Pre- and postnatal diagnosis of patients with CLN1 and CLN2 by assay of palmitoyl-protein thioesterase and tripeptidyl-peptidase I activities.

Palmitoyl-protein thioesterase (PPT) and tripeptidyl-peptidase I (TPP-I) activities were measured in leucocytes and fibroblasts. Fourteen patients were confirmed as having late infantile neuronal ceroid lipofuscinosis due to a deficiency of TPP-I activity. This included one patient with a milder and more protracted form of the disease. In addition this enzyme deficiency was found in a clinically normal younger sibling of a patient. Of particular importance was the finding of normal TPP-I activity in two patients who had been diagnosed as having classical late infantile neuronal ceroid lipofuscinosis. A deficiency of PPT was confirmed retrospectively in stored fibroblasts from two patients who had already died having been diagnosed with infantile neuronal ceroid lipofuscinosis. Palmitoyl-protein thioesterase or TPP-I activities were measured in chorionic villi and cultured chorionic villi cells in three pregnancies. The enzyme results were confirmed by mutational analysis if the mutations were known, or, in the case of the pregnancy at risk for infantile neuronal ceroid lipofuscinosis by electron microscopy of the chorionic villi. Our results show that assay of PPT and TPP-I is reliable in the diagnosis of patients with mutations in the CLN1 and CLN2 genes. It is imperative to assay these enzymes in all patients to confirm the diagnosis and ensure accurate genetic counselling of other family members. Once an enzyme deficiency has been confirmed reliable prenatal diagnosis is available even if both mutations have not been detected.

Successful treatment of carbohydrate deficient glycoprotein syndrome type 1b with oral mannose.

An Asian girl presented with failure to thrive, congenital hepatic fibrosis, protein losing enteropathy, and hypoglycaemia. Phosphomannose isomerase activity in skin fibroblasts was reduced. She is homozygous for a mutation, D131N, in the phosphomannose isomerase gene (PM1), consistent with the diagnosis of carbohydrate deficient glycoprotein syndrome type 1b. She responded to oral mannose treatment.

Early-onset fetal hydrops and muscle degeneration in siblings due to a novel variant of type IV glycogenosis.

We report on 3 consecutive sib fetuses, presenting at 13, 12, and 13 weeks of gestation, respectively, with fetal hydrops, limb contractures, and akinesia. Autopsy of the first fetus showed subcutaneous fluid collections and severe degeneration of skeletal muscle. Histologic studies demonstrated massive accumulation of diastase-resistant periodic acid-Schiff-positive material in the skeletal muscle cells and epidermal keratinocytes of all 3 fetuses. Enzyme studies of fibroblasts from the 3rd fetus showed deficient activity of glycogen brancher enzyme, indicating that this is a new, severe form of glycogenosis type IV with onset in the early second trimester.

Protective effects of carnosine against protein modification mediated by malondialdehyde and hypochlorite.

Malondialdehyde (MDA) and hypochlorite anions are deleterious products of oxygen free-radical metabolism. The effects of carnosine, a naturally occurring dipeptide (beta-alanyl-L-histidine), on protein modification mediated by MDA and hypochlorite have been studied. MDA and hypochlorite induced formation of carbonyl groups and high molecular weight and cross-linked forms of crystallin, ovalbumin and bovine serum albumin. The presence of carnosine effectively inhibited these modifications in a concentration-dependent manner. It is proposed that relatively non-toxic carnosine and related peptides might be explored as potential therapeutic agents for pathologies that involve protein modification mediated by MDA or hypochlorite.

Pluripotent protective effects of carnosine, a naturally occurring dipeptide.

Carnosine is a naturally occurring dipeptide (beta-alanyl-L-histidine) found in brain, innervated tissues, and the lens at concentrations up to 20 mM in humans. In 1994 it was shown that carnosine could delay senescence of cultured human fibroblasts. Evidence will be presented to suggest that carnosine, in addition to antioxidant and oxygen free-radical scavenging activities, also reacts with deleterious aldehydes to protect susceptible macromolecules. Our studies show that, in vitro, carnosine inhibits nonenzymic glycosylation and cross-linking of proteins induced by reactive aldehydes (aldose and ketose sugars, certain triose glycolytic intermediates and malondialdehyde (MDA), a lipid peroxidation product). Additionally we show that carnosine inhibits formation of MDA-induced protein-associated advanced glycosylation end products (AGEs) and formation of DNA-protein cross-links induced by acetaldehyde and formaldehyde. At the cellular level 20 mM carnosine protected cultured human fibroblasts and lymphocytes, CHO cells, and cultured rat brain endothelial cells against the toxic effects of formaldehyde, acetaldehyde and MDA, and AGEs formed by a lysine/deoxyribose mixture. Interestingly, carnosine protected cultured rat brain endothelial cells against amyloid peptide toxicity. We propose that carnosine (which is remarkably nontoxic) or related structures should be explored for possible intervention in pathologies that involve deleterious aldehydes, for example, secondary diabetic complications, inflammatory phenomena, alcoholic liver disease, and possibly Alzheimer's disease.

Protective effects of carnosine against malondialdehyde-induced toxicity towards cultured rat brain endothelial cells.

Malondialdehyde (MDA) is a deleterious end-product of lipid peroxidation. The naturally-occurring dipeptide carnosine (beta-alanyl-L-histidine) is found in brain and innervated tissues at concentrations up to 20 mM. Recent studies have shown that carnosine can protect proteins against cross-linking mediated by aldehyde-containing sugars and glycolytic intermediates. Here we have investigated whether carnosine is protective against malondialdehyde-induced protein damage and cellular toxicity. The results show that carnosine can (1) protect cultured rat brain endothelial cells against MDA-induced toxicity and (2) inhibit MDA-induced protein modification (formation of cross-links and carbonyl groups).

Changes in proteinase and peptidase activities during reticulocyte maturation.

The ability of rabbit reticulocytes to degrade puromycin-peptides and aminoethylcysteine-induced aberrant polypeptides decreased during cellular maturation. Cell-free studies indicate that the fall in proteolytic activity is not a consequence of accumulation of proteinase inhibitors or the conversion of all of the abnormal protein into undegradable forms. A decrease in peptidase activity using seven dipeptides and one tripeptide as substrates was also found to accompany reticulocyte maturation. Addition of the aminopeptidase B inhibitor bestatin to reticulocyte extracts did not inhibit the conversion of acid-precipitable puromycin-peptides to acid-soluble products; bestatin did induce the accumulation of very low molecular weight material (possibly di- or tripeptides) within the acid soluble fraction.

Protease and peptidase activities during reticulocyte maturation.

Rabbit reticulocytes were fractionated according to buoyant density on albumin gradients. The properties of fractionated cells were consistent with the thesis that the procedure separated the reticulocytes according to their degree of maturation. Catabolism of aminoethylcysteine-induced abnormal protein and puromycin-peptides declined with increasing cellular maturity. Cell-free extracts showed a similar decrease in degradative activity: addition of extracts of young cells stimulated proteolysis in extracts from old cells. Peptidase activity also declined during maturation. In the more mature cells abnormal protein was located increasingly in the cell-debris fraction, analogous to Heinz bodies.

Effects of ATP and cell development on the metabolism of high molecular weight aggregates of abnormal proteins in rabbit reticulocytes and cell-free extracts.

Abnormal proteins synthesized in rabbit reticulocytes in response to (i) the lysine analogue aminoethylcysteine and (ii) puromycin, form high molecular weight aggregates prior to degradation. Inhibitors of ATP synthesis partially inhibit catabolism of the aminoethylcysteine-induced abnormal protein; degradation of puromycin peptides synthesized after incubation with 25 micrograms/ml puromycin was not inhibited. Catabolism of the analogue-induced high molecular weight aggregate of abnormal protein in cell-free extracts was markedly stimulated by ATP, whereas proteolysis of the aggregated puromycin-peptides was ATP-independent. The ability of the reticulocytes to degrade the puromycin-peptide aggregates was found to decrease with cellular maturity. It is suggested that the energy-dependency for proteolysis is in some way related to the chain length of the abnormal protein synthesized.

Selective proteolysis of abnormal proteins of shortened chain length in rabbit reticulocytes.

Proteolysis of certain shortened globin chains can proceed in cell-free extracts of rabbit reticulocytes in the absence of ATP. Proteolysis of amino acid analogue-induced abnormal globin chains of normal chain length may be stimulated by ATP, however. The ATP independent degradation of globin cyanogen bromide-peptides appears to require the presence of free, unblocked amino groups in the substrate, has a pH optimum of 7.8 and is inhibited by phenanthroline, cobalt and zinc ions, N-ethylmaleimide and cystamine.

Inhibition of canavanyl-protein proteolysis in Escherichia coli by rifampin.

Rifampin inhibited the intracellular proteolysis of canavanine-induced, rapidly sedimenting protein complexes in Escherichia coli.