Biomedical subjects

Li Li

Publications and source records attributed to Li Li.

At least 487 records · Page 27Linked to original sources

PubMed2003

[Effects of long-term installation of intrauterine devices on intrauterine microenvironment].

OBJECTIVES: To study the effect of long-term installation of intrauterine devices (IUD) on the intrauterine microenvironment. METHODS: Eighty-nine healthy 26 - 50-year-old women undergoing physical examination or having their IUD removed were recruited. Among them 62 had used IUDs, including 32 inert IUD (I-IUD) and 30 copper releasing IUD (T-IUD), for 5 - 20 years, and 27 women without installation of IUD were used as controls. In the 3rd to 13th day of menstrual cycle, 3 ml of irrigation of intrauterine cavity were collected to examine the concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin 2 (IL-2) and secretary IgA (SIgA) by radioimmunoassay (RIA). Hysteroscopy was used to obtain 3 pieces of endometrium at the place where the IUD was attached for each subject. Three pieces of endometrium were curretted from each control during the proliferative stage. Thirty specimens of endometrium, including 10 cases with I-IUD, 10 cases with T-IUD, and 10 control cases were used to examine the distribution of T cell subset by immunohistochemistry. Fifteen specimens of endometrium, including 6 cases with I-IUD, 6 cases with T-IUD, and 3 control cases, were used to examine the endometrial ultrastructure by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Twenty-five specimens of endometrium, including 10 cases with I-IUD, 10 cases with T-IUD, and 5 control cases, were used to detect the expression and mutation of P16, P53 and K-ras by PCR-SSCP technique. RESULTS: There was no significant difference in TNF-alpha level in the irrigation among the three groups (P > 0.05). IL-2 and SIgA levels (0.39 +/- 0.18) microg/L and (2000 +/- 1224) microg/L respectively) in the T-IUD group were significantly lower than those in the control group (0.96 +/- 0.15) microg/L and (3377 +/- 1906) microg/L respectively, both (P < 0.05). There was a significant difference in the population of T lymphocytes (CD(8)(+) and CD(4)(+) lymphocytes) between the T-IUD group and control group (P < 0.05). SEM and TEM showed no necrosis and atypia in the endometrial cells of I-IUD group and T-IUD group. There was no positive expression of gene p16, p53 and K-ras in the endometrium of the three groups. CONCLUSION: It is safe and effective using I-IUD or T-IUD for a long time, however, T-IUD has some effects on intrauterine local immune function.

Adult↗

PubMed2003

Hypercapnic acidosis activates KATP channels in vascular smooth muscles.

ATP-sensitive K+ channels (KATP) couple intermediary metabolism to cellular activity, and may play a role in the autoregulation of vascular tones. Such a regulation requires cellular mechanisms for sensing O2, CO2, and pH. Our recent studies have shown that the pancreatic KATP isoform (Kir6.2/SUR1) is regulated by CO2/pH. To identify the vascular KATP isoform(s) and elucidate its response to hypercapnic acidosis, we performed these studies on vascular smooth myocytes (VSMs). Whole-cell and single-channel currents were studied on VSMs acutely dissociated from mesenteric arteries and HEK293 cells expressing Kir6.1/SUR2B. Hypercapnic acidosis activated an inward rectifier current that was K+-selective and sensitive to levcromakalim and glibenclamide with unitary conductance of approximately 35pS. The maximal activation occurred at pH 6.5 to 6.8, and the current was inhibited at pH 6.2 to 5.9. The cloned Kir6.1/SUR2B channel responded to hypercapnia and intracellular acidification in an almost identical pattern to the VSM current. In situ hybridization histochemistry revealed expression of Kir6.1/SUR2B mRNAs in mesenteric arteries. Hypercapnia produced vasodilation of the isolated and perfused mesenteric arteries. Pharmacological interference of the KATP channels greatly eliminated the hypercapnic vasodilation. These results thus indicate that the Kir6.1/SUR2B channel is a critical player in the regulation of vascular tones during hypercapnic acidosis.

ATP-Binding Cassette Transporters↗

PubMed2003

Synthesis of phosphoryl-tethered beta-cyclodextrins and their molecular and chiral recognition thermodynamics.

Two novel phosphoryl-bridged bis- and tris(beta-cyclodextrin)s of different tether lengths, i.e., bis[m-(N-(6-cyclodextryl)-2-aminoethylaminosulfonyl)phenyl]-m-(chlorosulfonyl)phenylphosphine oxide (5) and tris[m-(N-(6-cyclodextryl)-8-amino-3,6-diazaoctylaminosulfonyl)phenyl]phosphine oxide (6), have been synthesized by reactions of 6-oligo(ethylenediamino)-6-deoxy-beta-cyclodextrins with tris[m-(chlorosulfonyl)phenyl]phosphine oxide. The complex stability constants (K(S)), standard molar enthalpy (Delta H degrees ), and entropy changes (Delta S degrees ) were determined at 25 degrees C for the inclusion complexation of phosphoryl-modified bis- and tris-cyclodextrins (5 and 6, respectively), mono[6-O-(ethoxyhydroxyphosphoryl)]-beta-cyclodextrin (2), mono[6-O-(diethylamino-ethoxyphosphoryl)]-beta-cyclodextrin (3), and mono[6-O-(diphenoxyphosphoryl)]-beta-cyclodextrin (4) with representative alicyclic and N-Cbz-D/L-alanine guests in 0.1 M phosphate buffer solution at pH 7.2 by means of titration microcalorimetry. The thermodynamic parameters obtained indicate that the charge-dipole interaction between the phosphoryl moiety and the negatively charged guests, as well as the conformational difference of modified beta-cyclodextrins in aqueous solution, significantly contribute to the inclusion complexation and the enhanced chiral discrimination. The interactions and binding modes between the hosts and chiral guests were further studied by two-dimensional NMR spectroscopy to elucidate the influence of the structural features of hosts on their increased chiral recognition ability and to establish the correlation between the conformation of the resulting complexes and the thermodynamic parameters obtained.

Alanine↗

PubMed2003

Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells.

Human embryonic stem cells (hESCs) randomly differentiate into multiple cell types during embryoid body (EB) development. To date, characterization of specific factors capable of influencing hematopoietic cell fate from hESCs remains elusive. Here, we report that the treatment of hESCs during EB development with a combination of cytokines and bone morphogenetic protein-4 (BMP-4), a ventral mesoderm inducer, strongly promotes hematopoietic differentiation. Hematopoietic progenitors of multiple lineages were generated from EBs and were found to be restricted to the population of progeny expressing cell surface CD45. Addition of BMP-4 had no statistically significant effect on hematopoietic differentiation but enabled significant enhancement in progenitor self-renewal, independent of cytokine treatment. Hematopoietic commitment was characterized as the temporal emergence of single CD45+ cells first detectable after day 10 of culture and was accompanied by expression of hematopoietic transcription factors. Despite the removal of cytokines at day 10, hematopoietic differentiation of hESCs continued, suggesting that cytokines act on hematopoietic precursors as opposed to differentiated hematopoietic cells. Our study establishes the first evidence for the role of cytokines and BMP-4 in promoting hematopoietic differentiation of hESC lines and provides an unprecedented system to study early developmental events that govern the initiation of hematopoiesis in the human.

Bone Morphogenetic Protein 4↗

PubMed2003

An initiator and its flanking elements function as a core promoter driving transcription of the Hepatopoietin gene.

Hepatopoietin (HPO)/ALR (augmenter of liver regeneration), as a versatile hepatotrophic growth factor and a cellular thiol oxidase, is involved in a wide variety of basic processes of various tissues, especially in liver and testis. Here, we studied the regulation of HPO gene expression. By sequential deletion of the HPO 5'-flanking region, the minimal promoter of the HPO gene was shown to span positions -22 to +42 relative to the transcriptional start point. Further transfection assay and mutation analysis showed that the core promoter contains a functional initiator. Interestingly, three tandem repeats of a CTGGAGGC element, surrounding the transcription start site and bound by specific nuclear factors, were found to be pivotal for the promoter activity. This initiator flanking element functions in an initiator-dependent fashion and is present in many initiator-containing genes. Taken together, our findings revealed that the initiator-like element and its flanking repeat sequence comprise a core promoter and drive the transcriptional initiation of the HPO gene in a combinatorial manner. The HPO gene promoter might represent a novel architecture for core promoters.

Base Sequence↗

PubMed2003

Cytochrome b5 reductase and cytochrome b5 support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test.

With CYP2E1 in vitro both the first and the second electron of the catalytic cycle can come from cytochrome b(5) via either NADPH-cytochrome P450 reductase or NADH-cytochrome b(5) reductase, and the presence of cytochrome b(5) stimulates CYP2E1 turnover both in vitro and in vivo. To determine whether electron input via the NADH-dependent pathway was similarly functional in whole cells and necessary for the stimulation by cytochrome b(5), we constructed five plasmids designed to express human CYP2E1 in various combinations with cytochrome b(5) reductase, cytochrome b(5), and cytochrome P450 reductase. CYP2E1 activity in Salmonella typhimurium cells transformed with each plasmid was assessed by mutagenic reversion frequency in the presence of dimethylnitrosamine. A fivefold increase in reversion frequency when cytochrome b(5) was coexpressed with P450 reductase was abolished by disruption of heme-binding in cytochrome b(5) by site-directed mutagenesis (His68Ala), suggesting that electron transfer to cytochrome b(5) was necessary for the stimulation. Addition of cytochrome b(5) reductase to the cytochrome b(5)/P450 reductase coexpression plasmid did not further increase the stimulation by cytochrome b(5), but b(5) reductase could support CYP2E1 activity in the absence of P450 reductase at a level equivalent to that obtained with just CYP2E1 and P450 reductase. Neither cytochrome b(5) reductase nor cytochrome b(5) alone could support CYP2E1 activity. These results demonstrate that the cytochrome b(5) reductase/cytochrome b(5) pathway can support CYP2E1 activity in bacterial cells.

Cytochrome P-450 CYP2E1↗

PubMed2003

A novel anti-CD146 monoclonal antibody, AA98, inhibits angiogenesis and tumor growth.

The goal of our study was to raise monoclonal antibodies (mAbs) against endothelial cell-surface proteins specific for tumor vasculature. Here, we describe the generation and intensive characterization of mAb AA98, including its functional properties and its antigen identification. In our study, an enhanced mAb AA98 immunoreactivity was observed on stimulated human umbilical vein endothelial cells (HUVECs). In addition, mAb AA98 showed remarkably restricted immunoreactivity against intratumoral neovasculature compared with blood vessels of normal tissues. We identified the AA98 antigen as human CD146, an adhesion molecule belonging to the immunoglobulin superfamily. Data from in vitro experiments imply structural and signaling functions for endothelial CD146; however, the role of CD146 in vivo is largely unknown. Here, we show that mAb AA98 displays antiangiogenic properties in vitro and in vivo. Proliferation and migration of HUVECs were inhibited by mAb AA98 as was angiogenesis in chicken chorioallantoic membrane (CAM) assays and tumor growth in 3 xenografted human tumor models in mice. Our data provide new insights into the function of CD146 on endothelial cells, validate CD146 as a novel target for antiangiogenic agents, and demonstrate that mAb AA98 has potential as a diagnostic and therapeutic agent in vascular and cancer biology.

Animals↗

PubMed2003

Synthesis of novel bis(beta-cyclodextrin)s and metallobridged bis(beta-cyclodextrin)s with 2,2'-diselenobis(benzoyl) tethers and their molecular multiple recognition with model substrates.

To investigate quantitatively the cooperative binding ability of beta-cyclodextrin dimers, a series of bridged bis(beta-cyclodextrin)s with 2,2'-diselenobis(benzoyl) spacer connected by different lengths of oligo(ethylenediamine)s (2-5) and their platinum(IV) complexes (6-9) have been synthesized and their inclusion complexation behavior with selected substrates, such as Acridine Red, Neutral Red, Brilliant Green, Rhodamine B, ammonium 8-anilino-1-naphthalenesulfonate, and 6-p-toluidino-2-naphthalenesulfonic acid, were investigated by means of ultraviolet, fluorescence, fluorescence lifetime, circular dichroism, and 2D-NMR spectroscopy. The spectral titrations have been performed in aqueous phosphate buffer solution (pH 7.20) at 25 degrees C to give the complex stability constants (K(S)) and Gibbs free energy changes (-DeltaG degrees ) for the inclusion complexation of hosts 2-9 with organic dyes and other thermodynamic parameters (DeltaH degrees and TDeltaS degrees ) for the inclusion complexation of 2-5with fluorescent dyes ANS and TNS. The results obtained indicate that beta-cyclodextrin dimers 2-5 can coordinate with one or two platinum(IV) ions to form 1:1 or 1:2 stoichiometry metallobridged bis(beta-cyclodextrin)s. As compared with parent beta-cyclodextrin (1) and bis(beta-cyclodextrin)s 2-5, metallobridged bis(beta-cyclodextrin)s 6-9 can further switch the original molecular binding ability through the coordinating metal to orientate two beta-cyclodextrin cavities and an additional binding site upon the inclusion complexation with model substrates, giving the enhanced binding constants K(S) for both ANS and TNS. The tether length between two cyclodextrin units plays a crucial role in the molecular recognition with guest dyes. The binding constants for TNS decrease linearly with an increase in the tether length of dimeric beta-cyclodextrins. The Gibbs free energy change (-DeltaG degrees ) for the unit increment per ethylene is 0.32 kJ.mol(-)(1) for TNS. Thermodynamically, the higher complex stabilities of both ANS and TNS upon the inclusion complexation with 2-5 are mainly contributed to the favorable enthalpic gain (-DeltaH degrees ) by the cooperative binding of one guest molecule in the closely located two beta-cyclodextrin cavities as compared with parent beta-cyclodextrin. The molecular binding ability and selectivity of organic dyes by hosts 1-9 are discussed from the viewpoints of the multiple recognition mechanism and the size/shape-fitting relationship between host and guest.

Binding Sites↗

PubMed2003

Human SM22 alpha BAC encompasses regulatory sequences for expression in vascular and visceral smooth muscles at fetal and adult stages.

The SM22 alpha gene has widely been used to study the regulatory mechanisms of smooth muscle cell (SMC) gene expression during cardiovascular development. To determine the regulatory mechanisms for the evolutionarily conserved human SM22 alpha (hSM22 alpha) gene, we demonstrated that 445 bp upstream DNA sequences of hSM22 alpha gene exhibited a high transcriptional activity in arterial SMC, not in venous nor in visceral SMCs during embryogensis. However, this promoter was gradually turned off in adulthood. Inclusion of the first intron in this promoter suppressed the promoter activity in pulmonary trunk arterial SMCs, whereas the expression in other systemic vasculature remained similar to that of the hSM22-445 promoter during the fetal and adult stages. To determine whether additional sequences are required for SM22 alpha expression in all subtypes of SMCs, we examined the expression of a bacterial artificial chromosome containing the hSM22 alpha locus in transgenic mice. The hSM22 alpha transgene showed similar developmental expression patterns as the endogenous mouse SM22 alpha gene, suggesting that this bacterial artificial chromosome contains essential regulatory sequences for its expression in arterial, venous, and visceral tissues during development.

Animals↗

PubMed2003

PTEN regulation of neural development and CNS stem cells.

Even though phosphorylation of phosphatidylinositols by phosphoinositide 3-kinase (PI3K) has an important and pervasive role in the nervous system, there is little known about the phosphatases that reverse this reaction. Such a phosphatase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), was cloned as a tumor suppressor for gliomas. PTEN is expressed in most, if not all, neurons and is localized in the nucleus and cytoplasm. Recently, a series of papers using PTEN conditional knockouts has greatly extended our knowledge of PTEN's role during development. Loss of PTEN results in disorganization of the brain, probably due to a flaw in cell migration. In addition, there is a gradual increase in the size of neuronal soma, mimicking Lhermitte-Duclos disease. Recent experiments in our laboratory with adult PTEN +/- mice demonstrate that PTEN regulates migration of precursor cells in the subventricular zone to the olfactory bulb. We also found that PTEN haploinsufficiency can protect precursor cells from apoptosis in response to oxidative stress. Collectively, these studies demonstrate that PTEN does much more than suppressing tumors. It is a master regulator in developing and adult brain.

Animals↗

PubMed2003

ToxoDB: accessing the Toxoplasma gondii genome.

ToxoDB (http://ToxoDB.org) provides a genome resource for the protozoan parasite Toxoplasma gondii. Several sequencing projects devoted to T. gondii have been completed or are in progress: an EST project (http://genome.wustl.edu/est/index.php?toxoplasma=1), a BAC clone end-sequencing project (http://www.sanger.ac.uk/Projects/T_gondii/) and an 8X random shotgun genomic sequencing project (http://www.tigr.org/tdb/e2k1/tga1/). ToxoDB was designed to provide a central point of access for all available T. gondii data, and a variety of data mining tools useful for the analysis of unfinished, un-annotated draft sequence during the early phases of the genome project. In later stages, as more and different types of data become available (microarray, proteomic, SNP, QTL, etc.) the database will provide an integrated data analysis platform facilitating user-defined queries across the different data types.

Animals↗

PubMed2003

PlasmoDB: the Plasmodium genome resource. A database integrating experimental and computational data.

PlasmoDB (http://PlasmoDB.org) is the official database of the Plasmodium falciparum genome sequencing consortium. This resource incorporates the recently completed P. falciparum genome sequence and annotation, as well as draft sequence and annotation emerging from other Plasmodium sequencing projects. PlasmoDB currently houses information from five parasite species and provides tools for intra- and inter-species comparisons. Sequence information is integrated with other genomic-scale data emerging from the Plasmodium research community, including gene expression analysis from EST, SAGE and microarray projects and proteomics studies. The relational schema used to build PlasmoDB, GUS (Genomics Unified Schema) employs a highly structured format to accommodate the diverse data types generated by sequence and expression projects. A variety of tools allow researchers to formulate complex, biologically-based, queries of the database. A stand-alone version of the database is also available on CD-ROM (P. falciparum GenePlot), facilitating access to the data in situations where internet access is difficult (e.g. by malaria researchers working in the field). The goal of PlasmoDB is to facilitate utilization of the vast quantities of genomic-scale data produced by the global malaria research community. The software used to develop PlasmoDB has been used to create a second Apicomplexan parasite genome database, ToxoDB (http://ToxoDB.org).

Animals↗

PubMed2003

Endothelial lamellar keratoplasty using an artificial anterior chamber and a microkeratome.

OBJECTIVE: To compare postoperative astigmatic change and graft stability using 2 different donor button diameters in endothelial lamellar keratoplasty to treat corneal endothelial failure. METHODS: A 200- micro m-thick corneal flap keratectomy was performed in human donor corneoscleral rims (n = 20; 10 donors and 10 recipients) using an artificial anterior chamber and a manual microkeratome (ALTK System; Moria USA, Doylestown, Pa). After flap reflection, stromal bed trephination was performed to obtain a disc consisting of posterior stroma, Descemet membrane, and endothelium. Host beds of 7.0 mm and 7.25-mm (n = 5) or 7.50-mm (n = 5) donor buttons were obtained using a freehand trephine. The graft was secured with 8 interrupted sutures (10-0 nylon) in the stromal bed. The flap was sutured with 3 interrupted sutures. Transplanted corneas were submitted to increasing intrachamber pressures to detect graft stability, and preoperative and postoperative videokeratographic data were recorded to assess astigmatic change. RESULTS: The mean (SD) postoperative astigmatic change was 1.14 (3.17) diopters (D) in the 7.25-mm donor button group and 2.27 (1.77) D in the 7.50-mm donor button group (P =.69). Mean (SD) resisted pressures of 75.4 (44.81) mm Hg and 100.4 (46.86) mm Hg were observed in the 7.25-mm and 7.50-mm groups, respectively (P =.54). CONCLUSION: Both donor button sizes exhibited similar graft stability and astigmatic postoperative change in this experimental model. CLINICAL RELEVANCE: As endothelial lamellar keratoplasty becomes further developed as a clinical alternative to penetrating keratoplasty, this laboratory model system should be useful in evaluating different mechanical factors that contribute to graft success.

Adult↗

PubMed2003

Alternative promoters and polyadenylation regulate tissue-specific expression of Hemogen isoforms during hematopoiesis and spermatogenesis.

Hemogen is a nuclear protein encoded by HEMGN (also known as hemogen in mouse, EDAG in human and RP59 in rat). It is considered to be a hematopoiesis-specific gene that is expressed during the ontogeny of hematopoiesis. Herein, we characterize two distinct splicing variants of HEMGN mRNA with restricted expression to hematopoietic cells and to round spermatids in the testis, respectively. Expression of the testis-specific HEMGN mRNA (HEMGN-t) is developmentally regulated and is concurrent with the first wave of meiosis in prepuberal mice. Sequence analysis reveals that HEMGN-t and the hematopoietic HEMGN mRNA (HEMGN-h) share a common coding sequence with distinct 5' and 3' untranslated regions and that these two isoforms are transcribed from the same gene locus, HEMGN, through the use of alternative promoters and polyadenylation sites. Thus, HEMGN expression exemplifies a developmental regulatory mechanism by which the diversification of gene expression is achieved through using distinct regulatory sequences in different cell types. Moreover, the existence of a testis-specific isoform of HEMGN suggests a role in spermatogenesis. Finally, fluorescence in situ hybridization demonstrates that HEMGN is localized to chromosome 4 A5-B2 in mouse and to chromosome 9q22 in human, which is a region known to harbor a cluster of leukemia breakpoints.

3' Untranslated Regions↗

PubMed2003

Profiling of genes expressed by PTEN haploinsufficient neural precursor cells.

PTEN is a lipid phosphatase, and PTEN mutations are associated with gliomas, macrocephaly, and mental deficiencies. We have used PTEN +/- and PTEN +/+ mice to prepare subventricular zone (SVZ) precursor cells. Using DNA microarrays, we compared the expression profiles of PTEN +/+ and PTEN +/- cells and identified 91 differentially expressed genes in PTEN +/- precursor cells. Many of the PTEN-regulated genes are involved with signaling, cytoskeleton, extracellular matrix, metabolism, and transcription factors. Some of these changes are likely mediated by the transcription factor, HIF-1. We confirmed a subset of these changes by real-time PCR. In addition, we examined protein levels for two of the PTEN-up-regulated genes, vascular endothelial growth factor (VEGF) and doublecortin (DCX). PTEN haploinsufficiency increases immunostaining for VEGF for both cultured precursor cells and sections of the SVZ. PTEN haploinsufficiency shifted most of the DCX-positive cells from the SVZ to the olfactory bulb. These observations indicate that even a small decrease in PTEN levels results in substantial changes in gene expression and precursor cell function.

Animals↗

PubMed2003

Interaction of Smad3 and SRF-associated complex mediates TGF-beta1 signals to regulate SM22 transcription during myofibroblast differentiation.

Myofibroblasts play important roles in a variety of developmental and pathological processes, such as vascular remodeling, atherosclerosis and wound healing. In this study, we used the TGF-beta1-treated 10T1/2 cells as an in vitro model to understand how Smad-mediated TGF-beta1 signals regulate SM22 promoter transcription during myofibroblast differentiation. We found that TGF-beta1 transiently induces SRF and SM22 transcription, and that this process is accompanied by transient increases of SRF and Smad3 binding to the SM22 promoter. Interestingly, Smad3, not Smad2, is the primary mediator for TGF-beta1-induced transactivation of the SM22 promoter, while Smad6 and Smad7 repress such a transactivation. Smad3 can bind to a Smad-binding element (SBE) in the first exon of SM22, and directly associate with the SRF complex in response to TGF-beta1 treatment. Moreover, Smad3 and I-Smads regulate the SM22 promoter through CArG box-dependent transcription using dominant-negative SRF mutants and SRF-VP16. Although SBE as well as CArG boxes and TGF-beta control element are all important for the SM22 promoter activities, the promoters with mutations at either one or all of them still respond to TGF-beta1 treatment. Consistently, TGF-beta1 stimulates SM22 transcription in Smad3 null mouse embryonic fibroblasts. These findings provide the first evidence that Smad3 directly links TGF-beta1 signaling to an SRF-associated regulatory network in controlling SM22 transcription; it also implies that TGF-beta1 regulates the SM22 promoter via Smad3-dependent and Smad3-independent pathways.

Animals↗

PubMed2003

Selective binding of chiral molecules of cinchona alkaloid by beta- and gamma-cyclodextrins and organoselenium-bridged bis(beta-cyclodextrin)s.

The inclusion complexation behavior of chiral members of cinchona alkaloid with beta- and gamma-cyclodextrins (1 and 2) and 6,6(')-trimethylenediseleno-bridged bis(beta-cyclodextrin) (3) was assessed by means of fluorescence and 2D-NMR spectroscopy. The spectrofluorometric titrations have been performed in aqueous buffer solution (pH 7.20) at 25.0 degrees C to determine the stability constants of the inclusion complexation of 1-3 with guest molecules (i.e., cinchonine, cinchonidine, quinine, and quinidine) in order to quantitatively investigate the molecular selective binding ability. The stability constants of the resulting complexes of 2 with guest molecules are larger than that of 1. As a result of cooperative binding, the stability constants of inclusion complexation of dimeric beta-cyclodextrin 3 with cinchonidine and cinchonine are higher than that of parent 1 by factor of 4.5 and 2.4, respectively. These results are discussed from the viewpoint of the size-fit and geometric complementary relationship between the host and guest.

Binding Sites↗

PubMed2003

Hydrolysis of fibrinogen and plasminogen by immobilized earthworm fibrinolytic enzyme II from Eisenia fetida.

Earthworm fibrinolytic enzyme II (EFE-II) from Eisenia fetida has a broad hydrolytic specificity for peptide bonds. Our experiments show that EFE-II can hydrolyze the specific chromogenic substrates of thrombin (Chromozym TH), trypsin (Chromozym TRY) and elastase (Chromozym ELA). The Michaelis-Menten constant (K(m)) for Chromozym ELA (approximately 245 microM) is much higher than those for the thrombin (approximately 90 microM) and trypsin (approximately 60 microM) substrates. On the other hand, EFE-II is inhibited most strongly by soybean trypsin inhibitor (SBTI), and weakly inhibited by elastinal, suggesting that EFE-II has a trypsin-like activity. Degradation of plasminogen (PLg) and fibrinogen by EFE-II was investigated after EFE-II had been immobilized onto 1,1'-carboryl-diimidazole (CDI)-activated Sepharose CL-6B. The immobilized EFE-II has 55-60% activity of the native enzyme with a higher thermal and pH resistance. EFE-II cleaves PLg at four hydrolytic sites: Lys(77)-Arg(78), Arg(342)-Met(343), Ala(444)-Ala(445) and Arg(557)-Ile(558). The site Arg(557)-Ile(558) is also recognized and cleaved by tissue plasminogen activator (t-PA) and urokinase (UK), producing active plasmin. Cleaving Ala(444)-Ala(445) released mini-plasmin with secondary activity to hydrolyze fibrin. Immobilized EFE-II degrades not only the Aalpha chain of fibrinogen in the C-terminal region (like human neutrophil elastase, HNE), but also in the N-terminal region at the Val(21)-Glu(22) site.

Amino Acid Sequence↗