Clinical applications of fusion imaging in oncology

Recent developments in tumor imaging, made possible by advances in instrumentation and radiopharmaceuticals, has led to an increasing need for accurate anatomic correlation of single photon emission computed tomography (SPECT) and positron emission tomography (PET) images. Fusion imaging permits the functional strengths of SPECT and PET to be combined with the anatomic resolution of computed tomography (CT) and magnetic resonance imaging (MRI). Clinical applications of fusion imaging include the evaluation of brain tumors, lymphoma, hepatic lesions and monoclonal antibody studies. The continued development of these techniques will eventually allow fusion imaging to become a routine part of nuclear medicine practice.     

The interaction of the general anesthetic etomidate with the gamma-aminobutyric acid type A receptor is influenced by a single amino acid

The gamma-aminobutyric acid type A (GABAA) receptor is a transmitter-gated ion channel mediating the majority of fast inhibitory synaptic transmission within the brain. The receptor is a pentameric assembly of subunits drawn from multiple classes (alpha1-6, beta1-3, gamma1-3, delta1, and epsilon1). Positive allosteric modulation of GABAA receptor activity by general anesthetics represents one logical mechanism for central nervous system depression. The ability of the intravenous general anesthetic etomidate to modulate and activate GABAA receptors is uniquely dependent upon the beta subunit subtype present within the receptor. Receptors containing beta2- or beta3-, but not beta1 subunits, are highly sensitive to the agent. Here, chimeric beta1/beta2 subunits coexpressed in Xenopus laevis oocytes with human alpha6 and gamma2 subunits identified a region distal to the extracellular N-terminal domain as a determinant of the selectivity of etomidate. The mutation of an amino acid (Asn-289) present within the channel domain of the beta3 subunit to Ser (the homologous residue in beta1), strongly suppressed the GABA-modulatory and GABA-mimetic effects of etomidate. The replacement of the beta1 subunit Ser-290 by Asn produced the converse effect. When applied intracellularly to mouse L(tk-) cells stably expressing the alpha6beta3gamma2 subunit combination, etomidate was inert. Hence, the effects of a clinically utilized general anesthetic upon a physiologically relevant target protein are dramatically influenced by a single amino acid. Together with the lack of effect of intracellular etomidate, the data argue against a unitary, lipid-based theory of anesthesia.     

Cancer cells release a covalent complex containing disulfide-linked domains from urinary plasminogen activator, neural cell adhesion molecule, and haptoglobin alpha and beta chains

We have previously reported on the secretion of a family of high Mr plasminogen activators (PAs) by a human lung cancer cell line [Harvey et al. (1991) Biochim. Biophys. Acta 1078, 360-368]. We have now extended these studies to several human cancer cell lines and a human embryonic lung cell line. In the present study with HPL-SK-1 lung cancer, A431 epidermoid cancer, ovarian carcinoma, and embryonic lung cell lines, we show that the 900- and the 660-kDa PAs are disulfide-bonded multiprotein oligomeric complexes. They are functionally and immunologically related to human urinary PA (uPA). Their size and PA activity are not destroyed by strong denaturants such as 8 M urea or 2% sodium dodecyl sulfate (SDS), suggesting that the uPA moiety is covalently associated with the rest of the molecule. It is only under strong denaturing conditions with 1.4 M beta-mercaptoethanol and 2% SDS that the uPA moiety could be released as a 21- to 23-kDa fragment along with two major polypeptide chains of 70 and 40 kDa, respectively. The presence of the uPA active center in the reduced PA660 was demonstrated by [3H]diisopropylphosphorofluoridate labeling and by Western blot using a monoclonal antibody to uPA B chain. N-terminal amino acid sequencing of the 70- and 40-kDa polypeptides, respectively, showed homology to the neural cell adhesion molecule and the beta chain of haptoglobin. A minor fragment of 18 kDa obtained under strong reduction conditions was also sequenced and shown to share homology with the alpha chain of haptoglobin. Western blot analysis of the reduced PAs with monoclonal antibody to the neural cell adhesion molecule and rabbit anti-haptoglobin confirmed the homologies obtained by the sequence data. Further, immobilized monoclonal antibodies to the neural cell adhesion molecule, uPA B chain, and rabbit anti-haptoglobin bound the multiprotein complexes with uPA activity, from A431, ovarian cancer, and embryonic lung cell lines. The bound material, after dissociation, exhibited PA activity that was inhibited by monoclonal antibody to the uPA B chain. These data suggest that in tumor and embryonal cell lines, in addition to proper folding and assembly of proteins by intramolecular disulfide bond formation in the endomembrane compartment, intermolecular disulfide bonds could also occur, producing multiprotein oligomers as in the present case. Formation of such oligomers may have a selective advantage for such cells in the focalization of proteolytic activity through the interaction of the neural cell adhesion molecule domain with the extracellular matrix and in immunosuppression of lymphocytes by the haptoglobin portion of the complex.     

Evaluation of a programming algorithm for the third tachycardia zone in a fourth-generation implantable cardioverter-defibrillator

Lungs from eight goats of mixed sexes and breeds (Cashmere, Nubian and Toggenburg) aged between 10 and 48 months were used in this study. Tissues from lung parenchyma were minced and routinely prepared for transmission electron microscopy (TEM) after using different methods of fixation. Thick sections were examined with a light microscope and samples, to include terminal bronchioles, respiratory bronchioles, alveolar ducts and alveolar membrane, were selected for ultrathin sectioning. Six cell types, ciliated, non-ciliated bronchiolar epithelial, mucus-producing, alveolar Type I, alveolar Type II and capillary endothelial cell were identified and characterised cytologically. It was established that the cell population in the distal airways is similar to that observed in other domestic mammals. The mucus-producing cell, which appears to be a common cell type in the distal airways of man and Rhesus monkey, was encountered particularly in adult goats in the present study. This study has also established that the Clara cell of the goat shows some cytological differences from those of some other mammalian species by having a large amount of SER, particularly in the apical region. Lipid vacuoles were seen to be a feature of the alveolar Type II cells; these do not appear to have been reported in other mammalian species. The study has provided a basic understanding of the morphological features of the cell population of the epithelium lining the distal airways in the goat's respiratory tract. The difference in junctional complexes between the various alveolar epithelial cells perhaps signify a different pattern of intercellular transport, thus influencing the pathogenesis and resolution of alveolar pulmonary edema.     

Opposing actions of hepatocyte growth factor and basic fibroblast growth factor on cell contact, intracellular free calcium levels, and rat ovarian surface epithelial cell viability

Previous studies demonstrated that cell-to-cell contact stimulates a tyrosine phosphorylation signal transduction pathway that prevents rat ovarian surface epithelial (ROSE) cells from undergoing apoptosis. Hepatocyte growth factor (HGF), also know as scatter factor (SF), is expressed by ovarian stromal and thecal cells and has been shown to reduce cell contact in nonovarian tissues. The present studies were designed to determine whether HGF/SF promotes ROSE cells to dissociate and subsequently become apoptotic. Because an increase in intracellular free calcium ([Ca2+]i) is often an early event in the apoptotic cascade, the effects of HGF/SF on [Ca2+]i levels were also assessed. ROSE cells were cultured in serum-free medium with HGF/SF, basic fibroblast growth factor (bFGF), thapsigargin, Bay K, actinomycin D, cycloheximide, and/or BAPTA depending on the experimental design. Cell contact was assayed by time-lapse photography; [Ca2+]i levels were measured with Fluo-3, and apoptosis was assessed by in situ DNA staining. HGF/SF decreased cell contact within 1 h, increased [Ca2+]i levels by 3 h, and induced apoptosis by 6 h of culture. bFGF inhibited these HGF/SF-induced responses. The increase in [Ca2+]i appears to represent a point in the apoptotic cascade that commits ROSE cells to die. This concept is based on the observations that: 1) in the presence of the calcium chelator BAPTA, HGF/SF decreased cell contact but did not increase [Ca2+]i or apoptosis; 2) bFGF blocked HGF/SF-induced increase in [Ca2+]i; 3) bFGF did not attenuate HGF/SF's apoptotic action if exposed to cells after the increase in [Ca2+]i; and 4) RNA and protein synthesis were required for HGF/SF to increase [Ca2+]i, whereas the thapsigargin- and Bay K-induced increase in [Ca2+]i and apoptosis were independent of RNA/protein synthesis. These observations indicate that the components of the apoptotic cascade distal to the increase in [Ca2+]i are present within ROSE cells and are activated by a sustained elevation of [Ca2+]i. The present studies also show that when ROSE cells establish contact with 3T3 cells that express N-cadherin, [Ca2+]i levels are maintained at low basal levels. In contrast, cell contact with 3T3 cells that do not express N-cadherin results in elevated [Ca2+]i levels. Similarly, a synthetic N-cadherin peptide, which inhibits homophilic N-cadherin binding, increases [Ca2+]i levels. Taken together, these data indicate that homophilic N-cadherin binding between adhering cells plays an important role in maintaining calcium homeostasis. Further, these data support the concept that HGF/SF's ability to promote the dissociation of ROSE cells accounts in part for its ability to increase [Ca2+]i levels.     

Xenobiotics and xenoestrogens in fish: modulation of cytochrome P450 and carcinogenesis

The periaqueductal gray matter (PAG) serves as the midbrain link between forebrain emotional processing systems and motor pathways used in the defense reaction. Part of this response depends upon PAG efferent pathways that modulate cardiovascular-related sympathetic outflow systems, including those that regulate the heart. While it is known that the PAG projects to vagal preganglionic neurons, including possibly cardiovagal motoneurons, no information exists on the PAG circuits that may affect sympathetically mediated cardiac functions and, thus, the purpose of this study was to use neuroanatomical methods to identify these pathways. First, viral transneuronal retrograde tracing experiments were performed in which pseudorabies virus (PRV) was injected into the stellate ganglion of rats. After 4 days survival, five PAG regions contained transynaptically infected neurons; these included the dorsomedial, lateral and ventrolateral PAG columns as well as the Edinger-Westphal and precommissural nuclei. Second, the descending efferent PAG projections were studied with the anterograde axonal marker Phaseolus vulgaris leuco-agglutinin (PHA-L) with a particular focus on determining whether the PAG projects to the intermediolateral cell column (IML). Almost no axonal labeling was found throughout the thoracic IML suggesting that the PAG modulates sympathetic functions by indirect pathways involving synaptic relays through sympathetic premotor cell groups, especially those found in the medulla oblongata. This possibility was examined by a double tracing study. PHA-L was first injected into either the lateral or ventrolateral PAG and after 6 days, PRV was injected into the ipsilateral stellate ganglion. After an additional 4 days survival, a double immunohistochemical procedure for co-visualization of PRV and PHA-L was used to identify the sympathetic premotor regions that receive an input from the PAG. The PAG innervated specific groups of sympathetic premotor neurons in the hypothalamus, pons, and medulla as well as providing reciprocal intercolumnar connections within the PAG itself (Jansen et al., Brain Res. 784 (1998) 329-336). The major route terminates in the ventral medulla, especially within the medial region which contains sympathetic premotor neurons lying within the raphe magnus and gigantocellular reticular nucleus, pars alpha. Both serotonergic and non-serotonergic sympathetic premotor neurons in these two regions receive inputs from the PAG. Weak PAG projections to sympathetic premotor neurons were found in the rostral ventrolateral medulla (including to C1 adrenergic neurons), locus coeruleus, A5 cell group, paraventricular and lateral hypothalamic nuclei. In summary, both the lateral and ventrolateral PAG columns appear to be capable of modulating cardiac sympathetic functions via a series of indirect pathways involving sympathetic premotor neurons found in selected sites in the hypothalamus, midbrain, pons, and medulla oblongata, with the major outflow terminating in bulbospinal regions of the rostral ventromedial medulla.