Proadrenomedullin NH(2)-terminal 20 peptide (PAMP) and adrenomedullin bind to teratocarcinoma cells

Proadrenomedullin NH(2-)terminal 20 peptide (PAMP) and adrenomedullin (ADM) bind to teratocarcinoma cells. The effects of PAMP and ADM on teratocarcinoma cells were investigated. (125)I-PAMP bound to PA1 cells with moderate affinity (K(d) = 110 nM) to a single class of sites (B(max) = 110 000/cell). Specific (125)I-PAMP binding was inhibited by PAMP (IC(50) of 100 nM) but not ADM, calcitonin gene-related peptide (CGRP), or amylin. Specific (125)I-ADM binding was inhibited with high affinity by ADM, CGRP, and CGRP(8-37) (IC(50) values of 10, 10, and 15 nM respectively) but not PAMP or amylin. ADM elevated cAMP (ED(50) value of 100 nM), whereas PAMP had no effect on basal cAMP but inhibited the increase in cAMP caused by 10 nM ADM. Also, the increase in cAMP caused by ADM was inhibited CGRP(8-37), suggesting that ADM is binding to CGRP receptors. ADM (100 nM) stimulated transiently c-fos mRNA, whereas PAMP (1000 nM) had little effect; however, PAMP inhibited the increase in c-fos mRNA caused by ADM. ADM stimulated [(3)H]thymidine uptake into PA1 cells, whereas PAMP inhibited the increase in thymidine uptake caused by ADM. These results indicate that ADM and PAMP are both biologically active in teratocarcinoma cells.     

Novel mechanism that Trypanosoma cruzi uses to adhere to the extracellular matrix mediated by human galectin-3

Binding of Trypanosoma cruzi trypomastigotes to laminin is enhanced by galectin-3, a beta-galactoside binding lectin. The galectin-3 enhanced binding of trypanosomes to laminin is inhibited by lactose. Co-immunoprecipitations indicate that galectin-3 binds to the 45, 32 and 30 kDa trypanosome surface proteins. Binding of galectin-3 to the 45, 32 and 30 kDa surface proteins is inhibited by lactose. Polyclonal and a monoclonal antibodies to galectin-3 immunoprecipitated a major 64 kDa trypanosome surface protein. T. cruzi monoclonal antibody to mucin recognized the 45 kDa surface protein. The 45, 32 and 30 kDa surface proteins interact with galectin-3 in order to enhance trypanosome adhesion to laminin.     

Lipidomic Analysis Links Mycobactin Synthase K to Iron Uptake and Virulence in M. tuberculosis

The prolonged survival of Mycobacterium tuberculosis (M. tb) in the host fundamentally depends on scavenging essential nutrients from host sources. M. tb scavenges non-heme iron using mycobactin and carboxymycobactin siderophores, synthesized by mycobactin synthases (Mbt). Although a general mechanism for mycobactin biosynthesis has been proposed, the biological functions of individual mbt genes remain largely untested. Through targeted gene deletion and global lipidomic profiling of intact bacteria, we identify the essential biochemical functions of two mycobactin synthases, MbtK and MbtN, in siderophore biosynthesis and their effects on bacterial growth in vitro and in vivo. The deletion mutant, ΔmbtN, produces only saturated mycobactin and carboxymycobactin, demonstrating an essential function of MbtN as the mycobactin dehydrogenase, which affects antigenicity but not iron uptake or M. tb growth. In contrast, deletion of mbtK ablated all known forms of mycobactin and its deoxy precursors, defining MbtK as the essential acyl transferase. The mbtK mutant showed markedly reduced iron scavenging and growth in vitro. Further, ΔmbtK was attenuated for growth in mice, demonstrating a non-redundant role of hydroxamate siderophores in virulence, even when other M. tb iron scavenging mechanisms are operative. The unbiased lipidomic approach also revealed unexpected consequences of perturbing mycobactin biosynthesis, including extreme depletion of mycobacterial phospholipids. Thus, lipidomic profiling highlights connections among iron acquisition, phospholipid homeostasis, and virulence, and identifies MbtK as a lynchpin at the crossroads of these phenotypes.     

Testing Models of the APC Tumor Suppressor/β-Catenin Interaction Reshapes Our View of the Destruction Complex in Wnt Signaling

The Wnt pathway is a conserved signal transduction pathway that contributes to normal development and adult homeostasis, but is also misregulated in human diseases such as cancer. The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling inactivated in >80% of colorectal cancers. APC participates in a multiprotein “destruction complex” that targets the proto-oncogene β-catenin for ubiquitin-mediated proteolysis; however, the mechanistic role of APC in the destruction complex remains unknown. Several models of APC function have recently been proposed, many of which have emphasized the importance of phosphorylation of high-affinity β-catenin-binding sites [20-amino-acid repeats (20Rs)] on APC. Here we test these models by generating a Drosophila APC2 mutant lacking all β-catenin-binding 20Rs and performing functional studies in human colon cancer cell lines and Drosophila embryos. Our results are inconsistent with current models, as we find that β-catenin binding to the 20Rs of APC is not required for destruction complex activity. In addition, we generate an APC2 mutant lacking all β-catenin-binding sites (including the 15Rs) and find that a direct β-catenin/APC interaction is also not essential for β-catenin destruction, although it increases destruction complex efficiency in certain developmental contexts. Overall, our findings support a model whereby β-catenin-binding sites on APC do not provide a critical mechanistic function per se, but rather dock β-catenin in the destruction complex to increase the efficiency of β-catenin destruction. Furthermore, in Drosophila embryos expressing some APC2 mutant transgenes we observe a separation of β-catenin destruction and Wg/Wnt signaling outputs and suggest that cytoplasmic retention of β-catenin likely accounts for this difference.     

Reduced creatine kinase as a central and peripheral biomarker in Huntington's disease

A major goal of current clinical research in Huntington's disease (HD) has been to identify preclinical and manifest disease biomarkers, as these may improve both diagnosis and the power for therapeutic trials. Although the underlying biochemical alterations and the mechanisms of neuronal degeneration remain unknown, energy metabolism defects in HD have been chronicled for many years. We report that the brain isoenzyme of creatine kinase (CK-BB), an enzyme important in buffering energy stores, was significantly reduced in presymptomatic and manifest disease in brain and blood buffy coat specimens in HD mice and HD patients. Brain CK-BB levels were significantly reduced in R6/2 mice by ～ 18% to ～ 68% from 21 to 91 days of age, while blood CK-BB levels were decreased by ～ 14% to ～ 44% during the same disease duration. Similar findings in CK-BB levels were observed in the 140 CAG mice from 4 to 12 months of age, but not at the earliest time point, 2 months of age. Consistent with the HD mice, there was a grade-dependent loss of brain CK-BB that worsened with disease severity in HD patients from ～ 28% to ～ 63%, as compared to non-diseased control patients. In addition, CK-BB blood buffy coat levels were significantly reduced in both premanifest and symptomatic HD patients by ～ 23% and ～ 39%, respectively. The correlation of CK-BB as a disease biomarker in both CNS and peripheral tissues from HD mice and HD patients may provide a powerful means to assess disease progression and to predict the potential magnitude of therapeutic benefit in this disorder.     

Stopped-flow solution scattering using synchrotron radiation: Apparatus,data collection and data analysis

We have constructed an experimental system, under remote control, for stopped-flow X-ray scattering using synchrotron radiation. It has been used, in conjunction with an annular detector and its associated electronics, to obtain good scattering curves, with time-slices as short as 200 ms, in a new study of the dissociation of the enzyme complex aspartate transcarbamylase. The data have been analysed by new statistical methods, and they agree well with the results from parallel chemical quench experiments. For studying dissociation reactions, stopped-flow X-ray scattering is a quite practical method, which need not use very much more material than conventional stopped-flow experiments.     

Patterns of genetic diversity and candidate genes for ecological divergence in a homoploid hybrid sunflower, Helianthus anomalus

Natural hybridization accompanied by a shift in niche preference by hybrid genotypes can lead to hybrid speciation. Natural selection may cause the fixation of advantageous alleles in the ecologically diverged hybrids, and the loci experiencing selection should exhibit a reduction in allelic diversity relative to neutral loci. Here, we analyzed patterns of genetic diversity at 59 microsatellite loci associated with expressed sequence tags (ESTs) in a homoploid hybrid sunflower species, Helianthus anomalus. We used two indices, ln RV and ln RH, to compare variation and heterozygosity (respectively) at each locus between the hybrid species and its two parental species, H. annuus and H. petiolaris. Mean values of ln RV and ln RH were significantly lower than zero, which implies that H. anomalus experienced a population bottleneck during its recent evolutionary history. After correcting for the apparent bottleneck, we found six loci with a significant reduction in variation or with heterozygosity in the hybrid species, compared to one or both of the parental species. These loci should be viewed as a ranked list of candidate loci, pending further sequencing and functional analyses. Sequence data were generated for two of the candidate loci, but population genetics tests failed to detect deviations from neutral evolution at either locus. Nonetheless, a greater than eight-fold excess of nonsynonymous substitutions was found near a putative N-myristoylation motif at the second locus (HT998), and likelihood-based models indicated that the protein has been under selection in H. anomalus in the past and, perhaps, in one or both parental species. Finally, our data suggest that selective sweeps may have united populations of H. anomalus isolated by a mountain range, indicating that even low gene-flow species may be held together by the spread of advantageous alleles.