscopy on wild-type CcmF are consistent with a hexacoordinate low-spin b-heme with at least one endogeneous axial His ligand. Analysis of purified recombinant CcmF proteins from diverse prokaryotes reveals that the b-heme in CcmF is widely conserved. We have also determined the reduction potential of the CcmF b-heme (E(m,7) = -147 mV). We discuss these results in the context of CcmF structure and functions as a heme reductase and cytochrome c synthetase.     

Myxoma virus combined with rapamycin treatment enhances adoptive T cell therapy for murine melanoma brain tumors

Adoptive transfer of tumor-specific T cells has shown some success for treating metastatic melanoma. We evaluated a novel strategy to improve adoptive therapy by administering both T cells and oncolytic myxoma virus to mice with syngeneic B16.SIY melanoma brain tumors. Adoptive transfer of activated CD8⁺ 2C T cells that recognize SIY peptide doubled survival time, but SIY-negative tumors recurred. Myxoma virus killed B16.SIY cells in vitro, and intratumoral injection of virus led to selective and transient infection of the tumor. Virus treatment recruited innate immune cells to the tumor and induced IFNβ production in the brain, resulting in limited oncolytic effects in vivo. To counter this, we evaluated the safety and efficacy of co-administering 2C T cells, myxoma virus, and either rapamycin or neutralizing antibodies against IFNβ. Mice that received either triple combination therapy survived significantly longer with no apparent side effects, but eventually relapsed. Importantly, rapamycin treatment did not impair T cell-mediated tumor destruction, supporting the feasibility of combining adoptive immunotherapy and rapamycin-enhanced virotherapy. Myxoma virus may be a useful vector for transient delivery of therapeutic genes to a tumor to enhance T cell responses.     

The aryl hydrocarbon receptor-dependent deregulation of cell cycle control induced by polycyclic aromatic hydrocarbons in rat liver epithelial cells

Disruption of cell proliferation control by polycyclic aromatic hydrocarbons (PAHs) may contribute to their carcinogenicity. We investigated role of the aryl hydrocarbon receptor (AhR) in disruption of contact inhibition in rat liver epithelial WB-F344 'stem-like' cells, induced by the weakly mutagenic benz[a]anthracene (BaA), benzo[b]fluoranthene (BbF) and by the strongly mutagenic benzo[a]pyrene (BaP). There were significant differences between the effects of BaA and BbF, and those of the strongly genotoxic BaP. Both BaA and BbF increased percentage of cells entering S-phase and cell numbers, associated with an increased expression of Cyclin A and Cyclin A/cdk2 complex activity. Their effects were significantly reduced in cells expressing a dominant-negative AhR mutant (dnAhR). Roscovitine, a chemical inhibitor of cdk2, abolished the induction of cell proliferation by BbF. However, neither BaA nor BbF modulated expression of the principal cdk inhibitor involved in maintenance of contact inhibition, p27(Kip1), or pRb phosphorylation. The strongly mutagenic BaP induced apoptosis, a decrease in total cell numbers and significantly higher percentage of cells entering S-phase than either BaA or BbF. Given that BaP induced high levels of Cyclin A/cdk2 activity, downregulation of p27(Kip1) and hyperphosphorylation of pRb, the accumulation of cells in S-phase was probably due to cell proliferation, although S-phase arrest due to blocked replication forks can not be excluded. Both types of effects of BaP were significantly attenuated in dnAhR cells. Transfection of WB-F344 cells with siRNA targeted against AhR decreased induction of Cyclin A induced by BbF or BaP, further supporting the role of AhR in proliferative effects of PAHs. This suggest that activation of AhR plays a significant role both in disruption of contact inhibition by weakly mutagenic PAHs and in genotoxic effects of BaP possibly leading to enhanced cell proliferation. Thus, PAHs may increase proliferative rate and the likelihood of fixation of mutations.     

WOX gene phylogeny in Poaceae: a comparative approach addressing leaf and embryo development

The phylogeny based on the homeodomain (HD) amino acid sequence of the WOX (WUSCHEL-related homeobox gene family) was established in the 3 major radiations of the Poaceae family: Pooideae (Brachypodium distachyon), Bambusoideae (Oryza sativa), and Panicoideae (Zea mays). The genomes of all 3 grasses contain an ancient duplication in the WOX3 branch, and the cellular expression patterns in maize and rice indicate subfunctionalization of paralogues during leaf development, which may relate to the architecture of the grass leaf and the encircling of the stem. The use of maize WOX gene family members as molecular markers in maize embryo development for the first time allowed us to visualize cellular decisions in the maize proembryo, including specification of the shoot/root axis at an oblique angle to the apical-basal polarity of the zygote. All molecular marker data are compatible with the conclusion that the embryonic shoot/root axis comprises a discrete domain from early proembryo stages onward. Novel cell fates of the shoot and the root are acquired within this distinct morphogenic axis domain, which elongates and thus separates the shoot apical meristem and root apical meristem (RAM) anlagen in the maize embryo.     

Improved sensitivity and stability of amperometric enzyme microbiosensors by covalent attachment to gold electrodes

Covalent attachment of glucose oxidase to a pre-activated 16-mercaptohexadecanoic acid at a gold ultramicroelectrode surface improves sensitivity, stability, and reproducibility of enzyme-based amperometric microbiosensors. Self-assembled monolayers of the N-hydroxysuccinimide ester of 16-mercaptohexandecanoic acid (NHS-MHA) at gold electrodes enable spontaneous covalent linking of glucose oxidase to the gold surface of ultramicroelectrodes. By self-assembling NHS-MHA for 30 min, approximately 93% of the electrode surface is covered, thereby maximizing both the number of attachment sites for glucose oxidase, and sufficient diffusion of hydrogen peroxide to the gold electrode. The glucose oxidase reaction with NHS-MHA was optimized at pH of 6.5, and at a temperature of 43 degrees C, resulting in a surface concentration of 6.8+/-0.6 x 10(11) enzymemoleculescm(-2). Thus obtained amperometric microbiosensors were calibrated in the range of 1-10mM providing excellent correlation with the theoretical prediction of the microbiosensor response. The reported sensitivity of these microbiosensors documents an improvement by one order of magnitude compared to other approaches for covalent enzyme attachment. This is attributed to the NHS-MHA layer spacing the enzymatic recognition interface further from the electrode surface, thereby minimizing quenching of the enzyme activity.     

Enhancing recombinant protein quality and yield by protein stability profiling

The reliable production of large amounts of stable, high-quality proteins is a major challenge facing pharmaceutical protein biochemists, necessary for fulfilling demands from structural biology, for high-throughput screening, and for assay purposes throughout early discovery. One strategy for bypassing purification challenges in problematic systems is to engineer multiple forms of a particular protein to optimize expression, purification, and stability, often resulting in a nonphysiological sub-domain. An alternative strategy is to alter process conditions to maximize wild-type construct stability, based on a specific protein stability profile (PSP). ThermoFluor, a miniaturized 384-well thermal stability assay, has been implemented as a means of monitoring solution-dependent changes in protein stability, complementing the protein engineering and purification processes. A systematic analysis of pH, buffer or salt identity and concentration, biological metals, surfactants, and common excipients in terms of an effect on protein stability rapidly identifies conditions that might be used (or avoided) during protein production. Two PSPs are presented for the kinase catalytic domains of Akt-3 and cFMS, in which information derived from a ThermoFluor PSP led to an altered purification strategy, improving the yield and quality of the protein using the primary sequences of the catalytic domains.