Osteoclasts recycle via osteomorphs during RANKL-stimulated bone resorption

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Are Astrocytes the Missing Link Between Lack of Brain Aspartoacylase Activity and the Spongiform Leukodystrophy in Canavan Disease?

Canavan disease (CD) is a genetic degenerative brain disorder associated with mutations of the gene encoding aspartoacylase (ASPA). In humans, the CD syndrome is marked by early onset, hydrocephalus, macroencephaly, psychomotor retardation, and spongiform myelin sheath vacuolization with progressive leukodystrophy. Metabolic hallmarks of the disease include elevated N-acetylaspartate (NAA) levels in brain, plasma and CSF, along with daily excretion of large amounts of NAA and its anabolic metabolite, N-acetylaspartylglutamate (NAAG). Of the observed neuropathies, the most important appears to be the extensive demyelination that interferes with normal neuronal signaling. However, finding the links between the lacks of ASPA activity in oligodendrocytes, the buildup of NAA in white matter (WM) and the mechanisms underlying the edematous spongiform leukodystrophy have remained elusive. In this analytical review we consider what those links might be and propose that in CD, the pathological buildup of NAA in limited WM extracellular fluid (ECF) is responsible for increased ECF osmotic–hydrostatic pressure and initiation of the demyelination process. We also hypothesize that NAA is not directly liberated by neurons in WM as it is in gray matter, and that its source in WM ECF is solely as a product of the catabolism of axon-released NAAG at nodes of Ranvier by astrocyte NAAG peptidase after it has docked with the astrocyte surface metabotropic glutamate receptor 3. This hypothesis ascribes for the first time a possible key role played by astrocytes in CD, linking the lack of ASPA activity in myelinating oligodendrocytes, the pathological buildup of NAA in WM ECF, and the spongiform demyelination process. It also offers new perspectives on the cause of the leukodystrophy in CD, and on possible treatment strategies for this inherited metabolic disease. CD, a rare genetic disorder that compromises a physiologically important tri-cellular brain metabolic system.     

Design and applicability of DNA arrays and DNA barcodes in biodiversity monitoring

Background  

The rapid and accurate identification of species is a critical component of large-scale biodiversity monitoring programs. DNA arrays (micro and macro) and DNA barcodes are two molecular approaches that have recently garnered much attention. Here, we compare these two platforms for identification of an important group, the mammals.     

A direct selection strategy for shotgun cloning and sequencing in the bacteriophage M13.

A new cloning strategy is described which utilizes direct selection of recombinants for shotgun sequencing in the filamentous bacteriophage M13. Direct selection is accomplished by insertional inactivation of the M13 gene X protein, a powerful inhibitor of phage-specific DNA synthesis when overproduced. An extra copy of gene X was inserted into the intergenic region of M13 and placed under the control of the bacteriophage T7 gene 10 promoter and RBS. Random fragments are cloned into the EcoRV cloning site of the new gene X cistron and recombinants are selected in an E. coli male strain producing T7 RNA polymerase. Cloning efficiencies obtained with M13-100 or phosphatase-treated M13mp19 vector are comparable. The direct selection capability of M13-100 was demonstrated to have the following advantages: (a) consistently achieved high ratios of recombinants to religated vector in the libraries, averaging about 500:1 (0.2% background), and (b) the elimination of the need for phosphatase treatment of the vector to reduce background. The direct selection strategy significantly improves the efficiency of shotgun library construction in M13, and should therefore facilitate the cloning aspects of large scale sequencing projects.     

Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean

We have cloned and sequenced the gene encoding the largest subunit of RNA polymerase II (RPB1) from Arabidopsis thaliana and partially sequenced genes from soybean (Glycine max). We have also determined the nucleotide sequence for a number of cDNA clones which encode the carboxyl terminal domains (CTDs) of RNA polymerase II from both soybean and Arabidopsis. The Arabidopsis RPB1 gene encodes a polypeptide of approximately 205 kDa, consists of 12 exons, and encompasses more than 8 kb. Predicted amino acid sequence shows eight regions of similarity with the largest subunit of other prokaryotic and eukaryotic RNA polymerases, as well as a highly conserved CTD unique to RNA polymerase II.The CTDs in plants, like those in most other eukaryotes, consist of tandem heptapeptide repeats with the consensus amino acid sequence PTSPSYS. The portion of RPB1 which encodes the CTD in plants differs from that of RPB1 of animals and lower eukaryotes. All the plant genes examined contain 2–3 introns within the CTD encoding regions, and at least two plant genes contain an alternatively spliced intron in the 3 untranslated region. Several clustered amino acid substitutions in the CTD are conserved in the two plant species examined, but are not found in other eukaryotes. RPB1 is encoded by a multigene family in soybean, but a single gene encodes this subunit in Arabidopsis and most other eukaryotes.     

A GTPase Chimera Illustrates an Uncoupled Nucleotide Affinity and Release Rate,Providing Insight into the Activation Mechanism

The release of GDP from GTPases signals the initiation of a GTPase cycle, where the association of GTP triggers conformational changes promoting binding of downstream effector molecules. Studies have implicated the nucleotide-binding G5 loop to be involved in the GDP release mechanism. For example, biophysical studies on both the eukaryotic Gα proteins and the GTPase domain (NFeoB) of prokaryotic FeoB proteins have revealed conformational changes in the G5 loop that accompany nucleotide binding and release. However, it is unclear whether this conformational change in the G5 loop is a prerequisite for GDP release, or, alternatively, the movement is a consequence of release. To gain additional insight into the sequence of events leading to GDP release, we have created a chimeric protein comprised of Escherichia coli NFeoB and the G5 loop from the human Giα1 protein. The protein chimera retains GTPase activity at a similar level to wild-type NFeoB, and structural analyses of the nucleotide-free and GDP-bound proteins show that the G5 loop adopts conformations analogous to that of the human nucleotide-bound Giα1 protein in both states. Interestingly, isothermal titration calorimetry and stopped-flow kinetic analyses reveal uncoupled nucleotide affinity and release rates, supporting a model where G5 loop movement promotes nucleotide release.The hydrolysis of guanosine triphosphate (GTP) by GTPases, such as the oncoprotein p21 Ras and heterotrimeric Gα proteins, is a critical regulatory activity for cell growth and proliferation (1). Aberrant GTPases are consequently often implicated in tumorigenesis, developmental disorders, and metabolic diseases (2). Critical for the initiation of a GTPase cycle is the release of guanosine diphosphate (GDP), which allows GTP to bind and switch the protein from an inactive to an active conformation. The GTP is subsequently hydrolyzed to GDP and inorganic phosphate, returning the GTPase to an inactive conformation (3).Given that the release of GDP is the fundamental step in the initiation of a GTPase cycle, the detailed mechanism by which it is released has been under intense scrutiny. Studies using double electron-electron resonance, deuterium-exchange, Rosetta energy analysis, and electron paramagnetic resonance, have shown that the mechanism involves conformational changes in the nucleotide-coordinating G5 loop, one of five nucleotide recognition motifs (4, 5, 6, 7, 8, 9, 10, 11). Structural studies of eukaryotic Gα proteins and the intracellular TEES-type GTPase domain of the prokaryotic iron transporter FeoB (NFeoB) have also illustrated distinct conformations of the G5 loop, depending on the nucleotide-bound state (9, 12).Recently, we reported mutational studies of the G5 loop of Escherichia coli NFeoB, which illustrated a correlation between the sequence composition of the loop and the intrinsic GDP release rate (13). However, despite these observations, it is unclear whether the observed conformational changes in the G5 loop are a prerequisite for GDP release, or if the movement is a consequence of GDP release. To address this fundamental question, in this study we have used a combination of protein engineering and biophysical methods.Initially, to assess the relevance of conformational flexibility in the G5 loop, we aimed to create a protein chimera combining sequence and structural characteristics of both fast and slow GDP-releasing GTPases. We thus engineered a protein chimera using E. coli NFeoB as the scaffold (a protein with fast intrinsic GDP release) and substituted the G5 loop with that of a slow GDP-releasing protein (the human Giα1 protein; Gene ID 2770; Fig. 1 A (5)). GTP hydrolysis assays comparing wild-type (wt) NFeoB (wtNFeoB) and the protein chimera (ChiNFeoB) validated the integrity of the GTPase activities of both proteins (k_cat = 0.46 and 0.36 min⁻¹, respectively). To further assess the ChiNFeoB protein, we determined its crystal structure at 2.2 Å resolution (see Table S1 in the Supporting Material). The ChiNFeoB structure contains two molecules in the asymmetric unit, with molecule A bound to GDP. They are essentially identical to the nucleotide-bound wtNFeoB structure (root-mean-square deviation of 1.2 Å over 226 Cα atoms; Fig. 2).Open in a separate windowFigure 1Chimera model and structural comparison. (A) Illustration highlighting the chimera sequence change. (Orange) Sequence of the extended G5 loop from Giα1, which replaced the NFeoB sequence (gray). (B–F) Structural comparison of the G5 loop between (B) WT apo (PDB:3HYR) and nucleotide-bound (PDB:3HYT) NFeoB structures. (C) NFeoB nucleotide-bound and Giα1 (PDB:2ZJZ). (D) Nucleotide-bound NFeoB and chimera (Chi_GDP). (E) Nucleotide-bound chimera and Giα1. (F) Nucleotide-free (Chi_apo) and bound chimera protein. (G) Overview of the nucleotide binding site and structural overlay of chimera and Giα1 structures. To see this figure in color, go online.Open in a separate windowFigure 2Superimposition of nucleotide-bound NFeoB and chimera protein, with thermodynamic parameters. To see this figure in color, go online.However, the ChiNFeoB structure, when compared to the wtNFeoB structure, revealed an alteration in the conformation of the G5 loop, showing an extra turn on the N-terminal end of the α6 helix. This is structurally distinct from the wtFeoB protein, but with a conformation similar to that of the Giα1 protein (PDB:2ZJZ; Fig. 1, B–F). As in the crystal structures of wtNFeoB and Giα1, ChiNFeoB residues implicated in coordination of the nucleotide base maintain their positions in the G5 loop relative to GDP. In particular, residues Ala^∗150 and Thr^∗151 (NFeoB numbering, the asterisk indicates Giα1 chimera residue) are involved in electrostatic interactions with the nucleotide base moiety, analogous to the structures of both wtNFeoB and Giα1 (Fig. 1 G). Serendipitously, the second molecule in the asymmetric unit of ChiNFeoB (molecule B) was present in the nucleotide-free state. The two molecules (GDP-bound and nucleotide-free) are nearly identical (the superposition of molecules A and B yields a root-mean-square deviation of 0.36 Å over 229 Cα atoms), with the G5 loop adopting a nearly indistinguishable conformation compared to that of the GDP-bound molecule A (Fig. 1 F).Importantly, this conformation is independent of the crystallographic packing, inasmuch as the loop is not involved in any crystal contacts. In contrast, the structures of nucleotide-bound and nucleotide-free wtNFeoB illustrated a large conformational change in the G5 loop (Fig. 1 B). Hence, the substitution in the chimera extends the secondary structure of the α6 helix, and as hypothesized, the engineered ChiNFeoB protein has a G5 loop structure that is more conformationally stable than that of wtNFeoB.We subsequently measured the affinity of the ChiNFeoB protein for GDP using isothermal titration calorimetry (ITC). Nonlinear regression was used to attain the thermodynamic parameters (including the GDP binding affinity, K_a; the corresponding dissociation constant (K_d) was calculated from the equation K_d = 1/K_a). Interestingly, these measurements revealed the ChiNFeoB protein to have an almost 10-fold reduced affinity for GDP (82 vs. 9 μM measured for the WT protein; Fig. 2). In contrast, in a recent alanine scanning mutagenesis study of the G5 loop we observed a fivefold increase in affinity for GDP in a Ser¹⁵⁰Ala mutant (2 μM) (14). This mutant protein has a coordination environment for the GDP base analogous to that of the ChiNFeoB protein (Fig. 1 A), indicating that it is not the presence of an alanine at position 150 that causes the reduced GDP affinity observed for the chimera protein. Instead, the analysis by ITC and comparison with previous mutagenesis studies indicates that the GDP binding site is less accessible in the ChiNFeoB protein, likely due to the introduction of conformational rigidity that accompanies the extension of secondary structural elements within the loop (Fig. 1 D).To further evaluate the functional characteristics of the chimera protein, we used stopped-flow fluorescence assays to determine the rate of nucleotide dissociation (k_off) and association (k_on) for the ChiNFeoB protein. The association rate for the GTP analog mant-GMPPNP was determined from the slope of a linear plot of protein concentration versus the observed association constant (k_obs). The k_on for the chimera was determined to be 3.20 μM⁻¹ min⁻¹ (Supporting Material), the dissociation rate (k_off) of GDP for the chimera was determined to be 16.6 s⁻¹ (vs. 144 s⁻¹ for wtNFeoB;

Isolation and Properties of Nuclei from Control and Auxin-treated Soybean Hypocotyl

A quick procedure for the isolation of nuclei with good yield from soybean hypocotyl (Glycine max var. Wayne) was developed. The isolated nuclei appeared to retain their structural integrity. They were typically ellipsoidal with minima and maxima diameter of about 6 and 8 to 10 micrometers. While the nuclei were similar in size, the nucleoli were significantly larger in nuclei from auxin-treated tissue. The DNA content per nucleus was 4 ± 1 picograms for both untreated and auxin-treated tissues. The DNA: RNA: protein ratio of isolated nuclei in untreated and auxin-treated tissues was 1: 3.1: 11 and 1: 5.4: 21.7, respectively. The purified nuclei were active in RNA synthesis; the level of RNA polymerase II activity expressed in the nuclei from untreated tissue was 50 to 60% higher than RNA polymerase. I. The nuclei from auxin-treated tissues contained about 2.5 times as much RNA polymerase I activity as nuclei from untreated tissue. The purified nuclei from both untreated and auxin-treated tissues were also active in the incorporation of ³H-TTP into DNA.     

Auxin-induced changes in the population of translatable messenger RNA in elongating maize coleoptile sections

In-vitro translation products of polyadenylated RNA from untreated and indole-3-acetic acid (IAA)-treated elongating sections of maize (Zea mays L.) coleoptiles were analyzed by twodimensional polyacrylamide gel electrophoresis. Treatment with IAA results in an increased amount of at least four in-vitro translation products. The amounts of two of these translation products are increased within 10 min of IAA treatment.Abbreviation IAA indole-3-acetic acid