The crystal structure of human α(1)-microglobulin reveals a potential haem-binding site

We describe the 2.3 ? (1 ?=0.1?nm) X-ray structure of α1m (α1-microglobulin), an abundant protein in human blood plasma, which reveals the β-barrel fold typical for lipocalins with a deep pocket lined by four loops at its open rim. Loop #1 harbours the residue Cys34 which is responsible for covalent cross-linking with plasma IgA. A single disulfide bond between Cys72 and Cys169 connects the C-terminal segment to the β-barrel, as in many other lipocalins. The exposed imidazole side chains of His122 and His123 in loop #4 give rise to a double Ni2+-binding site together with a crystallographic neighbour. The closest structural relatives of α1m are the complement protein component C8γ, the L-prostaglandin D synthase and lipocalin 15, three other structurally characterized members of the lipocalin family in humans that have only distant sequence similarity. In contrast with these, α1m is initially expressed as a bifunctional fusion protein with the protease inhibitor bikunin. Neither the electron density nor ESI-MS (electrospray ionization MS) provide evidence for a chromophore bound to the recombinant α1m, also known as 'yellow/brown lipocalin'. However, the three side chains of Lys92, Lys118 and Lys130 that were reported to be involved in covalent chromophore binding appear to be freely accessible to ligands accommodated in the hydrophobic pocket. A structural feature similar to the well-known Cys-Pro haem-binding motif indicates the presence of a haem-binding site within the loop region of α1m, which explains previous biochemical findings and supports a physiological role in haem scavenging, as well as redox-mediated detoxification.     

Auxin-induced H+-pump stimulation does not depend on the presence of epidermal cells in corn coleoptiles

Cell-wall acidification and electrical reactions (depolarization and hyperpolarization) are typical auxin responses in maize (Zea mays L.) coleoptiles. In an attempt to test the role of the outer epidermis in these responses, they have been measured and compared in intact and peeled coleoptile fragments. To exclude interactions between parenchymal and epidermal cells, the coleoptile pieces were completely stripped of their outer epidermis. This preparation was monitored by means of a scanning electron microscope. When externally applied indole-3-acetic acid was tested, we found that neither cell-wall acidification nor the electrical membrane responses depended on the presence of intact epidermal cells.Abbreviations IAA Indole-3-acetic acid - MES 2-[N morpholino-ethane-sulfonic acid - TRIS 2-Amino-2-hydroxymethyl-1,3-propanediol We thank Kuki Kaethner for her excellent technical assistance. This work was supported by the Hessische Graduiertenförderung and the Deutsche Forschungsgemeinschaft.     

A novel flow based hollow-fiber blood-brain barrier in vitro model with immortalised cell line PBMEC/C1-2

A flow based hollow-fiber in vitro model of the blood-brain barrier (BBB) was established. The immortalised porcine brain microvascular endothelial cell line PBMEC/C1-2 was cultured in a pulsatile hollow-fiber cartridge system (Cellmax Quad). The usability of PBMEC/C1-2 in the flow based hollow-fiber model was increased from three days in the originally used Transwell model up to four months due to the application of shear stress and co-culturing with glioma cell line C6. It was shown that the tightness of PBMEC/C1-2 layers was enhanced significantly in astrocyte conditioned medium (ACM) and in co-culture. The morphology of PBMEC/C1-2 and C6 was visualised by environmental scanning electron microscopy (ESEM). Permeation studies were accomplished with a set of benzodiazepines. The raw data were processed with three different calculation models and the results were compared with permeability coefficients obtained with an established Transwell model. In summary a flow based hollow-fiber BBB in vitro model was developed, which can be used to perform experiments with physiological (e.g., regulation of BBB permeability), pharmacological (e.g., pharmacokinetics and dynamics) and pathophysiological (e.g., effects of diseases on BBB permeability and vice versa) objectives.     

Towards neural circuit reconstruction with volume electron microscopy techniques

Electron microscopy is the only currently available technique with a resolution adequate to identify and follow every axon and dendrite in dense neuropil. Reconstructions of large volumes of neural tissue, necessary to reconstruct even local neural circuits, have, however, been inhibited by the daunting task of serially sectioning and reconstructing thousands of sections. Recent technological developments have improved the quality of volume electron microscopy data and automated its acquisition. This opens up the prospect of reconstructing almost complete invertebrate and sizable fractions of vertebrate nervous systems. Such reconstructions of complete neural wiring diagrams could rekindle the tradition of relating neural function to the underlying neuroanatomical circuitry.     

Transport Rankings of Non-Steroidal Antiinflammatory Drugs across Blood-Brain Barrier In Vitro Models

The aim of this work was to conduct a comprehensive study about the transport properties of NSAIDs across the blood-brain barrier (BBB) in vitro. Transport studies with celecoxib, diclofenac, ibuprofen, meloxicam, piroxicam and tenoxicam were accomplished across Transwell models based on cell line PBMEC/C1-2, ECV304 or primary rat brain endothelial cells. Single as well as group substance studies were carried out. In group studies substance group compositions, transport medium and serum content were varied, transport inhibitors verapamil and probenecid were added. Resulted permeability coefficients were compared and normalized to internal standards diazepam and carboxyfluorescein. Transport rankings of NSAIDs across each model were obtained. Single substance studies showed similar rankings as corresponding group studies across PBMEC/C1-2 or ECV304 cell layers. Serum content, glioma conditioned medium and inhibitors probenecid and verapamil influenced resulted permeability significantly. Basic differences of transport properties of the investigated NSAIDs were similar comparing all three in vitro BBB models. Different substance combinations in the group studies and addition of probenecid and verapamil suggested that transporter proteins are involved in the transport of every tested NSAID. Results especially underlined the importance of same experimental conditions (transport medium, serum content, species origin, cell line) for proper data comparison.     

Herbicide effects on planktonic systems of different complexity

Bioassays of different complexity were compared with respect to their capability to predict the environmental impact of the herbicide atrazine in aquatic systems. Acute toxicity tests with Daphnia did not yield meaningful results. Sublethal tests with Daphnia (feeding inhibition, reduction of growth and reproduction) were more sensitive, but effective concentrations of atrazine were still rather high (2 mg/L). A relatively complicated artificial food chain system that incorporated direct and indirect effects on Daphnia yielded significant reduction of daphnid population growth at 0.1 mg/L. Enclosure experiments with natural communities were by far the most sensitive tools. Community responses could be measured at concentrations as low as 1 µg/L and 0.1 µg atrazine/L. At the lowest concentration, however, communities recovered after three weeks. We conclude that in complex systems indirect effects can be more important than direct effects, so that, contrary to the conditions in simple tests, non-target organisms may be the better indicators of herbicide stress to natural communities.     

Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein

Background

The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes.

Methods and Findings

We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal α-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions.

Conclusions

Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins.     

GFP tagging of sieve element occlusion (SEO) proteins results in green fluorescent forisomes

Forisomes are Ca(2+)-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as 'FOR' proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca(2+) and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that 'FOR'-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca(2+) binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism.     

Role of Maltogenic Amylase and Pullulanase in Maltodextrin and Glycogen Metabolism of Bacillus subtilis 168

The physiological functions of two amylolytic enzymes, a maltogenic amylase (MAase) encoded by yvdF and a debranching enzyme (pullulanase) encoded by amyX, in the carbohydrate metabolism of Bacillus subtilis 168 were investigated using yvdF, amyX, and yvdF amyX mutant strains. An immunolocalization study revealed that YvdF was distributed on both sides of the cytoplasmic membrane and in the periplasm during vegetative growth but in the cytoplasm of prespores. Small carbohydrates such as maltoheptaose and β-cyclodextrin (β-CD) taken up by wild-type B. subtilis cells via two distinct transporters, the Mdx and Cyc ABC transporters, respectively, were hydrolyzed immediately to form smaller or linear maltodextrins. On the other hand, the yvdF mutant exhibited limited degradation of the substrates, indicating that, in the wild type, maltodextrins and β-CD were hydrolyzed by MAase while being taken up by the bacterium. With glycogen and branched β-CDs as substrates, pullulanase showed high-level specificity for the hydrolysis of the outer side chains of glycogen with three to five glucosyl residues. To investigate the roles of MAase and pullulanase in glycogen utilization, the following glycogen-overproducing strains were constructed: a glg mutant with a wild-type background, yvdF glg and amyX glg mutants, and a glg mutant with a double mutant (DM) background. The amyX glg and glg DM strains accumulated significantly larger amounts of glycogen than the glg mutant, while the yvdF glg strain accumulated an intermediate amount. Glycogen samples from the amyX glg and glg DM strains exhibited average molecular masses two and three times larger, respectively, than that of glycogen from the glg mutant. The results suggested that glycogen breakdown may be a sequential process that involves pullulanase and MAase, whereby pullulanase hydrolyzes the α-1,6-glycosidic linkage at the branch point to release a linear maltooligosaccharide that is then hydrolyzed into maltose and maltotriose by MAase.Bacillus subtilis can utilize polysaccharides such as starch, glycogen, and amylose as carbon sources by hydrolyzing them into smaller maltodextrins via the action of extracellular α-amylase (AmyE) (14). In B. subtilis, α-glucosidase encoded by malL has been known to contribute to maltodextrin metabolism in the cell (40, 41). Schönert et al. (42) reported that maltose is transported by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in B. subtilis. They also reported that maltodextrins with degrees of polymerization (DP) of 3 to 7 (G3 to G7) are taken up via a maltodextrin-specific (Mdx) ATP-binding cassette (ABC) transport system (42). This system is made up of a maltodextrin-binding protein (MdxE) and two membrane proteins (MdxF and MdxG), as well as an ATPase (MsmX). The basic model proposed for the transport and metabolism of maltooligosaccharides includes a series of carbohydrate-hydrolyzing and -transferring enzymes. However, the enzymatic hydrolysis of maltodextrins and glycogen, providing a major energy reservoir in prokaryotes, was not reflected in the model, due probably to a lack of experimental analysis. Unlike those in Bacillus spp., the transport and metabolic systems for maltodextrins in Escherichia coli have been investigated extensively (7, 9, 10). A model for maltose metabolism involving an α-glucanotransferase (MalQ), a maltodextrin glucosidase (MalZ), and a maltodextrin phosphorylase (MalP) was proposed previously based on analyses of the breakdown of ¹⁴C-labeled maltodextrins in various knockout mutants (10).Ninety bacterial genomes were analyzed to identify the enzymes involved in sugar metabolism, and the results suggested that bacterial enzymes for the synthesis and degradation of glycogen belong to the glucosyltransferase and glycosidase/transglycosidase families, respectively. Free-living bacteria such as B. subtilis carry a minimal set of enzymes for glycogen metabolism, encoded by the glg operon of five genes. The four genes most proximal to the promoter encode enzymes for the synthesis of glycogen, including a branching enzyme (glgB), an ADP-glucose phyrophosphorylase (glgC and glcD), and a glycogen synthase (glgA). On the other hand, the most distal gene, glgP, encodes a glycogen phosphorylase (a member of glycosyltransferase family 35) (13, 18), which degrades glycogen branches by forming glucose-1-phosphate (glucose-1-P). B. subtilis carries two additional enzymes encoded at separate loci, a maltogenic amylase (MAase [YvdF, encoded at 304°]) and a pullulanase (AmyX, encoded at 262°), which have been known to degrade glycogen in vitro (15, 31). These two enzymes are ubiquitous among Bacillus spp. and may play an important role in glycogen and maltodextrin metabolism in the bacteria (see Table S1 in the supplemental material).The MAase YvdF in B. subtilis 168 and its homologue in B. subtilis SUH4-2 share 99% identity at both the nucleotide and amino acid sequence levels (4). MAase (EC 3.2.1.133) is a multisubstrate enzyme that acts on substrates such as cyclodextrin (CD), maltooligosaccharides, pullulan, starch, and glycogen (4). MAase belongs to a subfamily of glycoside hydrolase family 13, along with cyclodextrinase (EC 3.2.1.54), neopullulanase (EC 3.2.1.135), and Thermoactinomyces vulgaris R-47 α-amylase II (46). Although the catalytic properties and tertiary structure of MAase have been studied extensively (33), its physiological role in the bacterial cell is yet to be elucidated. The expression pattern of MAase in B. subtilis 168 has been investigated by monitoring the β-galactosidase activity expressed from the yvdF promoter in defined media containing various carbon sources (20). The yvdF promoter is induced in medium containing maltose, starch, or β-CD but is repressed in the presence of glucose, fructose, sucrose, or glycerol as the sole carbon source. In a previous study, Spo0A, a master regulator determining the life cycle of B. subtilis, was shown to be related to the expression of MAase in a positive manner (20). Kiel et al. (18) reported that the glycogen operon in B. subtilis is turned on during sporulation by RNA polymerase containing σ^E. This finding indicated that MAase, along with glycogen phosphorylase and pullulanase, might be involved in the metabolism of maltodextrin and glycogen in vivo.Pullulanases are capable of hydrolyzing the α-1,6-glycosidic linkages of pullulan to form maltotriose (2, 11, 15, 28, 31, 38). In particular, type I pullulanases have been reported to hydrolyze the α-1,6-glycosidic linkages in branched oligosaccharides such as starch, amylopectin, and glycogen, forming maltodextrins linked by α-1,4-glycosidic linkages (11). Pullulanase is also known as a debranching enzyme. The enzymatic properties and three-dimensional structure of AmyX from B. subtilis 168 were investigated by Malle et al. (28). However, to date, the physiological function of pullulanase encoded by amyX has not been investigated.The aim of this study was to elucidate the physiological functions of MAase and pullulanase, specifically concentrating on their roles in the degradation of maltodextrin and glycogen in B. subtilis. For this purpose, studies of the localization of the enzymes, the accumulation of glycogen, and the distribution of glycogen side chains were performed using the wild type and knockouts of MAase- and pullulanase-related genes.