Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase.

The structure of a 20-amino acid peptide inhibitor bound to the catalytic subunit of cyclic AMP-dependent protein kinase, and its interactions with the enzyme, are described. The x-ray crystal structure of the complex is the basis of the analysis. The peptide inhibitor, derived from a naturally occurring heat-stable protein kinase inhibitor, contains an amphipathic helix that is followed by a turn and an extended conformation. The extended region occupies the cleft between the two lobes of the enzyme and contains a five-residue consensus recognition sequence common to all substrates and peptide inhibitors of the catalytic subunit. The helical portion of the peptide binds to a hydrophobic groove and conveys high affinity binding. Loops from both domains converge at the active site and contribute to a network of conserved residues at the sites of magnesium adenosine triphosphate binding and catalysis. Amino acids associated with peptide recognition, nonconserved, extend over a large surface area.

Oxygen tension regulates the expression of angiogenesis factor by macrophages.

When cultured in a hypoxic environment similar to that found in the center of a wound, macrophages secreted active angiogenesis factor into the medium. Under conditions similar to those of well-oxygenated tissue, macrophages did not secrete active angiogenesis factor. Macrophages that secreted the factor at hypoxic conditions stopped secreting it when returned to room air. Thus the control of angiogenesis in wound healing may be the result of macrophages responding to tissue oxygen tension without the necessity of interacting with other cell types or biochemical signals.

Structural basis of the intrasteric regulation of myosin light chain kinases.

The smooth muscle myosin light chain kinase (smMLCK) catalytic core was modeled by using the crystallographic coordinates of the cyclic AMP-dependent protein kinase catalytic subunit (cAPK) and a bound pseudosubstrate inhibitor peptide, PKI(5-24). Despite only 30% identity in amino acid sequence, the MLCK sequence can be readily accommodated in this structure. With the exception of the short B-helix, all major elements of secondary structure in the core are very likely conserved. The active site of the modeled MLCK complements the known requirements for peptide substrate recognition. MLCK contains a pseudosubstrate sequence that overlaps the calmodulin binding domain and has been proposed to act as an intrasteric inhibitor and occupy the substrate binding site in the absence of Ca(2+)-calmodulin. The pseudosubstrate sequence can be modeled easily into the entire backbone of PKI(5-24). The results demonstrate that the intrasteric model for regulation of MLCK by intramolecular competitive inhibition is structurally plausible.

Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase.

The crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase complexed with a 20-amino acid substrate analog inhibitor has been solved and partially refined at 2.7 A resolution to an R factor of 0.212. The magnesium adenosine triphosphate (MgATP) binding site was located by difference Fourier synthesis. The enzyme structure is bilobal with a deep cleft between the lobes. The cleft is filled by MgATP and a portion of the inhibitor peptide. The smaller lobe, consisting mostly of amino-terminal sequence, is associated with nucleotide binding, and its largely antiparallel beta sheet architecture constitutes an unusual nucleotide binding motif. The larger lobe is dominated by helical structure with a single beta sheet at the domain interface. This lobe is primarily involved in peptide binding and catalysis. Residues 40 through 280 constitute a conserved catalytic core that is shared by more than 100 protein kinases. Most of the invariant amino acids in this conserved catalytic core are clustered at the sites of nucleotide binding and catalysis.

A template for the protein kinase family.

The crystal structure of the catalytic subunit of cAMP-dependent protein kinase, complexed with ATP and a 20-residue inhibitor peptide, is reviewed and correlated with chemical and genetic data. The striking convergence of the structure with the biochemistry and genetics provides for the first time a molecular basis for understanding how this enzyme functions, as well as an explanation for the highly conserved residues that are scattered throughout the molecule. Because these residues probably serve a common role in all eukaryotic protein kinases, this first protein kinase structure serves as a general template for the entire family of enzymes.

A three-dimensional model of the Cdc2 protein kinase: localization of cyclin- and Suc1-binding regions and phosphorylation sites.

The Cdc2 protein kinase requires cyclin binding for activity and also binds to a small protein, Suc1. Charged-to-alanine scanning mutagenesis of Cdc2 was used previously to localize cyclin A- and B- and Suc1-binding sites (B. Ducommun, P. Brambilla, and G. Draetta, Mol. Cell. Biol. 11:6177-6184, 1991). Those sites were mapped by building a Cdc2 model based on the crystallographic coordinates of the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) (D. R. Knighton, J. Zheng, L. F. Ten Eyck, V. A. Ashford, N.-H. Xuong, S. S. Taylor, and J. M. Sowadski, Science 253:407-414, 1991). On the basis of this model, additional mutations were made and tested for cyclin A and Suc1 binding and for kinase activity. Mutations that interfere with cyclin A binding are localized primarily on the small lobe near its interface with the cleft and include an acidic patch on the B helix and R-50 in the highly conserved PSTAIRE sequence. Two residues in the large lobe, R-151 and T-161, influence cyclin binding, and both are at the surface of the cleft near its interface with the PSTAIRE motif. Cyclin-dependent phosphorylation of T-161 in Cdc2 is essential for activation, and the model provides insights into the importance of this site. T-161 is equivalent to T-197, a stable phosphorylation site in cAPK. On the basis of the model, cyclin binding very likely alters the surface surrounding T-161 to allow for T-161 phosphorylation. The two major ligands to T-197 in cAPK are conserved as R-127 and R-151 in Cdc2. The equivalent of the third ligand, H-87, is T-47 in the PSTAIRE sequence motif. Once phosphorylated, T-161 is predicted to play a major structural role in Cdc2, comparable to that of T-197 in cAPK, by assembling the active conformation required for peptide recognition. The inhibitory phosphorylation at Y-15 also comes close to the cleft interface and on the basis of this model would disrupt the cleft interface and the adjacent peptide recognition site rather than prevent ATP binding. In contrast to cyclin A, both lobes influence Suc1 binding; however, the Suc1-binding sites are far from the active site. Several mutants map to the surface in cAPK, which is masked in part by the N-terminal 40 residues that lie outside the conserved catalytic core. The other Suc1-binding site maps to the large lobe near a 25-residue insert and includes R-215.

Electrostatic channeling in the bifunctional enzyme dihydrofolate reductase-thymidylate synthase.

The bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) carries out two distinct reactions, with the dihydrofolate produced by the TS-catalyzed reaction acting as the substrate for the DHFR-catalyzed reaction. Brownian dynamics simulation techniques were used to investigate the possible role of electrostatics in determining efficient channeling of the substrate, by explicitly simulating substrate diffusion between the two active sites. With a substrate charge of -2, almost all (> 95%) substrate molecules leaving the TS active site reached the DHFR active site at zero ionic strength. Under the same conditions, but in the absence of electrostatic effects, successful channeling was reduced to only around 6%: electrostatic effects therefore appear essential to explain the efficient channeling observed experimentally. The importance of substrate charge, the relative contributions of specific basic residues in the protein, the role played by the second monomer of the dimer in channeling and the effects of changing ionic strength were all investigated. Simulations performed for substrate transfer in the opposite direction suggest that channeling in DHFR-TS is not strongly directional and that the role of electrostatics is perhaps more one of restricting diffusion of the substrate than one of actively guiding it from the TS to the DHFR active site. The results demonstrate that electrostatic channeling can be a highly efficient means of transferring charged substrates between active sites in solvent-exposed environments.

Crystallization studies of cAMP-dependent protein kinase. Cocrystals of the catalytic subunit with a 20 amino acid residue peptide inhibitor and MgATP diffract to 3.0 A resolution.

Crystallographic studies of the catalytic subunit of cAMP-dependent protein kinase demonstrate that the presence of a 20 amino acid residue peptide inhibitor and MgATP during crystallization yields crystals with a different space group and, more significantly, makes an important difference in the quality of the resulting crystals. Under identical experimental conditions, the kinase crystallizes in a cubic space group P4(1)32 (a = b = c = 169.24 A), when no substrates or inhibitors are present, and in the hexagonal space group P6(1)22 (or P6(5)22) (a = b = 80.16 A, c = 288.07 A, alpha = beta = 90 degrees, gamma = 120 degrees) when a 20-amino acid residue peptide inhibitor and MgATP are present. Moreover, the hexagonal crystal diffracts to a resolution of 3.0 A, while the cubic crystals diffract to a resolution of 4.0 A.

Crystal structures of the myristylated catalytic subunit of cAMP-dependent protein kinase reveal open and closed conformations.

Three crystal structures, representing two distinct conformational states, of the mammalian catalytic subunit of cAMP-dependent protein kinase were solved using molecular replacement methods starting from the refined structure of the recombinant catalytic subunit ternary complex (Zheng, J., et al., 1993a, Biochemistry 32, 2154-2161). These structures correspond to the free apoenzyme, a binary complex with an iodinated inhibitor peptide, and a ternary complex with both ATP and the unmodified inhibitor peptide. The apoenzyme and the binary complex crystallized in an open conformation, whereas the ternary complex crystallized in a closed conformation similar to the ternary complex of the recombinant enzyme. The model of the binary complex, refined at 2.9 A resolution, shows the conformational changes associated with the open conformation. These can be described by a rotation of the small lobe and a displacement of the C-terminal 30 residues. This rotation of the small lobe alters the cleft interface in the active-site region surrounding the glycine-rich loop and Thr 197, a critical phosphorylation site. In addition to the conformational changes, the myristylation site, absent in the recombinant enzyme, was clearly defined in the binary complex. The myristic acid binds in a deep hydrophobic pocket formed by four segments of the protein that are widely dispersed in the linear sequence. The N-terminal 40 residues that lie outside the conserved catalytic core are anchored by the N-terminal myristylate plus an amphipathic helix that spans both lobes and is capped by Trp 30. Both posttranslational modifications, phosphorylation and myristylation, contribute directly to the stable structure of this enzyme.

Changes in the aortic wall oxygen tensions of hypertensive rabbits. Hypertension and aortic wall oxygen.

Hypertension is a known risk factor for atherosclerosis. We hypothesize that hypertension causes artery wall hypoxia that contributes to the formation of atherosclerotic lesions. Therefore, we examined the effect of hypertension on the transarterial wall oxygen gradient of the rabbit aorta. Hypertensive rabbits were created by unilateral nephrectomy and contralateral renal artery narrowing. Transarterial wall oxygen gradients of the infrarenal aorta were measured using an oxygen microelectrode 14-16 weeks (short-term hypertension) and 56-58 weeks (long-term hypertension) after the rabbits were made hypertensive. The transarterial wall oxygen gradients showed significant differences among the groups. Short-term hypertension caused significantly higher oxygen tensions in the outer 30% of the artery wall and significant thinning of the artery wall when compared with long-term hypertension and control groups. Long-term hypertension caused significantly lower oxygen tensions in the inner 40% of the artery wall and significant thickening of the artery wall when compared with short-term hypertension and control groups. These changes were noted despite no difference in the partial pressure of oxygen in arterial blood or visual evidence of atherosclerotic lesion formation in the three groups. These findings suggest that hypertension alters the transarterial wall oxygen gradient. This altered transarterial wall oxygen gradient may contribute to the formation of atherosclerotic lesions.

Proliferation of wound derived capillary endothelial cells: young versus aged.

The objective of this study was to compare the proliferative potential of wound derived capillary endothelial cells (WCEC) from aged and young rats. Endothelial cells were isolated from subcutaneously implanted sponges in 2- and 24-month-old rats. The identity of the cells as endothelial was confirmed by staining for Ac-LDL uptake. Aged and young WCEC (20,000/well) were stimulated with increasing concentrations of fetal calf serum (0, 2.5, 5, 10 and 15%). The increase in cell number was determined with a Coulter counter. At all serum concentrations, the proliferative capacity of WCEC from aged rats was significantly higher than that of WCEC from young rats.

Age-related alterations in the morphology of femoral artery vasa vasorum in the rat.

The objective of this study was to explore any age-related morphological changes in the vasa vasorum of the rat femoral artery. Vascular corrosion casts were prepared from 2, 12 and 24-month-old rats. Examination of the casts with the scanning electron microscope revealed dramatic differences in the appearance of the vessels of young and aged rats. The vasa vasorum of 2-month-old rats consisted of a dense network of capillaries. These vessels were dramatically reduced in number by 12 months, and even fewer capillaries were present at 24 months. This reduction in capillary density is consistent with the observed age-related decreases in oxygen tension and may explain why the aged are more prone to atherosclerosis.

Structure-based design of novel, urea-containing FKBP12 inhibitors.

The structure-based design and subsequent chemical synthesis of novel, urea-containing FKBP12 inhibitors are described. These compounds are shown to disrupt the cis-trans peptidylprolyl isomerase activity of FKBP12 with inhibition constants (Ki,app) approaching 0.10 microM. Analyses of several X-ray crystal structures of FKBP12-urea complexes demonstrate that the urea-containing inhibitors associate with FKBP12 in a manner that is similar to, but significantly different from, that observed for the natural product FK506.

Combined laser phototherapy and growth factor treatment of bronchial obstruction after lung transplantation.

Lung transplantation has resulted in dramatic functional improvement in patients with end-stage pulmonary diseases. Among the complications of lung transplantation are dehiscence and stenosis at the site of the bronchial or tracheal anastomosis. In this case report, we describe a single lung transplant recipient in whom partial bronchial dehiscence, followed by exuberant growth of granulation tissue, resulted in obstruction of the bronchial lumen. After mechanical dilation failed to produce lasting relief of bronchial obstruction, a novel approach to this problem was successfully employed: YAG laser phototherapy was used to remove obstructing granulation tissue, followed by application of a preparation derived from autologous blood platelets to promote epithelialization of the bronchial anastomosis. The bronchus remains patent and fully epithelialized six months after therapy.

Regulation of wound-healing angiogenesis-effect of oxygen gradients and inspired oxygen concentration.

Tissue hypoxia is a well-known stimulus to angiogenesis. The central dead space in healing wounds has been shown to be hypoxic (PO2 = 1 to 10). Angiogenesis is a necessary component of all healing wounds. Rabbit ear chambers were used to explore the contribution of O2 gradients and various inspired oxygen concentrations on wound healing and angiogenesis. These experiments demonstrate that: (1) A hypoxic tissue gradient is mandatory for wound-healing angiogenesis, (2) when the hypoxic gradient is destroyed capillary growth cases, and (3) inspired oxygen concentrations affect the rate and density of capillary growth.

Studies on inflammation and wound healing: angiogenesis and collagen synthesis stimulated in vivo by resident and activated wound macrophages.

Wound inflammatory cells were harvested by aspiration of fluid from the "dead space" of subcutaneous rabbit wounds and transplanted into the cornea where, compared with suitable controls, they stimulated healing (i.e., angiogenesis, fibroplasia, and new collagen synthesis, which led to formation of visible, vascularized scar tissue). Analysis of the data and the literature supports the conclusions that: (1) inflammatory cells control the continuation of the repair process after the immediate effects of injury subside; (2) macrophages, as opposed to granulocytes, appear to be the major contributors; (3) activated by their presence in the wound, macrophages release substances that stimulate fibroplasia, collagen synthesis, and angiogenesis in vivo; and (4) tissue injury is not a maximum stimulus to repair, since endotoxin-treated macrophages have increased capacity to stimulate collagen synthesis and angiogenesis. A hypothesis is offered to explain the notorious clinical discrepancy between the extent of injury and the extent of repair.

Oxygen as an antibiotic. The effect of inspired oxygen on infection.

Granulocytes' in vitro bactericidal capacity for certain bacteria depends on an adequate environmental oxygen supply. Oxygen available to granulocytes in infected tissue is decreased by local conditions and can be altered significantly by small changes in its inspired concentration. We modified Burke's and Miles' bacteria-injection model in animals to test the effect of breathing 12%, 21%, and 45% oxygen on the size of lesions produced by intradermal injections of Escherichia coli. Moderately increased inspired oxygen concentrations (fraction of inspired oxygen [Flo2]) significantly decreased the size and number of necrotic lesions, whereas hypoxia increased both. Increasing Flo2 to 45% after three and 24 hours of hypoxia also significantly decreased lesion size. Suppression of infection by moderate hyperoxia is comparable with that reported by Burke after timely, adequate doses of type-specific antibiotics.

Oxygen as an antibiotic. A comparison of the effects of inspired oxygen concentration and antibiotic administration on in vivo bacterial clearance.

Since prophylactic antibiotics and changes in tissue partial pressure of oxygen may affect bacterial clearance by different mechanisms, we tested the effects of hypoxia, hyperoxia, and normoxia with and without antibiotic administration on bacterial clearance. We found that improving tissue oxygenation by administration of normobaric oxygen decreased infectious necrosis as effectively as prophylactic antibiotic administration and that improved tissue oxygenation and antibiotic administration had an additive effect. We believe that a fraction of inspired oxygen of 45% should be added to prophylactic antibiotics as standard perioperative and postoperative care.

Oxygen as an antibiotic. The effect of inspired oxygen on bacterial clearance.

Previous experimentation with the guinea pig skin injection model showed that altering the fraction of inspired oxygen had a significant effect on infectious necrosis. Using the same model, we performed quantitative bacterial cultures to determine the number of viable injected bacteria 24 and 48 hours after injection. Animals were randomized to receive 12%, 21%, and 45% inspired oxygen. A significant decrease in bacterial number was seen at 45% inspired oxygen between 24 and 48 hours, and a significant decrease occurred at 48 hours between 12% and 45% inspired oxygen. These results demonstrated a prominent role for oxygen in bacterial clearance and host defense.

Definitions and guidelines for assessment of wounds and evaluation of healing.

BACKGROUND: Chronic wounds represent a worldwide problem. For laboratory and clinical research to adequately address this problem, a common language needs to exist. OBSERVATION: This language should include a system of wound classification, a lexicon of wound descriptors, and a description of the processes that are likely to affect wound healing and wound healing end points. CONCLUSIONS: The report that follows defines wound, acute wound, chronic wound, healing and forms of healing, wound assessment, wound extent, wound burden, and wound severity. The utility of these definitions is demonstrated as they relate to the healing of a skin wound, but these definitions are broadly applicable to all wounds.