Characterization of the 31P NMR spectrum of the schistosome vector Biomphalaria glabrata and of the changes following infection by Schistosoma mansoni

The 31P nuclear magnetic resonance (NMR) spectrum of the digestive gland-gonad complex (DGG) of the schistosome vector Biomphalaria glabrata was characterized and the effects of infection by Schistosoma mansoni noted. The in vivo spectrum was comprised of 11 peaks, 5 downfield and 6 upfield of an external 85% phosphoric acid standard. Based on a variety of analytical procedures, the upfield peaks from the standard were demonstrated to be composed of carbamoyl phosphate + a mixture of 3 phosphatides and sphingomyelin, the gamma + beta phosphorus resonances of nucleotide triphosphate (NTP) and nucleotide diphosphate (NDP), respectively, the alpha phosphorus resonances of NTP + NDP, NAD(H) + the phosphorus resonance of uridine phosphate from uridine diphosphoglucose (UDPG), the phosphorus resonance of glucose phosphate from UDPG and, last, the beta phosphorus resonance of NTP. The downfield peaks were assigned as glycerophosphoryl choline, intracellular inorganic phosphate (Pi), sugar phosphates + phosphoryl choline, aminoethyl phosphonate (AEP), and ceramide AEP. T1 values for the in vivo NMR components were determined by inversion recovery. Infection produced distinct alterations in the levels of nonnucleotide components of the in vivo 31P NMR spectrum and the spectra of tissue extracts. Specifically, the levels of phosphonate, phospholipids, and carbamoyl phosphate were markedly reduced, and the relative level of Pi was increased. The potential significance of these changes to the parasite-host relationship was discussed. In contrast, starvation resulted in a decreased level of phosphonate only. The pH of the intact DGG was estimated by titrating the inorganic phosphate component of tissue extracts. The mean pH was 6.9 for both control and infected material.     

NMR Analyses of the Interaction between the FYVE Domain of Early Endosome Antigen 1 (EEA1) and Phosphoinositide Embedded in a Lipid Bilayer

Phosphoinositides (PIs) are crucial lipid components of membranes and are involved in a number of cellular processes through interactions with their effector proteins. Recently, we have established a lipid-protein nanoscale bilayer (nanodisc) containing PIs, hereafter referred to as PI-nanodisc and demonstrated that it could be used for both qualitative and quantitative evaluations of protein-membrane interactions. Here, we report further NMR analyses for obtaining structural insights at the residue-specific level between PI-binding effector protein and PI-nanodisc, using the FYVE domain of early endosome antigen 1 (EEA1), denoted as EEA1 FYVE, and PI(3)P-nanodisc as a model system. We performed a combination of the NMR analyses including chemical shift perturbation, transferred cross-saturation, and paramagnetic relaxation enhancement experiments. These enabled an identification of the interaction surface, structural change, and relative orientation of EEA1 FYVE to the PI(3)P-incorporated lipid bilayer, substantiating that NMR analyses of protein-membrane interactions using nanodisc makes it possible to show the residue-specific interactions in the lipid bilayer environment.     

1H NMR Spectroscopy Profiling of Metabolic Reprogramming of Chinese Hamster Ovary Cells upon a Temperature Shift during Culture

We report an NMR based approach to determine the metabolic reprogramming of Chinese hamster ovary cells upon a temperature shift during culture by investigating the extracellular cell culture media and intracellular metabolome of CHOK1 and CHO-S cells during culture and in response to cold-shock and subsequent recovery from hypothermic culturing. A total of 24 components were identified for CHOK1 and 29 components identified for CHO-S cell systems including the observation that CHO-S media contains 5.6 times the level of glucose of CHOK1 media at time zero. We confirm that an NMR metabolic approach provides quantitative analysis of components such as glucose and alanine with both cell lines responding in a similar manner and comparable to previously reported data. However, analysis of lactate confirms a differentiation between CHOK1 and CHO-S and that reprogramming of metabolism in response to temperature was cell line specific. The significance of our results is presented using principal component analysis (PCA) that confirms changes in metabolite profile in response to temperature and recovery. Ultimately, our approach demonstrates the capability of NMR providing real-time analysis to detect reprogramming of metabolism upon cellular perception of cold-shock/sub-physiological temperatures. This has the potential to allow manipulation of metabolites in culture supernatant to improve growth or productivity.     

Intermolecular interactions and phase structures of plasticized wheat proteins materials

The intermolecular interactions and phase structures of thermally processed wheat proteins with glycerol and water as plasticizers were studied by dynamic mechanical analysis and solid-state high-resolution NMR spectroscopy. The results of phase structures at scales of molecular level to tens of nanometers were correlated with the mechanical properties of the materials. The strong hydrogen bonding intermolecular interactions between the components in wheat proteins and the plasticizers resulted in a significant change in molecular motions of wheat protein materials. The plasticized systems, however, still presented a wide distribution of chain mobility at a scale from the molecular level to 20-30 nm, and the plasticizing effect was different for each wheat protein system. High protein content systems tended to be plasticized relatively easily especially when lipid content is high, but the existence of residual starch would require more plasticizers to reach a similar level of chain mobility. On a scale of 20-30 nm, plasticized vital wheat gluten (WG) and the deamidated wheat proteins (WP-I) were heterogeneous with each component exhibiting its individual mobility, whereas the plasticized insoluble protein system (WP-II) with poor mechanical properties was homogeneous. Both WG and WP-I systems showed excellent mechanical polymeric properties in tensile strength and elasticity despite the heterogeneity. The strong intermolecular hydrogen bonding interactions and soluble protein components in the materials could provide an adhesion among different components and act as a continuous matrix in the systems. Therefore, these materials displayed excellent mechanical properties via coordination effects among different components.     

A pulsed H NMR study of the hydration properties of extruded maize-sucrose mixtures

The molecular mobility in maize and maize-sucrose extudates was studied as a function of the degree of hydration (up to 35% w/w dry basis) using proton spin-spin relaxation NMR. The extrusion of maize systems was found to enhance the molecular mobility of the constituents generating the rigid ‘solid-like’ and the mobile ‘liquid-like’ components of the NMR free induction decay signal.

As the moisture content was increased, the amplitude of the normalised NMR signal arising from the rigid component decreased dramatically for the samples containing sucrose, suggesting an increased mobility and thus a solution type behaviour of the sugar. This hypothesis was supported by the observation of shorter T₂ values for the liquid component of the signal recorded for the samples containing sugar. Sucrose was found to be miscible with the polysaccharide at moisture contents below 15%, but progressively dissolved into the additional water at higher moisture contents. This suggested the occurrence of phase separation over a short distance scale as the water content of the maize-sucrose system was modified. The results are discussed in terms of unequal water partitioning between the maize and the sugar components.     

Abnormal lipid metabolism in a rat model of arthritis: one possible pathway

Collagen-induced arthritis (CIA) animal model is associated with systemic manifestations, including alteration of lipid metabolism. In the present study, one possible pathway of altered lipid metabolism is proposed. Specimens of joint tissue and plasma were collected from the CIA and control rats, and quantitative analysis of lipid components was performed by nuclear magnetic resonance (NMR) spectroscopy technique. Correlation analysis was performed between the level of lipid components and antioxidant enzymes, lactate dehydrogenase (LDH), lipid peroxidation (LP), and cytokines in joint tissue and plasma. Differentiation between the CIA and control rats was established on the basis of the quantity of lipid components in the joint tissue and plasma. Positive correlation was observed for all the enzymes vs. lipid components as well as LP vs. lipid components in plasma and joint tissue. Positive correlation was observed for enzymes in plasma and joint tissue. A negative correlation was observed in between the plasma and joint tissue with the level of lipid components. Cytokine levels were also correlated with the level of lipid components and ratios of saturated fatty acids/unsaturated fatty acids in plasma and joint tissue. Inflammatory disease activity in CIA rats with synovitis brought about a significant change in lipid metabolism. Taken together, the results of our study are delineating a possible pathway of altered lipid metabolism in the CIA rat model, thereby contributing further to an understanding of the pathophysiology of rheumatoid arthritis (RA).     

Cancer-Associated Mutations Perturb the Disordered Ensemble and Interactions of the Intrinsically Disordered p53 Transactivation Domain

Intrinsically disordered proteins (IDPs) are key components of regulatory networks that control crucial aspects of cell decision making. The intrinsically disordered transactivation domain (TAD) of tumor suppressor p53 mediates its interactions with multiple regulatory pathways to control the p53 homeostasis during the cellular response to genotoxic stress. Many cancer-associated mutations have been discovered in p53-TAD, but their structural and functional consequences are poorly understood. Here, by combining atomistic simulations, NMR spectroscopy, and binding assays, we demonstrate that cancer-associated mutations can significantly perturb the balance of p53 interactions with key activation and degradation regulators. Importantly, the four mutations studied in this work do not all directly disrupt the known interaction interfaces. Instead, at least three of these mutations likely modulate the disordered state of p53-TAD to perturb its interactions with regulators. Specifically, NMR and simulation analysis together suggest that these mutations can modulate the level of conformational expansion as well as rigidity of the disordered state. Our work suggests that the disordered conformational ensemble of p53-TAD can serve as a central conduit in regulating the response to various cellular stimuli at the protein–protein interaction level. Understanding how the disordered state of IDPs may be modulated by regulatory signals and/or disease associated perturbations will be essential in the studies on the role of IDPs in biology and diseases.     

Magnetic resonance spectroscopy and metabolism. Applications of proton and 13C NMR to the study of glutamate metabolism in cultured glial cells and human brain in vivo.

Nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of cells from the central nervous system both in vitro on perchloric acid extracts obtained either from cultured tumoral cells (C6 rat glioma) or rat astrocytes in primary culture, and in vivo within the human brain. Analysis of carbon 13 NMR spectra of perchloric acid extracts prepared from cultured cells in the presence of NMR [1-13C] glucose as substrate allowed determination of the glutamate and glutamine enrichments in both normal and tumoral cells. Preliminary results indicated large changes in the metabolism of these amino acids (and also of aspartate and alanine) in the C6 cell as compared to its normal counterpart. Localized proton NMR spectra of the human brain in vivo were obtained at 1.5 T, in order to evaluate the content of various metabolites, including glutamate, in peritumoral edema from a selected volume of 2 x 2 x 2 cm3. N-acetyl aspartate, glutamate, phosphocreatine, creatine, choline and inositol derivative resonances were observed in 15 min spectra. N-acetyl-aspartate was found to be at a lower level in contrast to glutamate which was detected at a higher level in the injured area as compared to the contralateral unaffected side.     

Acute biochemical effects of La(NO3)3 on liver and kidney tissues by magic-angle spinning 1H nuclear magnetic resonance spectroscopy and pattern recognition

High-resolution magic-angle spinning (MAS) 1H nuclear magnetic resonance (NMR) spectroscopic and pattern recognition (PR) based methods have been applied to studies on the acute biochemical effects of La(NO3)3 on rats. Male Wistar rats were treated with various doses of La(NO3)3 (2, 10, and 50 mg/kg body weight), and MAS 1H NMR spectra of their intact liver and kidney tissues were analyzed using principal components analysis to extract metabolic information. The biochemical effects of La(NO3)3 were characterized by the increase of triglyceride and bile acid and the decrease of glycogen in liver tissue, together with a slight elevation of triglyceride level in kidney tissue. The target lesion of La(NO3)3 to liver was found by MAS NMR-PR methods. This study illustrated the power of the combination of MAS 1H NMR and pattern recognition for the analysis of biochemical effects of rare earths.     

31P NMR spectra of rod outer segment and sarcoplasmic reticulum membranes show no evidence of immobilized components due to lipid-protein interactions

31P NMR studies of rod outer segment (ROS) and sarcoplasmic reticulum (SR) membranes have been performed under conditions where broad and narrow spectral components can be clearly resolved. Control studies of an anhydrous, solid powder of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), as well as aqueous binary mixtures of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), demonstrate clearly that broad spectral components can be detected. For the codispersions of DSPC and DOPC in the mixed-phase region at 22 degrees C, the 31P NMR spectra consist of a superposition of a broad component and a narrow, axially symmetric component, due to coexisting solid and liquid-crystalline domains, which are in slow exchange on the 31P NMR time scale. The 31P NMR spectra of the native ROS and SR membranes, however, consist of only a narrow component, to within experimental error, indicating that most or all of the phospholipids are in the liquid-crystalline (L alpha) phase at 22 degrees C. The above conclusions are in agreement with many, but not all, previous studies [see, e.g., Yeagle, P.L. (1982) Biophys. J. 37, 227-239]. It is estimated that at most 10% of the phospholipids in the ROS and SR membranes could give rise to broad 31P NMR spectral components, similar to those seen for anhydrous or solid-phase lipids, corresponding to approximately 7 phospholipids/rhodopsin molecule and approximately 11 phospholipids/Ca2+-ATPase molecule, respectively.     

A natural-abundance 13C-NMR study of Dictyostelium discoideum metabolism. Monitoring of the spore germination process

Amoebae and spores of the cellular slime mold Dictyostelium discoideum have been investigated by natural-abundance proton-decoupled 13C-NMR spectroscopy. Axenically grown vegetative amoebae have been found to contain, as prominent metabolites, the polyamines 1,3-diaminopropane (3.2 mM), putrescine (9.4 mM) and spermidine (1.7 mM). We also detected lactic acid (4.4 mM) and the following amino acids as free metabolites in concentrations ranging over 1-3 mM: glycine, alanine, glutamine and glutamate. The glycogen level is highly dependent upon growth state, being below the level of NMR detection in early-exponential cells and reaching about 110 mM glucose equivalents in plateau-phase cells. Dormant spores contained high amounts of trehalose (50 mM), glutamine (73 mM) and glutamate (20 mM). The latter two compounds were not reported previously to be present in such high concentrations in Dictyostelium spores. Germination induced by heat-shock activation was monitored by 13C NMR. No change in the major components occurred during the activation step. The progressive disappearance of trehalose during germination correlated with the decrease of glutamine and glutamate. In general, the data suggest that germinated spores contain a composition of free metabolites very similar to that of starved vegetative amoebae.     

Characterization of the lipopolysaccharide and beta-glucan of the fish pathogen Francisella victoria

Lipopolysaccharide (LPS) and beta-glucan from Francisella victoria, a fish pathogen and close relative of highly virulent mammal pathogen Francisella tularensis, have been analyzed using chemical and spectroscopy methods. The polysaccharide part of the LPS was found to contain a nonrepetitive sequence of 20 monosaccharides as well as alanine, 3-aminobutyric acid, and a novel branched amino acid, thus confirming F. victoria as a unique species. The structure identified composes the largest oligosaccharide elucidated by NMR so far, and was possible to solve using high field NMR with cold probe technology combined with the latest pulse sequences, including the first application of H2BC sequence to oligosaccharides. The non-phosphorylated lipid A region of the LPS was identical to that of other Francisellae, although one of the lipid A components has not been found in Francisella novicida. The heptoseless core-lipid A region of the LPS contained a linear pentasaccharide fragment identical to the corresponding part of F. tularensis and F. novicida LPSs, differing in side-chain substituents. The linkage region of the O-chain also closely resembled that of other Francisella. LPS preparation contained two characteristic glucans, previously observed as components of LPS preparations from other strains of Francisella: amylose and the unusual beta-(1-6)-glucan with (glycerol)2phosphate at the reducing end.     

Determining the origin of the stabilization of DNA by 5-aminopropynylation of pyrimidines

DNA duplexes are stabilized by aminopropynyl modification of pyrimidines at the 5 position. A combination of thermodynamic analyses as a function of ionic strength, NMR, and molecular modeling has been applied to determine the origin of the stabilization. UV melting studies of a dodecamer bearing one, two, or three nonadjacent modified dU and dC and of a single dU(8) in the Dickerson-Drew dodecamer revealed that the modifications are essentially additive in terms of T(m), DeltaG, and DeltaH, and there is little difference between dU and dC. The free energy change was parsed into electrostatic and nonelectrostatic components, which showed a significant contribution from charge interactions at physiological ionic strength but also a nonelectrostatic contribution that arises in part from hydration. NMR spectroscopy of the modified Dickerson-Drew dodecamer revealed that the conformation of the duplexes is not significantly altered by the modifications, though (31)P NMR shows that the positive charge may affect ionic interactions with the oxygen atoms of the neighboring phosphates. The modified duplex showed significant hydration in both major and minor grooves. The single strands were also analyzed by NMR, which showed evidence of significant stacking interactions in the modified oligonucleotide. Parsing the energy contribution has shown that electrostatics and hydration can produce substantial increases in thermodynamic stability without significant changes in the conformation of the duplex state. These considerations have significance for the design of oligonucleotides used for hybridization.     

Aluminum binding to phosphatidylcholine lipid bilayer membranes: 27Al and 31P NMR spectroscopic studies

27Al and 31P nuclear magnetic resonance (NMR) spectroscopies were used to investigate aluminum interactions at pH 3.4 with model membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). A solution state 27Al NMR difference assay was developed to quantify aluminum binding to POPC multilamellar vesicles (MLVs). Corresponding one-dimensional (1D) fast magic angle spinning (MAS) 31P NMR spectra showed that aluminum induced the appearance of two new isotropic resonances for POPC shifted to -6.4 ppm and -9.6 ppm upfield relative to, and in slow exchange with, the control resonance at -0.6 ppm. Correlation of the (27)Al and (31)P NMR binding data revealed a 1:2 aluminum:phospholipid stoichiometry in the aluminum-bound complex at -9.6 ppm and a 1:1 aluminum:phospholipid stoichiometry in that at -6.4 ppm. Slow MAS 31P NMR spectra demonstrated shifts in the anisotropic chemical shift tensor components of the aluminum-bound POPC consistent with a close coordination of aluminum with phosphorus. A model of the aluminum-bis-phospholipid complex is proposed on the basis of these findings.     

Quantitation and characterization of process impurities and extractables in protein‐containing solutions using proton NMR as a general tool

The ability to detect and quantitate a variety of components in solution has become increasingly important in carrying out efficient and rigorous validation studies for biopharmaceutical manufacturing processes. Here, we demonstrate the general applicability of NMR spectroscopy for the identification and quantitation of leachables and other impurities in protein‐based drugs, at low levels previously unattainable in protein‐containing solutions. With improved NMR technology (i.e., CryoProbes) and the application of a Carr‐Purcell‐Meiboom‐Gill pulse sequence (CPMG) to attenuate protein signals, we have been able to use NMR to quantify impurities in a protein‐based biopharmaceutical product at ～1 μg mL^?1. The data indicate that NMR spectra can be used to quantitate a range of impurities, from small molecule components to higher molecular weight leachables, without removing protein from solution. Furthermore, quantitation of impurities by NMR is reliable and accurate enough for biopharmaceutical process validation, even for high molecular weight extractables whose structures are not precisely known. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Structural aspects in semicrystalline samples of the mannan II family

A series of samples having the mannan II character were prepared by either (i) desincrusting stems of Acetabularia crenulata, or (ii) acetylating these stems, followed by dissolution and recrystallization under deacetylation conditions, or (iii) recrystallizing at low temperature the alkali soluble fraction of ivory nut mannan. The samples were characterized by transmission electron microscopy, X-ray and electron diffraction analysis together with (13)C CP/MAS NMR spectroscopy. Whereas the A. crenulata stems consisted of a mixture of mannan I and mannan II, the recrystallized samples were all of the hydrated mannan II family and occurred in a ribbonlike morphology where the mannan chains were organized with their molecular axis perpendicular to the ribbon long axis. The recrystallized ivory nut mannan samples presented X-ray and electron diffraction diagrams, together with (13)C solid-state NMR spectra recorded at 95% RH, different from those of recrystallized A. crenulata recorded under the same RH conditions. They corresponded therefore to a new allomorph of the mannan II family. Despite this difference, when the recrystallized samples were in an aqueous environment, they revealed an additional well-defined perhydrated phase, which showed the same (13)C solid-state NMR spectrum for both samples. As this phase, which gave 6-band NMR spectra with narrow line-width and low T1, had no counterpart in X-ray diffraction, it was attributed to specific amorphous segments of mannan chains, gaining some mobility when swollen in water. When the samples were totally dried, their NMR spectra lost their resolution, thus indicating the role played by water for the structural organization of the crystalline and amorphous components of mannan II.     

Structure and dynamics of flour by solid state NMR: effects of hydration and wheat aging

The effects of accelerated aging of wheat seeds on structural and dynamic properties of dry and hydrated (ca 10 wt % H(2)O) flour at a molecular level were investigated by several high and low resolution solid-state NMR techniques. Identification and characterization of domains with different mobility was performed by (13)C direct excitation (DE) and cross-polarization (CP) magic angle spinning (MAS), as well as by (1)H static and MAS experiments. (1)H spin-lattice relaxation times (T(1) and T(1)(rho)) measurements were carried out to investigate molecular motions in different frequency ranges. Experimental data show that the main components of flour (starch and gluten proteins) are in a glassy phase, whereas the mobile fraction is constituted by lipids and, in hydrated samples, absorbed water. A lower proportion of rigid domains, as well as an increased dynamics of all flour components are observed after both seeds aging and flour hydration. Linear average dimensions between 20 and 200 A are estimated for water domains in hydrated samples.     

Structural determination of oligosaccharides derived from lipooligosaccharide of Neisseria gonorrhoeae F62 by chemical, enzymatic, and two-dimensional NMR methods

F62 LOS of Neisseria gonorrhoeae consists of two major LOS components; the higher and smaller molecular weight (MW) components were recognized by MAbs 1-1-M and 3F11 respectively. Base-line separation of the two major oligosaccharide (OS) components from F62 LOS was achieved by Bio-Gel P-4 chromatography after dephosphorylation of the OS mixture. The structures of the two major OSs were studied by chemical, enzymatic, and 2D NMR methods [double quantum filtered COSY (DQF-COSY), delayed COSY (D-COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), pure-absorption 2D NOE NMR] as well as methylation followed by GC/MS analysis. The OS component derived from the MAb 1-1-M defined LOS component was determined to have a V3-(beta-N-acetylgalactosaminyl)neolactotetraose structure (GalNAc is beta 1----3-linked to a neolactotetraose) at one of its nonreducing termini as shown below. The above pentaose is linked to a branched diheptose-KDO core in which a GlcNAc is alpha-linked. The OS component derived from the MAb 3F11 defined LOS component did not have a GalNAc residue. The rest of its structure was identical to that of the OS-1, and a neolactotetraose is exposed at its nonreducing terminus. [formula: see text]     

Membrane organization in soybean seeds during hydration

The ability of seeds to withstand dehydration indicates that their membranes may maintain structural integrity even when dry. Analysis of polar lipids (the principal lipidic constituents of the membranes) from soybean seeds (Glycine-max (L.) Merr.) by X-ray diffraction indicated that even in the dehydrated state the lipids retained a lamellar (bilayer) configuration. As the degree of hydration was raised, evidence of some structural alteration (apparent as an abrupt increase in bilayer spacing) was obtained from diffraction patterns of both the extracted lipid and particles of seed tissue. In seed tissue this increase in bilayer spacing occurred at a hydration level just above that at which free water could be detected by nuclear-magnetic-resonance analysis. The water content at which the increase in bilayer spacing occurred was higher in the seed tissue than in the extracted polar lipids, probably because other cell components restricted the availability of free water in the seed.Abbreviation NMR nuclear-magnetic resonance     

Effects of topographic variability on the scaling of plant species richness in gradient dominated landscapes

It is commonly assumed that variation in abiotic site conditions influences the number of niches, which in turn affects the potential species richness in an area. Based on theoretical considerations, abiotic variation is often used as an estimator of species richness at broad scales, while at finer landscape scales the diversity of habitat types is used. However, habitat estimators assume the landscape to be composed of discrete, homogeneous patches with sharp boundaries, and such a concept is hard to apply in gradient-dominated landscapes. The aim of this study was therefore to investigate the influence of topographic variability (TV) on species richness at the landscape level (gamma (γ) diversity) and on its components (alpha (α) and beta (β) diversity) at microsite and habitat group levels. Using floristic data from 12 "landscapes" of 1 km² we investigated the influence on diversity components of two simple and one complex measures of TV. While the standard deviation (SD) of altitude explained a high proportion of the variation in γ diversity (linear regression model, R²=0.63), the complex measure, SD of solar radiation explained it even better (R²=0.82). There were strong effects of TV on α and β diversity components at the microsite level, but only marginal increases of the diversity components at the habitat level. Further analyses revealed that the missing increase of the habitat level components was caused by differences between habitat groups and that only grassland diversity components increased significantly with TV. We conclude that TV at a landscape scale has strong effects on niche or microsite diversity and is an appropriate estimator of relative species richness in landscapes that are topographically heterogeneous and gradient dominated.