Structural Basis for Silicic Acid Uptake by Higher Plants

Many of the world's most important food crops such as rice, barley and maize accumulate silicon (Si) to high levels, resulting in better plant growth and crop yields. The first step in Si accumulation is the uptake of silicic acid by the roots, a process mediated by the structurally uncharacterised NIP subfamily of aquaporins, also named metalloid porins. Here, we present the X-ray crystal structure of the archetypal NIP family member from Oryza sativa (OsNIP2;1). The OsNIP2;1 channel is closed in the crystal structure by the cytoplasmic loop D, which is known to regulate channel opening in classical plant aquaporins. The structure further reveals a novel, five-residue extracellular selectivity filter with a large diameter. Unbiased molecular dynamics simulations show a rapid opening of the channel and visualise how silicic acid interacts with the selectivity filter prior to transmembrane diffusion. Our results will enable detailed structure–function studies of metalloid porins, including the basis of their substrate selectivity.     

Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

Pattern analysis of stem cell differentiation during <Emphasis Type="Italic">in vitro Arabidopsis</Emphasis> organogenesis

Plant somatic cells have the capability to switch their cell fates from differentiated to undifferentiated status under proper culture conditions, which is designated as totipotency. As a result, plant cells can easily regenerate new tissues or organs from a wide variety of explants. However, the mechanism by which plant cells have such remarkable regeneration ability is still largely unknown. In this study, we used a set of meristem-specific marker genes to analyze the patterns of stem cell differentiation in the processes of somatic embryogenesis as well as shoot or root organogenesis in vitro. Our studies furnish preliminary and important information on the patterns of the de novo stem cell differentiation during various types of in vitro organogenesis.     

The stimulus-secretion coupling of amino acid-induced insulin release metabolic interaction L-asparagine and L-leucine in pancreatic islets

1. Because L-asparagine augments insulin release evoked by L-leucine, the metabolism of these two amino acids was investigated in rat pancreatic islets. 2. L-Leucine inhibited the uptake and deamidation of L-asparagine, but failed to exert any obvious primary effect upon the further catabolism of aspartate derived from exogenous asparagine. 3. L-Asparagine augmented the oxidation of L-leucine, and effect possibly attributable to activaion of 2-ketoisocaproate dehydrogenase. 4. The association of L-asparagine and L-leucine exerted a sparing action on the utilization of endogenous amino acids, so that the integrated rate of nutrients oxidation was virtually identical in the sole presence of L-leucine and simultaneous presence of L-asparagine and L-leucine, respectively. 5. It is proposed that the enhancing action of L-asparagine upon insulin release evoked by L-leucine is attributable to an increased generation rate of cytosolic NADPH rather than any increase in nutrients oxidation.     

Inhibition of Membrane-Active Peptides by Fatty Acid–Peptide Hybrids

Nine fatty acid–peptide hybrid molecules were constructed using the general formula CH₃(CH₂) _n CO-Phe Asp Cys-amide and tested for their ability to inhibit cell lysis induced by the membrane-active peptide melittin. All of these molecules, where n = 4–14, inhibited the action of melittin to some extent, but the longer carbon chains were most effective. Several potential inhibitors were also constructed with conservative substitutions in the peptide portion of the molecule. All were effective to varying degrees. We concluded that in the hexapeptide inhibitor published by Blondelle et al. (1993), the role of the first three residues is only to provide hydrophobic interaction with the melittin and has no particular amino acid sequence specificity. Some of these inhibitors were found to inhibit the lytic activity of a melittin analogue which had only superficial sequence similarity to melittin and also a truncated form of melittin, indicating the generality of the action of the inhibitors.Deceased 5/4/98     

Isolation and Characterization of a Δ5-Desaturase from Oblongichytrium sp.

We isolated a cDNA clone with homology to known desaturase genes from Oblongichytrium sp., recently classified as a new genus of thraustochytrids (Labyrinthulomycetes), and found that it encoded Δ5-desaturase by its heterologous expression in yeast. The enzyme had higher activity toward 20:4n-3 than 20:3n-6, indicating that this Δ5-desaturase can be used in the production of n-3 polyunsaturated fatty acids in transgenic organisms.     

Depolymerization of Hyaluronic Acid by Low-molecular-weight Amadori-rearrangement Products and Glycated Polylysine

Depolymerization of hyaluronic acid (HA) by low-molecular-weight Amadori-rearrangement products in the presence of Cu^{2 +} was studied as an in vitro model for the glycated protein-mediated degradation of biopolymers. This oxygen radical-mediated depolymerization was found to be specifically accelerated by Cu^{2 +} , and significantly inhibited by catalase, hydroxyl radical scavengers, and metal ion chelators. Glycated polylysine also depolymerized HA. The difference in depolymerization rate between low- and high-molecular-weight Amadori products is discussed.     

MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.     

Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid

Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.     

5-Hydroxytryptamine stimulates Rb efflux from pancreatic β-cells

The effects of 5-hydroxytryptamine and 5-hydroxytryptophan on ⁸⁶Rb⁺ efflux from prelabelled ob/ob-mouse islets were studied to better understand the cellular mechanisms underlying the effects of 5-hydroxytryptamine and 5-hydroxytryptophan on insulin release. 5-Hydroxytryptophan (4 mM) had no effect on ⁸⁶Rb⁺ efflux either at a low (3 mM) or at a high (20 mM) d-glucose concentration, whereas 5-hydroxytryptamine (4 mM) stimulated ⁸⁶Rb⁺ efflux at both glucose concentrations. These results indicate that 5-hydroxytryptamine may reduce glucose-induced insulin release by inhibiting early steps in the β-cell stimulus-secretion coupling.