Molecular basis of secondary relaxation in stiff-chain glassy polymers.

Recent progress in establishing local order in polycarbonate-like glasses using rotational echo double resonance and centerband-only detection of exchange solid-state nuclear magnetic resonance (NMR) has stimulated a renewed attempt to connect molecular motion within glassy polymers and the mechanical properties of the glass. We have in fact established a correlation between molecular motion characterized by NMR and the mechanical secondary relaxation (tan δ) for nine polycarbonate-like glasses. All of the NMR and mechanical data are for T ≪ Tg. The resulting structural insights suggest that the chains of these polymers are simultaneously both Flory random coils and Vol'kenstein bundles. The cooperative motions of groups of bundles can be described qualitatively by a variety of constrained-kinetics models of the glass. All of the models share a common trait for large-amplitude motion: an exponential increase in the time required for an inter-bundle dilation event with a linear increase in bundle group size. This dependence and a locally ordered Vol'kenstein bundle lead to an understanding of the surprising 60° (K) shift of tan δ to higher temperature for ring-fluoro-polycarbonate relative to that of polycarbonate by the apparently minor substitution of a fluorine for a hydrogen on every fourth ring.

Long-term blood pressure lowering and cGMP-activating actions of the novel ANP analog MANP.

Based on the cardiac hormone atrial natriuretic peptide (ANP) and its seminal role in blood pressure (BP) homeostasis, we investigated the chronic BP lowering actions of a novel ANP analog currently entering clinical trials for hypertension. Previous reports demonstrate that this analog MANP activates the guanylyl cyclase A receptor (GC-A) and results in more potent biological actions compared with ANP; thus, it may represent a new therapeutic drug for hypertension. A major goal of this study was to establish that chronic subcutaneous delivery of MANP is feasible and hypotensive together with cGMP effects. We investigated the BP-lowering and cGMP-activating actions of acute and chronic subcutaneous delivery in normal and hypertensive rats. Furthermore, we explored vascular mechanisms of MANP in human aortic smooth muscle cells (HASMC) and ex vivo in isolated arteries. In normal rats with a single subcutaneous injection, MANP promoted robust dose-dependent BP-lowering actions and natriuresis, together with cGMP activation. Most importantly in hypertensive rats, once-a-day subcutaneous injection of MANP for 7 days induced cGMP elevation and long-term BP reduction compared with vehicle. Mechanistically, in HASMC, MANP activated cGMP and attenuated angiotensin II-mediated increases in intracellular Ca2+ levels while directly vasorelaxing arterial rings. Our study demonstrates for the first time the effectiveness of subcutaneous administration of MANP for 7 days and provides innovative, vascular mechanisms of BP regulation supporting its continued development as a novel therapeutic for hypertension.

Mechanistic Basis for ATP-Dependent Inhibition of Glutamine Synthetase by Tabtoxinine-ß-lactam.

Tabtoxinine-ß-lactam (TßL), also known as wildfire toxin, is a time- and ATP-dependent inhibitor of glutamine synthetase produced by plant pathogenic strains of Pseudomonas syringae. Here we demonstrate that recombinant glutamine synthetase from Escherichia coli phosphorylates the C3-hydroxyl group of the TßL 3-(S)-hydroxy-ß-lactam (3-HßL) warhead. Phosphorylation of TßL generates a stable, noncovalent enzyme-ADP-inhibitor complex that resembles the glutamine synthetase tetrahedral transition state. The TßL ß-lactam ring remains intact during enzyme inhibition, making TßL mechanistically distinct from traditional ß-lactam antibiotics such as penicillin. Our findings could enable the design of new 3-HßL transition state inhibitors targeting enzymes in the ATP-dependent carboxylate-amine ligase superfamily with broad therapeutic potential in many disease areas.

Surface proteins and the formation of biofilms by Staphylococcus aureus.

Staphylococcus aureus biofilms pose a serious clinical threat as reservoirs for persistent infections. Despite this clinical significance, the composition and mechanism of formation of S. aureus biofilms are unknown. To address these problems, we used solid-state NMR to examine S. aureus (SA113), a strong biofilm-forming strain. We labeled whole cells and cell walls of planktonic cells, young biofilms formed for 12-24h after stationary phase, and more mature biofilms formed for up to 60h after stationary phase. All samples were labeled either by (i) [15N]glycine and l-[1-13C]threonine, or in separate experiments, by (ii) l-[2-13C,15N]leucine. We then measured 13C-15N direct bonds by C{N} rotational-echo double resonance (REDOR). The increase in peptidoglycan stems that have bridges connected to a surface protein was determined directly by a cell-wall double difference (biofilm REDOR difference minus planktonic REDOR difference). This procedure eliminates errors arising from differences in 15N isotopic enrichments and from the routing of 13C label from threonine degradation to glycine. For both planktonic cells and the mature biofilm, 20% of pentaglycyl bridges are not cross-linked and are potential surface-protein attachment sites. None of these sites has a surface protein attached in the planktonic cells, but one-fourth have a surface protein attached in the mature biofilm. Moreover, the leucine-label shows that the concentration of ß-strands in leucine-rich regions doubles in the mature biofilm. Thus, a primary event in establishing a S. aureus biofilm is extensive decoration of the cell surface with surface proteins that are linked covalently to the cell wall and promote cell-cell adhesion.

Lactate metabolism is associated with mammalian mitochondria.

It is well established that lactate secreted by fermenting cells can be oxidized or used as a gluconeogenic substrate by other cells and tissues. It is generally assumed, however, that within the fermenting cell itself, lactate is produced to replenish NAD+ and then is secreted. Here we explore the possibility that cytosolic lactate is metabolized by the mitochondria of fermenting mammalian cells. We found that fermenting HeLa and H460 cells utilize exogenous lactate carbon to synthesize a large percentage of their lipids. Using high-resolution mass spectrometry, we found that both 13C and 2-2H labels from enriched lactate enter the mitochondria. The lactate dehydrogenase (LDH) inhibitor oxamate decreased respiration of isolated mitochondria incubated in lactate, but not of isolated mitochondria incubated in pyruvate. Additionally, transmission electron microscopy (TEM) showed that LDHB localizes to the mitochondria. Taken together, our results demonstrate a link between lactate metabolism and the mitochondria of fermenting mammalian cells.

Desleucyl-Oritavancin with a Damaged d-Ala-d-Ala Binding Site Inhibits the Transpeptidation Step of Cell-Wall Biosynthesis in Whole Cells of Staphylococcus aureus.

We have used solid-state nuclear magnetic resonance to characterize the exact nature of the dual mode of action of oritavancin in preventing cell-wall assembly in Staphylococcus aureus. Measurements performed on whole cells labeled selectively in vivo have established that des-N-methylleucyl-N-4-(4-fluorophenyl)benzyl-chloroeremomycin, an Edman degradation product of [19F]oritavancin, which has a damaged d-Ala-d-Ala binding aglycon, is a potent inhibitor of the transpeptidase activity of cell-wall biosynthesis. The desleucyl drug binds to partially cross-linked peptidoglycan by a cleft formed between the drug aglycon and its biphenyl hydrophobic side chain. This type of binding site is present in other oritavancin-like glycopeptides, which suggests that for these drugs a similar transpeptidase inhibition occurs.

Whole-Cell Detection of C-P Bonds in Bacteria.

Naturally produced molecules possessing a C-P bond, such as phosphonates and phosphinates, remain vastly underexplored. Although success stories like fosfomycin have reinvigorated small molecule phosphonate discovery efforts, bioinformatic analyses predict an enormous unexplored biological reservoir of C-P bond-containing molecules, including those attached to complex macromolecules. However, high polarity, a lack of chromophores, and complex macromolecular association impede phosphonate discovery and characterization. Here we detect widespread transcriptional activation of phosphonate biosynthetic machinery across diverse bacterial phyla and describe the use of solid-state nuclear magnetic resonance to detect C-P bonds in whole cells of representative Gram-negative and Gram-positive bacterial species. These results suggest that phosphonate tailoring is more prevalent than previously recognized and set the stage for elucidating the fascinating chemistry and biology of these modifications.

Characterization of the tertiary structure of the peptidoglycan of Enterococcus faecalis.

Solid-state NMR spectra of whole cells and isolated cell walls of Enterococcus faecalis grown in media containing combinations of 13C and 15N specific labels in d- and l-alanine and l-lysine (in the presence of an alanine racemase inhibitor alaphosphin) have been used to determine the composition and architecture of the cell-wall peptidoglycan. The compositional variables include the concentrations of (i) peptidoglycan stems without bridges, (ii) d-alanylated wall teichoic acid, (iii) cross-links, and (iv) uncross-linked tripeptide and tetra/pentapeptide stems. Connectivities of l-alanyl carbonyl­carbon bridge labels to d-[3-13C]alanyl and l-[ε-15N]lysyl stem labels prove that the peptidoglycan of E. faecalis has the same hybrid short-bridge architecture (with a mix of parallel and perpendicular stems) as the FemA mutant of Staphylococcus aureus, in which the cross-linked stems are perpendicular to one another and the cross-linking is close to the ideal 50% value. This is the first determination of the cell-wall chemical and geometrical architecture of whole cells of E. faecalis, a major source of nosocomial infections worldwide.

REDOR constraints on the peptidoglycan lattice architecture of Staphylococcus aureus and its FemA mutant.

The peptidoglycan of Gram-positive bacteria consists of glycan chains with attached short peptide stems cross-linked to one another by glycyl bridges. The bridge of Staphylococcus aureus has five glycyl units and that of its FemA mutant has one. These long- and short-bridge cross-links create totally different cell-wall architectures. S. aureus and its FemA mutant grown in the presence of an alanine-racemase inhibitor were labeled with d-[1-¹³C]alanine, l-[3-¹³C]alanine, [2-¹³C]glycine, and l-[5-¹9F]lysine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance (REDOR) NMR of isolated cell walls was used to measure internuclear distances between ¹³C-labeled alanines and ¹9F-labeled lysine incorporated in the peptidoglycan. The alanyl ¹³C labels in the parent strain were preselected for C{F} and C{P} REDOR measurement by their proximity to the glycine label using ¹³C¹³C spin diffusion. The observed ¹³C¹³C and ¹³C³¹P distances are consistent with a tightly packed architecture containing only parallel stems in a repeating structural motif within the peptidoglycan. Dante selection of d-alanine and l-alanine frequencies followed by ¹³C¹³C spin diffusion rules out scrambling of carbon labels. Cell walls of FemA were also labeled by a combination of d-[1-¹³C]alanine and l-[¹5N]alanine. Proximity of chains was measured by C{N} and N{C} REDOR distances and asymptotic plateaus, and both were consistent with a mixed-geometry model. Binding of an ¹9F-labeled eremomycin analog in the FemA cell wall matches that of binding to the parent-strain cell wall and reveals the proximity of parallel stems in the alternating parallel-perpendicular mixed-geometry model for the FemA peptidoglycan lattice.

Derivation of the freshwater fish fauna of Central America revisited: Myers's hypothesis in the twenty-first century.

Although attempts to understand Central American freshwater fish provincialism date to the 1960s, early efforts lacked the wealth of distributional data now available. Biogeographic work on Central American freshwater fishes has been largely descriptive and regional, and lacked a broader synthesis. Here we use parsimony analysis of endemicity (PAE) to elucidate faunistic relationships between major drainages and to delineate areas of endemism. We then perform a Brooks parsimony analysis (BPA) on the resulting areas. The PAE recovered a primary division between four Pacific and six Atlantic slope areas of endemism. In contrast, the BPA recovered two Central American geographic clades, one sharing a history with North America and the other with South America. Fish diversity is uneven across Central America, with greater diversity in areas adjacent to the more species-rich regions of North and South America. In northern and nuclear Central America, the paucity of ostariophysan freshwater fishes such as catfishes and characins (groups that dominate adjacent regions) contrasts with high species richness of poeciliids and cichlids. Results of this study are consistent with Myer's hypothesis that poeciliids and cichlids dispersed to Northern or Nuclear Middle America early in the Cenozoic, long before the Plio-Pleistocene rise of the Isthmus of Panama.

Cross-link formation and peptidoglycan lattice assembly in the FemA mutant of Staphylococcus aureus.

Staphylococcus aureus FemA mutant grown in the presence of an alanine-racemase inhibitor was labeled with d-[1-(13)C]alanine, l-[3-(13)C]alanine, [2-(13)C]glycine, and l-[5-(19)F]lysine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance (REDOR) NMR of isolated cell walls was used to measure internuclear distances between (13)C-labeled alanines and (19)F-labeled lysine incorporated in the peptidoglycan. The alanyl (13)C labels were preselected for REDOR measurement by their proximity to the glycine label using (13)C-(13)C spin diffusion. The observed (13)C-(13)C and (13)C-(19)F distances are consistent with a tightly packed, hybrid architecture containing both parallel and perpendicular stems in a repeating structural motif within the peptidoglycan.

Differential incorporation of glucose into biomass during Warburg metabolism.

It is well established that most cancer cells take up an increased amount of glucose relative to that taken up by normal differentiated cells. The majority of this glucose carbon is secreted from the cell as lactate. The fate of the remaining glucose carbon, however, has not been well-characterized. Here we apply a novel combination of metabolomic technologies to track uniformly labeled glucose in HeLa cancer cells. We provide a list of specific intracellular metabolites that become enriched after being labeled for 48 h and quantitate the fraction of consumed glucose that ends up in proteins, peptides, sugars/glycerol, and lipids.

Uniformity of glycyl bridge lengths in the mature cell walls of fem mutants of methicillin-resistant Staphylococcus aureus.

Peptidoglycan (PG) composition in intact cells of methicillin-resistant Staphylococcus aureus (MRSA) and its isogenic Fem mutants has been characterized by measuring the glycine content of PG bridge structures by solid-state nuclear magnetic resonance (NMR). The glycine content estimated from integrated intensities (rather than peak heights) in the cell walls of whole cells was increased by approximately 30% for the FemA mutant and was reduced by 25% for the FemB mutant relative to expected values for homogeneous structures. In contrast, the expected compositions were observed in isolated cell walls of the same mutants. For FemA mutant whole cells, the increase was due to the presence of triglycyl bridge PG units (confirmed directly by mass spectrometric analysis), which constituted 10% of the total PG. These species were coalesced in some sort of a lattice or aggregate with spatial proximity to other PG bridges. This result suggests that the triglycyl-bridged PG units form a PG-like structure that is not incorporated into the mature cell wall.

Staphylococcus aureus peptidoglycan stem packing by rotational-echo double resonance NMR spectroscopy.

Staphylococcus aureus grown in the presence of an alanine-racemase inhibitor was labeled with d-[1-(13)C]alanine and l-[(15)N]alanine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance NMR of intact whole cells was used to measure internuclear distances between (13)C and (15)N of labeled amino acids incorporated in the peptidoglycan, and from those labels to (19)F of a glycopeptide drug specifically bound to the peptidoglycan. The observed (13)C-(15)N average distance of 4.1-4.4 Å between d- and l-alanines in nearest-neighbor peptide stems is consistent with a local, tightly packed, parallel-stem architecture for a repeating structural motif within the peptidoglycan of S. aureus.

Locations of the hydrophobic side chains of lipoglycopeptides bound to the peptidoglycan of Staphylococcus aureus.

Glycopeptides whose aminosugars have been modified by attachment of hydrophobic side chains are frequently active against vancomycin-resistant microorganisms. We have compared the conformations of six such fluorinated glycopeptides (with side chains of varying length) complexed to cell walls labeled with d-[1-(13)C]alanine, [1-(13)C]glycine, and l-[ε-(15)N]lysine in whole cells of Staphylococcus aureus. The internuclear distances from (19)F of the bound drug to the (13)C and (15)N labels of the peptidoglycan, and to the natural abundance (31)P of lipid membranes and teichoic acids, were determined by rotational-echo double resonance NMR. The drugs did not dimerize, and their side chains did not form membrane anchors but instead became essential parts of secondary binding to pentaglycyl bridge segments of the cell-wall peptidoglycan.

The isotridecanyl side chain of plusbacin-A3 is essential for the transglycosylase inhibition of peptidoglycan biosynthesis.

Plusbacin-A3 (pb-A3) is a cyclic lipodepsipeptide that exhibits antibacterial activity against multidrug-resistant Gram-positive pathogens. Plusbacin-A3 is thought not to enter the cell cytoplasm, and its lipophilic isotridecanyl side chain is presumed to insert into the membrane bilayer, thereby facilitating either lipid II binding or some form of membrane disruption. Analogues of pb-A3, [(2)H]pb-A3 and deslipo-pb-A3, were synthesized to test membrane insertion as a key to the mode of action. [(2)H]pb-A3 has an isotopically (2)H-labeled isopropyl subunit of the lipid side chain, and deslipo-pb-A3 is missing the isotridecanyl side chain. Both analogues have the pb-A3 core structure. The loss of antimicrobial activity in deslipo-pb-A3 showed that the isotridecanyl side chain is crucial for the mode of action of the drug. However, rotational-echo double-resonance nuclear magnetic resonance characterization of [(2)H]pb-A3 bound to [1-(13)C]glycine-labeled whole cells of Staphylococcus aureus showed that the isotridecanyl side chain does not insert into the lipid membrane but instead is found in the staphylococcal cell wall, positioned near the pentaglycyl cross-bridge of the cell-wall peptidoglycan. Addition of [(2)H]pb-A3 during the growth of S. aureus resulted in the accumulation of Park's nucleotide, consistent with the inhibition of the transglycosylation step of peptidoglycan biosynthesis.

Shared and unique morphological responses of stream fishes to anthropogenic habitat alteration.

Understanding population-level responses to novel selective pressures can elucidate evolutionary consequences of human-altered habitats. Stream impoundments (reservoirs) alter riverine ecosystems worldwide, exposing stream fishes to uncommon selective pressures. Assessing phenotypic trait divergence in reservoir habitats will be a first step in identifying the potential evolutionary and ecological consequences of stream impoundments. We tested for body shape divergence in four stream-adapted fishes found in both habitats within three separate basins. Shape variation among fishes was partitioned into shared (exhibited by all species) and unique (species-specific) responses to reservoir habitats. All fishes demonstrated consistent significant shared and unique morphological responses to reservoir habitats. Shared responses were linked to fin positioning, decreased body depths and larger caudal areas; traits likely related to locomotion. Unique responses were linked to head shape, suggesting species-specific responses to abiotic conditions or changes to their trophic ecology in reservoirs. Our results highlight how human-altered habitats can simultaneously drive similar and unique trait divergence in native populations.

Contrasting phylogeographic histories between broadly sympatric topminnows in the Fundulus notatus species complex.

Sympatrically distributed closely related species provide opportunities for studying evolutionary patterns of diversification. Such studies must account for historical contingencies in interpreting contemporary patterns of variation. Topminnows in the Fundulus notatus species complex are distributed sympatrically across much of the southern and Midwestern United States. Throughout most of their ranges F. olivaceus is often found in headwater stream habitats, and F. notatus is more typically distributed along the margins of larger river habitats. However, in some drainages, ecological associations of the respective species are reversed, with F. notatus populations isolated in headwater streams and F. olivaceus in downstream river habitats. Phylogeographic analyses of AFLP marker and multi-locus sequence data detected historical isolation in F. notatus consistent with pre-Pleistocene drainage patterns. Four F. notatus clades corresponded to (i) the Western Gulf Slope, (ii) the southwestern Ouachita Highlands, (iii) the Mobile Basin, and (iv) central Coastal Plain and Mississippi River Basin. In contrast, a relative lack of range-wide geographic structure in F. olivaceus is consistent with recent range expansion over much of the same geographic area. The southwestern Ouachita Highlands and Mobile Basin F. notatus clades corresponded to regions where ecological associations between the two species are reversed, providing evidence of the independent evolution of variation in contemporary habitat associations. Fundulus olivaceus from several drainages demonstrated introgression of mitochondrial DNA from F. notatus, but none of the sites in this study included individuals with hybrid ancestry in their nuclear genome. Phylogenetic analyses that included only nuclear loci supported the reciprocal monophyly of F. notatus, F. olivaceus and a third narrowly endemic species, Fundulus euryzonus, and supported a sister relationship between F. olivaceus and F. euryzonus.

Variable rates of hybridization among contact zones between a pair of topminnow species, Fundulus notatus and F. olivaceus.

Pairs of species that exhibit broadly overlapping distributions, and multiple geographically isolated contact zones, provide opportunities to investigate the mechanisms of reproductive isolation. Such naturally replicated systems have demonstrated that hybridization rates can vary substantially among populations, raising important questions about the genetic basis of reproductive isolation. The topminnows, Fundulus notatus and F. olivaceus, are reciprocally monophyletic, and co-occur in drainages throughout much of the central and southern United States. Hybridization rates vary substantially among populations in isolated drainage systems. We employed genome-wide sampling to investigate geographic variation in hybridization, and to assess the possible importance of chromosome fusions to reproductive isolation among nine separate contact zones. The species differ by chromosomal rearrangements resulting from Robertsonian (Rb) fusions, so we hypothesized that Rb fusion chromosomes would serve as reproductive barriers, exhibiting steeper genomic clines than the rest of the genome. We observed variation in hybridization dynamics among drainages that ranged from nearly random mating to complete absence of hybridization. Contrary to predictions, our use of genomic cline analyses on mapped species-diagnostic SNP markers did not indicate consistent patterns of variable introgression across linkage groups, or an association between Rb fusions and genomic clines that would be indicative of reproductive isolation. We did observe a relationship between hybridization rates and population phylogeography, with the lowest rates of hybridization tending to be found in populations inferred to have had the longest histories of drainage sympatry. Our results, combined with previous studies of contact zones between the species, support population history as an important factor in explaining variation in hybridization rates.

Carbon partitioning in soybean (Glycine max) leaves by combined (11) C and (13) C labeling.

We labeled soybean (Glycine max) leaves with 200 and 600 ppm (13) CO(2) spiked with (11) CO(2) and examined the effects of light intensity and water stress on metabolism by using a combination of direct positron imaging and solid-state (13) C nuclear magnetic resonance (NMR) of the same leaf. We first made 60-min movies of the transport of photosynthetically assimilated (11) C labels. The positron imaging identified zones or patches within which variations in metabolism could be probed later by NMR. At the end of each movie, the labeled leaf was frozen in liquid nitrogen to stop metabolism, the leaf was lyophilized, and solid-state NMR was used either on the whole leaf or on various leaf fragments. The NMR analysis determined total (13) C incorporation into sugars, starch, proteins, and protein precursors. The combination of (11) C and (13) C analytical techniques has led to three major conclusions regarding photosynthetically heterogeneous soybean leaves: transient starch deposition is not the temporary storage of sucrose excluded from a saturated sugar-transport system; peptide synthesis is reduced under high-light, high CO(2) conditions; and all glycine from the photorespiratory pathway is routed to proteins within photosynthetically active zones when the leaf is water stressed and under high-light and low CO(2) conditions.