The Role of Mannosyl-phosphoryl-dihydropolyisoprenol in the Synthesis of Mammalian Glycoproteins

A mouse myeloma tumor was used as a model system to study the biochemical steps involved in the incorporation of mannose into glycoproteins. This tumor, MOPC-46B, synthesizes a K-type immunoglobulin light chain (K-46) which is a glycoprotein with a single oligosaccharide side chain containing mannose as one of its constituent sugars. MOPC-46B microsomal preparations contain enzymes which transfer mannose from the sugar nucleotide, GDPmannose, to endogenous lipid and protein acceptors. Formation of the mannolipid proceeds by the reversible transfer of mannose from GDP-mannose to an endogenous phospholipid. The mannolipid was purified and characterized by chemical methods and mass spectrometry as a mannosyl-monophosphoryl- dihydropolyisoprenol, containing at least 18 isoprene units, one of which is saturated. The mannolipid was implicated as an intermediate in the in vitro mannosylation of endogenous protein acceptors by three kinds of experiments. (a) Incorporation of [l%]mannose into protein was observed after the initial substrate, GDP-mannose, had been destroyed by sugar nucleotide hydrolases associated with the microsomal preparations. The continued increase in radioactivity in the protein fraction occurred concomitantly with a loss of radioactivity from the mannolipid fraction. (b) I ncorporation of [14C]mannose into both lipid and protein was inhibited by EDTA added at zero time. However, addition of EDTA after mannolipid synthesis had occurred resulted in cessation of mannolipid formation but continued incorporation of mannose into protein to an extent proportional to the amount of mannolipid originally formed. The increase in radioactivity in protein was again accompanied by a loss of radioactivity from the mannolipid. (c) When microsomes were pulsed briefly with GDP-[14C]mannose, which was then chased by a large excess of unlabeled GDP-mannose, incorporation of [‘XZ]mannose into lipid ceased immediately with the chase, while incorporation into protein continued afterwards to an extent proportional to the amount of mannolipid formed prior to the chase. Evidence that the mannolipid could function as a donor of mannose residues to protein was obtained by demonstrating that microsomes catalyze the transfer of [*4C]mannose from exogenously supplied mannolipid to endogenous protein acceptors. The amount of mannose transferred to protein was proportional to both microsomal protein and lipid concentrations. In addition, the amount of mannose transferred to protein from exogenous mannolipid is comparable to that incorporated from an equivalent amount of mannolipid generated endogenously from GDP-mannose. Gel filtration profiles of the [14C]mannose-containing protein formed in this system are essentially identical regardless of whether GDP-mannose or mannolipid is used as substrate. In both cases the radioactive protein fractionates in a manner similar to authentic K-46 (mol wt 24,000). The mannose-containing protein formed from either GDPmannose or mannolipid was degraded sequentially by Pronase and subtilisin. The products formed from either substrate appeared to be identical and exhibited chromatographic and electrophoretic characteristics of glycopeptides. It was concluded that mammalian microsomal preparations contain an endogenous phospholipid, characterized as a dihydropolyisoprenol-monophosphate, which serves as an acceptor of mannose from GDP-mannose, resulting in the formation of mannosyl-monophosphoryl-dihydropolyisoprenol, and that this mannolipid serves as a glycosyl donor for transfer of mannose residues to endogenous protein acceptors. The evidence indicates that the mannolipid is an essential intermediate in the in vitro transfer of mannose from GDP-mannose to protein

What if There Were Desktop Access to the Computer Science Literature?

What if there was an electronic computer science library? Consider the possibilities of having your favorite publications available within finger's reach. Consider project Envision, an ongoing effort to build a user-centered database from the computer science literature. This paper describes our first year progress, stressing the motivation underlying project Envision, user-centered development, and overall design

Structure of the first representative of Pfam family PF04016 (DUF364) reveals enolase and Rossmann-like folds that combine to form a unique active site with a possible role in heavy-metal chelation.

The crystal structure of Dhaf4260 from Desulfitobacterium hafniense DCB-2 was determined by single-wavelength anomalous diffraction (SAD) to a resolution of 2.01 Å using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). This protein structure is the first representative of the PF04016 (DUF364) Pfam family and reveals a novel combination of two well known domains (an enolase N-terminal-like fold followed by a Rossmann-like domain). Structural and bioinformatic analyses reveal partial similarities to Rossmann-like methyltransferases, with residues from the enolase-like fold combining to form a unique active site that is likely to be involved in the condensation or hydrolysis of molecules implicated in the synthesis of flavins, pterins or other siderophores. The genome context of Dhaf4260 and homologs additionally supports a role in heavy-metal chelation

Motile cilia create fluid-mechanical microhabitats for the active recruitment of the host microbiome

We show that mucociliary membranes of animal epithelia can create fluid-mechanical microenvironments for the active recruitment of the specific microbiome of the host. In terrestrial vertebrates, these tissues are typically colonized by complex consortia and are inaccessible to observation. Such tissues can be directly examined in aquatic animals, providing valuable opportunities for the analysis of mucociliary activity in relation to bacteria recruitment. Using the squid–vibrio model system, we provide a characterization of the initial engagement of microbial symbionts along ciliated tissues. Specifically, we developed an empirical and theoretical framework to conduct a census of ciliated cell types, create structural maps, and resolve the spatiotemporal flow dynamics. Our multiscale analyses revealed two distinct, highly organized populations of cilia on the host tissues. An array of long cilia (∼25 μm) with metachronal beat creates a flow that focuses bacteria-sized particles, at the exclusion of larger particles, into sheltered zones; there, a field of randomly beating short cilia (∼10 μm) mixes the local fluid environment, which contains host biochemical signals known to prime symbionts for colonization. This cilia-mediated process represents a previously unrecognized mechanism for symbiont recruitment. Each mucociliary surface that recruits a microbiome such as the case described here is likely to have system-specific features. However, all mucociliary surfaces are subject to the same physical and biological constraints that are imposed by the fluid environment and the evolutionary conserved structure of cilia. As such, our study promises to provide insight into universal mechanisms that drive the recruitment of symbiotic partners

Structure of a putative NTP pyrophosphohydrolase: YP_001813558.1 from Exiguobacterium sibiricum 255-15.

The crystal structure of a putative NTPase, YP_001813558.1 from Exiguobacterium sibiricum 255-15 (PF09934, DUF2166) was determined to 1.78 Å resolution. YP_001813558.1 and its homologs (dimeric dUTPases, MazG proteins and HisE-encoded phosphoribosyl ATP pyrophosphohydrolases) form a superfamily of all-α-helical NTP pyrophosphatases. In dimeric dUTPase-like proteins, a central four-helix bundle forms the active site. However, in YP_001813558.1, an unexpected intertwined swapping of two of the helices that compose the conserved helix bundle results in a `linked dimer' that has not previously been observed for this family. Interestingly, despite this novel mode of dimerization, the metal-binding site for divalent cations, such as magnesium, that are essential for NTPase activity is still conserved. Furthermore, the active-site residues that are involved in sugar binding of the NTPs are also conserved when compared with other α-helical NTPases, but those that recognize the nucleotide bases are not conserved, suggesting a different substrate specificity

Structure of the γ-D-glutamyl-L-diamino acid endopeptidase YkfC from Bacillus cereus in complex with L-Ala-γ-D-Glu: insights into substrate recognition by NlpC/P60 cysteine peptidases.

Dipeptidyl-peptidase VI from Bacillus sphaericus and YkfC from Bacillus subtilis have both previously been characterized as highly specific γ-D-glutamyl-L-diamino acid endopeptidases. The crystal structure of a YkfC ortholog from Bacillus cereus (BcYkfC) at 1.8 Å resolution revealed that it contains two N-terminal bacterial SH3 (SH3b) domains in addition to the C-terminal catalytic NlpC/P60 domain that is ubiquitous in the very large family of cell-wall-related cysteine peptidases. A bound reaction product (L-Ala-γ-D-Glu) enabled the identification of conserved sequence and structural signatures for recognition of L-Ala and γ-D-Glu and, therefore, provides a clear framework for understanding the substrate specificity observed in dipeptidyl-peptidase VI, YkfC and other NlpC/P60 domains in general. The first SH3b domain plays an important role in defining substrate specificity by contributing to the formation of the active site, such that only murein peptides with a free N-terminal alanine are allowed. A conserved tyrosine in the SH3b domain of the YkfC subfamily is correlated with the presence of a conserved acidic residue in the NlpC/P60 domain and both residues interact with the free amine group of the alanine. This structural feature allows the definition of a subfamily of NlpC/P60 enzymes with the same N-terminal substrate requirements, including a previously characterized cyanobacterial L-alanine-γ-D-glutamate endopeptidase that contains the two key components (an NlpC/P60 domain attached to an SH3b domain) for assembly of a YkfC-like active site

The structure of BVU2987 from Bacteroides vulgatus reveals a superfamily of bacterial periplasmic proteins with possible inhibitory function.

Proteins that contain the DUF2874 domain constitute a new Pfam family PF11396. Members of this family have predominantly been identified in microbes found in the human gut and oral cavity. The crystal structure of one member of this family, BVU2987 from Bacteroides vulgatus, has been determined, revealing a β-lactamase inhibitor protein-like structure with a tandem repeat of domains. Sequence analysis and structural comparisons reveal that BVU2987 and other DUF2874 proteins are related to β-lactamase inhibitor protein, PepSY and SmpA_OmlA proteins and hence are likely to function as inhibitory proteins

Counter-Insurgency against ‘kith and kin’?: the British Army in Northern Ireland, 1970–76

This article argues that state violence in Northern Ireland during the period 1970–1976—when violence during the Troubles was at its height and before the re-introduction of the policy of police primacy in 1976—was on a greatly reduced scale from that seen in British counterinsurgency campaigns in the colonies after the Second World War. When the army attempted to introduce measures used in the colonies—curfews, internment without trial—these proved to be extremely damaging to London's political aims in Northern Ireland, namely the conciliation of the Catholic minority within the United Kingdom and the defeat of the IRA. However, the insistence by William Whitelaw, secretary of state for Northern Ireland (1972–73), on ‘throttling back'—the release of internees and the imposition of unprecedented restrictions on the use of violence by the army—put a serious strain on civil-military relations in Northern Ireland. The relatively stagnant nature of the conflict—with units taking casualties in the same small ‘patch’ of territory without opportunities for the types of ‘positive actions’ seen in the colonies—led to some deviancy on the part of small infantry units who sought informal, unsanctioned ways of taking revenge upon the local population. Meanwhile, a disbelieving and defensive attitude at senior levels of command in Northern Ireland meant that informal punitive actions against the local population were often not properly investigated during 1970–72, until more thorough civilian and military investigative procedures were put in place. Finally, a separation of ethnic and cultural identity between the soldiers and the local population—despite their being citizens of the same state—became professionally desirable in order for soldiers to carry out difficult, occasionally distasteful work

Chapter 4 Design Options, Implementation Issues and Evaluating Success of Ecologically Engineered Shorelines

Human population growth and accelerating coastal development have been the drivers for unprecedented construction of artificial structures along shorelines globally. Construction has been recently amplified by societal responses to reduce flood and erosion risks from rising sea levels and more extreme storms resulting from climate change. Such structures, leading to highly modified shorelines, deliver societal benefits, but they also create significant socioeconomic and environmental challenges. The planning, design and deployment of these coastal structures should aim to provide multiple goals through the application of ecoengineering to shoreline development. Such developments should be designed and built with the overarching objective of reducing negative impacts on nature, using hard, soft and hybrid ecological engineering approaches. The design of ecologically sensitive shorelines should be context-dependent and combine engineering, environmental and socioeconomic considerations. The costs and benefits of ecoengineered shoreline design options should be considered across all three of these disciplinary domains when setting objectives, informing plans for their subsequent maintenance and management and ultimately monitoring and evaluating their success. To date, successful ecoengineered shoreline projects have engaged with multiple stakeholders (e.g. architects, engineers, ecologists, coastal/port managers and the general public) during their conception and construction, but few have evaluated engineering, ecological and socioeconomic outcomes in a comprehensive manner. Increasing global awareness of climate change impacts (increased frequency or magnitude of extreme weather events and sea level rise), coupled with future predictions for coastal development (due to population growth leading to urban development and renewal, land reclamation and establishment of renewable energy infrastructure in the sea) will increase the demand for adaptive techniques to protect coastlines. In this review, we present an overview of current ecoengineered shoreline design options, the drivers and constraints that influence implementation and factors to consider when evaluating the success of such ecologically engineered shorelines