Contractile properties of old rat muscles: effect of increased use

To examine how different kinds of activity affect the composition and contractile properties of aging skeletal muscle, old male rats were strength and swim trained. The mass of weights lifted during the strength training increased by 85 +/- 9% (P less than 0.05), which was accompanied by an increase by 32 +/- 5% (P less than 0.05) of the estimated force developed. The wet muscle weight of the soleus and the plantaris decreased significantly with age. The phenomenon was counteracted but not neutralized by the strength training. Twitch and tetanic tension also decreased significantly with age in both the soleus and plantaris muscle. This was avoided by the strength training. This training also significantly decreased time to peak tension and half-relaxation time of both muscles. The swim training increased the heart-to-body weight ratio by 21 +/- 5% (P less than 0.05) and the endurance of the soleus muscle. Time to peak tension and triosephosphate dehydrogenase activity of the plantaris muscle were strongly correlated (P less than 0.001) with myosin adenosinetriphosphatase activity. The results show that the composition and contractile properties of old skeletal muscle are considerably affected by strength training repeated during a substantial period of old age, whereas swim training only affects the endurance of the skeletal muscle.     

Conformational analysis of thioredoxin using organoarsenical reagents as probes. A time-resolved fluorescence anisotropy and size exclusion chromatography study

Reduced thioredoxin was subjected to chemical modification studies employing organoarsenical reagents specific for "spatially close" thiols. Modification was monitored by the loss in the free thiol content, by the percent incorporation of radiolabelled organoarsenical reagents, and by observing the changes in the amounts of the various thioredoxins by size exclusion chromatography. The rate of modification depends upon the polarity, rigidity, and size of the reagents. Small nonpolar organoarsenical reagents readily modified reduced thioredoxin, whereas polar and large reagents do not. Modifications resulted in the formation of stable 15-membered cyclodithioarsenite ring structures with no apparent changes in the secondary structure of the protein. Modification was reversed by the extrusion of the arsenical moiety by addition of 2,3-dimercaptopropanol. We have further characterized the oxidized, reduced, and modified thioredoxins by size exclusion chromatography and fluorescence anisotropy decay measurements. Both techniques show an increase in the hydrated volume of the protein upon reduction. Upon modification, the hydrodynamic volume of the protein further swells. Fluorescence anisotropy decay reveals that with modification there is loosening of the protein so that a "domain" containing the fluorophores can relax independently of the whole protein structure.     

A dynamic habitat selection game

A patch selection game is formulated and analyzed. Organisms can forage in one of H patches. Each patch is characterized by the cost of foraging, the density and value of food, the predation risk, and the density of conspecifics. The presence of conspecifics affects the finding and sharing of food, and the predation risk. Optimal foraging theory can be viewed as a "1-person" game against nature in which the optimal patch choice of a specific organism is analyzed assuming that the number of conspecifics in other patches is fixed. In the general game theoretic approach, the behavior of conspecifics is included in the determination of the distinguished organism's strategy. An iterative algorithm is used to compute the solution of the "n-person" game or dynamic ESS, which differs from the optimal foraging theory solution. Experiments to test the proposed theory using rodents and seed trays are briefly discussed.     

Regulation of the spatial order of cortical microtubules in developing guard cells ofAllium

The organization of microtubules (MTs) in the cortex of cells at interphase is an important element in morphogenesis. Mechanisms controlling the initiation of MTs and their spatial ordering, however, are largely unknown. Our recent study concerning the generation of a radial array of MTs in stomatal guard cells inAllium showed that the MTs initiate in a cortical MT-organizing zone adjacent to the ventral wall separating the two young guard cells (Marc, Mineyuki and Palevitz, 1989, Planta179, 516, 530). In an attempt to detect MT-ordering mechanisms separate from the sites of MT initiation, we now employ various drugs to manipulate the geometry and integrity of the ventral wall and thereby also the associated MT-organizing zone. In the presence of cytochalasin D the ventral wall is tilted away from its normal mid-longitudinal anticlinal alignment, while treatments with the herbicide chloroisopropyl-N-phenylcarbamate (CIPC) induce the formation of a branched ventral wall. Nonetheless, in either case the MTs still form a radial array, although this is asymmetric as it is centered in accordance with the misaligned or branched ventral wall. Since the MTs maintain their original course undisturbed as they extend beyond the abnormal ventral wall, there is no evidence for the presence of an inherent MT-ordering mechanism at locations remote from MT-initiation sites. Following treatments with caffeine, which abolishes the formation of the ventral wall, the MTs revert to a transversely oriented cylindrical array as in normal epidermal cells. Thus the presence of the ventral wall, and presumably also the associated MT-organizing zone, is essential for the establishment of the radial array. The MT-organizing zone is therefore involved not only in the initiation of MTs, but also in determining their spatial order throughout the cell cortex. We thank Drs. Richard J. Cyr and Yoshi Mineyuki for providing valueable suggestions during the course of this work, and Ms. Elizabeth Bruce printing some of the figures. This research was supported by Funds from the National Science Foundation grants DCB-8703292 to B.A.P. and DCB-8803286 to B.A.P. and J.M.     

Microwave fixation of the lung

A technique for microwave fixation of inflated rat lung is described. Conventional intratracheal fixation with instillation of fixative into the airways at a constant pressure results in pressure artifacts as well as flushing and disruption of cells and exudates. Microwave fixation fixes these elements in situ without disruption and thus is valuable when evaluating the distribution of inflammatory infiltrates. Exudative pneumonitis was produced in the rat using intratracheal instillations of either endotoxin or silica and comparisons were made between histologic sections fixed using either standard formalin fixation or microwave fixation.     

Standard specimens for stain calibration: application to Romanowsky-Giemsa staining.

Standardized specimens with reproducible staining properties were fabricated from extracts of biological objects (bovine liver, nucleoprotamine and defatted muscle). The standard specimens were stained with two formulations of the Romanowsky-Giemsa stain (RG), using the same azure B and eosin Y. One formulation used methanol and Sorensen's buffer and the other DMSO and Hepes buffer as solvents. The standard specimens were stained either in the composite stain or in the individual dyes dissolved in the same solvents and at the same concentration as the composite stain. Solution spectroscopy demonstrated different spectra for the two formulations with some wavelength regions varying by more than an order of magnitude. The RG spectra were also very different from those of the individual dyes dissolved at the RG concentration in the respective solvents. The stained standard specimens were analyzed by microspectrophotometry and were found to have spectra similar to those of cell smears. Furthermore, the standard specimens were shown to be a repeatable substrate for stain uptake. The transmitted light intensity from random fields of the same standardized specimen varied +/- 5%. When specimens were stained at the same time, the specimen-to-specimen variation depended on preparation conditions and the measurement wavelength, but was as good as +/- 5% for some conditions. The quantitative stain performance of both formulations was studied and compared. The standardized specimens provide a tool for the quantitative study of staining processes and specimen preparation procedures and for stain calibration.     

The protein Id: a negative regulator of helix-loop-helix DNA binding proteins

We have isolated a cDNA clone encoding a novel helix-loop-helix (HLH) protein, Id. Id is missing the basic region adjacent to the HLH domain that is essential for specific DNA binding in another HLH protein, MyoD. An in vitro translation product of Id can associate specifically with at least three HLH proteins (MyoD, E12, and E47) and attenuate their ability to bind DNA as homodimeric or heterodimeric complexes. Id is expressed at varying levels in all cell lines tested. In three cell lines that can be induced to undergo terminal differentiation, Id RNA levels decrease upon induction. Transfection experiments indicate that over-expression of Id inhibits the trans-activation of the muscle creatine kinase enhancer by MyoD. Based on these findings, we propose that HLH proteins lacking a basic region may negatively regulate other HLH proteins through the formation of nonfunctional heterodimeric complexes.     

Effect of Halide Ions on t-[35S]Butylbicyclophosphorothionate Binding

The binding of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to a site on the GABAA receptor complex is ion dependent. This study was conducted to determine the effects of ion species and concentration on the time course, affinity, and number of sites of [35S]TBPS binding. At a concentration of 200 mM ion, the time to equilibrium for [35S]TBPS binding was shortest for I-, followed by Br- less than Cl- less than F-. A similar rank order was observed for the concentration of ion required to produce half-maximal [35S]TBPS binding. Saturation binding experiments were conducted to evaluate the effect of increasing ion concentration on the KD and Bmax of [35S]TBPS binding. The Bmax was independent of both ion species and concentration. The receptor affinity, however, increased with increasing concentration for each ion. Calculated maximal affinity values were not different between ions; however, the EC50 to produce those values was different among ions and ranked in the same order as that for time course and maximal binding data. Association and dissociation rates for [35S]TBPS binding were greater in I- than in Cl-. These data emphasize the importance of ion selection and incubation times on [35S]TBPS binding.     

Use of diethyldithiocarbamate for quantitative determination of tellurite uptake by bacteria.

We have developed a simple method for the quantitative determination of tellurite in biological media. This assay is suitable for studying tellurite uptake in bacteria and overcomes the problems of older techniques which are time consuming and labor intensive. In earlier protocols diethyldithiocarbamate was reacted with tellurite and the resulting complex was extracted into organic solvents before spectrophotometric determination. In this study, diethyldithiocarbamate was incubated with tellurite at neutral pH to form a yellow colloidal solution. The absorbance of the aqueous yellow sol was used to determine tellurite concentrations in the range of 1 to 50 micrograms/ml (4 to 200 microM) without the need for solvent extraction.     

Calbindin D28k is essentially located in the colonic part of the toad intestine

Summary— The distribution of calbindin D28k in the digestive system and the urinary bladder of the toad was investigated using immunohistochemistry and Western blotting. By analogy with mammals and birds, the protein was expected to be located preferentially in the duodenal part of the intestine. Interestingly, absorptive cells of the duodenum were totally devoid of calbindin D28k while the colon contained high amounts of the calcium-binding protein. This reversed polarity of calbindin D28k content in the toad intestine should obviously correspond to a different scheme of calcium absorption regulation between amphibians and higher vertebrates. Calbindin D28k containing neuroendocrine-like cells were found scattered in the proximal parts of the gut with a similar distribution to what has been described in rat and chick intestine. The oesophagus, the stomach, and the intrinsic nervous sytem of the intestine were negative. No significant amounts of the proteins were found in the urinary bladder, which is known to be a site of Ca²⁺ active transport.