Immunohistochemical and ultrastructural study on effect of fibroblast growth factor on transformation of fibroblasts to regenerated mesothelial cells

To elucidate the effect of fibroblast growth factor on the phenotypical conversion of fibroblasts to mesothelial cells, both immunohistochemical and ultrastructural examinations were carried out on cultured spheroids that were composed of fibroblasts obtained from the parietal pleura of rats with and without addition of antifibroblast growth factor receptor antibody. In the present study, antifibroblast growth factor receptor antibody was employed to block the effect of the autocrine component of fibroblast growth factor in the culture medium. Phenotypical conversion from fibroblast to mesothelial cells was clearly blocked in the experimental group, to which culture medium had been added with antifibroblast growth factor receptor antibody, whereas the control group, cultured without addition of antifibroblast growth factor receptor antibody, showed phenotypical conversion of fibroblasts that was confirmed by the development of macula adherens, microvilli, and positive expression of cytokeratin. These results indicate the possibility that fibroblast growth factor plays a key role in the process of phenotypic conversion of fibroblasts to regenerated mesothelial cells.     

Stimulation-induced setting of postural muscle tone in the decerebrate rat

These studies demonstrate the presence of pontomedullary areas in the rat brainstem which, when stimulated electrically, serve to set postural muscle tone in the hindlimbs. Low amplitude stimulation of the dorsal tegmental field (DTF) was found to inhibit postural muscle tone and, in some rats, was found to decrease mean arterial pressure. Low amplitude stimulation of the ventral tegmental field (VTF) was found to increase postural muscle tone and, in all cases tested, was found to increase mean arterial pressure.     

Quantifying and visualizing weak interactions between anions and proteins

The molecular properties of proteins are influenced by various ions present in the same solution. While site-specific strong interactions between multivalent metal ions and proteins are well characterized, the behavior of other ions that are only weakly interacting with proteins remains elusive. In the current study, using NMR spectroscopy, we have investigated anion–protein interactions for three proteins that are similar in size but differ in overall charge. Using a unique NMR-based approach, we quantified anions accumulated around the proteins. The determined numbers of anions that are electrostatically attracted to the charged proteins were notably smaller than the overall charge valences and were consistent with predictions from the Poisson–Boltzmann theory. This NMR-based approach also allowed us to measure ionic diffusion and characterize the anions interacting with the positively charged proteins. Our data show that these anions rapidly diffuse while bound to the proteins. Using the same experimental approach, we observed the release of the anions from the protein surface upon the formation of the Antp homeodomain–DNA complex. Using paramagnetic relaxation enhancement (PRE), we visualized the spatial distribution of anions around the free proteins and the Antp homeodomain–DNA complex. The obtained PRE data revealed the localization of anions in the vicinity of the highly positively charged regions of the free Antp homeodomain and provided further evidence of the release of anions from the protein surface upon the protein–DNA association. This study sheds light on the dynamic behavior of anions that electrostatically interact with proteins.

Biological systems involve various inorganic and organic ions. Protein functions are influenced by the surrounding ions not only through the electrostatic screening effect (1), but also through direct interactions at the molecular surfaces (2). Compared to typical protein–ligand interactions, protein–ion interactions are weaker and more transient, yet ions can significantly influence various properties such as solubility, stability, and functional activities of proteins (3). The influences depend on ionic species. For example, when Cl⁻ ions are replaced with glutamate ions in biochemical experiments, some DNA-binding proteins exhibit substantially stronger (>100-fold for some cases) affinity for DNA (4, 5). To understand how ions affect the molecular properties of proteins, the behavior of ions around proteins should be elucidated.For DNA and RNA, ion-counting methods have greatly advanced experiment-based knowledge of ionic interactions (6, 7). These methods were successfully used to examine and validate theoretical models for ion–nucleic acid interactions (8, 9). However, ion-counting methods do not provide any information about the spatial distribution and dynamic properties of counterions around macromolecules. Even at high resolution in crystal structures, the vast majority of counterions are unresolved, suggesting that they are highly mobile. The dynamic nature of ions causes a major difficulty in studying the interactions between ions and biological macromolecules.Weak transient interactions of monovalent ions with proteins are particularly difficult to capture by experiments. Unlike nucleic acids that possess a negative charge at every residue, proteins typically contain both positively charged and negatively charged residues as well as many neutral residues. Consequently, proteins possess a far smaller overall charge than nucleic acids of similar molecular size. This implies that the electrostatic attraction of ions to proteins could be intrinsically weaker than that to nucleic acids. Furthermore, local environments around individual charged moieties of proteins are more diverse compared to those of nucleic acids. Although NMR spectroscopy is powerful for investigating various physicochemical properties of proteins (10, 11), there has been a lack of methods suited to quantitatively investigate ion–protein interactions. Experimental studies of weak ion–protein interactions have been challenging (3).In this work, using unique experimental methods, we study how anions behave in the vicinity of proteins. Our NMR-based approach allows us to determine how many anions are attracted to proteins. Our data show that the number is significantly smaller than the overall charge valence of each positively charged protein. We explain this observation using the concept of the ion atmosphere and theoretical calculations based on the Poisson–Boltzmann equation. Our experimental approach also reveals the diffusional properties of anions interacting with proteins and unravels the release of anions from the protein surface upon protein–DNA association. Furthermore, our solvent paramagnetic relaxation enhancement (PRE) data show how anions are spatially distributed around the protein surface and how their distribution changes when the protein binds to DNA. Our study sheds light on the dynamic behavior of counterions around proteins.     

Falsely elevated thyroid hormone levels caused by anti-ruthenium interference in the Elecsys assay resembling the syndrome of inappropriate secretion of thyrotropin

The syndrome of inappropriate secretion of thyrotropin (SITSH) is defined as the inappropriate non-suppression of serum TSH in the presence of elevated free thyroid hormone; TSH-secreting pituitary adenomas and the syndrome of resistance to thyroid hormone are the main etiologies of SITSH. In addition, erroneous thyroid function testing may result in the diagnosis of this syndrome. A 63-year-old woman was referred because of suspected SITSH. Laboratory tests showed a normal TSH (0.52 μIU/L; normal range: 0.5-5.0) measured by sandwich Elecsys, and elevated FT4 (3.8 ng/dL; normal range: 0.9-1.6) and FT3 (7.6 pg/mL; normal range: 2.3-4.0), determined by competitive Elecsys. To exclude possible assay interference, aliquots of the original samples were retested using a different method (ADVIA Centaur), which showed normal FT4 and FT3 levels. Eight hormone levels, other than thyroid function tests measured by competitive or sandwich Elecsys, were higher or lower than levels determined by an alternative analysis. Subsequent examinations, including gel filtration chromatography, suggested interference by substances against ruthenium, which reduced the excitation of ruthenium, and resulted in erroneous results. The frequency of similar cases, where the FT4 was higher than 3.2 ng/dL, in spite of a non-suppressed TSH, was examined; none of 10 such subjects appeared to have method-specific interference. Here, a patient with anti-ruthenium interference, whose initial thyroid function tests were consistent with SITSH, is presented. This type of interference should be considered when thyroid function is measured using the Elecsys technique, although the frequency of such findings is likely very low.     

Vasospasms of the radial artery after the transradial approach for coronary angiography and angioplasty

We examined vasospasms of the radial artery after a transradial approach was used for coronary angiography or angioplasty. In forty-eight patients (39 males and 9 females), arteriography of the radial artery was initially performed just after the transradial approach was used for coronary angiography and/or angioplasty. Then, five months later, a second arteriography of the radial artery was obtained after a transbrachial approach was used for coronary angiography. First and second arteriographies were compared to evaluate vaso-spasms of the radial artery. In the present study, more than 75% stenosis in the radial artery, 25-75% stenosis, and less than 25% stenosis were tentatively defined as severe spasms, moderate spasms, and mild spasms, respectively. In arteriographic studies on the radial artery, twenty-four patients (50%) had severe radial artery spasms, eleven patients (23%) had moderate spasms, and thirteen patients (27%) had mild spasms. The diameters of both the proximal and distal radial arteries in the severe spasm group were significantly smaller than those in the mild and moderate spasm groups (proximal site: severe group 2.39 +/- 0.70 mm versus mild group 2.98 +/- 0.46 mm, P < 0.05, and moderate group 2.96 +/- 0.77 mm, P < 0.05, distal site: severe group 2.26 +/- 0.60 mm versus mild group 2.73 +/- 0.47 mm, P < 0.05, and moderate group 2.86 +/- 0.71 mm, P < 0.05). We concluded that vasospasms of the radial artery occurred in most patients after the transradial approach. Furthermore, severe radial spasms were strongly correlated with the size of the diameter of the artery.     

De novo determination of near-surface electrostatic potentials by NMR

Electrostatic potentials computed from three-dimensional structures of biomolecules by solving the Poisson–Boltzmann equation are widely used in molecular biophysics, structural biology, and medicinal chemistry. Despite the approximate nature of the Poisson–Boltzmann theory, validation of the computed electrostatic potentials around biological macromolecules is rare and methodologically limited. Here, we present a unique and powerful NMR method that allows for straightforward and extensive comparison with electrostatic models for biomolecules and their complexes. This method utilizes paramagnetic relaxation enhancement arising from analogous cationic and anionic cosolutes whose spatial distributions around biological macromolecules reflect electrostatic potentials. We demonstrate that this NMR method enables de novo determination of near-surface electrostatic potentials for individual protein residues without using any structural information. We applied the method to ubiquitin and the Antp homeodomain–DNA complex. The experimental data agreed well with predictions from the Poisson–Boltzmann theory. Thus, our experimental results clearly support the validity of the theory for these systems. However, our experimental study also illuminates certain weaknesses of the Poisson–Boltzmann theory. For example, we found that the theory predicts stronger dependence of near-surface electrostatic potentials on ionic strength than observed in the experiments. Our data also suggest that conformational flexibility or structural uncertainties may cause large errors in theoretical predictions of electrostatic potentials, particularly for highly charged systems. This NMR-based method permits extensive assessment of near-surface electrostatic potentials for various regions around biological macromolecules and thereby may facilitate improvement of the computational approaches for electrostatic potentials.

Due to the fundamental importance of electrostatic interactions in chemistry and biology, electrostatic potentials are invaluable information for the understanding of molecular recognition, enzymatic catalysis, and other functions of proteins and nucleic acids (1–4). Quantification of electrostatics is also important for successful protein engineering (5) and structure-based drug design (6). Computational approaches based on the Poisson–Boltzmann theory are commonly used to calculate electrostatic potentials from three-dimensional (3D) molecular structures (1, 7). Owing to available software such as Adaptive Poisson-Boltzmann Solver (APBS) (8, 9) and DelPhi (10, 11), computation of the electrostatic potentials around biomolecules has gained widespread popularity in the fields of molecular biophysics, structural biology, and medicinal chemistry.However, the computed electrostatic potentials may not necessarily be accurate even if the 3D structures are precisely and accurately determined. Importantly, the Poisson–Boltzmann theory is approximate with known limitations. The electrostatic models based on this theory are valid under assumptions, which simplify the calculations (12). The lack of consideration of correlations between ions can diminish accuracy in calculations of electrostatic potentials for systems at high ionic strength (13). Due to the assumption of a dielectric continuum, the electrostatic potentials predicted with the Poisson–Boltzmann theory may be inaccurate for zones near the first hydration layer. Electrostatic potentials predicted for regions near highly charged molecular surfaces may also be inaccurate due to the assumption of linear dielectric response. Nonetheless, the Poisson–Boltzmann theory can accurately predict electrostatic interactions at longer range (7). The extent of validity for such electrostatic potentials near molecular surfaces remains to be addressed more rigorously through experiments.Despite the need, experimental validation of computed electrostatic potentials is rather rare and methodologically limited for biological macromolecules. The validity of electrostatic models has been examined using pK_a data on titratable side-chain moieties (14–16), redox potentials of redox-active groups (17, 18), and electron–electron double resonance (19). Among them, pK_a data have been most commonly used for the validation, but even fundamentally incorrect electrostatic models can reproduce pK_a data (20). Electrostatic fields can be experimentally determined by vibrational spectroscopy, for example, for nitrile groups that are conjugated to cysteine thiol moieties of proteins (21, 22). However, the approaches utilizing vibrational spectroscopy or electron–electron double resonance provide only limited information about the extrinsically introduced probes, which may perturb native systems.In this paper, we present a unique and powerful method for de novo determination of near-surface electrostatic potentials for many protein residues, regardless of their side-chain types, and without using any chemical modifications. In this method, data of NMR paramagnetic relaxation enhancement (PRE) arising from analogous charged paramagnetic cosolutes are analyzed for ¹H nuclear magnetizations of proteins (Fig. 1). The PRE data reflect the electrostatic biases in spatial distributions of charged paramagnetic cosolutes and permit the determination of near-surface electrostatic potentials around proteins without using any structural information. The de novo determination of near-surface electrostatic potentials can greatly facilitate the examination of theoretical models for electrostatics of biological macromolecules.Open in a separate windowFig. 1.NMR PRE arising from cationic amino-methyl-PROXYL or anionic carboxy-PROXYL reflects their spatial distribution bias due to near-surface electrostatic potentials around a biological macromolecule.     

Lumbar degenerative kyphosis. Clinical, radiological and epidemiological studies

We suggest that lumbar degenerative kyphosis be included as one of the abnormal sagittal curvatures in which a kyphosis or a marked loss of lordosis is seen in the lumbar spine, caused by degenerative changes in middle-aged and elderly. One hundred and five consecutive patients were investigated, most of whom complained of low-back pain, often with a long history. They all walked in a forward bending posture, either all the time or only when exhausted. In roentgenograms, most cases showed a marked loss of the sacral inclination, as well as multiple disc narrowing and/or vertebral wedging in the lumbar region. These subjects showed a definite weakness of the lumbar extensors compared to the flexors, and therefore a reversed ratio of extensors/flexors muscle power compared with normal controls and other types of spinal curvatures. Weakness of the lumbar extensors was clearly shown by isokinetic measurement and a marked atrophy of these muscles with fatty infiltration was demonstrated by CT scanning.     

Effects of atropine upon the hippocampal electrical activity in rats with special reference to paradoxical sleep.

In freely moving male rats with implanted electrodes, the influence of atropine sulphate (5-30 mg/kg, i.p.) on the hippocampal theta activity was studied with special emphasis on paradoxical sleep (PS). In accordance with previous work with cats and rabbits, atropine was found to inhibit PS, in delaying its first appearance as well as in decreasing the duration of the first PS episode. The hippocampal theta activity during PS was also changed after atropinization. In particular, when REM occurred often, theta activity was increased not only in frequency but also in amplitude; however, with no or little REM, theta frequency was not changed but the regularity of theta rhythms was markedly disturbed. These findings are in contrast with the theta activity associated with gross body movement, which was not affected by atropine as was previous reported by Vanderwolf (1975). Thus, in contrast to commonly held views, the hippocampal theta activity during gross body movement may be different in its underlying mechanisms from that during PS.