Structural basis of an embryonically lethal single Ala → Thr mutation in the vnd/NK-2 homeodomain

The structural and DNA binding behavior is described for an analog of the vnd/NK-2 homeodomain, which contains a single amino acid residue alanine to threonine replacement in position 35 of the homeodomain. Multidimensional nuclear magnetic resonance, circular dichroism, and electrophoretic gel retardation assays were carried out on recombinant 80-aa residue proteins that encompass the wild-type and mutant homeodomains. The mutant A35T vnd/NK-2 homeodomain is unable to adopt a folded conformation free in solution at temperatures down to −5°C in contrast to the behavior of the corresponding wild-type vnd/NK-2 homeodomain, which is folded into a functional three-dimensional structure below 25°C. The A35T vnd/NK-2 binds specifically to the vnd/NK-2 target DNA sequence, but with an affinity that is 50-fold lower than that of the wild-type homeodomain. Although the three-dimensional structure of the mutant A35T vnd/NK-2 in the DNA bound state shows characteristic helix–turn–helix behavior similar to that of the wild-type homeodomain, a notable structural deviation in the mutant A35T analog is observed for the amide proton of leucine-40. The wild-type homeodomain forms an unusual i,i-5 hydrogen bond with the backbone amide oxygen of residue 35. In the A35T mutant this amide proton resonance is shifted upfield by 1.27 ppm relative to the resonance frequency for the wild-type analog, thereby indicating a significant alteration of this i,i-5 hydrogen bond.

Imaging of H217O distribution in the brain of a live rat by using proton-detected 17O MRI

Imaging of H217O has a number of important applications. Mapping the distribution of H217O produced by oxidative metabolism of 17O-enriched oxygen gas may lead to a new method of metabolic functional imaging; regional cerebral blood flow also can be measured by measuring the H217O distribution after the injection of 17O-enriched physiological saline solution. Previous studies have proposed a method for indirect detection of 17O. The method is based on the shortening of the proton T2 in H217O solutions, caused by the residual 17O-1H scalar coupling and transferred to the bulk water via fast chemical exchange. It has been shown that the proton T2 of H217O solutions can be restored to that of H216O by irradiating the resonance frequency of the 17O nucleus. The indirect 17O image thus is obtained by taking the difference between two T2-weighted spin-echo images: one acquired after irradiation of the 17O resonance and one acquired without irradiation. It also has been established that, at relatively low concentrations of H217O, the indirect method yields an image that quantitatively reflects the H217O distribution in the sample. The method is referred to as PRIMO (proton imaging of oxygen). In this work, we show in vivo proton images of the H217O distribution in a rat brain after an i.v. injection of H217O-enriched physiological saline solution. Implementing the indirect detection method in an echo-planar imaging sequence enabled obtaining H217O images with good spatial and temporal resolution of few seconds.

Dissociation of response conflict, attentional selection, and expectancy with functional magnetic resonance imaging

Two different attentional networks have been associated with visuospatial attention and conflict resolution. In most situations either one of the two networks is active or both are increased in activity together. By using functional magnetic resonance imaging and a flanker task, we show conditions in which one network (anterior attention system) is increased in activity whereas the other (visuospatial attention system) is reduced, showing that attentional conflict and selection are separate aspects of attention. Further, we distinguish between neural systems involved in different forms of conflict. Specifically, we dissociate patterns of activity in the basal ganglia and insula cortex during simple violations in expectancies (i.e., sudden changes in the frequency of an event) from patterns of activity in the anterior attention system specifically correlated with response conflict as evidenced by longer response latencies and more errors. These data provide a systems-level approach in understanding integrated attentional networks.

Resonant tectorial membrane motion in the inner ear: its crucial role in frequency tuning.

The tectorial membrane has long been postulated as playing a role in the exquisite sensitivity of the cochlea. In particular, it has been proposed that the tectorial membrane provides a second resonant system, in addition to that of the basilar membrane, which contributes to the amplification of the motion of the cochlear partition. Until now, technical difficulties had prevented vibration measurements of the tectorial membrane and, therefore, precluded direct evidence of a mechanical resonance. In the study reported here, the vibration of the tectorial membrane was measured in two orthogonal directions by using a novel method of combining laser interferometry with a photodiode technique. It is shown experimentally that the motion of the tectorial membrane is resonant at a frequency of 0.5 octave (oct) below the resonant frequency of the basilar membrane and polarized parallel to the reticular lamina. It is concluded that the resonant motion of the tectorial membrane is due to a parallel resonance between the mass of the tectorial membrane and the compliance of the stereocilia of the outer hair cells. Moreover, in combination with the contractile force of outer hair cells, it is proposed that inertial motion of the tectorial membrane provides the necessary conditions to allow positive feedback of mechanical energy into the cochlear partition, thereby amplifying and tuning the cochlear response.

Object-related activity revealed by functional magnetic resonance imaging in human occipital cortex.

The stages of integration leading from local feature analysis to object recognition were explored in human visual cortex by using the technique of functional magnetic resonance imaging. Here we report evidence for object-related activation. Such activation was located at the lateral-posterior aspect of the occipital lobe, just abutting the posterior aspect of the motion-sensitive area MT/V5, in a region termed the lateral occipital complex (LO). LO showed preferential activation to images of objects, compared to a wide range of texture patterns. This activation was not caused by a global difference in the Fourier spatial frequency content of objects versus texture images, since object images produced enhanced LO activation compared to textures matched in power spectra but randomized in phase. The preferential activation to objects also could not be explained by different patterns of eye movements: similar levels of activation were observed when subjects fixated on the objects and when they scanned the objects with their eyes. Additional manipulations such as spatial frequency filtering and a 4-fold change in visual size did not affect LO activation. These results suggest that the enhanced responses to objects were not a manifestation of low-level visual processing. A striking demonstration that activity in LO is uniquely correlated to object detectability was produced by the "Lincoln" illusion, in which blurring of objects digitized into large blocks paradoxically increases their recognizability. Such blurring led to significant enhancement of LO activation. Despite the preferential activation to objects, LO did not seem to be involved in the final, "semantic," stages of the recognition process. Thus, objects varying widely in their recognizability (e.g., famous faces, common objects, and unfamiliar three-dimensional abstract sculptures) activated it to a similar degree. These results are thus evidence for an intermediate link in the chain of processing stages leading to object recognition in human visual cortex.