Structural role for Tyr-104 in Escherichia coli isopentenyl-diphosphate isomerase: site-directed mutagenesis, enzymology, and protein crystallography

Isopentenyl-diphosphate (IPP):dimethylallyl diphosphate isomerase is a key enzyme in the biosynthesis of isoprenoids. The mechanism of the isomerization reaction involves protonation of the unactivated carbon-carbon double bond in the substrate, but identity of the acidic moiety providing the proton is still not clear. Multiple sequence alignments and geometrical features observed in crystal structures of complexes with IPP isomerase suggest that Tyr-104 could play an important role during catalysis. A series of mutants was constructed by directed mutagenesis and characterized by enzymology. Crystallographic and thermal denaturation data for Y104A and Y104F mutants were obtained. Those data demonstrate the importance of residue Tyr-104 for proper folding of Escherichia coli type I IPP isomerase.     

Optical mapping of DNA: single-molecule-based methods for mapping genomes

The technologies associated with DNA sequencing are rapidly evolving. Indeed, single-molecule DNA sequencing strategies are cheaper and faster than ever before. Despite this progress, every sequencing platform to date relies on reading the genome in small, abstract fragments, typically of less than 1000 bases in length. The overarching aim of the optical map is to complement the information derived from DNA sequencing by providing long-range context on which these short sequence reads can be built. This is typically done using an enzyme to target and modify at short DNA sequences of, say, six bases in length throughout the genome. By accurately placing these short pieces of sequence on long genomic DNA fragments, up to several millions of bases in length, a scaffold for sequence assembly can be obtained. This review focuses on three enzymatic approaches to optical mapping. Optical mapping was first developed using restriction enzymes to sequence-specifically cleave DNA that is immobilized on a surface. More recently, nicking enzymes have found application in the sequence-specific fluorescent labeling of DNA for optical mapping. Such covalent modification allows the DNA to be imaged in solution, and this, in combination with developing nanofluidic technologies, is enabling new high-throughput approaches to mapping. And, finally, this review will discuss the recent development of mapping with subdiffraction-limit precision using methyltransferase enzymes to label the DNA with an ultrahigh density.     

Naegleria gruberi metabolism

The completion of the genome project for Naegleria gruberi provides a unique insight into the metabolic capacities of an organism, for which there is an almost complete lack of experimental data. The metabolism of Naegleria seems to be extremely versatile, as can be expected for a free-living amoeboflagellate, but although considered to be fully aerobic, its genome also predicts important anaerobic traits. Other predictions are that carbohydrates are oxidised to carbon dioxide and water when oxygen is not limiting and that in the absence of oxygen the end-products will be succinate, acetate and minor quantities of ethanol and d-lactate. The hybrid mitochondrion/hydrogenosome has both cytochromes and an [Fe] hydrogenase, but seems to lack pyruvate-ferredoxin oxidoreductase. Genomic information also provides the possibility to identify drugs with a possible mode of action in the fatal primary amoebic meningoencephalitis caused by the closely related opportunistic pathogen Naegleria fowleri.     

Mean residence time of O horizon carbon along a climatic gradient in Scandinavia estimated by 14C measurements of archived soils

We used two datasets of ¹⁴C analyses of archived soil samples to study carbon turnover in O horizons from spruce dominated old-growth stands on well-drained podzols in Scandinavia. The main data set was obtained from archived samples from the National Forest Soil Inventory in Sweden and represents a climatic gradient in temperature. Composite samples from 1966, 1972, 1983 and 2000 from four different regions in a latitude gradient ranging from 57 to 67°N were analysed for ¹⁴C content. Along this gradient the C stock in the O horizon ranges from 2.1 kg m^?2 in the north to 3.7 kg m^?2 in the southwest. The other data set contains ¹⁴C analyses from 1986, 1987, 1991, 1996 and 2004 from the O horizons in Birkenes, Norway. Mean residence times (MRT) were calculated using a two compartment model, with a litter decomposition compartment using mass loss data from the literature for the three-first years of decomposition and a humus decomposition compartment with a fitted constant turnover rate. We hypothesized that the climatic gradient would result in different C turnover in different parts of the country between northern and southern Sweden. The use of archived soil samples was very valuable for constraining the MRT calculations, which showed that there were differences between the regions. Longest MRT was found in the northernmost region (41 years), with decreasing residence times through the middle (36 years) and central Sweden (28 years), then again increasing in the southwestern region (40 years). The size of the soil organic carbon (SOC) pool in the O horizon was mainly related to differences in litter input and to a lesser degree to MRT. Because N deposition leads both to larger litter input and to longer MRT, we suggest that N deposition contributes significantly to the latitudinal SOC gradient in Scandinavia, with approximately twice as much SOC in the O horizon in the south compared to the north. The data from Birkenes was in good agreement with the Swedish dataset with MRT estimated to 34 years.     

Changing Realities—Perspectives on Balinese Rice Cultivation

This paper discusses issues of agrarian change in south-central Bali. The proximity to urban areas, especially the tourist centers along the southern coast, provides many off-farm employment opportunities for small-scale farming households. Although rice farming continues, for many households it has become a side business. The flexible nature of rice farming in terms of labor input and available casual off-farm work allows farming households to allocate their available labor to a variety of on-farm and off-farm income generating activities. The subak which unites farmers in the irrigation and cultivation of the rice crop plays an important role in supporting this flexibility. Still, the future of rice farming and the organization behind it looks rather dim with a younger generation unwilling to work in the “mud” and little appreciation of the many benefits the subak provides not only to the farming but to the wider community.     

Passive immunotherapies protect WRvFire and IHD-J-Luc vaccinia virus-infected mice from lethality by reducing viral loads in the upper respiratory tract and internal organs

Whole-body bioimaging was employed to study the effects of passive immunotherapies on lethality and viral dissemination in BALB/c mice challenged with recombinant vaccinia viruses expressing luciferase. WRvFire and IHD-J-Luc vaccinia viruses induced lethality with similar times to death following intranasal infection, but WRvFire replicated at higher levels than IHD-J-Luc in the upper and lower respiratory tracts. Three types of therapies were tested: licensed human anti-vaccinia virus immunoglobulin intravenous (VIGIV); recombinant anti-vaccinia virus immunoglobulin (rVIG; Symphogen, Denmark), an investigational product containing a mixture of 26 human monoclonal antibodies (HuMAbs) against mature virion (MV) and enveloped virion (EV); and HuMAb compositions targeting subsets of MV or EV proteins. Bioluminescence recorded daily showed that pretreatment with VIGIV (30 mg) or with rVIG (100 μg) on day -2 protected mice from death but did not prevent viral replication at the site of inoculation and dissemination to internal organs. Compositions containing HuMAbs against MV or EV proteins were protective in both infection models at 100 μg per animal, but at 30 μg, only anti-EV antibodies conferred protection. Importantly, the t statistic of the mean total fluxes revealed that viral loads in surviving mice were significantly reduced in at least 3 sites for 3 consecutive days (days 3 to 5) postchallenge, while significant reduction for 1 or 2 days in any individual site did not confer protection. Our data suggest that reduction of viral replication at multiple sites, including respiratory tract, spleen, and liver, as monitored by whole-body bioluminescence can be used to predict the effectiveness of passive immunotherapies in mouse models.     

Scavenger receptor class B type I is solely responsible for the selective uptake of cholesteryl esters from HDL by the liver and the adrenals in mice

Scavenger receptor class B type I (SR-BI) has been identified as a functional HDL binding protein that can mediate the selective uptake of cholesteryl ester (CE) from HDL. To quantify the in vivo role of SR-BI in the process of selective uptake, HDL was labeled with cholesteryl ether ([(3)H] CEt-HDL) and (125)I-tyramine cellobiose ([(125)I]TC-HDL) and injected into SR-BI knockout (KO) and wild-type (WT) mice. In SR-BI KO mice, the clearance of HDL-CE from the blood circulation was greatly diminished (0.043 +/- 0.004 pools/h for SR-BI KO mice vs. 0.106 +/- 0.004 pools/h for WT mice), while liver and adrenal uptake were greatly reduced. Utilization of double-labeled HDL ([(3)H]CEt and [(125)I]TC) indicated the total absence in vivo of the selective decay and liver uptake of CE from HDL in SR-BI KO mice. Parenchymal cells isolated from SR-BI KO mice showed similar association values for [(3)H]CEt and [(125)I]TC in contrast to WT cells, indicating that in parenchymal liver cells SR-BI is the only molecule exerting selective CE uptake from HDL. Thus, in vivo and in vitro, SR-BI is the sole molecule mediating the selective uptake of CE from HDL by the liver and the adrenals, making it the unique target to modulate reverse cholesterol transport.