Three-dimensional structure of the inclusion complex between phloridzin and beta-cyclodextrin

The inclusion of phloridzin into beta-cyclodextrin was studied as a model of molecular recognition in membranes. Effects on 1H NMR spectra and NOE correlational peaks between phloridzin and beta-cyclodextrin were observed in the complex. Strong NOEs were observed between hydrogens of a phenol group in phloridzin and beta-cyclodextrin. The three-dimensional structure of the inclusion complex between phloridzin and beta-cyclodextrin was simulated with distance constraints estimated by the intensity of NOE peaks using the DADAS90 programs. Two inclusion possibilities were suggested-the large rim of beta-cyclodextrin as an entrance of the inclusion and the small rim of beta-cyclodextrin as the entrance. In both cases, the phenol group of phloridzin was included in the hydrophobic space of beta-cyclodextrin.     

Genomic clones encoding two isoforms of pokeweed antiviral protein in seeds (PAP-S1 and S2) and the N-glycosidase activities of their recombinant proteins on ribosomes and DNA in comparison with other isoforms

Pokeweed antiviral proteins (PAPs) are single-chain ribosome-inactivating proteins (RIPs) isolated from several organs of Phytolacca americana (Pokeweed) that are characterized by their ability to depurinate not only ribosomes but also various nucleic acids. PAP-S is one of the isoforms found in seeds. In this study, we obtained three different genomic clones encoding two forms of PAP-S (here designated as PAP-S1 and PAP-S2) and alpha-PAP after PCR using a pair of degenerated primers based on the known N- and C-terminal amino acid sequences of PAP-S. The nucleotide sequences of the genomic clones contained no introns. The deduced amino acid sequences of PAP-S1 and PAP-S2, which showed 83% identity to each other, were found to correspond to sequences reported independently for PAP-S protein and cDNA, respectively, demonstrating that at least two forms of PAP-S actually exist in seeds of the same plant. The recombinant PAP-S1, PAP-S2, alpha-PAP, and PAP I (a form appearing in spring leaves) exhibit the same level of depurinating activity on rat ribosomes, while their efficiencies on Escherichia coli ribosomes and salmon sperm DNA differ substantially from one another in the order of PAP I > alpha-PAP > PAP-S1 > PAP-S2 and alpha-PAP > PAP I > PAP-S1 > PAP-S2. Structural comparisons suggest that the large difference in ribosome recognition between PAP-S1 (or S2) and PAP I is caused by the alteration of residues adjacent to the adenine-binding site.     

Real-time kinetic analyses of the interaction of ricin toxin A-chain with ribosomes prove a conformational change involved in complex formation

Ricin toxin A-chain (RTA), a ribosome-inactivating protein from seeds of the castor bean plant (Ricinus communis), inactivates eukaryotic ribosomes by hydrolyzing the N-glycosidic bond of a single adenosine residue in a highly conserved loop of 28S rRNA, but does not act on prokaryotic ribosomes. We investigated the interaction of rat liver 80S ribosomes with RTA using an optical biosensor based on surface plasmon resonance (BIAcore instrument), which allows real-time recording of the interaction. RTA was coupled to the dextran gel matrix on the sensor chip surface through a single thiol group that is not involved in the enzymatic action. The interaction of rat ribosomes with RTA, which was greatly affected by the Mg(2+) concentration and ionic strength, was usually measured at 5 mM Mg(2+), 50 mM KCl, and pH 7.5. The modes of interaction of intact and RTA-depurinated rat liver ribosomes with the immobilized RTA were virtually the same, while no considerable interaction was observed for Escherichia coli ribosomes. The interaction was not influenced by the presence of 5 mM adenine, which is higher than the reported dissociation constant (1 mM) for the adenine-RTA complex. These results demonstrate that binding of the target adenine with the active site of RTA does not contribute much to the total interaction of ribosomes and RTA. Global analyses of association and dissociation data with several binding models, taking account of mass transport, allowed us to conclude that the data were unable to fit a simple 1:1 binding model, but were best described by a model including a conformational change involved in high affinity complex formation.     

Endometriosis: the pathophysiology as an estrogen-dependent disease

Endometriosis, defined as the presence of endometrial glands and stroma outside of the uterine cavity, develops mostly in women of reproductive age and regresses after menopause or ovariectomy, suggesting that the growth is estrogen-dependent. Indeed, the lesions contain estrogen receptors (ER) as well as aromatase, an enzyme that catalyses the conversion of androgens to estrogens, suggesting that local estrogen production may stimulate the growth of lesions. The expression patterns of ER and progesterone receptors in endometriotic lesions are different from those in the eutopic endometrium. Moreover, estrogen metabolism, including the expression pattern of aromatase and the regulation of 17β-hydroxysteroid dehydrogenase type 2 (an enzyme responsible for the inactivation of estradiol to estrone), is altered in the eutopic endometrium of women with endometriosis, adenomyosis, and/or leiomyomas compared to that in the eutopic endometrium of women without disease. Immunostaining for P450arom in endometrial biopsy specimens diagnosed these diseases with sensitivity and specificity of 91 and 100%, respectively. This is applicable to the clinical diagnosis of endometriosis. The polymorphisms in the ER- gene, the CYP19 gene encoding aromatase, and several other genes are associated with the risk of endometriosis. Studies of these will lead to better understandings of the etiology and pathophysiology of endometriosis.     

Possible factors responsible for the toxicity of Cochlodinium polykrikoides,a red tide phytoplankton

Cochlodinium polykrikoides, a harmful red tide dinoflagellate, is highly toxic to fish, but the toxic mechanism is still unknown. Recent study has suggested that C. polykrikoides generates reactive oxygen species (ROS) such as superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)), and the ROS-mediated ichthyotoxicity has been proposed. In this study, we found that the levels of O(2)(-) and H(2)O(2) detected in C. polykrikoides were trace levels as compared with those of Chattonella marina which is well-known to produce ROS. Furthermore, no significant increase in O(2)(-) generation by C. polykrikoides was observed in the presence of lectins such as concanavalin A (Con A) and wheat germ agglutinin (WGA) or fish mucus prepared from skin and gill of yellowtail, whereas C. marina generated increased level of O(2)(-) responding to these stimuli. Interestingly, the cell-free aqueous extract prepared from C. polykrikoides showed toxic effect on the HeLa cells, but the extract of C. marina had no significant effect. Furthermore, gradual accumulation of polysaccharides in the medium was observed during the growth of C. polykrikoides, and the medium gradually became viscous, but no such changes were observed in the medium of C. marina. These results suggest that multiple factors may be responsible for the toxic mechanism of C. polykrikoides.     

Breeding of African green monkeys (Cercopithecus aethiops) under indoor individually-caged conditions.

This paper reports the results of reproduction with 45 wild African green monkeys (Cercopithecus aethiops) (36 females and 9 males) during the nine years from 1981 to 1989 under indoor individually-caged conditions. In 206 cases of menstruation observed, menstrual discharge lasted for 2.5 +/- 1.2 days in cycles of 22-48 days, and the length of each menstrual cycle was 31.2 +/- 6.5 days. Females who had regular menstrual cycles were subjected to "one-to-one timed mating"; females and males were put together on a one-to-one basis daily only for a certain period of time on and after the day of ovulation. Females who had irregular menstrual cycles or had no menstruation were subjected to "every-other-day mating"; females and males were put together on a one-to-one basis every other day for at least 16 weeks. The pregnancy rate (No. of pregnant females/No. of mated females) by one-to-one timed mating was 48.9% (116/237); 2.0 mating trials were needed to obtain one case of pregnancy. On the other hand, the pregnancy rate (No. of pregnant females/No. of mating trials) by every-other-day mating was 96% (48/50). Females who delivered normally totaled 129. The mean gestation period was 165 days when males, weighing 343 g on average at birth, were delivered, and 166 days when females, weighing 318 g on average at birth, were delivered. The male and female newborns were nursed for 131 and 138 days, respectively, on average. Details are summarized in Table 3. This paper also reports 23 cases of abortion, 6 stillbirths, and 6 cases of Caesarean section, by which three live fetuses and three dead fetuses were obtained.     

Mice with targeted deletion of eNOS develop hyperdynamic circulation associated with portal hypertension

Systemic vasodilation is the initiating event of the hyperdynamic circulatory state, being most likely triggered by increased levels of vasodilators, primarily nitric oxide (NO). Endothelial NO synthase (eNOS) is responsible for this event. We tested the hypothesis that gene deletion of eNOS and inducible NOS (iNOS) may inhibit the development of the hyperdynamic circulatory state in portal hypertensive animals. To test this hypothesis, we used mice lacking eNOS (eNOS-/-) or eNOS/iNOS (eNOS/iNOS-/-) genes. A partial portal vein ligation (PVL) was used to induce portal hypertension. Sham-operated animals were used as a control. Hemodynamic characteristics were tested 2 wk after surgery. As opposed to our hypothesis, PVL also caused significant reduction in peripheral resistance in eNOS-/- compared with sham animals (0.33 +/- 0.02 vs. 0.41 +/- 0.03 mmHg. min x kg body wt x ml(-1); P = 0.04) and in eNOS/iNOS-/- animals with PVL compared with that of the sham-operated group (0.44 +/- 0.02 vs. 0.54 +/- 0.04; P = 0.03). This demonstrates that, despite gene deletion of eNOS, the knockout mice developed hyperdynamic circulation. Compensatory vasodilator molecule(s) are upregulated in place of NO in the systemic and splanchnic circulation in portal hypertensive animals.     

Isolation and identification of EG-VEGF/prokineticins as cognate ligands for two orphan G-protein-coupled receptors

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF, identical to prokineticin 1) is a novel peptide recently identified as a selective mitogen for endocrine gland endothelial cells. The present study demonstrates that EG-VEGF/prokineticin 1 and a peptide closely related to EG-VEGF, prokineticin 2, are cognate ligands of two orphan G-protein-coupled receptors designated ZAQ (=EG-VEGF/PK-R1) and I5E (=EG-VEGF/PK-R2). EG-VEGF/prokineticin 1 and prokineticin 2 induced a transient increase in intracellular calcium ion concentration ([Ca(2+)](i)) with nanomolar potency in Chinese hamster ovary (CHO) cells expressing EG-VEGF/PK-R1 and -R2 and bind to these cells with high affinity and with different receptor selectivity. EG-VEGF/prokineticins provoke rapid phosphorylation of p44/42 MAP kinase and DNA synthesis in the bovine adrenal capillary endothelial cells (BACE). The mRNAs of both EG-VEGF/PK-R1 and -R2 were expressed in BACE. The identification of the receptors for EG-VEGF/prokineticins may provide a novel molecular basis for the regulation of angiogenesis in endocrine glands.     

Role of KIFC3 motor protein in Golgi positioning and integration

KIFC3, a microtubule (MT) minus end-directed kinesin superfamily protein, is expressed abundantly and is associated with the Golgi apparatus in adrenocortical cells. We report here that disruption of the kifC3 gene induced fragmentation of the Golgi apparatus when cholesterol was depleted. Analysis of the reassembly process of the Golgi apparatus revealed bidirectional movement of the Golgi fragments in both wild-type and kifC3-/- cells. However, we observed a markedly reduced inwardly directed motility of the Golgi fragments in cholesterol-depleted kifC3-/- cells compared with either cholesterol-depleted wild-type cells or cholesterol-replenished kifC3-/- cells. These results suggest that (a) under the cholesterol-depleted condition, reduced inwardly directed motility of the Golgi apparatus results in the observed Golgi scattering phenotype in kifC3-/- cells, and (b) cholesterol is necessary for the Golgi fragments to attain sufficient inwardly directed motility by MT minus end-directed motors other than KIFC3, such as dynein, in kifC3-/- cells. Furthermore, we showed that Golgi scattering was much more drastic in kifC3-/- cells than in wild-type cells to the exogenous dynamitin expression even in the presence of cholesterol. These results collectively demonstrate that KIFC3 plays a complementary role in Golgi positioning and integration with cytoplasmic dynein.