Polyphenols from some foodstuffs as inhibitors of ovalbumin permeation through caco-2 cell monolayers

Some spices showed high inhibitory activity against ovalbumin permeation through Caco-2 cell monolayers. Pimentol from allspice, rosmarinic acid and luteolin-7-O-beta-glucuronide from thyme, quercetin-3-O-beta-glucuronide from coriander and rutin from tarragon were identified as the active principles. A structure-activity relationship study among the active isolates and their related compounds indicated that the presence of a catechol structure played an important role in the inhibitory activity of each compound.     

Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis

Recent studies have suggested that apoptosis and necrosis share common features in their signaling pathway and that apoptosis requires intracellular ATP for its mitochondrial/apoptotic protease-activating factor-1 suicide cascade. The present study was, therefore, designed to examine the role of intracellular energy levels in determining the form of cell death in cardiac myocytes. Neonatal rat cardiac myocytes were first incubated for 1 h in glucose-free medium containing oligomycin to achieve metabolic inhibition. The cells were then incubated for another 4 h in similar medium containing staurosporine and graded concentrations of glucose to manipulate intracellular ATP levels. Under ATP-depleting conditions, the cell death caused by staurosporine was primarily necrotic, as determined by creatine kinase release and nuclear staining with ethidium homodimer-1. However, under ATP-replenishing conditions, staurosporine increased the percentage of apoptotic cells, as determined by nuclear morphology and DNA fragmentation. Caspase-3 activation by staurosporine was also ATP dependent. However, loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bax translocation, and cytochrome c release were observed in both apoptotic and necrotic cells. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition, attenuated staurosporine-induced apoptosis and necrosis through the inhibition of DeltaPsi(m) reduction, cytochrome c release, and caspase-3 activation. Our data therefore suggest that staurosporine induces cell demise through a mitochondrial death signaling pathway and that the presence of intracellular ATP favors a shift from necrosis to apoptosis through caspase activation.     

Characterization of P8 and J8/7 elements in the conserved core of the tetrahymena group I intron ribozyme

The universally conserved core region in the group I intron ribozymes is responsible for its catalytic activity. The structural elements in this region have been known to organize the active site of this class of ribozymes. However, it has been unclear whether all elements are requisite or some elements are dispensable for conducting the catalysis. To investigate the necessity of these elements in the catalysis, we prepared and examined a series of mutants having a nick or deletion in these elements. In this report, we show that two elements, P8 and 5' portion of J8/7, are nonessential for activity.     

The prodomain of caspase-1 enhances Fas-mediated apoptosis through facilitation of caspase-8 activation

Caspase-1 (interleukin-1beta converting enzyme) is produced in the form of a latent precursor, which is cleaved to yield a prodomain in addition to the p20 and p10 subunits. It has been established that the (p20/p10)(2) heterotetramer processes the latent precursor of interleukin-1beta into an active form during apoptosis, but the function of the residual prodomain of caspase-1 (Pro-C1) has not been established. To evaluate the involvement of Pro-C1 in apoptosis, a Pro-C1 expression vector was transfected into the HeLa cell line, which is susceptible to Fas-mediated apoptosis. Expression of recombinant Pro-C1 in HeLa cells enhanced apoptosis mediated by Fas, but not etoposide-induced apoptosis. This enhancement of Fas-mediated apoptosis was abolished by inhibitors of caspase-8 (Ile-Glu-Thr-Asp-fluoromethyl ketone) and caspase-3 (Asp-Glu-Val-Asp-aldehyde) but was only slightly diminished by an inhibitor of caspase-1 (acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone). During apoptosis induced by an agonistic anti-Fas antibody, the activation of caspase-8 and caspase-3 was more pronounced and occurred more rapidly in HeLa/Pro-C1 cells than in the empty vector transfectant (HeLa/vec) cells; in contrast, caspase-1 was not activated in either HeLa/Pro-C1 or HeLa/vec cells. These results demonstrate an additional and novel function for caspase-1 in which Pro-C1 acts to enhance Fas-mediated apoptosis, most probably through facilitation of the activation of caspase-8.     

SYNCRIP, a cytoplasmic counterpart of heterogeneous nuclear ribonucleoprotein R, interacts with ubiquitous synaptotagmin isoforms

Synaptotagmins (Syts) are a large family of membrane proteins consisted of at least 12 isoforms. They are categorized in neuron-specific isoforms (I-V, X, and XI) and ubiquitous isoforms (VI-IX) based on their expression patterns. Syt-I, a neuron-specific and abundant isoform, has been well characterized and postulated to be the exocytotic Ca(2+) sensor. However, the functions of other isoforms remain obscure. Here, we report that ubiquitous isoforms of synaptotagmins, Syt-VII, Syt-VIII, and Syt-IX, interacted with a cytoplasmic RNA-binding protein, SYNCRIP (Synaptotagmin-binding, cytoplasmic RNA-interacting protein), through their C2B domains. SYNCRIP was originally found in the Syt-II C2AB domain bound fraction from the mouse brain lysate. cDNA cloning of SYNCRIP cDNA revealed that the protein was highly homologous to heterogeneous nuclear ribonucleoprotein R (hnRNP R) recently identified. SYNCRIP protein was ubiquitously and constantly expressed in various tissues of mice parallel to hnRNP R. SYNCRIP indeed bound RNA with preference to poly(A) RNA; however, in contrast to the nuclear localization of hnRNP R, SYNCRIP was distributed predominantly in the cytoplasm as judged by both biochemical fractionation and immunohistochemical studies. In vitro binding experiments showed the potential interaction of SYNCRIP with C2B domains of Syts except for those of Syt-V, -VI, and -X. Furthermore, the interaction between SYNCRIP and Syt-VII, -VIII, or -IX was revealed by co-immunoprecipitation experiments using COS cells transiently expressing each Syt isoform. These findings suggested that SYNCRIP was a target of ubiquitous type of Syts and implied the involvement of ubiquitous Syts in the regulation of dynamics of the cytoplasmic mRNA.     

Molecular evidence of natural hybridization between abies veitchii and A. homolepis (Pinaceae) revealed by chloroplast, mitochondrial and nuclear DNA markers

Sub-alpine Abies veitchii and A. homolepis are distributed in the central part of Honshu Island, Japan, and their habitats are segregated vertically. These species sometimes form a mixed forest in the overlapping area of the two species, that is, in the upper limit of the A. homolepis habitat and the lower limit of A. veitchii. These species have been considered to be distantly related because they were classified into different sections by most conventional classifications. No natural hybridization has been reported between the two species. The aim of this study was to demonstrate, through the use of molecular markers, whether natural hybridization takes place between these two species at two experimental sites on Mt. Fuji, where the species occur naturally. DNA markers from paternally inherited chloroplast DNA (cpDNA), maternally inherited mitochondrial DNA (mtDNA) and biparentally inherited nuclear DNA (nDNA), were used for this study. As organelle DNA markers, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) markers were developed to determine the maternal and paternal species for each individual. Two of 334 individuals possessed a cpDNA haplotype derived from A. homolepis and a mtDNA haplotype from A. veitchii. Furthermore, the nDNA of these two individuals was analysed using the random amplified polymorphic DNA (RAPD) assay to investigate their genomic composition. RAPD analysis indicated that the nuclear genomes of the two individuals were derived from both species. We conclude that A. veitchii and A. homolepis produce natural hybrids, and that their systematic relationship should be re-evaluated.     

A small structural element, Pc-J5/5a, plays dual roles in a group IC1 intron RNA

The P4-P6 domain of group IC1 intron ribozymes such as that of the Tetrahymena autonomously folds into a hairpin-shaped structure in which the J5/5a region serves as a hinge. Phylogenetic comparisons of these IC1 introns suggested that the J5/5a region (termed Pc-J5/5a motif) in a subclass of IC1 introns such as the one from Pneumocystis carinii functions not only as a hinge but also as a receptor for a GAAA-tetraloop. We investigated the role of this motif by transplanting the structural unit, Pc-J5/5a motif, of Pneumocystis carinii into the P4-P6 domain of the Tetrahymena intron. The results showed that the Pc-J5/5a motif binds to a GAAA loop with high affinity and also facilitates the bending of the Tetrahymena P4-P6 domain more positively than the original J5/5a region.     

Effect of cisplatin on H+ transport by H+ -ATPase and Na+/H+ exchanger in rat renal brush-border membrane

The effect of the potent anticancer drug cisplatin, cis-diamminedichloroplatinum (II) (CDDP), on H+ -ATPase and Na+/H+ exchanger in rat renal brush-border membrane was examined. To measure H+ transport by vacuolar H+ -ATPase in renal brush-border membrane vesicles, we employed a detergent-dilution procedure, which can reorientate the catalytic domain of H+ -ATPase from an inward-facing configuration to outward-facing one. ATP-driven H+ pump activity decreased markedly in brush-border membrane prepared from rats two days after CDDP administration (5 mg/kg, i.p.). In addition, N-ethylmaleimide and bafilomycin A1 (inhibitors of vacuolar H+ -ATPase)-sensitive ATPase activity also decreased in these rats. The decrease in ATP-driven H+ pump activity was observed even at day 7 after the administration of CDDP. Suppression of ATP-driven H+ pump activity was also observed when brush-border membrane vesicles prepared from normal rats were pretreated with CDDP in vitro. In contrast with H+ -ATPase, the activity of Na+/H+ exchanger, which was determined by measuring acridine orange fluorescence quenching, was not affected by the administration of CDDP. These results provide new insights into CDDP-induced renal tubular dysfunctions, especially such as proximal tubular acidosis and proteinuria.