Polyamine cytotoxicity in the presence of bovine serum amine oxidase

The toxicity of extracellular spermine, determined in the presence of fetal calf serum, was studied using three cell lines: FM3A, L1210, and NIH3T3 cells. Amine oxidase in fetal calf serum produces aminodialdehyde generating acrolein spontaneously, H(2)O(2), and ammonia from spermine. Spermine toxicity was prevented by aldehyde dehydrogenase, but not by catalase. Similar concentrations of spermine and acrolein were needed to produce toxicity. Other aldehydes (formaldehyde, acetaldehyde, and propionaldehyde) and hydrogen peroxide were less toxic than acrolein. Spermidine and 3-aminopropanal, which produces acrolein, also exhibited severe cytotoxicity. The degree of cytotoxicity of spermine, spermidine, and 3-aminopropanal was nearly parallel with the amount of acrolein produced from each compound. Thus, it was deduced that acrolein is a major toxic compound produced from polyamines (spermine and spermidine) by amine oxidase.     

Lecithinized Cu, Zn-superoxide dismutase limits the infarct size following ischemia-reperfusion injury in rat hearts in vivo

Ghrelin is a novel gut-brain peptide that binds to the growth hormone secretagogue receptor (GHS-R), thereby functioning in the regulation of growth hormone (GH) release and food intake. Ghrelin-producing cells are most abundant in the oxyntic glands of the stomach. The regulatory mechanism that governs the biosynthesis and secretion of ghrelin has not been clarified. We report that ghrelin mRNA expression in the gastric fundus was increased, but that ghrelin peptide content decreased after a 48-h fast. Both values returned to control levels after refeeding. The ghrelin plasma concentration in the gastric vein and systemic venous blood increased after 24- and 48-h fasts. Furthermore, des-octanoylated ghrelin and n-octanoylated ghrelin were found in rat stomach, with the ratio of des-octanoylated ghrelin to n-octanoylated ghrelin markedly increased after fasting. The ghrelin mRNA level in the stomach also increased after administration of insulin and leptin. Conversely, db/db mice, which are deficient in the leptin receptor, had lower ghrelin mRNA levels than control mice. These findings suggest that this novel gastrointestinal hormone plays a role in the regulation of energy balance.     

Selective inhibition of beta-1,4- and alpha-1,3-galactosyltransferases: donor sugar-nucleotide based approach

A combined rational and library approach was used to identify bisphosphonates (IC50 = 20 microM) and galactose type 1-N-iminosugar (IC50=45 microM) as novel motifs for selective inhibition of beta-1,4-galactosyltransferase (beta-1,4-GalT) and alpha-1,3-galactosyltransferase (alpha-1,3-GalT), respectively. Our results demonstrate that, though these two galactosyltransferases both utilize the same donor sugar-nucleotide (UDP-Gal), the difference in their mechanisms can be utilized to design donor sugar or nucleotide analogues with inhibitory activities selective for only one of the galactosyltransferases. Investigation of beta-1,4-GalT inhibition using UDP-2-deoxy-2-fluorogalactose (UDP-2-F-Gal), UDP, and bisphosphonates, also led to the observation of metal dependent inhibition of beta-1,4-GalT. These observations and the novel inhibitor motifs identified in this study pave the way for the design and identification of even more potent and selective galactosyltransferase inhibitors.     

Processing and juxtacrine activity of membrane-anchored betacellulin

Betacellulin (BTC) was originally isolated as a secreted growth factor from a mouse pancreatic beta-tumor cell line, whereas the cDNA sequence predicts that BTC is synthesized as a larger transmembrane protein. In the present study, we have characterized the membrane-anchored forms of BTC, using Chinese hamster ovary (CHO) cells, mouse fibroblast A9 cells, and a human breast cancer cell line MCF-7, all of which were stably transfected with human BTC cDNA. A9 and MCF-7 transfectants produced membrane-anchored BTC isoforms of 21, 25, 29, and 40 kDa on the cell surface, as well as a secreted BTC isoform. CHO transfectants secreted little BTC but accumulated the membrane-anchored isoforms. The cleavage of the membrane-anchored forms to release a secreted from of BTC was not enhanced by biological mediators such as a phorbol ester, which stimulates the cleavage of other membrane-anchored growth factors. The membrane-anchored forms of BTC expressed on the transfected cells induced the insulin production and/or promoted the growth in subclones of AR42J rat pancreatic cells. These results suggest that the membrane-anchored BTC can function as a juxtacrine factor in regulating the growth and differentiation of pancreatic endocrine cells.     

Increases in ceramide levels in normal human mesangial cells subjected to different cellular stresses result from changes in distinct enzyme activities and can influence cellular responses to other stimuli

Sphingolipids, ceramide in particular, have come to be regarded as having roles in cellular signaling, most recently being associated with stress and the cellular responses to stress. In the present study we first examined the mechanisms involved in the changes in cellular ceramide levels in normal human mesangial cells (NHMC) in the growth, quiescent, and senescent phases as well as those resulting from environmental stimuli. We found that in NHMC total ceramide levels increase in response to cellular stresses as a result of a combination of enzyme activities. Furthermore, different stresses cause different alterations in various enzyme activities, with age and growth influencing acidic enzymes, but cell density affecting neutral, resulting in final ceramide level increases which most likely are associated with distinct pools of ceramide. Secondly, we examined the influence of changes in ceramide levels on apoptosis induced by sphingosine and its methylated derivative N, N-dimethylsphingosine. We found that increases in cellular ceramide levels prohibited the apoptosis and caused a quiescent state in the cells. The data presented here provide additional insight into the roles of ceramide and related enzymes in cellular responses to stress and suggest a possible relevance to in vivo disease states.     

Proteins recognized by antibodies against isolated cytological heterochromatin from rat liver cells change their localization between cell species and between stages of mitosis (interphase vs metaphase)

Heterochromatin in the cell nucleus seems to concentrate various proteins, such as Drosophila heterochromatin protein 1, which maintain the repressed state of gene expression. However, it still remains obscure how protein composition related to chromatin structure is different between heterochromatin and euchromatin in interphase nuclei. We isolated cytological heterochromatin from sonicated interphase nuclei obtained from rat liver cells and prepared antisera against it. The dense heterochromatic bodies seen in the preparation of intact nuclei were duplicated in a relatively pure form during the preparation of heterochromatin. In the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, differences between the fractions of heterochromatin and euchromatin were noted by their protein composition. Isolated heterochromatin was then digested by DNase after partial digestion with trypsin and its dense structure changed to become highly sensitive to DNase. The prepared antibodies reacted with the heterochromatin region of rat liver cell nuclei and isolated cytological heterochromatin; however, they did not react with euchromatin. Using immunohistochemistry, the antibodies bound to each cell nucleus in all tissues observed; some cell types were distinguished by their differential stainability (e.g. staining in the cytoplasm). Staining of the mitotic cells showed that the proteins recognized by the antibodies were localized in the cytoplasm and, in part, on the chromosomes. Based on the results of molecular cloning from rat liver cDNA library using the antibodies as a probe, it seemed that the antibodies mainly recognized two proteins similar to arginase and general vesicular transport factor p115, respectively. The results obtained from these experiments reveal that some proteins located in the heterochromatin of interphase liver cell nuclei seem to play important roles in condensing a portion of the chromatin structure during interphase and suggest that proteins composing heterochromatin might be changed according to cell types or the stage of the cell cycle.     

Protective immunity of gene-deleted SHIVs having an HIV-1 Env against challenge infection with a gene-intact SHIV

We constructed three simian-human immunodeficiency viruses (SHIVs) lacking regulatory gene(s) and analyzed their induction of protective immunity against challenge infection with gene-intact SHIV in rhesus macaques. Inoculation of SHIV-dn lacking nef and SHIV-drn lacking nef and vpr induced transient viremia, while that of SHIV-dxrn lacking nef, vpr, and vpx induced no viremia. The SHIVs with fewer deletions were more effective in inducing neutralizing antibodies and cytotoxic T lymphocyte responses. When these macaques were challenged with parental gene-intact SHIV-NM-3rN, all the SHIV-dn-vaccinated macaques and two of the four SHIV-drn-vaccinated macaques showed complete resistance. The other two SHIV-drn-vaccinated macaques and all SHIV-dxrn-vaccinated macaques did not show complete resistance, but they did show suppression of replication of the challenge virus. These results suggested that as more genes were deleted, protective immunity was decreased.     

Characterization of ADP-glucose pyrophosphorylase from Rhodobacter sphaeroides 2.4.1: evidence for the involvement of arginine in allosteric regulation

ADP-glucose pyrophosphorylase (ADPGlc PPase, EC 2.7.7.27) from Rhodobacter sphaeroides 2.4.1 has been purified to near homogeneity. The enzyme reacted in Western blots to polyclonal antibodies raised against other bacterial ADPGlc PPases. The purified enzyme was found to be activated by fructose 6-phosphate, fructose 1,6-bisphosphate, and pyruvate and inhibited by phosphate, phosphoenolpyruvate, ADP, and pyridoxal phosphate. Kinetic studies indicate that AMP, while having little effect on kinetic parameters at pH 8 in the absence of effectors, is a specific ligand for an allosteric site(s). Treatment of the purified enzyme with the arginyl reagents 2,3-butanedione and phenylglyoxal resulted in desensitization of the enzyme to both activation and inhibition by metabolites. Phosphate, fructose 6-phosphate, and AMP were found to protect the enzyme against allosteric desensitization supportive of these metabolites interacting at common site(s) or with a common enzyme form. As a first step in cloning the gene coding for this enzyme, a polymerase chain reaction fragment was generated from genomic DNA using primers based on amino terminal sequencing data and a highly conserved region in known ADPGlc PPases. The sequence of this fragment and position of amino terminal arginines in comparison to other known ADPGlc PPases is discussed in relation to the kinetic and chemical modification data.     

A mutant with constitutive sexual organ development in<Emphasis Type="Italic"> Marchantia polymorpha</Emphasis> L.

In lower land plants, genes controlling the transition from vegetative growth to sexual reproduction have not yet been identified. In the dioecious liverwort Marchantia polymorpha, the transition to sexual reproduction accompanied by the formation of sexual organs on the gametophytic thallus is initiated under long-day conditions. By particle bombardment-mediated mutagenesis, we generated a mutant of M. polymorpha that constitutively forms sexual organs. This mutant is fully fertile, showing that the mutation does not affect formation of male or female sexual organs per se. Genetic analysis reveals that this phenotype is caused by mutation of a single autosomal locus, suggesting that this mutation defines or controls a gene regulating the transition to sexual reproduction in M. polymorpha.