Aspects of the digestive gland cells of the mussel Mytilus galloprovincialis, in relation to lysosomal enzymes, lipofuscin presence and shell size: contribution in the assessment of marine pollution biomarkers.

The present study investigates the histochemical localization of N-acetyl-ß-hexozaminidase (Hex), acid phosphatase (AcP) and ß-glucuronidase (ß-Gus) in the digestive gland of mussels Mytilus galloprovincialis, as well as the clarification of suitable enzyme for biomarkers' application dealing with lysosomes. The results show more intense and homogenous localization of Hex, in relation to AcP and ß-Gus and, thus, Hex histochemistry is supported as more suitable procedure for the evaluation of "lysosomal membrane stability" and "morphometrical alterations of lysosomes". The affection of lipofuscin granules on lysosomal enzymes' activity is also discussed. Additionally, the present study examines the response of small- and large-sized mussels M. galloprovincialis by assessing the "lysosomal membrane stability", "morphometrical alterations of lysosomes", "lysosomal response index (LRI)" and "structural epithelial changes in digestive tubules". The results indicate appreciable alterations of the above parameters in large-sized mussels, supporting their greater influence by the environmental factors, in relation to small-sized ones.

Proteases inhibition assessment on PC12 and NGF treated cells after oxygen and glucose deprivation reveals a distinct role for aspartyl proteases.

Hypoxia is a severe stressful condition and induces cell death leading to neuronal loss both to the developing and adult nervous system. Central theme to cellular death is the activation of different classes of proteases such as caspases calpains and cathepsins. In the present study we investigated the involvement of these proteases, in the hypoxia-induced PC12 cell death. Rat PC12 is a model cell line for experimentation relevant to the nervous system and several protocols have been developed for either lethal hypoxia (oxygen and glucose deprivation OGD) or ischemic preconditioning (IPS). Nerve Growth Factor (NGF) treated PC12 differentiate to a sympathetic phenotype, expressing neurites and excitability. Lethal hypoxia was established by exposing undifferentiated and NGF-treated PC12 cells to a mixture of N(2)/CO(2) (93:5%) in DMEM depleted of glucose and sodium pyruvate for 16 h. The involvement of caspases, calpains and lysosomal cathepsins D and E to the cell death induced by lethal OGD was investigated employing protease specific inhibitors such as z-VAD-fmk for the caspases, MDL28170 for the calpains and pepstatin A for the cathepsins D and E. Our findings show that pepstatin A provides statistically significant protection from cell death of both naive and NGF treated PC12 cells exposed to lethal OGD. We propose that apart from the established processes of apoptosis and necrosis that are integral components of lethal OGD, the activation of cathepsins D and E launches additional cell death pathways in which these proteases are key partners.

Five novel mutations in the lysosomal sialidase gene (NEU1) in type II sialidosis patients and assessment of their impact on enzyme activity and intracellular targeting using adenovirus-mediated expression.

Sialidosis is an autosomal recessive disease resulting from a deficiency of lysosomal sialidase. Type II sialidosis is a rare disease characterized clinically by hydrops fetalis, hepatosplenomegaly, and severe psychomotor retardation. Genomic DNA from four unrelated sialidosis patients was screened for mutations within the sialidase gene NEU1. Five novel mutations were identified. Four are missense and one is nonsense: c.674G>C (p.R225P), c.893C>T (p.A298V), c.3G>A (p.M1?), c.941C>G (p.R341G), and c.69G>A (p.W23X). We have used our findings and diagnostic tools to confirm the presence of a homozygous null allele in a neonate sibling. Recombinant adenoviruses expressing the mutant sialidase alleles in primary cell cultures were utilized to assess the impact of each mutation on enzyme activity and intracellular localization. None of the mutant alleles expressed significant enzymatic activity. The p.R341G mutation exerts its pathological effect by perturbing substrate binding, while the p.A298V and p.R225P mutations appear to impair the folding of the sialidase enzyme. Our findings point to mutation-sensitive amino acids involved in catalytic function or structural stability and indicate the potential utility of these mutations for molecular diagnosis of this rare disease.

[Medical and biological assessment of the safety of genetically modified corn lines MON 810 and GA 21: a toxicological-biochemical study]. / Mediko-biologicheskaia otsenka bezopasnosti geneticheski modifitsirovannykh linii kukuruzy MON 810 i GA 21: toksikologo-biokhimicheskaia issledovaniia.

The rats were fed with the flour of corn from genetically modified corn MON 810 and from genetically modified corn GA 21 (Monsanto Co, USA) 3 g/rat/day for 6 months. Their blood, urea and liver were investigated to measure total protein and glucose levels, aminotransferase and alkaline phosphatase activities, pH, creatinine level as well as hepatic enzyme activity of the I and II phases of xenobiotic metabolism and whole and non-sedimentated lysosomal enzyme activities, the level of processes of lipids peroxidation and activity of antioxidant system.

Assessment of variability in some lysosomal and soluble enzyme activities in corneal epithelium from slaughterhouse bovine eyes.

The corneal epithelium was scraped from bovine eyes (Hereford or Holstein) within 3 h post mortem. The mitochondriallysosomal fraction of the cells was assayed for hexosaminidase (HEX) and acid phosphatase (AP), and the membrane-soluble fraction for lactate dehydrogenase (LDH) and leucine arylamidase (LAA) activity. Pairs of eyes with clear corneas showed variances (+/-1 SD, n = 10 pairs) of +/-62, +/-57, +/-72 and +/-31% in the four enzyme activities (HEX, AP, LDH, LAA). Selection based on age (corneal diameter) reduced the variance to +/-41, +/-37, +/-39 and +/-30%. Additional selection for corneal surface wettability reduced variances to +/-26, +/-21, +/-22 and +/-26% in the activities. The latency of the lysosomal enzymes (HEX, AP) increased substantially with eye selection.

[The diagnostic and prognostic assessment of the changes in lysosomal enzymatic activity in the blood of patients with acute myocardial infarct and unstable stenocardia]. / Diagnosticheskaia i prognosticheskaia otsenka izmeneii aktivnosti lizosomal'nykh fermentov v krovi u bol'nykh ostrym infarktom miokarda i nestabol'noi stenokardiei.

Serum activity of five lysosomal marker enzymes was measured in patients with acute myocardial infarction (AMI) and unstable angina pectoris (UAP) with consideration of the disease severity. A significant increase of enzyme activity was found both in patients with UAP and AMI on the first day of hospitalization. The enzymes activity was higher in patients with more severe form of AMI. Lysosomal enzyme activity was closely related to severity of the disease: clinical improvement occurred in patients with decreased activity (on day 7-10), while in aggravation of the disease lysosomal hyperenzymemia stayed high up to the end of the observation period (on day 30). The above findings demonstrate feasibility of using lysosomal enzymes test both for differentiation between AMI and UAP and for predicting the disease recurrences.

[Lysosomal storage disease: a group of genetic neurodegenerative disorders].

Lysosomal storage disease is a group of neurometabolic diseases mainly occurring in infancy and childhood. They were first recognized as new diseases on the basis of unique clinical manifestations or pathological findings, and then the stage of biochemical analysis of storage material and enzyme assays in tissues and cells from patients followed. Recent technological development has enabled us to look further into the molecular genetic basis of these inherited diseases. Protein analysis revealed intracellular events of the mutant enzyme molecule responsible for the pathogenesis of a disease, and more detailed information has been obtained about the mutant gene and its product. Clinical manifestations are not always uniform for a single disease with mutations in the same gene. Clinical subtypes have been proposed for many lysosomal diseases. At present, the molecular and metabolic basis of each phenotypic expression is not clear, although common mutations have been found for specific clinical forms in some diseases. In this article, the current status of lysosomal disease research was summarized, particularly focusing on molecular pathology and molecular diagnosis. Finally future prospects for pathogenetic analysis of neural dysfunction and possible gene therapy were briefly discussed.

Assessment of lysosomal function by quantitative histochemical and cytochemical methods.

Quantitative histochemistry and cytochemistry enables a direct link to be made between metabolic functions such as the activity of lysosomal enzymes and the morphology of a tissue or a type of cell. Several approaches exist such as microchemistry based on (bio)chemical analysis of a single cell or a small piece of tissue dissected from a freeze-dried section. This technique has been routinely used for prenatal diagnosis of inherited enzyme defects and especially of lysosomal storage diseases. Other approaches are cytofluorometry or cytophotometry, which are based on the principle that a fluorescent or coloured final reaction product is precipitated at the site of the enzyme. The amount of final reaction product is analysed per cell or per unit volume of tissue using either a microscope cytofluorometer or flow cytometer for fluorescence measurements or an image analysing system or scanning and integrating cytophotometer for absorbance measurements. In principle, fluorescence methods are to be preferred over chromogenic methods because they are more sensitive and enable multiparameter analysis. However, only a limited number of fluorogenic methods are at hand that give a final reaction product which is sufficiently water-insoluble to guarantee good localisation. The best results have been obtained with methods based on naphthol AS-TR derivatives and with methods for the demonstration of protease activity using methoxynaphthylamine derivatives as substrates and 5'-nitrosalicylaldehyde as coupling reagent. Chromogenic methods are far better with respect to localisation properties and, therefore, most commonly used for quantitative histochemical analysis of lysosomal enzyme activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of beta-glucuronidase and structural assessment of the carbohydrate chains by lectin affinity immunoelectrophoresis.

The purification of rat liver beta-glucuronidase from a lysosomal fraction by methods including affinity chromatography, chromatofocusing and preparative PAGE steps is described. Molecular weights of 300,000 and 150,000 were estimated by two dimensional gradient PAGE/immunoelectrophoresis of the lysosomal extract. Isoelectrofocusing in agarose gel followed by immunoelectrophoresis in the second dimension revealed the presence of at least five maxima in the range pH 4.3-7.4. The structural assessment of the carbohydrate chains of lysosomal and microsomal beta-glucuronidase was performed by lectin affinity immunoelectrophoresis. Reaction with Concanavalin A indicated the presence of bi-antennary complex, oligomannosidic and hybrid type structures, whereas the absence of tri- and tetra-antennary complex type structures was deduced from the lack of interaction with phytohemagglutinin-L. The reaction with Lens culinaris agglutinin, Pisum sativum agglutinin and Lotus tetragonolobus lectin revealed that part of the glycans contained a fucose alpha(1-6)-linked to the N-acetylglucosamine attached to asparagine. The presence of terminal beta(1-4)-galactose residues was detected with Ricinus communis agglutinin I.

Quantitative assessment of lysosomal size, number and enzyme activity in mouse kidney during maturational development.

Image and cytochemical analyses were undertaken to determine possible correlation between the number and size of acid phosphatase-positive granules (lysosomes), and variation in acid phosphatase (AcP) activity in the proximal tubule cells of mouse-kidney during growth and development. Eighteen ddY strain mice ages: 1 day, 1 and 2 weeks, and 1, 2 and 10 months were used. The lanthanide-based method for the ultrastructural localization of AcP-activity was employed. The number and size of AcP positive granules were quantitatively analyzed by image analysis, and AcP activity by X-ray microanalysis. Significance was evaluated by 2-tailed-Student's t-test for the difference between means. AcP activity was observed in the lysosomes and the reaction product appeared dense and heterogeneous. In some cells, it appeared apparently homogeneous. The results showed that the number and size of AcP Positive granules (lysosomes) increased significantly from the first day after birth, recorded a peak in one week time and thereafter, it gradually declined until the 10th month. The result of X-ray microanalysis demonstrated a variation in accordance with the degree of AcP activity at different ages of the animals studied. The AcP activity decreased significantly from day one and progressively until the 10th month. From the results of the present work, it could be inferred that the changes in size and number of AcP positive granules, at least, at the early stage, and/or the variation in AcP activity are related to the growth and development of the animal.

Rapid simultaneous assessment of neutrophil superoxide generation and lysosomal enzyme release.

A rapid method for the simultaneous measurement of neutrophil superoxide generation and beta-glucuronidase release is described. Assay of beta-glucuronidase using a fluorescent substrate is shown to be valid in the presence of reduced or unreduced ferricytochrome C, a prerequisite for the simultaneous assessment of this enzyme activity and O-2 generation.

An assessment of the importance of intralysosomal and of alpha-amylolytic glycogenolysis in the liver of normal rats and of rats with a glycogen-storage disease.

Mechanisms of glycogenolysis have been investigated in a comparative study with Wistar rats and gsd rats, which maintain a high glycogen concentration in the liver as a result of a genetic deficiency of phosphorylase kinase. In Wistar hepatocytes the rate of glycogenolysis, as modulated by glucagon and by glucose, was proportional to the concentration of phosphorylase a. In suspensions of gsd hepatocytes the rate of glycogenolysis was far too high as compared with the low level of phosphorylase a; in addition, only a minor fraction of the glycogen lost was recovered as glucose and lactate, owing to the accumulation of oligosaccharides. When the gsd hepatocytes were incubated in the presence of an inhibitor of alpha-amylase (BAY e 4609) glycogenolysis and the formation of oligosaccharides virtually ceased; the production of glucose plus lactate, already modest in the absence of BAY e 4609, was further decreased by 40%, owing to the suppression of a pathway for glucose production by the successive actions of alpha-amylase and alpha-glucosidase. Evidence was obtained that gsd hepatocytes are more fragile, and that amylolysis of glycogen occurred in damaged cells and/or in the extracellular medium. This may even occur in vivo, since quick-frozen liver samples from anesthetized gsd rats contained severalfold higher concentrations of oligosaccharides than did similar samples from Wistar rats. However, administration of a hepatotoxic agent (CCl4) caused hepatic glycogen depletion in Wistar rats, but not in gsd rats. The administration of phloridzin and of vinblastine, which have been proposed to induce glycogenolysis in the lysosomal system, did not decrease the hepatic glycogen level in gsd rats. Taken together, the data indicate that only the phosphorolytic degradation of glycogen is metabolically important, and that alpha-amylolysis is an indication of an increased fragility of gsd hepatocytes, which becomes prominent when these cells are incubated in vitro.

Biochemical investigation of the functional state of various subcellular structures as a criterion for the assessment of unfavourable effects of environmental factors.

Theoretical basis and results of biochemical studies of the enzymic systems of different localization in the cell--both bound with subcellular structures lysosomes--and dissolved in the cytosol--during experimental exposure to some environmental factors (carbon tetrachloride, carbon disulphide and others) are presented. It has been suggested to employ the assessment of the functional state of biomembranes of cellular organelles (e. g. lysosomes) and especially the effect of solubilization of membrane-bound enzymes and labilization of lysosomal membranes as one of the biochemical criteria of hygienic assessment of toxic effects and of some forms of delayed effects of environmental contaminants. Simultaneous biochemical investigation of enzyme-markers of subcellular organelles in the tissues of various organs and in biological fluids (blood, urine, seminal and amniotic fluids) permits us to elaborate and recommend for use in hygienic practice new biochemical criteria of unfavourable biological effects of chemical factors of the environment.

Considerations in the prenatal assessment of lysosomal enzyme deficiency in eight cases at risk.

Some of the factors which are to be considered in the antenatal diagnostic evaluation of lysosomal enzyme levels in cultured amniotic fluid cells are discussed in the light of eight consecutive cases in which the foetuses had a 1 in 4 chance of being homozygous for a lysosomal storage disease. There were 2 possible cases of GM1 gangliosidosis, 2 of neuropathic Gaucher's disease, 1 of Krabbe's disease and 2 of Pompe's disease. Each infant was predicted to be unaffected; the assessments were confirmed to be correct postnatally--7 by enzyme studies. Using a micro technique, 5 of the assessments were available in 28 days or less following amniocentesis. It is concluded that in certain circumstances skin fibroblasts may be used as valid controls and that the variability of assay results strongly militates against the confident assignment of heterozygous status and may cause difficulties in the identification of the homozygote in cases where 'residual' enzyme activity is high; concomitant family studies assist in the resolution of such problems.