Mechanical properties of the extracellular matrix of the aorta studied by enzymatic treatments

The microarchitecture of different components of the extracellular matrix (ECM) is crucial to our understanding of the properties of a tissue. In the study presented here, we used a top-down approach to understand how the interplay among different fibers determines the mechanical properties of real tissues. By selectively removing different elements of the arterial wall, we were able to measure the contribution of the different constituents of the ECM to the mechanical properties of the whole tissue. Changes in the network structure were imaged with the use of two-photon microscopy. We used an atomic force microscope to measure changes in the mechanical properties by performing nanoindentation experiments. We show that although the removal of a key element of the ECM reduced the local stiffness by up to 50 times, the remaining tissue still formed a coherent network. We also show how this method can be extended to study the effects of cells on real tissues. This new (to our knowledge) way of studying the ECM will not only help physicists gain a better understanding of biopolymers, it will be a valuable tool for biomedical researchers studying processes such as wound healing and cervix ripening.     

A large-scale survey of genetic copy number variations among Han Chinese residing in Taiwan

Background

Winter migration of immature brown trout (Salmo trutta) into freshwater rivers has been hypothesized to result from physiologically stressful combinations of high salinity and low temperature in the sea.

Results

We sampled brown trout from two Danish populations entering different saline conditions and quantified expression of the hsp70 and Na/K-ATPases α 1b genes following acclimation to freshwater and full-strength seawater at 2°C and 10°C. An interaction effect of low temperature and high salinity on expression of both hsp70 and Na/K-ATPase α 1b was found in trout from the river entering high saline conditions, while a temperature independent up-regulation of both genes in full-strength seawater was found for trout entering marine conditions with lower salinities.

Conclusion

Overall our results support the hypothesis that physiologically stressful conditions in the sea drive sea-run brown trout into freshwater rivers in winter. However, our results also demonstrate intra-specific differences in expression of important stress and osmoregulative genes most likely reflecting adaptive differences between trout populations on a regional scale, thus strongly suggesting local adaptations driven by the local marine environment.     

Heteromeric channel formation and Ca-free media reduce the toxic effect of the weaver Kir 3.2 allele

weaver mice have a severe hypoplasia of the cerebellum with an almost complete loss of the midline granule cells. Recent genetic studies of weaver mice have identified a mutation resulting in an amino acid substitution (G156S) in the pore of the inwardly rectifying potassium channel subunit K_ir 3.2. When expressed in Xenopus oocytes the weaver mutation alters channel selectivity from a potassium-selective to a nonspecific cation-selective pore. In this study we confirm by cell-attached patch-clamp recording that the mutation produces a non-selective cation channel. We also demonstrate that the cell death induced by weaver expression may be prevented by elimination of calcium from the extracellular solution as well as by coexpression with the wild-type K_ir 3.2 allele, or other members of the K_ir 3.0 subfamily. These results suggest that the weaver defect in K_ir 3.2 may cause cerebellar cell death by cell swelling and calcium overload. Cells which express the weaver subunit, but which normally survive, may do so because of heteromeric subunit assembly with wild-type subunits of the K_ir 3.0 subfamily.     

The effects of cholesterol inclusion on the molecular organisation of bimolecular lipid membranes

Dielectric measurements on planar egg phosphatidylcholine bilayers formed from n-hexadecane solutions indicate that these bilayers contain very low equilibrium concentrations of alkane. In 100 mM KCl the capacitance of the hydrophobic region was found to be 7.0 ±0.2 mF/m². The addition of cholesterol (at 2:1 mole ratio) was found to affect only marginally the capacitance of the hydrophobic region of such bilayers. Precise measurements of the frequency dependence of the bilayer impedance at very low frequencies now allow the resolution of several electrically distinct substructural regions within the bilayer. Examination of the effects of cholesterol inclusion upon the electrical parameters of these substructural regions indicate that cholesterol spans the acetyl region (i.e. the region containing the glycerol bridge of the phosphatidylcholine molecules in the bilayer) with the hydroxyl group of the cholesterol molecules located inbetween the phosphate group and the glycerol oxygens of the phosphatidylcholine molecules. The capacitance of the hydrophobic region of both phosphatidylcholine and phosphatidylcholine/cholesterol bilayers formed from n-hexadecane solutions was found to decrease slightly as the external KCl concentration was decreased.     

Effects of Ca2+, calmodulin and cyclic AMP on the phosphorylation of endogenous proteins by homogenates of rt islets of langerhans

Activation of Ca2+ -calmodulin- and cyclic AMP-dependent protein kinases has been suggested to be involved in stimulus-secretion coupling in the pancreatic beta-cell. To study the properties of suc kinases and their endogenous protein substrates homogenates of rat islets of Langerhans were incubated with [gamma-32P]ATP. Phosphorylated proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and detected by autoradiography. The phosphorylation of certain proteins could be enhanced by Ca2+ plus calmodulin or by cyclic AMP. The major effect of Ca2+ and calmodulin was to stimulate the phosphorylation of a protein (P53) of molecular weight 53,100 +/- 500 (n = 15). Maximum phosphorylation of protein P53 occurred within 2 min with 2 micrometers free Ca2+ and 0.7 micrometers calmodulin. Incorporation of label into protein P53 was inhibited by trifluoperazine or W7 but not by cyclic AMP-dependent protein kinase inhibitor. Phosphorylation of a proteins of similar molecular weight could be enhanced to a lesser extent in the absence of Ca2+ but in the presence of cyclic AMP and 3-isobutylmethylxanthine: this phosphorylation was blocked by cyclic AMP-dependent protein kinase inhibitor. Cyclic AMP also stimulated incorporation of label into polypeptides of molecular weights 55,000 and 70-80,000. The results are consistent with the hypothesis that protein phosphorylation mechanisms may play a role in the regulation of insulin secretion.     

Calcium currents of frog and insect skeletal muscle fibres measured during voltage clamp

Both vertebrate and invertebrate skeletal muscle fibres have Ca2+ permeability mechanisms which are turned on by depolarization of the surface membrane. In frog muscle, Ca currents are extremely slow and will be scarcely activated during the action potential that normally elicits a twitch. This Ca permeability cannot therefore play any substantial, direct role in excitation--contraction coupling. In insect (Carausius morosus) muscle, Ca currents activate within milliseconds of depolarization, even at low temperature, and may well play at least a triggering role in excitation--contraction coupling. These Ca currents show saturation with increasing [Ca]0, while the instantaneous current--voltage relation rectifies inwards, as expected from a very low [Ca]i. The Ca channel is permeable to Sr2+ and Ba2+. Inactivation of Ca currents under a maintained depolarization depends on Ca2+ carrying inward current, however, rather than on the depolarization itself.     

Perturbations to lipid bilayers by spectroscopic probes as determined by dielectric measurements

Dielectric measurements on lecithin/cholesterol bimolecular lipid membranes have indicated that the series of extrinsic fluorescent probe molecules, the $\text{n-(9-}\text{anthroyloxy}\text{)}$ fatty acids, cause significant perturbation to the bilayer structure at concentrations equivalent to those used in fluorescence experiments (0.1 mol%). Perturbations were observed in the capacitance and conductance of the electrically distinct substructural regions of the bilayer that were consistent with the putative location of the probe molecules. Inclusion of stearic acid decreased the thickness of the hydrocarbon region of the membrane, presumably by expanding the average surface area per unit membrane mass, and also significantly disrupted the surface regions. The attachment of the anthroyloxy moiety to position 2 of a fatty acid accentuated both these effects. Attachment at position 12 had the reverse effect by increasing the volume of the hydrocarbon region without further disturbance of the surface organisation. The 9-positioned probe had an intermediate effect. The degree of perturbation by the 2-positioned probe was dependent on the probe concentration within the range (probe:lipid) 1:1000 to 1:10 000. The technique therefore detects perturbation of structure at probe levels which are lower than those commonly used in fluorescence-labelling experiments.     

Song matching in the great tit Parus major L.

Great tits respond to playback of song from their own repertoire by singing the same song type as that on the tape. This is referred to as song matching. There is a good correlation between the probability of matching and response strength as indicated by latency, approach to the speaker and total number of songs. We propose that matching functions as a graded signal in territorial disputes.