Applications of new saturation transfer electron paramagnetic resonance methodology to the rotational dynamics of the Ca-ATPase in sarcoplasmic reticulum membranes.

The presence of small amounts of weakly immobilized probes can result in large systematic errors in the measurement of correlation times (tau r) from saturation transfer EPR spectra. However, we have recently developed experimental methodology to minimize these errors (Squier and Thomas, Biophys. J., 49:921-935). In the present study we have applied this methodology to the measurement of the rotational motion of the Ca-ATPase in sarcoplasmic reticulum. This analysis involves the estimate of tau r from line-shape parameters (spectral line-height ratios) and intensity parameters (spectral integral), coupled with digital subtractions to remove spectral components corresponding to weakly immobilized probes. We have analyzed the ST-EPR spectra of the Ca-ATPase over a range of temperatures and find that, unlike line-shape parameters, intensity parameters are little affected by the subtraction of the weakly immobilized spectral component (W). Thus, tau r values from intensity parameters are a more reliable measurement of rotational motion. As reported previously, an analysis with line-shape parameters yields a nonlinear Arrhenius plot of protein mobility. However, the plot is linear when intensity parameters or corrected spectra are used, consistent with the theory for the hydrodynamic properties of a membrane protein of unchanging size and shape in a fluid bilayer. An analysis with line-shape parameters yields different effective tau r values in different spectral regions, and these tau r values are temperature-dependent. However, correction of spectra for W yields temperature-independent tau r ratios, indicating that the motional anisotropy is temperature-independent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spatial relationships between fibroblasts during the growth of rat-tail tendon

Sections of tendons from the base of the tail of rats were taken at eight time intervals from 18 days in utero until 244 days after birth and were examined in the electron microscope. For each time period, measurements were made of the relative area of fibroblasts, collagen and interstitial material, of the number of fibroblasts per unit area of tendon and of the average area of individual fibroblasts. The spatial arrangement of fibroblasts in the tendon sections was described quantitatively using the "nearest neighbor" method. Initially there was a rapid increase in the area of collagen accompanied by a decrease in the area occupied by fibroblasts but after 104 days of age these values changed very little. The numbers of fibroblasts per unit area decreased steadily from the embryo until 104 days whereas the average size of each cell increased to reach a maximum area at 40 days of age and then declined. At all time intervals cells were arranged in a regular, dispersed pattern across the tendon fascicles. Growth in width of the rat tail appears to involve the secretion of collagen and other intercellular material symmetrically around each fibroblast, so as to gradually separate the cells until a stage is reached at which cells are sufficiently far apart that there is little contact between adjacent cell processes. This may interfere with the integration of metabolic activity in the tissue. As a consequence, there is shrinkage of the cell bodies and a reduction in secretory activity so that, between 55 and 104 days of age, the tendon enters a period of terminal senescence.     

An Ustilago maydis Mutant Partially Blocked in P45014DM Activity Is Hypersensitive to Azole Fungicides

An Ustilago maydis ergosterol biosynthesis mutant (A14) which is partially blocked in sterol 14alpha-demethylase (P45014DM) activity is described. This mutant accumulated the abnormal 14alpha-methyl sterols, eburicol, 14alpha-methylfecosterol, and obtusifoliol, along with significant amounts of ergosterol. Although the A14 mutant grew nearly as well as the wild type, it was impaired in cell extension growth, which indicated a dysfunction in apical cell wall synthesis. The mutant was also found to be hypersensitive to the azole fungicides penconazole and tebuconazole.     

Organization of the intercellular spaces of porcine epidermal and palatal stratum corneum: a quantitative study employing ruthenium tetroxide

Previous studies have demonstrated that the intercellular spaces of the stratum corneum contain multilamellar lipid sheets with variable ultrastructure in addition to desmosomes or desmosomal remnants. The intercellular lamellae are thought to provide a permeability barrier whereas the desmosomes are responsible for cell-cell cohesion. In this study, transmission electron microscopy of RuO₄-fixed tissue was used to compare the proportions of the intercellular spaces in epidermal and palatal stratum corneum occupied by desmosomes and by different patterns of lamellae. Desmosomes are more abundant in palatal than in epidermal stratum corneum (46.9 vs 15.0% length of intercellular space). In epidermis the most frequent lamellar arrangements involve 3 (23.5%) or 6 (24.2%) lucent bands with an alternating broad-narrow-broad pattern, whereas the most frequent lamellar arrangements in palatal tissue are 2 (17.2%) or 4 (10.5%) lucent bands of uniform width. Most of the nondesmosomal portion of the intercellular space in palatal stratum corneum was dilated and had elongated lamellae at the periphery and short disorganized lamellae and amorphous electron-dense material in the interior. It is concluded that the multilamellar lipid sheets are less extensive in palatal than in epidermal stratum corneum, which could explain the greater permeability of the palate.     

The effect of stretching on formation of myofibroblasts in mouse skin

Summary Wound contraction results from the contractile activity of modified fibroblasts, termed myofibroblasts, which are present in the granulation tissue of the healing wound. This study examines the relative role of mechanical tension (stretching) and wound healing as events capable of stimulating the formation of myofibroblasts in mouse skin. The skin of hairless mice was subjected to mechanical stretching and to a small incisional wound either separately or in combination. Animals were killed at intervals between 1 and 6 days and the dermis examined with the electron microscope. Stretching alone produced little evidence of inflammation at any time interval but cells with the ultrastructural characteristics of myofibroblasts were present at 4 days and abundant at 6 days. Skin that had been both stretched and wounded showed a marked inflammatory response and also contained myofibroblasts, but they were less frequent than in the skin subjected to stretching alone. Very few myofibroblasts were evident in skin that had only been wounded. It is suggested that the effect of mechanical tension alone may initiate formation of myofibroblasts in a tissue.     

Phosphorylation-dependent changes in the spatial relationship between Ca-ATPase polypeptide chains in sarcoplasmic reticulum membranes.

In order to investigate possible structural changes associated with the coupling mechanisms of the Ca-ATPase in sarcoplasmic reticulum membranes, we have utilized fluorescence resonance energy transfer between spectroscopic probes covalently bound to different domains of the ATPase. Using time-correlated single photon counting, we have directly measured the energy transfer efficiency between 5-[2-[(iodoacetyl)amino]ethyl]aminonaphthalene-1-sulfonic acid (IAEDANS), that is specifically bound to the B trypic fragment at cysteines 670 and 674 and acceptors covalently bound either near the nucleotide binding site, i.e. fluorescein 5-isothiocyanate at lysine 515, also on the B fragment, or maleimide-directed probes specifically located on the A1, tryptic fragment, i.e. 4-dimethylaminoazobenzene-4'-maleimide (DABmal) or fluorescein-5-maleimide (Fmal), probably at cysteines 344 and 364. All of these donor-acceptor pairs exhibit energy transfer both within and between Ca-ATPase molecules allowing us to investigate spatial relationships between the A1 and B domains and between different ATPase polypeptide chains. Differentiation between the intra- and intermolecular components of energy transfer was accomplished in two ways: 1) by comparing the transfer efficiencies in native membranes before and after detergent solubilization and 2) by reconstituting ATPase chains that have already been labeled with either the donor or acceptor chromophores. Using this approach, we find no significant change in the intramolecular transfer efficiency between any of these donor-acceptor pairs either upon binding of calcium to the high affinity sites or upon stabilization of the phosphoenzyme intermediate, indicating that there are no large structural changes within the B tryptic fragment or, alternatively, between the A1 and B fragments. With respect to intermolecular energy transfer, we observe no effect of calcium binding on the unliganded enzyme with either donor-acceptor pair. However, formation of the phosphoenzyme intermediate results in a measurable increase in the transfer efficiency between IAEDANS and DABmal (or Fmal); this increase is reversible upon phosphoenzyme destabilization by subsequent addition of calcium. There is no corresponding change in the intermolecular component of fluorescence resonance energy transfer between IAEDANS and fluorescein 5-isothiocyanate, indicating that the change in fluorescence resonance energy transfer probably occurs as a result of reorientation of associated ATPase polypeptide chains with respect to one another.     

A histological method for the visualization of the intercellular permeability barrier in mammalian stratified squamous epithelia

Summary Mammalian epidermis and oral epithelia possess an intercellular permeability barrier which is located in the superficial region of the tissue. This study reports a staining reaction which appears to demonstrate a histological correlate of this functional property. Specimens of ear skin, palate, buccal and oesophageal mucosa and of cornea and bladder were obtained from adult rabbits and rats, bisected and either incubatedin vitro with 2.5% horseradish peroxidase as a tracer or fixed and processed for light microscopy and stained with a modification of Hart's elastin stain. Examination of specimens prepared by each procedure showed a complementary staining pattern in the intercellular spaces of the stratum corneum or in the superficial region of the non-keratinized tissue. In the epidermis and oral and oesophageal epithelia, the region which excluded the tracer stained with the modified elastin stain. In contrast, the corneal and bladder epithelia neither excluded the tracer nor showed intercellular staining. This relationship between staining of the intercellular space and the exclusion of tracer suggests that the intercellular material in the superficial region of epithelia may be chemically altered to form a barrier substance, possibly as the result of the discharge of the contents of the membrane-coating granules which are present in all the epithelia examined except the cornea and bladder.     

Hexazonium pararosaniline as a fixative for animal tissues

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.