RESUMEN

BACKGROUND: In elastic and resistance arteries, an elastin-rich membrane, the Internal Elastic Lamina (IEL), separates the tunica intima from the underlying tunica media. The IEL often appears wrinkled or corrugated in histological images. These corrugations are sometimes ascribed to vessel contraction ex vivo, and to fixation artifacts, and therefore regarded as not physiologically relevant. We examine whether the IEL remains corrugated even under physiological conditions. METHODS: The diameters of carotid arteries of anesthetized pigs were measured by ultrasound. The arteries were then excised, inflated within a conical sleeve, fixed, and imaged by confocal microscopy. The conical sleeve allows fixing each artery across a wide range of diameters, which bracket its ultrasound diameter. Thus the study was designed to quantify how corrugations change with diameter for a single artery, and test whether corrugations exist when the fixed artery matches the ultrasound diameter. RESULTS: At diameters below the ultrasound diameter (i.e. when the artery was constricted as compared to ultrasound conditions), the IEL corrugations were found to decrease significantly with increasing diameter, but not fully flatten at the ultrasound diameter. The contour length of the IEL was found to be roughly 10% larger than the circumference of the artery measured by ultrasound. The physiological diameter is likely to be even smaller than the ultrasound diameter since ultrasound was conducted with the animal under general anesthesia, which leads to vasodilation, suggesting a higher level of corrugation under physiological conditions. For arterial cross sections constricted below the ultrasound diameter, the IEL contour length decreased roughly with the square root of the diameter. CONCLUSION: The primary conclusions of this study are: a) the IEL is corrugated when the artery is constricted and flattens as the artery diameter increases; b) the IEL is corrugated under physiological conditions and has a contour length at least 10% more than the physiological arterial diameter; and c) the IEL despite being relatively stiffer than the surrounding arterial layers, does not behave like an inextensible membrane.

RESUMEN

This paper reports a novel, physiologically significant, microfluidic phenomenon generated by nanomolar concentrations of drag-reducing polymers (DRP) dissolved in flowing blood, which may explain previously demonstrated beneficial effects of DRP on tissue perfusion. In microfluidic systems used in this study, DRP additives were found to significantly modify traffic of red blood cells (RBC) into microchannel branches as well as reduce the near-wall cell-free layer, which normally is found in microvessels with a diameter smaller than 0.3 mm. The reduction in plasma layer size led to attenuation of the so-called "plasma skimming" effect at microchannel bifurcations, increasing the number of RBC entering branches. In vivo, these changes in RBC traffic may facilitate gas transport by increasing the near vessel wall concentration of RBC and capillary hematocrit. In addition, an increase in near-wall viscosity due to the redirection of RBC in this region may potentially decrease vascular resistance as a result of increased wall shear stress, which promotes endothelium mediated vasodilation. These microcirculatory phenomena can explain the previously reported beneficial effects of DRP on hemodynamics in vivo observed in many animal studies. We also report here our finding that DRP additives reduce flow separations at microchannel expansions, deflecting RBC closer to the wall and eliminating the plasma recirculation zone. Although the exact mechanism of the DRP effects on RBC traffic in microchannels is yet to be elucidated, these findings may further DRP progress toward clinical use.

Asunto(s)

RESUMEN

Based upon the crystal structures of PcrA helicase, we have made and characterised mutations in a number of conserved helicase signature motifs around the ATPase active site. We have also determined structures of complexes of wild-type PcrA with ADPNP and of a mutant PcrA complexed with ADPNP and Mn2+. The kinetic and structural data define roles for a number of different residues in and around the ATP binding site. More importantly, our results also show that there are two functionally distinct conformations of ATP in the active site. In one conformation, ATP is hydrolysed poorly whereas in the other (activated) conformation, ATP is hydrolysed much more rapidly. We propose a mechanism to explain how the stimulation of ATPase activity afforded by binding of single-stranded DNA stabilises the activated conformation favouring Mg2+binding and a consequent repositioning of the gamma-phosphate group which promotes ATP hydrolysis. A part of the associated conformational change in the protein forces the side-chain of K37 to vacate the Mg2+binding site, allowing the cation to bind and interact with ATP.

Asunto(s)

RESUMEN

We have determined two different structures of PcrA DNA helicase complexed with the same single strand tailed DNA duplex, providing snapshots of different steps on the catalytic pathway. One of the structures is of a complex with a nonhydrolyzable analog of ATP and is thus a "substrate" complex. The other structure contains a bound sulphate ion that sits in a position equivalent to that occupied by the phosphate ion produced after ATP hydrolysis, thereby mimicking a "product" complex. In both complexes, the protein is monomeric. Large and distinct conformational changes occur on binding DNA and the nucleotide cofactor. Taken together, these structures provide evidence against an "active rolling" model for helicase action but are instead consistent with an "inchworm" mechanism.

Asunto(s)