Biochemical characterization of a thermophilic cellobiose 2-epimerase from a thermohalophilic bacterium, Rhodothermus marinus JCM9785

Cellobiose 2-epimerase (CE) reversibly converts glucose residue to mannose residue at the reducing end of β-1,4-linked oligosaccharides. It efficiently produces epilactose carrying prebiotic properties from lactose, but the utilization of known CEs is limited due to thermolability. We focused on thermoholophilic Rhodothermus marinus JCM9785 as a CE producer, since a CE-like gene was found in the genome of R. marinus DSM4252. CE activity was detected in the cell extract of R. marinus JCM9785. The deduced amino acid sequence of the CE gene from R. marinus JCM9785 (RmCE) was 94.2% identical to that from R. marinus DSM4252. The N-terminal amino acid sequence and tryptic peptide masses of the native enzyme matched those of RmCE. The recombinant RmCE was most active at 80 °C at pH 6.3, and stable in a range of pH 3.2-10.8 and below 80 °C. In contrast to other CEs, RmCE demonstrated higher preference for lactose over cellobiose.     

Synthesis, structure-activity relationships, and mechanism of action of anti-HIV-1 lamellarin α 20-sulfate analogues

Lamellarin α and six different types of lamellarin α 20-sulfate analogues were synthesized and their structure–activity relationships were investigated using a single round HIV-1 vector infection assay. All lamellarin sulfates having pentacyclic lamellarin core exhibited anti-HIV-1 activity at a 10 μM concentration range regardless of the number and position of the sulfate group. On the other hand, non-sulfated lamellarin α and ring-opened lamellarin sulfate analogues did not affect HIV-1 vector infection in similar concentrations. The lamellarin sulfates utilized in this study did not exhibit unfavorable cytotoxic effect under the concentrations tested (IC₅₀ > 100 μM). Confocal laser scanning microscopic analysis indicated that hydrophilic lamellarin sulfates were hardly incorporated in the cell. HIV-1 Env-mediated cell–cell fusion was suppressed by lamellarin sulfates. These results suggested that lamellarin sulfates have a novel anti-HIV-1 activity besides the previously reported integrase activity inhibition, possibly at a viral entry step of HIV-1 replication.     

A transmembrane glycoprotein, gp38, is a novel marker for immature hepatic progenitor cells in fetal mouse livers

Previously, we clarified the surface antigen profiles of hepatic progenitor cells (HPCs) in fetal liver tissue as the CD49f(+)CD45(-)Thy1(-) cell fraction. However, these cells were a heterogeneous cell population containing various stages of differentiation. This study aimed to detect more immature HPCs, using a novel surface antigen, gp38. After the collagenase digestion of fetal livers harvested from E13.5 to E18.5 fetal mice, HPCs were obtained and divided into two subpopulations using flow cytometry: gp38-positive HPCs, and gp38-negative HPCs. Both types of HPCs were characterized by immunocytochemistry and RT-PCR. The proliferative activity was compared by BrdU incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) assay. Furthermore, the comprehensive gene expression was investigated by DNA microarray. Both types of HPCs expressed alpha-fetoprotein. However, the gp38-positive HPCs derived from E13.5 fetal livers did not express albumin or cytokeratin 19, while the gp38-negative HPCs did. DNA microarray revealed that some genes related to the Wnt signal pathway were up-regulated in the gp38-positive HPCs. Furthermore, Wnt3a had a proliferative effect on the gp38-positive HPCs. In conclusion, the gp38-positive HPCs derived from fetal liver tissue until E13.5 could therefore be candidates for hepatic stem cells in the fetal liver.     

Spatial frequency-based analysis of mean red blood cell speed in single microvessels: investigation of microvascular perfusion in rat cerebral cortex

Background

Our previous study has shown that prenatal exposure to X-ray irradiation causes cerebral hypo-perfusion during the postnatal development of central nervous system (CNS). However, the source of the hypo-perfusion and its impact on the CNS development remains unclear. The present study developed an automatic analysis method to determine the mean red blood cell (RBC) speed through single microvessels imaged with two-photon microscopy in the cerebral cortex of rats prenatally exposed to X-ray irradiation (1.5 Gy).

Methodology/Principal Findings

We obtained a mean RBC speed (0.9±0.6 mm/sec) that ranged from 0.2 to 4.4 mm/sec from 121 vessels in the radiation-exposed rats, which was about 40% lower than that of normal rats that were not exposed. These results were then compared with the conventional method for monitoring microvascular perfusion using the arteriovenous transit time (AVTT) determined by tracking fluorescent markers. A significant increase in the AVTT was observed in the exposed rats (1.9±0.6 sec) as compared to the age-matched non-exposed rats (1.2±0.3 sec). The results indicate that parenchyma capillary blood velocity in the exposed rats was approximately 37% lower than in non-exposed rats.

Conclusions/Significance

The algorithm presented is simple and robust relative to monitoring individual RBC speeds, which is superior in terms of noise tolerance and computation time. The demonstrative results show that the method developed in this study for determining the mean RBC speed in the spatial frequency domain was consistent with the conventional transit time method.     

Phthalides from the rhizome of Ligusticum wallichii

Three phthalides, 3-butylidene-7-hydroxyphthalide, and cis and trans-6,7-dihydroxyligustilides, along with a dimeric phthalide wallichilide have been isolated from the hot water extract of the rhizome of Ligusticum wallichii. Their structures were established mainly on the basis of spectroscopic data. 3-Butyl-4,5-dihydro-3-hydroxy phthalide and adenosine were identified as active principles of the extract which are responsible for increase of coronary blood flow in the dog heart.     

Model platinum nucleobase and nucleoside complexes and antitumor activity: X-ray crystal structure of [PtIV(trans-1R,2R-diaminocyclohexane)trans-(acetate)2(9-ethylguanine)Cl]NO3.H2O

A series of platinum(II) and (IV) monoadducts of the type [Pt(II)(DACH)LCl]NO3 and [Pt(IV)(DACH)trans-(X)2LCl]NO3 (where DACH=trans-1R,2R-diaminocyclohexane, L=adenine, guanine, hypoxanthine, cytosine, adenosine, guanosine, inosine, cytidine, 9-ethylguanine (9-EtGua), or 1-methylcytosine and X=hydroxo or acetato ligand) have been synthesized and characterized by elemental analysis and by 1H and 195Pt nuclear magnetic resonance (NMR) spectroscopy. The crystal structure of the model nucleobase complex [Pt(IV)(trans-1R,2R-diaminocyclohexane)trans-(acetate)2(9-EtGua)Cl]NO3.H2O was determined using a single crystal X-ray diffraction method. The compound crystallized in the monoclinic space group P2(1), with a=10.446(2) A, b=22.906(5) A, c=10.978(2) A, Z=4, and R=0.0718, based upon the total of 11,724 collected reflections. In this complex, platinum had a slightly distorted octahedron geometry owing to the presence of a geometrically strained five-member ring. The two adjacent corners of the platinum plane were occupied by the two amino nitrogen of DACH, whereas, the other two equatorial positions occupied by chloride ion and 9-ethylguanine. The remaining two axial positions were occupied by the oxygen atoms of acetato ligands. The DACH ring was in a chair configuration. An intricate network of intermolecular hydrogen bonds held the crystal lattice together. Some of these synthesized models of DACH-Pt-DNA adducts have good in vitro cytotoxic activity against the cisplatin-sensitive human cancer ovarian A2780 cell line (IC50=1-8 microM). Interestingly, a substituted nucleobase (9-ethylguanine) adduct was over 6-fold more potent than regular adducts. The cross-resistance factor against the 44-fold cisplatin-resistant 2780CP/clone 16 cells was about 3-9; thus, the cytotoxicity of adducts was indicative of low potency, but the resistance factors were also substantially low. These results suggest that DNA adducts of DACH-Pt are cytotoxic with low cross-resistance.     

Black tea theaflavins suppress dioxin-induced transformation of the aryl hydrocarbon receptor

Dioxins cause various adverse effects through transformation of aryl hydrocarbon receptor (AhR). In this study, we investigated whether black tea extract and its components, theaflavins, suppress AhR transformation in vitro. First, we confirmed that black tea extract strongly suppressed AhR transformation compared to green and oolong tea, although the catechin contents did not change significantly among the extracts. Then we isolated four theaflavins as active compounds from black tea leaves. They suppressed 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AhR transformation in a dose-dependent manner. The IC(50) values of theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3,3'-digallate (Tfdg) were 4.5, 2.3, 2.2, and 0.7 muM, respectively. The suppressive effect of Tfdg was observed not only by pre-treatment but also by post-treatment. This suggests that theaflavins inhibit the binding of TCDD to the AhR and also the binding of the transformed AhR to the specific DNA-binding site as putative mechanisms.     

Characterization of structural and catalytic differences in rat intestinal alkaline phosphatase isozymes

To understand the differences between the rat intestinal alkaline phosphatase isozymes rIAP-I and rIAP-II, we constructed structural models based on the previously determined crystal structure for human placental alkaline phosphatase (hPLAP). Our models of rIAP-I and rIAP-II displayed a typical alpha/beta topology, but the crown domain of rIAP-I contained an additional beta-sheet, while the embracing arm region of rIAP-II lacked the alpha-helix, when each model was compared to hPLAP. The representations of surface potential in the rIAPs were predominantly positive at the base of the active site. The coordinated metal at the active site was predicted to be a zinc triad in rIAP-I, whereas the typical combination of two zinc atoms and one magnesium atom was proposed for rIAP-II. Using metal-depleted extracts from rat duodenum or jejunum and hPLAP, we performed enzyme assays under restricted metal conditions. With the duodenal and jejunal extract, but not with hPLAP, enzyme activity was restored by the addition of zinc, whereas in nonchelated extracts, the addition of zinc inhibited duodenal IAP and hPLAP, but not jejunal IAP. Western blotting revealed that nearly all of the rIAP in the jejunum extracts was rIAP-I, whereas in duodenum the percentage of rIAP-I (55%) correlated with the degree of AP activation (60% relative to that seen with jejunal extracts). These data are consistent with the presence of a triad of zinc atoms at the active site of rIAP-I, but not rIAP-II or hPLAP. Although no differences in amino acid alignment in the vicinity of metal-binding site 3 were predicted between the rIAPs and hPLAP, the His153 residue of both rIAPs was closer to the metal position than that in hPLAP. Between the rIAPs, a difference was observed at amino acid position 317 that is indirectly related to the coordination of the metal at metal-binding site 3 and water molecules. These findings suggest that the side-chain position of His153, and the alignment of Q317, might be the major determinants for activation of the zinc triad in rIAP-I.     

Comparative studies on the functional roles of N- and C-terminal regions of molluskan and vertebrate troponin-I

Vertebrate troponin regulates muscle contraction through alternative binding of the C-terminal region of the inhibitory subunit, troponin-I (TnI), to actin or troponin-C (TnC) in a Ca(2+)-dependent manner. To elucidate the molecular mechanisms of this regulation by molluskan troponin, we compared the functional properties of the recombinant fragments of Akazara scallop TnI and rabbit fast skeletal TnI. The C-terminal fragment of Akazara scallop TnI (ATnI(232-292)), which contains the inhibitory region (residues 104-115 of rabbit TnI) and the regulatory TnC-binding site (residues 116-131), bound actin-tropomyosin and inhibited actomyosin-tropomyosin Mg-ATPase. However, it did not interact with TnC, even in the presence of Ca(2+). These results indicated that the mechanism involved in the alternative binding of this region was not observed in molluskan troponin. On the other hand, ATnI(130-252), which contains the structural TnC-binding site (residues 1-30 of rabbit TnI) and the inhibitory region, bound strongly to both actin and TnC. Moreover, the ternary complex consisting of this fragment, troponin-T, and TnC activated the ATPase in a Ca(2+)-dependent manner almost as effectively as intact Akazara scallop troponin. Therefore, Akazara scallop troponin regulates the contraction through the activating mechanisms that involve the region spanning from the structural TnC-binding site to the inhibitory region of TnI. Together with the observation that corresponding rabbit TnI-fragment (RTnI(1-116)) shows similar activating effects, these findings suggest the importance of the TnI N-terminal region not only for maintaining the structural integrity of troponin complex but also for Ca(2+)-dependent activation.     

Characterization of the organic cation transporter SLC22A16: a doxorubicin importer

Specific efflux transporters, such as P-glycoprotein, have been shown to confer drug resistance by decreasing the intracellular accumulation of anticancer drugs. Understanding influx transporters, as well as efflux transporters, is essential to overcome this resistance. We report the expression profile and pharmacological characterization of an organic cation transporter, SLC22A16. The results of our experiments indicate that SLC22A16 is a mediator of doxorubicin uptake in cancer cells. Quantitative real-time RT-PCR analyses show that SLC22A16 is expressed in primary samples taken from patients with acute leukemia. Xenopus oocytes injected with SLC22A16 cRNA import doxorubicin, a widely used anticancer drug for hematological malignancies, in a saturable and dose-dependent manner. The apparent Km value for doxorubicin import was 5.2+/-0.4 microM. In cytotoxic assays, stable transfectants of leukemic Jurkat cells overexpressing SLC22A16 cells became significantly more sensitive to doxorubicin (2 microM) treatment. Characterization of SLC22A16 will help in designing novel therapies targeting hematological malignancies.