Effect of local sequence inversions on the crystalline antiparallel β-sheet lamellar structures of periodic polypeptides: implications for chain-folding

The crystal structure and texture of the monodisperse periodic polypeptide [(AG)₃EG(GA)₃EG]₁₀ (poly(±AG)₃EG; A=alanine, G=glycine, E=glutamic acid) were analyzed by X-ray diffraction, Fourier transform infrared spectroscopy, and electron microscopy. Structure determination was aided by comparison with the recently described structure for the related periodic polypeptide [(AG)₃EG]₃₆ by Krejchi et al. (Macromolecules 1997;30:5012). Texture-oriented samples of poly(±AG)₃EG were obtained by crystallization of the polymer from aqueous formic acid solution. The evidence supports an antiparallel (ap) β-sheet protein structure and the X-ray diffraction signals index on an orthorhombic unit cell with parameters: a=0.950 nm (hydrogen-bond direction), b=1.052 nm (apβ-sheet stacking direction), c=6.95 nm (chain direction). The absence of the (010) diffraction signal, a prominent signal in the poly(AG)₃EG diffraction pattern, implies that the apβ-sheets are ‘apolar', i.e. both surfaces are equally populated with alanyl methyl groups. Selective line broadening of wide-angle diffraction signals with ℓ≠0 gives an estimated crystal size of 4 nm in the chain direction. This observation, coupled with the appearance of low-angle particle interference peaks, indicates a crystal thickness considerably less than the chain length and suggests an adjacent-re-entry chain-folded lamellar structure incorporating the apβ-sheet architecture. The polypeptide folds through γ-turns, in-phase with the pseudo-octapeptide repeat; the glutamic acid residues occur on the lamellar surfaces. These results and those from the crystalline lamellae of poly(AG)₃EG suggest that β-turns are not compatible with these repetitively stacked apβ-sheet structures. This implies that intersheet interactions of alanyl methyl groups and glycyl -protons are not sufficiently strong to dictate the folding geometry in these structures.     

Relationships between conformation of β-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy

Attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) has been used to compare the structure of β-lactoglobulin, the major component of whey proteins, in solution and in its functional gel state. To induce variation in the conformation of β-lactoglobulin under a set of gelling conditions, the effect of heating temperature, pH, and high pressure homogenization on the conformation sensitive amide I band in the infrared spectra of both solutions and gels has been investigated. The results showed that gelification process has a pronounced effect upon β-lactoglobulin secondary structure, leading to the formation of intermolecular hydrogen-bonding β-sheet structure as evidenced by the appearance of a strong band at 1614 cm⁻¹ at the expense of other regular structures. These results confirm that this structure may be essential for the formation of a gel network as it was previously shown for other globular proteins. However, this study reveals, for the first time, that there is a close relationship between conformation of β-lactoglobulin in solution and its capacity to form a gel. Indeed, it is shown that conditions which promote predominance of intermolecular β-sheet in solution such as pH 4, prevent the formation of gel in conditions used by increasing thermal stability of β-lactoglobulin. On the basis of these findings, it is suggested that by controlling the extent of intermolecular β-structure of the protein in solution, it is possible to modify the ability of protein to form a gel and as a consequence to control the properties of gels.     

3D-structure of human estrogenic 17beta-HSD1: binding with various steroids.

Human estrogenic dehydrogenase (17β-HSD1) catalyses the last step in the biosynthesis of the active estrogens that stimulate the proliferation of breast cancer cells. While the primary substrate for the enzyme is estrone, the enzyme has some activity for the non-estrogenic substrates. To better understand the structure–function relationships of 17β-HSD1 and to provide a better ground for the design of inhibitors, we have determined the crystal structures of 17β-HSD1 in complex with different steroids.

The structure of the complex of estradiol with the enzyme determined previously (Azzi et al., Nature Structural Biology 3, 665–668) showed that the narrow active site was highly complementary to the substrate. The substrate specificity is due to a combination of hydrogen bonding and hydrophobic interactions between the steroid and the enzyme binding pocket. We have now determined structures of 17β-HSD1 in complex with dihydrotestosterone and 20-OH-progesterone. In the case of the C19 androgen, several residues within the enzyme active site make some small adjustments to accommodate the increased bulk of the substrate. In addition, the C19 steroids bind in a slightly different position from estradiol with shifts in positions of up to 1.4 Å. The altered binding position avoids unfavorable steric interactions between Leu 149 and the C19 methyl group (Han et al., unpublished). The known kinetic parameters for these substrates can be rationalized in light of the structures presented. These results give evidence for the structural basis of steroid recognition by 17β-HSD1 and throw light on the design of new inhibitors for this pivotal steroid enzyme.     

Thermally reversible acid-induced gelation of low-methoxy pectin

Gelation of low-methoxy pectin (DE 31.1) on cooling under acidic conditions in the absence of Ca²⁺ has been investigated by rheological measurements under low-amplitude oscillatory shear. The mechanical spectra obtained after 60 min at 5°C showed a progressive increase in solid-like response (increasing G′; decreasing tan δ; increasing frequency-dependence of η^*) as the pH was reduced from 4.0 to 1.6, with formation of a critically crosslinked network at pH 3.0 (for a polymer concentration of 3.0 wt%). By extrapolation from X-ray fibre diffraction analysis of pectic acid, it is suggested that crosslinking occurs by association of three-fold helices. At pH values between 3.5 and 2.5 there is no detectable thermal hysteresis between the sol–gel transition on cooling and gel–sol transition on heating, and both are accompanied by a sigmoidal change in optical rotation (attributed to formation and melting of three-fold order). Substantial hysteresis is, however, observed at lower and higher pH, and is attributed to extensive aggregation as electrostatic repulsion is suppressed (below pH 2.5) and slow formation of intermolecular hydrogen bonds by protonated carboxyl groups (above pH 3.5), respectively. The transition enthalpy from DSC heating scans has a maximum value of ΔH≈11 J/g at pH 3.0, but decreases sharply at lower and higher pH, with accompanying loss of a detectable transition in optical rotation. It is suggested that the chain conformation in solution at low pH is predominantly three-fold with, therefore, little conformational change on adoption of the ordered, intermolecular structure, whereas at high pH the solution conformation is predominantly two-fold, with only limited conversion to the three-fold (acid) form on cooling.     

Two non-reactive ternary complexes of estrogenic 17beta-hydroxysteroid dehydrogenase: crystallization and preliminary structural analysis.

Human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1, EC1.1.1.62) is an important enzyme that catalyses the last step of active estrogen formation. 17β-HSD1 plays a key role in the proliferation of breast cancer cells. The three-dimensional structures of this enzyme and of the enzyme-estradiol complex have been solved (Zhu et al., 1993, J. Mol. Biol. 234:242; Ghosh et al., 1995, Structure 3:503; Azzi et al., 1996, Nature Struct. Biol. 3:665). The determination of the non-reactive ternary complex structure, which could mimic the transition state, constitutes a further critical step toward the rational design of inhibitors for this enzyme (Ghosh et al. 1995, Structure 3:503; Penning, 1996, Endocrine-Related Cancer, 3:41).

To further study the transition state, two non-reactive ternary complexes, 17β-HSD1–EM519-NADP⁺ and 17β-HSD1–EM553-NADP⁺ were crystallized using combined methods of soaking and co-crystallization. Although they belong to the same C2 space group, they have different unit cells, with a=155.59 Å, b=42.82 Å, c=121.15 Å, β=128.5° for 17β-HSD1–EM519-NADP⁺, and a=124.01 Å, b=45.16 Å, c=61.40 Å, β=99.2° for 17β-HSD1–EM553-NADP⁺, respectively. Our preliminary results revealed that the inhibitors interact differently with the enzyme than do the natural substrates.     

Fourier-transform infrared spectroscopic study of globulin from Phaseolus angularis (red bean).

The conformation of red bean globulin dispersions (≈10% in D₂O or deuterated phosphate buffer pD 7.4) under the influence of pH, chaotropic salts, protein structure perturbants, and heating conditions was studied by Fourier-transform infrared (FTIR) spectroscopy. The FTIR spectrum of red bean globulin showed major bands from 1682 to 1637 cm⁻¹ in the amide I′ region, corresponding to the four types of secondary structures, i.e. β-turns, β-sheets, -helix and random coils. At extreme pH conditions, there were changes in intensity in bands attributed to β-sheet (1637 and 1618 cm⁻¹) and random coil (1644 cm⁻¹) structures, and shifts of these bands to lower or higher wavenumbers, indicating changes in protein conformation. Chaotropic salts caused progressive increases in random coil structures and concomitant decreases in β-sheet bands, following the lyotrophic series of anions. In the presence of sodium dodecyl sulfate and ethylene glycol, pronounced increases in the random coil band were observed, accompanied by slight shifts of the β-sheet band. Addition of dithiothreitol and N-ethylmaleimide did not cause marked changes in the FTIR spectra. Heating at increasing temperature led to progressive decreases in the intensity of the -helix and β-sheet bands and increases in random coil band intensity, leveling off at around 60 °C. The data suggest that re-organization of protein structure occurred at temperatures well below the denaturation temperature of red bean globulin (86 °C) as determined by differential scanning calorimetry. This was accompanied by pronounced increases in the intensity of the two intermolecular β-sheet bands (1682 and 1619–1620 cm⁻¹) associated with the formation of aggregated strands at higher temperatures (80–90 °C). Increases in intensity of the aggregation bands were also observed in the heat-induced buffer-soluble and insoluble aggregates.     

The type I and type II 11beta-hydroxysteroid dehydrogenase enzymes.

Local tissue concentrations of glucocorticoids are modulated by the enzyme 11β-hydroxysteroid dehydrogenase which interconverts cortisol and the inactive glucocorticoid cortisone in man, and corticosterone and 11-dehydrocorticosterone in rodents. The type I isoform (11β-HSD1) is a bidirectional enzyme but acts predominantly as a oxidoreductase to form the active glucocorticoids cortisol or corticosterone, while the type II enzyme (11β-HSD2) acts unidirectionally producing inactive 11-keto metabolites. There are no known clinical conditions associated with 11β-HSD1 deficiency, but gene deletion experiments in the mouse indicate that this enzyme is important both for the maintenance of normal serum glucocorticoid levels, and in the activation of key hepatic gluconeogenic enzymes. Other important sites of action include omental fat, the ovary, brain and vasculature. Congenital defects in the 11β-HSD2 enzyme have been shown to account for the syndrome of apparent mineralocorticoid excess (AME), a low renin severe form of hypertension resulting from the overstimulation of the non-selective mineralocorticoid receptor by cortisol in the distal tubule of the kidney. Inactivation of the 11β-HSD2 gene in mice results in a phenotype with similar features to AME. In addition, these mice show high neonatal mortality associated with marked colonic distention, and remarkable hypertrophy and hyperplasia of the distal tubule epithelia. 11β-HSD2 also plays an important role in decreasing the exposure of the fetus to the high levels of maternal glucocorticoids. Recent work suggests a role for 11β-HSD2 in non-mineralocorticoid target tissues where it would modulate glucocorticoid access to the glucocorticoid receptor, in invasive breast cancer and as a mechanism providing ligand for the putative 11-dehydrocorticosterone receptor. While previous homologies between members of the SCAD superfamily have been of the order of 20–30% phylogenetic analysis of a new branch of retinol dehydrogenases indicates identities of >60% and overlapping substrate specificities. The availability of crystal structures of family members has allowed the mapping of conserved 11β-HSD domains A–D to a cleft in the protein structure (cofactor binding domain), two parallel β-sheets, and an -helix (active site), respectively.     

The role of 11β-hydroxysteroid dehydrogenase in steroid hormone specificity

11β-hydroxysteroid dehydrogenase (11β-HSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the mineralocorticoid receptor (MR) from occupancy by endogenous glucocorticoids. In aldosterone target cells the type 2 11β-HSD is present, which, in contrast to the type 1 11β-HSD, has very high affinity for its substrate, is unidirectional and prefers NAD as cofactor. cDNAs encoding 11β-HSD2 have been recently cloned from different species, and the cell-specific expression of its mRNA and protein were determined. 11β-HSD2 is expressed in every aldosterone target tissue. Northern analysis revealed that the rabbit 11β-HSD2 is expressed at high levels in the renal collecting duct and at much lower levels in the colon. RT-PCR experiments demonstrated that 11β-HSD2 mRNA is present only in aldosterone target cells within the kidney. We determined the subcellular localization of the rabbit 11β-HSD2 using a chimera encoding 11β-HSD2 and the green fluorescent protein (GFP). This construct was stably transfected into CHO and MDCK cells. The expressed 11β-HSD2/GFP protein retained high enzymatic activity, and its characteristics were undistinguishable from those of the native enzyme. The intracellular localization of this protein was determined by fluorescence microscopy. 11β-HSD2-associated fluorescence was observed as a reticular network over the cytoplasm whereas the plasma membrane and the nucleus were negative, suggesting endoplasmic reticulum (ER) localization. Co-staining with markers for ER proteins, the Golgi membrane, mitochondria and nucleus confirmed that 11β-HSD2 is localized exclusively to the ER. To determine what structural motifs are responsible for the ER localization, we generated deletion mutants missing the C-terminal 42 and 118 amino acids, and fused them to GFP. Similarly as with the intact 11β-HSD2, these mutants localized exclusively to the ER. Both C-terminal deletion mutants completely lost dehydrogenase activity, independently whether activity was determined in intact cells or homogenates. These results indicate that 11β-HSD2 has a novel ER retrieval signal which is not localized to the C-terminal region. In addition, the C-terminal 118 amino acids are essential for NAD-dependent 11β-HSD activity.     

Hydrogen-bonded turns in proteins: the case for a recount

Beta-turns are sites at which proteins change their overall chain direction, and they occur with high frequency in globular proteins. The Protein Data Bank has many instances of conformations that resemble beta-turns but lack the characteristic N-H(i) --> O=C(i - 3) hydrogen bond of an authentic beta-turn. Here, we identify potential hydrogen-bonded beta-turns in the coil library, a Web-accessible database utility comprised of all residues not in repetitive secondary structure, neither alpha-helix nor beta-sheet (http://www.roselab.jhu.edu/coil). In particular, candidate turns were identified as four-residue segments satisfying highly relaxed geometric criteria but lacking a strictly defined hydrogen bond. Such candidates were then subjected to a minimization protocol to determine whether slight changes in torsion angles are sufficient to shift the conformation into reference-quality geometry without deviating significantly from the original structure. This approach of applying constrained minimization to known structures reveals a substantial population of previously unidentified, stringently defined, hydrogen-bonded beta-turns. In particular, 33% of coil library residues were classified as beta-turns prior to minimization. After minimization, 45% of such residues could be classified as beta-turns, with another 8% in 3(10) helixes (which closely resemble type III beta-turns). Of the remaining coil library residues, 37% have backbone dihedral angles in left-handed polyproline II structure.     

Regulation of sex steroid formation by interleukin-4 and interleukin-6 in breast cancer cells

Sex steroids play a predominant role in the development and differentiation of normal mammary gland as well as in the regulation of hormone-sensitive breast cancer growth. There is evidence suggesting that local intracrine formation of sex steroids from inactive precursors secreted by the adrenals namely, dehydroepiandrosterone (DHEA) and 4-androstenedione (4-dione) play an important role in the regulation of growth and function of peripheral target tissues, including the breast. Moreover, human breast carcinomas are often infiltrated by stromal/immune cells secreting a wide spectra of cytokines. These might in turn regulate the activity of both immune and neoplastic cells. The present study was designed to examine the action of cytokines on 17β-hydroxysteroid dehydrogenase (17β-HSD) and 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) activities in human breast cancer cells. The various types of human 17β-HSD (five types) and 3β-HSD (two types), because of their tissue- and cell-specific expression and substrate specificity, provide each cell with necessary mechanisms to control the level of intracellular active androgens and estrogens. We first investigated the effect of exposure to IL-4 and IL-6 on reductive and oxidative 17β-HSD activities in both intact ZR-75-1 and T-47D human breast cancer cells. In ZR-75-1 cells, a 6 d exposure to IL-4 and IL-6 decreased E₂-induced cell proliferation, the half maximal inhibitory effect being exerted at 88 and 26 pM, respectively. In parallel, incubation with IL-4 and IL-6 increased oxidative 17β-HSD activity by 4.4- and 1.9-fold, respectively, this potent activity being observed at ₅₀ values of 22.8 and 11.3 pM, respectively. Simultaneously, reductive 17β-HSD activity leading to E₂ formation was decreased by 70 and 40% by IL-4 and IL-6, respectively. Moreover, IL-4 and IL-6 exerted the same regulatory effects on 17β-HSD activities when testosterone and 4-dione were used as substrates, thus strongly suggesting the expression of the type 2 17β-HSD ZR-75-1 cells. In contrast, in T-47D cells, IL-4 increased the formation of E₂, whereas IL-6 exerts no effect on this parameter. However, we found that T-47D cells failed to convert testosterone efficiently into 4-DIONE, thus suggesting that there is little or no expression of type 2 17β-HSD in this cell line. The present findings demonstrate that the potent regulatory effects of IL-4 and IL-6 on 17β-HSD activities depend on the cell-specific gene expression of various types of 17β-HSD enzymes. We have also studied the effect of cytokines on the regulation of the 3β-HSD expression in both ZR-75-1 and T-47D human breast cancer cells. Under basal culture conditions, there is no 3β-HSD activity detectable in these cells. However, exposure to IL-4 caused a rapid and potent induction of 3β-HSD activity, whereas IL-6 failed to induce 3β-HSD expression. Our data thus demonstrate that cytokines may play a crucial role in sex steroid biosynthesis from inactive adrenal precursors in human breast cancer cells.     

β-Adrenoreceptor participation in the formation of the thermoregulatory and immune responses under the effect of rapid deep cooling

The effect of β-adrenoantagonist (obzidan) iontophoresis to skin on the thermoregulatory response and immune response to antigen was analyzed to elucidate the significance of β-adrenoceptors in formation of these responses at deep rapid cooling in rats.

On the background of β-adrenoceptors blockade in thermoneutral conditions the skin and core temperatures decreases; at rapid cooling non-shivering thermogenesis is attenuated and shivering thermogenesis is considerably enhanced.

Administration of β-adrenoantagonist affect the modulating influence of cold exposure on the immune response—the immunosuppressive effect of deep cooling on the immune response is abolished. This concerns both antigen binding function of spleen and peritoneal cells and antibody formation.

The results support the idea that β-adrenoceptors participates in the processes of the stimulation of thermogenesis and suppression of the immune response to antigen at rapid deep cooling.     

Cannulation in situ of equine umbilicus. Identification by gas chromatography-mass spectrometry (GC-MS) of differences in steroid content between arterial and venous supplies to and from the placental surface

Equine umbilicus was cannulated in utero and a series of cord plasma samples removed for analysis. After steroid extraction and derivatisation, gas chromatographic-mass spectrometric (GC-MS) analysis demonstrated large differences in steroid content between the plasma samples obtained from the umbilical artery and vein, the blood supplies leading to and from the placental surface, respectively. 3β-Hydroxy-5,7-androstadien-17-one, dehydroepiandrosterone, pregnenolone, 3β-hydroxy-5-pregnan-20-one, 5-pregnene-3β,20β-diol and 5β-pregnane-3β,20β-diol were identified as major constituents in extracts from umbilical arterial plasma samples, mostly as unconjugated steroids. Together with 5-pregnane-3,20-dione, these steroids were identified in extracts from umbilical venous plasma samples but at significantly reduced levels to those determined in arterial plasma samples. Oestradiol-17, dihydroequilin-17 and dihydroequilenin-17 were identified in extracts (mostly sulphate-conjugated) from both umbilical arterial and venous plasma samples, much larger amounts being detected in the plasma sampled from, rather than to, the placental surface. Equilin, equilenin, oestrone, oestradiol-17β, dihydroequilin-17β and dihydroequilenin-17β were not detected in the present studies. Isomers of 5(10)-oestrene-3,17β-diol together with 5(10),7-oestradiene-3,17β-diol and its possible oxidative artifact, 5(10),7,9-oestratriene-3,17β-diol, were tentatively identified only in sulphate-conjugated extracts from umbilical venous plasma samples. No glucuronic acid-conjugated steroids could be detected. The implications of this work in the elucidation of the biosynthetic pathways leading to both the formation of oestrogens and C₁₈ neutral steroids at the placental surface are discussed.     

Pustulan and branched β-galactofuranan from the phytopathogenic fungus Guignardia citricarpa, excreted from media containing glucose and sucrose

Guignardia citricarpa is a phytopathogenic fungus and the causal agent of citrus black spot. Incubation in a semi-defined media resulted in formation of exopolysaccharides [EPS(s)]. A medium containing glucose gave rise to a (1→6)-linked β-glucan (200 kD), pustulan, which was characterized by NMR and methylation analysis. A sucrose-containing medium provided a homogalactan (376 kD) and methylation analysis showed nonreducing end- (20%), 6-O- (53%) and 5,6-di-O-substituted Galf units (27%). An HMQC spectrum of the homogalactan showed C-1/H-1 signals at δ 108.2/4.820, 108.3/4.820 and 107.1/5.079, corresponding to three types of β- -Galf units. A DEPT analysis showed inverted signals (CH₂) at δ 67.8 and 67.2, corresponding to 6-O-substituted β- -Galf units, whereas a C-5 signal at δ 77.0 suggests 5-O-substitution, confirming a novel structure for a β-galactofuranan.     

Tissue- and temporal-specific regulation of 11beta-hydroxysteroid dehydrogenase type 1 by glucocorticoids in vivo.

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) catalyses the interconversion of active corticosterone and inert 11-dehydrocorticosterone. Short-term glucocorticoid excess upregulates 11β-HSD-1 in liver and hippocampus leading to suggestions that 11β-HSD-1 ameliorates the deleterious effects of glucocorticoid excess by its 11β-dehydrogenase activity. However the predominant activity of 11β-HSD-1 in vivo is 11β-reduction, thus generating active glucocorticoid. We have re-examined the time-course of glucocorticoid regulation of 11β-HSD-1 in the liver, hippocampus and kidney of adult male rats in vivo.

Sham operation markedly reduced 11β-HSD-1 mRNA expression in all tissues, and reduced 11β-HSD bioactivity in liver and hippocampus when compared to untouched controls. Adrenalectomy reduced 11β-HSD-1 expression in all tissues in the short-term (7 days), followed by subsequent recovery of enzyme activity by 21 days in liver and hippocampus. Dexamethasone replacement of adrenalectomised rats attenuated the initial decrease in hepatic 11β-HSD-1 activity, but by 21 days dexamethasone reduced activity compared to control levels.

Thus glucocorticoids regulate 11β-HSD-1 in a complex tissue- and temporal-specific manner. This pattern of regulation suggests glucocorticoids repress 11β-HSD-1 at least in the liver, a pattern of regulation more consistent with the evidence that 11β-HSD-1 is an 11β-reductase in vivo. Operational stress per se down-regulates 11β-HSD-1 which has implications for interpretation and design of in vivo studies of 11β-HSD-1.     

Transformation of steroids by Bacillus strains isolated from the foregut of water beetles (Coleoptera:Dytiscidae): I. Metabolism of androst-4-en-3,17-dione (AD)

Two Bacillus strains were isolated from the foregut of the water beetle Agabus affinis (Payk.) and tested for their steroid transforming ability. After incubation with androst-4-en-3,17-dione (AD), 13 different transformation products were detected. AD was hydroxylated at C₆, C₇, C₁₁ and C₁₄, resulting in formation of 6β-, 7-, 11- and 14-hydroxy-AD. One strain also produced small amounts of 6β,14-dihydroxy-AD. Partly, the 6β-hydroxy group was further oxidized to the corresponding 6-oxo steroids. In addition, a specific reduction of the Δ⁴-double bond was observed, leading to the formation of 5-androstane derivatives. In minor yields the carbonyl functions at C₃ and C₁₇ were reduced leading to the formation of 3ξ-OH or 17β-OH steroids. EI mass spectra of the trimethylsilyl and O-methyloxime trimethylsilyl ether derivatives of some transformation products are presented for the first time.     

Effects of landscape connectivity on the spatial distribution of insect diversity in agricultural mosaic landscapes

European agricultural landscapes are mosaics of intensively cultivated areas and semi-natural elements. Although comprising only a small fraction of the total area, semi-natural elements provide habitat for most of the landscape biodiversity. Agricultural intensification has increasingly fragmented semi-natural elements and species numbers are in decline. Insights into the effects of landscape structure on species’ distributions within and among semi-natural habitats are needed to conserve biodiversity in agricultural landscapes more effectively. We investigated the landscape- and habitat-specific diversity partitions of wild bees, true bugs, and carabid beetles in two differently structured agricultural landscapes in Switzerland. In each landscape, we partitioned the total species diversity (γ) into its additive components within (_P) and among patches (β_P) and among habitats (β_H). In the landscape characterized by a patchy, isolated distribution of habitat elements, among-patch diversity (β_P) explained 44% of the total species richness (γ) and was significantly higher than expected under a random distribution of samples among habitat patches; in the landscape with higher habitat connectivity, among-patch diversity (β_P) comprised 32% of the total species richness (γ) and did not differ from the random expectation. Habitat-specific within-patch contributions to species richness were similarly low across habitat types (_P=23–24%) in the patchy landscape, whereas in the more connected landscape within-patch partitions tended to be higher and differed among habitat types (_P=22–38%). Functionally different groups of bees, true bugs, and carabids also responded differently to landscape structure in a manner that was consistent with known differences in resource specialization and dispersal ability. Differences in diversity partitions among landscapes and taxa indicate the need for flexible conservation strategies. Conservation of habitat-specific diversity may require more habitat patches in landscapes that have lower habitat connectivity and low within-patch diversity (_P) than in landscapes with higher within-patch diversity (_P).     

Characterisation of the beta-tubulin gene of Cylicocyclus nassatus?

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the β-tubulin amino acid sequences of other species. To complete the coding sequence, the 3′ end was amplified with the 3′-RACE, and for amplification of the 5′ end the SL1-primer was used. The cDNA of the β-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the β-tubulin gene. The complete sequence of the genomic DNA of the β-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 β-tubulin gene of Haemonchus contortus range between 79% and 97%.