Identification of a cytoskeleton-bound form of phospholemman with unique C-terminal immunoreactivity.

Phospholemman (PLM) is a 72-amino acid transmembrane protein thought to function in Na,K-ATPase regulation or assembly, similar to other members of the FXYD family of proteins. Unique to PLM among these regulatory proteins are sites for C-terminal phosphorylation by PKA and PKC, although a role for phosphorylation in PLM function remains unclear. To study PLM phosphorylation, we used PLM phosphopeptides to generate antibodies to specifically detect phosphorylated PLM. Peptide affinity chromatography isolated two populations of antibodies: one reacting with standard PLM, a collection of closely-spaced 15-kDa protein bands by SDS-PAGE. About 20% of PLM antibodies reacted specifically with a single distinct form of PLM. Levels of this second immunological form (PLM-b) were increased with overexpression of PLM cDNA, and also reacted with a monoclonal antibody against the PLM N-terminus. In complete contrast to standard PLM, however, PLM-b was quantitatively insoluble in nonionic detergents and was released from tight binding by colchicine. Antibodies to PLM-b were present in two different antisera raised to the phosphorylated C-terminal peptide (residues 57-70), but not in antiserum raised to the non-phosphorylated C-terminal peptide. Despite an apparent relationship between PLM-b and phosphorylated PLM, PLM-b levels were not affected by treatment of heart cells with isoproterenol. PLM-b appears to represent a cytoskeleton-attached detergent-insoluble form of PLM with distinctive C-terminal immunoreactivity that might have implications for PLM structure and function.

Reproduction-associated immunoreactive peptides in the nervous systems of prosobranch gastropods.

Antibodies against reproductive peptides of Aplysia and Lymnaea were used to localize homologous immunoreactive peptides in the nervous systems of three prosobranch species: Busycon canaliculatum, Concholepas concholepas, and Tegula atra. Positive control experiments in L. stagnalis demonstrated the broad species range of the anti-egg-laying hormone (anti-ELH) antibody used in this study, and showed binding of anti-alpha-caudodorsal-cell peptide (anti-alpha-CDCP) to the same cells in cerebral and buccal ganglia. Dot immunoassays with synthetic ELH confirmed the reactivity and sensitivity (< 0.1 microgram) of the anti-ELH antibody. Experiments with preadsorbed antibody or no primary antibody confirmed its specificity. In B. canaliculatum, clusters of more than 300 neuronal cell bodies immunoreactive to both anti-ELH and anti-alpha-CDCP were observed along the medial margins of left and right cerebral ganglia. Anti-alpha-CDCP reacted with additional small populations of cerebral ganglion neurons not stained by anti-ELH. Anti-ELH and anti-alpha-CDCP also reacted with overlapping but different small populations of neurons in buccal ganglia. In C. concholepas and T atra, ELH-like immunoreactivity was found in cerebral ganglia, and in T. atra in fibers in the cerebral ganglia and cerebral-pedal connectives. Thus, cerebral ganglia are the major locus of the ELH-like immunoreactivity in prosobranchs.

Cholinergic and peptidergic regulation of siphon/mantle function in the zebra mussel, Dreissena polymorpha.

Neurotransmitter regulation of the siphon and adjacent mantle region of bivalves has not previously been examined. In the biofouling bivalve, Dreissena polymorpha, acetylcholine and FMRFamide both elicited contractions of siphon/mantle preparations. Hexamethonium bromide inhibited acetylcholine-elicited contractions but had no effect on FMRFamide-elicited contractions. FMRFamide-like immunoreactivity and chromatographic evidence for acetylcholine were found in central ganglia and the siphon/mantle region. Extracts of siphons, gonads, and gills, separated on Sephadex G-25, also contained macromolecules larger than acetylcholine and FMRFamide that caused siphon/mantle contraction. These results demonstrate regulation of contraction by several potential neurotransmitter agents in a new bivalve preparation, the siphon/mantle.

Structural investigation of catalytically modified F120L and F120Y semisynthetic ribonucleases.

The structures of two catalytically modified semisynthetic RNases obtained by replacing phenylalanine 120 with leucine and tyrosine have been determined and refined at a resolution of 2.0 A (R = 0.161 and 0.184, respectively). These structures have been compared with the refined 1.8-A structure (R = 0.204) of the fully active phenylalanine-containing enzyme (Martin PD, Doscher MS, Edwards BFP, 1987, J Biol Chem 262:15930-15938) and with the catalytically defective D121A (2.0 A, R = 0.172) and D121N (2.0 A, R = 0.186) analogs (deMel VSJ, Martin PD, Doscher MS, Edwards BFP, 1992, J Biol Chem 267:247-256). The movement away from the active site of the loop containing residues 65-72 is seen in all three catalytically defective analogs--F120L, D121A, and D121N--but not in the fully active (or hyperactive) F120Y. The insertion of the phenolic hydroxyl of Tyr 120 into a hydrogen-bonding network involving the hydroxyl group of Ser 123 and a water molecule in F120Y is the likely basis for the hyperactivity toward uridine 2',3'-cyclic phosphate previously found for this analog (Hodges RS, Merrifield RB, 1974, Int J Pept Protein Res 6:397-405) as well as the threefold increase in KM for cytidine 2',3'-cyclic phosphate found for this analog by ourselves.

Synthetic egg-laying hormone of Aplysia: quantitative studies of induction of egg laying in Stylocheilus and of the activation of Aplysia buccal neuron B16.

1. Synthetic Aplysia egg-laying hormone (ELH-lysine-amide) elicited egg laying in Stylocheilus at a threshold dose of 0.5 microgram per recipient, estimated to be a concentration in the circulation of Stylocheilus of approximately 70 nM. 2. Threshold level and size of egg mass produced by ELH-lysine-amide and bag cell extracts (containing biological ELH) were not significantly different. Latency to lay of recipients of 0.5-4.0 micrograms ELH-lysine-amide (30 +/- 1 min) was significantly longer (P less than 0.05) than for bag cell extract recipients (21 +/- 1 min). 3. ELH-lysine-amide depolarized and activated action potentials in Aplysia buccal neuron B16 in high magnesium, low calcium medium. 4. The lowest concentration of ELH-lysine-amide to activate a supra-threshold response of left and right B16 neurons ranged from 250 nM to 1 microM. 5. Threshold levels for responses to synthetic ELH-lysine-amide and to biological ELH were approximately the same in both egg-laying assay and electrophysiological assay, indicating the likely identity of synthetic and biological ELH. However, the shorter egg-laying latency with bag cell extract suggests that there may be additional factors in the extract that facilitate egg laying.

Prognosis and diagnosis of Reye syndrome by discriminant analysis.

Discriminant analysis was used to discriminate between Reye syndrome (RS) patients and non-RS cases based either on conventional blood chemistry data obtained upon admission, or on the activities of hepatic mitochondrial enzymes in biopsy or necropsy tissue. The control group for blood chemistry measurements contained children with upper respiratory tract infections, varicella, etc. who did not develop RS, as well as healthy children. Subjects with no liver disorder (e.g., accidental death, sudden infant death, etc.) or with non-RS liver disorders were used as controls for hepatic enzyme studies. Hepatic damage indicators (aspartate aminotransferase, AST; alanine aminotransferase, ALT; and bilirubin) correctly classified 86-96% of non-RS cases and 61-71% of RS. By contrast, AST and ALT had little prognostic value (63% overall correct). Ammonia effectively classified favorable outcome cases (95% correct) but not unfavorable (14% correct). However, when ammonia was included with stage of coma information 88% of the favorable and 85% of the unfavorable outcome cases were correctly classified. Discriminant analysis of hepatic enzymes (glutamate dehydrogenase and monoamine oxidase activity) for a RS and a non-RS group correctly classified 80% of non-RS and 95% of RS specimens. The function was suitable for the direct evaluation of RS-like mitochondrial enzyme changes in rat liver.

Hepatic and encephalopathic components of Reye's syndrome: factor analysis of admission data from 209 patients.

Factor analysis of admission data from 209 Reye's syndrome patients yielded three factors. Factor 1 was associated with encephalopathy, blood ammonia, creatinine kinase (CK), uric acid and, to a lesser extent, bilirubin. This factor was linked to the encephalopathy and hypermetabolic changes in muscle, possibly prostaglandin-mediated proteolysis. Factor 2 was associated with serum alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT), and was identified as a hepatic lesion component. These factors correspond to two etiologic components of Reye's syndrome. Salicylate was only weakly associated with neuropathic and hypercatabolic indicators and not at all associated with the hepatic damage indicators.

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.

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), D-glucose-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.

Comparison of cytosolic and mitochondrial hepatic enzyme alterations in Reye's syndrome.

The activities of nine enzymes in liver specimens obtained from four children who had died from Reye's syndrome were compared to the corresponding activities of a control group of four children who had died from unrelated causes. At the 95% significance level, the alterations could be classified into three groups. Five activities [lactate dehydrogenase, alanine aminotransferase, glucose 6-phosphatase, cytochrome oxidase, and malate dehydrogenase (mitochondrial plus cytosolic)] showed no change. Three enzymes [glutamate dehydrogenase, isocitrate dehydrogenase (NADP), and monoamine oxidase] were decreased. One activity (glucose 6-phosphate dehydrogenase) was increased. The malate dehydrogenase isozymes were resolved by electrophoresis, and the two bands were stained and measured. The ratio of cytosolic:mitochondrial enzyme was significantly greater in Reye's syndrome than in the control group. These results lend further support to the view that in Reye's syndrome the impairment of hepatic function is largely confined to the mitochondria. The lowered activity of monoamine oxidase means that the abnormalities extend to the outer mitochondrial membrane. Imbalances of the cytosolic:mitochondrial enzyme activities were evaluated in needle biopsy specimens from four other children under conditions where neurologic abnormalities were less severe. Two patients had elevated ratios of both glutamate:lactate dehydrogenase and cytosolic:mitochondrial malate dehydrogenase activities, and a third had only an abnormal malate dehydrogenase ratio. In contrast to these Reye's syndrome patients, a fourth case admitted with a provisional diagnosis of Reye's syndrome showed no abnormality in either ratio in stage IV coma.