Age-related increase of prostaglandin D2 synthase concentration and glycation in ovine cerebrospinal fluid

Prostaglandin D₂ synthase (PGDS) is a glycoprotein that is exclusively brain derived and is one of the most abundant proteins in the cerebrospinal fluid (CSF). Due to its high CSF specificity, it can be used as a tool for the diagnosis of central nervous system (CNS) disorders. However, several studies have yielded contradictory CSF PGDS concentrations in various CNS neurodegenerative disorders. Sheep CSF samples from different ages were used in this study and 2-dimensional electrophoresis (2-DE) was applied in PGDS identification and concentration calculation. SYPRO Ruby Protein Gel Stain was the staining method used to stain the 2-DE gel protein spots. Pro-Q Emerald 488 Staining for Glycoproteins was used for the staining of glycoproteins. A total of nine PGDS isoforms were identified and CSF total PGDS concentration was calculated to increase linearly by 44% from young (0.9323 ± 0.0637 mg dL⁻¹) to old (1.3669 ± 0.0558 mg dL⁻¹). However, the proportion of CSF total PGDS as a percentage of CSF total protein was discovered to decrease exponentially with age. This was due to the influence of larger age-related increase in CSF albumin concentration (>200% from young to old) as albumin is the most abundant protein in the CSF (>60% of total CSF proteins). Active deglycosylation was not observed in PGDS isoforms during healthy ageing. Some PGDS isoforms were observed to have age-related increase in glycation. These findings suggest that CSF PGDS concentration is increased during healthy ageing and must be taken into consideration when using PGDS as a potential biomarker in diagnosing CNS neurodegenerative disorders. Whether age-related increase in the glycation of some CSF PGDS isoforms will result in detrimental effects on the PGDS protein function needs further investigations.     

年龄和离体翻译后修饰对兔α-晶状体蛋白分子伴娘活性的影响

目的：探讨年龄和离体翻译后翻译对兔α－晶状体蛋白分子伴娘活性的影响。方法：（1）用凝胶过滤法分离幼年、成年和老年3个组兔水溶性晶状体蛋白,观察不同年龄兔α－晶状体蛋白蛋白抑制β-low晶状体蛋白凝集和变性作用的程度差异。（2）孵育α－晶状体蛋白使之氧化和糖基化,观察修饰和未修饰的α－晶状体蛋白抑制β-low晶状体蛋白凝集和变性作用的程度差异。结果：（1）3个年龄组α－晶状体蛋白抑制热诱导β-low晶状体蛋白抑制凝集和变性作用的程度差异有显著意义（F＝29．100,P＜0．01）,成年组兔α－晶状体蛋白的抑制作用强于老年组,弱于幼年组,差异有显著意义（q＝4．1924,4．339,P＜0．05）。（2）离体氧化和糖基化修饰的α－晶状体蛋白抑制β-low晶状体蛋白凝集和变性的作用均弱于未修饰的α－晶状体蛋白,差异有显著意义（q＝26．9171,11．6404;P＜0．01）。结论：（1）随着年龄的增加,兔α－晶状体蛋白的分子伴娘活性呈下降趋势。（2）离体翻译后修饰可使兔α－晶状体蛋白的分子伴娘活性下降。     

Sequence motif upstream of the Hendra virus fusion protein cleavage site is not sufficient to promote efficient proteolytic processing

The Hendra virus fusion (HeV F) protein is synthesized as a precursor, F(0), and proteolytically cleaved into the mature F(1) and F(2) heterodimer, following an HDLVDGVK(109) motif. This cleavage event is required for fusogenic activity. To determine the amino acid requirements for processing of the HeV F protein, we constructed multiple mutants. Individual and simultaneous alanine substitutions of the eight residues immediately upstream of the cleavage site did not eliminate processing. A chimeric SV5 F protein in which the furin site was substituted for the VDGVK(109) motif of the HeV F protein was not processed but was expressed on the cell surface. Another chimeric SV5 F protein containing the HDLVDGVK(109) motif of the HeV F protein underwent partial cleavage. These data indicate that the upstream region can play a role in protease recognition, but is neither absolutely required nor sufficient for efficient processing of the HeV F protein.     

Molecules affecting myelin stability: a novel hypothesis regarding the pathogenesis of multiple sclerosis

In this Mini-Review we present a new hypothesis in support of the neurodegenerative theory as a mechanism for the pathogenesis of multiple sclerosis (MS). The pathogenesis of MS results from changes in two distinct CNS compartments. These are the "myelin" and "nonmyelin" compartments. The myelin compartment is where primary demyelination, amidst attempts at remyelination, is superseded in the CNS by ongoing disease. Recent evidence obtained via magnetic resonance imaging and spectroscopy techniques supports the view that the normal-appearing white matter (NAWM) in the MS brain is altered. Several biochemical changes in NAWM have been determined. These include the cationicity of myelin basic protein (MBP) as a result of the action of peptidyl argininedeiminase (PAD) activity converting arginyl residues to citrulline. The accompanying loss of positive charge makes myelin susceptible to vesiculation and MBP more susceptible to proteolytic activity. An increase of MBP autocatalysis in the MS brain might also contribute to the generation of immunodominant epitopes. Accompanying the destruction of myelin in the myelin compartment is the activation of astrocytes and microglia. These contribute to the inflammatory response and T-cell activation leading to autoimmunity. The complex environment that exists in the demyelinating brain also affects the "nonmyelin" compartment. The inappropriate up-regulation of molecules, including those of the Jagged-1-Notch-1 signal transduction pathway, affects oligodendrocyte precursor cell (OPC) differentiation. Other effectors of oligodendrocyte maturation include stathmin, a microtubule-destabilizing protein, which prevents healing in the demyelinating brain. The hypothesis we present suggests a therapeutic strategy that should 1) target the effectors within the myelin compartment and 2) enable resident OPC maturation in the nonmyelin compartment, allowing for effective repair of myelin loss. The net effect of this new therapeutic strategy is the modification of the disease environment and the stimulation of healing and repair.     

Elevation of the level and activity of acid ceramidase in Alzheimer's disease brain

Protein glycosylation modifies the processing of several key proteins involved in the molecular pathogenesis of Alzheimer's disease (AD). Aberrant glycosylation of tau and down-regulation of sialyltransferase in AD brain suggest a possible dysregulation of protein glycosylation that may play a role in AD. We therefore isolated major glycoproteins from AD brain by using lectin-affinity chromatographies and ion-exchange chromatography and further separated them using SDS-polyacylamide gel electrophoresis. Mass spectrometry analysis of 11 isolated glycoproteins led to their identification as: neuronal cell adhesion molecule, beta-globin, IgM heavy chain VH1 region precursor, contactin precursor, dipeptidylpeptidase VI, CD81 partner 3, prenylcysteine lyase, adipocyte plasma-associated protein, acid ceramidase and two novel proteins. We found that the level and activity of acid ceramidase (AC), one of the major identified human brain glycoproteins, were significantly elevated in AD brain. Immunohistochemical staining indicated that AC was located mainly in the cell bodies of neurons and colocalized with neurofibrillary tangles. Our findings suggest that AC might play a role in controlling neuronal apoptosis and that AC-mediated signalling pathways might be involved in the molecular mechanism of AD.     

Cigarette smoke impairs neutrophil respiratory burst activation by aldehyde-induced thiol modifications

Exposure to airborne pollutants such as tobacco smoke is associated with increased activation of inflammatory-immune processes and is thought to contribute to the incidence of respiratory tract disease. We hypothezised that cigarette smoke (CS) could synergize with activated inflammatory/immune cells to cause oxidative injury or result in the formation of unique reactive oxidants. Isolated human neutrophils were exposed to gas-phase CS, and the production of nitrating and chlorinating oxidants following neutrophil stimulation was monitored using the substrate 4-hydroxyphenylacetate (HPA). Stimulation of neutrophils in the presence of CS resulted in a reduced oxidation and chlorination of HPA, suggesting inhibition of NADPH oxidase or myeloperoxidase (MPO), the two major enzymes involved in inflammatory oxidant formation. Peroxidase assays demonstrated that neutrophil MPO activity was not significantly affected after CS-exposure, leaving the NADPH oxidase as a likely target. The inhibition of neutrophil oxidant formation was found to coincide with depletion of cellular GSH, and a similar modification of critical cysteine residues, such as those in NADPH oxidase components, might be involved in reduced respiratory burst activity. As ,β-unsaturated aldehydes such as acrolein have been implicated in thiol modifications by CS, we exposed neutrophils to acrolein prior to stimulation, and observed inhibition of NADPH oxidase activation in relation to GSH depletion. Additionally, translocation of the cytosolic components of NADPH oxidase to the membrane, a necessary requirement for enzyme activation, was inhibited. Protein adducts of acrolein (or related aldehydes) could be detected in several neutrophil proteins, including NADPH oxidase components, following neutrophil exposure to either CS or acrolein. Alterations in neutrophil function by exposure to (environmental) tobacco smoke may affect inflammatory/infectious conditions and thereby contribute to tobacco-related disease.     

Reevaluation of the determinants of tyrosine sulfation

The posttranslational sulfation of tyrosine has been though to be initiated by the recognition of specific consensus features by the sulfating enzyme tyrosylprotein sulfotransferase (TPST). However, using these recognition features to identify new tyrosine sulfation sites misses recently characterized sites that lack these features. Rigorous analysis of the amino acids surrounding the target tyrosin using the position-specific scoring matrix (PSSM) demonstrates that a consensus sequence does not contain all the information necessary to predict tyrosine sulfation. Instead, accurate prediction requires consideration of all residues within five amino acids on either side of the target tyrosine. These results support the notion that secondary structure is the major determinant of sulfation and that other residues within the sulfation site can compensate for deviations from commonly observed features. This view implies that specific consensus features are not critical for TPST substrate recognition but that TPST may instead broadly recognize any sufficiently exposed tyrosine residue.     

From the Cover: Thuricin CD,a posttranslationally modified bacteriocin with a narrow spectrum of activity against Clostridium difficile

The last decade has seen numerous outbreaks of Clostridium difficile-associated disease (CDAD), which presented significant challenges for healthcare facilities worldwide. We have identified and purified thuricin CD, a two-component antimicrobial that shows activity against C. difficile in the nanomolar range. Thuricin CD is produced by Bacillus thuringiensis DPC 6431, a bacterial strain isolated from a human fecal sample, and it consists of two distinct peptides, Trn-α and Trn-β, that act synergistically to kill a wide range of clinical C. difficile isolates, including ribotypes commonly associated with CDAD (e.g., ribotype 027). However, this bacteriocin thuricin CD has little impact on most other genera, including many gastrointestinal commensals. Complete amino acid sequencing using infusion tandem mass spectrometry indicated that each peptide is posttranslationally modified at its respective 21st, 25th, and 28th residues. Solution NMR studies on [¹³C,¹⁵N] Trn-α and [¹³C,¹⁵N]Trn-β were used to characterize these modifications. Analysis of multidimensional NOESY data shows that specific cysteines are linked to the α-carbons of the modified residues, forming three sulfur to α-carbon bridges. Complete sequencing of the thuricin CD gene cluster revealed genes capable of encoding two S′-adenosylmethionine proteins that are characteristically associated with unusual posttranslational modifications. Thuricin CD is a two-component antimicrobial peptide system with sulfur to α-carbon linkages, and it may have potential as a targeted therapy in the treatment of CDAD while also reducing collateral impact on the commensal flora.     

Gold(III) complexes as a new family of cytotoxic and antitumor agents

In recent years, owing to the contributions of a few research groups, some new gold(III) compounds – either simple coordination complexes or organogold compounds – have been prepared that are sufficiently stable under physiological conditions and are promising candidates for pharmacological testing as cytotoxic and antitumor agents. In vitro pharmacological studies point out that some of these novel gold(III) complexes are highly cytotoxic toward cultured human tumor cell lines and are able to overcome resistance to platinum. Significant differences in the spectrum of action were observed compared with cisplatin. Studies are in progress to elucidate the mechanism of action of these compounds. The cellular effects of two representative gold(III) complexes are described. Preliminary results on binding to DNA in vitro are presented, pointing out that the interactions are generally weak. The implications of these results for the development of gold(III) complexes as a new family of cytotoxic and antitumor agents are discussed.