Monocyte-derived cells express CYP27A1 and convert vitamin D3 into its active metabolite

CYP27A1 catalyses hydroxylations in the biosynthesis of bile acids and the bioactivation of vitamin D3. We investigated the expression of CYP27A1 in human monocytes, monocyte-derived macrophages, and dendritic cells on mRNA and protein levels as well as its enzymatic activity in comparison with the expression of CYP27B1 and CYP24A1. Macrophages showed a strong expression of CYP27A1, whereas monocytes and dendritic cells expressed low levels of CYP27A1 mRNA. Immunohistochemistry revealed CYP27A1 and CYP27B1 protein expression in macrophages. Accordingly, macrophages converted vitamin D3 into the active metabolite 1,25(OH)2D3. Dendritic cells also metabolized vitamin D3 although to a lesser extent. This could be due to the high expression of CYP24A1, the enzyme that degrades 25(OH)D3 and 1,25(OH)2D3. Our results show that macrophages and dendritic cells are capable to perform both hydroxylation steps of the vitamin D3 metabolism suggesting a possible role of local 1,25(OH)2D3 synthesis by myeloid cells in the skin and gut.     

Innate mechanisms of epithelial host defense: spotlight on intestine

The single layerof epithelial cells lining the intestinal tract is charged with a mostdifficult task: protecting the underlying biological compartments fromboth the normal commensal flora that reside within the intestinal lumenas well as the uninvited pathogens. To such an end, the intestinalepithelial cells are equipped with a panoply of defense mechanisms,both constitutive and inducible. This review focuses only on thosedefense mechanisms that are initiated and executed by the intestinalepithelial cell. Fitting these strict criteria are three majorcategories of epithelial host defense: enhanced salt and watersecretion, expression of antimicrobial proteins and peptides, andproduction of intestinal mucins. Each of these areas is discussed inthis review.

     

Inhibitory effects of active vitamin D preparations on PTH secretion in rats

To study the effects of various vitamin D preparations on PTH secretion, serum calcium and urinary excretion of cAMP were monitored in conscious perfused rats, and the influences of a bolus iv injection of the preparations on these parameters were examined. Three hours after the administration of 0.25 microgram/kg (0.6 nmol/kg) of 1 alpha, 24(R)-dihydroxycholecalciferol [1 alpha, 24(OH)2D3], the urinary excretion of cAMP decreased to a level compatible with that of parathyroidectomized (PTX) rats (50% of initial value; p less than 0.05) with no change in the concentration of serum calcium (total and ionized). In PTX rats supplemented with bovine PTH (1 U/h), the vitamin D preparation showed no significant effects either on the urinary excretion of cAMP or on serum calcium. These effects were rather specific for active vitamin D preparations, i.e. 1 alpha, 25(OH)2D3 (0.25 micrograms/kg) and 1 alpha OHD3 (1.25-6.25 micrograms/kg). However, 24,25(OH)2D3 (up to 25 micrograms/kg) had no significant effect on these parameters. These results suggest that, in rats, active vitamin D preparations specifically inhibit PTH secretion without causing a significant increase in the serum calcium concentration, reflecting a direct feedback mechanism between active vitamin D metabolite and the parathyroid glands.     

Nitric oxide production by human intestinal epithelial cells and competition for arginine as potential determinants of host defense against the lumen-dwelling pathogen Giardia lamblia

Giardia lamblia infection of the human small intestine is a common protozoan cause of diarrheal disease worldwide. Although infection is luminal and generally self-limiting, and secretory Abs are thought to be important in host defense, other defense mechanisms probably affect the duration of infection and the severity of symptoms. Because intestinal epithelial cells produce NO, and its stable end products, nitrite and nitrate, are detectable mainly on the apical side, we tested the hypothesis that NO production may constitute a host defense against G. lamblia. Several NO donors, but not their control compounds, inhibited giardial growth without affecting viability, suggesting that NO is cytostatic rather than cytotoxic for G. lamblia. NO donors also inhibited giardial differentiation induced by modeling crucial environmental factors, i. e., encystation induced by bile and alkaline pH, and excystation in response to gastric pH followed by alkaline pH and protease. Despite the potent antigiardial activity of NO, G. lamblia is not simply a passive target for host-produced NO, but has strategies to evade this potential host defense. Thus, in models of human intestinal epithelium, G. lamblia inhibited epithelial NO production by consuming arginine, the crucial substrate used by epithelial NO synthase to form NO. These studies define NO and arginine as central components in a novel cross-talk between a luminal pathogen and host intestinal epithelium.     

A method of active conformation search based on active and inactive analogues and its application to allylamine antimycotics

A new program ACSBAIA (Active Conformation Search Based on Active and Inactive Analogues) for determination of the active conformations was developed based on the rationales that specific functional groups of active analogues could reach and interact with the active site of target receptor by means of the change of conformations, but that of inactive analogues could not interact with the active site owing to conformational restriction. The program consisted of 4 sub-programs: conformation sampling system, active conformation constraint system, inactive conformation exclusion system, and activity prediction system. Pharmacophoric conformation of allylamine antimycotics was studied by this method. Activities of 2 analogues were predicted and tested. The results suggested that the method was scientific and practical. The application of this method was not restricted by the three-dimensional structural knowledge of target receptor. In the absence of structural information about the receptor, the method was     

A method of active conformation search based on active and inactive analogues and its application to allylamine antimycotics

A new program ACSBAIA (Active Conformation Search Based on Active and Inactive Analogues) for determination of the active conformations was developed based on the rationales that specific functional groups of active analogues could reach and interact with the active site of target receptor by means of the change of conformations, but that of inactive analogues could not interact with the active site owing to conformational restriction. The program consisted of 4 sub-programs: conformation sampling system, active conformation constraint system, inactive conformation exclusion system, and activity prediction system. Pharmacophoric conformation of allylamine antimycotics was studied by this method. Activities of 2 analogues were predicted and tested. The results suggested that the method was scientific and practical. The application of this method was not restricted by the three-dimensional structural knowledge of target receptor. In the absence of structural information about the receptor, the method was particularly applicable.     

Cell density dependent regulation of phenylalanine hydroxylase activity in hepatoma cells : Evidence for both an active and inactive enzyme form

The cell density dependent regulation of phenylalanine hydroxylase activity in Reuber hepatoma (H4) cells growing in monolayer culture has been examined in detail. We found that 48 h or more after subculture phenylalanine hydroxylase activity in the cells is an exponential function of cell density (cells/cm²). No discontinuity in the relationship is seen with the formation of a confluent monolayer.A rapid loss or a rapid gain in enzyme activity in the cells is observed after diluting or concentrating the cell cultures. The two processes appear qualitatively different. The loss in activity is a first order process which starts at the time of subculture with the rate of loss dependent on the density of subculture. The gain in activity begins 6–8 h after subculture to a higher density; it can be blocked by cycloheximide and has a maximum rate of increase that is about 10% of the maximum rate of loss of activity.Using immunochemical procedures, we found the same amount of phenylalanine hydroxylase associated antigen in Reuber cells from low density as from high density cultures, over a range of phenylalanine hydroxylase specific activities from 0.2 to 4.2. After concentrating cells to a higher density, no increase in enzyme antigen was observed, despite a several-fold increase in enzyme activity and a requirement for protein synthesis during the process. These observations imply the presence of an active and inactive phenylalanine hydroxylase with the relative amounts of each determined by the cell density. The effects of db-cAMP are discussed. Evidence is presented here that the hydrocortisone stimulation of phenylalanine hydroxylase activity works through a different mechanism than the cell density dependent process.     

Interaction of vitamin D analogs with signaling pathways leading to active cell death in breast cancer cells

Induction of apoptosis is a feature of the anti-tumor effects of certain vitamin D analogs. The aim of this study was to identify if common effectors are involved in cell death mediated by serum starvation, vitamin D analogs and tumor necrosis factor (TNF) alpha in 3 human breast cancer cell lines: MCF-7, T47-D and Hs578T. Incubation of cells in serum-free medium induced apoptosis as assessed by loss of cell viability and increased DNA fragmentation. Addition of IGF-I (30 ng/ml) protected against loss of cell viability in MCF-7 cells and co-treatment with two synthetic analogs (CB1093 and EB1089, 50 nM for 4 days) prevented these anti-apoptotic effects of IGF-I. Pretreatment of MCF-7 and Hs578T cells with the vitamin D analogs substantially potentiated the cytotoxic effects of TNFalpha. This cytokine was not cytotoxic for T47-D cells but co-incubation with CB1093 led to loss of cell viability. Potentiation by CB1093 of TNFalpha-induced apoptosis in MCF-7 cells was accompanied by increased activation of cytosolic phospholipase A2 and arachidonic acid release, which was partially inhibited by AACOCF3, a specific cPLA2 inhibitor. The broad-spectrum caspase inhibitor z-VAD-fmk prevented TNFalpha but not CB1093 mediated cell death and activation of cPLA2. Serum starvation induced apoptosis was accompanied by cPLA2 activation, which was inhibited by IGF-I and by z-VAD-fmk. However, the ability of these agents to suppress cPLA2 activation was abrogated by co-treatment with CB1093, suggesting a role for arachidonic acid release in the caspase-independent mechanism by which vitamin D analogs prevent the protective effects of IGF-I on breast cancer cell survival.     

A novel cytostatic form of autophagy in sensitization of non-small cell lung cancer cells to radiation by vitamin D and the vitamin D analog,EB 1089

The standard of care for unresectable lung cancer is chemoradiation. However, therapeutic options are limited and patients are rarely cured. We have previously shown that vitamin D and vitamin D analogs such as EB 1089 can enhance the response to radiation in breast cancer through the promotion of a cytotoxic form of autophagy. In A549 and H460 non-small cell lung cancer (NSCLC) cells, 1,25-D₃ (the hormonally active form of vitamin D) and EB 1089 prolonged the growth arrest induced by radiation alone and suppressed proliferative recovery, which translated to a significant reduction in clonogenic survival. In H838 or H358 NSCLC cells, which lack VDR/vitamin D receptor or functional TP53, respectively, 1,25-D₃ failed to modify the extent of radiation-induced growth arrest or suppress proliferative recovery post-irradiation. Sensitization to radiation in H1299 NSCLC cells was evident only when TP53 was induced in otherwise tp53-null H1299 NSCLC cells. Sensitization was not associated with increased DNA damage, decreased DNA repair or an increase in apoptosis, necrosis, or senescence. Instead sensitization appeared to be a consequence of the conversion of the cytoprotective autophagy induced by radiation alone to a novel cytostatic form of autophagy by the combination of 1,25-D₃ or EB 1089 with radiation. While both pharmacological and genetic suppression of autophagy or inhibition of AMPK phosphorylation sensitized the NSCLC cells to radiation alone, inhibition of the cytostatic autophagy induced by the combination treatment reversed sensitization. Evidence for selectivity was provided by lack of radiosensitization in normal human bronchial cells and cardiomyocytes. Taken together, these studies have identified a unique cytostatic function of autophagy that appears to be mediated by VDR, TP53, and possibly AMPK in the promotion of an enhanced response to radiation by 1,25-D₃ and EB 1089 in NSCLC.     

A novel cytostatic form of autophagy in sensitization of non-small cell lung cancer cells to radiation by vitamin D and the vitamin D analog,EB 1089

The standard of care for unresectable lung cancer is chemoradiation. However, therapeutic options are limited and patients are rarely cured. We have previously shown that vitamin D and vitamin D analogs such as EB 1089 can enhance the response to radiation in breast cancer through the promotion of a cytotoxic form of autophagy. In A549 and H460 non-small cell lung cancer (NSCLC) cells, 1,25-D₃ (the hormonally active form of vitamin D) and EB 1089 prolonged the growth arrest induced by radiation alone and suppressed proliferative recovery, which translated to a significant reduction in clonogenic survival. In H838 or H358 NSCLC cells, which lack VDR/vitamin D receptor or functional TP53, respectively, 1,25-D₃ failed to modify the extent of radiation-induced growth arrest or suppress proliferative recovery post-irradiation. Sensitization to radiation in H1299 NSCLC cells was evident only when TP53 was induced in otherwise tp53-null H1299 NSCLC cells. Sensitization was not associated with increased DNA damage, decreased DNA repair or an increase in apoptosis, necrosis, or senescence. Instead sensitization appeared to be a consequence of the conversion of the cytoprotective autophagy induced by radiation alone to a novel cytostatic form of autophagy by the combination of 1,25-D₃ or EB 1089 with radiation. While both pharmacological and genetic suppression of autophagy or inhibition of AMPK phosphorylation sensitized the NSCLC cells to radiation alone, inhibition of the cytostatic autophagy induced by the combination treatment reversed sensitization. Evidence for selectivity was provided by lack of radiosensitization in normal human bronchial cells and cardiomyocytes. Taken together, these studies have identified a unique cytostatic function of autophagy that appears to be mediated by VDR, TP53, and possibly AMPK in the promotion of an enhanced response to radiation by 1,25-D₃ and EB 1089 in NSCLC.     

Sialoglycoconjugates in Herpetomonas megaseliae: role in the adhesion to insect host epithelial cells

Herpetomonas megaseliae is a monoxenic trypanosomatid isolated from the phorid fly Megaselia scalaris . In the present report, the expression of cell surface sialoglycoconjugates in this parasite was analyzed by Western blotting, flow cytometry and fluorescence microscopy analyses using lectins that specifically recognize sialic acid residues. A strong reaction was detected when parasites were treated with Limax flavus, Maackia amurensis and Sambucus nigra lectins. Analysis of crude protein extracts by Western blotting revealed that bands with molecular masses ranging from 19 to 80 kDa were reactive to these lectins, which showed a sugar-inhibited recognition with the parasite extract. These results indicated that molecules containing α2,3- and α2,6-sialylgalactosyl sequences are present in this protozoan. The role of the surface sialomolecules in the interaction with explanted guts from Aedes aegypti was assessed. The interaction of H. megaseliae with the insect gut was strongly inhibited in the presence of mucin (71%), fetuin (68%) and sialyllactose (68%). Collectively, our results suggest a possible involvement of sialomolecules in the interaction between this insect trypanosomatid and the invertebrate host.     

Effects of 1,25-dihydroxyvitamin D3 on the distribution of androgen and vitamin D receptors in human prostate neonatal epithelial cells

Although many studies have examined the mechanisms of 1,25-dihydroxyvitamin D(3) (calcitriol or 1,25 D) action in different prostate cancer cell lines, little is known regarding the influence of this steroid on the normal prostate. The presence of both VDR and AR in normal prostatic tissues raises the distinct possibility of an important role for this hormone in the normal gland. In order to ascertain the possible role of 1,25 D on both AR and VDR in the normal prostate, the effects of calcitriol and dihydrotestosterone (DHT) on the normal human neonatal prostatic epithelial cell line, 267B-1, were examined. These studies were approached by focusing on how 1,25 D in the presence or absence of DHT affects the distribution of AR and VDR in the cytoplasmic and nuclear compartments of the cells in terms of their protein levels and DNA binding activities. Immunoblot analyses show that 1,25 D increases the AR protein level in both the cytoplasmic and nuclear fractions but not the VDR protein level. On the other hand, the gel shift assays demonstrate that 1,25 D increases both the AR- and VDR-DNA binding activities in the nuclear fraction, whereas there is no increase in DNA binding activities in the cytoplasmic fraction. Addition of DHT along with 1,25 D does not affect the DNA binding activities of both AR and VDR. Overall, these studies suggest that 1,25 D actions on the normal prostate cells may be mediated independently through AR and VDR, respectively.     

Synthesis of vitamin D5: its biological activity relative to vitamins D3 and D2.

The chemical synthesis, spectral characterization, and biological activity of vitamin D₅ in vitamin D-deficient rats is reported. Vitamin D₅ is about 180-fold less active than vitamin D₃ in calcification of rachitic cartilage and about 100- to 200-fold less active in induction of bone-calcium mobilization. In stimulation of intestinal-calcium transport, vitamin D₅ is about 80-fold less active than vitamin D₃. Vitamins D₂ and D₃ appear to be equiactive in all three responses when low doses are administered.     

Studies on the acid unfolded and molten globule states of catalytically active stem bromelain: a comparison with catalytically inactive form

We report the accumulation of an acid unfolded (UA) state and a molten globule (MG) state in the acid induced unfolding pathway of unmodified preparation of stem bromelain (SB) [EC 3.4.22.32], a cystein protease from Ananas cosmosus. The conformation of SB was examined over the pH 0.8-3 regions by circular dichroism, tryptophanyl fluorescence, 1-anilino-8-naphthalenesulfonate (ANS) binding, and tryptophanyl fluorescence quenching study. The pH 0.8-3.0 regions were selected to study the acid induced unfolding of SB because no autolysis of the enzyme was observed in these pH regions. The results show that SB at pH 2.0 is maximally unfolded and characterizes by significant loss of secondary structure ( approximately 80%) and almost complete loss of tertiary contacts. However, on further decreasing the pH to 0.8 a MG state was observed, with secondary structure content similar to that of native protein but no tertiary structure. We also made a comparative study of these acid induced states of SB with acid induced states of modified stem bromelain (mSB), reported by our group earlier [Eur. J. Biochem. (2002) 269, 47-52]. We have shown that modification of SB for inactivation significantly affects the N-UA transition but neither affects the UA-MG transition nor the stability of the MG state.     

Refractoriness of eryptotic red blood cells to Plasmodium falciparum infection: a putative host defense mechanism limiting parasitaemia

Recently, we have described that apoptosis-like process of red blood cells (RBC) - eryptosis - in malaria is not restricted to parasitized cells, occurring also in non-parasitized RBC (nRBC). Besides to pathogenic proprieties, apoptosis also participates in the innate defense trough restriction of intracellular pathogens propagation. In the present study, we investigated the capacity of P. falciparum parasites to infect eryptotic RBC. Schizont parasitized RBC concentrated by magnetic separation were cultured with eryptotic RBC obtained by ionomycin treatment and, then, parasite growth was evaluated in Giemsa-stained thin blood smears. While parasites infected and developed normally in control non-eryptotic RBC, cultures performed with eryptotic RBC had a marked decrease in parasitaemia. It was noteworthy a great number of free merozoites in eryptotic RBC cultures, indicating that these cells were not susceptible to invasion. We suggest that although eryptosis could be involved in malaria pathogenesis, it could also acting protectively by controlling parasite propagation.