The convergent point of the endocytic and autophagic pathways in leydig cells.
(9/671)
Endocytic tracers and marker enzyme of lysosomes were used in the present study to analyze the processes of autophagocytosis and endocytosis, and the convergent point of these two pathways in Leydig cells. The endocytic and autophagic compartments can be easily identified in Leydig cells, which makes easier to define the stages of two pathways than was possible before. The evidences indicated that the late endosomes (dense MVBs) deliver their endocytosed gold tracers together with lysosomal enzymes to the early autophagosomes and they are the convergent point of the two pathways. During this convergent process, the early autophagosomes transform into late autophagosomes and the late endosomes transform into mature lysosomes. (+info)
Identification of BFN1, a bifunctional nuclease induced during leaf and stem senescence in Arabidopsis.
(10/671)
Nuclease I enzymes are responsible for the degradation of RNA and single-stranded DNA during several plant growth and developmental processes, including senescence. However, in the case of senescence the corresponding genes have not been reported. We describe the identification and characterization of BFN1 of Arabidopsis, and demonstrate that it is a senescence-associated nuclease I gene. BFN1 nuclease shows high similarity to the sequence of a barley nuclease induced during germination and a zinnia (Zinnia elegans) nuclease induced during xylogenesis. In transgenic plants overexpressing the BFN1 cDNA, a nuclease activity of about 38 kD was detected on both RNase and DNase activity gels. Levels of BFN1 mRNA were extremely low or undetectable in roots, leaves, and stems. In contrast, relatively high BFN1 mRNA levels were detected in flowers and during leaf and stem senescence. BFN1 nuclease activity was also induced during leaf and stem senescence. The strong response of the BFN1 gene to senescence indicated that it would be an excellent tool with which to study the mechanisms of senescence induction, as well as the role of the BFN1 enzyme in senescence using reverse genetic approaches in Arabidopsis. (+info)
A novel target of lithium therapy.
(11/671)
Phosphatases converting 3'-phosphoadenosine 5'-phosphate (PAP) into adenosine 5'-phosphate are of fundamental importance in living cells as the accumulation of PAP is toxic to several cellular systems. These enzymes are lithium-sensitive and we have characterized a human PAP phosphatase as a potential target of lithium therapy. A cDNA encoding a human enzyme was identified by data base screening, expressed in Escherichia coli and the 33 kDa protein purified to homogeneity. The enzyme exhibits high affinity for PAP (K(m)<1 microM) and is sensitive to subtherapeutic concentrations of lithium (IC(50)=0.3 mM). The human enzyme also hydrolyzes inositol-1, 4-bisphosphate with high affinity (K(m)=0.4 microM), therefore it can be considered as a dual specificity enzyme with high affinity (microM range) for both PAP and inositol-1,4-bisphosphate. Hydrolysis of inositol-1,4-bisphosphate was also inhibited by lithium (IC(50)=0.6 mM). Thus, we present experimental evidence for a novel target of lithium therapy, which could explain some of the side effects of this therapy. (+info)
Hydrolysis of extracellular adenine nucleotides by cultured bovine endocardial endothelial cells.
(12/671)
AIM: To characterize the ATP diphosphohydrolase (apyrase) of bovine endocardial endothelial cells, and to compare ecto-adeninenucleotidase activity between bovine endocardial and aortic endothelial cells (BEEC and BAEC). METHODS: The nucleotide was analyzed by reversed phase HPLC and apyrase activity was assayed by inorganic phosphate release. RESULTS: Apyrase inhibitors, both NaN3 10 mmol.L-1 and NaF 20 mmol.L-1, inhibited BEEC apyrase activity by 51% and 38%, respectively. The inhibitor for Na+/K(+)-ATPase, ouabain, did not affect the enzyme activity. Edetic acid 5 mmol.L-1 completely inhibited the enzyme activity. H2O2 0.5 mmol.L-1 downregulated BEEC apyrase activity in a time-dependent manner. The apyrases activities in BAEC were higher than those in BEEC, while the ecto-AMPase activity in BAEC was much weaker than that in BEEC. CONCLUSION: BEEC have NaN3- and NaF-sensitive, ouabain-insensitive apyrase activity. BEEC had high ecto-AMPase activities, and low apyrases activities as compared with BAEC. (+info)
Mammalian 5'(3')-deoxyribonucleotidase, cDNA cloning, and overexpression of the enzyme in Escherichia coli and mammalian cells.
(13/671)
5'(3')-Deoxyribonucleotidase is a ubiquitous enzyme in mammalian cells whose physiological function is not known. It was earlier purified to homogeneity from human placenta. We determined the amino acid sequences of several internal peptides and with their aid found an expressed sequence tag clone with the complete cDNA for a murine enzyme of 23.9 kDa. The DNA was cloned into appropriate plasmids and introduced into Escherichia coli and ecdyson-inducible 293 and V79 cells. The recombinant enzyme was purified to homogeneity from transformed E. coli and was found to be identical with the native enzyme. After induction with ponasterone, the transfected mammalian cells showed a gradual increase of enzyme activity. A human expressed sequence tag clone contained a large part of the cDNA of the human enzyme but lacked the 5'-end corresponding to 51 amino acids of the murine enzyme. Several polymerase chain reaction-based approaches to find this sequence met with no success. A mouse/human hybrid cDNA that had substituted the missing human 5'-end with the corresponding mouse sequence coded for a fully active enzyme. (+info)
Dissection of the functional domains of the Leishmania surface membrane 3'-nucleotidase/nuclease, a unique member of the class I nuclease family.
(14/671)
Class I nucleases are a family of enzymes that specifically hydrolyze single-stranded nucleic acids. Recently, we characterized the gene encoding a new member of this family, the 3'-nucleotidase/nuclease (Ld3'NT/NU) of the parasitic protozoan Leishmania donovani. The Ld3'NT/NU is unique as it is the only class I nuclease that is a cell surface membrane-anchored protein. Currently, we used a homologous episomal expression system to dissect the functional domains of the Ld3'NT/NU. Our results showed that its N-terminal signal peptide targeted this protein into the endoplasmic reticulum. Using Ld3'NT/NU-green fluorescent protein chimeras, we showed that the C-terminal domain of the Ld3'NT/NU functioned to anchor this protein into the parasite cell surface membrane. Further, removal of the Ld3'NT/NU C-terminal domain resulted in its release/secretion as a fully active enzyme. Moreover, deletion of its single N-linked glycosylation site showed that such glycosylation was not required for the enzymatic functions of the Ld3'NT/NU. Thus, using the fidelity of a homologous expression system, we have defined some of the functional domains of this unique member of the class I nuclease family. (+info)
Intrinsic interrelation of lymphatic endothelia with nerve elements in the monkey urinary bladder.
(15/671)
Histochemical staining techniques for 5'-nucleotidase (5'-Nase) and acetylcholinesterase (AChE) were undertaken to localize the lymphatic network and nerve plexus in the monkey urinary bladder. Abundant 5'-Nase-positive lymphatic networks were characterized by increased number of valve-like structures and decreased calibre of blind-ends from the subepithelium to the subserosa. AChE-positive nerve fibers were visible throughout the vesical walls as fine plexuses, the densest being the neuromuscular plexus among the detrusor muscle cells or in each muscle bundle. AChE-positive nerve fibers or terminals were more frequently discernible around blood vessels than around lymphatics, and showed more intimate association with the lymphatics in the muscularis than those in the subepithelium. The nerve terminals in the subepithelium were frequently separated from attenuated lymphatic endothelium by the long processes of fibroblasts or some connective tissue cells. An ultrastructural observation revealed that unmyelinated nerve fibers with numerous neurofilaments and neurotubules run in close apposition to the lymphatic endothelium. Noteworthily, fewer terminal varicosities containing numerous small agranular vesicles (30-50 nm) and mitochondria, partially or completely bare of their Schwann cell covering in the vicinity of the lymphatic endothelium, were found in the subendothelium of initial lymphatics than in collecting ones. These terminals were occasionally identified at a distance of 120-350 nm from the subendothelial aspect of valve-originating roots, although no direct innervation of the vascular muscle cells could be found. A loose fibro-elastic connective tissue was usually interlaced between glial cell covering and lymphatic endothelium. The intrinsic interrelation of the lymphatic wall with the nerve plexus implies that the twisted subendothelial nerve terminals might be involved in intramural lymph drainage of the bladder. (+info)
Characterization of diphosphonucleotide phosphatase/phosphodiesterase from yellow lupin (Lupinus luteus) seeds.
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A phosphatase cleaving the pyrophosphate bond in diphosphonucleotides and phosphodiester bond in various phosphodiesters (pH optimum at 6.25) was purified from yellow lupin (Lupinus luteus L.) seeds. The enzyme is 75 kDa monomeric glycoprotein (pI=6.4) with 4.4% of carbohydrate (mannose, N-acetylglucosamine, fucose and xylose). Analysis of its partial amino acid sequence (8 peptides, 101 amino acid residues) together with no divalent cation requirements for catalysis points out that the purified enzyme is different from known plant pyrophosphate cleaving enzymes (apyrases and inorganic pyrophosphatases). Its physiological role could be related to a regulation of diphosphonucleotides level in plant metabolism. (+info)