Biomedical subjects
Publications and source records attributed to J R Stone.
Following Coulomb excitation of the radioactive ion beam (RIB) 132Te at HRIBF we report the first use of the recoil-in-vacuum (RIV) method to determine the g factor of the 2(+)(1) state: g(973.9 keV 2(+) 132Te) = (+)0.35(5). The advantages offered by the RIV method in the context of RIBs and modern detector arrays are discussed.
Additional evidence for the rare kaon decay K+-->pi+nu(nu) has been found in a new data set with comparable sensitivity to the previously reported result. One new event was observed in the pion momentum region examined, 211 pi+nu(nu)) = 1.57(+1.75)(-0.82)x10(-10).
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BACKGROUND: The mechanisms of extracellular matrix changes accompanying myxomatous valvular degeneration are uncertain. METHODS AND RESULTS: To test the hypothesis that valvular interstitial cells mediate extracellular matrix degradation in myxomatous degeneration by excessive secretion of catabolic enzymes, we examined the functional characteristics of valvular interstitial cells in 14 mitral valves removed for myxomatous degeneration from patients with mitral regurgitation and in 11 normal mitral valves obtained at autopsy. Immunohistochemical staining assessed (1) cell phenotype using antibodies to alpha-actin (microfilaments), vimentin and desmin (intermediate filaments), smooth muscle myosin (SM1), and SMemb (a nonmuscle myosin produced by activated mesenchymal cells) and (2) the expression of proteolytic activity using antibodies to collagenases (matrix metalloproteinase [MMP]-1, MMP-13), gelatinases (MMP-2, MMP-9), cysteine endoproteases (cathepsin S and K), and interleukin-1beta, a cytokine that can induce secretion of proteolytic enzymes. Although interstitial cells in normal valves stained positively for vimentin, but not alpha-actin or desmin, cells in myxomatous valves contained both vimentin and alpha-actin or desmin (characteristics of myofibroblasts). Moreover, cells in myxomatous valves strongly expressed SMemb, MMPs, cathepsins, and interleukin-1beta, which were weakly stained in controls. Nevertheless, interstitial cells in both groups strongly expressed procollagen-I mRNA (in situ hybridization), suggesting preserved ability to synthesize collagen in myxomatous valves. CONCLUSIONS: Interstitial cells in myxomatous valves have features of activated myofibroblasts and express excessive levels of catabolic enzymes, without altered levels of interstitial collagen mRNA. We conclude that valvular interstitial cells regulate matrix degradation and remodeling in myxomatous mitral valve degeneration.
Using a heterotopic model of transplantation, we investigated the role of T cell activation in vivo during allograft rejection in I-kappaB(DeltaN)-transgenic mice that express a transdominant inhibitor of NF-kappaB in T cells. Our results show indefinite prolongation of graft survival in the I-kappaB(DeltaN)-transgenic recipients. Interestingly, at the time of rejection of grafts in wild-type recipients, histology of grafts in the I-kappaB(DeltaN)-transgenic recipients showed moderate rejection; nevertheless, grafts in the I-kappaB(DeltaN) recipients survived >100 days. Analysis of acute phase cytokines, chemokine, chemokine receptors, and immune responses shows that the blockade of NF-kappaB activation in T cells inhibits up-regulation of many of these parameters. Interestingly, our data also suggest that the T cell component of the immune response exerted positive feedback regulation on the expression of multiple chemokines that are produced predominantly by non-T cells. In conclusion, our studies indicate NF-kappaB activation in T cells is necessary for acute allograft rejection.
Our laboratory has shown that traumatically induced axonal injury (TAI) is significantly reduced by posttraumatic hypothermia followed by slow rewarming. Further, TAI can be exacerbated by rapid rewarming, and the damaging consequences of rapid rewarming can be reversed by cyclosporin A, which is believed to protect via blunting mitochondrial permeability transition (MPT). In this communication, we continue investigating the damaging consequences of rapid posthypothermic rewarming and the protective role of immunophilin ligands using another member of the immunophilin family, FK506, which does not affect MPT but rather inhibits calcineurin. Rats were subjected to impact-acceleration brain injury followed by the induction of hypothermia with subsequent rapid or slow posthypothermic rewarming. During rewarming, animals received either FK506 or its vehicle. Three hours postinjury, animals were prepared for the visualization of TAI via antibodies targeting impaired axoplasmic transport (APP) and/or overt neurofilament alteration (RMO-14). Rapid rewarming exacerbated TAI, which was attenuated by FK506. This protection was statistically significant for the APP-immunoreactive fibers but not for the RMO-14-positive fibers. Combined labeling, using one chromagen to visualize both axonal changes, suggested that these two immunoreactive profiles revealed two distinct pathologies not occurring along the same axon. Collectively, these studies confirmed previous observations identifying the adverse consequences of rapid rewarming while also showing the complexity of the pathobiology of TAI. Additionally, the demonstration that FK506 is protective suggests that calcineurin may be a major target for neuroprotection.
Traumatic axonal injury (TAI) contributes to morbidity and mortality following traumatic brain injury (TBI). Single-label immunocytochemical studies employing antibodies to neurofilament compaction (NFC), RM014, and antibodies to APP, a marker of impaired axonal transport (AxT), have shown that TAI involves both NFC and disruption of AxT. Although it may be hypothesized that both events occur within the same injured axon, this has not been confirmed. To determine the relationship between NFC and impaired AxT, dual-label immunofluorescence was employed. To compare and contrast specific changes associated with these two markers of TAI, single-label electron microscopy was also used. Rats were subjected to an impact acceleration injury (30 min-6 h survival), and their brains were prepared for dual-label immunofluorescence and single-label electron microscopy. APP and RM014 were consistently found in two distinct classes of TAI. One, which showed only RM014 immunoreactivity, was thin and elongate, was sometimes vacuolated, and revealed little progressive change over time. The second was distinguished by focal axonal swellings containing APP immunoreactivity alone in small-caliber axons or in combination with RM014 immunoreactivity in large-caliber axons. These swellings were localized to either nodal or internodal loci and underwent progressive swelling over time, ultimately leading to secondary axotomy. Ultrastructural examination of these two classes of TAI revealed NFC together with mitochondrial dilation without organelle pooling in the RM014 single-labeled axons. However, the APP single-labeled small-caliber axons and APP/RM014 dual-labeled large-caliber axons revealed a progressive accumulation of organelles associated with increased axonal swelling over time. In contrast to previous thought, it now appears that NFC may occur independent of impaired AxT in TAI. This finding underscores the complexity of TAI, suggesting the need for multiple immunocytochemical approaches to fully assess the overall axonal response to TBI.
The application of elementary equations from information theory to the elements involved in cladistic analysis is formalized mathematically. An equation is derived that quantifies the amount of information obtained by constructing a cladogram from a cladistic data matrix. Given particular conditions, the amount of information obtained increases monotonically with increases of the number of taxa involved and, so, may be used directly as a comparative measure of species richness for sister groups; in general, however, the amount of information obtained is related to the distribution of character states on the cladogram(s) deduced. An example is presented in which clades representing 11 phyla in the animal kingdom are compared in terms of information yielded. The amount of information obtained is consistent for different numbers of taxa and characters used in classifications. Speculative evolutionary explanations are presented for differences of information yielded among the phyla analyzed.
The immunophilin ligand, cyclosporin A (CsA), is effective in reducing the axonal damage associated with traumatic brain injury (TBI). Based upon extensive ultrastructural and immunohistochemical studies, the neuroprotection afforded by CsA appeared to be mediated via mitochondrial protection, specifically, the prevention of mitochondrial swelling and inhibition of mitochondrial permeability transition (MPT). However, the potential that CsA could also be neuroprotective via the immunophilin-mediated inhibition of the protein phosphatase, calcineurin (CN) has not been directly assessed. To address this issue, the current study assessed the ability of FK506, another immunophilin ligand that inhibits CN with no effect on MPT, to attenuate axonal damage in a rat impact-acceleration model of TBI. Traumatic axonal injury (TAI), detected via an antibody against beta-amyloid precursor protein (APP), a specific marker of axonal injury, was significantly reduced at 24 hr postinjury in Sprague-Dawley rats receiving intravenous FK506 (2 mg/kg; n = 5) 30 min prior to injury compared to vehicle controls (n = 3). While not rejecting the established efficacy of CsA in providing neuroprotection via its targeting of MPT, this study does underscore the potential importance of CN in the progressive pathobiology of TAI, suggesting that CN may constitute another important therapeutic target.
Although the evolution of protein-coding sequences within genomes is well understood, the same cannot be said of the cis-regulatory regions that control transcription. Yet, changes in gene expression are likely to constitute an important component of phenotypic evolution. We simulated the evolution of new transcription factor binding sites via local point mutations. The results indicate that new binding sites appear and become fixed within populations on microevolutionary timescales under an assumption of neutral evolution. Even combinations of two new binding sites evolve very quickly. We predict that local point mutations continually generate considerable genetic variation that is capable of altering gene expression.
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Antibodies to the amyloid precursor protein (APP) are commonly used to detect traumatic axonal injury (TAI). Carried by fast anterograde axoplasmic transport, APP will pool at regions of impaired transport associated with TAI. Based primarily upon commercial antibody availability, previous studies have targeted the N-terminus of APP, which, with respect to antigen detection, is suboptimally located within anterogradely transported vesicles. Recently, antibodies to the APP C-terminus, located on the external surface of anterogradely transported vesicles, have become available, allowing for the exploration of their utility in detecting TAI. To this end, rats were subjected to an impact acceleration injury, surviving 30 min to 24 h post-injury. They were then perfused, their brains sectioned and prepared for dual label immunofluorescent microscopy, single label bright field microscopy, and electron microscopy (EM). Antibodies to the APP C-terminus yielded the ready detection of intensely labeled TAI with significantly reduced diffuse background staining in comparison to antibodies to the APP N-terminus in both dual label immunofluorescent and single label bright-field approaches. EM examination of antibodies to the APP C-terminus in TAI revealed intense labeling of pooled intra-axonal vesicular profiles, confirming the anterogradely transported vesicular source of the APP seen in TAI. Interestingly, in addition to providing a technically superior approach and new detailed information on the subcellular localization of APP, antibodies to the APP C-terminus also proved more cost effective. Immunofluorescent studies of APP C-terminus immunoreactivity involved 1/3 the cost of targeting the N-terminus, while bright field APP C-terminus studies were performed for 1/20 the cost.
Fluorescent immunocytochemistry (FICC) allows multiple labeling approaches when enzyme-based techniques are difficult to combine, such as in double-labeling experiments targeting small-caliber axonal segments. Nevertheless, the conversion of FICC to a product visible at the electron microscopic (EM) level requires labor-intensive procedures, thus justifying the development of more user-friendly conversion methods. This study was initiated to simplify the conversion of FICC to EM by employing the unique properties of tyramide signal amplification (TSA), which allowed the simultaneous targeting of a fluorescent tag and biotin label to the same antigenic site. Briefly, one of two antigenic sites typically co-localized in damaged axonal segments was visualized by the application of a fluorescent secondary antibody, with the other tagged via a biotinylated antibody. Next, an ABC kit was used, followed by the simultaneous application of fluorophore-tyramide and biotin-tyramide. After temporary mounting for fluorescent digital photomicroscopy, sections were incubated in ABC and reacted with diaminobenzidine before EM analysis. Double-labeling fluorescent immunocytochemistry with TSA clearly delineated damaged axonal segments. In addition, these same axonal segments yielded high-quality EM images with discrete electron-dense reaction products, thereby providing a simple and reproducible means for following fluorescent analysis with EM.
Antibodies to the amyloid precursor proteins (APP) have become routine markers for detecting traumatically induced axonal injury (AI) in animals and man. Unfortunately, the techniques used to visualize these proteins are not compatible with routine electron microscopic (EM) analysis. In the current communication, we describe a method for the ultrastructural visualization of antibodies to APP and, using this method, we identify a previously unrecognized population of traumatically injured axons. Rats were subjected to an impact acceleration traumatic brain injury and allowed to survive 30 min to 3h postinjury. The animals were then perfused, their brains sectioned on a vibratome and the sections prepared for immunocytochemistry using a computer-controlled microwave capable of temperature regulation. The use of temperature-controlled microwave energy unmasked APP antigenic epitopes without sacrificing ultrastructural detail. The APP antibody was found in two distinct populations of reactive axons that differed in size, morphology, location, and temporal progression. Comparable to previous descriptions, one population showed traumatically related reactive changes that led to swelling and disconnection. The other population, however, revealed unanticipated changes reflected in nodal and paranodal swelling of small continuous fibers that showed no evidence of disconnection during the time periods assessed. These studies provide new insight into the complexity of the pathobiology of AI, while describing a novel approach for enhancing APP immunoreactivity at the EM level.
Subplate neurons, the first neurons of the cerebral cortex to differentiate and mature, are thought to be essential for the formation of connections between thalamus and cortex, such as the system of ocular dominance columns within layer 4 of visual cortex. To learn more about the requirement for subplate neurons in the formation of thalamocortical connections, we have sought to identify the neurotransmitters and peptides expressed by the specific class of subplate neurons that sends axonal projections into the overlying visual cortex. To label retrogradely subplate neurons, fluorescent latex microspheres were injected into primary visual cortex of postnatal day 28 ferrets, just prior to the onset of ocular dominance column formation. Subsequently, neurons were immunostained with antibodies against glutamate, glutamic acid decarboxylase (GAD-67), parvalbumin, neuropeptide Y (NPY), somatostatin (SRIF), or nitric oxide synthase (NOS). Retrograde labeling results indicate that the majority of subplate neurons projecting into the cortical plate reside in the upper half of the subplate. Combined immunostaining and microsphere labeling reveal that about half of cortically projecting subplate neurons are glutamatergic; most microsphere-labeled subplate neurons do not stain for GAD-67, parvalbumin, NPY, SRIF, or NOS. These observations suggest that subplate neurons can provide a significant glutamatergic synaptic input to the cortical plate, including the neurons of layer 4. If so, excitation from the axons of subplate neurons may be required in addition to that from lateral geniculate nucleus neurons for the activity-dependent synaptic interactions that lead to the formation of ocular dominance columns during development.
BACKGROUND: Nitric oxide (.NO) is used in biology as both an intercellular signaling agent and a cytotoxic agent. In signaling, submicromolar quantities of .NO stimulate the soluble isoform of guanylate cyclase (sGC) in the receptor cell. .NO increases the Vmax of this heterodimeric hemoprotein up to 400-fold by interacting with the heme moiety of sGC to form a 5-coordinate complex. Carbon monoxide (CO) binds to the heme to form a 6-coordinate complex, but only activates the enzyme 5-fold, YC-1 is a recently discovered compound that relaxes vascular smooth muscle by stimulating sGC. RESULTS: In the presence of YC-1, CO activates sGC to the same specific activity as attained with .NO. YC-1 did not affect the NO-stimulated activity. The on-rate (kon) and off-rate (koff) of CO for binding to sGC in the presence of YC-1 were determined by stopped-flow spectrophotometry. Neither the kon nor the koff varied from values previously obtained in the absence of YC-1, indicating that YC-1 has no effect on the affinity of CO for the heme. In the presence of YC-1, the visible spectrum of the sGC-CO complex has a Soret peak at 423 nm, indicating the complex is 6-coordinate. CONCLUSIONS: YC-1 has no effect on the affinity of CO for the heme of sGC. In the presence of YC-1, maximal activation of sGC by CO is achieved by formation of a 6-coordinate complex between CO and the heme indicating that cleavage of the Fe-His bond is not required for maximal activation of sGC.
Applications of the concept of a morphological space to the study of biological form during the past three decades are reviewed. Common features of the various types of morphological spaces constructed by morphometricians during this period are identified and used to construct a space of morphological spaces. This semiquantitative analysis reveals that the use of empirical morphospace by contemporary researchers is similar to Thompson's classic method of coordinate grid transformations, in the description of biological form.