Arginine deficiency, hyperammonemia and Reye's syndrome in ferrets

Young male ferrets developed hyperammonemia and encephalopathy shortly after eating a diet lacking in arginine. The dietary supplementation of arginine or intraperitoneal injection of ornithine prevented hyperammonemia and shortened the duration of encephalopathy. Therefore, young ferrets were assumed to be unable to meet their ornithine needs from sources other than arginine. Adult ferrets did not develop hyperammonemia and encephalopathy after eating arginine-free diet. Because young ferrets are also susceptible to human influenza infections, they were further tested as animal model of Reye's syndrome. Reye's syndrome is a serious childhood disorder that develops following influenza infections and is characterized in part by an encephalopathy, hyperammonemia and elevated serum transaminases. In young ferrets, concurrent administration of aspirin with human influenza inoculation and an arginine-free diet produced symptoms similar to those seen in humans with Reye's syndrome. The ferret model appears to be useful for studying the roles of various etiologic agents and their interactions in producing Reye's syndrome-like disorders. The ammonia metabolism in ferrets is reviewed and the ferret model for Reye's syndrome and its applications for the better understanding of this disorder in humans are discussed.     

PI3K/mTORC1 activation in hamartoma syndromes: Therapeutic prospects

Dysregulated activity of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin complex 1 (mTORC1) is characteristic feature of hamartoma syndromes. Hamartoma syndromes, dominantly inherited cancer predisposition disorders, affect multiple organs and are manifested by benign tumors consisting of various cell types native to the tissues in which they arise. In the past few years, three inherited hamartoma syndromes, Cowden syndrome (CS), tuberous sclerosis complex (TSC) syndrome, and Peutz-Jeghens syndrome (PJS), have all been linked to a common biochemical pathway: the hyperactivation of PI3K/mTORC1 intracellular signaling. Three tumor suppressors, PTEN (phosphatases and tensin homolog), tuberous sclerosis complex TSC1/TSC2, and LKB1, are negative regulators of PI3K/mTORC1 signaling; disease-related inactivation of these tumor suppressors results in the development of PTEN-associated hamartoma syndromes, TSC, and PJS, respectively. The goal of this review is to provide a roadmap for navigating the inherently complex regulation of PI3K/mTORC1 signaling while highlighting the progress that has been made in elucidating the cellular and molecular mechanisms of hamartoma syndromes and identificating potential therapeutic targets for their treatment. Importantly, because the PI3K/mTORC1 pathway is activated in the majority of common human cancers, the identification of novel molecular target(s) for the treatment of hamartoma syndromes may have a broader translational potential, and is critically important not only for therapeutic intervention in hamartoma disorders, but also for the treatment of cancers.     

Hepatic polyamine metabolism in children with Reye's syndrome.

Acute mitochondrial insult has been suggested as a primary reason for the clinical, histopathological and biochemical abnormalities seen in Reye's syndrome. However, the etiology of mitochondrial dysfunction has not been identified. Polyamines have been known to alter the mitochondrial structure and function. Influenza infection may cause an increase in ornithine decarboxylase activity and thereby channel ornithine for polyamine biosynthesis, leading to mitochondrial dysfunction in Reye's syndrome. To test this hypothesis, the hepatic concentrations of polyamines, polyamine-metabolizing enzymes and urea cycle enzyme activities in Reye's syndrome patients were determined and compared with patients who died from illnesses other than Reye's syndrome. The hepatic concentration of putrescine, spermidine and spermine were increased in Reye's syndrome patients. The activity of ornithine decarboxylase was elevated but, due to the small number of samples, these values did not reach statistical significance. Ornithine carbamoyltransferase activity was decreased in the liver of Reye's syndrome patients. Our results suggest that increased synthesis of polyamines from ornithine may initiate mitochondrial injury in Reye's syndrome.     

Identification of 5-hydroxyhexanoic acid in the urine of twin siblings with a Reye's-like syndrome associated with dicarboxylic aciduria and hypoglycaemia and with similarities to Jamaican vomiting sickness

Twin male infant siblings who presented in Harrow, UK, with a Reye's-like syndrome associated with profound hypoglycaemia, vomiting, diarrhoea, coma and death in one child, with dicarboxylic aciduria, and similarities to Jamacian vomiting sickness (hypoglycin toxicity) have been shown to excrete large amounts of a previously unrecorded urinary organic acid. This has been identified as 5-hydroxyhexanoic acid by gas chromatography mass spectrometry using a synthesized standard. Concentrations observed were 340 and 330 mg g-1 creatinine in the two patients. The metabolic precursor of the urinary acid is suggested to be hex-4-enoic acid, a probable chemical toxin closely related to the active organic acid metabolite of hypoglycin. The possibility of omega - 1 oxidation of hexanoic acid to 5-hydroxyhexanoic acid in these and other patients with dicarbocylic aciduris is also discussed.     

Total and peroxisomal fatty acid oxidation by liver homogenates from autopsied Reye's and control subjects

To determine whether the accumulation of liver triglyceride in Reye's syndrome could be due to a block in beta-oxidation of the fatty acids, the ability of Reye's and control liver homogenates from samples obtained at autopsy to oxidize fatty acids was examined. Total fatty acid oxidation as measured by oxidation of [1-14C]oleoyl CoA, which mostly represents mitochondrial activity, was comparable between the groups. Peroxisomal fatty acid oxidation was, likewise, similar despite the reported increase in the numbers and sizes of these organelles. This disparity could not be explained by an artifactual dilution of product by accumulated endogenous substrate. Inference is made that active peroxisomal beta-oxidation may contribute to the increased short chain fatty CoA content of liver which was reported earlier.     

Liver fatty acid-binding protein in two cases of human lipid storage

Summary FABPs in the various tissues play an important role in the intracellular fatty acid transport and metabolism. Reye's syndrome (RS) and multisystemic lipid storage (MLS) are human disorders characterized by a disturbance of lipid metabolism of unknown etiology. We investigated for the first time L-FABP in these two conditions. Affinity purified antibodies against chicken L-FABP were raised in rabbits, and found to cross-react specifically with partially purified human L-FABP. L-FABP content in liver samples of two patients with RS and MLS was investigated by immuno-histochemistry, SDS-PAGE and ELISA. L-FABP immuno-histochemistry showed increased reactivity in the liver of RS patient and normal reactivity in MLS liver. L-FABP increase in RS liver was confirmed by densitometry of SDS-PAGE and ELISA method. By these two methods the increase amounted to 180% and 199% (p < 0.02), respectively, as compared to controls. A possible role of L-FABP in the pathogenesis of RS is discussed.     

Infantile disease with microvesicular fatty infiltration of viscera spontaneously occurring in the C3H-H-2(0) strain of mouse with similarities to Reye's syndrome

The clinical, biochemical and histopathological findings of an infantile disease occurring in the C3H-H-2 degree strain of mice, which has similarities with Reye's syndrome in children, is described.     

Formation and degradation of dicarboxylic acids in relation to alterations in fatty acid oxidation in rats.

Dicarboxylic acids are excreted in urine when fatty acid oxidation is increased (ketosis) or inhibited (defects in beta-oxidation) and in Reye's syndrome. omega-Hydroxylation and omega-oxidation of C6-C12 fatty acids were measured by mass spectrometry in rat liver microsomes and homogenates, and beta-oxidation of the dicarboxylic acids in liver homogenates and isolated mitochondria and peroxisomes. Medium-chain fatty acids formed large amounts of medium-chain dicarboxylic acids, which were easily beta-oxidized both in vitro and in vivo, in contrast to the long-chain C16-dicarboxylic acid, which was toxic to starved rats. Increment of fatty acid oxidation in rats by starvation or diabetes increased C6:C10 dicarboxylic acid ratio in rats fed medium-chain triacylglycerols, and increased short-chain dicarboxylic acid excretion in urine in rats fed medium-chain dicarboxylic acids. Valproate, which inhibits fatty acid oxidation and may induce Reye like syndromes, caused the pattern of C6-C10-dicarboxylic aciduria seen in beta-oxidation defects, but only in starved rats. It is suggested, that the origin of urinary short-chain dicarboxylic acids is omega-oxidized medium-chain fatty acids, which after peroxisomal beta-oxidation accumulate as C6-C8-dicarboxylic acids. C10-C12-dicarboxylic acids were also metabolized in the mitochondria, but did not accumulate as C6-C8-dicarboxylic acids, indicating that beta-oxidation was completed beyond the level of adipyl CoA.     

Octanoic Acid Inhibits Astrocyte Volume Control: Implications for Cerebral Edema in Reye's Syndrome

Abstract Octanoic acid has been implicated in the pathogenesis of cytotoxic cerebral edema in Reye's syndrome. Using astrocytes from primary culture, we studied the dose-dependent effects of octanoate on cellular volume regulation and metabolism. Astrocyte volume recovery following hypoosmotic swelling was stimulated by 1.0 m M octanoate and inhibited by 3.0 m M octanoate. Parallel effects were obtained at these concentrations on the activity of the Na⁺,K⁺-depen-dent ATPase. Cellular ATP concentrations also were reduced 36% with the higher octanoate concentration. These effects of octanoate may contribute to the severe astrocyte swelling observed in the brains of Reye's syndrome patients.     

Analysis of risk epitopes of anti-neutrophil antibody MPO-ANCA in vasculitis in Japanese population

Autoantibodies to myeloperoxidase (MPO) are a subset of anti-neutrophil cytoplasmic antibody (ANCA, MPO-ANCA) detected in the sera of some patients with primary systemic vasculitis. The titer of MPO-ANCA does not always reflect disease activity and this inconsistency may be attributable to differences in epitopic specificity by MPO-ANCA among various patients with vasculitis. Epitope analysis may also explain the occurrence of MPO-ANCA in different vasculitic syndromes. We screened the sera of 148 MPO-ANCA positive patients from six vasculitic syndromes: rapidly progressive gromerulonephritis (RPGN), microscopic polyangiitis (MPA), idiopathic crescentic glomerulonephritis (I-CrGN), classic polyangiitis nodosa (cPAN), Churg-Strauss syndrome (CSS), Kawasaki disease (KD); and from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The sera were collected by the Intractable Vasculitis Research Project Group in Japan. No serum showed epitopes La and Lb of light chain of MPO, and sera with 68.6% of patients showed a positive reaction to one or more epitopes in heavy chain of MPO. Analysis of binding level showed that RPGN, I-CrGN and MPA sera mainly reacted to the Ha epitope at the N-termimus of the MPO heavy chain, CSS sera reacted to Ha and the Hf epitope close to the C-terminus of the MPO heavy chain, KD reacted mainly to Hf, while SLE and RA sera reacted to all epitopes. These results suggest that MPO-ANCA recognizing specific regions of the N-terminus of the MPO H-chain confer an increased risk of vasculitis RPGN, I-CrGN, MPA and CSS. Furthermore, the epitopic specificity of MPO-ANCA differentiates vasculitic from non-vasculitic syndromes associated with MPO-ANCA positivity and differentiates in the cirtain type of vasculitis from various vasculitic syndromes. In particular, vasculitic syndromes associated with kidney involvement had similar epitopic reactivity which suggests that this pattern confers an increased risk of vasculitis.     

Accumulation of mini-plasmin in the cerebral capillaries causes vascular invasion of the murine brain by a pneumotropic influenza A virus: implications for influenza-associated encephalopathy

The infectivity and pathogenicity of influenza virus are primarily determined by host cellular trypsin-type processing proteases which cleave the viral membrane fusion glycoprotein hemagglutinin (HA). Therefore the distribution of the processing protease is a major determinant of the infectious organ tropism. The common epidemic human influenza A virus is pneumotropic and the HA processing proteases tryptase Clara, mini-plasmin, tryptase TC30 and ectopic anionic trypsin have all been isolated from mammalian airways. However, the pneumotropic influenza virus occasionally causes severe brain edema, particularly in children presenting with Reye's syndrome treated with aspirin, or in children with influenza-associated encephalopathy without antipyretic treatment. We have observed that, after influenza virus infection, the accumulation of mini-plasmin in the cerebral capillaries in mice with a congenital or acquired abnormality of mitochondrial beta-oxidation mimicking the pathological findings of Reye's syndrome, causes an invasion and multiplication of the pneumotropic influenza virus at these same locations. From these findings, we hypothesize that the accumulated mini-plasmin modifies the brain capillaries from a non-permissive to a permissive state, thereby allowing multiplication of pneumotropic influenza virus. In addition, mini-plasmin proteolytically destroys the blood-brain barrier. These pathologic findings, consistent with encephalopathy in mice with a systemic impairment of beta-oxidation, may have implications for human influenza encephalopathy.     

Liquid-liquid Phase Separation in Viral Function

An emerging set of results suggests that liquid-liquid phase separation (LLPS) is the basis for the formation of membrane-less compartments in cells. Evidence is now mounting that various types of virus-induced membrane-less compartments and organelles are also assembled via LLPS. Specifically, viruses appear to use intracellular phase transitions to form subcellular microenvironments known as viral factories, inclusion bodies, or viroplasms. These compartments - collectively referred to as viral biomolecular condensates - can be used to concentrate replicase proteins, viral genomes, and host proteins that are required for virus replication. They can also be used to subvert or avoid the intracellular immune response. This review examines how certain DNA or RNA viruses drive the formation of viral condensates, the possible biological functions of those condensates, and the biophysical and biochemical basis for their assembly.     

Understanding the molecular basis of Apert syndrome

Apert syndrome, first described in 1906, is one of the most severe of the craniosynostosis syndromes and is further characterized by midface hypoplasia, syndactyly, and other visceral abnormalities. Affected individuals generally require lifelong management by a multidisciplinary team of health care specialists. Apert syndrome results almost exclusively from one or the other of two point mutations in fibroblast growth factor receptor 2. Tremendous scientific advances have been made recently in understanding the molecular basis for Apert syndrome through clinical genetic, biochemical, and structural approaches. In this review, the authors provide the clinician with a basic overview of these findings and their therapeutic implications.     

Influenza A viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (IRE1) stress pathway

Known therapies for influenza A virus infection are complicated by the frequent emergence of resistance. A therapeutic strategy that may escape viral resistance is targeting host cellular mechanisms involved in viral replication and pathogenesis. The endoplasmic reticulum (ER) stress response, also known as the unfolded protein response (UPR), is a primitive, evolutionary conserved molecular signaling cascade that has been implicated in multiple biological phenomena including innate immunity and the pathogenesis of certain viral infections. We investigated the effect of influenza A viral infection on ER stress pathways in lung epithelial cells. Influenza A virus induced ER stress in a pathway-specific manner. We showed that the virus activates the IRE1 pathway with little or no concomitant activation of the PERK and the ATF6 pathways. When we examined the effects of modulating the ER stress response on the virus, we found that the molecular chaperone tauroursodeoxycholic acid (TUDCA) significantly inhibits influenza A viral replication. In addition, a specific inhibitor of the IRE1 pathway also blocked viral replication. Our findings constitute the first evidence that ER stress plays a role in the pathogenesis of influenza A viral infection. Decreasing viral replication by modulating the host ER stress response is a novel strategy that has important therapeutic implications.     

The "peripheral-type" benzodiazepine (omega 3) receptor in hyperammonemic disorders

Increased levels of brain ammonia occur in both congenital and acquired hyperammonemic syndromes including hepatic encephalopathy, fulminant hepatic failure, Reye's syndrome and congenital urea cycle disorders. In addition to its effect on neurotransmission and energy metabolism, ammonia modulates the expression of various genes including the astrocytic "peripheral-type" benzodiazepine (or omega 3) receptor (PTBR). Increased expression of the isoquinoline carboxamide binding protein (IBP), one of the components of the PTBR complex, is observed in brain and peripheral tissues following chronic liver failure as well as in cultured astrocytes exposed to ammonia. Increased densities of binding sites for the PTBR ligand [3H]-PK11195 are also observed in these conditions as well as in brains of animals with acute liver failure, congenital urea cycle disorders and in patients who died in hepatic coma. The precise role of PTBR in brain function has not yet fully elucidated, but among other functions, PTBR mediates the transport of cholesterol across the mitochondrial membrane and thus plays a key role in the biosynthesis of neurosteroids some of which modulate major neurotransmitter systems such as the gamma-aminobutyric acid (GABA(A)) and glutamate (N-methyl-D-aspartate (NMDA)) receptors. Activation of PTBR in chronic and acute hyperammonemia results in increased synthesis of neurosteroids which could lead to an imbalance between excitatory and inhibitory neurotransmission in the CNS. Preliminary reports suggest that positron emission tomography (PET) studies using [11C]-PK11195 may be useful for the assessment of the neurological consequences of chronic liver failure.     

Carbohydrate-deficient glycoprotein syndrome type IA (phosphomannomutase-deficiency)

The carbohydrate-deficient glycoprotein or CDG syndromes (OMIM 212065) are a recently delineated group of genetic, multisystem diseases with variable dysmorphic features. The known CDG syndromes are characterized by a partial deficiency of the N-linked glycans of secretory glycoproteins, lysosomal enzymes, and probably also membranous glycoproteins. Due to the deficiency of terminal N-acetylneuraminic acid or sialic acid, the glycan changes can be observed in serum transferrin or other glycoproteins using isoelectrofocusing with immunofixation as the most widely used diagnostic technique. Most patients show a serum sialotransferrin pattern characterized by increased di- and asialotransferrin bands (type I pattern). The majority of patients with type I are phosphomannomutase deficient (type IA), while in a few other patients, deficiencies of phosphomannose isomerase (type IB) or endoplasmic reticulum glucosyltransferase (type IC) have been demonstrated. This review is an update on CDG syndrome type IA.     

Histochemical Methods for Dissociated Muscle Fibers

Skeletal or cardiac muscle fibers can be separated by brief (3-5 second) dissociation of formalin-fixed pieces with a Willems Polytron (Brinkmann Instrument Co.). Such separated fibers are useful for demonstration of abnormal accumulations of lipids, carbohydrates, proteins and minerals in metabolic diseases. Staining techniques for demonstration of various stored materials include: 1) toluidine blue at pH 2.8 for acid mucopolysaccharide in skeletal muscle fibers in Pompe's glycogenesis 2,2) one-step trichrome stain for nemaline myopathy and for abnormal mitochondria in X-linked infantile cardiomyopathy, 3) periodic acid-methenamine silver stain for glycolipid-containing lysosomes in I-cell disease (mucolipidosis 2), 4) Sudan black B stain for lipid in skeletal muscle fibers in Reye's syndrome, infantile lactic acidosis, Leigh's infantile subacute necrotizing encephalopathy and Jansky-Bielschowsky late infantile ceroid lipofuscinosis, 5) iron stain for iron in cardiac and skeletal muscle fibers in thalassemia with advanced hemosiderosis, and 6) autofluorescence for “ceroid” in skeletal muscle fibers in Jansky-Bielschowsky disease.     

Emulsifiers that enhance susceptibility to virus infection: increased virus penetration and reduced interferon response.

An emulsifier which had an environmental relationship to Reye's syndrome, when used to treated L-929 cultures, was shown to increase the rate of encephalomyocarditis virus penetration and uncoating while having no effect on the attachment of virus or on the replication of infectious ribonucleic acid. This treatment also rendered L-929 cells unable to respond normally to interferon inducers and reversed an already established interferon antiviral state. It is proposed that one or more of these actions result in the cellular enhancement of virus susceptibility.