Targeted disruption of the ubiquitous CNC-bZIP transcription factor, Nrf-1, results in anemia and embryonic lethality in mice.

The CNC-basic leucine zipper (CNC-bZIP) family is a subfamily of bZIP proteins identified from independent searches for factors that bind the AP-1-like cis-elements in the beta-globin locus control region. Three members, p45-Nf-e2, Nrf-1 and Nrf-2 have been identified in mammals. Expression of p45-Nf-e2 is largely restricted to hematopoietic cells while Nrf-1 and Nrf-2 are expressed in a wide range of tissues. To determine the function of Nrf-1, targeted disruption of the Nrf-1 gene was carried out. Homozygous Nrf-1 mutant mice are anemic due to a non-cell autonomous defect in definitive erythropoiesis and die in utero.     

Perspective: peptides as mimics of transmembrane segments in proteins.

Peptide-based approaches to protein structure within membranes have proven enormously valuable. When one focusses on the detailed manner through which membrane proteins actually traverse the cell bilayer, a simple observation emerges: helical peptide segments of 20 amino acids each constitute the only tangible connection between the inside and outside of the cell. Thus, a major step towards understanding the key relationships between biological function and membrane protein structure can be taken through characterization, by composition, sequence, chain length, hydrophobicity and conformation, of hydrophobic peptides designed as mimics of transmembrane segments.     

Life cycle inventories of electricity generation and power supply in version 3 of the ecoinvent database—part I: electricity generation

Purpose

Life cycle inventories (LCI) of electricity generation and supply are among the main determining factors regarding life cycle assessment (LCA) results. Therefore, consistency and representativeness of these data are crucial. The electricity sector has been updated and substantially extended for ecoinvent version 3 (v3). This article provides an overview of the electricity production datasets and insights into key aspects of these v3 inventories, highlights changes and describes new features.

Methods

Methods involved extraction of data and analysis from several publically accessible databases and statistics, as well as from the LCA literature. Depending on the power generation technology, either plant-specific or region-specific average data have been used for creating the new power generation inventories representing specific geographies. Whenever possible, the parent–child relationship was used between global and local activities. All datasets include a specific technology level in order to support marginal mixes used in the consequential version of ecoinvent. The use of parameters, variables and mathematical relations enhances transparency. The article focuses on documentation of LCI data on the unlinked unit process level and presents direct emission data of the electricity-generating activities.

Results and discussion

Datasets for electricity production in 71 geographic regions (geographies) covering 50 countries are available in ecoinvent v3. The number of geographies exceeds the number of countries due to partitioning of power generation in the USA and Canada into several regions. All important technologies representing fossil, renewable and nuclear power are modelled for all geographies. The new inventory data show significant geography-specific variations: thermal power plant efficiencies, direct air pollutant emissions as well as annual yields of photovoltaic and wind power plants will have significant impacts on cumulative inventories. In general, the power plants operating in the 18 newly implemented countries (compared to ecoinvent v2) are on a lower technology level with lower efficiencies and higher emissions. The importance of local datasets is once more highlighted.

Conclusions

Inventories for average technology-specific electricity production in all globally important economies are now available with geography-specific technology datasets. This improved coverage of power generation representing 83 % of global electricity production in 2008 will increase the quality of and reduce uncertainties in LCA studies worldwide and contribute to a more accurate estimation of environmental burdens from global production chains. Future work on LCI of electricity production should focus on updates of the fuel chain and infrastructure datasets, on including new technologies as well as on refining of the local data.

     

Case Series: Report of the First Two Human Indigenous Cases of <Emphasis Type="Italic">Cryptococcus gattii</Emphasis> Infection in Eastern Canada

We report the two first cases of human C. gattii meningoencephalitis acquired on the Canadian east coast, from the province of Quebec. Unlike C. neoformans, C. gattii is not known to have an established ecological niche on the North American east coast. C. gattii has recently been responsible for major outbreaks in British Columbia, Canada, and in the American pacific northwest. However, no human cases acquired in other Canadian provinces have been reported to our knowledge. The source of acquisition remains unclear for both patients but since neither had traveled outside of the province of Quebec, we discuss the possibilities of environmental and animal-associated acquisition, as well as the possible established endemicity in new areas. These cases add to the growing reported human and animal cases in areas previously not thought to be endemic for C. gattii.     

A Unified Scheme for Initiation and Conformational Adaptation of Human Apolipoprotein E N-terminal Domain upon Lipoprotein Binding and for Receptor Binding Activity

We report here a high-resolution NMR structure of the complete receptor-binding domain of human apolipoprotein E3 (apoE3-NT). Similar to the crystal structure of apoE-NT, the NMR structure displayed an elongated four-helix bundle. However, additional unique structural features were also observed. The segments in the N and C termini, which were missing in the crystal structure, formed α-helices having extensive tertiary contacts with the bundle, which oriented these short helices at specific positions for receptor binding activity. Several buried hydrophilic residues observed in the bundle were located strategically between helices 1 and 2 and between helices 3 and 4, significantly destabilizing these helix-helix interfaces. In addition, these buried hydrophilic residues formed buried H-bonds, which may play a key role in specific lipid-free helix bundle recovery. A short helix, nHelix C, was fully solvent-exposed and nearly perpendicular to the bundle. This short helix likely plays a critical role in initiating protein-lipid interaction, causing a preferred conformational adaptation of the bundle at the weaker helix-helix interfaces. This produces an open conformation with two lobes of helices, helices 1 and 4 and helices 2 and 3, which may be the competent conformation for receptor binding activity. Thus, the NMR structure suggests a unified scheme for the initiation and helix bundle opening of apoE-NT upon lipoprotein-binding and for receptor binding activity.Human apolipoprotein E (apoE)² is a 299-residue plasma-exchangeable apolipoprotein with the primary function of transporting lipids from one tissue to another. ApoE performs its functions via interactions with the low-density lipoprotein receptor (LDLR) superfamily (1). The high affinity binding of apoE to the receptors allows apoE-associated lipoprotein particles to be targeted for endocytosis and intracellular degradation. As a subclass of high-density lipoprotein, apoE also influences both cholesterol efflux and influx, thus playing an important role in reverse cholesterol transport (2, 3). Three major isoforms of apoE have been identified: ApoE3 has a cysteine at position 112 and an arginine at position 158, whereas apoE2 has cysteines and apoE4 has arginines at both positions. Although these isoforms differ in only two residues, they show profound functional differences. Recent evidence indicates that apoE is also critical in several other important biological processes, including Alzheimer disease, cognitive functioning, immunoregulation, cell signaling, and infectious diseases (4).ApoE is a two-domain protein that contains a 22-kDa N-terminal domain (residues 1-191) and a 10-kDa C-terminal domain (residues 216-299) linked by a protease sensitive hinge region. Although the N-terminal domain of apoE (apoE-NT) is primarily responsible for LDL-receptor binding, the C-terminal domain (apoE-CT) binds to lipoprotein with a high affinity (1). The x-ray crystal structure of lipid-free apoE-NT reveals a globular up-and-down four-helix bundle (5). The major receptor-binding region, residues 130-150, is located on the fourth helix. The positively charged residues (Lys and Arg) in this region are critical for interacting with the negatively charged residues in the receptor (1, 6). This structure only contains residues 24-164, whereas the rest of the regions are disordered. However, experimental evidence indicates that regions beyond residues 24-164 are also critical for LDLR binding activity. For example, deletion of residues 167-185 reduces the apoE3 LDLR binding activity to 15%, and a mutation at position Arg-172 reduces the LDLR binding activity to only ∼2% (7). In addition, an E3K mutant of apoE3 enhances the LDLR binding activity by 2-fold (8). Although the x-ray crystal structure of apoE-NT provides a structural explanation of the major receptor-binding domain of apoE, this structure does not explain the above described important experimental data. Thus, our understanding of the structural basis of the receptor binding activity of apoE remains incomplete.Previous studies using truncation mutants have shown that apoE(1-183) displays nearly 100% LDLR binding activity (9), suggesting that residues beyond position 183 are not important in LDLR binding. We report here a high-resolution NMR structure of the complete LDLR-binding domain of apoE3. Interestingly, our NMR structure shows that the N and C termini form α-helical structures that have extensive contacts with the helix bundle, orienting the two termini at specific positions for potential receptor binding. The NMR structure also displays several novel structural features that may provide the structural basis of a unified scheme for initiation and conformational adaptation of apoE-NT upon lipoprotein binding.     

On the possible methods for the mathematical description of the ball and chain model of ion channel inactivation

Ion channels are large transmembrane proteins that are able to conduct small inorganic ions. They are characterized by high selectivity and the ability to gate, i.e. to modify their conductance in response to different stimuli. One of the types of gating follows the ball and chain model, according to which a part of the channel’s protein forms a ball connected with the intracellular side of the channel by a polypeptide chain. The ball is able to modify the conductance of the channel by properly binding to and plugging the channel pore. In this study, the polypeptide ball is treated as a Brownian particle, the movements of which are limited by the length of the chain. The probability density of the ball’s position is resolved by different diffusional operators — parabolic (including the case with drift), hyperbolic, and fractional. We show how those different approaches shed light on different aspects of the movement. We also comment on some features of the survival probabilities (which are ready to be compared with electrophysiological measurements) for issues based on the above operators. Paper authored by participants of the international conference: International Workshop on Ionic Channels, Szczyrk, Poland, May 27 – June 01, 2007. Publication cost was covered by the organisers of this meeting.     

Apoptosis stalks <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> maintained in continuous culture condition

Background

Growth kinetic of Plasmodium falciparum in culture or in the host fall short of expected growth rate considering that there are 4 x 10⁶/µL red blood cell (RBCs) available for invasion and about 16 merozoites growing in each infected RBC. This study determined whether apoptotic machinery is operable to keep the parasite population under check.

Methods

A synchronized culture of P. falciparum (Dd2 strain) was initiated at 0.5% ring stage parasitaemia and kept under conditions not limiting for RBCs and nutrient by adjusting hematocrit to 5% at each schizogony and changing growth media daily. Parasite growth pattern and morphology was evaluated by blood smear microscopy and flow-cytometry using SYBR green. The apoptotic processes were evaluated for evidence of: DNA fragmentation by TUNEL, collapse of mitochondria membrane potential (ΔΨ_m) by TMRE, expression of metacaspse gene by RT-qPCR and by probing parasite proteins with anti-caspase antibodies.

Results

From the seeding parasitaemia of 0.5%, the parasites doubled every 48 hours to a parasitaemia of 4%. Thereafter, the growth stagnated and the culture consistently crashed at about 6% parasitaemia. ΔΨ_m potential collapsed as the parasite density increased and DNA fragmentation increased steadily from 0.2% to ~6%. The expression of metacaspase gene and protein was observed in all stages, but their abundance was variable among the stages.

Conclusion

These findings suggest existence of P. falciparum quorum sensing that keep the parasite population under check.

     

Mechanical properties of the tracheal mucosal membrane in the rabbit. I. steady-state stiffness as a function of age.

Airway responsiveness is exaggerated in infancy and declines with maturation. These age-related differences (R.S. Tepper, T. Du, A. Styhler, M. Ludwig, and J.G. Martin. Am. J. Respir. Crit. Care Med. 151: 836-840, 1995; R.S. Tepper, S.J. Gunst, C.M. Doerschuk, Y. Shen, and W. Bray. J. Appl. Physiol. 78: 505-512, 1995; R.S. Tepper, J. Stevens, and H. Eigen. Am. J. Respir. Crit. Care Med. 149: 678-681, 1994) could be due to changes in the smooth muscle, the lung, and/or the airway wall. Folding of the mucosal membrane can provide an elastic load (R.K. Lambert, J. Appl. Physiol. 71: 666-673, 1991), which impedes smooth muscle shortening. We hypothesized that increased stiffness of the mucosal membrane occurs during aging, causing an increased mechanical load on airway smooth muscle and a decrease in airway responsiveness. Forty female New Zealand White rabbits between 0.75 and 35 mo of age were studied. Rectangular mucosal membrane strips oriented both longitudinally and circumferentially to the long axis of the trachea were dissected, and the stress-strain relationships of each strip were tested. The results showed that the membrane was stiffer in the longitudinal than in the circumferential direction of the airway. However, there was no significant change with age in either orientation. We conclude that the mechanical properties of the airway mucosal membrane did not change during maturation and were not likely to influence age-related changes in airway responsiveness.