The LRP5 high-bone-mass G171V mutation disrupts LRP5 interaction with Mesd

The mechanism by which the high-bone-mass (HBM) mutation (G171V) of the Wnt coreceptor LRP5 regulates canonical Wnt signaling was investigated. The mutation was previously shown to reduce DKK1-mediated antagonism, suggesting that the first YWTD repeat domain where G171 is located may be responsible for DKK-mediated antagonism. However, we found that the third YWTD repeat, but not the first repeat domain, is required for DKK1-mediated antagonism. Instead, we found that the G171V mutation disrupted the interaction of LRP5 with Mesd, a chaperone protein for LRP5/6 that is required for transport of the coreceptors to cell surfaces, resulting in fewer LRP5 molecules on the cell surface. Although the reduction in the number of cell surface LRP5 molecules led to a reduction in Wnt signaling in a paracrine paradigm, the mutation did not appear to affect the activity of coexpressed Wnt in an autocrine paradigm. Together with the observation that osteoblast cells produce autocrine canonical Wnt, Wnt7b, and that osteocytes produce paracrine DKK1, we think that the G171V mutation may cause an increase in Wnt activity in osteoblasts by reducing the number of targets for paracrine DKK1 to antagonize without affecting the activity of autocrine Wnt.     

Heat-inactivated C. pneumoniae organisms are not atherogenic

We have previously shown that infection with Chlamydia pneumoniae can significantly exacerbate atherosclerotic lesions in LDLR-/- mice concurrently fed a high cholesterol diet in 6 or 9 months. We now report that a period of 4 month was sufficient for demonstrating the C. pneumoniae atherogenicity. However, heat inactivation of C. pneumoniae organisms completely abolished the ability of C. pneumoniae to exacerbate the atherosclerotic lesions, suggesting that viable organism infection may be required for the C. pneumoniae atherogenicity.     

Cloud-point temperature and liquid-liquid phase separation of supersaturated lysozyme solution

The detailed understanding of the structure of biological macromolecules reveals their functions, and is thus important in the design of new medicines and for engineering molecules with improved properties for industrial applications. Although techniques used for protein crystallization have been progressing greatly, protein crystallization may still be considered an art rather than a science, and successful crystallization remains largely empirical and operator-dependent. In this work, a microcalorimetric technique has been utilized to investigate liquid-liquid phase separation through measuring cloud-point temperature T(cloud) for supersaturated lysozyme solution. The effects of ionic strength and glycerol on the cloud-point temperature are studied in detail. Over the entire range of salt concentrations studied, the cloud-point temperature increases monotonically with the concentration of sodium chloride. When glycerol is added as additive, the solubility of lysozyme is increased, whereas the cloud-point temperature is decreased.     

Adenine auxotrophic mutants of Aspergillus oryzae: development of a novel transformation system with triple auxotrophic hosts

adeA and adeB genes homologous to Saccharomyces cerevisiae ADE1 and ADE2, respectively, were cloned from Aspergillus oryzae. AdeA and AdeB share 62.8% and 52.5% identities with S. cerevisiae Ade1 and Ade2, respectively. In order to obtain triple auxotrophic mutants from A. oryzae, 12 red-colored mutant colonies were isolated by UV mutagenesis of a double auxotrophic host, NS4 (niaD(-), sC(-)), as a parent strain. All the mutants exhibited adenine auxotrophy and showed fluorescence in the vacuoles due to accumulation of a purine biosynthetic pathway precursor. Adenine auxotrophy of all the mutants was restored by introduction of either A. oryzae adeA or adeB genes. Sequence analysis demonstrated that substitutions or deletions of a single base pair occurred, inducing substitutions or frame shifts of amino acid sequences in both ade genes complementing the mutants. This study provides a novel host-vector system with triple auxotrophy in A. oryzae.     

Prevention of lethal respiratory vaccinia infections in mice with interferon-alpha and interferon-gamma

The antiviral efficacy of interferons (IFNs) was evaluated using a vaccinia intranasal infection model in mice in this study. We provide evidence that intranasal administration of IFN-alpha and IFN-gamma (days -1 to +3) resulted in 100 and 90% survival against a lethal respiratory vaccinia infection (8 LD50) in mice, respectively; whereas no animals in the placebo group survived through the study period (21 days). The IFN treatment consisted of a single daily dose of 5x10(3) U per mouse for 5 consecutive days. The efficacy of IFN-gamma was evident even when the IFN-gamma treatments started 1-2 days after infection and when a lower dose (2x10(3) U per mouse) was used. The treatment of IFN-alpha and IFN-gamma reduced the virus titers in the lungs of infected mice by 1000-10,000-fold, when the administration started 1 day after infection. Our data suggest that IFN-alpha and IFN-gamma are effective in protecting vaccinia-infected mice from viral replication in lungs and mortality, and may be beneficial in other human orthopoxvirus infections.     

Mobilization of neutrophil sialidase activity desialylates the pulmonary vascular endothelial surface and increases resting neutrophil adhesion to and migration across the endothelium

The amount of sialic acid on the surface of the neutrophil (PMN) influences its ability to interact with other cells. PMN activation with various stimuli mobilizes intracellular sialidase to the plasma membrane, where it cleaves sialic acid from cell surfaces. Because enhanced PMN adherence, spreading, deformability, and motility each are associated with surface desialylation and are critical to PMN diapedesis, we studied the role of sialic acid on PMN adhesion to and migration across pulmonary vascular endothelial cell (EC) monolayers in vitro. Neuraminidase treatment of either PMN or EC increased adhesion and migration in a dose-dependent manner. Neuraminidase treatment of both PMNs and ECs increased PMN adhesion to EC more than treatment of either PMNs or ECs alone. Moreover, neuraminidase treatment of ECs did not change surface expression of adhesion molecules or release of IL-8 and IL-6. Inhibition of endogenous sialidase by either cross-protective antineuraminidase antibodies (45.5% inhibition) or competitive inhibition with pseudo-substrate (41.2% inhibition) decreased PMN adhesion to ECs; the inhibitable sialidase activity appeared to be associated with activated PMNs. Finally, EC monolayers preincubated with activated PMNs became hyperadhesive for subsequently added resting PMNs, and this hyperadhesive state was mediated through endogenous PMN sialidase activity. Blocking anti-E-selectin, anti-CD54 and anti-CD18 antibodies decreased PMN adhesion to tumor necrosis factor-activated ECs but not to PMN-treated ECs. These data implicate desialylation as a novel mechanism through which PMN-EC adhesion can be regulated independent of de novo protein synthesis or altered adhesion molecule expression. The ability of activated PMNs, through endogenous sialidase activity, to render the EC surface hyperadherent for unstimulated PMNs may provide for rapid amplification of the PMN-mediated host response.     

Mutual dependence of VIP/PACAP and CCK receptor signaling for a physiological role in duck exocrine pancreatic secretion

Unlike in rodents, CCK has not been established as a physiological regulator in avian exocrine pancreatic secretion. In the isolated duck pancreatic acini, 1 nM CCK was required for stimulation of amylase secretion, maximal effect being achieved at 10 nM; picomolar CCK was without effect. Vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase activating peptide (PACAP) receptor (VPAC) agonists PACAP-38 and PACAP-27 (10(-12)-10(-7) M) alone had no effect, but made picomolar CCK effective. VPAC agonist VIP 10(-10)-10(-7) M stimulated amylase secretion marginally, but made CCK 10(-12)-10(-10) M effective also. PACAP-27 and VIP both shifted the maximal CCK concentration from 10(-8) to 10(-9) M. This sensitizing effect was mimicked by forskolin. CCK dose dependently induced intracellular Ca2+ concentration ([Ca2+]i) oscillations. PACAP-38 (1 nM), PACAP-27 (1 nM), VIP (10 nM), or forskolin (10 microM) alone did not stimulate [Ca2+]i increase, neither did they modulate CCK (1 nM)-induced oscillations; but when they were added to cells simultaneously exposed to subthreshold CCK (10 pM), calcium spikes emerged. Amylase secretion induced by the simultaneous presence of 10 pM CCK and VPAC agonists was completely blocked by removing extracellular calcium, but the protein kinase C inhibitor staurosporine (1 microM) was without effect. CCK (10 nM)-induced secretion was inhibited by CCK1 receptor antagonist FK480 (1 microM). Gastrin from 10(-12) to 10(-6) M did not stimulate amylase secretion nor did it (100 nM) induce [Ca2+]i increase. The above data suggest that duck pancreatic acini possess both CCK1 and VPAC receptors; simultaneous activation of both is required for each to play a physiological role.