Mashenka Dimitrova

Comparison of the activity levels and localization of dipeptidyl peptidase IV in normal and tumor human lung cells

Dipeptidyl peptidase IV (DPPIV) was studied in three human lung cells - P (fetal lung-derived cells), A549 (lung adenocarcinoma) and SK-MES-1 (squamous cell carcinoma) using a fluorescent cytochemical procedure developed on the basis of the substrate 4-(glycyl-L-prolyl hydrazido)-N-hexyl-1,8-naphthalimide. The observed differences in the enzyme expression were confirmed by measuring the enzyme hydrolysis of glycyl-L-prolyl-para-nitroanilide. The surface and total dipeptidyl peptidase activities of P cells were correspondingly 7-8 and 3-10 times higher than those of SK-MES-1 and A549 cells. The ratio surface per total activity showed that in P (95%) and A549 (93%) cells the enzyme is associated with the plasmalemma while in SK-MES-1 cells (35%) it is bound to intracellular membranes. In order to compare the results from cell cultures with those in human tumor, the enzyme activity was investigated in cryo-sections of three cases of diagnosed squamous lung carcinoma. DPPIV activity was restricted to the connective tissue stroma surrounding the DPPIV-negative tumor foci.

Developmental study of tripeptidyl peptidase I activity in the mouse central nervous system and peripheral organs

Tripeptidyl peptidase I (TPPI) — a lysosomal serine protease — is encoded by the CLN2 gene, mutations that cause late-infantile neuronal ceroid lipofuscinosis (LINCL) connected with profound neuronal loss, severe clinical symptoms and early death at puberty. Developmental studies of TPPI activity levels and distribution have been done in the human and rat central nervous systems (CNS) and visceral organs. Similar studies have not been performed in mouse. In this paper, we follow up on the developmental changes in the enzyme activity and localization pattern in the CNS and visceral organs of mouse over the main periods of life — embryonic, neonate, suckling, infantile, juvenile, adult and aged — using biochemical assays and enzyme histochemistry. In the studied peripheral organs (liver, kidney, spleen, pancreas and lung) TPPI is present at birth but further its pattern is not consistent in different organs over different life periods. TPPI activity starts to be expressed in the brain at the 10th embryonic day but in most neuronal types it appears at the early infantile period, increases during infancy, reaches high activity levels in the juvenile period and is highest in adult and aged animals. Thus, in mice TPPI activity becomes crucial for the neuronal functions later in development (juvenile period) than in humans and does not decrease with aging. These results are essential as a basis for comparison between normal and pathological TPPI patterns in mice. They can be valuable in view of the use of animal models for studying LINCL and other neurodegenerative disorders.

Neurodegenerative changes in rat produced by lithium treatment.

Lithium is extensively used in psychiatric practice for the prevention and treatment of manic-depressive disorders. However, neurotoxicity attributed to lithium salts within therapeutic doses was also reported in patients, manifested by transient or persistent neurological deficits. In this study, morphological changes were examined in rats treated acutely and chronically with lithium. Pathological changes were observed in different brain regions including cerebral cortex, cerebellum, medulla oblongata, mesencephalon, thalamus, and pons, using a silver–copper impregnation technique for neurodegeneration. Vacuolization of brain tissue with subsequent formation of spongiosis was the prominent morphological feature following lithium administration. The zones of spongiosis were irregularly distributed throughout the brain. More intensive compact areas with spongiform changes were found in the cerebral cortex and medulla oblongata. Less pronounced vacuolization was noted in the pons and thalamic region. The cerebellum and mesencephalon appeared least affected. Vacuolization in the cerebellar cortex was found at loci with Purkinje cells, but the classical picture of spongiosis was not apparent. Data indicate that both acute and chronic lithium intoxication accelerated neurodegenerative changes normally seen with normal brain aging.

Effect of acute hypoxic shock on the rat brain morphology and tripeptidyl peptidase I activity

Hypoxic events are known to cause substantial damage to the hippocampus, cerebellum and striatum. The impact of hypoxic shock on other brain parts is not sufficiently studied. Recent studies show that tripeptidyl peptidase I (TPPI) activity in fish is altered after a hypoxic stress pointing out at a possible enzyme involvement in response to hypoxia. Similar studies are not performed in mammals. In this work, the effect of sodium nitrite-induced acute hypoxic shock on the rat brain was studied at different post-treatment periods. Morphological changes in cerebral cortex, cerebellum, medulla oblongata, thalamus, mesencephalon and pons were assessed using silver-copper impregnation for neurodegeneration. TPPI activity was biochemically assayed and localized by enzyme histochemistry. Although less vulnerable to oxidative stress, the studied brain areas showed different histopathological changes, such as neuronal loss and tissue vacuolization, dilatation of the smallest capillaries and impairment of neuronal processes. TPPI activity was strictly regulated following the hypoxic stress. It was found to increase 12-24h post-treatment, then decreased followed by a slow process of recovery. The enzyme histochemistry revealed a temporary enzyme deficiency in all types of neurons. These findings indicate a possible involvement of the enzyme in rat brain response to hypoxic stress.

Tripeptidyl Peptidase I and Its Role in Neurodegenerative and Tumor Diseases

Tripeptidyl peptidase I (TPPI) is a lysosomal enzyme widely distributed in mammals and humans. Its genetically determined deficiency causes the classical late-infantile form of neuronal ceroid lipofuscinosis, a fatal hereditary neurodegenerative disease associated with severe symptoms and early death, usually in the second decade of life. Many studies also show that TPPI is differentially regulated under various pathological conditions such as malignancy, neurodegeneration, ischemia, and inflammation, pointing at possible enzyme involvement in the pathogeneses of these entities. This chapter focuses on the TPPI participation in neurodegenerative and neoplastic diseases.

Histochemical Demonstration of Tripeptidyl Aminopeptidase I

Enzyme histochemical methods are valuable for the studies on the enzyme involvement in different pathological processes. Here we describe two protocols for chromogenic and fluorogenic histochemical demonstration of tripeptidyl aminopeptidase I (TPPI), a protease that is crucial for neuronal functions. The procedures are based on newly synthesized substrates for TPPI-glycyl-L-prolyl-L-metionyl-5-chloro-1-anthraquinonylhydrazide (GPM-CAH) and glycyl-L-prolyl-L-metionyl-4-hydrazido-N-hexyl-1,8-naphthalimide (GPM-HHNI). Using such protocols, precise enzyme localization can be obtained in tissue sections of mammalian organs.

Gamma-Glutamyl Transpeptidase Activity in Human Fetal Lung-Derived Cells

ABSTRACT Gamma-glutamyl transpeptidase (GGT, EC 2.3.2.2) - a membrane-associated enzyme is a main regulator of glutathione (GSH) levels. The enzyme is not only the inhibitor of apoptosis induced by oxidative stress, but it is engaged in anti-tumor drug cell resistance and is considered as a marker for neoplastic changes, cell differentiation and aging. The surface and total activities of GGT in unfixed, fixed with para-formaldehyde vapors P cells (human fetal lung-derived cells) and cells lyzates were measured and compared using the γ-Glu-4-nitroanilide substrate. Our results showed that the surface activity was higher than the total, which might be due to the desegregation of the enzyme molecules upon mechanical cell homogenization and at the same time, after cell fixation in para-formaldehyde vapors it was decreased twice. Thus, methods including homogenization and/or aldehyde fixation are not suitable for the study of GGT activity. In addition, visualization of GGT activity was tested using the fluorogenic substrate γ-Glu-4-hydrazido-N-hexyl-1,8- naphthalimide (γ-Glu-HHNI). Our results pointed out that GGT could not be visualized in fixed P (normal diploid) cells using this substrate probably as a result of the lower reactivity of the enzyme towards hydrazide substrates in comparison to amide substrates. On the other hand, GGT activity was well demonstrated with the same substrate in SK-MES-1 cells (human squamous cell carcinoma). Consequently, the novel fluorogenic substrate γ-Glu-HHNI could be used for diagnostic purposes on the basis of not-detectable/detectable GGT activity in normal and pathologically altered lung epithelial cells respectively.