Differential expression and localization of calmodulin-dependent phosphodiesterase genes during ontogenesis of chick dorsal root ganglion

The level and characteristics of 3'-5'-cyclic nucleotide phosphodiesterase (PDE) activity in chick dorsal root ganglion (DRG) extracts of 5-day posthatching chicken (P5) and E10 and E18 embryos were studied. At all stages, PDE activity is stimulated by calcium and calmodulin. A 5-fold increase in basal cAMP and cGMP PDE activity is evident from E10 to E18, while from E18 to P5 basal PDE activity remains constant. Ion exchange chromatography elution profile indicates that PDE1 isoforms represent the bulk of the PDE activity present. Inhibition studies were performed in order to distinguish the activity due to PDE1A, B and C. Western blot analysis using anti-mammalian PDE1A, B and C specific antibodies was also performed. Densitometric analysis of the stained bands reveals that PDE1B and PDE1C display a prominent increase between day 10 and day 18 of development (eight- and 3.6 fold, respectively) while a more limited increase (1.6- and 1.5-fold) is observed between E18 and P5; on the other hand PDE1A shows continuously increasing levels throughout development. Immunohistochemical analysis was performed with isoform specific antibodies used for western blot analysis. PDE1A immunoreactivity is found in the cytoplasm and fibers of several neurons differing in size and distributed throughout the ganglion. PDE1B staining is evident on all neurons, however, fibers appear very faintly labelled. All neurons appear stained by PDE1C antibody, although the intensity of immunostaining is always heterogeneous in different neuronal populations: no staining was evident on fibers or in non-neural cells. The distinct spatial and temporal expression patterns of PDE1 isoforms may indicate their different physiological roles in developing and mature chick DRG.     

Increase in ceramide level alters the lysosomal targeting of cathepsin D prior to onset of apoptosis in HT-29 colon cancer cells

Ceramide has been suggested as an important mediator of apoptosis. In HT-29 colorectal cancer cells increased ceramide levels, induced by exogenous N-acetylsphingosine (NAS, also known as C2-ceramide) or by 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol (PDMP), inhibited the transport and processing of cathepsin D (CD), a lysosomal protease implicated in apoptosis of tumour cells. C2-dihydroceramide (DH-C2), an inactive analogue of NAS, had no effect on CD transport and maturation. The treatment with either NAS or PDMP was revealed to be cytotoxic for HT-29 cells and led to cell death with classical features of apoptosis. Morphological signs of apoptosis and DNA fragmentation became apparent only between 24 and 48 h of incubation and poly(ADP ribose)-polymerase cleavage, a hallmark of caspase 3 activity, occurred no earlier than 8 h from incubation. Secretion of proCD was almost abolished and the formation of double-chain mature CD was reduced and delayed by NAS, whereas PDMP largely inhibited the lysosomal targeting and maturation of proCD. NAS- and PDMP-induced alteration of proCD transport and maturation were apparent already 2 h after incubation with the drugs, which is much earlier than when classical biochemical and morphological evidence of apoptosis could be detected. These data indicate that alteration of CD (and possibly of other glycoproteins) transport along the secretory pathway due to increased levels of cell-associated ceramide is an early event in cells undergoing apoptosis.     

DNA fragmentation and morphological changes in apoptotic human lymphocytes

Cell suspensions enriched in cells at various stages of apoptosis were obtained by separation of irradiated human peripheral blood lymphocytes on density gradients at different post-irradiation times. The state of DNA fragmentation in the cells was determined by comet assay and pulsed field gel electrophoresis. The morphologically distinguishable features of apoptosis such as chromatin condensation and cell shrinkage correlated with discrete stages of DNA fragmentation. It was found that >/=50 kbp fragmentation of DNA occurs already in cells of normal density whereas the subsequent DNA fragmentation onto fragments <50 kbp occurs in parallel with cell shrinkage and simultaneous increase in cell density.The observed stages of DNA fragmentation seem to be separated in time that could allow in case of abortive apoptosis formation of chromosomal aberrations.     

NMDA receptor stimulation induces temporary alpha-tubulin degradation signaled by nitric oxide-mediated tyrosine nitration in the nervous system of Sepia officinalis

Biochemical and immunohistochemical evidence is reported, showing basal protein nitration in specific regions of the optic lobes of Sepia officinalis, mainly in the fiber layers of the plexiform zone. SDS-PAGE analysis of optic lobe extracts revealed an intense 3-nitrotyrosine immunoreactive band identified as alpha-tubulin by immunoprecipitation and partial purification. Stimulation of NMDA receptors resulted in a selective decrease in alpha-tubulin levels within 30 min with partial recovery after 4 h. The effect was suppressed by the NO synthase (NOS) inhibitor L-nitroarginine. Incubation of optic lobes with free 3-nitrotyrosine resulted likewise in a selective loss of alpha-tubulin, due apparently to incorporation of the amino acid into the C-terminus of detyrosinated alpha-tubulin to give the nitrated protein purportedly more susceptible to degradation. Overall, these results point to a novel potential physiologic role of NO and free 3-nitrotyrosine in the control of the alpha-tubulin tyrosination/detyrosination cycle and turnover in Sepia nervous tissue.     

Structure of FAD-bound L-aspartate oxidase: insight into substrate specificity and catalysis

L-Aspartate oxidase (Laspo) catalyzes the conversion of L-Asp to iminoaspartate, the first step in the de novo biosynthesis of NAD(+). This bacterial pathway represents a potential drug target since it is absent in mammals. The Laspo R386L mutant was crystallized in the FAD-bound catalytically competent form and its three-dimensional structure determined at 2.5 A resolution in both the native state and in complex with succinate. Comparison of the R386L holoprotein with the wild-type apoenzyme [Mattevi, A., Tedeschi, G., Bacchella, L., Coda, A., Negri, A., and Ronchi, S. (1999) Structure 7, 745-756] reveals that cofactor incorporation leads to the ordering of two polypeptide segments (residues 44-53 and 104-141) and to a 27 degree rotation of the capping domain. This motion results in the formation of the active site cavity, located at the interface between the capping domain and the FAD-binding domain. The structure of the succinate complex indicates that the cavity surface is decorated by two clusters of H-bond donors that anchor the ligand carboxylates. Moreover, Glu121, which is strictly conserved among Laspo sequences, is positioned to interact with the L-Asp alpha-amino group. The architecture of the active site of the Laspo holoenzyme is remarkably similar to that of respiratory fumarate reductases, providing strong evidence for a common mechanism of catalysis in Laspo and flavoproteins of the succinate dehydrogenase/fumarate reductase family. This implies that Laspo is mechanistically distinct from other flavin-dependent amino acid oxidases, such as the prototypical D-amino acid oxidase.     

Biochemical and molecular study of mentally retarded patient with partial deficiency of hypoxanthine-guanine phosphoribosyltransferase

Nucleotide metabolism was studied in erythrocytes of a mentally retarded child and family members. Partial hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency was found in the propositus and an asymptomatic maternal uncle. Studies in crude lysates demonstrated decreased apparent V(max) and slightly decreased apparent K(m) for hypoxanthine in both HPRT-deficient subjects. Genomic DNA analysis revealed a single nucleotide change with leucine-147 to phenylalanine substitution in both subjects; mother and grandmother were heterozygous carriers of the same defect. This new variant has been termed HPRT(Potenza). Increased erythrocyte concentration of NAD and rate of synthesis by intact erythrocytes were found in the patient; increased activities of nicotinic acid phosphoribosyltransferase (NAPRT) and NAD synthetase (NADs) were demonstrated in erythrocyte lysates, with normal apparent K(m) for their substrates and increased V(max). These alterations were not found in any member of the family, including the HPRT-deficient uncle. These findings show multiple derangement of nucleotide metabolism associated with partial HPRT deficiency. The enzyme alteration was presumably not the cause of neurological impairment since no neurological symptoms were found in the HPRT-deficient uncle, whereas they were present in the propositus' elder brother who had normal HPRT activity.     

Intracellular pH regulation in U-2 OS human osteosarcoma cells transfected with P-glycoprotein

The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma cell line were investigated by loading with 2',7'-bis(2-carboxyethyl)-5(6) carboxyfluorescein ester and manipulation of Cl(-) and Na(+) gradients, both in HEPES- and HCO(3)(-)/CO(2)-buffered media. Both acidification and alkalinisation were poorly sensitive to 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulfonic acid, inhibitor of the anion exchanger, but sensitive to amiloride, inhibitor of the Na(+)/H(+) exchanger. In addition to the amiloride-sensitive Na(+)/H(+) exchanger, another H(+) extruding mechanism was detected in U-2 OS cells, the Na(+)-dependent HCO(3)(-)/Cl(-) exchanger. No significant difference in resting pH(i) and in the rate of acidification or alkalinisation was observed in clones obtained from U-2 OS cells by transfection with the MDR1 gene and overexpressing P-glycoprotein. However, both V(max) and K' values for intracellular [H(+)] of the Na(+)/H(+) exchanger were significantly reduced in MDR1-transfected clones, in the absence and/or presence of drug selection, in comparison to vector-transfected or parental cell line. NHE1, NHE5 and at a lower extent NHE2 mRNA were detected in similar amount in all U2-OS clones. It is concluded that, although overexpression of P-glycoprotein did not impair pH(i) regulation in U-2 OS cells, the kinetic parameters of the Na(+)/H(+) exchanger were altered, suggesting a functional relationship between the two membrane proteins.     

Carbocysteine lysine salt monohydrate (SCMC-LYS) is a selective scavenger of reactive oxygen intermediates (ROIs)

Carbocysteine lysine salt monohydrate (SCMC-Lys) is a well-known mucoactive drug whose therapeutic efficacy is commonly related to the ability of SCMC-Lys to replace fucomucins by sialomucins. The aim of this study was to determine if SCMC-Lys could exert an anti-oxidant action by scavenging reactive oxygen intermediates (ROIs). Our results show that SCMC-Lys proved effective as a selective scavenger of hypochlorous acid (HOCl) and hydroxyl radical (OH.), this effect being related to the reactivity of the SCMC tioether group. The scavenger activity of SCMC-Lys was observed in free cellular system as well as in activated human polymorphonuclear neutrophils (PMNs). SCMC-Lys scavenger activity on HOCl was paralleled by a powerful protection from HOCl-mediated inactivation of alpha1-antitripsin (alpha1-AT) inhibitor, the main serum protease inhibitor. Production of interleukin-(IL-)8, a major mediator of PMN recruitment in inflammatory diseases, is known to be mediated by intracellular OH. SCMC-Lys significantly reduced IL-8 production on stimulated human peripheral blood mononuclear cells (PBMCs) in the same range of concentrations affecting OH. activity. It is concluded that SCMC-Lys could exert, in addition to its mucoactive capacity, an anti-oxidant action, thus contributing to the therapeutic efficacy of SCMC-Lys.     

Interleukin-6 N-terminal peptides modulate the expression of junB protooncogene and the production of fibrinogen in HepG2 cells

Interleukin-6 (IL-6) is a helical cytokine exerting pleiotropic activities including the regulation of hematopoiesis, B cell activation and acute-phase reaction. The structure-function relationship of the molecule is the subject of intensive investigation using point and deletion mutants. Our objective was to analyse the role of the N-terminal 18-46 region in IL-6-mediated expression of junB protooncogene and fibrinogen production, reflecting the acute phase response, with synthetic overlapping peptides. mRNA expression of junB was monitored by competitive RT-PCR, while sandwich ELISA was used for the detection of fibrinogen in the supernatant of HepG2 human hepatoma cells. We found that even short synthetic octapeptides can be stimulatory (in the absence of IL-6) or inhibitory (in the presence of IL-6) in both assays. To establish the molecular mechanism by which synthetic peptides exert their biological effects electromobility shift assay was carried out using HepG2 nuclear extracts. Peptides inducing junB expression initiate gel shifts of STAT3/DNA complexes, which may indicate the involvement of this signal transduction pathway. Circular dicroism spectroscopy data suggest that 8-11-mer peptides representing different parts of the 18-46 region have a marked tendency to adopt ordered conformations in a water/trifluoroethanol (1:1 v/v) mixture. Competition studies with rhIL-6 and selected fluorophore-labelled peptides indicate the presence of more than one binding site on soluble IL-6 receptor. Considering the possible multiple etiologic role of IL-6 in the pathogenesis of various diseases, these peptides could be useful for dissection of IL-6 related biological effects.