Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology

To gauge the experimental variability associated with Biacore analysis, 36 different investigators analyzed a small molecule/enzyme interaction under similar conditions. Acetazolamide (222 g/mol) binding to carbonic anhydrase II (CAII; 30,000 Da) was chosen as a model system. Both reagents were stable and their interaction posed a challenge to measure because of the low molecular weight of the analyte and the fast association rate constant. Each investigator created three different density surfaces of CAII and analyzed an identical dilution series of acetazolamide (ranging from 4.1 to 1000 nM). The greatest variability in the results was observed during the enzyme immobilization step since each investigator provided their own surface activating reagents. Variability in the quality of the acetazolamide binding responses was likely a product of how well the investigators’ instruments had been maintained. To determine the reaction kinetics, the responses from the different density surfaces were fit globally to a 1:1 interaction model that included a term for mass transport. The averaged association and dissociation rate constants were 3.1 ± 1.6 × 10⁶ M⁻¹ s⁻¹ and 6.7 ± 2.5 × 10⁻² s⁻¹, respectively, which corresponded to an average equilibrium dissociation constant (K_D) of 2.6 ± 1.4 × 10⁻⁸ M. The results provide a benchmark of variability in interpreting binding constants from the biosensor and highlight keys areas that should be considered when analyzing small molecule interactions.     

Territorial defense against various food competitors in the Tanganyikan benthophagous cichlid <Emphasis Type="Italic">Neolamprologus tetracanthus</Emphasis>

Territorial defense of nonbreeding female Neolamprologus tetracanthus, a shrimp-eating Tanganyikan cichlid, was investigated. Females defended territories (=home ranges, ca. 1m across) against a variety of intruding fishes. Conspecific females were usually attacked outside the territories, heterospecific benthivores (shrimp eaters) and omnivores near the border of the territories, and piscivores, algae and detritus feeders, and herbivores inside the territories. Females used some parts of the sandy substrate in the territories for foraging (foraging areas). Territorial defense prevented most of the conspecific females and benthivores from intruding into the foraging areas. In omnivores, piscivores, and algae and detritus feeders, about half the intruders were repelled from the foraging areas, although herbivores were infrequently repelled in the areas. Soon after removal of the resident females, many food competitors invaded the foraging areas and eagerly devoured prey, suggesting that the territories are maintained for food resource protection from these competitors. Females are likely to discriminate intruding fishes and change their territorial defense primarily on the basis of the degree of dietary overlap, resulting in monofunctional serial territories.     

Avidin related protein 2 shows unique structural and functional features among the avidin protein family

Background  

The chicken avidin gene family consists of avidin and several avidin related genes (AVRs). Of these gene products, avidin is the best characterized and is known for its extremely high affinity for D-biotin, a property that is utilized in numerous modern life science applications. Recently, the AVR genes have been expressed as recombinant proteins, which have shown different biotin-binding properties as compared to avidin.     

The effect of hydrogen bonds on the conformation of glycosphingolipids. Methylated and unmethylated cerebroside studied by X-ray single crystal analysis and model calculations

The conformation and molecular packing of permethylated beta-D-galactosyl-N-octadecanoyl-D-spingosine (cerebroside) was determined by X-ray single crystal analysis at 185 K (R = 0.16). The lipid crystallizes in the orthorhombic space group P2(1)2(1)2(1) with the unit cell dimensions a = 8.03, b = 7.04 and c = 88.10 A. The four molecules in the unit cell pack in a bilayer arrangement with tilting (48 degrees) hydrocarbon chains. The direction of the chain tilt alternates in the two bilayer halves and in adjacent bilayers. In order to define the effect of hydrogen bonds on the molecular conformation the structural features of the permethylated cerebroside are compared with that of unsubstituted cerebroside (I. Pascher and S. Sundell (1977) Chem. Phys. Lipids 20, 179). It is shown that methylation of the hydrogen donor groups does not affect the conformation of the ceramide part. However, by abolishing the intramolecular hydrogen bond between the amide N--H group and the glycosidic oxygen the galactose ring changes its orientation from layer-parallel to layer-perpendicular. Calculations using molecular mechanics, MM2(87), show that in natural cerebroside the intramolecular hydrogen bond stabilizes the theta 1 = -syn-clinal conformation about the C(1)--C(2) sphingosine bond by 2-2.5 kcal/mol compared to other staggered conformations. The significance of the L shape of the native cerebroside, making both the carbohydrate and polar ceramide groups accessible as a binding epitope in recognition processes, is discussed.     

Developmental immunohistology of melanotrophs in Xenopus laevis tadpoles

Summary The adenohypophysial primordium of Xenopus laevis tadpoles at stages 33/34 to 46 (Nieuwkoop and Faber, 1956) were examined immuno-histologically for -MSH, -MSH and ACTH. -MSH was demonstrated from stage 37/38 onwards, and -MSH from stage 39. No signs of ACTH production were detected. -MSH and -MSH occurred in the same cells. No differences were found in the intensity of immunofluorescence between tadpoles which were kept on a black and a white background. The present study lends no support to the hypothesis concerning the derivation of -MSH from ACTH. The observations made suggest that the morphological formation of the pars intermedia is accomplished during stages 37/38 to 39. Acknowledgement. The authors express warm thanks to Dr. M.P. Dubois (Laboratoire de Physiologie de la Reproduction, INRA, Nouzilly, France), who prepared and verified the antibodies. Grants from Swedish Natural Science Research Council and Landshovding Per Westlings minnesfond, Lund, Sweden are gratefully acknowledged     

Nonlamellar-Phase-Promoting Colipids Enhance Segregation of Palmitoyl Ceramide in Fluid Bilayers

Ceramide is an important intermediate in sphingolipid homeostasis. We examined how colipids, with negative intrinsic curvature and which may induce curvature stress in the bilayers, affected the segregation of palmitoyl ceramide (PCer). Such colipids include 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and tetra-linoleoyl cardiolipin (CL). In 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers, PCer formed ordered, gel-like domains at concentrations above 10 mol% at 23°C, as evidenced by the change in the average lifetime of the trans-parinaric acid emission. When POPE or DOPE were included in the DOPC bilayer (at 20:80 or 40:60 POPE or DOPE to DOPC, by mol), the lateral segregation of PCer was facilitated in a concentration-dependent manner, and less PCer was required for the formation of the ordered ceramide-rich domains. Inclusion of CL in the DOPE bilayer (at 10:90 or 20:80 CL to PC, by mol) also caused a similar facilitation of the lateral segregation of PCer. The PCer-rich domains formed in the presence of POPE, DOPE, or CL in DOPC bilayers were slightly more thermostable (by 2–10°C) when compared to PCer-rich domains in DOPC-only bilayers. Nonlamellar phases were not present in bilayers in which the effects of POPE or DOPE on PCer segregation were the largest, as verified by ³¹P NMR. When palmitoyl sphingomyelin was added to the different bilayer compositions at 5 mol%, relative to the phospholipids, PCer segregated into gel domains at lower concentrations (2–3 mol% PCer), and the effect of POPE on PCer segregation was eliminated. We suggest that the effects of POPE, DOPE, and CL on PCer segregation was in part influenced by their effects on membrane curvature stress and in part because of unfavorable interactions with PCer due to their unsaturated acyl chains. These lipids are abundant in mitochondrial membranes and are likely to affect functional properties of saturated ceramides in them.     

High-capacity conductive nanocellulose paper sheets for electrochemically controlled extraction of DNA oligomers

Highly porous polypyrrole (PPy)-nanocellulose paper sheets have been evaluated as inexpensive and disposable electrochemically controlled three-dimensional solid phase extraction materials. The composites, which had a total anion exchange capacity of about 1.1 mol kg(-1), were used for extraction and subsequent release of negatively charged fluorophore tagged DNA oligomers via galvanostatic oxidation and reduction of a 30-50 nm conformal PPy layer on the cellulose substrate. The ion exchange capacity, which was, at least, two orders of magnitude higher than those previously reached in electrochemically controlled extraction, originated from the high surface area (i.e. 80 m(2) g(-1)) of the porous composites and the thin PPy layer which ensured excellent access to the ion exchange material. This enabled the extractions to be carried out faster and with better control of the PPy charge than with previously employed approaches. Experiments in equimolar mixtures of (dT)(6), (dT)(20), and (dT)(40) DNA oligomers showed that all oligomers could be extracted, and that the smallest oligomer was preferentially released with an efficiency of up to 40% during the reduction of the PPy layer. These results indicate that the present material is very promising for the development of inexpensive and efficient electrochemically controlled ion-exchange membranes for batch-wise extraction of biomolecules.     

Probing the lipid-protein interface using model transmembrane peptides with a covalently linked acyl chain

The aim of this study was to gain insight into how interactions between proteins and lipids in membranes are sensed at the protein-lipid interface. As a probe to analyze this interface, we used deuterium-labeled acyl chains that were covalently linked to a model transmembrane peptide. First, a perdeuterated palmitoyl chain was coupled to the Trp-flanked peptide WALP23 (Ac-CGWW(LA)₈LWWA-NH₂), and the deuterium NMR spectrum was analyzed in di-C18:1-phosphatidylcholine (PC) bilayers. We found that the chain order of this peptide-linked chain is rather similar to that of a noncovalently coupled perdeuterated palmitoyl chain, except that it exhibits a slightly lower order. Similar results were obtained when site-specific deuterium labels were used and when the palmitoyl chain was attached to the more-hydrophobic model peptide WLP23 (Ac-CGWWL₁₇WWA-NH₂) or to the Lys-flanked peptide KALP23 (Ac-CGKK(LA)₈LKKA-NH₂). The experiments showed that the order of both the peptide-linked chains and the noncovalently coupled palmitoyl chains in the phospholipid bilayer increases in the order KALP23 < WALP23 < WLP23. Furthermore, changes in the bulk lipid bilayer thickness caused by varying the lipid composition from di-C14:1-PC to di-C18:1-PC or by including cholesterol were sensed rather similarly by the covalently coupled chain and the noncovalently coupled palmitoyl chains. The results indicate that the properties of lipids adjacent to transmembrane peptides mostly reflect the properties of the surrounding lipid bilayer, and hence that (at least for the single-span model peptides used in this study) annular lipids do not play a highly specific role in protein-lipid interactions.