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Systemic amyloidosis is a serious disease which is caused when normal circulating proteins misfold and aggregate extracellularly as insoluble fibrillary deposits throughout the body. This commonly results in cardiac, renal and neurological damage. The tissue target, progression and outcome of the disease depends on the type of protein forming the fibril deposit, and its correct identification is central to determining therapy. Proteomics is now used routinely in our centre to type amyloid; over the past 7 years we have examined over 2000 clinical samples. Proteomics results are linked directly to our patient database using a simple algorithm to automatically highlight the most likely amyloidogenic protein. Whilst the approach has proved very successful, we have encountered a number of challenges, including poor sample recovery, limited enzymatic digestion, the presence of multiple amyloidogenic proteins and the identification of pathogenic variants. Our proteomics procedures and approaches to resolving difficult issues are outlined.

Apolipoprotein A-IV amyloidosis is an uncommon form of the disease normally resulting in renal and cardiac dysfunction. ApoA-IV amyloidosis was identified in 16 patients attending the National Amyloidosis Centre and in eight clinical samples received for histology review. Unexpectedly, proteomics identified the presence of ApoA-IV signal sequence residues (p.18-43 to p.20-43) in 16/24 trypsin-digested amyloid deposits but in only 1/266 non-ApoA-IV amyloid samples examined. These additional signal residues were also detected in the cardiac sample from the Swedish patient in which ApoA-IV amyloid was first described, and in plasma from a single cardiac ApoA-IV amyloidosis patient. The most common signal-containing peptide observed in ApoA-IV amyloid, p.20-43, and to a far lesser extent the N-terminal peptide, p.21-43, were fibrillogenic in vitro at physiological pH, generating Congo red-positive fibrils. The addition of a single signal-derived alanine residue to the N-terminus has resulted in markedly increased fibrillogenesis. If this effect translates to the mature circulating protein in vivo, then the presence of signal may result in preferential deposition as amyloid, perhaps acting as seed for the main circulating native form of the protein; it may also influence other ApoA-IV-associated pathologies. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Introduction: Hereditary fibrinogen Aα-chain (AFib) amyloidosis is a relatively uncommon renal disease associated with a small number of pathogenic fibrinogen Aα (FibA) variants; wild-type FibA normally does not result in amyloid deposition. Proteomics is now routinely used to identify the amyloid type in clinical samples, and we report here our algorithm for identification of FibA in amyloid. Methods: Proteomics data from 1001 Congo red–positive patient samples were examined using the Mascot search engine to interrogate the Swiss-Prot database and generate protein identity scores. An algorithm was applied to identify FibA as the amyloid protein based on Mascot scores. FibA variants were identified by appending the known amyloidogenic variant sequences to the Swiss-Prot database. Results: AFib amyloid was identified by proteomics in 64 renal samples based on the Mascot scores relative to other amyloid proteins, the presence of a pathogenic variant, and coverage of the p.449-621 sequence. Contamination by blood could be excluded from a comparison of the FibA score with that of the fibrinogen β and γ chains. The proteomics results were consistent with the clinical diagnosis. Four additional renal samples did not fulfill all the criteria using the algorithm but were adjudged as AFib amyloid based on a full assessment of the clinical and biochemical results. Conclusion: AFib amyloid can be identified reliably in glomerular amyloid by proteomics using a score-based algorithm. Proteomics data should be used as a guide to AFib diagnosis, with the results considered together with all available clinical and laboratory information. Keywords: amyloid, amyloidosis, fibrinogen, mass spectrometry, proteomics

Amyloidosis is a relatively rare human disease caused by the deposition of abnormal protein fibres in the extracellular space of various tissues, impairing their normal function. Proteomic analysis of patients’ biopsies, developed by Dogan and colleagues at the Mayo Clinic, has become crucial for clinical diagnosis and for identifying the amyloid type. Currently, the proteomic approach is routinely used at National Amyloidosis Centre (NAC, London, UK) and Istituto di Tecnologie Biomediche-Consiglio Nazionale delle Ricerche (ITB-CNR, Milan, Italy). Both centres are members of the European Proteomics Amyloid Network (EPAN), which was established with the aim of sharing and discussing best practice in the application of amyloid proteomics. One of the EPAN’s activities was to evaluate the quality and the confidence of the results achieved using different software and algorithms for protein identification. In this paper, we report the comparison of proteomics results obtained by sharing NAC proteomics data with the ITB-CNR centre. Mass spectrometric raw data were analysed using different software platforms including Mascot, Scaffold, Proteome Discoverer, Sequest and bespoke algorithms developed for an accurate and immediate amyloid protein identification. Our study showed a high concordance of the obtained results, suggesting a good accuracy of the different bioinformatics tools used in the respective centres. In conclusion, inter-centre data exchange is a worthwhile approach for testing and validating the performance of software platforms and the accuracy of results, and is particularly important where the proteomics data contribute to a clinical diagnosis.

The nematode Caenorhabditis elegans exhibits rapid senescence that is promoted by the insulin/IGF-1 signalling (IIS) pathway via regulated processes that are poorly understood. IIS also promotes production of yolk for egg provisioning, which in post-reproductive animals continues in an apparently futile fashion, supported by destructive repurposing of intestinal biomass that contributes to senescence. Here we show that post-reproductive mothers vent yolk which can be consumed by larvae and promotes their growth. This implies that later yolk production is not futile; instead vented yolk functions similarly to milk. Moreover, yolk venting is promoted by IIS. These findings suggest that a self-destructive, lactation-like process effects resource transfer from postreproductive C. elegans mothers to offspring, in a fashion reminiscent of semelparous organisms that reproduce in a single, suicidal burst. That this process is promoted by IIS provides insights into how and why IIS shortens lifespan in C. elegans., It is unclear why C. elegans continues to produce large quantities of yolk after reproduction. Here the authors show that post-reproductive C. elegans mothers vent yolk which supports their offspring’s growth, serving as a form of primitive lactation.

AdultC. eleganshermaphrodites exhibit severe senescent pathology that begins to develop within days of reaching sexual maturity (Ezcurra et al., 2018; Garigan et al., 2002; Herndon et al., 2002; Wang et al., 2018). For example, after depletion of self-sperm, intestinal biomass is converted into yolk leading to intestinal atrophy and yolk steatosis (pseudocoelomic lipoprotein pools, PLPs) (Ezcurra et al., 2018; Garigan et al., 2002; Herndon et al., 2002; Sornda et al., 2019). These senescent pathologies are promoted by insulin/IGF-1 signalling (IIS), which also shortens lifespan (Ezcurra et al., 2018; Kenyon, 2010). This pattern of rapid and severe pathology in organs linked to reproduction is reminiscent of semelparous organisms where massive reproductive effort leads to rapid death (reproductive death) as in Pacific salmon (Finch, 1990; Gems et al., 2020). Moreover, destructive conversion of somatic biomass to support reproduction is a hallmark of reproductive death (Gems et al., 2020). Yet arguing against the occurrence of reproductive death inC. elegansis the apparent futility of post-reproductive yolk production. Here we show that this effort is not futile, since post-reproductive mothers vent yolk through their vulva, which is consumed by progeny and supports their growth; thus vented yolk functions as a milk, andC. elegansmothers exhibit a form of lactation. Moreover, IIS promotes lactation, thereby effecting a costly process of resource transfer from postreproductive mothers to offspring. These results support the view thatC. eleganshermaphrodites exhibit reproductive death involving a self-destructive process of lactation that is promoted by IIS. They also provide new insight into how the strongly life-shortening effects of IIS inC. elegansevolved.

Human lysozyme is a bacteriolytic enzyme synthesised by gastrointestinal (GI) tract macrophages and hepatocytes. It is found in many different tissues and body fluids including the liver, articular...

Proteomics is becoming the de facto gold standard for identifying amyloid proteins and is now used routinely in a number of centres. The technique is compound class independent and offers the added ability to identify variant and modified proteins. We re-examined proteomics results from a number of formalin-fixed paraffin-embedded amyloid samples, which were positive for transthyretin (TTR) by immunohistochemistry and proteomics, using the UniProt human protein database modified to include TTR variants. The amyloidogenic variant, V122I TTR, was incorrectly identified in 26/27 wild-type and non-V122I variant samples due to its close mass spectral similarity with the methyl lysine-modified WT peptide [126KMe]105–127 (p.[146 KMe]125–147) generated during formalin fixation. Similarly, the methyl lysine peptide, [50KMe]43–59, from immunoglobulin lambda light chain constant region was also misidentified as arising from a rare myeloma-derived lambda variant V49I. These processing-derived modifications are not present in fresh cardiac tissue, non-fixed fat nor serum and do not materially affect the identification of amyloid proteins. They could result in the incorrect assignment of a variant, and this may have consequences for the immediate family who will require genetic counselling and potentially early clinical intervention. As proteomics becomes a routine clinical test for amyloidosis, it becomes important to be aware of potentially confounding issues such as formalin-mediated lysine methylation, and how these may influence diagnosis and possibly treatment.

The systemic amyloidoses are a group of rare diseases, in which extracellular deposition of a variety of proteins in an abnormal fibrillar confirmation results in life-threatening organ dysfunction...

Background The systemic amyloidoses are a group of rare diseases, in which extracellular deposition of a variety of proteins in an abnormal fibrillar confirmation results in life-threatening organ dysfunction. Acquired and hereditary amyloidoses differ in their precursor proteins and predilection for specific organ involvement. Objectives To describe the history of two types of amyloidosis developing consecutively in a single individual. Methods Targeted biopsies were used to confirm the presence of amyloid by Congo red staining viewed under polarized light, while immunohistochemistry and mass spectrometry were used to characterize the amyloid fibril type. I123labeled serum amyloid P component (SAP) scintigraphy was performed to map the distribution of amyloid deposits. Results We report a woman of Sudanese origin who presented aged 31 with dysuria and haematuria. She was found to have an estimated Glomerular Filtration Rate of 38 ml/min and no proteinuria, and a renal biopsy demonstrated AA amyloid deposition. An I-123 labelled SAP scan demonstrated a small amount of amyloid confined to the kidneys. She had no overt underlying inflammatory disease, an infectious diseases work up, including blood borne viruses, was negative and serial measurement of serum amyloid A protein showed no significant elevation with a median of 5 mg/L. Management was blood pressure control only, and her inflammatory markers and renal function remained stable until she was lost to follow up 3 years later. Thirteen years after her renal biopsy she represented in end stage renal failure with a history of weight loss, deranged liver function tests and marked easy bleeding. Further investigation demonstrated well controlled C reactive and serum amyloid A proteins, and an IgG lambda M-band with no serum free light chain bias. A bone marrow demonstrated 7% neoplastic plasma cells and was complicated by a retroperitoneal bleed. An SAP scan now showed a large amyloid load with amyloid deposition in the liver and spleen obscuring the kidneys. Review of the bone marrow and a duodenal biopsy demonstrated amyloid deposition which was AL (lambda) type by both immunohistochemistry and proteomic analysis after laser dissection and mass spectroscopy. Review of the original biopsy confirmed AA type amyloid by both immunohistochemistry and proteomics. Six-cycle chemotherapy for AL amyloidosis was administered with complete clonal response. She remained on dialysis and died four years later of a cerebrovascular accident. Conclusion This is the first reported case of two types of amyloidosis in a single patient. The underlying inflammatory driver of her AA amyloidosis was never identified and given that she had migrated some years earlier from Africa, previous chronic infection that has resolved or responded to non-disclosed prior treatment was thought to be the most likely cause. Whether the subsequent development of AL amyloidosis was pure chance remains unclear. Theoretically chronic inflammation/infection may drive generation of oligoclonal bands with the potential for monoclonal breakthrough. Whether her AA amyloid deposits played a role by providing a template for deposition of subsequent AL amyloidosis derived from an entirely separate precursor protein is also unknown although this theoretically possible and has been shown in reverse in mice models. References [1] Varga J, Flinn MSM, Shirahama T, Rodgers OG, Cohen AS. The induction of accelerated murine amyloid with human splenic extract. Virchows Archiv B. 1986;51(1):177-85. Acknowledgement The presenting author would like to thank the European Federation of Immunology (EFIS) for the short-term fellowship bursary Disclosure of Interests Riccardo Papa: None declared, Janet A. Gilbertson: None declared, Nigel Rendell: None declared, Ashutosh Wechalekar: None declared, Julian D. Gillmore: None declared, Philip N Hawkins: None declared, Helen J. Lachmann Grant/research support from: SOBI, Novartis, Consultant for: Novartis, Takeda, Speakers bureau: SOBI. Novartis

Diagnosis and treatment of systemic amyloidosis depend on accurate identification of the specific amyloid fibril protein forming the tissue deposits. Confirmation of monoclonal immunoglobulin light chain amyloidosis (AL), requiring cytotoxic chemotherapy, and avoidance of such treatment in non-AL amyloidosis, are particularly important. Proteomic analysis characterises amyloid proteins directly. It complements immunohistochemical staining of amyloid to identify fibril proteins and gene sequencing to identify mutations in the fibril precursors. However, proteomics sometimes detects more than one potentially amyloidogenic protein, especially immunoglobulins and transthyretin which are abundant plasma proteins. Ambiguous results are most challenging in the elderly as both AL and transthyretin (ATTR) amyloidosis are usually present in this group. We have lately described a procedure for tissue decellularisation which retains the structure, integrity and composition of amyloid but removes proteins that are not integrated within the deposits. Here we show that use of this procedure before proteomic analysis eliminates ambiguity and improves diagnostic accuracy. Significance Unequivocal identification of the protein causing amyloidosis disease is crucial for correct diagnosis and treatment. As a proof of principle, we selected a number of cardiac and fat tissue biopsies from patients with various types of amyloidosis and show that a classical procedure of decellularisation enhances the specificity of the identification of the culprit protein reducing ambiguity and the risk of misdiagnosis., Graphical abstract Image 4, Highlights • Clinical management of systemic amyloidosis depends on the fibrillar protein. • Decellularisation of tissue biopsies preserves amyloid. • Decellularisation of biopsies enhances specificity in MS-based proteomic diagnosis. • This procedure eliminates ambiguity between two potential amyloidogenic proteins.

In their article entitled “Sex-Related Discordance Between Aortic Valve Calcification and Hemodynamic Severity of Aortic Stenosis: Is Valvular Fibrosis the Explanation?,” Simard et al1 report that in severe aortic stenosis, women (compared with men) have less valvular calcification (by computed tomography), but more valve fibrosis (by histology: Picrosirius red and Masson’s trichrome) than men. Aortic stenosis is common and therapies are required that alter the trajectory of valve narrowing. Our understanding of the pathophysiology is of mechanical valve leaflet stress triggering endothelial damage, and cascade of inflammation, fibrosis and calcification—but this explanation seems incomplete, and it does not explain the sex dimorphism and discordance of valve calcification and fibrosis. We wondered whether valve amyloid …

Hereditary transthyretin (ATTR) amyloidosis is caused by inheritance of an abnormal TTR gene in an autosomal dominant fashion. In its native state, TTR is a homotetramer consisting of four identical polypeptides. Mutations in the TTR gene contribute to destabilization and dissociation of the TTR tetramer, enabling abnormally folded monomers to self-assemble as amyloid fibrils. Currently, over 120 TTR variants have been described, with varying geographic distributions, degrees of amyloidogenicity and organ involvement. We report here a large Irish family with familial amyloid polyneuropathy (FAP), consisting of multiple affected generations, caused by a novel TTR mutation; p.H110D (H90D). The demonstration, by immunohistochemistry and laser micro dissection-mass spectrometry (LMD/MS) that the amyloid fibrils were composed of TTR, in conjunction with a typical FAP phenotype, indicates that the novel TTR mutation was the cause of amyloidosis. We used a molecular visualization tool PyMOL to analyze the effect of the p.H110D (H90D) replacement on the stability of the TTR molecule. Our data suggest that the loss of two hydrogen bonds and the presence of an additional negative charge in the core of a cluster of acidic residues significantly perturb the tetramer stability and likely contribute to the pathogenic role of this variant.

Introduction: Renal biopsy series from North America suggest that leucocyte chemotactic factor 2 (ALECT2) amyloid is the third most common type of renal amyloid. We report the first case series from a European Centre of prevalence, clinical presentation and diagnostic findings in ALECT2 amyloidosis and report long-term patient and renal outcomes for the first time. Methods: We studied the clinical features, diagnostic investigations and the outcome of all patients with ALECT2 amyloidosis followed systematically at the UK National Amyloidosis Centre (NAC) between 1994 and 2015. Results: Twenty-four patients, all non-Caucasian, were diagnosed with ALECT2 amyloidosis representing 1.3% of all patients referred to the NAC with biopsy-proved renal amyloid. Diagnosis was made at median age of 62 years, usually from renal histology; immunohistochemical staining was definitive for ALECT2 fibril type. Median estimated glomerular filtration rate (GFR) at diagnosis was 33 mL/min/1.73 m2 and median proteinuria was 0.5 g/24 h. Hepatic amyloid was evident on serum amyloid P component (SAP) scintigraphy in 11/24 cases but was not associated with significant derangement of liver function. No patient had evidence of cardiac amyloidosis or amyloid neuropathy. Median follow-up was 4.8 (range 0.5–15.2) years, during which four patients died and four progressed to end-stage renal disease. The mean rate of GFR loss was 4.2 (range 0.5–9.6) mL/min/year and median estimated renal survival from diagnosis was 8.2 years. Serial SAP scans revealed little or no change in total body amyloid burden. Conclusions: ALECT2 amyloidosis is a relatively benign type of renal amyloid, associated with a slow GFR decline, which is reliably diagnosed on renal histology. Neither the molecular basis nor the factors underlying the apparent restriction of ALECT2 amyloidosis to non-Caucasian populations have been determined.

To the editor: Amyloidosis is a heterogeneous group of diseases, in which amyloidogenic precursor proteins misfold and adopt a β-pleated sheet conformation.[1][1] Several proteins can form amyloid fibrils in vivo including transthyretin,[2][2] apolipoprotein A-I and A-II, lysozyme, fibrinogen

A 70-year-old gentleman, originally from Tanzania, was referred to our centre after an echocardiogram incidentally revealed findings suggestive of cardiac amyloidosis. At presentation he was dyspno...

The influence of low density lipoproteins (LDL) in the plasma on the regulation of cholesterol biosynthesis is not clear. We studied the changes in plasma mevalonic acid (MVA) concentration and the lathosterol/cholesterol (L/C) ratio, which are well established indices of whole body cholesterol synthesis, in four normocholesterolaemic subjects after each had undergone LDL apheresis on two occasions. LDL apheresis of 75% of the calculated plasma volume reduced LDL-cholesterol by 44% to 1.5±0.2 mmol/l without changing plasma MVA levels or L/C ratios. Apheresis of 125% of the calculated plasma volume decreased plasma LDL-cholesterol by 69% to 0.9±0.2 mmol/l, with significant increases in plasma MVA and L/C ratio on the day after the procedure. These results imply that LDL-cholesterol is an integral part of the sterol regulatory pool and suggest that plasma levels cannot be lowered below 1–1.4 mmol/l in normal subjects without upregulating cholesterol biosynthesis.

HMG-CoA reductase inhibitors or statins are effective in both the primary and secondary prevention of coronary heart disease, the extent of benefit being proportional to the reduction in low density lipoprotein (LDL) cholesterol achieved. Atorvastatin, a newly licensed compound, reportedly lowers LDL with greater efficacy than other statins. The mechanism of this action was, therefore, explored in twenty patients with refractory familial hypercholesterolemia who received in a single-blind sequence simvastatin 40 mg/day, placebo and atorvastatin 10 mg/day each for 4 weeks. At the end of the placebo period the effects of single 40-mg doses of simvastatin and atorvastatin on plasma levels and urinary excretion of mevalonic acid, indices of HMG-CoA reductase activity, were compared. Administration of atorvastatin 10 mg daily for 1 month lowered LDL cholesterol by 32.5%, compared with placebo (P = 0.0001), which was 4.5% less than the decrease after simvastatin 40 mg daily (P = 0.33). The area under the plasma curve and urinary mevalonic acid/ creatinine ratio were both significantly less during the 24 h after a single dose of atorvastatin 40 mg than after a single dose of simvastatin 40 mg (P < 0.01). These findings suggest that the greater efficacy of atorvastatin compared with simvastatin is due to more prolonged inhibition of HMG-CoA reductase, presumably reflecting longer residence of atorvastatin or its active metabolites in the liver.

18-Hydroxy-11-deoxycorticosterone forms a non-polar dimer under acidic conditions. The dimer was purified by high performance liquid chromatography and further analysed by electrospray mass spectrometry. The purified dimer has a molecular weight of 656 Da with two free ketone groups and no free hydroxyl functions. The mass spectrometric data suggest a 20,21,20',21'-anhydro dimer structure for the dimer. Microbore high performance liquid chromatography/electrospray mass spectrometry of rat adrenal extracts indicated that both 18-OH-B and 18-OH-DOC were present, although no 18-OH-DOC dimer was defected. (C) 1997 by John Whey & Sons, Ltd.

With the increasing abuse of steroids in both athletic and equine sports, there is a growing need for more sensitive and specific methods of detection of these compounds. Steroid sulphates are products of steroid metabolism and may be used as markers of steroid administration as well as endogenous steroid production. Electrospray negative ion mass spectra of 23 steroid monosulphates, 5 steroid disulphates and 4 bile acid sulphates have been recorded. The most intense ions in the mass spectra are due to molecular anions or dianions and in most cases these are the only ions present. The daughter ion mass spectra of these compounds vary depending on the location of the sulphate group in the molecule. Alicyclic steroid sulphates have daughter ion mass spectra containing a single ion at m/z 97 (HSO 4 − ) while, for aromatic sulphates, m/z 97 is absent. Combined microbore HPLC-electrospray ionisation mass spectrometry was used for the separation and identification of the individual components of a mixture of seven steroid monosulphates and detection of these compounds by scanning and multiple reaction monitoring is reported.

1Mass spectrometry (MS) has played a vital role in the research of the department of clinical pharmacology for over 25 years. 2MS has been used for trace analysis of endogenous compounds and xenobiotics in plasma and urine, and also for a wide range of structural studies. 3Examples of current applications are reported, including data from gas chromatography-mass spectrometry (GC-MS) assays for mevalonic acid, the identification of an antibiotic produced by Pseudomonas aeruginosa which is active against Helicobacter pylori, high pressure liquid chromatography (h.p.l.c)-electrospray MS studies on steroid sulphates, the aspergillus ciliotoxin, gliotoxin, and ADP ribosyltransferase activity in human polymorphonuclear neutrophil leucocytes (PMNs). The value of electrospray MS in the molecular weight determination of proteins is exemplified by the analysis of human serum amyloid component P.

1. Chemotaxis of human neutrophils is mediated by numerous agents [e.g. N-formyl-methionyl-leucyl-phenylalanine (FMLP) and platelet activating factor (PAF)] whose receptors are coupled to phospholipase C. However, the subsequent transduction pathway mediating cell movement remains obscure. We now propose involvement of mono(ADP-ribosyl)transferase activity in receptor-dependent chemotaxis. 2. Human neutrophils were isolated from whole blood and measurements were made of FMLP or PAF-dependent actin polymerization and chemotaxis. The activity of cell surface Arg-specific mono(ADP-ribosyl)transferase was also measured. Each of these activities was inhibited by vitamin K3 and similar IC50 values obtained (4.67 +/- 1.46 microM, 2.0 +/- 0.1 microM and 4.7 +/- 0.1 microM respectively). 3. There were similar close correlations between inhibition of (a) enzyme activity and (b) actin polymerization or chemotaxis by other known inhibitors of mono(ADP-ribosyl)transferase, namely vitamin K1, novobiocin, nicotinamide and the efficient pseudosubstrate, diethylamino(benzylidineamino)guanidine (DEA-BAG). 4. Intracellular Ca2+ was measured by laser scanning confocal microscopy with two fluorescent dyes (Fluo-3 and Fura-Red). Exposure of human neutrophils to FMLP or PAF was followed by transient increases in intracellular Ca2+ concentration, but the inhibitors of mono(ADP-ribosyl)transferase listed above had no effect on the magnitude of the response. 5. A panel of selective inhibitors of protein kinase C, tyrosine kinase, protein kinases A and G or phosphatases 1 and 2A showed no consistent inhibition of FMLP-dependent polymerization of actin. 6. We conclude that eukaryotic Arg-specific mono(ADP-ribosyl)transferase activity may be implicated in the transduction pathway mediating chemotaxis of human neutrophils, with involvement in the assembly of actin-containing cytoskeletal microfilaments.

1. Mono(ADP-ribosyl)transferase activity has been identified on the external surface of human polymorphonuclear neutrophil leucocytes (PMNs). The enzyme is released from the plasma membrane by phosphoinositide-specific phospholipase C, suggesting a glycosylphosphatidylinositol (GPI) linkage of the enzyme to the plasma membrane. Partial sequence of cDNA encoding the enzyme suggests that it is identical to the GPI-linked mono(ADP-ribosyl)-transferase identified previously on human skeletal muscle. 2. A panel of inhibitors of mono(ADP-ribosyl)transferase (including vitamins K1 and K3, novobiocin and nicotinamide) showed a rank order of inhibitory potency similar to that described for other mono(ADP-ribosyl)transferases. Furthermore, the mono(ADP-ribosyl)ation of agmatine was inhibited also by diethylamino (benzylidineamino)guanidine (DEA-BAG), another substrate of the enzyme related structurally to arginine. 3. There was a close linear correlation between the IC50 values for inhibition of mono(ADP-ribosyl)ation of agmatine by DEA-BAG or the enzyme inhibitors and their IC50 values for inhibition of receptor-dependent polymerization of cytoskeletal actin and chemotaxis. 4. These results suggest a role for mono(ADP-ribosyl)transferase in the transduction pathway involved in receptor-dependent re-alignment of the cytoskeleton during neutrophil chemotaxis.

An Arg-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leucocytes (PMNs) was confirmed by the use of diethylamino(benzylidineamino)guanidine (DEA-BAG) as an ADP-ribose acceptor. Two separate HPLC systems were used to separate ADP-ribosyl-DEA-BAG from reaction mixtures, and its presence was confirmed by electrospray mass spectrometry. ADP-ribosyl-DEA-BAG was produced in the presence of PMNs, but not in their absence. Incubation of DEA-BAG with ADP-ribose (0.1-10 mM) did not yield ADP-ribosyl-DEA-BAG, which indicates that ADP-ribosyl-DEA-BAG formed in the presence of PMNs was not simply a product of a reaction between DEA-BAG and free ADP-ribose, due possibly to the hydrolysis of NAD(+) by an NAD(+) glycohydrolase. The assay of mono(ADP-ribosyl)transferase with agmatine as a substrate was modified for intact PMNs, and the activity was found to be approx. 50-fold lower than that in rabbit cardiac membranes. The K-m of the enzyme for NAD(+) was 100.1 +/- 30.4 mu M and the V-max 1.4 +/- 0.2 pmol of ADP-ribosylagmatine/h per 10(6) cells. The enzyme is likely to be linked to the cell surface via a glycosylphosphatidylinositol anchor, since incubation of intact PMNs with phosphoinositol-specific phospholipase C (PI-PLC) led to a 98 %, decrease in mono(ADP-ribosyl)transferase activity in the cells. Cell surface proteins were labelled after exposure of intact PMNs to [P-32]NAD(+). Their molecular masses were 79, 67, 46, 36 and 26 kDa. The time course for labelling was non-linear under these conditions over a period of 4 h. The labelled products were identified as mono(ADP-ribosyl)ated proteins by hydrolysis with snake venom phosphodiesterase to yield 5'-AMP.

Prostaglandin E2 (PGE2) is important in the early stages of human labour, leading particularly to cervical ripening and dilatation. The source of PGE2 is thought to be either the amnion or the decidua, but the chorion interposes between the amnion and the target tissues, namely the myometrium and cervix. In order to investigate the role of the chorion in modulating prostanoid production, [3H]PGE2 was added to the amnion side of fetal membranes, and the production of metabolites on both sides of the fetal membrane followed by HPLC. The major metabolite was 13,14-dihydro-15-oxo-PGE2 with smaller amounts of 13,14-dihydro-15-oxo-PGA2 and PGB2. The production of all metabolites of PGE2 was time dependent. [3H]PGF2 alpha, which is normally produced by the decidua, was also added to fetal membranes and found to be metabolised to 13,14-dihydro-15-oxo-PGF2 alpha and PGE2. These results suggest that the metabolic enzymes in the chorion may determine intra-uterine levels of prostaglandins, and may also determine the identity of the eicosanoids released by intact fetal membranes.

The Gram negative organism, Pseudomonas aeruginosa, is often found in the lungs of patients with cystic fibrosis and other forms of severe bronchiectasis, where it secretes a number of extracellular toxins including the mono- and dirhamnolipids. The principal monorhamnolipid from P. aeruginosa has previously been identified as rhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoate (Rh-C10.C10). A number of related mono- and dirhamnolipids have been purified from cultures of a clinical isolate of P. aeruginosa and identified by fast atom bombardment and electron impact mass spectrometry: these contain the 3-hydroxyoctanoyl-3-hydroxydecanoate (C8.C10) and 3-hydroxydecanoyl-3-hydroxydodecanoate (C10.C12) homologues. Structural isomers were also present where the order of the lipid linkage was transposed (Rh-C10.C8 and Rh-C12.C10). Unsaturated mono- and dirhamnolipids containing the 3-hydroxydecanoyl-3-hydroxydodec-5-enoate (C10.C12:1) lipid were also present.

Lethal necrotizing fasciitis caused by Streptococcus pyogenes is characterized by a paucity of neutrophils at the site of infection. Interleukin (IL)-8, which is important for neutrophil transmigration and activation, can be degraded by S. pyogenes. Blood isolates of S. pyogenes were better able to degrade human IL-8 than throat isolates. Degradation of IL-8 was the result of a single specific cleavage between 59glutamine and 60arginine within the IL-8 C-terminal alpha helix. Cleaved IL-8 reduced neutrophil activation and migration. IL-8-cleaving activity was found in partially purified supernatant of a necrotizing fasciitis isolate, and this activity was associated with an approximately 150-kDa fraction containing S. pyogenes cell envelope proteinase (SpyCEP). IL-8-cleaving activity corresponded with the presence of SpyCEP in the supernatant. Cleavage of IL-8 by S. pyogenes represents an unprecedented mechanism of immune evasion, effectively preventing IL-8 C-terminus-mediated endothelial translocation and subsequent recruitment of neutrophils.

Background and Aim To compare the cholesterol-lowering efficacy and other metabolic effects of plant sterol and stanol esters, both of which are commonly used in the dietary management of hypercholesterolaemia. Methods and Results The cholesterol-lowering efficacy of equivalent intakes of sterol and stanol esters and of different intakes of stanol esters were compared at 1 and 2 months, both in normal subjects and treated patients with familial hypercholesterolaemia. Systemic effects were assessed by measuring serum levels of plant sterols and of lathosterol and 7α-hydroxy-cholestenone, indices of sterol absorption and of cholesterol and bile acid synthesis respectively. There were no significant differences during the study between 1.6g daily of sterol and stanol esters in reducing total cholesterol (by 3–7%) or low density lipoprotein cholesterol (by 4–8%), nor between 1.6 and 2.6 g daily of stanol. However, the cholesterol-lowering effect of plant sterol esters was attenuated between 1 and 2 months. This was accompanied by increased serum plant sterols and decreased levels of 7α-hydroxy-cholestenone, especially in statin-treated hypercholesterolaemic patients not taking bile acid sequestrants. Conclusions These findings suggest that absorption of dietary plant sterols suppressed bile acid synthesis, thereby diminishing their cholesterol-lowering efficacy. In contrast, plant stanols reduced plant sterol absorption and maintained their cholesterol-lowering efficacy.

An assay based on combined microbore high-performance liquid chromatography–positive ion electrospray ionisation mass spectrometry with selected ion recording has been developed for the measurement of the antihistamine drug terfenadine in human plasma. A deuterated analogue of terfenadine was synthesised for use as an internal standard and extraction of terfenadine was carried out on C18 solid phase extraction columns. The limit of detection of terfenadine in plasma is 0.1 ng/ml and the intra-assay coefficient of variation at 1 ng/ml is 10.1%. Plasma concentrations of terfenadine measured in six normal subjects following a 120 mg oral dose are reported.

Mono(ADP-ribosyl)transferase activity has been detected on the external surface of human polymorphonuclear neutrophil leucocytes (PMNs). The corresponding cDNA has been cloned and shown to be identical to that derived from human skeletal muscle. Our results suggest that mono(ADP-ribosyl)transferase is involved in the transduction pathway mediating (i) receptor-dependent re-alignment of cytoskeletal actin and (ii) chemotaxis of PMNs.

Fasting plasma mevalonic acid (MVA), an indicator of in vivo cholesterol synthesis, was measured in 35 patients with familial hypercholesterolaemia (FH) of whom 7 were treated with pravastatin 10-40 mg/day, 7 with simvastatin 10-40 mg/day and 21 with atorvastatin 80 mg/day. Reductions in low density lipoprotein (LDL) cholesterol and MVA on maximal dose therapy differed significantly between the three drugs: 34.7%, 42.9% and 54.0% (P = 0.0001), and 31.6%, 48.9% and 58.8% (P = 0.004), respectively. Patients on atorvastatin were subdivided according to whether their reduction in LDL cholesterol on treatment was above or below the mean percentage change for the whole group. Basal values of LDL cholesterol did not differ significantly, but above average responders had a significantly higher mean pre-treatment level of MVA (6.2 +/- 0.60 vs. 4.3 +/- 0.61 ng/ml, P < 0.05) than below average responders. When all three drug groups were pooled above average responders showed a significantly greater absolute decrease in MVA on treatment than below average responders (3.85 +/- 0.48 vs. 2.33 +/- 0.40 ng/ml, P < 0.05). However, there was no significant correlation between the magnitude of the decreases in LDL cholesterol and MVA. These findings suggest that FH patients who responded well to statins had a higher basal level of plasma MVA, i.e. a higher rate of cholesterol synthesis, which was more susceptible to pharmacological inhibition. The more marked cholesterol lowering effect of atorvastatin 80 mg/day presumably reflects, at least in part, its ability to inhibit HMG-CoA reductase to a greater extent than maximal recommended doses of pravastatin and simvastatin of 40 mg/day.

Human granulosa-luteal cells cultured in the presence of arachidonic acid produced low levels of the epoxygenase metabolite 14,15-epoxy-5,8,11-(Z,Z,Z)-eicosatrienoic acid (14,15-EpETrE) as determined by HPLC analysis and gas chromatography mass spectrometry. When authentic 14,15-[3H]EpETrE was incubated with these cells in the absence of serum it was metabolised initially to the dihydroxy derivative (14,15-dihydroxy-5,8,11-eicosatrienoic acid, 14,15-DiHETrE) and subsequently to a number of more polar metabolites as determined by HPLC. Fetal calf serum protected 14,15-EpETrE from metabolism for at least 2 h. A similar pattern of metabolism was obtained when 14,15-[3H]EpETrE was incubated with a human choriocarcinoma cell line (BeWo). Microsomes from this cell line converted arachidonic acid to a large number of radioactive metabolites including 14,15-DiHETrE and 11,12-DiHETrE although there was no evidence for the parent epoxides. These results extend earlier findings that human reproductive tissues produce epoxygenase metabolites, and demonstrate the rapid metabolism of these compounds by intact cells in the absence of serum.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant of the heterocyclic aromatic amines formed during the cooking of meat, is genotoxic and carcinogenic in rodents. MeIQx requires metabolic activation by P450 before it can exert these effects. Whilst there is indirect evidence that the mutagenic product is N-hydroxy-MeIQx (-OHMeIQx), we have now identified this unequivocally following incubation of the amine with human hepatic microsomal fraction. A mixture of unlabelled MeIQx, [C-13, N-15(2)]MeIQx and [C-14]MeIQx was used as substrate and the products analysed by HPLC - thermospray mass spectrometry. Characteristic doublet ions, 3 mass units apart, were found at m/z 214/217 ([M + H]+) from the parent compound, MeIQx and at 230/233 ([M + H]+) from N-OHMeIQx. The presence of a doublet ion at m/z 214/217 with the doublet at 230/233 [M + H+] provided additional evidence that this was N-OHMeIQx, as facile loss of 'O' is characteristic of N-hydroxylamines. Further evidence for the identity of the major metabolite, which accounted for approximately 90% of all microsomal metabolism, was obtained by comparing the mutagenicity of the HPLC eluate using Salmonella typhimurium YG1024, which is particularly sensitive to N-hydroxylamines, and TA98/1,8-DNP6 which is resistant to most N-hydroxylamines. Ninety-five per cent of direct-acting mutagenicity present in the reaction mixture was associated with a single peak, which co-eluted with N-OHMeIQx, as indicated by mass spectrometry. In the presence of a metabolic activation system, only one additional mutagenic peak, corresponding to unchanged MeIQx, could be detected. MeIQx (5 muM was N-hydroxylated at a rate of 77 +/- 11 pmol/mg/min (mean +/- SEM, n = 4) by human liver microsomes. The specific inhibitor of human CYP1A2, furafylline (5 muM) inhibited the N-hydroxylation of MeIQx by >90%. These data show that N-OHMeIQx is both the major oxidation product and the major genotoxic product of MeIQx generated by microsomal fractions of human liver and that the reaction is catalysed almost exclusively by CYP1A2.

Pseudomonas aeruginosa colonizes the lower respiratory tracts of patients with severe bronchiectasis, including cystic fibrosis, a condition associated with increased airway mucus output. We have shown that an extract containing chloroform-soluble extracellular products of P. aeruginosa releases glycoconjugates into the cat trachea in vivo. This activity was not related to pyocyanin, a major component of the extract, but was associated with the rhamnolipids. Purified monorhamnolipid (100 micrograms/ml) released radiolabeled and periodic acid-Schiff (PAS)-reactive glycoconjugates (delta 3H = +490 +/- 70%, delta 35S = +170 +/- 40%, delta PAS = +8.6 +/- 1.7 micrograms/min; n = 6, P less than 0.02 for each). Dirhamnolipid (200 micrograms/ml) was also effective (delta 3H = +640 +/- 70%, delta 35S = +130 +/- 20%, delta PAS = +9.3 +/- 1.5 micrograms/min; n = 6, P less than 0.02 for each). Monorhamnolipid (100 micrograms/ml) also released 35S-labeled and PAS-reactive glycoconjugates from human bronchial tissue in vitro (delta 35S = +189 +/- 47%, delta PAS = +26.3 +/- 8.5 micrograms/min; n = 7, P less than 0.001 versus control tissues in which no stimulus was given). The cat tracheal glycoconjugates released by the rhamnolipids differed from those released by pilocarpine 50 microM, in having a higher 3H:35S ratio (P less than 0.001). After gel chromatography on a Sepharose CL-4B column, the void volume fractions of the glycoconjugates also had different profiles in a cesium chloride density gradient. Those released by rhamnolipid banded at 1.62 g/ml, while those released by pilocarpine banded mainly at 1.50 g/ml, with some of the higher density material also present.(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating concentrations of mevalonic acid (MVA) change in parallel with, and may be used as a marker of cholesterol biosynthesis. Plasma MVA levels have been quantified using a sensitive and specific capillary gas chromatography-electron capture mass spectrometric assay. The detection limit for MVA in plasma is 100 pg/ml; the intra-assay variation is 5.11%; the inter-assay variation is 7.7%. Using this assay, the mean plasma MVA in 15 normolipidemic subjects was 2.37 +/- 1.2 ng/ml (range 0.41-5.31 ng/ml). Administration of 40 mg of simvastatin (an HMG-CoA reductase inhibitor) significantly accenutated the diurnal decrease in plasma MVA levels. This assay may be useful in investigating cholesterol synthesis rates in different dyslipidemias and individual responses of HMG-CoA reductase-inhibiting drugs.

Toxocara excretory-secretory antigens (TES) were isolated from the culture media of T.canis and T.cati larvae and their O-glycan content was investigated using fast atom bombardment-mass spectrometry (FAB-MS), gas chromatography and electron impact mass spectrometry. The major oligosaccharides released by reductive elimination of T.canis TES glycoproteins were shown to be two, approximately equi-abundant, trisaccharides: 2-O-Me-Fucp(alpha 1----2)-4-O-Me-Galp(beta 1----3)GalNAcitol and 2-O-Me-Fucp(alpha 1----2)-Galp(beta 1----3)GalNAcitol. In contrast T.cati TES O-glycans are predominantly one component, shown by FAB-MS to be a di-O-methylated trisaccharide, which is probably identical to the di-O-methylated trisaccharide from T.canis. The O-methylated trisaccharides are strong candidates for the carbohydrate epitopes recognized by a panel of monoclonal antibodies which exhibit multiple reactivity against TES antigens. This study constitutes the first rigorous characterization of glycans from a parasitic nematode.