Voltage-dependent channel formation by rods of helical polypeptides

Summary The voltage-dependence of channel formation by alamethicin and its natural analogues can be described by a dipole flip-flop gating model, based on electric field-induced transbilayer orientational movements of single molecules. These field-induced changes in orientation result from the large permanent dipole moment of alamethicin, which adopts -helical conformation in hydrophobic medium. It was, therefore, supposed that the only structural requirement for voltage-dependent formation of alamethicin-type channels might be a rigid lipophilic helical segment of minimum length.In order to test this hypothesis we synthesized a family of lipophilic polypeptides—Boc-(Ala-Aib-Ala-Aib-Ala) _n -OMe,n=1–4—which adopt -helical conformation forn=2–4 and studied their interaction with planar lipid bilayers. Surprisingly, despite their large difference in chain length, all four polypeptides showed qualitatively similar behavior. At low field strength of the membrane electric field these polypeptides induce a significant, almost voltage-independent increase of the bilayer conductivity. At high field strength, however, a strongly voltage-dependent conductance increase occurs similar to that observed with alamethicin. It results from the opening of a multitude of ion translocating channels within the membrane phase.The steady-state voltage-dependent conductance depends on the 8^th–9^th power of polypeptide concentration and involves the transfer of 4–5 formal elementary charges. From the power dependences on polypeptide concentration and applied voltage of the time constants in voltage-jump current-relaxation experiments, it is concluded that channels could be formed from preexisting dodecamer aggregates by the simultaneous reorientation of six formal elementary charges. Channels exhibit large conductance values of several nS, which become larger towards shorter polypeptide chain length. A mean channel diameter of 19 Å is estimated corresponding roughly to the lumen diameter of a barrel comprised of 10 -helical staves. Similar to experiments with the N-terminal Boc-derivative of alamethicin we did not observe the burst sequence of nonintegral conductance steps typical of natural (N-terminal Ac-Aib)-alamethicin. Saturation in current/voltage curves as well as current inactivation in voltage-jump current-relaxation experiments are found. This may be understood by assuming that channels are generated as dodecamers but, while reaching the steady state, reduce their size to that of an octamer or nonamer. We conclude that the overall behavior of these synthetic polypeptides is very similar to that of alamethicin. They exhibit the same concentration and voltage-dependences but lack the stabilizing principle of resolved channel states characteristic of alamethicin.     

Pilot study: potential transcription markers for adult attention-deficit hyperactivity disorder in whole blood

Attention-deficit hyperactivity disorder (ADHD) is a common behavioural disorder that affects not only children and adolescents but also adults; however, diagnosis of adult ADHD is difficult because patients seem to have reduced externalized behaviour. ADHD is a multifactorial disorder in which many genes, all with small effects, are thought to cause the disorder in the presence of unfavourable environmental conditions. Therefore, in this pilot study, we explored the expression profile of a list of previously established candidate genes in peripheral blood samples from adult ADHD subjects (n = 108) and compared these results with those of healthy controls (n = 35). We demonstrate that combining the gene expression levels of dopamine transporter (SLC6A3), dopamine D5 receptor, tryptophan hydroxylase-1, and SNAP25 as predictors in a regression model resulted in sensitivity and specificity of over 80 % (ROC: max R(2) = 0.587, AUC = 0.917, P < 0.001, 95 % CI: 0.900-0.985). In conclusion, the combination of these four genes could represent a potential method for estimating risk and could be of diagnostic value for ADHD. Nevertheless, further investigation in a larger independent population including different subtypes of ADHD (inattentive, hyperactive, or combined type) patients is required to obtain more specific sets of biomarkers for each subtype as well as to differentiate between child, adolescent, and adulthood forms.     

Isolation and characterization of a collagen fibril-associated dermatan sulphate proteoglycan from bovine lung

Dermatan sulphate proteoglycans have been extracted from bovine lung with 2.0 M CaCl2 and isolated using CsCl density gradient centrifugation, DEAE ion-exchange chromatography, gel chromatography and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Ultrastructurally these proteoglycans are specifically associated with collagen fibrils. Dermatan sulphate (Mr 15.10(3)-35.10(3), with a strong prevalence for the higher Mr) is link via an O-glycosidic bond to a protein core, which is rich in Asx, Glx and Leu. Of the total uronic acid, 91% is iduronic acid. A part of the glucuronic acid residues is located near the protein core and a large cluster of disaccharides is devoid of glucuronic acid residues. An inhibition enzyme immunoassay has been developed to quantitate the proteoglycan. A model for the interaction between dermatan sulphate proteoglycans and collagen fibrils is proposed.     

Regulation of amidase formation in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity

The formation of amidase was studied in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity. It appeared that catabolite repression of amidase synthesis by succinate was partially relieved when cellular growth was limited by glutamine. Under these conditions, a correlation between amidase and urease formation was observed. The results suggest that amidase formation in strain PAO is subject to nitrogen control and that glutamine or some compound derived from it mediates the nitrogen repression of amidase.     

Identification of SH3 Domain Proteins Interacting with the Cytoplasmic Tail of the A Disintegrin and Metalloprotease 10 (ADAM10)

The a disintegrin and metalloproteases (ADAMs) play a pivotal role in the control of development, adhesion, migration, inflammation and cancer. Although numerous substrates of ADAM10 have been identified, the regulation of its surface expression and proteolytic activity is still poorly defined. One current hypothesis is that both processes are in part modulated by protein-protein interactions mediated by the intracellular portion of the protease. For related proteases, especially proline-rich regions serving as docking sites for Src homology domain 3 (SH3) domain-containing proteins proved to be important for mediating regulatory interactions. In order to identify ADAM10-binding SH3 domain proteins, we screened the All SH3 Domain Phager library comprising 305 human SH3 domains using a GST fusion protein with the intracellular region of human ADAM10 as a bait for selection. Of a total of 291 analyzed phage clones, we found 38 SH3 domains that were precipitated with the ADAM10-derived fusion protein but not with GST. We verified the binding to the cytosolic portion of ADAM10 for several candidates by co-immunoprecipitation and/or pull down analyses. Intriguingly, several of the identified proteins have been implicated in regulating surface appearance and/or proteolytic activity of related ADAMs. Thus, it seems likely that they also play a role in ADAM10 biology.     

A numerical analysis of steady flow in a three-dimensional model of the carotid artery bifurcation

A finite element approximation of steady flow in a rigid three-dimensional model of the carotid artery bifurcation is presented. A Reynolds number of 640 and a flow division ratio of about 50/50, simulating systolic flow, was used. To limit the CPU- and I/O-times needed for solving the systems of equations, a mesh-generator was developed, which gives full control over the number of elements into which the bifurcation is divided. A mini-supercomputer, based on parallel and vector processing techniques, was used to solve the system of equations. The numerical results of axial and secondary flow compare favorably with those obtained from previously performed laser-Doppler velocity measurements. Also, the influence of the Reynolds number, the flow division ratio, and the bifurcation angle on axial and secondary flow in the carotid sinus were studied in the three-dimensional model. The influence of the interventions is limited to a relatively small variation in the region with reversed axial flow, more or less pronounced C-shaped axial velocity contours, and increasing or decreasing axial velocity maxima.     

Gap junction protein connexin-43 interacts directly with microtubules.

Gap junctions are specialized cell-cell junctions that mediate intercellular communication. They are composed of connexin proteins, which form transmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating by growth factors [3-5]. The Cx43 tail contains various protein interaction sites, but little is known about binding partners. To identify Cx43-interacting proteins, we performed pull-down experiments using the C-terminal tail of Cx43 fused to glutathione-S-transferase. We find that the Cx43 tail binds directly to tubulin and, like full-length Cx43, sediments with microtubules. Tubulin binding to Cx43 is specific in that it is not observed with three other connexins. We established that a 35-amino acid juxtamembrane region in the Cx43 tail, which contains a presumptive tubulin binding motif, is necessary and sufficient for microtubule binding. Immunofluorescence and immunoelectron microscopy studies reveal that microtubules extend to Cx43-based gap junctions in contacted cells. However, intact microtubules are dispensable for the regulation of Cx43 gap-junctional communication. Our findings suggest that, in addition to its well-established role as a channel-forming protein, Cx43 can anchor microtubule distal ends to gap junctions and thereby might influence the properties of microtubules in contacted cells.     

The influence of the non-Newtonian properties of blood on the flow in large arteries: steady flow in a carotid bifurcation model.

Laser Doppler anemometry experiments and finite element simulations of steady flow in a three dimensional model of the carotid bifurcation were performed to investigate the influence of non-Newtonian properties of blood on the velocity distribution. The axial velocity distribution was measured for two fluids: a non-Newtonian blood analog fluid and a Newtonian reference fluid. Striking differences between the measured flow fields were found. The axial velocity field of the non-Newtonian fluid was flattened, had lower velocity gradients at the divider wall, and higher velocity gradients at the non-divider wall. The flow separation, as found with the Newtonian fluid, was absent. In the computations, the shear thinning behavior of the analog blood fluid was incorporated through the Carreau-Yasuda model. The viscoelastic properties of the fluid were not included. A comparison between the experimental and numerical results showed good agreement, both for the Newtonian and the non-Newtonian fluid. Since only shear thinning was included, this seems to be the dominant non-Newtonian property of the blood analog fluid under steady flow conditions.     

Fitness Alters the Associations of BMI and Waist Circumference with Total and Abdominal Fat

Objective: We tested the following hypotheses in black and white men and women: 1) for a given BMI or waist circumference (WC), individuals with moderate cardiorespiratory fitness (CRF) have lower amounts of total fat mass and abdominal subcutaneous and visceral fat compared with individuals with low CRF; and 2) exercise training is associated with significant reductions in total adiposity and abdominal fat independent of changes in BMI or WC. Research Methods and Procedures: The sample included 366 sedentary male (111 blacks and 255 whites) and 462 sedentary female (203 blacks and 259 whites) participants in the HERITAGE Family Study. The relationships between BMI and WC with total fat mass (determined by underwater weighing) and abdominal subcutaneous and visceral fat (determined by computed tomography) were compared in subjects with low (lower 50%) and moderate (upper 50%) CRF. The effects of a 20‐week aerobic exercise training program on changes in these adiposity variables were examined in 86% of the subjects. Results: Individuals with moderate CRF had lower levels of total fat mass and abdominal subcutaneous and visceral fat than individuals with low CRF for a given BMI or WC value. The 20‐week aerobic exercise program was associated with significant reductions in total adiposity and abdominal fat, even after controlling for reductions in BMI and WC. With few exceptions, these observations were true for both men and women and blacks and whites. Discussion: These findings suggest that a reduction in total adiposity and abdominal fat may be a means by which CRF attenuates the health risk attributable to obesity as determined by BMI and WC.     

Beetle diversity in a matrix of old-growth boreal forest: influence of habitat heterogeneity at multiple scales

The relative contribution of compositional and structural heterogeneity on biodiversity is currently ambiguous because field studies generally integrate these two sources of habitat heterogeneity into a single index. We established the relationship between species richness of ground-dwelling and flying beetles and compositional and structural attributes of forest heterogeneity. The relationship was evaluated at two spatial scales: the scale of forest stand, corresponding to an 11.3  m radius, and the scale of landscape, corresponding to either a 400 or 800  m radius. Seventy stands were sampled in the matrix of old-growth boreal forest of the North Shore region of Québec, Canada, during the summers of 2004 and 2005. A total of 133 ground-dwelling beetle species (range: 4–42 species per site) were captured in the pitfall traps and 251 flying species (range 16–58 species per site) in flight-interception traps. We found that the most relevant type of heterogeneity to explain variations in species richness and the significance of landscape scale information varied between groups of beetles. Compositional heterogeneity (i.e. the number of species of forest trees and shrubs) at the stand scale best predicted species richness in ground-dwelling beetles. On the other hand, it was the combined influence of structural and compositional habitat heterogeneity at stand and landscape scales that best explained richness patterns in flying beetles. Our study outlines the significance of considering multiple types and spatial scales of habitat heterogeneity when describing patterns of species richness.