Glycophospholipid membrane anchor attachment. Molecular analysis of the cleavage/attachment site.

The COOH terminus of decay accelerating factor (DAF) contains a signal that directs attachment of a glycophosphatidylinositol (GPI) membrane anchor in a process involving proteolytic removal of 17-31 COOH-terminal residues. Previous work suggested that two elements are required for anchor addition, a COOH-terminal hydrophobic domain (the GPI signal) and an element located NH2-terminal to it, postulated to be the cleavage/attachment site. Using [3H]ethanolamine (a component of the anchor) to tag the COOH terminus, we isolated and sequenced a COOH-terminal tryptic peptide, thereby identifying Ser-319 as the COOH-terminal residue attached to the GPI anchor. This indicates that a 28-residue peptide is removed during processing and localizes the cleavage/attachment site precisely to the region previously shown to be required for anchor attachment (between 10 and 20 residues NH2-terminal to the hydrophobic domain). Since DAF contains multiple cryptic cleavage/attachment sites, we used a GPI-linked human growth hormone-DAF fusion to study the structural requirements for cleavage/attachment. Our results show that while sequences immediately NH2-terminal to the attachment site are not required for anchor addition, deletion of Ser-319 abolishes both anchor attachment and transport to the cell surface. Systematic replacement of the attachment site serine with all possible amino acids indicated that alanine, aspartate, asparagine, glycine, or serine efficiently support GPI anchor attachment while valine and glutamate are partially effective. All other substitutions including cysteine (permitted at the attachment site in other GPI-anchored proteins) abolish both GPI anchor attachment and transport to the cell surface, resulting in accumulation of uncleaved fusion protein in internal compartments (endoplasmic reticulum and Golgi). These results support the general rule that the residue at the cleavage/attachment site must be small. Further, addition of a GPI anchor appears to be necessary for transport to the cell surface in transfected COS cells.     

Neuronal nitric oxide synthase signaling within cardiac myocytes targets phospholamban

Studies have shown that neuronal nitric oxide synthase (nNOS, NOS1) knockout mice (NOS1^–/–) have increased or decreased contractility, but consistently have found a slowed rate of intracellular Ca²⁺ ([Ca²⁺]_i) decline and relengthening. Contraction and [Ca²⁺]_i decline are determined by many factors, one of which is phospholamban (PLB). The purpose of this study is to determine the involvement of PLB in the NOS1-mediated effects. Force-frequency experiments were performed in trabeculae isolated from NOS1^–/– and wild-type (WT) mice. We also simultaneously measured Ca²⁺ transients (Fluo-4) and cell shortening (edge detection) in myocytes isolated from WT, NOS1^–/–, and PLB^–/– mice. NOS1^–/– trabeculae had a blunted force-frequency response and prolonged relaxation. We observed similar effects in myocytes with NOS1 knockout or specific NOS1 inhibition with S-methyl-L-thiocitrulline (SMLT) in WT myocytes (i.e., decreased Ca²⁺ transient and cell shortening amplitudes and prolonged decline of [Ca²⁺]_i). Alternatively, NOS1 inhibition with SMLT in PLB^–/– myocytes had no effect. Acute inhibition of NOS1 with SMLT in WT myocytes also decreased basal PLB serine16 phosphorylation. Furthermore, there was a decreased SR Ca²⁺ load with NOS1 knockout or inhibition, which is consistent with the negative contractile effects. Perfusion with FeTPPS (peroxynitrite decomposition catalyst) mimicked the effects of NOS1 knockout or inhibition. β-Adrenergic stimulation restored the slowed [Ca²⁺]_i decline in NOS1^–/– myocytes, but a blunted contraction remained, suggesting additional protein target(s). In summary, NOS1 inhibition or knockout leads to decreased contraction and slowed [Ca²⁺]_i decline, and this effect is absent in PLB^–/– myocytes. Thus NOS1 signaling modulates PLB serine16 phosphorylation, in part, via peroxynitrite. NOS1; peroxynitrite; force-frequency response     

Hip joint replacement surgery for idiopathic osteoarthritis aggregates in families

In order to determine whether there is a genetic component to hip or knee joint failure due to idiopathic osteoarthritis (OA), we invited patients (probands) undergoing hip or knee arthroplasty for management of idiopathic OA to provide detailed family histories regarding the prevalence of idiopathic OA requiring joint replacement in their siblings. We also invited their spouses to provide detailed family histories about their siblings to serve as a control group. In the probands, we confirmed the diagnosis of idiopathic OA using American College of Rheumatology criteria. The cohorts included the siblings of 635 probands undergoing total hip replacement, the siblings of 486 probands undergoing total knee replacement, and the siblings of 787 spouses. We compared the prevalence of arthroplasty for idiopathic OA among the siblings of the probands with that among the siblings of the spouses, and we used logistic regression to identify independent risk factors for hip and knee arthroplasty in the siblings. Familial aggregation for hip arthroplasty, but not for knee arthroplasty, was observed after controlling for age and sex, suggesting a genetic contribution to end-stage hip OA but not to end-stage knee OA. We conclude that attempts to identify genes that predispose to idiopathic OA resulting in joint failure are more likely to be successful in patients with hip OA than in those with knee OA.     

Ancient large-scale genome duplications: phylogenetic and linkage analyses shed light on chordate genome evolution

Paralogous genes from several families were found in four human chromosome regions (4p16, 5q33-35, 8p12-21, and 10q24-26), suggesting that their common ancestral region underwent several rounds of large- scale duplication. Searches in the EMBL databases, followed by phylogenetic analyses, showed that cognates (orthologs) of human duplicated genes can be found in other vertebrates, including bony fishes. In contrast, within each family, only one gene showing the same high degree of similarity with all the duplicated mammalian genes was found in nonvertebrates (echinoderms, insects, nematodes). This indicates that large-scale duplications occurred after the echinoderms/chordates split and before the bony vertebrate radiation. It has been suggested that two rounds of gene duplication occurred in the vertebrate lineage after the separation of Amphioxus and craniate (vertebrates + Myxini) ancestors. Before these duplications, the genes that have led to the families of paralogous genes in vertebrates must have been physically linked in the craniate ancestor. Linkage of some of these genes can be found in the Drosophila melanogaster and Caenorhabditis elegans genomes, suggesting that they were linked in the triploblast Metazoa ancestor.      

Encounter with New Resources Causes Polarized Growth of the Cord-Forming Basidiomycete Phanerochaete velutina on Soil

Abstract The development and physiology of cord-forming saprotrophic basidiomycetes, which form extensive and persistent mycelial networks in woodland ecosystems, can be conveniently studied on non-sterile soil in laboratory microcosms mimicking field conditions. Morphological responses of Phanerochaete velutina mycelial systems to resource encounters, and decay partitioning following encounters, varied according to whether simulated woody litter was unsterile or autoclaved and on whether encounter took place at the mycelial foraging front or behind the margin (simulating litter fall onto established systems in the field). Results show that encounter of discrete resources by P. velutina is rapidly communicated to the entire mycelial system; that resource capture takes high priority at the expense of continued system extension and decay-derived carbon reallocation; and that polarized growth toward newly encountered resources, previously considered to occur infrequently with this species, may be readily detected using image analysis techniques. Potential advantages of polarized development of P. velutina are discussed.     

Cultured endothelial cells produce heparinlike inhibitor of smooth muscle cell growth

Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 μg/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.     

Ensemble attribute profile clustering: discovering and characterizing groups of genes with similar patterns of biological features

Background  

Ensemble attribute profile clustering is a novel, text-based strategy for analyzing a user-defined list of genes and/or proteins. The strategy exploits annotation data present in gene-centered corpora and utilizes ideas from statistical information retrieval to discover and characterize properties shared by subsets of the list. The practical utility of this method is demonstrated by employing it in a retrospective study of two non-overlapping sets of genes defined by a published investigation as markers for normal human breast luminal epithelial cells and myoepithelial cells.     

Anti-Angiogenic/Vascular Effects of the mTOR Inhibitor Everolimus Are Not Detectable by FDG/FLT-PET

Noninvasive functional imaging of tumors can provide valuable early-response biomarkers, in particular, for targeted chemotherapy. Using various experimental tumor models, we have investigated the ability of positron emission tomography (PET) measurements of 2-deoxy-2-[¹⁸F]fluoro-glucose (FDG) and 3′-deoxy-3′-[¹⁸F]fluorothymidine (FLT) to detect response to the allosteric mammalian target of rapamycin (mTOR) inhibitor everolimus. Tumor models were declared sensitive (murine melanoma B16/BL6 and human lung H596) or relatively insensitive (human colon HCT116 and cervical KB31), according to the IC₅₀ values (concentration inhibiting cell growth by 50%) for inhibition of proliferation in vitro (<10 nM and >1 µM, respectively). Everolimus strongly inhibited growth of the sensitive models in vivo but also significantly inhibited growth of the insensitive models, an effect attributable to its known anti-angiogenic/vascular properties. However, although tumor FDG and FLT uptake was significantly reduced in the sensitive models, it was not affected in the insensitive models, suggesting that endothelial-directed effects could not be detected by these PET tracers. Consistent with this hypothesis, in a well-vascularized orthotopic rat mammary tumor model, other antiangiogenic agents also failed to affect FDG uptake, despite inhibiting tumor growth. In contrast, the cytotoxic patupilone, a microtubule stabilizer, blocked tumor growth, and markedly reduced FDG uptake. These results suggest that FDG/FLT-PET may not be a suitable method for early markers of response to antiangiogenic agents and mTOR inhibitors in which anti-angiogenic/vascular effects predominate because the method could provide false-negative responses. These conclusions warrant clinical testing.     

Uric acid is a strong independent predictor of renal dysfunction in patients with rheumatoid arthritis

Introduction  

Recent evidence suggests that uric acid (UA), regardless of crystal deposition, may play a direct pathogenic role in renal disease. We have shown that UA is an independent predictor of hypertension and cardiovascular disease (CVD), and that CVD risk factors associate with renal dysfunction, in patients with rheumatoid arthritis (RA). In this study we investigated whether UA associates with renal dysfunction in patients with RA and whether such an association is independent or mediated through other comorbidities or risk factors for renal impairment.