Repeated hypergravity: morphologic investigations of pituitary, thyroid, blood and bone marrow in rats

To elicit the mechanism of facilitating the mammals' adaptation to repeated changed gravity influence, the pituitary, thyroid, blood and bone marrow were investigated morphologically in rats exposed to single and repeated hypergravity (2 g) and Coriolis accelerations for 5 days during rotation on centrifuge. No distinct difference in blood and bone marrow cytology was determined after single and repeated exposure to 2 g and the Coriolis accelerations. Compared to single and in contrast to single and repeated exposures to the Coriolis accelerations, a repeated 2 g influence produced some structural changes in somatotropic cells of the pituitary and thyroid parenchyma of the thyroid. These changes were indicative of a significant intensification of synthesis and secretion of somatotropic and thyroid hormones. Elevated functional activity of the somatotropic cells and thyroid parenchyma during repeated exposure to 2 g appears to be a part of mechanism that makes adaptation to repeated hypergravity easier and points to the ability of mammals "to remember" changed gravity. It also advocates for the potentiality of intermittent centrifugation as a means of generating artificial gravity forces in space flight.     

Two modes of ligand binding in maltose-binding protein of Escherichia coli. Electron paramagnetic resonance study of ligand-induced global conformational changes by site-directed spin labeling

Binding of ligands to the maltose-binding protein (MBP) of Escherichia coli often causes a global conformational change involving the closure of its two lobes. We have introduced a cysteine residue onto each of these lobes by site-directed mutagenesis and modified these residues with spin labels. Using EPR spectroscopy, we examined the changes, caused by the ligand binding, in distance between the two spin labels, hence between the two lobes. The binding of both maltose and maltotetraose induced a considerable closure of the N- and C-terminal lobes of MBP. Little closure occurred upon the binding of maltotetraitol or beta-cyclodextrin. Previous study by fluorescence and UV differential absorbance spectroscopy (Hall, J. A., Gehring, K., and Nikaido, H. (1997) J. Biol. Chem. 272, 17605-17609) showed that maltose and a large portion of maltotetraose bound to MBP via one mode (R mode or "end-on" mode), which is physiologically active and leads to the subsequent transport of the ligands across the cytoplasmic membrane. In contrast, maltotetraitol and beta-cyclodextrin bound to MBP via a different mode (B mode or "middle" mode), which is physiologically inactive. The present work suggests that the B mode is nonproductive because ligands binding in this manner prevent the closure of the two domains of MBP, and, as a result, the resulting ligand-MBP complex is incapable of interacting properly with the inner membrane-associated transporter complex.     

New gene assignments using a complete, characterized sheep-hamster somatic cell hybrid panel

OBJECTIVE: To assess the ability of the International Association for the Study of Pain Complex Regional Pain Syndrome (CRPS) diagnostic criteria and associated features to discriminate between CRPS patients and patients with painful diabetic neuropathy. DESIGN: Prospective assessment of signs and symptoms in a series of CRPS and diabetic neuropathy patients. SETTING: University of Washington Multidisciplinary Pain Center. PATIENTS: A consecutive series of 18 CRPS patients and 30 diabetic neuropathy patients. INTERVENTIONS: Patients completed a 10-item patient history questionnaire assessing symptoms of CRPS prior to medical evaluation. The evaluating physician completed a 10-item patient examination questionnaire assessing objective signs of CRPS. OUTCOME MEASURES: The analyses conducted were designed to test the ability of CRPS signs and symptoms and associated features to discriminate between CRPS patients and diabetic neuropathy patients. RESULTS: Data analysis suggested that CRPS decision rules may lead to overdiagnosis of the disorder. Diagnosis based on self-reported symptoms can be diagnostically useful in some circumstances. The addition of trophic tissue changes, range of motion changes, and "burning" quality of pain did not improve diagnostic accuracy, but the addition of motor neglect signs did. Test of a CRPS scoring system resulted in improved accuracy relative to current criteria and decision rules. CONCLUSIONS: Poorly understood disorders lacking prototypical signs/symptoms and diagnostic laboratory testing must rely on the development of reliable diagnostic guidelines. The results of this study should assist in the further refinement of the CRPS diagnostic criteria.     

C3a is a chemotaxin for human eosinophils but not for neutrophils. I. C3a stimulation of neutrophils is secondary to eosinophil activation

Inflammatory action of the potent chemotaxin C5a has been well characterized on a variety of human cell types, including neutrophils, monocytes, basophils, and eosinophils. The cellular effects of C3a are less well defined. Contradictory reports have been published for C3a activation of neutrophils. Recent reports that C3a activates both basophils and eosinophils prompted us to reinvestigate the effects of C3a stimulation on eosinophils. We hypothesized that C3a activation of eosinophils, cells that are present in most neutrophil preparations, might lead to neutrophil activation. Using neutrophils of 98% purity, we observed no evidence of cellular activation after stimulation with either C3a, recombinant human C3a (rhC3a), or the synthetic C3a analogue C3a 57-77, Y57. Eosinophils purified to > 98% purity displayed concentration-dependent polarization, chemotaxis, and enzyme release by stimulation with C3a, rhC3a, and the synthetic C3a analogue. An inactive form of C3a, C3adesArg, failed to stimulate either eosinophils or neutrophils. Using neutrophil preparations containing 5-9% eosinophils, up to 20% of neutrophils became polarized after exposure to C3a. Likewise, we demonstrated that supernatant from C3a-stimulated eosinophils promotes neutrophil chemotaxis. Eosinophil polarization experiments were repeated in the presence of antibody to the C5a receptor (C5aR) to show that C3a and C5a interact with different receptors. C3a activates eosinophils in the presence of anti-C5aR antibody at concentrations that fully block C5a activation. We conclude that eosinophils are directly activated by either C3a or C5a, whereas C3a failed to activate neutrophils. C3a acts on eosinophils via a receptor that is distinct from C5aR. Since neutrophils are indirectly stimulated by C3a, eosinophils contaminating neutrophil preparations may explain earlier reports that C3a activates human neutrophils.     

Esophagitis in Sprague-Dawley rats is mediated by free radicals

Free radical-mediated esophagitis was studied during duodenogastroesophageal reflux (mixed reflux) or acid reflux in rats. The influence of reflux on esophageal glutathione levels was also examined. Mixed reflux caused more gross mucosal injury than acid reflux. Gross mucosal injury occurred in the mid-esophagus. Total glutathione (GSH) in the esophageal mucosa of control rats was highest in the distal esophagus. The time course of esophageal GSH in rats treated by mixed reflux showed a significant decrease 4 hr after initiation of reflux, followed by a significant increase from the 12th hour on. Mucosal GSH was increased in both reflux groups after 24 hr but significantly more so in the mixed than in the acid reflux group. The free radical scavenger superoxide dismutase (SOD) prevented esophagitis and was associated with decreased GSH levels. GSH depletion by buthionine sulfoximine (BSO) prevented esophagitis and stimulated SOD production in the esophageal mucosa. It is concluded that gastroesophageal reflux is associated with oxidative stress in the esophageal mucosa. The lower GSH levels in the mid-esophagus may predispose to damage in this area. Duodenogastroesophageal reflux causes more damage than pure acid reflux. Oxidative stress leads to GSH depletion of the esophageal mucosa in the first few hours following damage but then stimulates GSH production. GSH depletion by BSO does not worsen esophagitis since it increases the esophageal SOD concentration.     

Regulation and mechanisms of gene amplification

Amplification in rodent cells usually involves bridge-breakage-fusion (BBF) cycles initiated either by end-to-end fusion of sister chromatids, or by chromosome breakage. In contrast, in human cells, resistance to the antimetabolite N-(phosphonacetyl)-L-aspartate (PALA) can be mediated by several different mechanisms that lead to overexpression of the target enzyme carbamyl-P synthetase, aspartate transcarbamylase, dihydro-orotase (CAD). Mechanisms involving BBF cycles account for only a minority of CAD amplification events in the human fibrosarcoma cell line HT 1080. Here, formation of a 2p isochromosome and overexpression of CAD by other types of amplification events (and even without amplification) are much more prevalent. Broken DNA is recognized by mammalian cells with intact damage-recognition pathways, as a signal to arrest or to die. Loss of these pathways by, for example, loss of p53 or pRb tumour suppressor function, or by increased expression of ras and myc oncogenes, causes non-permissive rat and human cells to become permissive both for amplification and for other manifestations of DNA damage. In cells that are already permissive, amplification can be stimulated by overexpressing oncogenes such as c-myc or ras, or by damaging DNA in a variety of ways. To supplement genetic analysis of amplification in mammalian cells, an amplification selection has been established in Schizosaccharomyces pombe. Selection with LiCl yields cells with amplified sod2 genes in structures related to those observed in mammalian cells. The effect on amplification in S. pombe can now be tested for any mutation in a gene involved in repair of damaged DNA or in normal cellular responses to DNA damage.