Function and cytochemical characteristics of postural limb muscles of the rhesus monkey: A telemetered EMG and immunofluorescence study

Using telemetered electromyography and immunocytochemical fibre typing (of both fresh frozen and preserved specimens), the present paper demonstrates clearly that at the elbow, knee, and ankle joints, the rhesus monkey (Macaca mulatta) is endowed with one extensor-muscle head specialized for posture. These postural heads are distinguished by (a) recruitment at low levels to maintain joint position against the effect of gravity, (b) recruitment near maximum levels during walking, and (c) high content and relatively large size of slow, fatigue-resistant (type I) muscle fibres. The nonpostural heads of the investigated muscles were recruited at levels correlated to the strenuousness of the effort and are notable by the small percentage and size of slow muscle fibres. The postmortem stability of the structural properties of myosin makes immunocytochemical fibre typing suitable for the study of preserved cadavers.     

Design of an MRI-compatible robotic stereotactic device for minimally invasive interventions in the breast

The objective of this work was to develop a robotic device to perform biopsy and therapeutic interventions in the breast with real-time magnetic resonance imaging (MRI) guidance. The device was designed to allow for (i) stabilization of the breast by compression, (ii) definition of the interventional probe trajectory by setting the height and pitch of a probe insertion apparatus, and (iii) positioning of an interventional probe by setting the depth of insertion. The apparatus is fitted with five computer-controlled degrees of freedom for delivering an interventional procedure. The entire device is constructed of MR compatible materials, i.e. nonmagnetic and non-conductive, to eliminate artifacts and distortion of the MR images. The apparatus is remotely controlled by means of ultrasonic motors and a graphical user interface, providing real-time MR-guided planning and monitoring of the operation. Joint motion measurements found probe placement in less than 50 s and sub-millimeter repeatability of the probe tip for same-direction point-to-point movements. However, backlash in the rotation joint may incur probe tip positional errors of up to 5 mm at a distance of 40 mm from the rotation axis, which may occur for women with large breasts. The imprecision caused by this backlash becomes negligible as the probe tip nears the rotation axis. Real-time MR-guidance will allow the physician to correct this error Compatibility of the device within the MR environment was successfully tested on a 4 Tesla MR human scanner     

Imaging the pharmacodynamics of HER2 degradation in response to Hsp90 inhibitors

The development of therapeutic inhibitors of key signaling pathways has been hampered by the inability to assess the effect of a drug on its target in the patient. 17-allylaminogeldanamycin (17-AAG) is the first Hsp90 inhibitor to be tested in a clinical trial. It causes the degradation of HER2 and other Hsp90 targets, and has antitumor activity in preclinical models. We have developed a method for imaging the inhibition of Hsp90 by 17-AAG. We labeled an F(ab')2 fragment of the anti-HER2 antibody Herceptin with 68Ga, a positron emitter, which allows the sequential positron-emission tomographic imaging of HER2 expression. We have used this method to quantify as a function of time the loss and recovery of HER2 induced by 17-AAG in animal tumors. This approach allows noninvasive imaging of the pharmacodynamics of a targeted drug and will facilitate the rational design of combination therapy based on target inhibition.     

The "Goldilocks Effect" in Cystic Fibrosis: identification of a lung phenotype in the <Emphasis Type="Italic">cftr</Emphasis> knockout and heterozygous mouse

Background  

Cystic Fibrosis is a pleiotropic disease in humans with primary morbidity and mortality associated with a lung disease phenotype. However, knockout in the mouse of cftr, the gene whose mutant alleles are responsible for cystic fibrosis, has previously failed to produce a readily, quantifiable lung phenotype.     

Proximal airway mucous cells of ovalbumin-sensitized and -challenged Brown Norway rats accumulate the neuropeptide calcitonin gene-related peptide

Mucous cell hypersecretion and increased neuropeptide production play a role in the exacerbation of symptoms associated with asthma. The source of these neuropeptides have been confined to the contributions of small afferent nerves or possibly neuroendocrine cells. We tested the hypothesis that repeated exposure to allergen would alter the sources and abundance of neuropeptides in airways. Right middle lobes from rats (8 wk old) exposed to 2.5% ovalbumin (OVA) for five episodes (30 min each) or filtered air were inflation fixed with paraformaldehyde. The lobes were dissected to expose the airway tree, permeabilized with DMSO, and incubated in antibody to rat calcitonin gene-related peptide (CGRP), followed with a fluorochrome-labeled second antibody. CGRP-positive structures were imaged via confocal microscopy. Airways were later embedded in plastic and sectioned for cell identification. In animals challenged with OVA, CGRP-positive cells, not neuroendocrine or neuronal in origin (confirmed by a lack of protein gene product 9.5 signal), were recorded along the axial path. In section, this fluorescent signal was localized to granules within epithelial cells. Alcian blue/periodic acid-Schiff staining of these same sections positively identify these cells as mucous cells. Mucous cells of animals not challenged with OVA were not positive for CGRP. We conclude that episodic allergen exposure results in the accumulation of CGRP within mucous cells, creating a new source for the release of this neuropeptide within the airway.     

Alpha4beta1 integrin affinity changes govern cell adhesion

Integrin alpha4beta1 is a receptor for vascular cell adhesion molecule-1 and fibronectin. It is important in lymphopoiesis, inflammatory recruitment of leukocytes, and other situations that require cell adhesion to the vascular endothelium. The avidity of the cells expressing alpha4beta1 integrin can be rapidly changed by chemokines and chemoattractants. Different mechanisms, including changes in the number of interacting molecules due to the alteration of the receptor topology or changes in the affinity of the individual bonds, have been proposed to explain the nature of these fast changes in avidity. Recently, we described a fluorescent LDV-containing small molecule, which we used to monitor the affinity changes on live cells in real time (Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, J. M., Edwards, B. S., Prossnitz, E. R., Larson, R. S. et al. (2001) J. Biol. Chem. 276, 48670-48678). Here we show that the affinity of the small molecule probe as well as the native ligand vascular cell adhesion molecule-1 varies in parallel when the integrin is modulated with divalent cations and that the affinity modulation leads to the changes in cell avidity. Using formyl peptide receptor-transfected U937 cells, we further show that the time course of avidity changes in response to the receptor activation coincides with the time course of the affinity changes. Taken together, these data are consistent with the idea that affinity regulation is a major factor that governs the avidity of cell adhesion mediated by the alpha4 integrin.     

Characterization of the metal ion-binding domains from rat alpha- and beta-parvalbumins

We have examined the metal ion-binding domains from rat alpha and beta parvalbumin. We find that the CD-EF fragments differ markedly in their tendency to self-associate. Whereas Ca(2+)-free alpha CD-EF is monomeric, the Ca(2+)-free beta peptide dimerizes weakly (K(2) = 2400 +/- 200 M(-1)). In buffer containing 1.0 mM Ca(2+), the apparent dimerization constant for beta CD-EF (191,000 +/- 29,000 M(-1)) is more than 50 times that of alpha (3400 +/- 200 M(-1)). Alpha CD-EF binds two Ca(2+) with positive cooperativity. Titration calorimetry data afford binding constants of 3.7(0.1) x 10(3) M(-1) and 8.6(0.2) x 10(4) M(-1). Beta CD-EF also binds two Ca(2+) cooperatively but with lower affinity. Equilibrium dialysis yields Adair constants of 4.2(0.1) x 10(3) and 6.1(0.2) x 10(3) M(-1). Significantly, the difference in Ca(2+) affinity is substantially smaller than that observed for the full-length proteins-suggesting that the AB domain can modulate divalent ion affinity. Analysis of beta calorimetry data requires explicit consideration of the self-association behavior. Data collected at low CD-EF concentration are consistent with preferential occupation of the EF site, dimerization of singly bound monomers, and cooperative filling of the CD sites. At higher concentrations, apo-protein dimerization can apparently precede cooperative occupation of the EF sites. In the presence of Ca(2+), alpha CD-EF exhibits higher thermal stability, consistent with its higher Ca(2+) affinity. However, the beta melting temperature shows greater concentration dependence, consistent with its greater tendency to dimerize. Neither fragment exhibits a sigmoidal melting curve in the Ca(2+)-free state, suggesting that the apo-peptides are disordered.