Characterization of the tryptophan fluorescence from sarcoplasmic reticulum adenosinetriphosphatase by frequency-domain fluorescence spectroscopy

We examined the tryptophan decay kinetics of sarcoplasmic reticulum Ca2+-ATPase using frequency-domain fluorescence. Consistent with earlier reports on steady-state fluorescence intensity, our intensity decays reveal a reproducible and statistically significant 2% increase in the mean decay time due to calcium binding to specific sites involved in enzyme activation. This Ca2+ effect could not be eliminated with acrylamide quenching, which suggests a global effect of calcium on the Ca2+-ATPase, as opposed to a specific effect on a single water-accessible tryptophan residue. The tryptophan anisotropy decays indicate substantial rapid loss of anisotropy, which can be the result of either intramolecular energy transfer or a change in segmental flexibility of the ATPase protein. Energy transfer from tryptophan to TNP-ATP in the nucleotide binding domain, or to IEADANS on Cys-670 and -674, indicates that most tryptophan residues are 30 A or further away from these sites and that this distance is not decreased by Ca2+. In light of known structural features of the Ca2+-ATPase, the tryptophan fluorescence changes are attributed to stabilization of clustered transmembrane helices resulting from calcium binding.     

Enhancement of tissue expansion by anticontractile agents

To evaluate the effect of anticontractile agents on the rate of tissue expansion, guinea pig back skin was expanded while being treated with various anticontractile agents. Expansion was carried out using standard percutaneous inflatable skin expanders modified by the addition of a catheter to deliver the anticontractile agents papaverine or cytochalasin D. Expansion proceeded for 25 days with one or other of the substances being infused through the catheter; saline was used in a separate control group. Measurements of the rate and extent of expansion showed that there was a statistically significant increase in these parameters for the experimental groups as compared with saline controls. Histologic examination of the expanded tissue suggests that the cellular basis for this phenomenon may involve the relaxation or inactivation of contractile fibroblasts in the fibrous capsule surrounding the expander.     

(Iodoacetamido)fluorescein labels a pair of proximal cysteines on the Ca2+-ATPase of sarcoplasmic reticulum

Previous energy transfer studies [Squier, T. C., Bigelow, D. J., de Ancos, J. G., & Inesi, G. (1987) J. Biol. Chem. 262, 4748-4754] have utilized fluorescent iodoacetamide derivatives covalently bound to the Ca2+-ATPase of sarcoplasmic reticulum (SR), using labeling conditions that completely modify the most reactive of the protein's surface sulfhydryls to a final level of 9 nmol/mg of SR protein. Unambiguous interpretation of these results requires localization of these labeling sites with respect to the primary structure of the Ca2+-ATPase. In the present study, we have used the probe 6-(iodoacetamido)fluorescein (IAF) as a marker for these sites. The IAF-labeled Ca2+-ATPase was completely proteolyzed with trypsin, followed by centrifugation to remove (unlabeled) membrane-associated portions. The soluble IAF-labeled tryptic peptides were purified by size-exclusion and reverse-phase high-performance liquid chromatography. Two IAF-peptides resulted. The major (4.1 nmol of IAF/mg of starting protein) and minor (1.9 nmol/mg) IAF-peptides were sequenced and were identified, respectively, as Ala673-IAF-Cys674-Cys675-Phe676-Ala677+ ++-Arg678 and as Glu668-Ala669-IAF-Cys670-Arg671. A model is proposed to explain the selectivity of IAF for Cys670 and Cys674 of the approximately 14 surface sulfhydryls of the Ca2+-ATPase. The labeling region, Arg667 through Arg678, has been predicted to be alpha-helical; Cys670 and Cys674 would be adjacent in the helix and imbedded in an Arg cluster. The Arg residues would both attract the anionic IAF and enhance sulfhydryl reactivities by lowering their pK values.     

Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

Abstract Two chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was approximately 250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: approximately 1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.     

Electron microscopic immunochemical localization of actin in fibroblasts in healing skin and palate wounds of beagle dog

Wound contraction in soft tissue has been attributed to the activity of contractile fibroblasts containing actin microfilaments. Immunochemical staining at the electron microscopic level was used to demonstrate the presence of such cells in healing wounds from skin and oral mucosa. Biopsies of granulation tissue from 10 and 16 day old excision wounds in beagle palate mucoperiosteum and skin were fixed and 10 micrometer sections were treated with antiactin serum, peroxidase-anti peroxidase (PAP) and then incubated to reveal the localization of actin. Controls were prepared using non-immune serum or preabsorbed immune serum. Thin sections examined with the electron microscope revealed the presence of PAP particles associated with microfilament bundles beneath the plasma membrane and in processes of fibroblasts. Reaction was also associated with micropinocytotic vesicles at the cell surface. More reactive cells were seen in 16 day than in 10 day old wounds and there were greater numbers of these cells in skin than in oral mucoperiosteum. The results indicate that actin containing cells with the ultrastructural characteristics of contractile fibroblasts (myofibroblasts) are present in the granulation tissue of healing skin and oral mucosal wounds. Such cells may be responsible for the wound contraction observed clinically in the healing palatal mucoperiosteum.     

Three-dimensional organization of fibroblasts and collagen fibrils in rat tail tendon

Summary The orderly arrangement of fibroblasts and collagen in tendons and ligaments suggests that these cells may have precise relationships with one another and with the collagen fibrils. The spatial organization of rat tail tendon was therefore examined using scanning and transmission electron microscopy and by reconstructing a 35-m long segment of tendon from serial transmission electron micrographs. Fibroblasts were regularly arranged in columns and showed more intimate association in the longitudinal than in the transverse plane. Thin cytoplasmic sheets extended up to 3 m transversely, frequently forming junctional attachments with similar processes from adjacent cells or from the same cell. Longitudinal processes were longer, often extending for more than 20 m and forming junctional attachments with other cells in the same column. Such processes often exhibited invaginations in which there were single fibrils or small groups of fibrils; this arrangement may be indicative of fibril elongation or may serve to transmit tension between the fibroblast and the collagen fibrils. This organization has interesting implications for the growth and function of other fibrous connective tissue, such as the periodontal ligament.     

Calcium occupancy of N-terminal sites within calmodulin induces inhibition of the ryanodine receptor calcium release channel

Calmodulin (CaM) regulates calcium release from intracellular stores in skeletal muscle through its association with the ryanodine receptor (RyR1) calcium release channel, where CaM association enhances channel opening at resting calcium levels and its closing at micromolar calcium levels associated with muscle contraction. A high-affinity CaM-binding sequence (RyRp) has been identified in RyR1, which corresponds to a 30-residue sequence (i.e., K3614-N3643) located within the central portion of the primary sequence. However, it is presently unclear whether the identified CaM-binding sequence in association with CaM (a) senses calcium over the physiological range of calcium concentrations associated with RyR1 regulation or alternatively, (b) plays a structural role unrelated to the calcium-dependent modulation of RyR1 function. Therefore, we have measured the calcium-dependent activation of the individual domains of CaM in association with RyRp and their relationship to the CaM-dependent regulation of RyR1. These measurements utilize an engineered CaM, permitting the site-specific incorporation of N-(1-pyrene)maleimide at either T34C (PyN-CaM) or T110C (PyC-CaM) in the N- and C-domains, respectively. Consistent with prior measurements, we observe a high-affinity association of both apo-CaM and calcium-activated CaM with RyRp. Upon association with RyRp, fluorescence changes in PyN-CaM or PyC-CaM permit the measurement of the calcium-dependent activation of these individual domains. Fluorescence changes upon calcium activation of PyC-CaM in association with RyRp are indicative of high-affinity calcium-dependent activation of the C-terminal domain of CaM at resting calcium levels; at calcium levels associated with muscle contraction, activation of the N-terminal domain occurs with concomitant increases in the fluorescence intensity of PyC-CaM that is associated with structural changes within the CaM-binding sequence of RyR1. Occupancy of calcium-binding sites in the N-domain of CaM mirrors the calcium dependence of RyR1 inhibition observed at activating calcium levels, where [Ca]1/2 = 4.3 +/- 0.4 microM, suggesting a direct regulation of RyR1 function upon the calcium-dependent activation of CaM. These results indicate that occupancy of the N-terminal domain calcium binding sites in CaM bound to the identified CaM-binding sequence K3614-N3643 induces conformational rearrangements within the complex between CaM and RyR1 responsible for the CaM-dependent modulation of the RyR1 calcium release channel.     

Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes

Dissimilatory reduction of metal (e.g. Fe, Mn) (hydr)oxides represents a challenge for microorganisms, as their cell envelopes are impermeable to metal (hydr)oxides that are poorly soluble in water. To overcome this physical barrier, the Gram-negative bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens have developed electron transfer (ET) strategies that require multihaem c-type cytochromes (c-Cyts). In S. oneidensis MR-1, multihaem c-Cyts CymA and MtrA are believed to transfer electrons from the inner membrane quinone/quinol pool through the periplasm to the outer membrane. The type II secretion system of S. oneidensis MR-1 has been implicated in the reduction of metal (hydr)oxides, most likely by translocating decahaem c-Cyts MtrC and OmcA across outer membrane to the surface of bacterial cells where they form a protein complex. The extracellular MtrC and OmcA can directly reduce solid metal (hydr)oxides. Likewise, outer membrane multihaem c-Cyts OmcE and OmcS of G. sulfurreducens are suggested to transfer electrons from outer membrane to type IV pili that are hypothesized to relay the electrons to solid metal (hydr)oxides. Thus, multihaem c-Cyts play critical roles in S. oneidensis MR-1- and G. sulfurreducens-mediated dissimilatory reduction of solid metal (hydr)oxides by facilitating ET across the bacterial cell envelope.     

Lipids of epidermis and keratinized and non-keratinized oral epithelia

Lipid compositions have been determined by quantitative thin-layer chromatography for epidermis, floor of mouth, gingiva, palate and buccal epithelium of the pig (Sus scrofa). All of the epithelia contained phospholipids, glycosylceramides, cholesteryl sulfate, cholesteryl esters, free sterols, fatty acids and triglycerides. Epidermis, palate and gingiva had similar lipid compositions, which were characterized by the presence of O-acylglycosylceramides, O-acylceramides and relatively high proportions of ceramides. Floor of mouth and buccal epithelium, which are not keratinized, contained no O-acylglucosylceramides or O-acylceramides and very little ceramide. The higher water permeability of the non-keratinized oral regions may reflect the absence of acylglucosylceramides and the low proportions of ceramides.