Human placental N-acetyl-beta-D-hexosaminidase isozymes. Activity toward native hyaluronic acid.

The activity of purified human hexosaminidases A and B toward hyaluronic acid (HA) isolated from cultured human skin fibroblasts was investigated. The cleavage of N-acetylglucosaminyl residues to monosaccharide N-acetylglucosamines by hexosaminidase isozymes was determined in the presence and absence of purified human β-glucuronidase. The pH optima of this reaction, with and without β-glucuronidase, were 4.5 for hexosaminidase A and 4.0 for hexosaminidase B. The hydrolysis of HA by both hexosaminidase isozymes proceeds linearily for at least 18 h in the presence of β-glucuronidase. Concentrations of 0.5–5 units of either isozyme showed a linear relationship with rate of hydrolysis. Without β-glucuronidase, hexosaminidase only cleaved the terminal N-acetylglucosamine residue. However, under optimal conditions, with β-glucuronidase, the hydrolytic activity of hexosaminidase B was about 30% as efficient as that of hexosaminidase A. Approximately 70% of the HA could be degraded by 5 units of hexosaminidase A in the presence of 0.5 unit of β-glucuronidase, as opposed to 25% degraded by hexosaminidase B. These results probably reflect intrinsic differences in the activities of the two isozymes. Since the substrate (HA) did not inhibit the hydrolysis of a synthetic substrate (4-methylumbelliferyl-β-glucosaminide) by hexosaminidase B, the linear kinetics of HA hydrolysis implies no product inhibition. These data indicate that native HA can be hydrolyzed by the combined activities of β-glucuronidase with hexosaminidase A or hexoaminidase B.     

c-fos expression in the amygdala:In vivo antisense modulation and role in anxiety

Summary 1. The amygdaloid complex is a key structure in mechanisms of fear and anxiety. Expression of the immediate-early gene c-fos has been reported in the central nucleus of the amygdala following various stressors, but the functional role of this phenomenon has remained unknown.2. c-fos expression was observed in the central nucleus when rats were subjected to a pharmacologically validated animal model of anxiety, the Vogel conflict test, but not after mere exposure to the test apparatus. Bilateral amygdala injection of a 15-mer phosphorothioate c-fos antisense oligodeoxynucleotide prior to testing blocked conflict-induced c-fos expression and had behavioral effects similar to those of established antianxiety drugs.3. Separate experiments determined that antisense treatment did not affect conflict behavior by acting on shock thresholds or drinking motivation.4. These findings provide evidence that neuronal activation and c-fos induction in the amygdala may be of importance for mechanisms of fear and anxiety.     

Mutations of Factor H Impair Regulation of Surface-bound C3b by Three Mechanisms in Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy associated with mutations in complement proteins, most frequently in the main plasma alternative pathway regulator factor H (FH). The hotspot for the FH mutations is in domains 19–20 (FH19–20) that are indispensable for FH activity on C3b bound covalently to host cells. In aHUS, down-regulation of cell-bound C3b by FH is impaired, but it is not clear whether this is due to an altered FH binding to surface-bound C3b or to cell surface structures. To explore the molecular pathogenesis of aHUS we tested binding of 14 FH19–20 point mutants to C3b and its C3d fragment, mouse glomerular endothelial cells (mGEnC-1), and heparin. The cell binding correlated well, but not fully, with heparin binding and the cell binding site was overlapping but distinct from the C3b/C3d binding site that was shown to extend to domain 19. Our results show that aHUS-associated FH19–20 mutants have different combinations of three primary defects: impaired binding to C3b/C3d, impaired binding to the mGEnC-1 cells/heparin, and, as a novel observation, an enhanced mGEnC-1 cell or heparin binding. We propose a model of the molecular pathogenesis of aHUS where all three mechanisms lead eventually to impaired control of C3b on the endothelial cell surfaces. Based on the results with the aHUS patient mutants and the overlap in FH19–20 binding sites for mGEnC-1/heparin and C3b/C3d we conclude that binding of FH19–20 to C3b/C3d is essential for target discrimination by the alternative pathway.Atypical hemolytic uremic syndrome (aHUS)2 is a familial disease characterized by erythrocyte fragmentation and hematuria, damaged renal endothelium, vascular microthrombi, and thrombocytopenia (1). The syndrome leads ultimately to end-stage renal disease with a high mortality rate (2). In aHUS cases point mutations have been found in complement components C3, factor B, CD46, factor I, and factor H (FH), all of which play a role in the activation or control of the alternative pathway (3–8). More than half of the mutations have been found to originate in the HF1 gene that encodes FH and FH-like protein 1.The alternative pathway is initiated spontaneously by hydrolysis of C3 to C3_H2O that forms the C3-convertase C3_H2OBb (9, 10). This enzyme complex converts numerous C3 molecules to C3b that are covalently bound onto practically any nearby surface (11). On a so-called activator surface, such as a microbe, the surface-bound C3b molecules are not efficiently eliminated and therefore new C3bBb complexes are formed leading to more C3b depositions and eventually effective opsonization or damage of the target cell. On non-activator surfaces, such as viable self (host) cells, factor I cleaves C3b to inactive C3b (iC3b) in the presence of one of the cofactors (CD46, CD35, FH, and FHL-1) (12–16). FH is the only one of these cofactors that mediates recognition of self-surfaces making the alternative pathway capable of discriminating between activating and non-activating surfaces (17–19).The two main functions of FH are to prevent the alternative pathway activation in plasma and on self-surfaces. This 150-kDa glycoprotein consists of 20 tandemly arranged short consensus repeat domains that are composed of ∼60 amino acids. Domains 1–4 are essential for the cofactor and decay accelerating activity (20). In the middle region of FH (domains 5–15) there are two binding sites for C-reactive protein (21), one or two sites for glycosaminoglycans (GAGs) (22–25), and one site for C3c part of C3b (C3b/C3c) (25, 26). The C-terminal domains 19–20 (FH19–20) possess binding sites for the thiol ester domain of C3b (C3d or C3dg, TED domain) and GAGs (26, 27).The most common types of mutations found in aHUS are FH missense mutations located within FH19–20 that was recently solved as crystal and NMR structures (2, 28, 29). The C terminus of FH is crucial in self-cell protection as demonstrated by the severity of the aHUS cases and also in a recent mouse model of aHUS where domains 16–20 had been deleted (30, 31). Histopathology of aHUS in these mice had all the characteristics of human aHUS being concordant with the similarity of binding sites for C3b, heparin, and human umbilical vein endothelial cells between human and mouse FH domains 18–20 (32). Binding of mouse or human FH to glomerular endothelial cells has not been characterized despite the fact that in aHUS damage occurs mainly in the small vessels, especially in the glomeruli.The molecular pathogenesis leading to the clinical aHUS in patients with FH mutations remains elusive. The suggested molecular mechanisms for some aHUS-associated mutations include defective binding of the mutated FH to GAGs, endothelial cells, or C3b/C3d (28, 29, 33, 34). The aim of this study was to define the effects of nine aHUS-associated FH mutations and five other structurally closely located mutations on binding of FH19–20 to C3b, C3d, mouse glomerular endothelial cells, and heparin. We identified three primary defects of the mutants: impaired C3b/C3d binding, enhanced mGEnC-1/heparin binding, and impaired mGEnC-1/heparin binding that could lead via three mechanisms to incapability of FH to eliminate C3b on plasma-exposed self-cells. The results clarify the mechanism of target discrimination of the alternative pathway by the C terminus of FH.     

Establishment of a chimeric, replication-deficient influenza A virus vector by modulation of splicing efficiency

Segment 8 of the influenza A virus codes for two proteins (NS1 and NS2/NEP) via splicing. Here, we developed a viral vector expressing a cytokine or chemokine instead of the interferon antagonist NS1. To achieve both the desired genetic stability and high transgene expression levels, NS2/NEP mRNA splicing efficacy had to be fine-tuned by modification of splicing elements. Expression levels of secreted foreign proteins could be further enhanced by fusing the N-terminal 13 amino acids of NS1 with an IgK-derived secretion signal peptide. Thus, the first start codon was used for translation initiation of both NS2/NEP and the foreign protein.     

A unique automation platform for measuring low level radioactivity in metabolite identification studies

Generation and interpretation of biotransformation data on drugs, i.e. identification of physiologically relevant metabolites, defining metabolic pathways and elucidation of metabolite structures, have become increasingly important to the drug development process. Profiling using (14)C or (3)H radiolabel is defined as the chromatographic separation and quantification of drug-related material in a given biological sample derived from an in vitro, preclinical in vivo or clinical study. Metabolite profiling is a very time intensive activity, particularly for preclinical in vivo or clinical studies which have defined limitations on radiation burden and exposure levels. A clear gap exists for certain studies which do not require specialized high volume automation technologies, yet these studies would still clearly benefit from automation. Use of radiolabeled compounds in preclinical and clinical ADME studies, specifically for metabolite profiling and identification are a very good example. The current lack of automation for measuring low level radioactivity in metabolite profiling requires substantial capacity, personal attention and resources from laboratory scientists. To help address these challenges and improve efficiency, we have innovated, developed and implemented a novel and flexible automation platform that integrates a robotic plate handling platform, HPLC or UPLC system, mass spectrometer and an automated fraction collector.     

ORAI-mediated calcium influx in T cell proliferation, apoptosis and tolerance

Ca²⁺ homeostasis controls a diversity of cellular processes including proliferation and apoptosis. A very important aspect of Ca²⁺ signaling is how different Ca²⁺ signals are translated into specific cell functions. In T cells, Ca²⁺ signals are induced following the recognition of antigen by the T cell receptor and depend mainly on Ca²⁺ influx through store-operated CRAC channels, which are mediated by ORAI proteins following their activation by STIM proteins. The complete absence of Ca²⁺ influx caused by mutations in Stim1 and Orai1 leads to severe immunodeficiency. Here we summarize how Ca²⁺ signals are tuned to regulate important T cell functions as proliferation, apoptosis and tolerance, the latter one being a special state of immune cells in which they can no longer respond properly to an otherwise activating stimulus. Perturbations of Ca²⁺ signaling may be linked to immune suppressive diseases and autoimmune diseases.     

Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice

The Lyme disease vaccine is based on the outer-surface lipoprotein (OspA) of the pathogen Borrelia burgdorferi, and 95% of vaccine recipients develop substantial titers of antibodies against OspA. Here, we identified seven individuals with very low antibody titers after vaccination (low responders). The macrophages of low responders produced less tumor necrosis factor-alpha and interleukin-6 after OspA stimulation and had lower cell-surface expression of Toll-like receptor (TLR) 1 as compared to normal cells, but normal expression of TLR2. TLRs activate innate responses to pathogens, and TLR2 recognizes lipoproteins and peptidoglycan (PGN). After OspA immunization, mice genetically deficient in either TLR2 (TLR2(-/-)) or TLR1 (TLR1(-/-)) produced low titers of antibodies against OspA. Notably, macrophages from TLR2(-/-) mice were unresponsive to OspA and PGN, whereas those from TLR1(-/-) mice responded normally to PGN but not to OspA. These data indicate that TLR1 and TLR2 are required for lipoprotein recognition and that defects in the TLR1/2 signaling pathway may account for human hyporesponsiveness to OspA vaccination.