Showing papers on "Proteolytic enzymes published in 2010"

The role of synovitis in pathophysiology and clinical symptoms of osteoarthritis

Abstract: Osteoarthritis (OA), one of the most common rheumatic disorders, is characterized by cartilage breakdown and by synovial inflammation that is directly linked to clinical symptoms such as joint swelling, synovitis and inflammatory pain The gold-standard method for detecting synovitis is histological analysis of samples obtained by biopsy, but the noninvasive imaging techniques MRI and ultrasonography might also perform well The inflammation of the synovial membrane that occurs in both the early and late phases of OA is associated with alterations in the adjacent cartilage that are similar to those seen in rheumatoid arthritis Catabolic and proinflammatory mediators such as cytokines, nitric oxide, prostaglandin E(2) and neuropeptides are produced by the inflamed synovium and alter the balance of cartilage matrix degradation and repair, leading to excess production of the proteolytic enzymes responsible for cartilage breakdown Cartilage alteration in turn amplifies synovial inflammation, creating a vicious circle As synovitis is associated with clinical symptoms and also reflects joint degradation in OA, synovium-targeted therapy could help alleviate the symptoms of the disease and perhaps also prevent structural progression

RTX proteins: a highly diverse family secreted by a common mechanism.

Abstract: Repeats-in-toxin (RTX) exoproteins of Gram-negative bacteria form a steadily growing family of proteins with diverse biological functions. Their common feature is the unique mode of export across the bacterial envelope via the type I secretion system and the characteristic, typically nonapeptide, glycine- and aspartate-rich repeats binding Ca2+ ions. In this review, we summarize the current state of knowledge on the organization of rtx loci and on the biological and biochemical activities of therein encoded proteins. Applying several types of bioinformatic screens on the steadily growing set of sequenced bacterial genomes, over 1000 RTX family members were detected, with the biological functions of most of them remaining to be characterized. Activities of the so far characterized RTX family members are then discussed and classified according to functional categories, ranging from the historically first characterized pore-forming RTX leukotoxins, through the large multifunctional enzymatic toxins, bacteriocins, nodulation proteins, surface layer proteins, up to secreted hydrolytic enzymes exhibiting metalloprotease or lipase activities of industrial interest.

The complex extracellular biology of Streptomyces

Abstract: Streptomycetes, soil-dwelling mycelial bacteria that form sporulating aerial branches, have an exceptionally large number of predicted secreted proteins, including many exported via the twin-arginine transport system. Their use of noncatalytic substrate-binding proteins and hydrolytic enzymes to obtain soluble nutrients from carbohydrates such as chitin and cellulose enables them to interact with other organisms. Some of their numerous secreted proteases participate in developmentally significant extracellular cascades, regulated by inhibitors, which lead to cannibalization of the substrate mycelium biomass to support aerial growth and sporulation. They excrete many secondary metabolites, including important antibiotics. Some of these play roles in interactions with eukaryotes. Surprisingly, some antibiotic biosynthetic enzymes are extracellular. Antibiotic production is often regulated by extracellular signalling molecules, some of which also control morphological differentiation. Amphipathic proteins, assembled with the help of cellulose-like material, are required for both hyphal attachment to surfaces and aerial reproductive growth. Comparative genomic analysis suggests that the acquisition of genes for extracellular processes has played a huge part in speciation. The rare codon TTA, which is present in the key pleiotropic regulatory gene adpA and many pathway-specific regulatory genes for antibiotic production, has a particular influence on extracellular biology.

Biochemical features of microbial keratinases and their production and applications.

Abstract: Keratinases are exciting proteolytic enzymes that display the capability to degrade the insoluble protein keratin. These enzymes are produced by diverse microorganisms belonging to the Eucarya, Bacteria, and Archea domains. Keratinases display a great diversity in their biochemical and biophysical properties. Most keratinases are optimally active at neutral to alkaline pH and 40–60°C, but examples of microbial keratinolysis at alkalophilic and thermophilic conditions have been well documented. Several keratinases have been associated to the subtilisin family of serine-type proteases by analysis of their protein sequences. Studies with specific substrates and inhibitors indicated that keratinases are often serine or metalloproteases with preference for hydrophobic and aromatic residues at the P1 position. Keratinolytic enzymes have several current and potential applications in agroindustrial, pharmaceutical, and biomedical fields. Their use in biomass conversion into biofuels may address the increasing concern on energy conservation and recycling.

Proinflammatory cytokine expression profile in degenerated and herniated human intervertebral disc tissues

Abstract: Acute low back pain is among the most common reasons for which patients seek medical care, with estimates of annual economic impact as high as $200 billion in the US (1). Degeneration of the intervertebral disc (IVD) is a common cause of such pain and disability, characterized by anatomic, morphologic, and biochemical changes including altered expression of both matrix metalloproteinases and proinflammatory cytokines (2). Elevated levels of molecular mediators of inflammation have been described in pathologic disc tissue, increasing with grade of degeneration (3,4). Such findings have been observed for both interleukin-1 (IL-1) and tumor necrosis factor α (TNFα), both of which have established roles in regulating nitric oxide (NO) and prostaglandin production, metalloproteinase expression, and apoptosis, all changes that may contribute to progressive pathology of the IVD. The inflammatory and immune activation profile exhibited by a herniated disc may be distinct from that observed in IVD degeneration and may differ further among different extents of herniation from protrusion through sequestration. Histologic evaluation of herniated disc tissue has revealed prominent infiltration of inflammatory cells, most markedly macrophages (5–10). Surgical specimens of herniated disc tissue also exhibit a greater presence of TNFα, IL-1β, and IL-6 compared with autopsy control tissue specimens (3,4), and explant cultures demonstrate a heightened release of IL-6, IL-8, prostaglandin E2 (PGE2), and NO from herniated disc tissue compared with control tissue (11). Recent studies have also revealed greater IL-12 and interferon-γ (IFNγ) expression in herniated disc fragments compared with bulging discs that remain contained within the disc space by an intact anulus fibrosus (12). Most of these cytokines are macrophage products, which is consistent with the greater presence of macrophages documented for herniated disc tissues (9,10). These cytokines can promote lymphocyte activation and differentiation while also recruiting additional macrophages and activating phagocytosis and secretion of proteolytic enzymes. Activated Th1 lymphocytes produce IFNγ that assists in further macrophage recruitment and activation, and the mechanism eliciting the heightened IFNγ expression in herniated disc fragments may represent a specific immune response against herniated nucleus pulposus tissue. The immune privilege of the nucleus pulposus occurs by both vascular isolation and a biochemical phenotype with the Fas ligand causing infiltrating T lymphocyte apoptosis. Support for this theory is found in regional lymph node accumulation of lymphocytes after exposure to autologous nucleus pulposus (13), lymphocyte accumulation in the IVD after anulus fibrosus injury (6), and immunoglobulin and complement membrane attack complex deposition in human herniated disc tissue (14–16). Recent studies have revealed the importance of Th17 lymphocytes in a range of inflammatory pathologies and autoimmune diseases, including rheumatoid arthritis, Crohn’s disease, and multiple sclerosis (17,18). IL-17 is a proinflammatory cytokine produced primarily by Th17 lymphocytes activated in response to IL-23 production. IL-17 can promote inflammation by different signaling cascades, some modulated by other lymphocyte products such as IFNγ (19), and thereby promote the synthesis of other cytokines, proteases, NO, and PGE2. Given the demonstrated presence of infiltrating and activated lymphocytes in herniated disc fragments and the potential importance of immune system activation by herniated disc tissues, it is of interest to determine a potential role of Th17 lymphocytes in mediating this process. The objective of this study was to evaluate differences in the immunophenotype of the IVD fragments obtained from patients undergoing surgery for treatment of degenerative disc disease and lumbar disc herniation. The presence of Th17 lymphocyte markers in IVD pathology has not yet been investigated, and a primary goal of this study was to use immunohistochemistry to evaluate the molecular markers of different inflammatory and immune cells alongside expression of the lymphocyte product IL-17 in this surgically obtained tissue. Macrophage and lymphocyte markers as well as other mediators of inflammation known to be of relevance to IVD disease, including IL-6, IFNγ, IL-12, IL-10, and IL-4, were also evaluated to more comprehensively characterize the inflammation pattern. All markers were evaluated in degenerated and herniated disc fragments as well as in cadaveric nucleus pulposus and anulus fibrosus tissues with little to no evidence of degeneration. Results of this study reveal a high expression level of IL-17 in surgical tissues obtained from both degenerated and herniated IVDs, but not from autopsy tissue, suggesting a role of Th17-mediated inflammatory processes in IVD pathology. Additional results reveal the heightened presence of immune-activating IFNγ in herniated disc fragments, with concomitant greater macrophage infiltration and IL-6 expression as compared with degenerated tissues. These findings illustrate a pattern of immune activation by herniated disc tissue involving macrophage infiltration and activation, and further suggest that a novel lineage of Th17 lymphocytes may also be involved in both forms of IVD pathology.

Molecular mediators of angiogenesis.

Abstract: Angiogenesis, or the formation of new blood vessels from the preexisting vasculature, is a key component in numerous physiologic and pathologic responses and has broad impact in many medical and surgical specialties. In this review, we discuss the key cellular steps that lead to the neovascularization of tissues and highlight the main molecular mechanisms and mediators in this process. We include discussions on proteolytic enzymes, cell-matrix interactions, and pertinent cell signaling pathways and end with a survey of the mechanisms that lead to the stabilization and maturation of neovasculatures.

The pathogenesis of tendinopathy: balancing the response to loading

Abstract: Tendons are designed to withstand considerable loads. Mechanical loading of tendon tissue results in upregulation of collagen expression and increased synthesis of collagen protein, the extent of which is probably regulated by the strain experienced by the resident fibroblasts (tenocytes). This increase in collagen formation peaks around 24 h after exercise and remains elevated for about 3 days. The degradation of collagen proteins also rises after exercise, but seems to peak earlier than the synthesis. Despite the ability of tendons to adapt to loading, repetitive use often results in injuries, such as tendinopathy, which is characterized by pain during activity, localized tenderness upon palpation, swelling and impaired performance. Tendon histological changes include reduced numbers and rounding of fibroblasts, increased content of proteoglycans, glycosaminoglycans and water, hypervascularization and disorganized collagen fibrils. At the molecular level, the levels of messenger RNA for type I and III collagens, proteoglycans, angiogenic factors, stress and regenerative proteins and proteolytic enzymes are increased. Tendon microrupture and material fatigue have been suggested as possible injury mechanisms, thus implying that one or more 'weak links' are present in the structure. Understanding how tendon tissue adapts to mechanical loading will help to unravel the pathogenesis of tendinopathy.

Dichotomy of gingipains action as virulence factors: from cleaving substrates with the precision of a surgeon’s knife to a meat chopper‐like brutal degradation of proteins

Abstract: Arguably periodontitis is the most prevalent bacteria-driven chronic inflammatory disease of the humankind. The incidence of periodontal disease has been reported to range from 30% of the population in developed countries (154) to over 70% of the population in developing countries (198) with severe disease inflicting 7% to 15% of human population worldwide (41, 67, 154). Periodontitis is a major cause of loss of teeth, and is also implicated in the onset, development and/or progression of systemic diseases such as cardiovascular diseases (68), rheumatoid arthritis (27, 207, 224) and Alzheimer disease (87-89). The involvement of bacteria in the etiology of periodontitis is unquestionable but the composition of the causative species is still being debated. Presently, over 500 bacterial species have been detected in periodontal plaque (160) but only a handful, including Porphyromonas gingivalis has been implicated as major pathogens of chronic periodontitis (208), the most prevailing form of periodontal disease. This bacterium produces a variety of virulence factors including, bioactive metabolic products, fimbriae, and an array of proteolytic enzymes (107). Among the proteolytic enzymes, cysteine proteases referred to as gingipains are responsible for at least 85% of total proteolytic activity exerted by various strains of P. gingivalis (171) and are considered major contributors to the pathogenic potential of P. gingivalis. Indeed, three lines of evidence, including immunization, analysis of knockout strains, and testing of specific protease inhibitors using murine models of infection or bone loss clearly indicate that gingipains are absolutely essential for bacterial survival/proliferation in vivo and for the pathological outcome of the experimental infection (Table 1). Table 1 Gingipains are essential virulence factor of P. gingivalis indispensable for pathological outcome of experimental infection. Gingipains, including arginine-specific gingipains (Arg-gingipain-A, RgpA and Arg-gingipain-B, RgpB) and lysine-specific gingipain (Lys-gingipain, Kgp), are encoded by three different genes referred to as rgpA, rgpB and kgp, which are conserved among laboratory and clinical strains of P. gingivalis (26). The rgpA and rgpB genes translation products, RgpA and RgpB, share a basically identical caspase-like protease domain with specificity restricted to Arg-Xaa peptide bonds and an Ig-like domain. In RgpA, the protease and the Ig-like domains are followed by a large C-terminal extension known as the hemagglutinin-adhesin domains. Similarly, the kgp gene translation product, Kgp, consists of a catalytic domain with selectivity for Lys-Xaa peptide bonds followed by a C-terminal extension similar to that in RgpA. Except for a short C-terminal domain shared with RgpA and Kgp and several other secretory proteins (136, 189), the hemagglutinin-adhesin domains are absent in RgpB. During the secretion process, progingipains are subjected to extensive posttranslational proteolytic processing and glycosylation. Finally, they are secreted either as a monomeric form (RgpB) or as a non-covalent but stable complexes of protease and hemagglutinin-adhesin domains (RgpA and Kgp), which are either predominantly attached to the bacterial surface or released into the medium in a soluble form depending on P. gingivalis strain (Fig. 1). The varied external localization of these multi-functional gingipain complexes makes them the perfect tool for manipulation of the host immune system by P. gingivalis. Fig. 1 Schematic diagram of discrete steps in gingipain secretion, processing, posttranslational maturation, and assembly of extracellular gingipain complexes. Western blot analysis (upper corners) of the whole culture of P. gingivalis strain HG66 and W83 using ... In this review, we will discuss how gingipains participate in sequential phases of the disease process starting from (i) adherence and colonization, through (ii) nutrient acquisition, (iii) neutralization of host defenses and manipulation of inflammatory response, to (iv) tissue destruction, invasion and dissemination to systemic sites. Over the past 15 years, gingipains have been the subject of more than 20 reviews, including a few truly outstanding comprehensive compendiums on the role of these enzymes as virulence factors of P. gingivalis (35, 84, 193). Consequently, in this paper, we will focus on the most recent investigations and cite the previous reviews where appropriate. For references on the early work on the gingipains, we invite the reader to peruse reviews by Imamura (69, 73), Kadowaki & Yamamoto (84), Potempa and colleagues (169, 172), Nakayama (131), and O'Brien-Simpson and colleagues (144).

Extracellular Matrix, Inflammation, and the Angiogenic Response

Abstract: Inflammation and angiogenesis are frequently coupled in pathological situations such as atherosclerosis, diabetes, and arthritis. The inflammatory response increases capillary permeability and induces endothelial activation, which, when persistent, results in capillary sprouting. This inflammation-induced angiogenesis and the subsequent remodelling steps are in large part mediated by extracellular matrix (ECM) proteins and proteases. The focal increase in capillary permeability is an early consequence of inflammation, and results in the deposition of a provisional fibrin matrix. Subsequently, ECM turnover by proteases permits an invasive program by specialized endothelial cells whose phenotype can be regulated by inflammatory stimuli. ECM activity also provides specific mechanical forces, exposes cryptic adhesion sites, and releases biologically active fragments (matrikines) and matrix-sequestered growth factors, all of which are critical for vascular morphogenesis. Further matrix remodelling and vascular regression contribute to the resolution of the inflammatory response and facilitate tissue repair.

Tumor-Associated Macrophages (TAM) as Major Players of the Cancer-Related Inflammation

Abstract: The microenvironment of solid tumors is characterized by a reactive stroma with an abundance of inflammatory mediators and leukocytes, dysregulated vessels and proteolytic enzymes. TAM, major players in the connection between inflammation and cancer, summarize a number of functions (e.g., promotion of tumor cell proliferation and angiogenesis, incessant matrix turnover, repression of adaptive immunity), which ultimately have an important impact on disease progression. Thus, together with other myeloid-related cells present at the tumor site (Tie2 macrophages and MDSCs), TAM represent an attractive target of novel biological therapies of tumors.

Lung dysfunction following cardiopulmonary bypass.

Abstract: Background: It is well known by now that during open heart surgery many detrimental factors are involved in lung injury. The influence mainly of cardiopulmonary bypass (CPB), anesthesia, hypothermia, operation itself, as well as medication and transfusion, can cause a diffuse injury in the lungs, which most often leads to a postoperative pulmonary edema and abnormal gas exchange. Methods: We performed an unrestricted search of Pubmed Medline and EMBASE from 1966 through 2008. Clinical, experimental, basic science, and review papers were included. Results: The hypothesis that a systemic inflammatory reaction takes place after the use of CPB, could explain most of these effects influences in the lung. On the other hand, the release of various pro-inflammatory cytokines like TNF-a, IL-1, IL-2, IL-6, IL-8, and endotoxin during CPB can lead to the entrapment of neutrophils in the pulmonary capillaries. Consequently, the following chain of reactions is likely to occur: an endothelial cell swelling, plasma and protein extravasation into the interstitial tissue, release of proteolytic enzymes, congestion of the alveoli with plasma, erythrocytes and inflammatory debris. Conclusion: In this review we highlight the possible pathophysiological mechanisms implicated in the observed postoperative lung dysfunction.(J Card Surg 2010;25:47-55)

Important role of integrins in the cancer biology

Abstract: Adhesion of breast cancer cells is supported by various integrins. Cell adhesion is critical for maintenance of both three-dimensional and normal function of these tissues. Several integrins have been shown to have higher expression levels in metastatic cancers and have been implicated in degrading basement membrane by interacting with proteolytic enzymes. This suggests that a group of integrins plays an important role in migration and invasion through the remodeling of the extracellular matrix. In this review, we highlight recent advances in our understanding of how integrins regulate breast cancer through modulation of the actin cytoskeleton and the mechanisms that regulate this process. Also, we highlight the importance of integrin-binding proteins in cell migration and mechanisms that operate in invasive cells, during breast cancer progression.

Using enzymes to remove biofilms of bacterial isolates sampled in the food-industry

Abstract: The aim of this study was to analyze the cleaning efficiency of polysaccharidases and proteolytic enzymes against biofilms of bacterial species found in food industry processing lines and to study ...

Rapid and sustained improvement in bone and cartilage turnover markers with the anti-interleukin-6 receptor inhibitor tocilizumab plus methotrexate in rheumatoid arthritis patients with an inadequate response to methotrexate: results from a substudy of the multicenter double-blind, placebo-controlled trial of tocilizumab in inadequate responders to methotrexate alone.

Abstract: Objective To investigate the effects of tocilizumab (TCZ) added to a stable dosage of methotrexate (MTX) on biochemical markers of bone and cartilage metabolism in patients in the multicenter double-blind, placebo-controlled OPTION (Tocilizumab Pivotal Trial in Methotrexate Inadequate Responders) study who have moderate-to-severe rheumatoid arthritis (RA) and an inadequate response to MTX. Methods Included in this study were 416 of the 623 patients with active RA enrolled in the OPTION study. Patients were randomized to receive TCZ (4 mg/kg or 8 mg/kg) or placebo intravenously every 4 weeks, with MTX continued at the stable prestudy doses (10–25 mg for 20 weeks, with a final followup at week 24). Serum biochemical markers of bone formation (osteocalcin, N-terminal propeptide of type I collagen [PINP]), bone resorption (C-terminal crosslinking telopeptide of type I collagen [CTX-I] and C-terminal crosslinking telopeptide of type I collagen generated by matrix metalloproteinases [ICTP]), cartilage metabolism (N-terminal propeptide of type IIA collagen [PIIANP]), collagen helical peptide [HELIX-II]), and matrix metalloproteinase 3 (MMP-3) were measured at baseline and at weeks 4, 16, and 24. Results TCZ induced marked dose-dependent reductions in PIIANP, HELIX-II, and MMP-3 levels at week 4 that were maintained until week 24, an effect associated with increased levels of bone formation markers that were significant as compared with placebo only for PINP and only at 4 weeks (P < 0.01 for both TCZ doses). TCZ induced significant decreases in the bone degradation markers CTX-I and ICTP, providing initial evidence of a beneficial effect on bone turnover. TCZ-treated patients who met the American College of Rheumatology 50% improvement criteria (achieved an ACR50 response) or achieved clinical remission (as determined by a Disease Activity Score in 28 joints <2.6) at week 24 had greater reductions in ICTP, HELIX-II, and MMP-3 levels as compared with ACR50 nonresponders. Conclusion TCZ combined with MTX reduces systemic bone resorption, cartilage turnover, and proteolytic enzyme MMP-3 levels, which provides evidence of a limitation of joint damage and possible beneficial effects on skeletal structure in patients with established moderate-to-severe RA.

Influenza Virus—Cytokine-Protease Cycle in the Pathogenesis of Vascular Hyperpermeability in Severe Influenza

Abstract: Background Severe influenza is characterized by cytokine storm and multiorgan failure with edema. The aim of this study was to define the impact of the cytokine storm on the pathogenesis of vascular hyperpermeability in severe influenza. Methods Weanling mice were infected with influenza A WSN/33(H1N1) virus. The levels of proinflammatory cytokines, tumor necrosis factor (TNF) alpha, interleukin (IL) 6, IL-1beta, and trypsin were analyzed in the lung, brain, heart, and cultured human umbilical vein endothelial cells. The effects of transcriptional inhibitors on cytokine and trypsin expressions and viral replication were determined. Results Influenza A virus infection resulted in significant increases in TNF-alpha, IL-6, IL-1beta, viral hemagglutinin-processing protease trypsin levels, and viral replication with vascular hyperpermeability in lung and brain in the first 6 days of infection. Trypsin upregulation was suppressed by transcriptional inhibition of cytokines in vivo and by anti-cytokine antibodies in endothelial cells. Calcium mobilization and loss of tight junction constituent, zonula occludens-1, associated with cytokine- and trypsin-induced endothelial hyperpermeability were inhibited by a protease-activated receptor-2 antagonist and a trypsin inhibitor. Conclusions The influenza virus-cytokine-protease cycle is one of the key mechanisms of vascular hyperpermeability in severe influenza.

Design, synthesis, and imaging of an activatable photoacoustic probe.

Abstract: Photoacoustic tomography is a rapidly growing imaging modality that can provide images of high spatial resolution and high contrast at depths up to 5 cm. We report here the design, synthesis, and evaluation of an activatable probe that shows great promise for enabling detection of the cleaved probe in the presence of high levels of nonactivated, uncleaved probe, a difficult task to attain in absorbance-based modality. Before the cleavage by its target, proteolytic enzyme MMP-2, the probe, an activatable cell-penetrating peptide, Ceeee[Ahx]PLGLAGrrrrrK, labeled with two chromophores, BHQ3 and Alexa750, shows photoacoustic signals of similar intensity at the two wavelengths corresponding to the absorption maxima of the chromophores, 675 and 750 nm. Subtraction of the images taken at these two wavelengths makes the probe effectively photoacoustically silent, as the signals at these two wavelengths essentially cancel out. After the cleavage, the dye associated with the cell-penetrating part of the probe, BHQ3, accumulates in the cells, while the other dye diffuses away, resulting in photoacoustic signal seen at only one of the wavelengths, 675 nm. Subtraction of the photoacoustic images at two wavelengths reveals the location of the cleaved (activated) probe. In the search for the chromophores that are best suited for photoacoustic imaging, we have investigated the photoacoustic signals of five chromophores absorbing in the near-infrared region. We have found that the photoacoustic signal did not correlate with the absorbance and fluorescence of the molecules, as the highest photoacoustic signal arose from the least absorbing quenchers, BHQ3 and QXL 680.

Immunoelectron microscopic evidence for Tetherin/BST2 as the physical bridge between HIV-1 virions and the plasma membrane.

Abstract: Tetherin/BST2 was identified in 2008 as the cellular factor responsible for restricting HIV-1 replication at a very late stage in the lifecycle. Tetherin acts to retain virion particles on the plasma membrane after budding has been completed. Infected cells that express large amounts of tetherin display large strings of HIV virions that remain attached to the plasma membrane. Vpu is an HIV-1 accessory protein that specifically counteracts the restriction to virus release contributed by tetherin. Tetherin is an unusual Type II transmembrane protein that contains a GPI anchor at its C-terminus and is found in lipid rafts. The leading model for the mechanism of action of tetherin is that it functions as a direct physical tether bridging virions and the plasma membrane. However, evidence that tetherin functions as a physical tether has thus far been indirect. Here we demonstrate by biochemical and immunoelectron microscopic methods that endogenous tetherin is present on the viral particle and forms a bridge between virion particles and the plasma membrane. Endogenous tetherin was found on HIV particles that were released by partial proteolytic digestion. Immunoelectron microscopy performed on HIV-infected T cells demonstrated that tetherin forms an apparent physical link between virions and connects patches of virions to the plasma membrane. Linear filamentous strands that were highly enriched in tetherin bridged the space between some virions. We conclude that tetherin is the physical tether linking HIV-1 virions and the plasma membrane. The presence of filaments with which multiple molecules of tetherin interact in connecting virion particles is strongly suggested by the morphologic evidence.

A synergistic approach to protein crystallization: combination of a fixed-arm carrier with surface entropy reduction.

Abstract: Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier-driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail.

Runx2 transcriptome of prostate cancer cells: insights into invasiveness and bone metastasis

Abstract: Prostate cancer (PCa) cells preferentially metastasize to bone at least in part by acquiring osteomimetic properties. Runx2, an osteoblast master transcription factor, is aberrantly expressed in PCa cells, and promotes their metastatic phenotype. The transcriptional programs regulated by Runx2 have been extensively studied during osteoblastogenesis, where it activates or represses target genes in a context-dependent manner. However, little is known about the gene regulatory networks influenced by Runx2 in PCa cells. We therefore investigated genome wide mRNA expression changes in PCa cells in response to Runx2. We engineered a C4-2B PCa sub-line called C4-2B/Rx2dox, in which Doxycycline (Dox) treatment stimulates Runx2 expression from very low to levels observed in other PCa cells. Transcriptome profiling using whole genome expression array followed by in silico analysis indicated that Runx2 upregulated a multitude of genes with prominent cancer associated functions. They included secreted factors (CSF2, SDF-1), proteolytic enzymes (MMP9, CST7), cytoskeleton modulators (SDC2, Twinfilin, SH3PXD2A), intracellular signaling molecules (DUSP1, SPHK1, RASD1) and transcription factors (Sox9, SNAI2, SMAD3) functioning in epithelium to mesenchyme transition (EMT), tissue invasion, as well as homing and attachment to bone. Consistent with the gene expression data, induction of Runx2 in C4-2B cells enhanced their invasiveness. It also promoted cellular quiescence by blocking the G1/S phase transition during cell cycle progression. Furthermore, the cell cycle block was reversed as Runx2 levels declined after Dox withdrawal. The effects of Runx2 in C4-2B/Rx2dox cells, as well as similar observations made by employing LNCaP, 22RV1 and PC3 cells, highlight multiple mechanisms by which Runx2 promotes the metastatic phenotype of PCa cells, including tissue invasion, homing to bone and induction of high bone turnover. Runx2 is therefore an attractive target for the development of novel diagnostic, prognostic and therapeutic approaches to PCa management. Targeting Runx2 may prove more effective than focusing on its individual downstream genes and pathways.

Cathepsin B in synovial cells at the site of joint destruction in rheumatoid arthritis

Abstract: Based on the concept that proteolytic enzymes, like cathepsins, are associated with tissue destruction, we investigated the expression of the matrix-degrading cysteine proteinase cathepsin B in synovial tissues from the joints of patients with rheumatoid arthritis. The data indicate an enhanced transcription of cathepsin B in synovial cells when compared with normal fibroblasts, cathepsin B-producing epithelial tumor cells (SW1116), or fibroblasts derived from inflamed tonsils. Immunolocalization of cathepsin B appeared to be restricted mainly to the synovial cells attached to cartilage and bone at sites of rheumatoid joint erosion.

Secretion and Dipeptidyl Peptidase-4-Mediated Metabolism of Incretin Hormones after a Mixed Meal or Glucose Ingestion in Obese Compared to Lean, Nondiabetic Men.

Abstract: Context: Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are cleaved by dipeptidyl peptidase-4 (DPP-4); plasma activity of DPP-4 may be increased in obesity. The impact of this increase on incretin hormone secretion and metabolism is not known. Objective: The aim of the study was to assess incretin hormone secretion and degradation in lean and obese nondiabetic subjects. Design, Settings, and Participants: We studied the ingestion of a mixed meal (560 kcal) or oral glucose (2 g/kg) in healthy lean (n = 12; body mass index, 20-25 kg/m(2)) or obese (n = 13; body mass index, 30-35 kg/m(2)) males at a University Clinical Research Unit. Main Outcome Measures: We measured the area under the curve of plasma intact (i) and total (t) GIP and GLP-1 after meal ingestion and oral glucose. Results: Plasma DPP-4 activity was higher in the obese subjects (38.5 +/- 3.0 vs. 26.7 +/- 1.6 mmol/min . mul; P = 0.002). Although GIP secretion (AUCtGIP) was not reduced in obese subjects after meal ingestion or oral glucose, AUCiGIP was lower in obese subjects (8.5 +/- 0.6 vs. 12.7 +/- 0.9 nmol/liter x 300 min; P < 0.001) after meal ingestion. GLP-1 secretion (AUCtGLP-1) was reduced in obese subjects after both meal ingestion (7.3 +/- 0.9 vs. 10.0 +/- 0.6 nmol/liter x 300 min; P = 0.022) and oral glucose (6.6 +/- 0.8 vs. 9.6 +/- 1.1 nmol/liter x 180 min; P = 0.035). iGLP-1 was reduced in parallel to tGLP-1. Conclusions: 1) Release and degradation of the two incretin hormones show dissociated changes in obesity: GLP-1 but not GIP secretion is lower after meal ingestion and oral glucose, whereas GIP but not GLP-1 metabolism is increased after meal ingestion. 2) Increased plasma DPP-4 activity in obesity is not associated with a generalized augmented incretin hormone metabolism.

Molecular Pathogenesis of Necrotizing Fasciitis

Abstract: Necrotizing fasciitis, also known as the flesh-eating disease, is a severe invasive infection associated with very high rates of human morbidity and mortality. It is most commonly caused by group A Streptococcus(GAS), a versatile human pathogen that causes diseases ranging in severity from uncomplicated pharyngitis (or strep throat) to life-threatening infections such as necrotizing fasciitis. Herein, we review recent discoveries bearing on the molecular pathogenesis of GAS necrotizing fasciitis. Importantly, the integration of new technologies and the development of human-relevant animal models have markedly expanded our understanding of the key pathogen-host interactions underlying GAS necrotizing fasciitis. For example, we now know that GAS organisms secrete a variety of proteases that disrupt host tissue and that these proteolytic enzymes are regulated by multiple transcriptional and posttranslational processes. This pathogenesis knowledge will be crucial to supporting downstream efforts that seek to ...

Cleavage of IgGs by proteases associated with invasive diseases: an evasion tactic against host immunity?

Abstract: The effective functioning of immunoglobulins and IgG mAbs in removing pathological cells requires that the antigen binding regions and the Fc (effector) domain act in concert. The hinge region that connects these domains itself presents motifs that engage Fc receptors on immune effector cells to achieve cell lysis. In addition, sequences in the lower hinge/CH2 and further down the CH2 region are involved in C1q binding and complement-mediated cell killing. Proteolytic enzymes of little relevance to human physiology were successfully used for decades to generate fragments of IgGs for reagent and therapeutic use. It was subsequently noted that tumor-related and microbial proteases also cleaved human IgG specifically in the hinge region. We have shown previously that the “nick” of just one of the lower hinge heavy chains of IgG unexpectedly prevented many effector functions without impacting antigen binding. Of interest, related single-cleaved IgG breakdown products were detected in breast carcinoma extracts...