Jacqueline S. Partin

Association of reovirus proteins with the structural matrix of infected cells

The interaction of reovirus with the cytoskeleton was investigated. The soluble components of infected cells were extracted with the nonionic detergent NP-40 in a physiological buffer, and a cytoskeletal extract was prepared from the detergent-insoluble fraction. We observed a selective association of viral-specified products with the cytoskeleton that was temporally controlled. Viral dsRNA appeared first on the framework but after several hours was found also in the soluble phase, encapsidated in mature virions. The initial viral translation products were associated exclusively with the soluble fraction, but concomitant with the appearance of dsRNA, viral proteins microNS and sigma 3 were detected on the cytoskeleton. Several hours later, all viral proteins were detected on the framework. Viral polypeptide microNS exhibited unique spatial distribution patterns that correlated with viral assembly: Before dsRNA replication, it appeared as diffusely distributed protein; a few hours later, it was detected in punctate foci interconnected by tiny filaments; several hours later, it appeared as an extensive fiber network that traversed the foci. The other viral proteins were detected only within viral foci. MicroNS remained bound to the matrix fraction after treatment with DNase, Mg2+, and high salt, treatments that released other viral proteins. This distribution pattern was virus-directed because passage of virus at high multiplicity of infection induced mutations that prevented assembly of the microNS-coated filament organization. A small fraction of the viral-specified products that included polypeptide microNS, but not viral dsRNA, was coprecipitated from cytoskeletal extracts with proteins of mol wt approximately 55K by monoclonal antibodies that recognized tubulin and vimentin. Disruption of this interaction by long exposure to colchicine did not prevent association of viral proteins or RNA with the matrix, indicating that viral products were not transported through these interactions. The results indicate that reovirus morphogenesis includes temporal and spatial controls not described previously.

Brain Ultrastructure in Reye's Disease. II.Acute Injury and Recovery Processes in Three Children

Acute and recovery biopsies of three patients with Reyes Disease are described. Pleomorphic changes of neuronal mitochondria were identified in all of the acute biopsies, similar in appearance to the characteristic alterations of hepatic mitochondria. Distinctive myelin bleb formation may be directly attributable to the mitochondrial injury. The mitochondrial lesion is reversible. There is morphologic evidence for regeneration and repair of myelin; but the presence of myelin ovoids at long intervals after recovery indicates a loss of some myelinated fibers. The neuronal mitochondrial changes, pleomorphism with matrix expansion, and myelin bleb formation, reflect a specific biochemical injury be attributable to ischemic injury secondary to brain edema.

Grade I Reye's Syndrome — Outcome and Predictors of Progression to Deeper Coma Grades

Abstract We studied 83 biopsy-proved cases of Grade I Reyes syndrome to determine the outcome, possible clinical or laboratory predictors of progression to deeper coma grades, and hepatic ultrastructural findings. Seventy-eight patients had no change in coma grade during hospitalization, whereas five (6 per cent) had progression to deeper coma grades. All the patients survived without sequelae except one who sustained severe brain damage. The mean (±S.E.) level of serum ammonia on admission was significantly higher (P = 0.005) in patients whose disease progressed to deeper neurologic grades (291±42 μg per deciliter) than in those whose disease did not so progress (53±5 μg per deciliter), and the corrected prothrombin time was significantly more prolonged (P = 0.005) in patients with progressing coma (3.9±0.5 seconds) than in those whose coma grade did not change (1.6±0.2 seconds). The combination of a prothrombin time 3 seconds or longer than that of the control and a serum level of ammonia on admission ...

MICRONODULAR CIRRHOSIS IN ABETALIPOPROTEINEMIA: POSSIBLE EXACERBATION BY MEDIUM CHAIN TRIGLYCERIDE |[lpar]|MCT|[rpar]| FEEDING

Electron microscopical (EM) and lipid histochemical studies have been made of liver biopsies from 3 abetalipoproteinemic (AB-LP) and 2 hypobetalipoproteinemic (HypoB-LP) children. All AB-LP livers were enlarged and grossly fatty (38% lipid, 91% triglyceride [TG] ). By EM, endogenous tri-glyceride particles were absent. Trans-Golgi vesicles and circum-Golgi smooth endoplasmic reticulum were absent. Most lipid vacuoles were non-membrane-bounded. This Golgi lesion has not been seen in other childhood fatty livers. One AB-LP infant was biopsied at 10, 12, 24, 30 and 38 months, before, during and after treatment with a low-fat MCT diet. During the interval of observation, he had persistent elevation of transaminases and he developed severe micronodular cirrhosis. Alcoholic hyalin (AH) appeared in hepatocytes after MCT treatment; mitochondrial dense bodies increased in size and density. Eight months after MCT was stopped, AH was greatly reduced and mitochondrial dense bodies normalized. It is speculated the alcoholic hyalin was related to accumulation of acetate from MCT-fatty acids in the face of defective TG secretion in a manner biochemically analogous to hepatic ethanol metabolism. MCT should be used cautiously in AB-LP infants until more data is available. Support: NIH #RR-123

Quantitative evaluation of the extent of hepatic enzyme changes in Reye syndrome compared with normal liver or with non-Reye liver disorders: objective criteria for animal models.

ABSTRACT: Five enzymes were measured in 50 liver specimens (18 normal liver, 20 Reye liver, 12 diverse liver disorders other than Reye syndrome). The enzymes were: glutamic dehydrogenase (E.C. 1.4.1.3), monoamine oxidase (E.C. 1.4.3.4), lactate dehydrogenase (E.C. 1.1.1.27), DgIucose- 6-phosphate dehydrogenase (E.C. 1.1.1.49), catalase (E.C. 1.11.1.6). The Reye syndrome group showed significant decreases in glutamic dehydrogenase (56%) and monoamine oxidase (70%) compared to normal control tissue and these changes were not characteristic of the non-Reye liver disorder group as a whole. Neither catalase nor lactate dehydrogenase appeared to be altered significantly in the Reye or in the abnormal control group compared with normal controls. Thus, only the prominent decreases in the mitochondrial enzyme activities appeared to be highly characteristic of Reye syndrome. Paradoxically, the means of the five hepatic enzymes and the admission levels of two serum enzymes indicative of liver damage (alanine and aspartate aminotransferase) were remarkably similar for both survivors and nonsurvivors of Reye syndrome.

Hypocomplementemia in Reye Syndrome: Relationship to Disease Stage, Circulating Immune Complexes, and C3b Amplification Loop Protein Synthesis

Summary: Measurement of C1q, C2, C4, C5, C6, factor B, properdin, β1H, and C3bINA were made in acute sera from 31 patients with Reye syndrome. Abnormalities were found in 18 patients. The magnitude of the complement component depression correlated with disease severity. Sera from patients with stage IV illness had significantly lower complement levels than did sera from patients with stage 1 (P < 0.001), II (P < 0.005), and III (P < 0.05) disease. Circulating immune complex measurements were performed on all 31 acute sera and were present in six (19%). However, from the results of the present study, it would appear that in the majority of the patients circulating immune complexes are not the cause of the lowered levels of, at least, C3 and factor B. Rather, these low levels could be explained as secondary to reductions in the levels of the C3b amplification loop control proteins β1H and C3bINA.Speculation: In Reye syndrome, the exact mechanism responsible for reduction of many complement proteins including β1H and C3bINA remains obscure. Although circulating immune complexes are present transiently, the observed low levels of at least C3 and factor B are best explained by hyposynthesis of their control proteins, β1H and C3bINA. Although the mechanism of this hyposynthesis is unknown, the absence of a pattern of complement depression typical of severe liver disease suggests a generalized cellular disturbance resulting in impaired complement protein synthesis.

Fate of the sea urchin egg receptor for sperm following fertilization

The sea urchin egg receptor for sperm is a 350 kDa glycoprotein containing a large extracellular domain that contains the sperm binding site, a transmembrane domain and a short COOH‐ terminal intracellular domain. During oogenesis, the receptor protein is first detected in Golgi‐associated vesicles and cortical granules. Not until the egg is mature does the receptor appear on the cell surface; at this stage the intact receptor is found in approximately equal quantities on the egg cell surface and in cortical granules. As a potentially unique type of receptor, we were interested in its fate following fertilization. Several techniques have revealed that, following sperm binding, the amount of receptor markedly decreases. Using western blot analysis as well as direct measurement of the receptor protein, it was found that the membrane‐bound form of the receptor rapidly disappeared following sperm binding to the egg, with only 3% of the receptor remaining after 30 s. Analysis by immupoelectron microscopy revealed that 30 s after sperm binding, 30% of the initial level of receptor was present. This remaining 30% was found mostly within the perivitelline space formed by the raised fertilization envelope. The disparity between these two sets of results (i.e. 3 vs 30%) is most likely accounted for by the exocytosis of receptor molecules from cortical granules; this fraction of the receptor would have been lost during isolation of the membrane‐bound form of the receptor. Thus, unlike other cell surface receptors, the sea urchin egg receptor for sperm is not endocytosed and recycled following ligand binding. Rather, it disappears, presumably as a result of proteolysis. Transiently, the cortical granule form of the receptor is found released into the perivitelline space where it may bind to sperm and thereby prevent polyspermy. Despite the apparent secretion of this form of the receptor, experiments with antibodies to the extracellular and intracellular domains indicate that the receptors in cortical granules and in the plasmic membrane are similar, if not identical.

MUSCLE ULTRASTRUCTURE IN REYE'S SYNDROME (RS): EVIDENCE FOR A MYOPATHY

Biochemical studies have shown elevation of muscle creatine phosphokinase (Roe et al., Ped. 55:119, 1975) and excessive urinary nitrogen losses up to 0.64 g/kg/day (Snodgrass and DeLong, N. Eng. J. Med. 294:855, 1976) in RS, suggest ing skeletal muscle injury. We report electronmicroscopy of muscle biopsies obtained from 15 RS cases on the day of admission and at followup 4 days to 23 weeks later. A myopathy of variable severity was present in each case acutely. In severely affected specimens about 1/40 muscle cells were necrotic with dissolution of the sarcolemma,loss of striations and dispersion of myofilaments, producing a homogenous matrix which contained vesicular fragments of sarcoplasmic reticulum, autophagic vacuoles and spherical mitochondria with expanded matrix and frequently ruptured outer membranes. Surrounding muscle cells were abnormal, displaying glycogen loss, ribosome disorganization, dilation of sarcoplasmic reticulum and intramyo-fibrillar edema. There were 2 populations of mitochondria in muscle cells: Individual mitochondria demonstrated extreme matrix expansion suggestive of that seen in brain and liver in RS, but the majority of muscle cell mitochondria demonstrated only slight matrix expansion. Muscle cell triglyceride was increased and many cells contained myelin figures. Inflammation was absent but influenza virus was isolated from 3 of 5 biopsies in which cultivation was attempted. These studies confirm the existence of a myopathy in RS. The relationship between the myopathy of RS and post-influenzal myopathy of childhood should be investigated.

CONGENITAL CHOLESTASIS: CLINICAL AND ULTRASTRUCTURAL STUDY*

Byler’s disease is a recessively inherited form of progressive intrahepatic cholestasis first described by Clayton in an extensive Amish kindred descended from Jacob Byler born in the United States in 1799 (2). Clinical manifestations are steatorrhea, conjugated hyperbilirubinemia which is initially intermittent and later persistent, pruritus, and hepatosplenomegaly. Alkaline phosphatase is elevated but serum cholesterol is normal. Serum bile acid levels are elevated and an increased level of lithocholic acid has been described in one patient. The transport maximum (Tm) for bromsulphalein and storage capacity (S) are both reduced. The liver architecture early in the disease is normal and normal numbers of interlobular bile ducts are present (6). Progressive hepatic cirrhosis occurs; the longest survivor was 14 3/12 years of age and had severe cirrhosis and mental and physical retardation (6). Defective excretion of conjugated bile salts and bilirubin across the canalicular membrane has been postulated (2, 6).

1147 LIVER ULTRASTRUCTORE IN NEUROLOGICAL GRADE I (SO-CALLED MILD) REYE'S SYNDROME (RS)

Of 106 consecutive cases of RS, 23 were neurological Grade I upon admission. Fourteen cases, including 2 who progressed to coma, received diagnostic liver biopsy for examination by lipid histochemistry, and electron microscopy (EM). Initial clinical chemical measures were SGOT 130-1524 IU (mean 609), SGOT 50-2300 IU (mean 761), blood NH3 15-339 μgm/dl (mean 103), CPK 0-10 Bioscience units (mean 2.3). By light microscopy, all liver specimens were typical of RS, demonstrating heavy panlobular micro-vesicular neutral fat. There was substantial variation in organelle damage from patient to patient; all demonstrated universal mitochondrial matrix expansion and pleomorphism in osmium-fixed liver, but matrix dense bodies were not universally absent, being present in most mitochondria of least damaged livers and absent in 2 livers demonstrating the most severe EM changes. Peroxisomes and smooth endoplasmic reticulum (ER) were greatly increased; glycogen was reduced and golgi lipoprotein particles were reduced or absent in all but 1 case. Rough ER was normal. Bile stasis was absent. Some disintegrating liver cells were present in all samples.Conclusion: In RS, Grade I neurological status is not synonymous with “mild” disease because: 1) some Grade I children have unexpected severe alteration of liver ultrastructure; and 2) certain children unexpectedly progress to severe encephalopathy. All Grade I cases should be hospitalized for glucose infusion and careful observation.