Molecular recognition of aldehydes by aldehyde dehydrogenase and mechanism of nucleophile activation

Experimental structural data on the state of substrates bound to class 3 Aldehyde Dehydrogenases (ALDH3A1) is currently unknown. We have utilized molecular mechanics (MM) simulations, in conjunction with new force field parameters for aldehydes, to study the atomic details of benzaldehyde binding to ALDH3A1. Our results indicate that while the nucleophilic Cys243 must be in the neutral state to form what are commonly called near-attack conformers (NACs), these structures do not correlate with increased complexation energy calculated with the MM-Generalized Born Molecular Volume (GBMV) method. The negatively charged Cys243 (thiolate form) of ALDH3A1 also binds benzaldehyde in a stable conformation but in this complex the sulfur of Cys243 is oriented away from benzaldehyde yet yields the most favorable MM-GBMV complexation energy. The identity of the general base, Glu209 or Glu333, in ALDHs remains uncertain. The MM simulations reveal structural and possible functional roles for both Glu209 and Glu333. Structures from the MM simulations that would support either glutamate residue as the general base were further examined with Hybrid Quantum Mechanical (QM)/MM simulations. These simulations show that, with the PM3/OPLS potential, Glu209 must go through a step-wise mechanism to activate Cys243 through an intervening water molecule while Glu333 can go through a more favorable concerted mechanism for the same activation process.     

Cutting edge: homeostatic proliferation of peripheral T lymphocytes is regulated by clonal competition

Homeostatic proliferation functions to maintain peripheral T cell numbers and is regulated by cytokines. In this study, we provide evidence that T cell homeostasis is also regulated by clonal competition. Naive polyclonal T cells divided when transferred to TCR transgenic hosts, as did monoclonal T cells when transferred to TCR transgenic hosts of differing clonotype. However, T cells did not divide in hosts of identical clono-type. Transgenic T cell proliferation was inhibited in irradiated hosts of the same clonotype, while cotransferred nontransgenic T cells proliferated extensively. These results show that clonal competition is a component of homeostasis that may contribute to selection of the peripheral T cell repertoire.     

Reproductive behavior in the squid Sepioteuthis australis from South Australia: ethogram of reproductive body patterns

Squids use a diverse range of body patterns for communication. Each pattern consists of a series of chromatic, postural, and locomotor components that are under neural control and can change within fractions of a second. Here we describe an ethogram of 48 body pattern components from in situ observations of reproductively active Sepioteuthis australis. In addition, we identify the total time and average duration that each component is shown, to a resolution of 1 s. Our results suggest that only a few components (e.g., "Golden epaulettes," "Stitchwork fins," and "Rigid arms") are temporally common, that is, shown for more than 80% of the time. In contrast to the component classification reported for other species of squid, for this species we suggest a classification system consisting of "short acute" (lasting for < 10 s); some of these same components were also classified as "medium acute" (11-60 s) or "chronic" (> 60 s). Several body patterning components were previously unreported, as were some of the combinations observed. The significance of these patterning components is discussed within the context of the associated behaviors of the squid on the spawning grounds.     

Design and synthesis of 4,5-disubstituted-thiophene-2-amidines as potent urokinase inhibitors

A study of the S1 binding of lead 5-methylthiothiophene amidine 3, an inhibitor of urokinase-type plasminogen activator, was undertaken by the introduction of a variety of substituents at the thiophene 5-position. The 5-alkyl substituted and unsubstituted thiophenes were prepared using organolithium chemistry. Heteroatom substituents were introduced at the 5-position using a novel displacement reaction of 5-methylsulfonylthiophenes and the corresponding oxygen or sulfur anions. Small alkyl group substitution at the 5-position provided inhibitors equipotent with but possessing improved solubility.     

Normal conjunctival flora in the North American opossum (Didelphis virginiana) and raccoon (Procyon lotor)

We documented the normal conjunctival bacterial flora from 17 opossums (Didelphis virginiana) and 10 raccoons (Procyon lotor) trapped in Manhattan, Kansas (USA) from November 1999 to January 2000. Both raccoons and opossums were free of apparent ocular disease. The inferior conjunctival sacs of each animal were swabbed for aerobic bacterial and Mycoplasma culture and polymerase chain reaction (PCR) for Mycoplasma and Chlamydia detection. All conjunctival samples were positive for one or more species of aerobic bacteria. The most common isolate from opossums was Staphylococcus spp. Other isolates included Streptococcus spp., Bacillus spp., Corynebacterium spp., and Enterococcus faecalis. The most common isolates in raccoons was Bacillus spp. Other isolates included Streptococcus spp., Staphylococcus spp., non-hemolytic Escherichia coli, and Enterococcus faecalis. Mycoplasma culture was negative in samples from opossums and raccoons. Evidence of Mycoplasma and Chlamydia presence was detected by PCR.     

Global analysis of the Brucella melitensis proteome: Identification of proteins expressed in laboratory-grown culture

Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.     

Ischemic preconditioning exaggerates cardiac damage in PKC-delta null mice

Ischemic preconditioning confers cardiac protection during subsequent ischemia-reperfusion, in which protein kinase C (PKC) is believed to play an essential role, but controversial data exist concerning the PKC-delta isoform. In an accompanying study (26), we described metabolic changes in PKC-delta knockout mice. We now wanted to explore their effect on early preconditioning. Both PKC-delta(-/-) and PKC-delta(+/+) mice underwent three cycles of 5-min left descending artery occlusion/5-min reperfusion, followed by 30-min occlusion and 2-h reperfusion. Unexpectedly, preconditioning exaggerated ischemia-reperfusion injury in PKC-delta(-/-) mice. Whereas ischemic preconditioning increased superoxide anion production in PKC-delta(+/+) hearts, no increase in reactive oxygen species was observed in PKC-delta(-/-) hearts. Proteomic analysis of preconditioned PKC-delta(+/+) hearts revealed profound changes in enzymes related to energy metabolism, e.g., NADH dehydrogenase and ATP synthase, with partial fragmentation of these mitochondrial enzymes and of the E(2) component of the pyruvate dehydrogenase complex. Interestingly, fragmentation of mitochondrial enzymes was not observed in PKC-delta(-/-) hearts. High-resolution NMR analysis of cardiac metabolites demonstrated a similar rise of phosphocreatine in PKC-delta(+/+) and PKC-delta(-/-) hearts, but the preconditioning-induced increase in phosphocholine, alanine, carnitine, and glycine was restricted to PKC-delta(+/+) hearts, whereas lactate concentrations were higher in PKC-delta(-/-) hearts. Taken together, our results suggest that reactive oxygen species generated during ischemic preconditioning might alter mitochondrial metabolism by oxidizing key mitochondrial enzymes and that metabolic adaptation to preconditioning is impaired in PKC-delta(-/-) hearts.     

Environmental impacts of hybrid and electric vehicles—a review

Purpose

A literature review is undertaken to understand how well existing studies of the environmental impacts of hybrid and electric vehicles (EV) address the full life cycle of these technologies. Results of studies are synthesized to compare the global warming potential (GWP) of different EV and internal combustion engine vehicle (ICEV) options. Other impacts are compared; however, data availability limits the extent to which this could be accomplished.

Method

We define what should be included in a complete, state-of-the-art environmental assessment of hybrid and electric vehicles considering components and life cycle stages, emission categories, impact categories, and resource use and compare the content of 51 environmental assessments of hybrid and electric vehicles to our definition. Impact assessment results associated with full life cycle inventories (LCI) are compared for GWP as well as emissions of other pollutants. GWP results by life cycle stage and key parameters are extracted and used to perform a meta-analysis quantifying the impacts of vehicle options.

Results

Few studies provide a full LCI for EVs together with assessment of multiple impacts. Research has focused on well to wheel studies comparing fossil fuel and electricity use as the use phase has been seen to dominate the life cycle of vehicles. Only very recently have studies begun to better address production impacts. Apart from batteries, very few studies provide transparent LCIs of other key EV drivetrain components. Estimates of EV energy use in the literature span a wide range, 0.10?C0.24?kWh/km. Similarly, battery and vehicle lifetime plays an important role in results, yet lifetime assumptions range between 150,000?C300,000?km. CO₂ and GWP are the most frequently reported results. Compiled results suggest the GWP of EVs powered by coal electricity falls between small and large conventional vehicles while EVs powered by natural gas or low-carbon energy sources perform better than the most efficient ICEVs. EV results in regions dependant on coal electricity demonstrated a trend toward increased SO _x emissions compared to fuel use by ICEVs.

Conclusions

Moving forward research should focus on providing consensus around a transparent inventory for production of electric vehicles, appropriate electricity grid mix assumptions, the implications of EV adoption on the existing grid, and means of comparing vehicle on the basis of common driving and charging patterns. Although EVs appear to demonstrate decreases in GWP compared to conventional ICEVs, high efficiency ICEVs and grid-independent hybrid electric vehicles perform better than EVs using coal-fired electricity.     

Negative Regulation of TGFβ Signaling by Stem Cell Antigen-1 Protects against Ischemic Acute Kidney Injury

Acute kidney injury, often caused by an ischemic insult, is associated with significant short-term morbidity and mortality, and increased risk of chronic kidney disease. The factors affecting the renal response to injury following ischemia and reperfusion remain to be clarified. We found that the Stem cell antigen-1 (Sca-1), commonly used as a stem cell marker, is heavily expressed in renal tubules of the adult mouse kidney. We evaluated its potential role in the kidney using Sca-1 knockout mice submitted to acute ischemia reperfusion injury (IRI), as well as cultured renal proximal tubular cells in which Sca-1 was stably silenced with shRNA. IRI induced more severe injury in Sca-1 null kidneys, as assessed by increased expression of Kim-1 and Ngal, rise in serum creatinine, abnormal pathology, and increased apoptosis of tubular epithelium, and persistent significant renal injury at day 7 post IRI, when recovery of renal function in control animals was nearly complete. Serum creatinine, Kim-1 and Ngal were slightly but significantly elevated even in uninjured Sca-1-/- kidneys. Sca-1 constitutively bound both TGFβ receptors I and II in cultured normal proximal tubular epithelial cells. Its genetic loss or silencing lead to constitutive TGFβ receptor—mediated activation of canonical Smad signaling even in the absence of ligand and to KIM-1 expression in the silenced cells. These studies demonstrate that by normally repressing TGFβ-mediated canonical Smad signaling, Sca-1 plays an important in renal epithelial cell homeostasis and in recovery of renal function following ischemic acute kidney injury.     

Evolution of E. coli on [U-13C]Glucose Reveals a Negligible Isotopic Influence on Metabolism and Physiology

¹³C-Metabolic flux analysis (¹³C-MFA) traditionally assumes that kinetic isotope effects from isotopically labeled compounds do not appreciably alter cellular growth or metabolism, despite indications that some biochemical reactions can be non-negligibly impacted. Here, populations of Escherichia coli were adaptively evolved for ~1000 generations on uniformly labeled ¹³C-glucose, a commonly used isotope for ¹³C-MFA. Phenotypic characterization of these evolved strains revealed ~40% increases in growth rate, with no significant difference in fitness when grown on either labeled (¹³C) or unlabeled (¹²C) glucose. The evolved strains displayed decreased biomass yields, increased glucose and oxygen uptake, and increased acetate production, mimicking what is observed after adaptive evolution on unlabeled glucose. Furthermore, full genome re-sequencing revealed that the key genetic changes underlying these phenotypic alterations were essentially the same as those acquired during adaptive evolution on unlabeled glucose. Additionally, glucose competition experiments demonstrated that the wild-type exhibits no isotopic preference for unlabeled glucose, and the evolved strains have no preference for labeled glucose. Overall, the results of this study indicate that there are no significant differences between ¹²C and ¹³C-glucose as a carbon source for E. coli growth.