Deficit in neuraminidase associated with mucolipidosis II (I-cell disease)

Using a tritiated sialyloligosaccharide as a substrate, the authors showed that mucolipidosis II is characterized by a lack of neuraminidase activity in leucocytes, while the other acidic hydrolases activities are normal. According to Ashwell, terminal galactose is the required signal for glycoproteins uptake by the cells. Thus, a neuraminidase deficit may explain the increase of sialylated hydrolases activities in the plasma and the non-recognition of these enzymes by cultured fibroblasts.     

Calcium-activated neutral protease activity is decreased in PC12 cells after ethanol exposure

Calcium-activated neutral protease activity was determined in PC12 cells exposed to ethanol for 96 h using a fluorescence-based assay with N-succinyl-Leu-Tyr 7-amido-4-methylcoumarin as the substrate. Stimulated activity was measured at high (1,400 microM) or low (140 microM) Ca2+ concentrations in the presence of 20 microM ionomycin. Kinetic parameters were derived by fitting a model relating fluorescence intensity to time: F(t) = F(final)*(1 - e(-k(obs)t). Cell extracts were subjected to nondenaturing gel electrophoresis and casein zymography with quantification of the activity of the two calpain isoforms. Exposure to ethanol significantly decreased whole cell calpain activity measured by k(obs) beginning at 20 mM, to 27.8% of control at 1,400 microM Ca2+ and 29.2% of control at 140 microM Ca2+ in the presence of 20 microM ionomycin. No changes in mu-calpain or m-calpain activities were found in cell extracts from cells exposed to 20 mM ethanol, whereas at 40 and 80 mM ethanol, significant decreases in both mu-calpain and m-calpain activities were discovered.     

Activation of phosphatidylinositol-specific phospholipase C in response to HDL3 and LDL is markedly reduced in cultured fibroblasts from Tangier patients

We compared HDL3- and LDL-induced signal transduction in normal and Tangier fibroblasts to elucidate whether impaired signal transduction responses to lipoproteins might contribute to disturbed cellular lipid and lipoprotein metabolism in Tangier disease, a rare autosomal disorder of cellular lipid and lipoprotein metabolism. In several cell types HDL and LDL activate a currently unknown isoform of phosphatidylinositol-specific phospholipase C (PI-PLC) that results in the generation of 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. Compared with normal fibroblasts, Tangier fibroblasts stimulated with HDL3 or LDL resulted in a significantly reduced accumulation of inositol phosphates and 1,2-diacylglycerol formation. Furthermore, in Tangier fibroblasts both lipoproteins failed to mobilize calcium from internal pools, and the cytosol-to-membrane redistribution of protein kinase C (in both the alpha and epsilon isoforms) was markedly reduced. Thus, the data indicate an impaired PI-PLC activation in response to lipoproteins in Tangier fibroblasts.     

Association of N- and C-terminal domains of phospholipase D is required for catalytic activity

Rat brain phospholipase D1 (rPLD1) belongs to a superfamily defined by the highly conserved catalytic motif (H(X)K(X)4D, denoted HKD. RPLD1 contains two HKD domains, located in the N- and C-terminal regions. Deletion mutants of rPLD1 that contained only an N- or C-terminal HKD domain exhibited no catalytic activity when expressed in COS 7 cells. However, when N-terminal fragments containing one of the HKD domains were cotransfected with a C-terminal fragment containing the other HKD domain, PLD activity was restored. Furthermore, immunoprecipitation assays showed that the N- and C-terminal halves of rPLD1 were physically associated when expressed in COS 7 cells. In addition, deletion of 168 amino acids from the N terminus of rPLD1 significantly enhanced basal PLD activity while inhibiting the response to phorbol ester. Likewise, the coexpression of this truncated N-terminal half with the C-terminal half resulted in increased PLD activity. In summary, this study provides direct evidence that the enzymatic activity of rPLD1 requires the presence of the HKD domains in both the N- and C-terminal regions of the molecule. More importantly, the two halves of rPLD1 can associate, and this may be essential to bring the two HKD domains together to form an active catalytic center. These findings provide new insights into the catalytic mechanism of enzymes of the PLD superfamily.     

Type II phospholipase A2 in human gestational tissues: extractable immuno- and enzymatic activity in fetal membranes

In this study, we have established the presence of immunoreactive (ir) Type II PLA2 in human amnion and choriodecidua obtained from women at term prior to the onset of labour. The content of irType II PLA2 present in 1 M NaCl extracts of choriodecidua and amnion averaged 3.5 +/- 3.1 and 10.6 +/- 5.2 ng/mg tissue protein (n = 3), respectively. PLA2 enzymatic activity present in the same tissues averaged 1.3 +/- 0.2 and 1.9 +/- 0.7 nmol phosphatidylethanolamine (PE) hydrolysed/mg tissue protein per h (n = 3), respectively. To allow intra-patient comparison of the relative distribution in gestational tissues, irType II PLA2 and PLA2 enzymatic activity was also determined in placenta obtained from the same group of women, and averaged 26.0 +/- 7.0 ng/mg tissue protein and 3.5 +/- 1.0 nmol PE hydrolysed/mg protein per h (n = 3), respectively. As has been previously reported for human placenta, the recovery of Type II PLA2 and PLA2 enzymatic activity from amnion and choriodecidua was increased between 16- and 25-fold when tissues were homogenized in high-ionic strength media (i.e., 10% (w/v) ammonium sulphate or 1 M NaCl) compared with that recovered when tissues were homogenized in low-ionic strength media (i.e., 0.32 M sucrose-20 mM Hepes). The data obtained represent the first quantitative estimates of immunoreactive Type II PLA2 in human amnion and choriodecidua, and support the conclusion that previous analyses of the PLA2 enzymatic activity present in gestational tissues have essentially excluded the contribution made by this PLA2 isozyme to net enzymatic activity. We suggest that this isozyme represents a major component of the PLA2 enzymatic activity present in human gestational tissues at term and that it contributes significantly to the phospholipid metabolism and arachidonic acid release which occurs during late pregnancy and at the time of labour.     

Anemia in children with cancer is associated with decreased erythropoietic activity and not with inadequate erythropoietin production

A defect in erythropoietin (EPO) production has been advocated as being the main cause of anemia presented at time of diagnosis or during treatment by adults with solid tumors. On the basis of this defect, anemic cancer patients, both adults and children, have been treated with recombinant human EPO (rHuEPO). To further elucidate the pathophysiology of anemia in children with cancer, we measured serum soluble transferrin receptor (sTfR), a quantitative marker of erythropoiesis, and serum EPO at time of diagnosis and during chemotherapy in children suffering from solid tumor or leukemia. We determined serum EPO in 111 children (55 leukemia, 56 solid tumors) at time of diagnosis. In the last 44 patients (23 leukemia and 21 solid tumors), sTfR levels were also measured. Serum EPO together with sTfR levels were also determined in 60 children receiving chemotherapy (29 leukemia, 31 solid tumors). These results were compared with those obtained from appropriate control groups. In all patients, we found a highly significant correlation between the logarithm of EPO (log[EPO]) and the hemoglobin (Hb) level. In all subsets of patients, sTfR levels were inappropriately low for the degree of anemia. Neither leukemic nor solid tumor groups showed a significant inverse relationship between log(sTfR) and the Hb level as would be expected in anemic patients with appropriate marrow response. Thus, in children with cancer, anemia is associated with a decreased total bone marrow erythropoietic activity which, in contrast to what has been reported in anemic cancer adults, is not related to defective EPO production.     

Cerebral cortical phospholipase A2 activity of senescence-accelerated mouse is increased in an age-dependent manner

An immunogenetic examination of 86 cases of stomach cancer established a correlation between predisposition and resistance, on the one hand, and the distribution of allele sets of HLA-genes (classes I and II), on the other. The relationship was found to vary according to sex and age. The most significant relationships with respect to predisposition were identified for HLA-B51 (RR = 19.82) alleles and allele combinations of HLA-DRI-DR7 (RR = 25.52) and HLA-A9-DRI (RR = 33.67). High relative risk of stomach cancer was attributed to the absence of relevant alleles in 91 patients included into the group of comparison. Also, combinations of allele sets were identified in healthy subjects which never occur in stomach cancer patients. The results provide a substantiation for developing an automated system of interpreting HLA-typing data which are instrumental in evaluating the patient's predisposition, resistance and prognosis.     

Telomerase activity of sarcoma cell lines and fibroblasts is independent of p53 status

Telomerase activity is necessary for the stabilization of telomeres, which function to overcome cellular senescence and are linked to unlimited cell proliferation. Activation of telomerase is characteristic of immortalized cell lines and most tumors. The p53 gene has been implicated as a crucial barrier to unlimited cell proliferation, and its absence has been shown to allow direct immortalization of cells by certain oncogenes. The p53 gene may have an additional function of signaling cell growth arrest in response to telomere shortening, which occurs with repeated cellular divisions and ultimately threatens chromosomal stability. This prompted us to consider whether the enzyme telomerase, responsible for adding new telomeres to chromosomal ends, may be affected by the p53 status of normal and malignant cells. We investigated whether a relationship between telomerase and p53 could be demonstrated in a human sarcoma cell line containing a missense p53 mutation and several stable transfectants that express the wild-type p53 gene or a temperature-sensitive mutant of p53. All cell lines had readily detectable telomerase activity regardless of p53 status. In addition, murine fibroblast cell strains established from tissues of p53+/+ and p53-/- (p53 knockout) mice expressed telomerase regardless of the p53 status of their tissue of origin. Levels of telomerase subunit mRNA (hEST2) were comparable among cell lines and tissues with different p53 status. These results imply that p53 status is not associated with telomerase activity per se and that activation of telomerase can occur either in cells completely devoid of p53 or in cells that have functional p53.     

Corticosteroid-binding globulin is not decreased in depressed patients

We studied corticosteroid-binding globulin (CBG) in 25 drug-free depressed patients and 33 healthy controls over a wide age-range. CBG was measured at 0800, 1400, 2000 and 2400 h in all subjects. Analysis of variance (ANOVA) with repeated measurement design revealed a significant effect of gender and time, but not of diagnosis (depressed patients vs healthy controls) or age group (< 50/> 50 years). In females, regardless of diagnosis, CBG plasma concentrations were significantly increased, when compared with their male counterparts. Although as a group depressed patients had significantly higher plasma cortisol concentrations (108.0 +/- 23.1 vs 70.7 +/- 10.9 micrograms/l), CBG levels did not differ between the two groups. Thus we did not find hypercortisolemia in depression to be paralleled by a decrease in CBG. However, the exaggerated activity of the hypothalamus-pituitary-adrenocortical system in healthy and depressed females is associated with an increase in plasma CBG.     

N-glycosylation is requisite for the enzyme activity and Golgi retention of N-acetylglucosaminyltransferase III

UDP-N-acetylglucosamine: beta-D-mannoside beta-1,4N-acetylglucosaminyltransferase III (GnT-III, EC 2.4.1.144) is a glycoprotein involved in the biosynthesis of N-linked oligosaccharides. Rat GnT-III contains three potential N-glycosylation sites, which have been predicted to be Asn243, Asn261, and Asn399. To study the roles of N-glycosylation in the GnT-III function, rat GnT-III was expressed in COS-1 cells under tunicamycin or castanospermine treatment. The tunicamycin-treated GnT-III, which was not N-glycosylated, had almost no activity. The castanospermine-treated GnT-III was not localized in the Golgi, but glucosylation did not affect its activity. To clarify the role of individual N-glycosylations, we obtained a series of mutant cDNAs in which some or all of the potential glycosylation sites were eliminated by site-directed mutagenesis, and expressed them in COS-1 cells. All the mutants exhibited lower enzyme activity than the wild-type, but deglycosylation at individual sites had different effects on the enzyme activity. The deglycosylation at Asn243 or Asn261 was more effective on the activity than that at Asn399. The enzyme activity decreased as the number of glycosylation sites decreased. The null glycosylation mutant had no activity, corresponding to the case of tunicamycin-treated wild-type GnT-III. Kinetic analysis revealed that the deglycosylation at Asn243 or Asn261 resulted in slightly lower affinity for the donor substrate, but the other mutation did not significantly change the K(m) value for either the donor or acceptor. None of the mutant GnT-IIIs showed perinuclear localization or Golgi retention, that was observed for the wild-type protein. This is the first demonstration that the glycosyltransferase localized in the Golgi apparatus requires N-glycosylation for its activity and retention.     

Increased phospholipase D activity in human breast cancer

Direct resolution of enantiomers has acquired greater analytical importance. Of various chromatographic techniques, TLC continues to enjoy considerable reputation for the purpose. The present paper deals with an important and yet less explored aspect of TLC i.e. impregnation. Methods of impregnation of thin layer material with a variety of reagents and the role of impregnating reagents in resolving compounds on these layers, with special reference to main/basic approaches of impregnation as applied to direct enantiomeric resolution, are discussed.     

Evidence that phospholipase A2 activity is required for Golgi complex and trans Golgi network membrane tubulation

Membrane tubules of uniform diameter (60-80 nm) and various lengths (up to several micrometers) emanate from elements of the Golgi stack and trans Golgi network (TGN). These organelle membrane tubules are thought to be involved in membrane trafficking and maintenance of Golgi/TGN architecture. The number of these tubules, and their frequency of formation, can be greatly enhanced by the fungal metabolite brefeldin A (BFA), an inhibitor of Golgi/TGN-associated coated vesicle formation. We show here that BFA stimulation of Golgi and TGN membrane tubulation, and the resultant retrograde transport of resident Golgi enzymes to the endoplasmic reticulum, was potently inhibited by a number of membrane-permeant antagonists of phospholipase A2 (PLA2; EC 3.1.1.4) activity. In addition, PLA2 inhibitors on their own caused a reversible fragmentation of the Golgi complex into juxtanuclear, stacked cisternal elements. We conclude from these observations that tubulation of Golgi complex and TGN membranes requires a PLA2 activity, and that this activity may participate not only in Golgi tubule-mediated retrograde trafficking to the endoplasmic reticulum, but also in the maintenance of Golgi complex architecture.     

Lysosomal enzyme activity in the serum of rats with experimental lead poisoning

Examination of the effect of experimental lead poisoning on permeability of lysosomal membranes in albino rats demonstrated activation of lysosomal enzymes (alpha-manosidase and beta-acetylglucosaminidase) in the blood serum as soon as the third day after daily administration of lead acetate (20 mg/kg). Apparently damage of the lysosomal membrane played an important role in the pathogenesis of lead poisoning.     

Lactase phlorizin hydrolase synthesis is decreased in protein-malnourished pigs

Epidermal Langerhans cells (LC) may occur in subsets with different phenotypic and functional characteristics. In this work give further evidence that the CD1a-positive LC population in the normal human epidermis may be heterogeneous. We found that one of our monoclonal antibodies (TE4B) to stratum corneum chymotryptic enzyme (SCCE) stained a population of dendritic cells in the normal epidermis in addition to high suprabasal keratinocytes. The staining of the dendritic cells was seen only when the biopsies had been fixed with formaldehyde and when the sections had been pretreated, either with proteolytic enzymes or with Triton X-100. The binding of the antibody was mediated through its antigen binding site, as it could be inhibited by adsorption with recombinant pro-SCCE. Experiments with double labelling showed that the TE4B-positive dendritic cells were also CD1a-positive. On the other hand, not all CD1a-positive cells were TE4B-positive. By means of confocal microscopy of double-labelled cells, the TE4B binding site could be localized intracellularly. SCCE-mRNA could be detected by in situ hybridization in high suprabasal keratinocytes only. A possible explanation may be that there is a subset of LC which have taken up SCCE secreted by high suprabasal keratinocytes. Alternatively, TE4B may bind to an epitope present in a subgroup of epidermal LC which cross-reacts immunologically with SCCE. It is suggested that the demonstrated heterogeneity of the population of LC in the normal epidermis should be taken into account in studies on the possible role of epidermal autoantigens in the development of immune-mediated skin diseases.     

Biologically active heteroarotinoids exhibiting anticancer activity and decreased toxicity

A series of retinoids, containing heteroatoms in a cyclic ring and called heteroarotinoids, were synthesized, and their biological activity was evaluated using tissue culture lines that have measurable responses to trans-retinoic acid (t-RA). Transglutaminase (TGase) was assessed in the human erythroleukemia cell line (GMO6141A) as an indicator of differentiation and apoptosis. Proliferation was evaluated in a human cervical cell line, CC-1, which exhibits dose-dependent alterations in growth rate in response to treatment with trans-retinoic acid. Activation of nuclear retinoic acid receptors was determined in a reporter cell line established from CC-1. The reporter line, called CC-B, contains a reporter gene controlled by a retinoic acid responsive element (RARE) and a thymidine kinase (tk) promoter. Treatment of the CC-B line with the heteroarotinoids resulted in a dose-responsive and retinoid-dependent regulation of reporter gene expression. The heteroarotinoids exhibited activity in all assays and correlated in a statistically significant manner between assays. RARE transactivation activity in CC-B cells correlated with induction of TGase in GMO6141A (R = 0.96) and with a decrease in the growth rate of CC-1 cells (R = -0.90). The ability of the selected heteroarotinoids to induce differentiation, inhibit proliferation, and activate nuclear receptors demonstrates the chemotherapeutic potential of these agents. In view of the biological activity cited, an in vivo toxicity study was conducted on male B6D2F1 mice with three heteroarotinoids, namely 8 [(2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimeth ylthiochroman-6-yl)-2,4,6-heptatrienoic acid], 10 [(2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimeth ylchroman-6-yl)-2, 4,6-heptatrienoic acid], and 13 [(E)-p-[2-(4,4-dimethylchroman-6-yl)propenyl]benzoic acid]. The mice were used with gavage of heteroarotinoids in corn oil [0.1, 0.2, 0.4, or 0.8 mg/kg] and with 0.01 or 0.05 mg/kg of TTNPB (5) [(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1- propenyl]benzoic acid] as reference controls. The target organs affected in the mice by the three heteroarotinoids were those typically associated with t-RA (1) toxicity. The maximum tolerated dose (MTD) of 13 was 9.4 mg/kg/day, which was equal in toxicity to that of t-RA (1) and 1000-fold less toxic than TTNPB (5). The MTDs of 8 and 10 were 34 and 32 mg/kg/day, respectively, which is 3-fold less toxic than t-RA (1) and 3000-fold less toxic than TTNPB (5). The 3000-fold reduced toxicity, compared with only a 27% reduction biological activity of 8 and 10 with respect to that of TTNPB, observed in our assays indicates a good therapeutic ratio of these heteroarotinoids over the parent compound. The biological activity and reduced toxicity of these heteroartinoids demonstrate the potential efficacy as anticancer agents.     

Phospholipid dependence of membrane-bound phospholipase A2 in ras-transformed NIH 3T3 fibroblasts

Although the activation of phospholipase A2 (PLA2) in ras-transformed cells has been well documented, the mechanisms underlying this activation are poorly understood. In this study we tried to elucidate whether the membrane phospholipid composition and physical state influence the activity of membrane-associated PLA2 in ras-transformed fibroblasts. For this purpose membranes from non-transfected and ras-transfected NIH 3T3 fibroblasts were enriched with different phospholipids by the aid of partially purified lipid transfer protein. The results showed that of all tested phospholipids only phosphatidylcholine (PC) increased PLA2 activity in the control cells, whereas in their transformed counterparts both PC and phosphatidic acid (PA) induced such effect. Further we investigated whether the activatory effect was due only to the polar head of these phospholipids, or if it was also related to their acyl chain composition. The results demonstrated that the arachidonic acid-containing PC and PA molecules induced a more pronounced increase of membrane-associated PLA2 activity in ras-transformed cells compared to the corresponding palmitate-stearate- or oleate- containing molecular species. However, we did not observe any specific effect of the phospholipid fatty acid composition in non-transformed NIH 3T3 fibroblasts. In ras-transformed cells incubated with increasing concentrations of arachidonic acid, PLA2 activity was altered in parallel with the changes of the cellular content of this fatty acid. The role of phosphatidic and arachidonic acids as specific activators of PLA2 in ras-transformed cells is discussed with respect to their possible role in the signal transduction pathways as well as in the processes of malignant transformation of cells.     

DOCA administration increases renal phospholipase activity in the rat

The phospholipase activity of renal tissue has been evaluated in controls and in DOCA treated rats. DOCA treated animal showed a higher than normal enzyme activity. Since a phospholipase is the key step in prostaglandin biosynthesis, it is suggested that the increased prostaglandin release promoted by mineraloactive steroids is mediated by an activation of this key enzyme.     

Evidence for cell surface and internal phospholipase activity in ascidian eggs

Glutaredoxin, also known as thioltransferase, was purified from Cryptococcus neoformans by procedures including DEAE-cellulose ion exchange chromatography, Q-Sepharose ion-exchange chromatography, and gel filtration on Sephadex G-50. Its purity was confirmed by SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 12,000 Da. The purified enzyme has a K(m) value of 1.03 mM with 2-hydroxyethyl disulfide as a substrate. The enzyme also utilizes L-sulfocysteine, L-cystine, and bovine serum albumin as substrates in the presence of reduced glutathione. The enzyme has K(m) values of 0.34-2.50 mM for these substrates. It was greatly activated by thiol compounds such as reduced glutathione, dithiothreitol, L-cysteine and beta-mercaptoethanol. It is partially inactivated at 60 degrees C or higher temperatures. It plays an important role in thiol-disulfide exchange in Cryptococcus neoformans.