Regulation of Ca2+ transport by sarcoplasmic reticulum Ca2+-ATPase at limiting [Ca2+

The factors regulating Ca2+ transport by isolated sarcoplasmic reticulum (SR) vesicles have been studied using the fluorescent indicator Fluo-3 to monitor extravesicular free [Ca2+]. ATP, in the presence of 5 mM oxalate, which clamps intravesicular [Ca2+] at approximately 10 microM, induced a rapid decline in Fluo-3 fluorescence to reach a limiting steady state level. This corresponds to a residual medium [Ca2+] of 100 to 200 nM, and has been defined as [Ca2+]lim, whilst thermodynamic considerations predict a level of less than 1 nM. This value is similar to that measured in intact muscle with Ca2+ fluophores, where it is presumed that sarcoplasmic free [Ca2+] is a balance between pump and leaks. Fluorescence of Fluo-3 at [Ca2+]lim was decreased 70% to 80% by histidine, imidazole and cysteine. The K0.5 value for histidine was 3 mM, suggesting that residual [Ca2+]lim fluorescence is due to Zn2+. The level of Zn2+ in preparations of SR vesicles, measured by atomic absorption, was 0.47+/-0.04 nmol/mg, corresponding to 0.1 mol per mol Ca-ATPase. This is in agreement with findings of Papp et al. (Arch. Biochem. Biophys., 243 (1985) 254-263). Histidine, 20 mM, included in the buffer, gave a corrected value for [Ca2+]lim of 49+/-1.8 nM, which is still higher than predicted on thermodynamic grounds. A possible 'pump/leak' mechanism was tested by the effects of varying active Ca2+ transport 1 to 2 orders with temperature and pH. [Ca2+]lim remained relatively constant under these conditions. Alternate substrates acetyl phosphate and p-NPP gave similar [Ca2+]lim levels even though the latter substrate supported transport 500-fold slower than with ATP. In fact, [Ca2+]lim was lower with 10 mM p-NPP than with 5 mM ATP. The magnitude of passive efflux from Ca-oxalate loaded SR during the steady state of [Ca2+]lim was estimated by the unidirectional flux of 45Ca2+, and directly, following depletion of ATP, by measuring release of 40Ca2+, and was 0.02% of Vmax. Constant infusion of CaCl2 at [Ca2+]lim resulted in a new steady state, in which active transport into SR vesicles balances the infusion rate. Varying infusion rates allows determination of [Ca2+]-dependence of transport in the absence of chelating agents. Parameters of non-linear regression were Vmax=853 nmol/min per mg, K0.5(Ca)=279 nM, and nH(Ca)=1.89. Since conditions employed in this study are similar to those in the sarcoplasm of relaxed muscle, it is suggested that histidine, added to media in studies of intracellular Ca2+ transients, and in the relaxed state, will minimise contribution of Zn2+ to fluophore fluorescence, since it occurs at levels predicted in this study to cause significant overestimation of cytoplasmic free [Ca2+] in the relaxed state. Similar precautions may apply to non-muscle cells as well. This study also suggests that [Ca2+]lim in the resting state is a characteristic feature of Ca2+ pump function, rather than a balance between active transport and passive leakage pathways.     

Specificity of the dynorphin-processing endoprotease: comparison with prohormone convertases

The cleavage specificity of a monobasic processing dynorphin converting endoprotease is examined with a series of quench fluorescent peptide substrates and compared with the cleavage specificity of prohormone convertases. A dynorphin B-29-derived peptide, Abz-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-Arg-Ser-Glneddnp (where Abz is o-aminobenzoyl and eddnp is ethylenediamine 2,4-dinitrophenyl), that contains both dibasic and monobasic cleavage sites is efficiently cleaved by the dynorphin converting enzyme and not cleaved by two propeptide processing enzymes, furin and prohormone convertase 1. A shorter prorenin-related peptide, Dnp-Arg-Met-Ala-Arg-Leu-Thr-Leu-eddnp, that contains a monobasic cleavage site is cleaved by the dynorphin converting enzyme and prohormone convertase 1 and not by furin. Substitution of the P1' position by Ala moderately affects cleavage by the dynorphin-processing enzyme and prohormone convertase 1. It is interesting that this substitution results in efficient cleavage by furin. The site of cleavage, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry, is N-terminal to the Arg at the P1 position for the dynorphin converting enzyme and C-terminal to the Arg at the P1 position for furin and prohormone convertase 1. Peptides with additional basic residues at the P2 and at P4 positions also serve as substrates for the dynorphin converting enzyme. This enzyme cleaves shorter peptide substrates with significantly lower efficiency as compared with the longer peptide substrates, suggesting that the dynorphin converting enzyme prefers longer peptides that contain monobasic processing sites as substrates. Taken together, these results suggest that the cleavage specificity of the dynorphin converting enzyme is distinct but related to the cleavage specificity of the prohormone convertases and that multiple enzymes could be involved in the processing of peptide hormones and neuropeptides at monobasic and dibasic sites.     

Insights into strand displacement and processivity from the crystal structure of the protein-primed DNA polymerase of bacteriophage phi29

The DNA polymerase from phage phi29 is a B family polymerase that initiates replication using a protein as a primer, attaching the first nucleotide of the phage genome to the hydroxyl of a specific serine of the priming protein. The crystal structure of phi29 DNA polymerase determined at 2.2 A resolution provides explanations for its extraordinary processivity and strand displacement activities. Homology modeling suggests that downstream template DNA passes through a tunnel prior to entering the polymerase active site. This tunnel is too small to accommodate double-stranded DNA and requires the separation of template and nontemplate strands. Members of the B family of DNA polymerases that use protein primers contain two sequence insertions: one forms a domain not previously observed in polymerases, while the second resembles the specificity loop of T7 RNA polymerase. The high processivity of phi29 DNA polymerase may be explained by its topological encirclement of both the downstream template and the upstream duplex DNA.     

Modeling Sustainability of Arctic Communities: An Interdisciplinary Collaboration of Researchers and Local Knowledge Holders

How will climate change affect the sustainability of Arctic villages over the next 40 years? This question motivated a collaboration of 23 researchers and four Arctic communities (Old Crow, Yukon Territory, Canada; Aklavik, Northwest Territories, Canada; Fort McPherson, Northwest Territories, Canada; and Arctic Village, Alaska, USA) in or near the range of the Porcupine Caribou Herd. We drew on existing research and local knowledge to examine potential effects of climate change, petroleum development, tourism, and government spending cutbacks on the sustainability of four Arctic villages. We used data across eight disciplines to develop an Arctic Community Synthesis Model and a Web-based, interactive Possible Futures Model. Results suggested that climate warming will increase vegetation biomass within the herd’s summer range. However, despite forage increasing, the herd was projected as likely to decline with a warming climate because of increased insect harassment in the summer and potentially greater winter snow depths. There was a strong negative correlation between hypothetical, development-induced displacement of cows and calves from utilized calving grounds and calf survival during June. The results suggested that climate warming coupled with petroleum development would cause a decline in caribou harvest by local communities. Because the Synthesis Model inherits uncertainties associated with each component model, sensitivity analysis is required. Scientists and stakeholders agreed that (1) although simulation models are incomplete abstractions of the real world, they helped bring scientific and community knowledge together, and (2) relationships established across disciplines and between scientists and communities were a valuable outcome of the study. Additional project materials, including the Web-based Possible Futures Model, are available at http://www.taiga.net/sustain.     

Altered bleomycin-induced lung fibrosis in osteopontin-deficient mice

Osteopontin is a multifunctional matricellular protein abundantly expressed during inflammation and repair. Osteopontin deficiency is associated with abnormal wound repair characterized by aberrant collagen fibrillogenesis in the heart and skin. Recent gene microarray studies found that osteopontin is abundantly expressed in both human and mouse lung fibrosis. Macrophages and T cells are known to be major sources of osteopontin. During lung fibrosis, however, osteopontin expression continues to increase when inflammation has receded, suggesting alternative sources of ostepontin during this response. In this study, we demonstrate immunoreactivity for osteopontin in lung epithelial and inflammatory cells in human usual interstitial pneumonitis and murine bleomycin-induced lung fibrosis. After treatment with bleomycin, osteopontin-null mice develop lung fibrosis characterized by dilated distal air spaces and reduced type I collagen expression compared with wild-type controls. There is also a significant decrease in levels of active transforming growth factor-beta(1) and matrix metalloproteinase-2 in osteopontin null mice. Type III collagen expression and total collagenase activity are similar in both groups. These results demonstrate that osteopontin expression is associated with important fibrogenic signals in the lung and that the epithelium may be an important source of osteopontin during lung fibrosis.     

The Protein Data Bank and structural genomics

The Protein Data Bank (PDB; http://www.pdb.org/) continues to be actively involved in various aspects of the informatics of structural genomics projects--developing and maintaining the Target Registration Database (TargetDB), organizing data dictionaries that will define the specification for the exchange and deposition of data with the structural genomics centers and creating software tools to capture data from standard structure determination applications.     

A desert octodontid rodent,Tympanoctomys barrerae,uses modified hairs for stripping epidermal tissue from leaves of halophytic plants

Desert rodents that consume halophytic plants must have adaptations for coping with the high salt content of the leaves. A kidney capable of excreting very concentrated urine is one method. Another is removal of the hypersaline epidermis by means of chisel-like incisors prior to ingestion of the leaves. Tympanoctomys barrerae has evolved a unique refinement of the latter adaptation. It possesses two bundles of stiffened hairs on either side of the palate just caudal to the incisors. The bundles vibrate against the lower incisors, removing the epidermis from the leaves. The efficiency of the operation is significantly greater than with the use of incisors alone. Such a device has not been described in any other mammal. The facial muscles associated with the lips, the cheek vibrissae, and the oral cavity are described in T. barrerae and the nonhalophilic octodontid Octomys mimax. M. buccinatorius pars intermaxillaris is the only muscle in direct contact with the bristle bundles. Other anatomical features found in T. barrerae that may be associated with this feeding device are: 1) a much enlarged and mobile lower labial pad operated by Mm. buccinatorius pars orbicularis oris, pars longitudinalis profunda, and mandibularis cranialis profunda; 2) two oral glands not described in other rodents; and 3) a shortened tongue. Although, taken as a whole, this epidermal stripping device is unique to T. barrerae, most of its features have evolved by modification of structures present in the facial region of more generalized rodents.