Ligand Binding and Crystal Structures of the Substrate-Binding Domain of the ABC Transporter OpuA

Background

The ABC transporter OpuA from Lactococcus lactis transports glycine betaine upon activation by threshold values of ionic strength. In this study, the ligand binding characteristics of purified OpuA in a detergent-solubilized state and of its substrate-binding domain produced as soluble protein (OpuAC) was characterized.

Principal Findings

The binding of glycine betaine to purified OpuA and OpuAC (K_D = 4–6 µM) did not show any salt dependence or cooperative effects, in contrast to the transport activity. OpuAC is highly specific for glycine betaine and the related proline betaine. Other compatible solutes like proline and carnitine bound with affinities that were 3 to 4 orders of magnitude lower. The low affinity substrates were not noticeably transported by membrane-reconstituted OpuA. OpuAC was crystallized in an open (1.9 Å) and closed-liganded (2.3 Å) conformation. The binding pocket is formed by three tryptophans (Trp-prism) coordinating the quaternary ammonium group of glycine betaine in the closed-liganded structure. Even though the binding site of OpuAC is identical to that of its B. subtilis homolog, the affinity for glycine betaine is 4-fold higher.

Conclusions

Ionic strength did not affect substrate binding to OpuA, indicating that regulation of transport is not at the level of substrate binding, but rather at the level of translocation. The overlap between the crystal structures of OpuAC from L.lactis and B.subtilis, comprising the classical Trp-prism, show that the differences observed in the binding affinities originate from outside of the ligand binding site.     

Effect of bread containing resistant starch on postprandial blood glucose levels in humans

We examined the inhibitory effect of a single ingestion of bread containing resistant starch (bread containing about 6 g of resistant starch derived from tapioca per 2 slices) (test food) on the postprandial increase in blood glucose in male and female adults with a fasting blood glucose level between 100 and 140 mg/dl. Bread not containing resistant starch (placebo) was used as the control.The study was conducted in 20 subjects (9 men and 11 women with a mean age of 50.5+/-7.5 years) using the crossover method, with a single ingestion of either bread containing resistant starch or the placebo. Blood glucose and insulin were measured before ingestion, and at 0.5, 1, 1.5, and 2 h after ingestion. The blood glucose level before ingestion was stratified into a borderline group (blood glucose level >/= 111 mg/dl) and a normal group (blood glucose level 相似文献   

Synthesis of 6-Sulfur Analogues of Oxanosine and Closely Related Derivatives Thereof

Abstract

Novel β-D-ribofuranosides having a 5-substituted imidazo [4,5-d] [1,3]thiazine ring, including the S⁶-congener 3 of oxanosine 2, were synthesized for screening their anticancer and antiviral activities.     

Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells

The eukaryotic sliding DNA clamp, proliferating cell nuclear antigen (PCNA), is essential for DNA replication and repair synthesis. In order to load the ring-shaped, homotrimeric PCNA onto the DNA double helix, the ATPase activity of the replication factor C (RFC) clamp loader complex is required. Although the recruitment of PCNA by RFC to DNA replication sites has well been documented, our understanding of its recruitment during DNA repair synthesis is limited. In this study, we analyzed the accumulation of endogenous and fluorescent-tagged proteins for DNA repair synthesis at the sites of DNA damage produced locally by UVA-laser micro-irradiation in HeLa cells. Accumulation kinetics and in vitro pull-down assays of the large subunit of RFC (RFC140) revealed that there are two distinct modes of recruitment of RFC to DNA damage, a simultaneous accumulation of RFC140 and PCNA caused by interaction between PCNA and the extreme N-terminus of RFC140 and a much faster accumulation of RFC140 than PCNA at the damaged site. Furthermore, RFC140 knock-down experiments showed that PCNA can accumulate at DNA damage independently of RFC. These results suggest that immediate accumulation of RFC and PCNA at DNA damage is only partly interdependent.     

ADVENTIVE EMBRYOGENESIS IN CITRUS I. THE OCCURRENCE OF ADVENTIVE EMBRYOS WITHOUT POLLINATION OR FERTILIZATION

Anatomical studies of unfertilized undeveloped seeds from open- and control-pollinated fruits of ten facultative apomictic Citrus cultivars were carried out with the aid of light and epifluorescence microscopes. With or without pollination, adventive embryos autonomously developed at all positions in the nucellus in all cultivars. The adventive embryos initiated at the chalazal end of the nucellus were more vigorous than those initiated at the micropylar end. Because of the lack of endosperm and poor seed development, however, all adventive embryos within the unfertilized seeds terminated their development at the globular or early cotyledonary stages and were unable to germinate under natural conditions. The capability of unfertilized seeds to develop varied from species to species. Growth of the adventive embryos was dependent on nucellus size, but the growth rate of adventive embryos relative to nucellus size was different in different species. Neither pollination, fertilization nor subsequent zygote and endosperm development further stimulated adventive embryo initiation. Conversely, pollination and subsequent fertilization of other seeds in the same fruit slightly, but significantly, suppressed adventive embryo growth in the unfertilized seeds. These facts concerning adventive embryogenesis in unfertilized seeds indicate that neither pollination nor fertilization is essential for in vivo adventive embryogenesis and that normal endosperm is necessary for perfect development of adventive embryos initiated only in the micropylar half of the nucellus.     

Development of a novel tocopheryl ester for suppression of lipid accumulation without cytotoxicity by optimization of dicarboxylic ester moiety

Tocopheryl succinate (Tsuc) is a succinic acid ester of the well-known antioxidant α-tocopherol (T). Tsuc exhibits various biological activities, including tumor growth suppression via activation of cell signaling and prevention of lipid accumulation in mouse adipocyte 3T3-L1 cells. The latter findings suggest that Tsuc may be a drug candidate for the treatment of obesity. However, Tsuc was found to induce apoptosis of normal cells (in addition to cancer cells), demonstrating the need to reduce the cytotoxicity of Tsuc without losing the suppression effect on lipid accumulation. Based on our previous findings, we focused on the ester structure of Tsuc for controlling cytotoxicity. Herein, we examined the cytotoxicity and lipid accumulation suppression effect of various T ester derivatives. We found that the terminal carboxylic group is necessary for suppression of lipid accumulation. We synthesized tocopheryl glutarate (Tglu) and tocopheryl adipate (Tadi) by elongation of carbon atoms 1 and 2 of the dicarboxylic moiety, respectively. Tglu and Tadi did not show any cytotoxicity, and both esters suppressed lipid accumulation, although their suppression activities were weaker than that of Tsuc. Tadi showed a more potent lipid accumulation inhibitory effect than Tglu. Although Tadi inhibited lipogenesis and promoted lipolysis, lipolysis was induced at lower concentrations than inhibition of lipogenesis, suggesting that Tadi mainly affects lipolysis. Taken together, we succeeded in the reduction of cytotoxicity, without loss of the suppression effect on lipid accumulation, by elongation of the dicarboxylic moiety of Tsuc. Tadi may be a promising candidate as an anti-obesity drug.     

Function of Pax1 and Pax9 in the sclerotome of medaka fish

We examined the expression and functions of Pax1 and Pax9 in a teleost fish, the medaka Oryzias latipes. While Pax1 and Pax9 show distinct expression in the sclerotome in amniotes, we could not detect the differential expression of Pax1 and Pax9 in the developing sclerotome of the medaka. Furthermore, unlike the mouse, in which Pax1 is essential for development of the vertebral body, and where the neural arch is formed independent of either Pax1 or Pax9, our morpholino knockdown experiments revealed that both Pax1 and Pax9 are indispensable for the development of the vertebral body and neural arch. Therefore, we conclude that after gene duplication, Pax1 and Pax9 subfunctionalize their roles in the sclerotome independently in teleosts and amniotes. In Stage-30 embryo, Pax9 was strongly expressed in the posterior mesoderm, as was also observed for mouse Pax9. Since this expression was not detected for Pax1 in the mouse or fish, this new expression in the posterior mesoderm likely evolved in Pax9 of ancestral vertebrates after gene duplication. Two-month-old fish injected with Pax9 morpholino oligonucleotide showed abnormal morphology in the tail hypural skeletal element, which may have been related to this expression.     

Regulated spatial expression of fusion gene constructs with the 5′ upstream region of Halocynthia roretzi muscle actin gene in Ciona savignyi embryos

pHrMA4a-Z is a recombinant plasmid in which about 1.4 kb of the 5 flanking region of a gene for muscle actin HrMA4a from the ascidian Halocynthia roretzi is fused with the coding sequence of a bacterial gene for -galactosidase (lac-Z). In this study, we examined the expression of the fusion gene construct when it was introduced into eggs of another ascidian, namely Ciona savignyi. When a moderate amount of linearized pHrMA4a-Z was introduced into fertilized Ciona eggs, the expression of the reporter gene was evident in muscle cells of the larvae, suggesting that both species share a common machinery for the expression of muscle actin genes. The 5 upstream region of HrMA4a contains several consensus sequences, including a TATA box at -30, a CArG box at -116 and four E-boxes within a region of 200 bp. A deletion construct, in which only the 216-bp 5 flanking region of HrMA4a was fused with lac-Z, was expressed primarily in larval muscle cells. However, another deletion construct consisting of only the 61-bp upstream region of HrMA4a fused with lac-Z was not expressed at all. When pHrMA4a-Z or pHrMA4a-Z (–216) was injected into each of the muscle-precursor blastomeres of the 8-cell embryo, expression of the reporter gene was observed in larval muscle cells in a lineage-specific fashion. However, expression of the reporter gene was not observed when the plasmid was injected into non-muscle lineage. Therefore, the expression of the reporter gene may depend on some difference in cytoplasmic constituents between blastomeres of muscle and non-muscle lineage in the 8-cell embyo.