Changes In Macromolecular Structure Research Articles

Quantitative MRI Identifies Heterogeneous Bone Marrow Treatment Responses in a Mouse Model of Myelofibrosis

Myeloproliferative neoplasms (MPNs), a class of blood cancers, typically arise from mutations in the thrombopoietin receptor (MPL), JAK2, or calreticulin that constitutively activate JAK/STAT signaling in hematopoietic stem and progenitor cells. Myelofibrosis (MF), the most aggressive of the MPNs, can arise as a primary neoplasm or secondary to other MPNs. MF is characterized by progressive fibrosis of bone marrow (BM), destruction of the hematopoietic niche, and eventual BM failure. Recently, the focus of treatment in MF has shifted toward restoration of healthy BM architecture and a functional hematopoietic niche rather than focusing solely on alleviating symptoms. Accomplishing this paradigm shift in drug development and therapy requires new approaches to monitor spatial and temporal changes in BM in patients during treatment. We present a quantitative magnetic resonance imaging (MRI) approach to analyze key pathologic changes in BM in a mouse model of MPN driven by MPLW515L. To evaluate disease extent, progression, and response to therapy in mice, we use anatomical MRI for spleen volume and three quantitative MRI metrics for BM: 1) apparent diffusion coefficient (ADC), 2) proton density fat fraction (PDFF), and 3) magnetization transfer ratio (MTR). PDFF and ADC assess the fat content and water movement of the BM and are useful in identifying cellularity changes such as hypercellularity in MPNs and MF. MTR probes changes in macromolecular structure, such as those observed with increasing fibrosis in MF. We used quantitative MRI to measure response to treatment in BM and change in spleen volume in response to three drugs: ruxolitinib (JAK1/2 inhibitor), fedratinib (JAK2 inhibitor), and navitoclax (BCL-2/BCL-xL inhibitor). Within 10 days after starting treatment, diseased spleen volumes in the ruxolitinib group (~3.5x healthy volume) had the greatest average reduction in spleen volume measured by MRI (-73.5% ± 11.4%) followed by the fedratinib group (-52.7% ± 16.7%). Interestingly, the spleens of every ruxolitinib-treated mouse displayed consistent and prolonged spleen volume reductions, while only a subset of mice in the other treatment groups (~70% fedratinib mice; ~33% navitoclax mice) demonstrated reductions in spleen volume. Despite the robust spleen response in ruxolitinib-treated mice, only some of the mice (~60%) exhibited sustained changes in BM ADC, MTR, and PDFF signals expected with reversion to healthy BM (decreased ADC, decreased MTR, and increased PDFF). In mice treated with fedratinib or navitoclax, even fewer mice (~40% and ~33% respectively) showed reversion toward healthy BM. Importantly, we observed spatial heterogeneity of BM response within the tibia of single mice, where proximal and distal tibia regions often responded disparately (~49% of cases). Because improving BM cellularity and fibrosis is increasingly the target of therapy in MPNs and in MF specifically, there is a distinct need for better techniques for longitudinally evaluating variations in treatment efficacy in living subjects. In this study, we demonstrate that the combination of three quantitative MRI metrics (ADC, PDFF, and MTR) can detect the impact of therapy on the BM in a preclinical mouse model of MPN/MF. Interestingly, we identified pronounced heterogeneity at multiple levels during treatment: 1) differences in changes in spleen volume versus BM; 2) disparate treatment responses of mice within a treatment group; and 3) contrasting regional treatment responses of the BM within a single mouse. Because these quantitative MRI metrics are directly translatable to clinical medicine and can identify variations in treatment efficacy in BM, both between and within subjects, this study sets the stage for future drug development and analysis of therapeutic interventions on the BM of patients with MF.

Differences in Macromolecular Structure Evolution during the Pyrolysis of Vitrinite and Inertinite Based on In Situ FTIR and XRD Measurements

An accurate understanding of molecular structure evolution during pyrolysis is essential for the clean utilization of coal. In this study, middle-rank coal was taken as the research subject, and vitrinite and inertinite samples were stripped from coal using a hand picking and sink–float separation process. In situ Fourier transform infrared (FTIR) spectroscopy and in situ X-ray diffraction (XRD) were performed to compare the macromolecular structure changes between vitrinite and inertinite during pyrolysis. The results show that the aromaticity (I), the polycondensation degree of aromatic rings (DOC), the average lateral sizes (La) of basic structure unit (BSU), and the stacking heights (Lc) of BSU in both vitrinite and inertinite during pyrolysis increase continuously with increasing temperature. The values of these parameters for inertinite are higher than those of vitrinite, suggesting that the aromatization degree of inertinite has always been higher than that of vitrinite. In situ FTIR spectroscopy shows that the macromolecular structure evolution of vitrinite and inertinite could be divided into three stages based on temperature: 30–200 °C, 200–300 °C, and 300–500 °C. The content of C–O–C, aromatic C=O, O–H groups, and aromatic ring C=C gradually decreases, while that of the CH2 in aliphatic hydrocarbons increases between 30 °C and 200 °C. The 200–300 °C stage is mainly filled by the synergistic effects of aliphatic and aromatic groups. The content of aliphatic groups, C–O–C groups, aromatic C=O, and aromatic ring C=C of both vitrinite and inertinite decreases greatly. The 300–500 °C stage is dominated by the aromatization and condensation of macromolecules. The substituents of the aromatic system gradually detach, leading to an increase in I and DOC. From 30 °C to 1000 °C, in situ XRD results reveal a difference in macromolecular structural evolution between vitrinite and inertinite. The arrangement of aromatic layers in vitrinite tends to be ordered during pyrolysis, whereas there are no significant changes in the inertinite. However, the aromatic layers of inertinite are always more compact than that of vitrinite. In addition, the aliphatic side chains of inertinite are more stable than that of vitrinite during the pyrolysis process.

Macromolecular structural changes in contact metamorphosed inertinite-rich coals from the No. 2 Seam, Witbank Coalfield (South Africa): Insights from petrography, NMR and XRD

The response of inertinite-rich Witbank coals (South Africa) to rapid heating was evaluated using petrography, selected Nuclear Magnetic Resonance (NMR) and X-Ray Diffraction (XRD) structural parameters. A total of thirteen (13) coal samples were collected from the No. 2 Seam; a set of six (6) samples from the western and the eastern sides of a ~ 0.4 m thick dolerite dyke emplaced into the coal seam at 0.25 m intervals. An unaltered coal (Un-C; the 13th sample), collected away from the dyke but within the same seam at the mine, was included for comparison. All 13 coal samples were dominated by inertinite. Semifusinite was most abundant in the samples from the western side of the dyke, whereas the proportion of inertodetrinite was highest in samples from the eastern side. Due to very low vitrinite contents, mean random total reflectance (%RoTmr) was used to compare the maturity of the inertinite-rich coals. A decrease in volatile matter was correlated with an increase in mean random total reflectance (%RoTmr; measured on semifusinite, inertodetrinite, and vitrinite), the latter used as a potential maturity indicator for the thermally altered inertinite-rich coals. Mean random total reflectance rose from a background value of 1.52%RoTmr (for Un-C) to 2.83%RoTmr for the coal sample at the western coal-dyke contact (0.0 m). The corresponding reflectance values for the thermally altered coals from the eastern side were less consistent, decreasing closest to the dyke (1.93%RoTmr for the sample at 0.0 m); this trend is also reflected in the NMR and XRD parameters. Eight (8) samples were selected for the NMR analysis, representative of the maceral composition and maturities of the coals investigated. For Un-C, the fraction of aromatic carbons (fa) was 0.88 (average number of carbons per aromatic cluster (C) = 17), and the interplanar spacing (d002) was 3.48 Å (with a crystallite height of 22.09 Å). For the thermally altered coals from the western side, fa decreased from 0.96 at the coal-dyke contact (C = 26 for sample at 0.0 m) to 0.92 for the sample at 1.25 m from the dyke (C = 20). For samples from the eastern side, fa increased from 0.91 (C = 19 for the sample at 0.25 m) to 0.93 (C = 23 for sample at 0.5 m), before decreasing to 0.89 further from the dyke (C = 18 for sample at 1.25 m). In general, the d002 values for the thermally altered coal samples were larger than for graphite. Similarly, only marginal increases in crystallite heights were observed. Although the dyke induced coal maturation and the resultant increase in fa, the overall lack of graphitic order in the thermally altered coals was ascribed to the limited thermal energy provided by the relatively thin dyke; increased heat input would be required to effect significant structural changes in the inertinite-rich coals. Differences between the two sets of thermally altered coal samples were attributed to the emplacement dynamics of the igneous intrusion, which require further investigation.

New high-throughput endstation to accelerate the experimental optimization pipeline for synchrotron X-ray footprinting.

Synchrotron X-ray footprinting (XF) is a growing structural biology technique that leverages radiation-induced chemical modifications via X-ray radiolysis of water to produce hydroxyl radicals that probe changes in macromolecular structure and dynamics in solution states of interest. The X-ray Footprinting of Biological Materials (XFP) beamline at the National Synchrotron Light Source II provides the structural biology community with access to instrumentation and expert support in the XF method, and is also a platform for development of new technological capabilities in this field. The design and implementation of a new high-throughput endstation device based around use of a 96-well PCR plate form factor and supporting diagnostic instrumentation for synchrotron XF isdescribed. This development enables a pipeline for rapid comprehensive screening of the influence of sample chemistry on hydroxyl radical dose using a convenient fluorescent assay, illustrated here with a study of 26 organic compounds. The new high-throughput endstation device and sample evaluation pipeline now available at the XFP beamline provide the worldwide structural biology community with a robust resource for carrying out well optimized synchrotron XF studies of challenging biological systems with complex samplecompositions.

Macromolecular Structure Changes of Tectonically Deformed Coal: Evidence from Coal Pyrolysis, 13C NMR, and XRD Experiments

Research on tectonically deformed coal (TDC) is of significance to gas outburst prevention and coalbed methane development. Despite numerous works focusing on the structural characteristics of TDC, the influence of tectonic stress on coalification is a long-term controversial issue. In this study, the X-ray diffraction (XRD), 13C solid-state nuclear magnetic resonance (NMR), and coal pyrolysis techniques were applied to compare the macromolecular structural characteristics and gas generation properties between weak TDC (WTDC) and strong TDC (STDC). The results show that the strong tectonic stress obviously promotes the evolution of coalification in advance. The basic structural unit (BSU) of the WTDC and STDC both increases with increasing coal rank, but the d(002) values of the STDC are usually less than those of the WTDC, and the Lc, La, and N values of the STDC are larger than those of the WTDC. Furthermore, the impact of tectonic deformation on the BSU of coals becomes weaker as the coal rank increases. The aromaticity in coals is accelerating and increasing with the enhancement of deformation degree. The fractions of protonated aromatic carbon (fH a) and aromaticity (fa) in the STDC are higher than those in the WTDC of the same coal rank, while the relative contents of the oxygen-substituted aromatic carbon (faO), aliphatic-substituted aromatic carbon (faS), and aliphaticity (fal) in the STDC are lower than those in the WTDC. The gas generation potential of the STDC is lower than that of the WTDC of the same coal rank from the coal pyrolysis data. Furthermore, with an increase of temperature, the difference in the generation capacity of CH4 and H2 between STDC and WTDC increases. The research results help us to illustrate the effect of tectonic stress on coalification and supply guidance on the prevention and management of gas outburst disasters.

The degradation and performance of electrospun supramolecular vascular scaffolds examined upon in vitro enzymatic exposure

To maintain functionality during in situ vascular regeneration, the rate of implant degradation should be closely balanced by neo-tissue formation. It is unknown, however, how the implant’s functionality is affected by the degradation of the polymers it is composed of. We therefore examined the macro- and microscopic features as well as the mechanical performance of vascular scaffolds upon in vitro enzymatic degradation. Three candidate biomaterials with supramolecularly interacting bis-urea (BU) hard blocks (‘slow-degrading’ polycarbonate-BU (PC-BU), ‘intermediate-degrading’ polycarbonate-ester-BU (PC(e)-BU), and ‘fast-degrading’ polycaprolactone-ester-BU (PCL-BU)) were synthesized and electrospun into microporous scaffolds. These materials possess a sequence-controlled macromolecular structure, so their susceptibility to degradation is tunable by controlling the nature of the polymer backbone. The scaffolds were incubated in lipase and monitored for changes in physical, chemical, and mechanical properties. Remarkably, comparing PC-BU to PC(e)-BU, we observed that small changes in macromolecular structure led to significant differences in degradation kinetics. All three scaffold types degraded via surface erosion, which was accompanied by fiber swelling for PC-BU scaffolds, and some bulk degradation and a collapsing network for PCL-BU scaffolds. For the PC-BU and PC(e)-BU scaffolds this resulted in retention of mechanical properties, whereas for the PCL-BU scaffolds this resulted in stiffening. Our in vitro study demonstrates that vascular scaffolds, electrospun from sequence-controlled supramolecular materials with varying ester contents, not only display different susceptibilities to degradation, but also degrade via different mechanisms. Statement of SignificanceOne of the key elements to successfully engineer vascular tissues in situ, is to balance the rate of implant degradation and neo-tissue formation. Due to their tunable properties, supramolecular polymers can be customized into attractive biomaterials for vascular tissue engineering. Here, we have exploited this tunability and prepared a set of polymers with different susceptibility to degradation. The polymers, which were electrospun into microporous scaffolds, displayed not only different susceptibilities to degradation, but also obeyed different degradation mechanisms. This study illustrates how the class of supramolecular polymers continues to represent a promising group of materials for tissue engineering approaches.

The Degradation and Performance of Electrospun Supramolecular Vascular Scaffolds Examined Upon <i>In Vitro</i> Enzymatic Exposure

To maintain functionality during in situ regeneration of load-bearing tissues, a balance between the rates of implant degradation and neo-tissue formation is required. Strictly segmented thermoplastic elastomers with supramolecularly interacting bis-urea (BU) hard blocks are attractive biomaterials for vascular tissue engineering, as these polymers are soft, tough, and biodegradable. Moreover, these materials possess a sequence-controlled macromolecular structure, so their susceptibility to degradation is tunable by controlling the nature of the polymer backbone. It is unknown, however, how the implant’s functionality is affected by the degradation of the polymers it is composed of. We therefore examined the macro- and microscopic features as well as the mechanical performance of vascular scaffolds upon in vitro enzymatic degradation. Three candidate biomaterials (‘slow-degrading’ polycarbonate-BU (PC-BU), ‘intermediate-degrading’ polycarbonate-ester-BU (PC(e)-BU), and ‘fast-degrading’ polycaprolactone-ester-BU (PCL-BU)) were synthesized and electrospun into microporous scaffolds. The scaffolds were incubated in lipase and monitored for changes in physical, chemical, and mechanical properties. Remarkably, comparing PC-BU to PC(e)-BU, we observed that small changes in macromolecular structure led to significant differences in degradation kinetics. All three scaffold types degraded via surface erosion, which was accompanied by fiber swelling for PC-BU scaffolds, and some bulk degradation and a collapsing network for PCL-BU scaffolds. For the PC-BU and PC(e)-BU scaffolds this resulted in retention of mechanical properties, whereas for the PCL-BU scaffolds this resulted in stiffening. Our in vitro study demonstrates that vascular scaffolds, electrospun from sequence-controlled supramolecular materials with varying ester contents, not only display different susceptibilities to degradation, but also degrade via different mechanisms.

Monitoring Metallo-Macromolecular Assembly Equilibria by Ion Mobility-Mass Spectrometry.

Ion mobility-mass spectrometry (IM-MS) allows the separation of isomeric and isobaric species on the basis of their size, shape, and charge. The fast separation timescale (ms) and high sensitivity of these measurements make IM-MS an ideally suitable method for monitoring changes in macromolecular structure, such as those occurring in interconverting terpyridine-based metallosupramolecular self-assemblies. IM-MS is used to verify the elemental composition (size) and architecture (shape) of the self-assembled products. Additionally, this article demonstrates its applicability to the elucidation of concentration-driven association-dissociation (fusion-fission) equilibria between isobaric structures. IM-MS enables both quantitative separation and identification of the interconverting complexes as well as derivation of the corresponding equilibrium constants (i.e., thermodynamic information) from extracted IM-MS abundance data.

Choline chloride vs choline ionic liquids for starch thermoplasticization

Native starch containing 12% water was melt processed in presence of 23% of various plasticizers at 120°C, either by simple compression molding or by extrusion using a laboratory scale microcompounder. Glycerol, a typical starch plasticizer, was used as a reference and compared to three choline salts: raw choline chloride (which is a solid in dry state with a melting point above 300°C), and two ionic liquids synthesized from this precursor (choline acetate and choline lactate, liquids below 100°C). These ionic plasticizers were shown to allow a more efficient melting of native starch in both processes. The investigation of macromolecular structure changes during processing shows that this efficiency can be ascribed to a starch chain scission mechanism, resulting in lower specific mechanical energy input need for starch thermoplasticization compared to glycerol plasticized starch. Compared to the synthesized ionic liquids, raw commercial choline chloride leads to a good compromise between limited chain scission, and final water uptake and thermomechanical properties.

Analysis of Structural Changes in Jute Fibers after Peracetic Acid Treatment

Peracetic acid (PAA) is one of the most commonly used bleaching agents in the textile industry. The structural changes of jute fibers after PAA treatment are important in creating industrial applications. An investigation was carried out to determine the structural changes resulting from the removal of noncellulosic materials from jute fibers by PAA. The surface features of jute fiber were studied using a scanning electron microscope. PAA was effective in separateingindividual cellulose fibers from raw jute. However, fibers were damaged when the treatment time reached 80 minutes, PAA concentration was 10 wt. % and the pH was in the 4~5 range. The delignification by PAA treatment was confirmed by Fourier transform infrared spectroscopy. CP/MAS13C-NMR spectroscopy showed that the PAA did cause some macromolecular structure changes in the cellulose fibers. X-ray diffraction technique demonstrated that there were four stages of crystallinity change, due to the removal of noncellulosic materials. These four stages include attack at the surfaces of cellulose crystallites, the dissolution of amorphous zones, attack of the inner cellulose crystallite and oxygen interference, respectively.

Molecular and macromolecular structure changes in polyamide 11 during thermal oxidation – Kinetic modeling

The oxidation kinetics of unstabilized polyamide 11 thin film under oxygen pressures up to 2.0 MPa was studied by means of ferrous ion method for hydroperoxides titration, size exclusion chromatography (SEC) for molar weight measurements and in situ FTIR monitoring for carbonyl build up. Oxidation was shown not to be limited by oxygen diffusion which militates in favor of the use of a simple kinetic model for simulating experimental data. The kinetic parameters of this model were estimated from literature for initiation and propagation, and from selective experiments such as aging under several oxygen pressures for the estimation of the termination rate constants, or exposures under inert atmosphere to investigate the solid state polymerization effects and tentatively check the boundary conditions of the model. The kinetic model simulates satisfyingly the overall experimental results using the minimal number of adjustable parameters (except for some reactions difficult to be experimentally isolated and studied).

Molecular and macromolecular structure changes in polyamide 11 during thermal oxidation

The oxidation kinetics of unstabilized polyamide 11 thin film under oxygen pressures up to 2.0 MPa was studied by means of ferrous ion method for hydroperoxides titration, size exclusion chromatography (SEC) for molar weight measurements and in situ FTIR monitoring for carbonyl build up. Oxidation was shown not to be limited by oxygen diffusion which militates in favor of the use of a simple kinetic model for simulating experimental data. The kinetic parameters of this model were estimated from literature for initiation and propagation, and from selective experiments such as aging under several oxygen pressures for the estimation of the termination rate constants, or exposures under inert atmosphere to investigate the solid state polymerization effects and tentatively check the boundary conditions of the model. The kinetic model simulates satisfyingly the overall experimental results using the minimal number of adjustable parameters (except for some reactions difficult to be experimentally isolated and studied).

Comparing Conformational Ensembles Using the Kullback–Leibler Divergence Expansion

We present a thermodynamical approach to identify changes in macromolecular structure and dynamics in response to perturbations such as mutations or ligand binding, using an expansion of the Kullback-Leibler Divergence that connects local population shifts in torsion angles to changes in the free energy landscape of the protein. While the Kullback-Leibler Divergence is a known formula from information theory, the novelty and power of our implementation lies in its formal developments, connection to thermodynamics, statistical filtering, ease of visualization of results, and extendability by adding higher-order terms. We present a formal derivation of the Kullback-Leibler Divergence expansion and then apply our method at a first-order approximation to molecular dynamics simulations of four protein systems where ligand binding or pH titration is known to cause an effect at a distant site. Our results qualitatively agree with experimental measurements of local changes in structure or dynamics, such as NMR chemical shift perturbations and hydrogen-deuterium exchange mass spectrometry. The approach produces easy-to-analyze results with low background, and as such has the potential to become a routine analysis when molecular dynamics simulations in two or more conditions are available. Our method is implemented in the MutInf code package and is available on the SimTK website at https://simtk.org/home/mutinf.

Biocrystallography: past, present, future.

The evolution of biocrystallography from the pioneers' time to the present era of global biology is presented in relation to the development of methodological and instrumental advances for molecular sample preparation and structure elucidation over the last 6 decades. The interdisciplinarity of the field that generated cross-fertilization between physics- and biology-focused themes is emphasized. In particular, strategies to circumvent the main bottlenecks of biocrystallography are discussed. They concern (i) the way macromolecular targets are selected, designed, and characterized, (ii) crystallogenesis and how to deal with physical and biological parameters that impact crystallization for growing and optimizing crystals, and (iii) the methods for crystal analysis and 3D structure determination. Milestones that have marked the history of biocrystallography illustrate the discussion. Finally, the future of the field is envisaged. Wide gaps of the structural space need to be filed and membrane proteins as well as intrinsically unstructured proteins still constitute challenging targets. Solving supramolecular assemblies of increasing complexity, developing a "4D biology" for decrypting the kinematic changes in macromolecular structures in action, integrating these structural data in the whole cell organization, and deciphering biomedical implications will represent the new frontiers.

Influence of thermally induced scission and crosslinking on the post-scission inflation of circular elastomeric membranes

When an elastomeric material is deformed and subjected to temperatures above some characteristic value T cr (near 100 °C for natural rubber), macromolecular chains and crosslinks undergo scission, recoiling and re-crosslinking. The process depends on time and temperature and continues until the temperature decreases below T cr. Compared to the virgin material, the new material system has modified properties (reduced stiffness) and permanent set on removal of the applied load. A constitutive theory that accounts for these changes in macromolecular structure is used to simulate an experiment involving the inflation of an initially flat circular membrane. The membrane is initially at a temperature T < T cr. A prescribed volume of fluid inflates the membrane to a surface of revolution. The temperature of the fluid and membrane is increased above T cr for some period of time. Attention is restricted to the case when scission is independent of the deformation of the membrane. It is shown that the time dependence of the pressure in the inflating fluid must satisfy a specific relation involving a time dependent property measured during scission under uniaxial extension. This result provides a useful experimental means for determining when scission is independent of deformation in biaxial deformations. The temperature is returned to its original value and the pressure is gradually removed. It is shown that compressive stresses may develop as the pressure is reduced. If the membrane is removed from its support, it will have developed a permanently curved shape. Results of a numerical simulation are presented that compares the initial inflated shape to the permanently curved shape.

Effect of laccase and manganese peroxidase on wheat gluten and pentosans during mixing

Wheat gluten and water extractable pentosans (WEP) were mixed in a batch-mixer in the presence of laccase or manganese peroxidase (MnP). The enzymes provoked different changes in mixing behaviour. Doughs were analysed at time to peak ( t) and over two t (2 t or overmixed). The effects of the oxidative enzymes were studied on ferulic acid (FA), arabinoxylans (AX), proteins and possible intermolecular associations in these dough systems. Changes in macromolecular structures were followed using reverse phase-high performance liquid chromatography of phenolic compounds, AX extractability and the viscosity measurements, size exclusion-high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins. The oxidative enzymes increased dough consistency, accelerated the loss in total FA, decreased AX water extractability and water extractable AX apparent intrinsic viscosity in the dough water extracts. The effects of laccase and MnP were similar on feruloylated AX when compared on a reaction time basis. No covalent complex between AX and protein was detected in mixed and oxidised gluten/WEP.

Unique poly(glycerophosphate) lipoteichoic acid and the glycolipids of a Streptococcus sp. closely related to Streptococcus pneumoniae.

The Streptococcus sp. studied here is closely related to Streptococcus pneumoniae with 98.6% 16S rRNA similarity and 65% DNA/DNA homology. We isolated the lipoteichoic acid and the membrane glycolipids whose structures were established using conventional procedures and NMR spectroscopy. The lipoteichoic acid contains a linear 1,3-linked poly(glycerophosphate) chain which is partly substituted with D-alanine ester and is phosphodiester-linked to O6 of beta-D-Galf(1-->3)acyl2Gro. This lipoteichoic acid is the first example in which a monohexosylglycerol serves as the glycolipid anchor; and with an average chain length of 10 glycerophosphate residues it is the shortest known to date. MS analysis, applied for the first time to a native acylated lipoteichoic acid, revealed a continuous increase in chain length from seven to 17 glycerophosphate residues with a maximum at 10, and allowed identification of the fatty acid combinations. Membrane glycolipids consisted of beta-D-Galf(1-->3)acyl2Gro (9%), alpha-D-Glcp(1-->3)acyl2Gro (22%), alpha-D-Galp(1-->2)-alpha-D-Glcp(1-->3)acyl2Gro (64%) and alpha-D-Galp(1-->2)-(6-O-acyl)-alpha-D-Glcp(1-->3)acyl2Gro (5%). It is noteworthy that in lipoteichoic acid biosynthesis, Galfacyl2Gro, a less abundant membrane glycolipid, is selected as the lipid anchor. Despite the genetic relatedness to Streptococcus pneumoniae, the lipoteichoic acid structure is quite different to the complex structure of pneumococcal lipoteichoic acid [T. Behr et al. (1992) Eur. J. Biochem. 207, 1063-1075], thus providing an example that minor differences in DNA sequence exert major changes in macromolecular structure.

Imaging of humic substance macromolecular structures in water and soils

Humic substances (HSs) are the natural organic polyelectrolytes formed from the biochemical weathering of plant and animal remains. Their macromolecular structure and chemistry determine their role in biogeochemical processes. In situ spectromicroscopic evidence showed that the HS macromolecular structures (size and shape) vary as a function of HS origin (soil versus fluvial), solution chemistry, and the associated mineralogy. The HSs do not simply form coils in acidic or strong electrolyte solutions and elongated structures in dilute alkaline solutions. The macromolecular structural changes of HSs are likely to modify contaminant solubility, biotransformation, and the carbon cycle in soils and sediments.

The effect of humic acids on the water solubility and water — Organic carbon partitioning of fluorene and its NSO-heteroanalogues: Carbazole, dibenzofuran, and dibenzothiophene

The solubilizing effect of humic acids on fluorene and its NSO analogues carbazole, dibenzofuran and dibenzothiophene has been studied. The interaction between these substances and humic acids was found to depend significantly on the actual humic acid concentration. A pronounced decrease in the interaction constant, as well as in the water - organic matter partitioning coefficient was observed with increasing humic acid concentration. The effects are discussed in terms of concentration dependent macromolecular structural changes in the humic acids. A linear free energy relation for the interaction is suggested.

Time-Resolved Analysis of Macromolecular Structures During Reactions by Stopped-Flow Electrooptics

A stopped-flow field-jump instrument and its use for the analysis of macromolecular structure changes during reactions is described. The operation of the new instrument is simple and reliable, owing to a new type of cell construction with electrodes directly integrated in a quartz cuvette: major advantages are the relatively low demand on sample quantities and a high time resolution. The stopped flow is characterized by a dead time of ∼0.5 ms. Electric field pulses with field strengths up to 20 kV/cm and rise times in the nanosecond range are applied at adjustable times after stop of the flow. The time resolution of the optical detection is up to the nanosecond time range. The instrument may be used for the combination of stopped flow with temperature-jump and field-jump experiments. A particularly useful new application is the analysis of macromolecular reactions by electrooptical measurements, because electrooptical data provide information about structures. This is demonstrated for the intercalation of ethidium into double-helical DNA. The transients, measured at 313 nm, where the signal is exclusively due to ethidium bound to the DNA, demonstrate a relatively high negative dichroism at 0.5 ms after mixing. The absolute value of this negative dichroism increases in the millisecond time range and approaches the equilibrium value within about a second. The dichroism decay time constants demonstrate a clear increase of the effective DNA length due to ethidium binding, already 0.5 ms after mixing; a further increase to the equilibrium value is found in the millisecond time range. The analysis of these data demonstrate the existence of up to three relaxation processes, depending on the conditions of the experiments. The dichroism amplitudes, together with the decay time constants, indicate that all the reaction states found in the present investigation are complexes with insertion of ethidium residues between basepairs. Moreover, the data clearly show the degree of intercalation in the intermediate states, which is very useful information for the quantitative assignment of the mechanism.