Lysosomal Marker Enzyme Research Articles

Levels of marker lysosomal hydrolases in women with coronary heart disease depending on age and sex hormone level

Aim of the study was to evaluate the serum concentrations of three marker lysosomal hydrolases (cathepsin D, acid phosphatase (AP) and acid DNase (aDNAase)) in women with coronary heart disease (CHD) depending on the level of follicle-­stimulating hormone (FSH), testosterone (T), age and find if those parameters associated with anthropometric parameters, glycemia, insulinemia and HOMA-IR index, biomarkers of atherosclerosis. The study included 285 women aged 35–65 years (median age was 54.4 years (25% and 75% percentiles — 43.2 and 61.3 years, respectively) who had had myocardial infarction no earlier than 30 days before the examination. Patients were divided into the following age groups: 35–55 and 56–65 years (first and second age groups, respectively), and into groups according to the levels of sex hormones: FSH ≥ and <30 mIU/mL and testosterone ≥ and <3 nmol/L. Results of comparative and correlation analyzes demonstrates that in women 35–65 years old with FSH ≥30 mIU/mL, the levels of cathepsin D are higher (p<0.05) than in patients with FSH <30 mIU/mL, and in women 35–55 years old, the content of AP was also higher (p=0.025). Associations of a high level of androgen with lysosomal hyperenzymemia were demonstrated only in the second age group, where at a level of T ≥3 nmol/L, higher values of all three lysosomal enzymes were recorded. Multivariate analysis in both age groups is confirmed direct impact of periand postmenopausal periods on the levels of lysosomal enzymemia and, accordingly, a negative effect on the state of lysosomal membranes. Thus, FSH levels directly determined the concentrations of AP and cardiotropic cathepsin D. The levels of aDNAase in women with CHD of 56–65 years of age were positively correlated with indicators that determine insulin-­glucose homeostasis: glycemia (p<0.001), HOMA-IR index (p<0.001). Such associations of three marker lysosomal enzymes demonstrate the primary contribution of FSH ≥30 mIU/mL to an increase in the concentration of lysosomal hydrolases in women with CHD35–65 years old and the correlation of aDNAase with the processes triggered by insulin resistance.

Isolation and characterization of mitochondria and lysosome from isoproterenol induced cardiotoxic rats


 
 
 Mitochondrial and lysosomal membranes are prominent membranes of cardiac cells and are the factors that determine membrane function in myocardial ischemia. In this study, isolation of mitochondria and lysosome from heart tissue under the control, isoproterenol (ISO) (8.5mg/100g) induced cardiotoxic rats and oral pretreatment with Z. armatum fruit (200, 400mg/kg body weight) treated rats. Further characterization of marker enzymes was done. A decreased in the activity of all the mitochondrial and lysosomal marker enzymes in ISO administered cardiotoxic rats when compared to control rats which indicate ISO decreased the stability of the membrane. Pretreatment with hydroethanolic extract of Z. armatum fruit to ISO induced rats significantly reverted these biochemical alterations near to normal. The possible mechanism for the protection of heart mitochondria and lysosome against oxidative damage induced by ISO might be due to quenching of free radicals and enhancing the action of marker enzymes.
 
 

Chemopreventive and anti-breast cancer activity of compounds isolated from leaves of Abrus precatorius L.

The present study focuses on isolation and evaluation of the anti-cancer activity of compounds from the leaves of Abrus precatorius. The bioassay-directed strategy was adopted using chromatographic, gas chromatographic-mass spectrum analysis, nuclear magnetic resonance and X-ray crystallography techniques for purification and characterization of active cytotoxic compounds. Further, MDA-MB-231 breast cancer cell lines and 7,12-dimethylbenz (a) anthracene (DMBA) induced virgin female Sprague Dawley (SD) rats were used for in vitro and in vivo cytotoxicity evaluation. Stigmasterol hemihydrate and 9,12-Octadecadienoic acid (Z,Z)-2-hydroxy-1-(hydroxymethyl)ethyl ester or (β-monolinolein) were the two main cytotoxic constituents of leaf extract of A. precatorius, with an IC50 value of 74.2 and 13.2µg/ml, respectively, in MDA-MB-231 cells. Additionally, the treatment with the stigmasterol and β-monolinolein as a combinatorial drug therapy in DMBA-induced female SD rats led to recovery of body weight, decreased tumor weight and volume, without any toxic side effects. Immunohistochemical examination showed extensive cell death and low proliferation in the treated tumor tissues that was confirmed by results from H and E staining, TUNEL assay and Ki-67 index as compared to control animal group. The reversion of glycoprotein, lysosomal and tumor marker enzyme levels back to near-normal levels after treatment with the plant compounds clearly demonstrated the reduction of tumor burden in these animals. This is the first report on isolation and characterization of the two active cytotoxic components from leaves of A. precatorius. Additionally, the profound cytotoxic and tumor-suppressive effect of these two compounds as a combinatorial therapy provide an alternative option for breast cancer treatment.

Steviol slows renal cyst growth by reducing AQP2 expression and promoting AQP2 degradation

Overexpression of aquaporin 2 (AQP2) was observed and suggested to be involved in fluid secretion leading to cyst enlargement in polycystic kidney disease (PKD). The cyst expansion deteriorates the renal function and, therefore, therapies targeting cyst enlargement are of clinical interest. Of note, inhibition of vasopressin function using vasopressin 2 receptor (V2R) antagonist which decreased cAMP production along with AQP2 production and function can slow cyst growth in ADPKD. This finding supports the role of AQP2 in cyst enlargement. Steviol, a major metabolite of the sweetening compound stevioside, was reported to retard MDCK cyst growth and enlargement by inhibiting CFTR activity. Interestingly, its efficacy was found to be higher than that of CFTRinh-172. Since steviol was also found to produce diuresis in rodent, it is likely that steviol might have an additional effect in retarding cyst progression, such as inhibition of AQP2 expression and function. Here, we investigated the effect of steviol on AQP2 function and on cyst growth using an in vitro cyst model (MDCK and Pkd1−/− cells). We found that steviol could markedly inhibit cyst growth by reducing AQP2 expression in both Pkd1−/− and MDCK cells. Real-time PCR also revealed that steviol decreased AQP2 mRNA expression level as well. Moreover, a proteasome inhibitor, MG-132, and the lysosomotropic agent, hydroxychloroquine (HCQ) were found to abolish the inhibitory effect of steviol in Pkd1−/− cells. Increased lysosomal enzyme marker (LAMP2) expression following steviol treatment clearly confirmed the involvement of lysosomes in steviol action. In conclusion, our finding showed for the first time that steviol slowed cyst growth, in part, by reducing AQP2 transcription, promoted proteasome, and lysosome-mediated AQP2 degradation. Due to its multiple actions, steviol is a promising compound for further development in the treatment of PKD.

Postnatal Organellogenesis in Pyramidal Neurons in the Cerebral Cortex in Rats

We report here quantitative structural and histochemical evaluation of the postnatal development of organelles in neurons in the inner pyramidal layer of the frontal cortex in rat pups aged 5, 20, and 45 days form birth (four animals of each age). Growth of the perikaryon was seen during postnatal ontogeny, along with the consistent development of organelles within neurons. With age there was a progressive increase in the number of mitochondria, which became more extended in shape. Mitochondria showed progressive increases in the number and lengths of cristae. These changes were accompanied by increases in the activities of mitochondrial marker enzymes SDH and NADHDH. On day 5 after birth, the neuron cytoplasm was dominated by free ribosomes and then by rough endoplasmic reticulum. Cistern size increased progressively. Golgi complexes were not yet formed on day 5 after birth, though they were present by day 20. Lysosome numbers and size increased significantly by day 20; the activity of the lysosomal marker enzyme acid phosphatase showed a similar pattern.

Steviol stabilizes polycystin 1 expression and promotes lysosomal degradation of CFTR and β-catenin proteins in renal epithelial cells

Malfunction of polycystin 1 (PC1) is linked to abnormally high epithelial cell proliferation and fluid secretion, eventually leading to renal cyst development and declined renal function as found in autosomal dominant polycystic kidney disease (ADPKD). Currently, there is no effective therapy for ADPKD. Recent studies report PC1 regulates CFTR chloride channels and β-catenin levels in normal renal epithelial cells. Concurrently, our previous study found steviol retarded renal cyst enlargement in an in vitro and in an in vivo models by reducing CFTR expression and activity. Therefore, a potential relationship between steviol and PC1 is worthy of exploration. The present study was aimed to determine the effect of steviol on PC1, CFTR, and β-catenin levels in renal epithelial cells with defective PC1 biogenesis and expression (Prkcsh−/− cell) and postnatal Pkd1 homozygous cell (Pkd1−/− cells). Using western blot analysis, it was found that steviol treatment at 100μM for 24–48h substantially enhanced and stabilized PC1 C-terminal expression, while decreasing CFTR and β-catenin protein expression in both Prkcsh−/− and Pkd1−/− cells. In addition, steviol promoted LAMP2 expression, a lysosomal enzyme marker. Interestingly, hydroxychloroquine (a lysosome inhibitor) treatment abolished steviol’s effect in reducing CFTR and β-catenin protein expression. Taken together, these findings suggest steviol slows cyst progression in cells and animal models of PKD, in part, by enhancing and stabilizing PC1 protein expression as well as by promoting lysosomal degradation of CFTR and β-catenin. Therefore, steviol may represent a promising compound for treatment of polycystic kidney disease.

Acid Phosphatase and Vitamin A Induced Abnormal Tail Regeneration in Frog Tadpoles: an Immunohistochemical Study

Application of vitamin A palmitate or retinoic acid causes abnormal tail regeneration and sometimes leads to formation of ectopic hind limbs along with pelvic girdles at the cut end of tail. Such a phenomenon referred to as homeotic transformation, has been reported in different anuran species. Acid phosphatase, a lysosomal marker enzyme is known to be associated with regeneration in amphibians. However, role of this enzyme during homeotic transformation of tail of an anuran tadpole into limbs is not explored. Thus, aim of the present study is immunohistochemical localization of acid phosphatase in non-amputated, normally regenerated and vitamin A treated abnormally regenerated tails (a prerequisite for ectopic organ development) of tadpoles of the Indian tree frog Polypedates maculatus. In the non-amputated and normally regenerated tails, acid phosphatase is majorly restricted to the epidermis and muscle patches although in the normally regenerated tails notochordal sheath and spinal cord also stain for acid phosphatase. In vitamin A treated tails, acid phosphatase is mostly localized in the epidermis, notochord precursor cells and undifferentiated cells of the mesenchyme. Notochordal cells and notochordal sheath also show positive staining. Since, acid phosphatase is majorly expressed by tissue forming precursor cells, this enzyme is suggested to be involved in tissue remodelling processes.

An Active 32-kDa Cathepsin L Is Secreted Directly from HT 1080 Fibrosarcoma Cells and Not via Lysosomal Exocytosis.

Cathepsin L [EC 3.4.22.15] is secreted via lysosomal exocytosis by several types of cancer cells, including prostate and breast cancer cells. We previously reported that human cultured fibrosarcoma (HT 1080) cells secrete cathepsin L into the medium; this secreted cathepsin is 10-times more active than intracellular cathepsin. This increased activity was attributed to the presence of a 32-kDa cathepsin L in the medium. The aim of this study was to examine how this active 32-kDa cathepsin L is secreted into the medium. To this end, we compared the secreted active 32-kDa cathepsin L with lysosomal cathepsin L by using a novel gelatin zymography technique that employs leupeptin. We also examined the glycosylation and phosphorylation status of the proteins by using the enzymes endoglycosidase H [EC 3.2.1.96] and alkaline phosphatase [EC 3.1.3.1]. Strong active bands corresponding to the 32-kDa and 34-kDa cathepsin L forms were detected in the medium and lysosomes, respectively. The cell extract exhibited strong active bands for both forms. Moreover, both forms were adsorbed onto a concanavalin A-agarose column. The core protein domain of both forms had the same molecular mass of 30 kDa. The 32-kDa cathepsin L was phosphorylated, while the 34-kDa lysosomal form was dephosphorylated, perhaps because of the lysosomal marker enzyme, acid phosphatase. These results suggest that the active 32-kDa form does not enter the lysosomes. In conclusion, our results indicate that the active 32-kDa cathepsin L is secreted directly from the HT 1080 cells and not via lysosomal exocytosis.

Thioautotrophic bacterial endosymbionts are degraded by enzymatic digestion during starvation: Case study of two lucinids Codakia orbicularis and C. orbiculata.

The Caribbean bivalves Codakia orbicularis (Linné, 1758) and C. orbiculata (Montagu, 1808) live in seagrass beds of Thalassia testudinum and harbor intracellular sulfur-oxidizing gamma-proteobacteria. These bacterial symbionts fix CO2 via the Calvin Benson cycle and provide organic compounds to the bivalve. During experimentally induced starvation, no reduced sulfur compounds and no organic particle food are available; the symbionts could be considered as the sole nutrient source of the host bivalve. A previous study has shown that the intracellular bacterial population decreased considerably during starvation and that bacterial endosymbionts were not released by the bivalves. In this study, the activity of two lysosomal marker enzymes (acid phosphatase and arylsulfatase) was detected using cytochemical experiments coupled with energy-dispersive X-ray transmission electron microscopy during sulfide and organic particle starvation. The degradation of bacterial endosymbionts began after 2 weeks of starvation in C. orbiculata and after 3 weeks in C. orbicularis. Degradation processes seem to be continuous over several months and could be responsible for the disappearance of the bacterial endosymbionts within the gills during starvation. These data suggest that the host use symbionts as a nutrient source to survive a hunger crisis. The carbon transfer from the symbionts to the host could be flexible and could consist in transfer of organic matter, "milking," under normal feeding conditions and digestion of the symbionts under starved conditions.

Therapeutic effect of Ananus comosus peel on breast cancer induced by 7, 12- dimethylbenz(α)anthracene

Ananus comosus , has demonstrated a wide variety of biological activities which make it a good plant source for the treatment of many oxidative stress mediated diseases. The present study was aimed to evaluate therapeutic potential by assaying the activities of liver marker and lysosomal enzymes in 7, 12 dimethylbenz(α)anthracene induced mammary cancer bearing rats. Animals were divided into five groups of six numbers each. Group 1 served as control, group 2 induced mammary carcinogenesis, group 3 and 4 were treated with plant extract and tamoxifen and group 5 served as animals treated with plant extract alone. All the animals were sacrificed after 30 days treatment, serum and mammary tissue are used for the analysis of liver marker and lysosomal marker enzymes using standard protocols. The rats were induced with mammary cancer by DMBA showed altered the levels of liver markers and lysosomal marker enzymes in serum and mammary tissue. On the other hand, oral administrations of ethanolic extract of Ananus comosus peel and standard drug tamoxifen to mammary cancer bearing rats for 30 days, those levels were brought back to near normal. The histology of mammary tissues inevitably supports the biochemical alterations and this was attributed due to the interaction of Ananus comosus peel through the induction or inhibition of metabolism and also the modulating property in the marker and lysosomal enzymes.

The biological clock and the molecular basis of lysosomal storage diseases.

The lysosomal storage disorders encompass nearly fifty diseases provoked by lack or deficiency of enzymes essential for the breakdown of complex molecules and hallmarked by accumulation in the lysosomes of metabolic residues. Histochemistry and cytochemistry studies evidenced patterns of circadian variation of the lysosomal marker enzymes, suggesting that lysosomal function oscillates rhythmically during the 24-h day. The circadian rhythmicity of cellular processes is driven by the biological clock ticking through transcriptional/translational feedback loops hardwired by circadian genes and proteins. Malfunction of the molecular clockwork may provoke severe deregulation of downstream gene expression regulating a complex array of cellular functions leading to anatomical and functional changes. In this review we highlight that all the genes mutated in lysosomal storage disorders encode circadian transcripts suggesting a direct participation of the biological clock in the pathophysiological mechanisms underlying cellular and tissue derangements hallmarking these hereditary diseases. The 24-h periodicity of oscillation of gene transcription and translation could lead in physiological conditions to circadian rhythmicity of fluctuation of enzyme levels and activity, so that gene transfer could be envisaged to reproduce 24-h periodicity of variation of enzymatic dynamics and circadian rhythmicity could have an impact on the schedule of enzyme replacement therapy.

Zonation in the digestive tract of Eisenia fetida: Implications in biomarker measurements for toxicity assessment

Eisenia fetida is a model species for soil health assessment and different biomarkers that detect either the presence of bioavailable contaminants or their biological effect have been developed. These parameters are performed in a target tissue or whole earthworm, without considering the marked zonation in histological organisation, enzyme activities and gene expression pattern existing along the body. Thus, the present work was aimed at (a) characterising the morphofunctional heterogeneity along the digestive tract of E. fetida in tissue morphology and turnover, lysosomal enzyme markers (β-glucuronidase, β-GUS; hexosaminidase, HEX), lipofuscin contents (LPF) and metallothionein (MT) and catalase (CAT) gene expression; and (b) determining whether the responsiveness to Cd exposure varies among tissues and along the digestive tract. HEX and β-GUS exhibited a heterogeneous distribution pattern along and across the digestive tract and Cd exposure caused a marked decrease of HEX and an increase of β-GUS activity. Likewise, the significant decrease of cell turnover and the induction of MT transcription were was zone-dependent. Therefore, in was concluded that the consideration of the zonation when applying biomarker for toxicity assessment would reduce the intrinsic variability that results from overlooking the marked morphofunctional heterogeneity that exists in annelids along their body axis.

Time-course study of the early lysosomal responses to pollutants in mussel digestive cells using acid phosphatase as lysosomal marker enzyme

Lysosomal biomarkers are early warning signals of the biological effects caused by environmental pollutants but the promptness of lysosomal responses to pollutants has not been investigated yet. This work is aimed to determine the response-time of digestive cell lysosomes in mussels exposed to metals and hydrocarbons. Mussels, Mytilus galloprovincialis, were exposed, under laboratory conditions to Cd and to the water-accommodated fraction of a lubricant oil. One mussel per experimental group was sacrificed and processed every hour from 0 h to 30 h. Changes in AcP activity, immunoreactivity and LMS test based on AcP histochemistry, discriminates significantly control and exposed mussels within 5 h exposure. The present results suggested that after 15–20 h exposure digestive cell loss might be accompanied by increased AcP activity (extralysosomal) without a parallel increase in the levels of immunoreactive AcP protein, especially after Cd-exposure. The reduced labilisation period of lysosomal membrane constitute a cost effective early warning signal that, however, is not necessarily correlated with the exposure time. The routine application of immunochemical techniques deserves more research efforts before its implementation although, these techniques are very valuable to understand and interpret correctly lysosomal responses to pollutants.

β-Glucuronidase and hexosaminidase are marker enzymes for different compartments of the endo-lysosomal system in mussel digestive cells

In environmental toxicology, the most commonly used techniques used to visualise lysosomes in order to determine their responses to pollutants (LSC test: lysosomal structural changes test; LMS test: lysosomal membrane stability test) are based on the histochemical application of lysosomal marker enzymes. In mussel digestive cells, the marker enzymes used are beta-glucuronidase (beta-Gus) and hexosaminidase (Hex). The present work has been aimed at determining the distribution of these lysosomal marker enzymes in the various compartments of the endo-lysosomal system (ELS) of mussel digestive cells and at exploring whether intercellular transfer of lysosomal enzymes occurs between digestive and basophilic cells. Immunogold cytochemistry has allowed us to conclude that beta-Gus is present in every compartment of the digestive cell ELS, whereas Hex is not so widely distributed. Moreover, Hex is intimately linked to the lysosomal membrane, whereas beta-Gus appears to be not necessarily membrane-bound. Therefore, two populations of heterolysosomes with different enzyme load and membrane stability have been distinguished in the digestive cell. In addition, heterolysosomes of different electron density have been commonly observed merging together by contact; we suggest that some might act as storage granules for lysosomal enzymes. On the other hand, beta-Gus seems to be released to the digestive alveolar lumen in secretory lysosomes produced by basophilic cells and endocytosed by digestive cells. Regarding the implications of the present study on the interpretation of lysosomal biomarkers, we conclude that beta-Gus, but not Hex, histochemistry provides an appropriate marker for the LSC test and that, although both lysosomal marker enzymes can be employed in the LMS test, different values would be obtained depending on the marker enzyme employed.

Morphofunctional changes in Leydig cells throughout the continuous spermatogenesis of the freshwater teleost fish, Serrasalmus spilopleura (Characiformes, Characidae): an ultrastructural and enzyme study

The freshwater fish Serrasalmus spilopleura (piranha) has a continuous type of reproduction; gametes are constantly produced and released during the reproductive cycle. The testes do not undergo seasonal morphological changes but exhibit two constant regions throughout the year: the medullar region (involved with spermatogenesis) and the cortical region (involved with spermiation and sperm storage). We have evaluated the ultrastructure of the Leydig cells and the activity of 3beta-HSD (an essential enzyme related to steroid hormone biosynthesis) and acid phosphatase (AcPase; lysosomal marker enzyme) in these two regions. The activity of 3beta-HSD is stronger in the medullar region, and the Leydig cells in this region have a variety of cytological features that reflect differences in hormone synthesis and/or that could be linked to steroidogenic cells under various degrees of hormonal activity. In the cortical region, 3beta-HSD activity is weak and the Leydig cells exhibit signs of degeneration, as confirmed by their ultrastructure and intense AcPase activity. These degenerative signs are indicative of cytoplasmic remodelling to degrade steroidogenic enzymes, such as 3beta-HSD, that could lead to senescence or even to autophagic cell degeneration. S. spilopleura thus constitutes an interesting model for increasing our understanding of steroidogenesis control in freshwater teleost fish.

Characterization of a Rat Subcutaneous Malignant Fibrous Histiocytoma and Its Tumor Lines, with Reference to Histiocytic Features

Malignant fibrous histiocytoma (MFH) is regarded as soft tissue-derived undifferentiated pleomorphic sarcoma, of which the histogenesis remains to be proven. To investigate the cellular characteristics, a homotransplantable tumor line (KJ) was established from a spontaneous MFH that developed in the subcutis of an aged F344 rat. KJ tumors have been produced in syngeneic rats by serial subcutaneous implantation of tissue fragments. The original and KJ tumors consisted of oval and fusiform cells arranged in interlacing bundles with fibrous stroma. Occasional giant cells with bizarre nuclei were observed. Enzyme/immunohistochemically, neoplastic cells reacted to ED1 and ED2 (antibodies specific for rat histiocytes/macrophages), and showed a positive reaction to vimentin and lysosomal enzyme markers such as acid phosphatase (ACP) and nonspecific esterase (Non-SE). Electron microscopically, neoplastic cells possessed lysosomal granules in cytoplasm. A cloned cell line (KJ-A) was isolated from a KJ tumor. KJ-A cells showed positive reactions to ED1, ED2, ACP, and Non-SE, and had cytoplasmic lysosomal granules. Tumors induced by KJ-A cells exhibited histologic and enzyme/immunohistochemical findings similar to those of KJ tumors. Lipopolysaccharide (LPS) treatment increased the number of ED1-positive cells and the expression of tumor necrosis factor-alpha mRNA by reverse transcription-polymerase chain reaction. Collectively, it is likely that rat MFH cells originally possess histiocyte/macrophage-like features that may be enhanced by LPS. Because tumor lines are useful for in vivo and in vitro studies concerning different characteristics of the original neoplasms. KJ and KJ-A should prove useful for studies concerning the morphogenesis of MFH.

Identification and Characterization of a Lysosomal Membrane Protein ofDictyostelium discoideumwith Monoclonal Antibodies

Lysosomes are responsible for the degradation of macromolecules derived from the cell exterior by endocytosis, or from within the cell by autophagy. While our knowledge of the biosynthesis and targeting of lysosomal hydrolases is considerable, much less is known about the lysosomal membrane itself. To identify the lysosomal membrane proteins that mediate these functions, we have isolated lysosomes from amebae and injected them into mice to produce monoclonal antibodies (MAbs). We produced nine MAbs against Dictyostelium lysosomes from the batches of fused cells. Among them, three MAbs were specific to lysosomal membrane and gave a strong signal, and thus used in this study. The MAbs specifically reacted with a single protein band of 27 kd and stained a lysosome-like structure by immunofluorescence microscopy. To identify the antigen that the MAbs recognize, we processed differential centrifugation with whole-cell extract of Dictyostelium and traced p27 protein by activity assay of organelle marker enzyme. We showed that p27 is one component of the lysosomal system on the basis of comigration with a lysosomal marker enzyme. We also demonstrated that the 27-kd lysosomal protein is a tightly bound integral membrane protein by using a phase separation method of Triton X-114.

Alteration in Cellular and Biochemical Markers of Pulmonary Toxicity in Rat Lung Exposed to Carpet Dusts

Epidemiological studies of workers in weaving units in carpet industries have shown relationships between the airborne dust concentrations and pulmonary ill health. Therefore, to predict the health risk of carpet weavers, this preliminary experiment was conducted to evaluate the effect of carpet dust (knotted, tufted) on cellular and biochemical mediators considered as potential biological markers of lung injury. Lung cytoplasmic (lactate dehydrogenase, LDH), lysosomal (acid phosphatase, ACP), type II (alkaline phoshatase, ALP) and Clara-cell marker enzymes (gamma-glutamyl transferase, GGT) were monitored in rat cell-free lung lavage (BAL) during postexposure days 1, 4, 8, and 16. Furthermore, lung microsomal cytochrome P-450 (CYP450) and Clara-cell secretory protein (CC16) content in BAL was also evaluated. These pulmonary marker enzymes were significantly elevated during the postexposure period over the respective untreated control; however, tufted carpet dust shows more responses than knotted carpet dust. Lung CYP450 content was reduced significantly at early days; the pattern shows the reoccurrence of CYP450 content in the later stage of postexposure to carpet dust. Clara-cell secretory protein in BAL shows decline in the carpet-treated group; however, tufted carpet shows more decline than knotted carpet. Thus, reduction in CC16 level may have important implication in the development of chronic lung inflammation and diseases. Present investigation found that modulation of these cellular marker enzymes is clear evidence of pulmonary damage caused by exposure to carpet dust.

Phosphoinositide 3-kinase regulates maturation of lysosomes in rat hepatocytes

To obtain information about the role of phosphoinositide 3-kinase (PI3K) in the endocytic pathway in hepatocytes, the uptake and intracellular transport of asialo-orosomucoid (ASOR) was followed in cells treated with wortmannin or LY294002. The two inhibitors, at concentrations known to inhibit the enzyme, did not affect internalization or the number of surface asialoglycoprotein receptors, but they caused a paradoxical increase (approx. 50% above control values) in the degradation of ASOR labelled with [(125)I]tyramine cellobiose ([(125)I]TC). Wortmannin or LY204002 inhibited the autophagic sequestration of lactate dehydrogenase very effectively, and the enhanced degradation of [(125)I]TC-ASOR could be an indirect effect of reduced autophagy, as an amino acid mixture known to inhibit autophagy also caused increased degradation of [(125)I]TC-ASOR, and its effect was not additive to that of wortmannin or LY294002. Wortmannin or LY294002 had pronounced effects on the late parts of the endocytic pathway in the hepatocytes: first, dense lysosomes disappeared and were replaced by swollen vesicles; secondly, degradation of [(125)I]TC-ASOR took place in an organelle of lower buoyant density (in a sucrose gradient) than the bulk of lysosomes (identified in the gradient by lysosomal marker enzymes). With increasing length of incubation with wortmannin or LY294002, the density distributions of the lysosomal markers also shifted to lower density and gradually approached that of the labelled degradation products. The labelled degradation products formed from [(125)I]TC-labelled proteins were trapped at the site of formation, because they did not penetrate the vesicle membranes. The results obtained indicate that internalization and intracellular transport of ASOR to lysomes may take place in the absence of PI3K activity in rat hepatocytes. On the other hand, fusion of late endosomes with lysosomes seems to produce 'hybrid organelles' (active lysosomes) that are unable to mature into dense lysosomes.

In vivo activation of pro-form Bombyx cysteine protease (BCP) in silkmoth eggs: localization of yolk proteins and BCP, and acidification of yolk granules

The present study was designed to investigate the process of acidification of yolk granules during embryogenesis. In oocytes of mature Bombyx mori silkmoth, yolk proteins and a cysteine protease (pro-form BCP) were found in yolk granules. BCP was localized in small sized yolk granules (SYG, 3–6 μm in diameter) and yolk proteins in large sized granules (LYG, 6–11 μm in diameter), which might result in a spatial separation of protease and its substrates to avoid unnecessary hydrolysis. The granules were isolated on Percoll density gradient centrifugation. Although separation of LYG and SYG was incomplete, the granules sedimented in different fractions when using unfertilized egg extract, in which LYG was recovered from heavier fractions and BCP from lighter fractions. Acid phosphatase, as well as other lysosomal marker enzymes tested, was recovered from LYG-containing fractions. When extracts were prepared from developing eggs (day 3), some BCP-containing granules co-sedimented with LYG. The inactive pro-form BCP was activated in vivo, in parallel with yolk protein degradation, and as demonstrated previously in vitro under acidic conditions ( Takahashi et al., 1993). These results suggest that acidification occurs in yolk granules during embryogenesis. This was also confirmed using acridine orange fluorescent dye. In early development, most yolk granules were neutral, but became acidic during embryonic development. SYG were progressively recovered in heavier density fractions, displaying acidic interior. In this fraction, BCP-containing granules seem to be associated with larger granules (6–11 μm in size). In addition, SYG (BCP containing granules) were likely to be acidified earlier than LYG. Our results suggest that acidification initiates yolk degradation through activation of pro-form BCP.