Effects of Suramin on PMN Interactions with Different Surfaces

Human polymorphonuclear leukocytes (PMN) were found to tightly adhere on endothelial (lines EAhy926 and ECV304) and collagen surfaces under the influence of the chemotherapeutic drug suramin. This was observed by scanning electron microscopy and quantitated by myeloperoxidase assays. Suramin also inhibited Ca²⁺ ionophore A23187-stimulated leukotriene (LT) synthesis in PMN interaction with endothelial cells or with collagen surface. Suramin decreased the release of radiolabeled arachidonic acid (AA) and 5-lip-oxygenase (5-LO) metabolites by prelabeled PMN stimulated with A23187. Using agents releasing the suramin-stimulated adhesion namely jasplakonolide and dextran sulfate, we observed a reversal of the suramin effect on leukotriene synthesis. Jasplakonolide released the adhesion of PMN on endothelial and collagen-coated surfaces and restored 5-LO activity. Dextran-sulfate released adhesion on collagen-coated surfaces and abolished suramin inhibition. Arachidonate could also overcome adhesion and inhibition of 5-LO. We conclude that suramin-induced tight attachment of PMN on to solid surfaces lead to decreased leukotriene synthesis during subsequent A23187 stimulation in the absence of exogenous substrates.     

Were inefficient mitochondrial haplogroups selected during migrations of modern humans? A test using modular kinetic analysis of coupling in mitochondria from cybrid cell lines

We introduce a general test of the bioenergetic importance of mtDNA (mitochondrial DNA) variants: modular kinetic analysis of oxidative phosphorylation in mitochondria from cybrid cells with constant nuclear DNA but different mtDNA. We have applied this test to the hypothesis [Ruiz-Pesini, Mishmar, Brandon, Procaccio and Wallace (2004) Science 303, 223-226] that particular mtDNA haplogroups (specific combinations of polymorphisms) that cause lowered coupling efficiency, leading to generation of less ATP and more heat, were positively selected during radiations of modern humans into colder climates. Contrary to the predictions of this hypothesis, mitochondria from Arctic haplogroups had similar or even greater coupling efficiency than mitochondria from tropical haplogroups.     

A reality check for alignments and trees

Making multiple sequence alignments is one of the more commonplace procedures in modern biology. Multiple alignments are typically generated by feeding sequences into the alignment program from the N-terminus to the C-terminus. Recent results show that if the same sequences are processed from the C- to the N-terminus, a different alignment is often obtained. Because phylogenetic trees are built from alignments, the resulting trees can also differ. The new findings highlight sequence alignment as a crucial step in molecular evolutionary studies and provide straightforward measures to assess alignment reliability.     

Extra-renal 25-hydroxyvitamin D3-1α-hydroxylase in human health and disease

Although ectopic expression of 25-hydroxyvitamin D₃-1α-hydroxylase (1α-OHase) has been recognized for many years, the precise function of this enzyme outside the kidney remains open to debate. Three specific aspects of extra-renal 1α-OHase have attracted most attention: (i) expression and regulation in non-classical tissues during normal physiology; (ii) effects on the immune system and inflammatory disease; (iii) expression and function in tumors. The most well-recognized manifestation of extra-renal 1α-OHase activity remains that found in some patients with granulomatous diseases where locally synthesized 1α,25(OH)₂D₃ has the potential to spill-over into the general circulation. However, immunohistochemistry and mRNA analyses suggest that 1α-OHase is also expressed by a variety of normal human tissues including the gastrointestinal tract, skin, vasculature and placenta. This has promoted the idea that autocrine/paracrine synthesis of 1,25(OH)₂D₃ contributes to normal physiology, particularly in mediating the potent effects of vitamin D on innate (macrophage) and acquired (dendritic cell) immunity. We have assessed the capacity for synthesis of 1,25(OH)₂D₃ in these cells and the functional significance of autocrine responses to 1α-hydroxylase. Data suggest that local synthesis of 1,25(OH)₂D₃ may be a preferred mode of response to antigenic challenge in many tissues.     

Expression of novel extracellular sulfatases Sulf-1 and Sulf-2 in normal and osteoarthritic articular cartilage

Introduction  

Changes in sulfation of cartilage glycosaminoglycans as mediated by sulfatases can regulate growth factor signaling. The aim of this study was to analyze expression patterns of recently identified extracellular sulfatases Sulf-1 and Sulf-2 in articular cartilage and chondrocytes.     

Biotransformation and reduction of estrogenicity of bisphenol A by the biphenyl-degrading Cupriavidus basilensis

The biphenyl-degrading Gram-negative bacterium Cupriavidus basilensis (formerly Ralstonia sp.) SBUG 290 uses various aromatic compounds as carbon and energy sources and has a high capacity to transform bisphenol A (BPA), which is a hormonally active substance structurally related to biphenyl. Biphenyl-grown cells initially hydroxylated BPA and converted it to four additional products by using three different transformation pathways: (a) formation of multiple hydroxylated BPA, (b) ring fission, and (c) transamination followed by acetylation or dimerization. Products of the ring fission pathway were non-toxic and all five products exhibited a significantly reduced estrogenic activity compared to BPA. Cell cultivation with phenol and especially in nutrient broth (NB) resulted in a reduced biotransformation rate and lower product quantities, and NB-grown cells did not produce all five products in detectable amounts. Thus, the question arose whether enzymes of the biphenyl degradation pathway are involved in the transformation of BPA and was addressed by proteomic analyses.

     

Localization of the delta-like-1-binding site in human Notch-1 and its modulation by calcium affinity

The Notch signaling pathway plays a key role in a myriad of cellular processes, including cell fate determination. Despite extensive study of the downstream consequences of receptor activation, very little molecular data are available for the initial binding event between the Notch receptor and its ligands. In this study, we have expressed and purified a natively folded wild-type epidermal growth factor-like domain (EGF) 11-14 construct from human Notch-1 and have used flow cytometry and surface plasmon resonance analysis to demonstrate a calcium-dependent interaction with the human ligand Delta-like-1. Site-directed mutagenesis of three of the calcium-binding sites within the Notch-(11-14) fragment indicated that only loss of calcium binding to EGF12, and not EGF11 or EGF13, abrogates ligand binding. Further mapping of the ligand-binding site within this region by limited proteolysis of Notch wild-type and mutant fragments suggested that EGF12 rather than EGF11 contains the major Delta-like-1-binding site. Analysis of an extended fragment EGF-(10-14), where EGF11 is placed in a native context, surprisingly demonstrated a reduction in ligand binding, suggesting that EGF10 modulates binding by limiting access of ligand. This inhibition could be overcome by the introduction of a calcium binding mutation in EGF11, which decouples the EGF-(10-11) module interface. This study therefore demonstrates that long range calcium-dependent structural perturbations can influence the affinity of Notch for its ligand, in the absence of any post-translational modifications.     

Identification of diagnostic biomarkers for infection in premature neonates

Infection is a leading cause of neonatal morbidity and mortality worldwide. Premature neonates are particularly susceptible to infection because of physiologic immaturity, comorbidity, and extraneous medical interventions. Additionally premature infants are at higher risk of progression to sepsis or severe sepsis, adverse outcomes, and antimicrobial toxicity. Currently initial diagnosis is based upon clinical suspicion accompanied by nonspecific clinical signs and is confirmed upon positive microbiologic culture results several days after institution of empiric therapy. There exists a significant need for rapid, objective, in vitro tests for diagnosis of infection in neonates who are experiencing clinical instability. We used immunoassays multiplexed on microarrays to identify differentially expressed serum proteins in clinically infected and non-infected neonates. Immunoassay arrays were effective for measurement of more than 100 cytokines in small volumes of serum available from neonates. Our analyses revealed significant alterations in levels of eight serum proteins in infected neonates that are associated with inflammation, coagulation, and fibrinolysis. Specifically P- and E-selectins, interleukin 2 soluble receptor alpha, interleukin 18, neutrophil elastase, urokinase plasminogen activator and its cognate receptor, and C-reactive protein were observed at statistically significant increased levels. Multivariate classifiers based on combinations of serum analytes exhibited better diagnostic specificity and sensitivity than single analytes. Multiplexed immunoassays of serum cytokines may have clinical utility as an adjunct for rapid diagnosis of infection and differentiation of etiologic agent in neonates with clinical decompensation.     

Characterization of Ser338 phosphorylation for Raf-1 activation

Raf kinases are essential for regulating cell proliferation, survival, and tumorigenesis. However, the mechanisms by which Raf is activated are still incompletely understood. Phosphorylation plays a critical role in Raf activation in response to mitogens. The present study characterizes phosphorylation of Ser338, a crucial event for Raf-1 activation. Here we report that mutation of Lys375 to Met diminishes phosphorylation of Ser338 on both wild type Raf-1 in cells treated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA) and a constitutively active mutant in which Tyr340/Tyr341 are replaced by 2 aspartic acids, a conserved substitution present in natural B-Raf. The loss of Ser338 phosphorylation in these Raf mutants is not engendered by a mutation-induced conformational change, inasmuch as mutation of another site (Ser471 to Ala) in the activation segment also abolishes Ser338 phosphorylation, whereas both the kinase-dead mutants of Raf-1 are phosphorylated well by active Pak1. Furthermore, our data demonstrate that EGF-stimulated phosphorylation of Ser338 is inhibited by Sorafenib, a Raf kinase inhibitor, but not by the MEK inhibitor U0126. Interestingly, a kinase-dead mutation and Sorafenib also markedly reduce phosphorylation of Ser445 on B-Raf, a site equivalent to Raf-1 Ser338. Finally, our data reveal that Ser338 is phosphorylated on inactive Raf-1 by an active mutant of Raf-1 when they are dimerized in cells and that artificial dimerization of Raf-1 causes Ser338 phosphorylation, accompanied by activation of ERK1/2. Altogether, our data suggest that Ser338 on Raf-1 is autophosphorylated in response to mitogens.