A real-time method of imaging glucose uptake in single, living mammalian cells

This protocol details a method for monitoring glucose uptake into single, living mammalian cells using a fluorescent D-glucose derivative, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), as a tracer. The specifically designed chamber and superfusion system for evaluating 2-NBDG uptake into cells in real time can be combined with other fluorescent methods such as Ca2+ imaging and the subsequent immunofluorescent classification of cells exhibiting divergent 2-NBDG uptake. The whole protocol, including immunocytochemistry, can be completed within 2 d (except for cell culture). The procedure for 2-NBDG synthesis is also presented.     

Crystal Structures of Human Dipeptidyl Peptidase Ⅳ in its Apo and Diprotin B-complexed Forms

Dipeptidyl peptidase Ⅳ (DPPIV), which belongs to the prolyl oligopeptidase family of serine proteases, is known to have a variety of regulatory biological functions and has been shown to be implicated in type 2 diabetes. It is therefore important to develop selective human DPPIV (hDPPIV) inhibitors. In this study, we determined the crystal structure of apo hDPPIV at 1.9 A resolution. Our high-resolution crystal structure of apo hDPPIV revealed the presence of sodium ion and glycerol molecules at the active site. In order to elucidate the hDPPIV binding mode and substrate specificity, we determined the crystal structure of hDPPIV-diprotin B (Val-Pro-Leu) complex at 2.1 A resolution, and clarified the difference in binding mode between diprotin B and diprotin A (Ile-Pro-Ile) into the active site of hDPPIV. Comparison between our crystal structures and the reported apo hDPPIV structures revealed that positively charged functional groups and conserved water molecules contributed to the interaction of ligands with hDPPIV. These results are useful for the design of potent hDPPIV inhibitors.     

The Membrane Topology of ALMT1, an Aluminum-Activated Malate Transport Protein in Wheat (Triticum aestivum)

The wheat ALMT1 gene encodes an aluminum (Al)-activated malate transport protein which confers Al-resistance. We investigated the membrane topology of this plasma-membrane localized protein with immunocytochemical techniques. Several green fluorescent protein (GFP)-fused and histidine (His)-tagged chimeras of ALMT1 were prepared based on a computer-predicted secondary structure and transiently expressed in cultured mammalian cells. Antibodies raised to polypeptide epitopes of ALMT1 were used in conjunction with the antibody to the His-tags to determine the topology of ALMT1. This study shows that the ALMT1 protein contains six transmembrane domains with the amino and carboxyl termini located on the extracellular side of the plasma membrane.Key Words: ALMT1, aluminum resistance, immunofluorescent staining, malate transporter, topology, Triticum aestivum     

Production and characterization of recombinant P1 adhesin essential for adhesion,gliding, and antigenic variation in the human pathogenic bacterium,Mycoplasma pneumoniae

Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6?×?His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications.     

Variation in morphological and behavioral traits among isofemale strains of Drosophila prolongata (Diptera: Drosophilidae)

Drosophila prolongata, a member of the rhopaloa subgroup of the melanogaster species group, occurs in Southeast Asia. Drosophila prolongata is known to have unique and prominent sexual dimorphism, with extraordinarily thick and elongated forelegs only in males. Mating behavior of D. prolongata is also characteristic: males perform “leg vibration” in their courtship toward females, in which the elongated forelegs play an important role. Comparisons with closely related species suggest that these morphological and behavioral traits have evolved rapidly after the divergence of D. prolongata. In the present study, variation in morphological and behavioral traits was examined among D. prolongata strains derived from single females collected in their natural habitats. Significant variations were detected in the size of various body parts, aggressiveness of interactions between males, and mating behavior. However, no obvious relationship was observed between morphological and behavioral traits. These results suggested that genetic factors contribute to the variation in morphological and behavioral traits in D. prolongata. The strains characterized in this study are useful for studies on the genetic mechanisms underlying the evolution of characteristic traits in D. prolongata.     

Akhirin regulates the proliferation and differentiation of neural stem cells in intact and injured mouse spinal cord

Although the central nervous system is considered a comparatively static tissue with limited cell turnover, cells with stem cell properties have been isolated from most neural tissues. The spinal cord ependymal cells show neural stem cell potential in vitro and in vivo in injured spinal cord. However, very little is known regarding the ependymal niche in the mouse spinal cord. We previously reported that a secreted factor, chick Akhirin, is expressed in the ciliary marginal zone of the eye, where it works as a heterophilic cell‐adhesion molecule. Here, we describe a new crucial function for mouse Akhirin (M‐AKH) in regulating the proliferation and differentiation of progenitors in the mouse spinal cord. During embryonic spinal cord development, M‐AKH is transiently expressed in the central canal ependymal cells, which possess latent neural stem cell properties. Targeted inactivation of the AKH gene in mice causes a reduction in the size of the spinal cord and decreases BrdU incorporation in the spinal cord. Remarkably, the expression patterns of ependymal niche molecules in AKH knockout (AKH?/?) mice are different from those of AKH+/+, both in vitro and in vivo. Furthermore, we provide evidence that AKH expression in the central canal is rapidly upregulated in the injured spinal cord. Taken together, these results indicate that M‐AKH plays a crucial role in mouse spinal cord formation by regulating the ependymal niche in the central canal. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 494–504, 2015     

NADPH production from NADP+ with a glucose dehydrogenase system involving whole cells and immobilized cells of Gluconobacter suboxydans

Summary A convenient and efficient method of NADPH production from NADP⁺ has been established using a glucose dehydrogenase system involving whole cells and immobilized cells of Gluconobacter suboxydans IFO 3172. Using airdried cells of the bacterium, the optimum conditions for NADPH production were examined, including the cell and glucose concentrations, NADP⁺ concentration, pH, buffer and reaction temperature. Under suitable conditions, the conversion ratio and the amount of NADPH accumulated reached about 100% and 73 mg/ml of the reaction mixture, respectively, after 1-h reaction. Intact cells of the bacterium also showed high NADPH production even in the reaction mixture without a surfactant. The addition of Triton X-100 to the reaction mixture and freeze-thawing treatment of intact cells enhanced the production. The NADPH production method was further improved by using cells of the bacterium immobilized by entrapment in a -carrageenan gel lattice. The immobilized cells had almost the same enzymatic properties as the air-dried cells. The conditions for the continuous production of NADPH with an immobilized cell column were also investigated. NADPH was produced in a good yield (about 95%) with this continuous process.