Caldesmon: a common actin-linked regulatory protein in the smooth muscle and nonmuscle contractile system

Caldesmon was originally purified from gizzard smooth muscle as a major calmodulin-binding protein which also interacts with actin filaments. It has an alternative binding ability to either calmodulin or actin filaments depending upon the concentration of Ca2+ ("flip-flop binding"). Two forms of caldesmon (Mr's in the range of 120-150 kDa and 70-80 kDa) have been demonstrated in a wide variety of smooth muscles and nonmuscle cells. Immunohistochemical studies suggest that caldesmon is colocalized with actin filaments in vivo. Considering its abundance, the Ca2+-dependent flip-flop binding ability to either calmodulin or actin filaments, and its intracellular localization, caldesmon is expected to be involved in contractile events. Recent results from our laboratory have led to the conclusion that caldesmon regulates the smooth muscle and nonmuscle actin-myosin interaction and the smooth muscle actin-high Mr actin-binding protein (ABP or filamin) interactin in a flip-flop manner. It might function in cell motility by regulating the contractile system.     

Cerebral ischemia: Changes in monoamines are independent of energy metabolism

The relationship of neurotransmitters and neuroeffectors to the energy state of the brain was examined in the gerbil model of ischemia after 5 and 15 min of bilateral common carotid artery occlusion only or with 1 hr of reperfusion. The gerbil brains were fixed by microwave irradiation and a total of 15 metabolites were measured from a single piece of tissue from either the hippocampus or the striatum. The rapid alterations in energy-related compounds and cyclic nucleotides appeared to be directly related both to the loss of oxygen and glucose during ischemia and the resupply of these nutrients during reflow. Significant reduction in the level of monoamines occurred prinicipally during reflow, at a time when the energy-related metabolites were restored. It is proposed that the changes in monoamines were triggered by other ischemic-induced events unrelated to energy depletion.Presented in part at the Nineteenth Annual Meeting of the American Society for Neurochemistry, 1988     

Increment in the Ta1+ cells in the peripheral blood and thyroid tissue of patients with Graves' disease

The present study utilized the anti-Ta1 mAb to characterize the cell surface phenotypes of peripheral blood and intrathyroidal lymphocytes in patients with Graves' disease. We found an increase in PBL bearing the Ta1 Ag in untreated patients. The euthyroid patients in remission, induced by antithyroidal drugs, radioisotope therapy, and subtotal thyroidectomy, had lower percentages of Ta1+ cells than did untreated patients. An increased percentage of Ta1+ cells in untreated patients was found in both CD4+ cells and CD8+ cells. The ratio of CD4+Ta1+ cells to CD8+Ta1+ cells in untreated patients was significantly higher than that of normal subjects. There was a positive correlation between the percentage of Ta1+ cells and the level of anti-TSH receptor antibody. In this prospective study, the proportion of Ta1+ cells was decreased in parallel with the reduction in anti-TSH receptor antibody and free T3 levels. In the chronically treated patients, the proportion of Ta1+ cells in the thyroid tissue was, yet similar to that in the peripheral blood, markedly increased in comparison to that of normal subjects. In contrast to Ta1+ cells, the thyroid tissue had a significantly higher percentage of HLA-DR+ T cells than did the paired peripheral blood. The proliferative responses of the Ta1+ cell-enriched population isolated from untreated patients toward thyroglobulin and microsomal Ag were markedly higher than those in a Ta1+ cell-depleted population, but both populations were able to respond equally to PHA. These results suggest that the Ta1+ cells may include Ag-triggered memory cells that are reactive with thyroid-specific Ag. Furthermore, monitoring such cells may provide an objective measure of abnormal immunologic activity.     

Synchrotron X-ray scattering study of chromatin condensation induced by monovalent salt: analysis of the small-angle scattering data

Small-angle X-ray scattering experiments were carried out on rat thymus chromatin in "native" and "H1-depleted" states at various NaCl concentrations using synchrotron radiation. From the analysis of cross-sectional Guinier plots, the radius of gyration of the cross section (Rc) and the mass per unit length (Mc) of native chromatin were evaluated. In the absence of NaCl, the cross section of chromatin filament has a radius of gyration of 3.44 nm, suggesting the structure corresponding to the "10 nm" filament. With increasing NaCl concentration, the Rc value increases steeply to 6.74 nm at 5 mM NaCl and then gradually to 8.82 nm at 50 mM NaCl, whereas the Mc value, which is determined relative to that of tobacco mosaic virus (TMV), increases steadily from 1.58 nucleosomes per 10 nm in the absence of NaCl to 7.66 nucleosomes per 10 nm at 50 mM NaCl. However, since calibration with TMV tends to overestimate the Mc value, the actual Mc values may be less than those values. Above about 40 mM NaCl, aggregation of chromatin is suggested. Similar analysis of H1-depleted chromatin confirmed that H1-depleted chromatin takes a more disordered structure than native chromatin at low ionic strength and does not undergo a definite structure change upon further addition of NaCl.     

Binding of both Ca2+ and mastoparan to calmodulin induces a large change in the tertiary structure

The technique of small-angle X-ray scattering has been employed to examine the solution conformation of calmodulin and its complexes with Ca2+ alone, and with both Ca2+ and mastoparan. The radius of gyration decreased by 3.1 +/- 0.3 A upon binding of both 4 mol Ca2+/mol of protein and 1 mol mastoparan/mol of protein to form the ternary complex. A smaller increase was found for the separate binding of 4 mol Ca2+/mol of protein in the absence of mastoparan (0.6 +/- 0.3 A). The analyses of pair distance distribution function showed that the maximal pair distance in calmodulin complex with both Ca2+ and mastoparan decreased by 20-30% in comparison with calmodulin or its complex with Ca2+, and a shoulder near 40 A, which characterizes the dumbbell-shaped molecule of calmodulin, disappeared. These results indicate that the two globular domains of the calmodulin complex with Ca2+ and mastoparan come close together by 8.0-9.5 A on average, if the size and the overall shape of the globular domains are the same in Ca2+-calmodulin-mastoparan complex as in calmodulin or Ca2+-calmodulin complex.     

Calcium-induced shape change of calmodulin with mastoparan studied by solution X-ray scattering

Solution x-ray scattering using synchrotron radiation as an x-ray source was used to analyze the Ca2+-dependent shape change of pig brain calmodulin in detail. The radius of gyration of calmodulin at 10 mg/ml was increased by 0.9 A. The increase was nearly completed when 2.5 mol of Ca2+/mol of calmodulin was added, whereas the radius of gyration of calmodulin with mastoparan decreased by about 3 A with an increasing Ca2+ concentration up to 4 mol of Ca2+/mol of calmodulin. At a moderate angle of region, both scattering profiles from calmodulin with or without Ca2+ displayed clear humps at s = 0.03 A-1 which are characteristic of a dumbbell structure. However, in the presence of mastoparan, the hump in the scattering profile became obscure and later disappeared with the third and fourth Ca2+ binding to calmodulin. These findings are attributable to the Ca2+-induced shape change of calmodulin with mastoparan from a dumbbell structure to a non-dumbbell structure in which the distance between the two lobes of calmodulin become closer by a bend in the central helix.     

Circular dichroic study of conformational changes in ovalbumin

By simulation of the circular dichroic spectra (Greenfield and Fasman (1969)) and using reference spectra of Chen et al. (1974), native ovalbumin was estimated to contain 33% -helix, 5% -structure, and 62% random coil. Ovalbumin resisted conformational changes in solutions of urea and of SDS. However, guanidine induced transition, starting at about 2 M and completing at about 4.5 M. At concentrations exceeding 4.5 M guanidine, ovalbumin existed as 6–7% -helical, 12–13% -structure, and 80–81% random coil. Ovalbumin after denaturation in 6 M guanidine or in 8 M urea (incubated at 4°C for 24 hr) did not recover the native conformation but acquired a new conformation in each case, with a somewhat destabilized helical structure.Abbreviation used CD circular dichroism - SDS sodium dodecyl sulfate     

Is an endothelin a neurotoxic factor in the brain?

A possible role for endothelin in the pathogenesis of hypoglycemic brain damage in rats was evaluated using an in vitro model with which we could directly monitor the release of dopamine from striatal slices. There was no evidence of impairment in case of non-exposure of the slices to endothelin-3 during 20–40 min of hypoglycemia. The response all but disappeared in striatal slices stimulated twice with 10⁻⁵ M endothelin-3 during 20 min of hypoglycemia. Hypoglycemic damage triggered by endothelin-3 was not observed in the absence of extracellular Ca²⁺. Nifedipine (10⁻⁶ M), but not verapamil (10⁻⁵ M) nor diltiazem (10⁻⁵M), protected striatal tissue from this damage. Our findings provide evidence that endothelins might be etiological factors in the development of hypoglycemic/ischemic brain injury by stimulating dihydropyridine-regulated Ca²⁺ influx.     

Neonatal repetitive maternal separation causes long-lasting alterations in various neurotrophic factor expression in the cerebral cortex of rats

AimsThis study was carried out to examine the effects of early postnatal maternal separation stress on the development of the cerebral cortex with respect to time-dependent fluctuations of neurotrophic factor ligand and receptor expression.Main methodsWistar rats were separated from their mothers for 3 h per day during postnatal days (PND) 10 to 15. The cerebral cortex was analyzed by real-time RT-PCR for the evaluation of the expression of mRNA for brain-derived neurotrophic factor (BDNF), TrkB, insulin-like growth factor-1 (IGF-1), and type 1 IGF receptor (IGF-1R) on PND16, 20, 30, and 60.Key findingsThe expression of these neurotrophic factor ligands and receptors in the cerebral cortex was enhanced on PND16 and PND20, and then it returned to baseline levels on PND30. By PND60, however, the expression levels were attenuated.SignificanceThe important implication of this study is the persistent abnormal fluctuation of neurotrophic factor expression for a prolonged period, triggered even after the brain growth spurt. Given that neurotrophic factors play important roles in brain development, it can be speculated that the altered expression of these factors induced by maternal separation may interrupt normal brain development and ultimately lead to functional disruption. However, the possibility of such changes leading to various functional disruptions and the underlying mechanisms involved require further study.